Europ. J. Cancer Vol. 13, pp. 71-79. Pergamon Press 1977. Printed in O-teat Britain
Role of Fetal Antigens in Tumor Immunity SAVITA GAUTAM and B. K. AIKAT Department of Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India Abstract Studies were undertaken to demonstrate the role of fetal antigens in tumor immunity. Viable fetal cells inoculation foUowed by challenge with transplanted methylcholanthrene induced tumor cells after 7, 14 and 21 days significantly delayed the time of appearance of tumors. Transfer of lymphoid cells from mice immunized with fetal cells followed by tumor challenge resulted in delayed appearance of tumors. Inoculation of viable tumor cells pre-treated in vitro with serum from mice immunized with fetal cells shortened the time of appearance of tumors in mice.
INTRODUCTION
challenge and they possessed cytotoxic lymph node cells that destroyed several viral and methylcholanthrene-induced tumor ceils in vitro and in vivo. By means of blocking antibody studies, Baldwin et al. [6, 13], Hellstrom and Hellstrom [17, 18] demonstrated that serum from multiparous rats blocked the cytotoxicity of lymph node cells from multiparous rats both to tumour cells as well as to fetal cells. The present studies were undertaken to explore the possible immunological similarity between the antigens of fetus and methylcholanthrene-induced fibrosarcoma in inbred mice of C57 strain. The effect of fetal cells inoculation followed by tumor challenge at different intervals, the effect of lymphoid cells and sera from mice immunized with fetal cells and the effect of lymphoid cell transfer from multiparous pregnant mice on the time of appearance of tumors was studied in different groups and compared with appropriate control groups.
THE DEMONSTRATION of tumor specific transplantation antigens in tumors and the demonstration of both humoral and cell-mediated immunity to these antigens have opened up great possibilities of immunologic control of cancer [1, 2]. Several workers from time to time have suggested that animal and h u m a n tumor antigens are, in fact, reexpressed fetal components which appear in early fetal life
[3-6]. However, the role of these tumor-associated fetal antigens in tumor rejection response remains controversial. Prehn [7], Lemevel and Wells [8], Bendich et al. [9] reported protection of C57B1/6 and C57 BL mice against methylcholanthrene-induced tumors by pre-immunizing with embryo cells or by exposure to fetal antigens during pregnancy. Grant et al. [10] also reported significant protective effect to chemically induced tumor challenge following immunization with fetal tissue. However, Blair [11], Castro et al. [12], Baldwin et al. [13], Parmiani and Lembo [14] reported that immunization with fetal tissue does not elicit resistance to challenge with chemically induced hepatomas and sarcomas. Brawn [15], Girardi et al. [16]. Grant et al. [10] showed that multiparous female animals were resistant to tumor
MATERIAL A N D M E T H O D S Animals
All animals used belonged to an inbred strain C57 (Jet black, ICRC) mice maintained by of continuous single line brother to sister mating. The purity of inbred lines was tested routinely by acceptance of the reciprocal skin grafts. Tumors
Accepted 16 August 1976.
Primary fibrosarcomas were produced by 20-methylcholanthrene (MC) according to the method of Reiner and Southam [19]. Tumors were then routinely passaged by subcutaneous
Correspondence to: Dr. Savita Gautam, Division of Surgical Sciences, Section of Transplantation Biology, Clinical Research Centre, Watford Road, Harrow, HAl 3UJ, Middlesex, England. 71
72
Savita Gautam and B. K. Aikat
transplantation in syngeneic mice. Inoculation of C57 inbred mice with 1 x 106 viable tumor cells has always given 100% tumor incidence in a large group of animals challenged. The tumors become palpable within 8-10 days. All experiments were conducted on passaged tumors. The demonstration of transplantation immunity in syngeneic animals after surgical removal of the tumor was in support of the fact that this tumor is antigenic.
