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Cell-mediated immunity in polyoma oncogenesis
Europ. d. Cancer Vol. 8, pp. 1-8. Pergamon Press 1972. Printed in Great Britain.
Cell-Mediated Immunity in Polyoma Oncogenesis* M. VANDEPUTTE and S. K. DATTA Rega Institute for Medical Research, University of I_,awen, Belgium Absta'act--Primary polyoma virus-induced tumors in rats were prevented by the transfer of sensitized lymphoid cells. Sensitized lymphoid cellsfrom donors infected with the virus or immunized with aUogeneicpolyoma tumors were both effective. The e~icaey of the transfer gradually decreased with the increase in the period of time between the virus inoculation and the injection of the immune cells. Non-sensitized lymphoid cells were only effective when given before the virus inoculation. By this in vivo transfer assay the gradual development of the cell-mediated immunity directed against polyoma T S T A could be demonstrated, indicating the absence of tolerance or unresponsiveness to the tumor antigens.
INTRODUCTION POLYOMA virus-induced tumors in the rat contain tumor-specific transplantation antigen(s) (TSTA) foreign to the normal tissues of the host [1]. These T S T A evoke in the host an immunologic response, chiefly of the homograft-rejection type, which can be detected in vivo as well as in vitro [2]. Furthermore, the strength of this cellular immunologic response seems of paramount importance in determining whether or not a neoplastic clone will lead to the development of a clinical tumor. To assess the role of cell-mediated immune mechanisms in polyoma oncogenesis two main approaches are used. One consists in verifying the influence of immunodepression (X-irradiation, thymectomy, ALS) on the tumor incidence or tumorlatency period [3-7]. The alternate approach is to test the tumor repression effectiveness of an increased cellular immunologic response obtained by non-specific stimulation such as BCG [8] or poly I : C [9], or by specific stimulation as achieved by transfer of normal or sensitized lymphoid cells [ 10-12]. In the present study we tried to evaluate by the transfer of sensitized syngeneic lymphoid cells (a) the period after virus inoculation during which Accepted 5 October 1971. *This work was supported by a grant from the Belgian "Algemene Spaar- en Lijfrentekas".
tumor development can be prevented in nonimmunosuppressed syngeneic rats, and (b) the appearance of cellular immunity during the process of primary polyoma oncogenesis.
MATERIAL AND M E T H O D S Animals Rats of the inbred strains Wistar R and Brown Norway (BN) were used, They were kept on a standard diet of pellets and water ad libitum. Virus Polyoma virus was grown on mouse embryo fibroblast cultures (MEF) and prepared by the method of Crawford [ 13]. The virus was titrated with the plaque method of Dulbecco and Freeman on MEF cells [14]. Transplantable tumors and immunization procedures Adult female R rats were inoculated 3 times subcutaneously (s.c.) at weekly intervals with a BN transplantable polyoma fibrosarcoma. This tumor was free of demonstrable virus as determined by the absence of hemagglutinationinhibiting antibodies in the recipients and by in vitro culturing. Control R rats were immunized with an allogeneic (BN) transplantable 7,12-dimethylbenz(a) anthracene (DMBA) sar-
2
M. Vandeputteand S. K. Datta
coma following the same schedule as for the BN polyoma tumor. Five to six days after the last immunization, dissociated lymphoid cells were obtained from the spleen and the combined cervical, inguinal and axillary lymph nodes and prepared by the method described elsewhere [2]. The lymphoid cells were counted and the percentage of living cells determined by trypan blue exclusion. A suspension was made up in Eagle medium to contain 10 x 106-100 x 106 living lymphoid cells in 0" l ml and was inoculated either intraperitoneaUy (i.p.) or intravenously (i.v.) in the lateral tail vein at the concentration of 2-5 x 106 cells/g of body weight. Serum was obtained by cardiac puncture from the same immunized syngeneic donor rats. The sera were stored at - 20°C. Another group of adult female R rats was immunized by high doses ofpolyoma virus, having a titer of 1 × 109 plaque-forming units (PFU) per ml, administered s.c. on the right and left flank alternatively and i.p. twice a week for approximately 8 weeks. Lymph nodes and spleen were removed 4 to 6 days after the last inoculation and processed as described above. The serum obtained by cardiac puncture was inactivated for ½ hr at 56°C. This hyperimmune serum had a titer of 1 : 16.384, as determined by t h e hemagglutination-inhibition test [ 15].