Cell viability test Viability of cells was tested by trypan blue dye exclusion test. Immunization with fetal cells First litters from 12 week old C57 mice were used as a source of fetal tissue. Timed matings were achieved and the number of vaginal plugs was recorded. The day the plug was recorded was considered day 0. Embryos 8-10 days old, from primiparous female mice, were removed under aseptic conditions, dissected free of placenta and membranes with sharp forceps and were placed in cold Eagle's minimum essential medium (MEM). The fetuses were then homogenized gently in glass homogenizer. Cells were sieved through a sterile stainless steel (80 mesh) gauze and washed twice in MEM. Trypan blue dye exclusion test was performed for cell viability, 2x108 viable cells/0.1 ml were then inoculated under the kidney capsule of adult male mice, with a very fine needle. These mice were then divided into five groups. Mice of these different groups were then challenged with 1 x 106 viable tumor cells at interval of 0, 7, 14, 21 and 28 days respectively by subcutaneous route. Simultaneously, a control group was challenged with tumor cells only. In another set of experiments the mice were inoculated with 2x108/0.1 ml normal adult spleen cells under the kidney capsule and challenged at different intervals with 1 x 106 viable tumor cells. The initial time of appearance of tumors in mice of different groups was recorded and statistically compared. In addition, unchallenged recipients injected with fetal cells were sacrificed at intervals to study the histology of the growing fetal tissues under the kidney capsule. Lymphoid cell transfer To study the effect of embryonic antigen sensitized lymphoid cells, lymph node cell suspensions were prepared separately from mice of following three groups: Group I :
Normal virgin mice.
Group II : Group I I I :
Mice immunized with fetal cells (after 8 days of immunization). Multiparous pregnant mice previously having had more than 4 pregnancies.
The cell suspensions were passed through 80 mesh stainless steel screen and washed twice in BSS. Cells were finally suspended in MEM. Normal adult male mice were divided iato three groups and each mouse of each group received two doses of 4 x 108/0.5 ml lymphnode cells from mice of above groups respectively at four days interval by intra-peritoneal route. After 7 days of last transfer, these mice were challenged with I x 106 viable tumor cells. Initial time of appearance of tumors in all mice was noted and compared.
Inoculation of tumor cells treated with serum To study the effect of serum from fetal cells immunized mice, normal syngeneic mice were injected subcutaneously in three different groups with 1 x 105 viable tumor cells/mouse incubated previously for 30 min. at 37°C with 0.5 ml of M E M medium, with 0.5 ml of normal virgin mice serum and with 0.5 ml of serum from mice immunized with fetal cells respectively. These mice were then observed to record the initial time of appearance of tumors. Serum was collected after 28 days of fetal cells injection. RESULTS
Histological studies Normal adult male mice were inoculated under the kidney capsule with cells from syngeneic 8-10 days old fetuses. These mice were then divided in 4 groups of 4 mice in each group and sacrificed at intervals of 7 days, 14 days and 28 days. Their kidneys were examined macroscopically and microscopically. Figure 1 shows a mouse kidney with a fetal growth on its surface. Kidney sections from mice killed after seven days of fetal cells inoculation revealed proliferation of undifferentiated cells under capsule of kidney (Fig. 2). After fourteen days of fetal cells injection, there was beginning of formation of more organized structures with cavity formation (Fig. 3). Kidney sections from mice sacrificed after 28 days of fetal cells inoculation, showed the development of teratomatous elements consisting of mature bone with bone marrow cells, cavities lined by columnar epithelium, cartilage and undifferentiated cells showing more organized pattern of development (Fig. 4).
Role of Fetal Antigens in Tumor Immunity
Fig. 1.
73
A mouse kidney showing a subcapsular growth of fetal cells.
Fig. 2.
Microphotograph of a kidney section from mouse sacrificed after 7 days of fetal cell inoculation showing proliferation of undifferentiated cells under kidney capsule (H and E x 110).
Fig. 3.
Microphotograph of a kidney section of mouse sacrificed after 14 days of fetal cell inoculation showing cavity formation (H and E x 440).
Fig. 4. Microphotograph of a kidney section of mouse sacrificed after 28 days of fetal cell inoculation showing a mature bone with bone marrow cells, cavities lined by columnar epithelium, cartilage and undifferentiated cells (H and E x 110).
Role of Fetal Antigens in Tumor Immunity Effect offetal cell inoculation Figure 5 compares the time of appearance of tumors in mice injected with fetal cells followed by tumor challenge at different intervals. Mean time of appearance of tumors in control mice was 8.62_+0-93 and in mice inoculated with fetal cells followed by tumor challenge after 7, 14 and 21 days, the mean time of appearance of tumors was 22.9 _+5.17 days, 17.6+3.75 days and 15.1_+2.68 days respectively. These mean values are significantly different when compared to those of control mice (P < 0.05). The mean time of appearance of tumors in mice challenged on 0 day and after 28 days of fetal cell inoculation was 10.5_+ 2.14 and 10.8_+ 1.76 days respectively. These values are not significantly different from the control mice (P > 0.05). The mean time of tumor appearance in mice inoculated with normal adult spleen cells followed by tumor challenge after 0, 7, 14, 21 and 28 days was 8.25+0.78 days, 9.25_+ 1.03 days, 9.0_+ 1.32 days, 8.75 + 1.03 days and 9.0 _+1.32 days respectively. These values are not significantly different as compared to that of control mice (P > 0.05). Except for 0 and 28 days, all these mean values are significantly different (P < 0.05) as compared to the mean values of corresponding groups of mice inoculated with fetal cells under the kidney capsule. Effect of lymphoid cells from fetal cells immunized from multiparous mice Figure 6 compares the effect of lymphoid cells from fetal cells inoculated mice and from multiparous pregnant mice for their capacity to adoptively transfer resistance to tumor challenge. Mean time of appearance of tumors in control mice challenged with tumor cells only was 9.4 _+ 1.35 days. Normal lymphoid cells did not affect the time of appearance of tumors since the mean time of appearance of tumors in these mice was 9.6_+ 2.33 days. Mean time of tumor appearance in mice transferred with lymphoid cells from fetal cells immunized mice and from multiparous pregnant mice was 17.2 +2.4 days and 14.0_+ 1-76 respectively. These mean values are significantly different as compared to both the control groups (P < 0.05). Effect of serum The results of this experiment are presented in Fig. 7. Pre-treatment of tumor cells with serum from fetal cells injected mice did not delay the tumor appearance. O n the other hand, it shortened the time of appearance of tumors. The mean time of appearance of tumors in mice injected with tumor cells treated
75
with serum from fetal cells immunized mice was 14.0_+1-5 days and in mice injected with normal serum treated tumor cells and M E M medium treated tumor cells was 18.25_+ 1.7 days and 19.25+ 1.3 days respectively. These mean values are significantly different as compared to that of experimental animals (P < 0.05). DISCUSSION
There is considerable evidence to suggest the existence of a variety of fetal antigens in association with h u m a n tumors [3, 4, 20, 21]. Experimental studies have further proved the association of embryonic antigen with chemically induced tumors [6, 22, 23]. Tumors induced by viruses have also been shown to share antigens with embryos [24, 25]. Prehn [7] for the first time found suppression of transplanted MC induced tumor growth in mice pre-immunized with viable embryo cells. Blair [11], Ting et al. [26], Castro et al. [12], Baldwin et al. [13], Parmiani and Lembo [14] failed to find transplantation immunity against virus induced m a m m a r y tumors, SV40 induced tumors and chemically induced tumors after immunization with fetal cells. But studies of Buttle and Frayn [27], Coggin et al. [24], Girardi et al. [16] Lemevel and Wells [8], Bendich et al. [9] and Grant et al. [10] encouraged various workers to carry on further studies with other tumor types since these workers showed that preimmunization with fetal antigens produced significant resistance against development and suppression in growth of imferon induced sarcoma, SV40 induced tumors, adeno virus induced tumors, Rouscher leukemia virus injected cells, plasma cell tumors and MC induced tumors. In the present studies, inoculation of fetal cells followed by tumor challenge after 7, 14 and 21 days resulted in significant delay in tumor appearance as compared to the control mice. Tumor challenge on 0 day and after 28 days of fetal cells inoculation did not affect the time of appearance of tumors (Fig. 5). Failure of different investigators to induce immunity against various types of tumors following immunization with fetal cells may be attributed to several factors and different experimental conditions. First and most important may be the strain dependent sensitivity. Whether the host responds to these fetal antigens or not will determine their suitability for immunotherapy. Ting et al. [25] could produce anti fetal serum in C57 mice which was found to be highly reactive. Antisera
76
Savita Gautam and B. K. Aikat
produced by the same schedule in C3H mice was weakly reactive whereas repeated hyperimmunization of BALB/C or CBFI mice with X irradiated fetal cells failed to produce serum that would react with tumor antigen or fetal antigen. Several workers reported the induction of immunity against tumors in C57 mice by preimmunizing them with fetal cells [8, 9], while Blair [11], Ting et al. [26], Parmiani and Lembo [14] failed to induce such immunity in BALB/C mice. Similarly hamsters have been found to be good responders to fetal antigen as compared to BALB/C mice [25, 10]. 32 o
28 24 20 ~, 16
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o
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...~o
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Fig. 5. Figure showing the effect of fetal cells inoculation followed by tumor challenge at different intervals on time of appearance of tumors in mice.