Histology Eight to nine weeks after inoculation all surviving rats were killed after bleeding by cardiac puncture. Rats developing clinical signs of tumor growth before this period were processed in the same way. The kidney and brain, as well as the lymphoid organs, were fixed in Bouin's fluid and further processed for histological examination. RESULTS
Transfer of lymphoid cells or serum from polyoma virus immunized syngeneic donors Litters of R rats were inoculated i.p. with 5 x 106 PFU of polyoma virus, 5 days after birth. They were divided into different experimental groups depending upon the time interval after birth or virus inoculation when transfer of lymphoid cells or hyperimmune serum was effected. The results shown in Table 1 indicate that the transfer of 2 x 106 sensitized (immunized) lymphoid ceils per gram of body weight protected the recipients against tumor development when the transfer was effected either before (group 1A) or 8 days after virus inoculation (group 2A). Sensitized lymphoid cells transferred 14 or 21 days after virus inoculation had no
protective effect. Normal non-sensitized lymphoid cells were effective only when given neonatally and at the level of 5 × 106 cells/g of body weight (group 1C). Hyperimmune serum injected i.p. in 0.1 ml amounts at 1, 3, and 4 days after birth was very effective in preventing tumor development (group 5). Serum treatment started 5 days after the virus inoculation (i.e. 10 days after birth); however, it was totally ineffective (group 6).
Transfer of lymphoid cells from syngeneic donOrs immunized by allogeneicpolyoma tumor or aUogeneic DMBA tumor This experiment was carried out to check the effectiveness of lymphoid cells sensitized against polyoma T S T A or unrelated (DMBA) T S T A in the prevention of the induction of polyoma tumors in rats. The design of the experiment was the same as in the previous one. Yet to increase the sensitivity of the system the recipients were infected with a lower dose of virus, 106 P F U instead of 5 x 106 P F U and the number of transferred lymphoid cells was increased from 2 × 106-5 x 106/gofbodyweight intherecipients treated 14 and 21 days after virus inoculation. As shown in Table 2, a significant reduction in the tumor incidence was observed in all the experimental groups receiving sensitized (antipolyoma TSTA) lymphoid ceils. However, the reduction gradually decreased with the increase of the time period between virus infection and transfer of lymphoid cells. The transfer of lymphoid cells not sensitized against polyoma T S T A was only effective when administered neonatally and had no protective effect given after the virus inoculation. The serum obtained from the donors of antipolyoma TSTA sensitized lymphoid cells and inoculated i.p. in 0.2 ml amounts at 5, 7, and 9 days after the virus inoculation had no protective effect. This serum did not contain antipolyoma virus antibodies (HAI titer < 1/120). In the groups transferred with DMBA tumor sensitized lymphoid cells and, in the control group, about 10% of the rats (11/128) had to be killed, having clinical symptoms of tumor development before the end of the experiment (8-9 weeks after virus inoculation). In the groups treated with anti-polyoma TSTA sensitized lymphoid cells, 3 out of 105 rats were autopsied with tumors 5 to 6 weeks after the virus inoculation. As the overall tumor incidence was also much lower in the experimental groups compared to the control groups, one cannot conclude from these small numbers that the tumor progression was slower in rats transferred
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Cell-Mediated Immunity in Polyoma Oneogenesis with anti-polyoma TSTA sensitized lymphoid cells. The histopathological examination of the tumors did not reveal differences in tumor histology between the experimental and the control groups. In only two cases (1 rat in group 1A and 1 rat in group 2B) did the histology reveal neoplastic foci in the kidney which had not been noted at the macroscopic examination as tumor.
Development of anti-polyoma TSTA cellular immunity during the process of polyoma oncogenesisin rats To verify the development of cellular immunity directed against polyoma TSTA during the process of primary polyoma oncogenesis, we used the same experimental model as described above. The rats (donor animals) in which the appearance of this cellular mediated immunity was verified were inoculated 3 days after birth i.p. with 5 × 106 PFU polyoma virus. Previous experiments had shown that such a dose induced polyoma tumors in 95-100% of the animals. Lymphoid cells were prepared from these donors respectively 6, 15, and 21 days after the virus inoculation and transferred i.v. into 13-day old syngeneic recipients rats inoculated 8 days previously at the age of 5 days with 106 PFU polyoma virus i.p. We chose the experimental model of transferring the lymphoid cells into recipients inoculated 8 days before with the virus for the following reasons: (a) as shown in the previous experiments, the transfer of lymphoid ceils 8 days after virus inoculation is a very sensitive system to detect antipolyoma TSTA cellular immunity; (b) the protective effect of the transferred lymphoid cells, if present, cannot be caused by an antiviral action; (c) 13-day old recipients do not necessitate as many donors as older recipient animals would do. Taking into account the number of lymphoid cells required for transfer and the relatively small numbers recovered from young donors transfer into older animals would require too great a number of donors; (d) normal, non-sensitized lymphoid cells do not exert any protective effect when transferred after polyoma virus inoculation. The results of this experiment, as shown in Table 3, indicate that recipient rats were significantly protected by lymphoid cells removed from donors 15 or 21 days after polyoma virus inoculation. Donor lymphoid cells removed earlier, e.g. 6 days after virus inoculation, were apparently not able to protect the recipients.