Whether sex plays any significant role in response to fetal antigens is again a controversial question. Some workers found that males responded better to fetal antigen immunization [24, 16]. However, Grant et al. [10] did not find any such sex difference in case of M C induced tumors. The degree of immunity also depends upon the immunization schedule, latent period etc. In the present study, failure of viable fetal cells to induce resistance in mice challenged with tumor cells after 28 days may be explained on the basis that fetal tissues become mature in adult hormonal environment as has been discussed by Coggin et al. [24]. This is confirmed by the histological study of fetal cell growth below the kidney capsule in mice sacrificed after 28 days of fetal cell injection which constituted almost all mature structures like bone with bone marrow, cartilage, cavities lined by columnar epithelium (Fig. 4). However, it is difficult to suggest whether the tumor associated fetal antigens were present at this stage or not. Appearance of blocking antibodies by this time may be another factor. Buttle and Frayn [27] found that degree of tumor inhibition was highest if the interval between fetal cells injec-
tion and tumor challenge was 7 days and the effect gradually reduced as this interval was increased. Under natural conditions only females are exposed to fetal antigens during pregnancy. Blair et al. [28] reported that spontaneous tumor development in the virgin females occurs at a later time than in the parous females. On the other hand, Fraumeni et al. [29] after studying a large group of patients concluded that unmarried females are more susceptible to tumors of the colon, breast, corpus uteri and ovary. Apart from hormonal influences, the cause of this difference may be that during pregnancy females are exposed to fetal antigens which sensitize the lymphoid cells. Baldwin et al. [5, 6], Steele and Sjogren [23], Hellstrom and Hellstrom [17, 18] reported that multiparous female animals possess cytotoxic lymph node cells that destroy several viral and MC induced tumors in vitro. Very few investigators have tried to demonstrate the in vivo role of fetal antigen sensitized lymphocytes in tumor rejection response. Only few contradictory reports are available. In the present investigation studies were undertaken to examine the capacity of lymphoid cells from fetal cells injected mice and from muhiparous pregnant mice to transfer adoptively the resistance to tumor challenge. It was found that lymphoid cells from fetal cells immunized mice and from multiparous pregnant mice significantly delayed the time of appearance of tumors as compared to the control mice whereas the normal lymphoid cells did not (Fig. 6). 28 24
0 o
I 20 16
® ®
Ib
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t.
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o
o
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4
Control mice
© Tronsferred o Transferred ® Tronsferred with normol with lymphoid with lymphoid lymphoidcells
cells from cells from fetol cells mulqiporous injected mice preqnon~r mice
Fig. 6. This figure shows the effect of lymphoid cell transfer from mice immunized with fetal cells and from multiparous pregnant mice on appearance of tumors.
Hanna et al. [30] reported that transfer of spleen cells from multiparous mice protected the normal mice from infection with Rouse leukemia virus. Girardi et al. [16] showed that
Role o f Fetal Antigens in Tumor Immunity
lymphoid cells from primiparous and multiparous hamsters protected normal male hamsters against SV40 induced tumor challenge. O n the other hand, studies of Baldwin et al. [13], Parmiani and Lembo [14] showed that lymphoid cells from multiparous rats and mice were ineffective in adoptively transferring tumor immunity to normal animals. In the next set of experiments, three groups of normal mice received tumor cells incubated previously with M E M , with serum from normal virgin female mice, with serum from fetal cells immunized mice respectively. Inoculation of mice with tumor cells treated with serum from fetal cells immunized mice did not protect them against tumor formation (Fig. 7). Surprisingly, the tumor appearance time was shortened in these mice as compared to the control groups.
77
studies, Baldwin et al. [6, 13] have shown that lymph node cells from multiparous pregnant mice were cytotoxic in vitro for cultured tumor cells and fetal cells. But this cytotoxicity for tumor cells and fetal cells was blocked ill presence of serum from multiparous mice (Fig. 8). Similar findings have been given by Hellstrom and Hellstrom [17, 18] who ascribed the inability of fetal antigens to induce tumor resistance in body to these blocking antibodies.
Multiparous pregnant mice
Cytotoxic
28
/
m
o
\
Cultured fe*al cells
Cultured tumor cells
24 2O
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.Qe :
16
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o
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-.... %%+
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j
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4 Tumor cells
Tumor cells
treated ~
treated ~
mere
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Tumor ceils
treated serum of mice inj. ~ fetal cells
Multiporous pregnant mice
Fig. 7. This figure shows the effect of in vitro treatment of tumor cells with serum from normal mice and from mice immunized with fetal cells on time of appearance of tumors.
Coggin and Anderson [31] demonstrated that females splenectomized to reduce the antibody forming capacity react protectively to fetal vaccine. Antibody response against fetal antigens is more because these antigens contain more antigenic sites as compared to the tumor antigen. Recently in a series of their in vitro
Fig. 8. Diagrammaticpresentation of ln vitro cytotoxicity of lymphoid cells from multiparous mice against tumor cells, fetal cells and blocking of this cytotoxicity by serum from multiparous mice.
Recently, Tamerius et al. [32] described that fetal antigens have a special property to form blocking factors. These factors prevent either the development of cell mediated immunity or act against the expression of an already developed cell mediated immunity.
REFERENCES
1. 2. 3. 4. 5.
Y. H. PILCI-I and R. S. RlOOlNS, Antibodies to spontaneous and methylcholanthrene induced tumors in inbred mice. Cancer Res. 26, 871 (1966). J . E . DELORME and P. ALEXANDER,Treatment of primary fibrosarcoma in the rat with immune lymphocytes. Lancet ii, 117 (1964). G . I . ABELEV, Production of embryonal serum alpha-globulin by hepatomas. Review of experimental and clinical data. Cancer Res. 28, 1344 (1968). E. ~/[. EDYNAK, L. J. O L D , ~/~. VRANA and M. P. LARDIS, A fetal antigen associated with human neoplasia. New Engl. J. Med. 286, 1178 (1972). R . W . BALDWIN, D. GLAVES, l~. V. PIMM and B. M. VasE, Tumor specific and embryonic antigen expression on chemically induced rat tumors. Ann. Inst. Pasteur 122, 715 (1972).
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Savita Gautam and B. K. Aikat
6.
7.
8. 9.
10.
I 1.
12. 13. 14.
15.
16.
17.
18.
19. 20.
21. 22.
23. 24.
25. 26. 27.
R . W . BALDWIN, D. GLAVES and B. M. VOSE, Differentiation between the embryonic and tumour specific antigens of chemically induced rat tumours. Int. d. Cancer 29, 1 (1974). R . T . PREHN, The significance of tumor distinctive histo-compatibility antigens. In: Cross Reacting Antigens and Neoantigens. (Edited by J. J. TRENTIN), p. 105. Williams Wilkins Co., Baltimore (1967). B . P . LEMEVEL and S. A. WELLS, Foetal antigens cross reactive with tumour specific transplantation antigens. Nature New Biol. 244, 183 (1973). A. BENDICH, E. BOREN FREUND and E. H. STONEHILL,Protection of adult mice against tumor challenge by immunization with irradiated adult skin or embryo cells, d. Immunol. 111, 284 (1973). J . P . GRANT, S. LADISCHand S. A. WELLS, Immunologic similarities between fetal cell antigens and tumor cell antigens in guinea pigs. Cancer (Philad.) 