5
DISCUSSION
Our results illustrate the efficacy of syngeneic lymphoid cells sensitized against polyoma TSTA (by allogeneic polyoma tumor cells) in the prevention of primary polyoma virus induced neoplasms. This is in accordance with the results obtained in immunosuppressed mice injected with polyoma virus or with Moloney sarcoma virus and afterwards immunologically restored with sensitized lymphoid cells [12, 16, 17]. Transfer of sensitized thoracic duct cells was also effective in protecting against chemical carcinogen induced tumors [18]. However, in the latter case the experiments were carried out in a non-syngeneic system. Whereas lymphoid cells from donors sensitized with allogeneic polyoma tumors gave a good protection, the serum from the same animals was ineffective. Lymphoid cells from donors immunized with polyoma virus were also found to be effective, whether transferred before or 8 days after the virus inoculation. In the latter case their efficacy cannot be due to a transfer of an antiviral serum-mediated immunity as shown by the inability of high doses of hyperimmune antipolyoma virus antiserum to inhibit the tumor development when injected after the virus. Moreover, previous studies have shown that in the rat the virus persists only for a very short period of time after its inoculation [6]. Hence, it is very likely that the polyoma virus inoculated into the donor rats induces both antiviral humoral response and an immunologic reaction directed against the virus induced TSTA. Evidence for this is also given by the resistance against syngeneic polyoma tumor grafts found in animals pretreated with the virus [1]. Although lymphoid cells sensitized against polyoma TSTA seem to give a better protection than lymphoid cells from donors inoculated with the virus, no definite conclusion can be drawn from our experiments as the dose of virus and the number of lymphoid cells transferred were different in the two experiments. The explanation of this protective effect could be that administration of sensitized, actively immune lymphoid cells in polyoma virus infected rats, upon seeding out and becoming established in the new host, increased their immunologic reactivity of cell mediated type, and thus enabled them to mount an effective immune response against arising neoplastic clones. But, there seems to be a critical limit of the size of neoplastic cell mass, which if attained during oncogenesis, outflanks the efficient rejection responses of the host, even if they are
6
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Cell-Mediated Immunity in Polyoma Oncogenesis strengthened through passive transfer of sensitized lymphoid cells, and leads to progressive development of tumor. This is indicated by the decrease in efficacy as the period of transfer of sensitized lymphoid cells, after virus infection is gradually increased (Table 2). Lymphoid cells sensitized against an antigenic unrelated DMBA tumor as well as control lymphoid cells were only active when inoculated before the virus and at a very high dose level (Tables 1 and 2). Presumably, the control lymphoid cells transferred soon after birth, after getting adjusted to new environment, need enough time to get sensitized and take part in cell-mediated reaction to the stimulus provided by the T S T A of the neoplastic clones. Because in our experiments the transfer was done in normal rats, the results are difficult to compare with those obtained in thymectomized animals in which non-sensitized normal lymphoid cells were also found to be effective after the virus inoculation [16]. Moreover, in the latter case, a strain of mice was used which is only sensitive to polyoma oncogenesis when under immunosuppressive treatment. It may be concluded that in the process of oncogenesis, the animals which display efficient cell mediated responses resist the development of tumor, whereas others which fail to acquire
enough immunological reactivity succumb to tumorous state. Whether serum factor(s), which block the inhibitory effect of sensitized lymphocytes in vitro and thereby nullify the useful effect of cell-mediated immune reaction, also play an important role in vivo is still an open question [2, 19]. The clinical outcome seems to be based on a delicate balance in which the strength and the rapidity in the development of an anti-TSTA cell-mediated immunity are of paramount importance. From the results obtained with transferred lymphoid cells, prepared from donors at different times after the various inoculation, it is clear that the cell-mediated anti-TSTA immunity is gradually built up and is easily demonstrable 2 and 3 weeks after the virus inoculation (Table 3). Moreover, there exists a good correlation between the demonstration of antipolyoma TSTA sensitized ceils as assayed by the in vivo transfer system and by the colony-inhibition test in vitro [unpublished observation]. As shown by Allison [20], the much more rapid development of an efficient cell-mediated immunity in adults compared to newborn animals may well be the main reason why the former are resistant to polyoma ontogenesis. However, it is clear that in neither ease is there any evidence of tolerance or unresponsiveness to the polyoma TSTA.