33, 376 (1974). P . B . BLAIR, Search for cross reacting antigenicity between m a m m a r y tumor virus induced m a m m a r y tumors and embryonic antigens: Effect of immunization on development of spontaneous m a m m a r y tumors. Cancer Res. 30, 1199 (1970). J. E. CASTRO, E. M. LANCE, P. B. MEDAWAR, J. ZANELLI and R. HUNT, Foetal antigens and cancer. Nature (Lond.) 243, 225 (1973). R . W . BALDWIN, D. GLAVES and B. M. VOSE, Immunogenicity of embryonic antigens associated with chemically induced rat tumors. Int. d. Cancer 13, 135 (1974). G. PARMIANI and R. LEMBO, Effect of antiembryo immunization on Methylcholanthrene induced sarcoma growth in BALB/C mice. Int. J. Cancer 14, 555 (1974). l~. J. BRAWN, Possible association of embryonal antigen(s) with several primary 3-methyl cholanthrene induced murine sarcomas. Int. J. Cancer 6, 245 (1970). A . J . GIRARDI, P. PEPPUCCI, P. DIERLAM, W. RUTALA and J. H. COGOlN, Prevention of Simian Virus 40 tumors by hamster fetal tissue, influence of parity status of donor females on immunogenicity of fetal tissue and on immune cell cytotoxicity. Proc. nat. Acad. Sci. (Wash.) 70, 183 (1973). I. HELLSTROMand K. E. HELLSTROM, Cytotoxic effect of lymphocytes from pregnant mice on cultivated tumor cells. II. Blocking and unblocking of cytotoxicity. Int. J. Cancer 15, 30 (1975). I. I-IELLSTROMand K. E. HELLSTROM, Cytotoxic effect of lymphocytes from pregnant mice on cultivated tumor cells. I. Specificity, nature of effector cells and blocking by serum. Int. d. Cancer 15, 1 (1975). J. REINER and C. M. SOUTHAM,Evidence of common antigenic properties in chemically induced sarcomas of mice. Cancer Res. 27, 1243 (1967). P. GOLDMANand S. O. FREEDMAN,Specific carcino embryonic antigens of the human digestive system, d. exp. Med. 122, 467 (1965). D. BUFFE, C. RIMBAUT, J. LEMERLE, O. SCHWEISGUTCHand P. BURTIN, Presence of a ferroprotein of tissular origin, the .. 2H globulin in sera of children with tumours. Int. J. Cancer 5, 85 (1970). D . M . P . THOMSON and P. ALEXANDER, A cross reacting embryonic antigen in the membrane of rat sarcoma cells which is immunogenic in the syngeneic host. Brit. J. Cancer 27, 35 (1973). G. STEELEand H. O. SJOOREN, Embryonic antigens associated with chemically induced colon carcinoma in rats. Int. J. Cancer 14, 435 (1974). J . H . COGOIN, K. R. AMBROSE,B. B. BELLAMYand N. G. ANDERSON, T u m o r immunity in hamsters immunized with fetal tissues. J. Immunol. 107, 526 (1971). C.C. TING,J. R. ORTALDOand R. B. HERBERMAN,Expression of fetal antigens and tumor specific antigens in SV40-transformed cells. I. Serological analysis of the antigenic specificities. Int. J. Cancer 12, 511 (1973). C.C. TING, D. RODRIGUESand K. B. HERBERMAN,Expression of fetal antigens and tumor specific antigens in SV40-transformed cells. II. T u m o r transplantation studies. Int. J. Cancer 12, 519 (1973). G . A . H . BUTTLE and A. FRAYN, Effect of previous injection of homologous embryonic tissue on the growth of certain transplantable mouse tumours. Nature (Lond.) 215, 1495 (1967).
Role of Fetal Antigens in Tumor Immunity 28.
29. 30.
31.
39.
P . B . BLAIR, S. M. BLAIR, W. R. LYONS, H. A. BERN and C. H. LI, Effect of hormones and of parity on the occurrence of hyperplastic alveolar nodules and tumors in m a m m a r y glands of female A/Crgl mice. Cancer Res. 20, 1640 (1960). J . F . FRAUMENI,J. W. LLOYD, E. M. SMITH and J. K. WAGONER, Cancer mortality among nuns: Role of marital status in etiology of neoplastic disease in women. J. nat. Cancer Inst. 42, 455 (1969). !k/[. G. HANNA, R. W. TENNANT and J. H. COGOIN, Suppressive effect of immunization with mouse fetal antigens on growth of cells infected with Rauscher leukemia virus and on plasma cell tumors. Proc. nat. Acad. Sci. (Wash.) 68, 1748 (1971). J. H. COGGIN and N. G. ANDERSON, Phase specific autoantigens (fetal) in model tumor systems. In: Embryonic and Fetal Antigens in Cancer. (Edited by N. G. ANDERSON,J. H. COGGIN,E. COLE a n d J . W. HOLLEMAN)Vo1. II, p. 91. U.S. Department of Commerce, Springfield. J. TAMERIUS, I. HELLSTROMand K. E. HELLSTROM,Evidence that blocking factors in the sera of multiparous mice are associated with immuno-globulins. Int. d. Cancer20, 456 (1975).