REFF.I~N~S 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
7
M. VAr~PUWW~. and P. DE So~llZR,Transplantation of polyoma tumours in rats. Nature (Lond.) 1 ~ , 391 (1963). S.K. DAa~rAand M. V~¢D~Pur'r~, Studies on cellular and humoral immunity to tumor-specific antigens in polyoma virus-induced tumors of rats. CancerRes. 31,882 (1971). L.W. LAW and C. J. DAW~., Influence of total body X-irradiation on tumor induction by parotid tumor agent in adult mice. Proc. Soc. exp. Biol. (N.Y.) 1059 414 (1960). M. V~a~.Pua'rE, P. DEters, R. LnYa~N and P. DE So,mR, The oncogenic activity of the polyoma virus in thymectomized rats. Life Sci. 2, 475 (1963). H . D . Aor~w, Immunologic restoration of neonatally thymectomized arts with thoracic duct lymphocytes. Proc. Soc. exp. Biol. (N. Y.) 125~ 132 (1967). M. V ~ P ~ r r r ~ , Antilymphocytic serum and polyoma oneogenesis in rats. Transplant. Proc. Is 100 (1969). A.C. ALLISONand L. W. LAw, Effects of antilymphocytic serum and virus oncogenesis. Proc. Soc. exp. Biol. (N.Y.) 127, 207 (1968). J. L. AmsL and M. BERARDET, Essais de traitement de la leuc~mie EG2 associant chimiothrrapie et immunothrrapie active non sp6cifique et sp6cifique. Rev.fran~. Etud. din. biol. 14, 685 (1969). M. VANDEPUTTE, S. K. DATTA,A. BILLIAUand P. DE SOMER, Inhibition of polyoma virus oncogenesis in rats by polyriboinosinic-ribocytidylic acid. Europ. J. Cancer6~ 323 (1970). V. DEF~-NDI and H. KOPROWSKI, Influence of adult lymphoid tissues on polyoma induced tumours. Nature (L0nd.) 184, 1579 (1959). L . W . LAW, Studies of the significance of tumor antigens in induction and repression of neoplastic diseases. CancerRes. 29, 1 (1969). A. C. ALLISON, Tumour development following immunosuppression. Proc. roy. Soc. Med. 63, 1077 ( 1970).
8
M. Vandeputte and S. K. Datta 13. 14. 15.
L.V. C~WFO~, The adsorption ofpolyoma virus. Virology 1~ 177 (1962). R. Dut.n~cco and G. Fn~zsm~a~, Plaque production by the polyoma virus. Virology$, 396 (1959). W.P. RowE, J. W. H.~atTL~'¢,J. D. ESTESand R. J. HU~Bm~R,Growth curves ofpolyoma virus in mice and hamsters. Nat. Cancer Inst. Monogr. no. 4, 189
(1960). 16. 17. 18. 19. 20.
L.W. LAW,R. C. TINO and E. LECKBAND,Prevention of virus-induced neoplasms in mice through passive transfer of immunity by sensitized syngeneic lymphoid cells. Proc. nat. Acad. Sci. (Wash.) 579 I068 (1967). A. FP.FER, Immunotherapy of primary Moloney sarcoma-virus-induced tumors. Int. J. Cancer5, 327 (1970). P. ALEXANDER,E.J. D~-LORM~and J. G. HALL, The effect of lymphoid cells from the lymph of specifically immunised sheep on the growth of primary sarcomata in rats. Lancet i~ 1186 (1966). H . O . SJ6OPaZNand K. BoRtnw, Tumor-specific immunity in the course of primary polyoma and Rous tumor development in intact and immunosuppressed rats. CancerRes. 31, 890 (1971). A.G. ALLISON,On the absence of tolerance in virus oncogenesis. In Immunity and Tolerance in Oncogenesis, Proceedings of the 4th Perugia Quadrennial International Conference on Cancer (Edited by L. SEVERI),pp. 563--574 (1970).
Cell-Mediated Immunity in Polyoma Oncogenesis* M. VANDEPUTTE and S. K. DATTA Rega Institute for Medical Research, University of I_,awen, Belgium Absta'act--Primary polyoma virus-induced tumors in rats were prevented by the transfer of sensitized lymphoid cells. Sensitized lymphoid cellsfrom donors infected with the virus or immunized with aUogeneicpolyoma tumors were both effective. The e~icaey of the transfer gradually decreased with the increase in the period of time between the virus inoculation and the injection of the immune cells. Non-sensitized lymphoid cells were only effective when given before the virus inoculation. By this in vivo transfer assay the gradual development of the cell-mediated immunity directed against polyoma T S T A could be demonstrated, indicating the absence of tolerance or unresponsiveness to the tumor antigens.
INTRODUCTION POLYOMA virus-induced tumors in the rat contain tumor-specific transplantation antigen(s) (TSTA) foreign to the normal tissues of the host [1]. These T S T A evoke in the host an immunologic response, chiefly of the homograft-rejection type, which can be detected in vivo as well as in vitro [2]. Furthermore, the strength of this cellular immunologic response seems of paramount importance in determining whether or not a neoplastic clone will lead to the development of a clinical tumor. To assess the role of cell-mediated immune mechanisms in polyoma oncogenesis two main approaches are used. One consists in verifying the influence of immunodepression (X-irradiation, thymectomy, ALS) on the tumor incidence or tumorlatency period [3-7]. The alternate approach is to test the tumor repression effectiveness of an increased cellular immunologic response obtained by non-specific stimulation such as BCG [8] or poly I : C [9], or by specific stimulation as achieved by transfer of normal or sensitized lymphoid cells [ 10-12]. In the present study we tried to evaluate by the transfer of sensitized syngeneic lymphoid cells (a) the period after virus inoculation during which Accepted 5 October 1971. *This work was supported by a grant from the Belgian "Algemene Spaar- en Lijfrentekas".
tumor development can be prevented in nonimmunosuppressed syngeneic rats, and (b) the appearance of cellular immunity during the process of primary polyoma oncogenesis.
MATERIAL AND M E T H O D S Animals Rats of the inbred strains Wistar R and Brown Norway (BN) were used, They were kept on a standard diet of pellets and water ad libitum. Virus Polyoma virus was grown on mouse embryo fibroblast cultures (MEF) and prepared by the method of Crawford [ 13]. The virus was titrated with the plaque method of Dulbecco and Freeman on MEF cells [14]. Transplantable tumors and immunization procedures Adult female R rats were inoculated 3 times subcutaneously (s.c.) at weekly intervals with a BN transplantable polyoma fibrosarcoma. This tumor was free of demonstrable virus as determined by the absence of hemagglutinationinhibiting antibodies in the recipients and by in vitro culturing. Control R rats were immunized with an allogeneic (BN) transplantable 7,12-dimethylbenz(a) anthracene (DMBA) sar-
2
M. Vandeputteand S. K. Datta
coma following the same schedule as for the BN polyoma tumor. Five to six days after the last immunization, dissociated lymphoid cells were obtained from the spleen and the combined cervical, inguinal and axillary lymph nodes and prepared by the method described elsewhere [2]. The lymphoid cells were counted and the percentage of living cells determined by trypan blue exclusion. A suspension was made up in Eagle medium to contain 10 x 106-100 x 106 living lymphoid cells in 0" l ml and was inoculated either intraperitoneaUy (i.p.) or intravenously (i.v.) in the lateral tail vein at the concentration of 2-5 x 106 cells/g of body weight. Serum was obtained by cardiac puncture from the same immunized syngeneic donor rats. The sera were stored at - 20°C. Another group of adult female R rats was immunized by high doses ofpolyoma virus, having a titer of 1 × 109 plaque-forming units (PFU) per ml, administered s.c. on the right and left flank alternatively and i.p. twice a week for approximately 8 weeks. Lymph nodes and spleen were removed 4 to 6 days after the last inoculation and processed as described above. The serum obtained by cardiac puncture was inactivated for ½ hr at 56°C. This hyperimmune serum had a titer of 1 : 16.384, as determined by t h e hemagglutination-inhibition test [ 15].