79
Role of Fetal Antigens in Tumor Immunity SAVITA GAUTAM and B. K. AIKAT Department of Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India Abstract Studies were undertaken to demonstrate the role of fetal antigens in tumor immunity. Viable fetal cells inoculation foUowed by challenge with transplanted methylcholanthrene induced tumor cells after 7, 14 and 21 days significantly delayed the time of appearance of tumors. Transfer of lymphoid cells from mice immunized with fetal cells followed by tumor challenge resulted in delayed appearance of tumors. Inoculation of viable tumor cells pre-treated in vitro with serum from mice immunized with fetal cells shortened the time of appearance of tumors in mice.
INTRODUCTION
challenge and they possessed cytotoxic lymph node cells that destroyed several viral and methylcholanthrene-induced tumor ceils in vitro and in vivo. By means of blocking antibody studies, Baldwin et al. [6, 13], Hellstrom and Hellstrom [17, 18] demonstrated that serum from multiparous rats blocked the cytotoxicity of lymph node cells from multiparous rats both to tumour cells as well as to fetal cells. The present studies were undertaken to explore the possible immunological similarity between the antigens of fetus and methylcholanthrene-induced fibrosarcoma in inbred mice of C57 strain. The effect of fetal cells inoculation followed by tumor challenge at different intervals, the effect of lymphoid cells and sera from mice immunized with fetal cells and the effect of lymphoid cell transfer from multiparous pregnant mice on the time of appearance of tumors was studied in different groups and compared with appropriate control groups.
THE DEMONSTRATION of tumor specific transplantation antigens in tumors and the demonstration of both humoral and cell-mediated immunity to these antigens have opened up great possibilities of immunologic control of cancer [1, 2]. Several workers from time to time have suggested that animal and h u m a n tumor antigens are, in fact, reexpressed fetal components which appear in early fetal life
[3-6]. However, the role of these tumor-associated fetal antigens in tumor rejection response remains controversial. Prehn [7], Lemevel and Wells [8], Bendich et al. [9] reported protection of C57B1/6 and C57 BL mice against methylcholanthrene-induced tumors by pre-immunizing with embryo cells or by exposure to fetal antigens during pregnancy. Grant et al. [10] also reported significant protective effect to chemically induced tumor challenge following immunization with fetal tissue. However, Blair [11], Castro et al. [12], Baldwin et al. [13], Parmiani and Lembo [14] reported that immunization with fetal tissue does not elicit resistance to challenge with chemically induced hepatomas and sarcomas. Brawn [15], Girardi et al. [16]. Grant et al. [10] showed that multiparous female animals were resistant to tumor
MATERIAL A N D M E T H O D S Animals
All animals used belonged to an inbred strain C57 (Jet black, ICRC) mice maintained by of continuous single line brother to sister mating. The purity of inbred lines was tested routinely by acceptance of the reciprocal skin grafts. Tumors
Accepted 16 August 1976.
Primary fibrosarcomas were produced by 20-methylcholanthrene (MC) according to the method of Reiner and Southam [19]. Tumors were then routinely passaged by subcutaneous
Correspondence to: Dr. Savita Gautam, Division of Surgical Sciences, Section of Transplantation Biology, Clinical Research Centre, Watford Road, Harrow, HAl 3UJ, Middlesex, England. 71
72
Savita Gautam and B. K. Aikat
transplantation in syngeneic mice. Inoculation of C57 inbred mice with 1 x 106 viable tumor cells has always given 100% tumor incidence in a large group of animals challenged. The tumors become palpable within 8-10 days. All experiments were conducted on passaged tumors. The demonstration of transplantation immunity in syngeneic animals after surgical removal of the tumor was in support of the fact that this tumor is antigenic.
Cell viability test Viability of cells was tested by trypan blue dye exclusion test. Immunization with fetal cells First litters from 12 week old C57 mice were used as a source of fetal tissue. Timed matings were achieved and the number of vaginal plugs was recorded. The day the plug was recorded was considered day 0. Embryos 8-10 days old, from primiparous female mice, were removed under aseptic conditions, dissected free of placenta and membranes with sharp forceps and were placed in cold Eagle's minimum essential medium (MEM). The fetuses were then homogenized gently in glass homogenizer. Cells were sieved through a sterile stainless steel (80 mesh) gauze and washed twice in MEM. Trypan blue dye exclusion test was performed for cell viability, 2x108 viable cells/0.1 ml were then inoculated under the kidney capsule of adult male mice, with a very fine needle. These mice were then divided into five groups. Mice of these different groups were then challenged with 1 x 106 viable tumor cells at interval of 0, 7, 14, 21 and 28 days respectively by subcutaneous route. Simultaneously, a control group was challenged with tumor cells only. In another set of experiments the mice were inoculated with 2x108/0.1 ml normal adult spleen cells under the kidney capsule and challenged at different intervals with 1 x 106 viable tumor cells. The initial time of appearance of tumors in mice of different groups was recorded and statistically compared. In addition, unchallenged recipients injected with fetal cells were sacrificed at intervals to study the histology of the growing fetal tissues under the kidney capsule. Lymphoid cell transfer To study the effect of embryonic antigen sensitized lymphoid cells, lymph node cell suspensions were prepared separately from mice of following three groups: Group I :
Normal virgin mice.