Histology Eight to nine weeks after inoculation all surviving rats were killed after bleeding by cardiac puncture. Rats developing clinical signs of tumor growth before this period were processed in the same way. The kidney and brain, as well as the lymphoid organs, were fixed in Bouin's fluid and further processed for histological examination. RESULTS
Transfer of lymphoid cells or serum from polyoma virus immunized syngeneic donors Litters of R rats were inoculated i.p. with 5 x 106 PFU of polyoma virus, 5 days after birth. They were divided into different experimental groups depending upon the time interval after birth or virus inoculation when transfer of lymphoid cells or hyperimmune serum was effected. The results shown in Table 1 indicate that the transfer of 2 x 106 sensitized (immunized) lymphoid ceils per gram of body weight protected the recipients against tumor development when the transfer was effected either before (group 1A) or 8 days after virus inoculation (group 2A). Sensitized lymphoid cells transferred 14 or 21 days after virus inoculation had no
protective effect. Normal non-sensitized lymphoid cells were effective only when given neonatally and at the level of 5 × 106 cells/g of body weight (group 1C). Hyperimmune serum injected i.p. in 0.1 ml amounts at 1, 3, and 4 days after birth was very effective in preventing tumor development (group 5). Serum treatment started 5 days after the virus inoculation (i.e. 10 days after birth); however, it was totally ineffective (group 6).
Transfer of lymphoid cells from syngeneic donOrs immunized by allogeneicpolyoma tumor or aUogeneic DMBA tumor This experiment was carried out to check the effectiveness of lymphoid cells sensitized against polyoma T S T A or unrelated (DMBA) T S T A in the prevention of the induction of polyoma tumors in rats. The design of the experiment was the same as in the previous one. Yet to increase the sensitivity of the system the recipients were infected with a lower dose of virus, 106 P F U instead of 5 x 106 P F U and the number of transferred lymphoid cells was increased from 2 × 106-5 x 106/gofbodyweight intherecipients treated 14 and 21 days after virus inoculation. As shown in Table 2, a significant reduction in the tumor incidence was observed in all the experimental groups receiving sensitized (antipolyoma TSTA) lymphoid ceils. However, the reduction gradually decreased with the increase of the time period between virus infection and transfer of lymphoid cells. The transfer of lymphoid cells not sensitized against polyoma T S T A was only effective when administered neonatally and had no protective effect given after the virus inoculation. The serum obtained from the donors of antipolyoma TSTA sensitized lymphoid cells and inoculated i.p. in 0.2 ml amounts at 5, 7, and 9 days after the virus inoculation had no protective effect. This serum did not contain antipolyoma virus antibodies (HAI titer < 1/120). In the groups transferred with DMBA tumor sensitized lymphoid cells and, in the control group, about 10% of the rats (11/128) had to be killed, having clinical symptoms of tumor development before the end of the experiment (8-9 weeks after virus inoculation). In the groups treated with anti-polyoma TSTA sensitized lymphoid cells, 3 out of 105 rats were autopsied with tumors 5 to 6 weeks after the virus inoculation. As the overall tumor incidence was also much lower in the experimental groups compared to the control groups, one cannot conclude from these small numbers that the tumor progression was slower in rats transferred
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Cell-Mediated Immunity in Polyoma Oneogenesis with anti-polyoma TSTA sensitized lymphoid cells. The histopathological examination of the tumors did not reveal differences in tumor histology between the experimental and the control groups. In only two cases (1 rat in group 1A and 1 rat in group 2B) did the histology reveal neoplastic foci in the kidney which had not been noted at the macroscopic examination as tumor.
Development of anti-polyoma TSTA cellular immunity during the process of polyoma oncogenesisin rats To verify the development of cellular immunity directed against polyoma TSTA during the process of primary polyoma oncogenesis, we used the same experimental model as described above. The rats (donor animals) in which the appearance of this cellular mediated immunity was verified were inoculated 3 days after birth i.p. with 5 × 106 PFU polyoma virus. Previous experiments had shown that such a dose induced polyoma tumors in 95-100% of the animals. Lymphoid cells were prepared from these donors respectively 6, 15, and 21 days after the virus inoculation and transferred i.v. into 13-day old syngeneic recipients rats inoculated 8 days previously at the age of 5 days with 106 PFU polyoma virus i.p. We chose the experimental model of transferring the lymphoid cells into recipients inoculated 8 days before with the virus for the following reasons: (a) as shown in the previous experiments, the transfer of lymphoid ceils 8 days after virus inoculation is a very sensitive system to detect antipolyoma TSTA cellular immunity; (b) the protective effect of the transferred lymphoid cells, if present, cannot be caused by an antiviral action; (c) 13-day old recipients do not necessitate as many donors as older recipient animals would do. Taking into account the number of lymphoid cells required for transfer and the relatively small numbers recovered from young donors transfer into older animals would require too great a number of donors; (d) normal, non-sensitized lymphoid cells do not exert any protective effect when transferred after polyoma virus inoculation. The results of this experiment, as shown in Table 3, indicate that recipient rats were significantly protected by lymphoid cells removed from donors 15 or 21 days after polyoma virus inoculation. Donor lymphoid cells removed earlier, e.g. 6 days after virus inoculation, were apparently not able to protect the recipients.
5
DISCUSSION
Our results illustrate the efficacy of syngeneic lymphoid cells sensitized against polyoma TSTA (by allogeneic polyoma tumor cells) in the prevention of primary polyoma virus induced neoplasms. This is in accordance with the results obtained in immunosuppressed mice injected with polyoma virus or with Moloney sarcoma virus and afterwards immunologically restored with sensitized lymphoid cells [12, 16, 17]. Transfer of sensitized thoracic duct cells was also effective in protecting against chemical carcinogen induced tumors [18]. However, in the latter case the experiments were carried out in a non-syngeneic system. Whereas lymphoid cells from donors sensitized with allogeneic polyoma tumors gave a good protection, the serum from the same animals was ineffective. Lymphoid cells from donors immunized with polyoma virus were also found to be effective, whether transferred before or 8 days after the virus inoculation. In the latter case their efficacy cannot be due to a transfer of an antiviral serum-mediated immunity as shown by the inability of high doses of hyperimmune antipolyoma virus antiserum to inhibit the tumor development when injected after the virus. Moreover, previous studies have shown that in the rat the virus persists only for a very short period of time after its inoculation [6]. Hence, it is very likely that the polyoma virus inoculated into the donor rats induces both antiviral humoral response and an immunologic reaction directed against the virus induced TSTA. Evidence for this is also given by the resistance against syngeneic polyoma tumor grafts found in animals pretreated with the virus [1]. Although lymphoid cells sensitized against polyoma TSTA seem to give a better protection than lymphoid cells from donors inoculated with the virus, no definite conclusion can be drawn from our experiments as the dose of virus and the number of lymphoid cells transferred were different in the two experiments. The explanation of this protective effect could be that administration of sensitized, actively immune lymphoid cells in polyoma virus infected rats, upon seeding out and becoming established in the new host, increased their immunologic reactivity of cell mediated type, and thus enabled them to mount an effective immune response against arising neoplastic clones. But, there seems to be a critical limit of the size of neoplastic cell mass, which if attained during oncogenesis, outflanks the efficient rejection responses of the host, even if they are
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Cell-Mediated Immunity in Polyoma Oncogenesis strengthened through passive transfer of sensitized lymphoid cells, and leads to progressive development of tumor. This is indicated by the decrease in efficacy as the period of transfer of sensitized lymphoid cells, after virus infection is gradually increased (Table 2). Lymphoid cells sensitized against an antigenic unrelated DMBA tumor as well as control lymphoid cells were only active when inoculated before the virus and at a very high dose level (Tables 1 and 2). Presumably, the control lymphoid cells transferred soon after birth, after getting adjusted to new environment, need enough time to get sensitized and take part in cell-mediated reaction to the stimulus provided by the T S T A of the neoplastic clones. Because in our experiments the transfer was done in normal rats, the results are difficult to compare with those obtained in thymectomized animals in which non-sensitized normal lymphoid cells were also found to be effective after the virus inoculation [16]. Moreover, in the latter case, a strain of mice was used which is only sensitive to polyoma oncogenesis when under immunosuppressive treatment. It may be concluded that in the process of oncogenesis, the animals which display efficient cell mediated responses resist the development of tumor, whereas others which fail to acquire
enough immunological reactivity succumb to tumorous state. Whether serum factor(s), which block the inhibitory effect of sensitized lymphocytes in vitro and thereby nullify the useful effect of cell-mediated immune reaction, also play an important role in vivo is still an open question [2, 19]. The clinical outcome seems to be based on a delicate balance in which the strength and the rapidity in the development of an anti-TSTA cell-mediated immunity are of paramount importance. From the results obtained with transferred lymphoid cells, prepared from donors at different times after the various inoculation, it is clear that the cell-mediated anti-TSTA immunity is gradually built up and is easily demonstrable 2 and 3 weeks after the virus inoculation (Table 3). Moreover, there exists a good correlation between the demonstration of antipolyoma TSTA sensitized ceils as assayed by the in vivo transfer system and by the colony-inhibition test in vitro [unpublished observation]. As shown by Allison [20], the much more rapid development of an efficient cell-mediated immunity in adults compared to newborn animals may well be the main reason why the former are resistant to polyoma ontogenesis. However, it is clear that in neither ease is there any evidence of tolerance or unresponsiveness to the polyoma TSTA.
REFF.I~N~S 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
7
M. VAr~PUWW~. and P. DE So~llZR,Transplantation of polyoma tumours in rats. Nature (Lond.) 1 ~ , 391 (1963). S.K. DAa~rAand M. V~¢D~Pur'r~, Studies on cellular and humoral immunity to tumor-specific antigens in polyoma virus-induced tumors of rats. CancerRes. 31,882 (1971). L.W. LAW and C. J. DAW~., Influence of total body X-irradiation on tumor induction by parotid tumor agent in adult mice. Proc. Soc. exp. Biol. (N.Y.) 1059 414 (1960). M. V~a~.Pua'rE, P. DEters, R. LnYa~N and P. DE So,mR, The oncogenic activity of the polyoma virus in thymectomized rats. Life Sci. 2, 475 (1963). H . D . Aor~w, Immunologic restoration of neonatally thymectomized arts with thoracic duct lymphocytes. Proc. Soc. exp. Biol. (N. Y.) 125~ 132 (1967). M. V ~ P ~ r r r ~ , Antilymphocytic serum and polyoma oneogenesis in rats. Transplant. Proc. Is 100 (1969). A.C. ALLISONand L. W. LAw, Effects of antilymphocytic serum and virus oncogenesis. Proc. Soc. exp. Biol. (N.Y.) 127, 207 (1968). J. L. AmsL and M. BERARDET, Essais de traitement de la leuc~mie EG2 associant chimiothrrapie et immunothrrapie active non sp6cifique et sp6cifique. Rev.fran~. Etud. din. biol. 14, 685 (1969). M. VANDEPUTTE, S. K. DATTA,A. BILLIAUand P. DE SOMER, Inhibition of polyoma virus oncogenesis in rats by polyriboinosinic-ribocytidylic acid. Europ. J. Cancer6~ 323 (1970). V. DEF~-NDI and H. KOPROWSKI, Influence of adult lymphoid tissues on polyoma induced tumours. Nature (L0nd.) 184, 1579 (1959). L . W . LAW, Studies of the significance of tumor antigens in induction and repression of neoplastic diseases. CancerRes. 29, 1 (1969). A. C. ALLISON, Tumour development following immunosuppression. Proc. roy. Soc. Med. 63, 1077 ( 1970).
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M. Vandeputte and S. K. Datta 13. 14. 15.
L.V. C~WFO~, The adsorption ofpolyoma virus. Virology 1~ 177 (1962). R. Dut.n~cco and G. Fn~zsm~a~, Plaque production by the polyoma virus. Virology$, 396 (1959). W.P. RowE, J. W. H.~atTL~'¢,J. D. ESTESand R. J. HU~Bm~R,Growth curves ofpolyoma virus in mice and hamsters. Nat. Cancer Inst. Monogr. no. 4, 189
(1960). 16. 17. 18. 19. 20.
L.W. LAW,R. C. TINO and E. LECKBAND,Prevention of virus-induced neoplasms in mice through passive transfer of immunity by sensitized syngeneic lymphoid cells. Proc. nat. Acad. Sci. (Wash.) 579 I068 (1967). A. FP.FER, Immunotherapy of primary Moloney sarcoma-virus-induced tumors. Int. J. Cancer5, 327 (1970). P. ALEXANDER,E.J. D~-LORM~and J. G. HALL, The effect of lymphoid cells from the lymph of specifically immunised sheep on the growth of primary sarcomata in rats. Lancet i~ 1186 (1966). H . O . SJ6OPaZNand K. BoRtnw, Tumor-specific immunity in the course of primary polyoma and Rous tumor development in intact and immunosuppressed rats. CancerRes. 31, 890 (1971). A.G. ALLISON,On the absence of tolerance in virus oncogenesis. In Immunity and Tolerance in Oncogenesis, Proceedings of the 4th Perugia Quadrennial International Conference on Cancer (Edited by L. SEVERI),pp. 563--574 (1970).