Group II : Group I I I :
Mice immunized with fetal cells (after 8 days of immunization). Multiparous pregnant mice previously having had more than 4 pregnancies.
The cell suspensions were passed through 80 mesh stainless steel screen and washed twice in BSS. Cells were finally suspended in MEM. Normal adult male mice were divided iato three groups and each mouse of each group received two doses of 4 x 108/0.5 ml lymphnode cells from mice of above groups respectively at four days interval by intra-peritoneal route. After 7 days of last transfer, these mice were challenged with I x 106 viable tumor cells. Initial time of appearance of tumors in all mice was noted and compared.
Inoculation of tumor cells treated with serum To study the effect of serum from fetal cells immunized mice, normal syngeneic mice were injected subcutaneously in three different groups with 1 x 105 viable tumor cells/mouse incubated previously for 30 min. at 37°C with 0.5 ml of M E M medium, with 0.5 ml of normal virgin mice serum and with 0.5 ml of serum from mice immunized with fetal cells respectively. These mice were then observed to record the initial time of appearance of tumors. Serum was collected after 28 days of fetal cells injection. RESULTS
Histological studies Normal adult male mice were inoculated under the kidney capsule with cells from syngeneic 8-10 days old fetuses. These mice were then divided in 4 groups of 4 mice in each group and sacrificed at intervals of 7 days, 14 days and 28 days. Their kidneys were examined macroscopically and microscopically. Figure 1 shows a mouse kidney with a fetal growth on its surface. Kidney sections from mice killed after seven days of fetal cells inoculation revealed proliferation of undifferentiated cells under capsule of kidney (Fig. 2). After fourteen days of fetal cells injection, there was beginning of formation of more organized structures with cavity formation (Fig. 3). Kidney sections from mice sacrificed after 28 days of fetal cells inoculation, showed the development of teratomatous elements consisting of mature bone with bone marrow cells, cavities lined by columnar epithelium, cartilage and undifferentiated cells showing more organized pattern of development (Fig. 4).
Role of Fetal Antigens in Tumor Immunity
Fig. 1.
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A mouse kidney showing a subcapsular growth of fetal cells.
Fig. 2.
Microphotograph of a kidney section from mouse sacrificed after 7 days of fetal cell inoculation showing proliferation of undifferentiated cells under kidney capsule (H and E x 110).
Fig. 3.
Microphotograph of a kidney section of mouse sacrificed after 14 days of fetal cell inoculation showing cavity formation (H and E x 440).
Fig. 4. Microphotograph of a kidney section of mouse sacrificed after 28 days of fetal cell inoculation showing a mature bone with bone marrow cells, cavities lined by columnar epithelium, cartilage and undifferentiated cells (H and E x 110).
Role of Fetal Antigens in Tumor Immunity Effect offetal cell inoculation Figure 5 compares the time of appearance of tumors in mice injected with fetal cells followed by tumor challenge at different intervals. Mean time of appearance of tumors in control mice was 8.62_+0-93 and in mice inoculated with fetal cells followed by tumor challenge after 7, 14 and 21 days, the mean time of appearance of tumors was 22.9 _+5.17 days, 17.6+3.75 days and 15.1_+2.68 days respectively. These mean values are significantly different when compared to those of control mice (P < 0.05). The mean time of appearance of tumors in mice challenged on 0 day and after 28 days of fetal cell inoculation was 10.5_+ 2.14 and 10.8_+ 1.76 days respectively. These values are not significantly different from the control mice (P > 0.05). The mean time of tumor appearance in mice inoculated with normal adult spleen cells followed by tumor challenge after 0, 7, 14, 21 and 28 days was 8.25+0.78 days, 9.25_+ 1.03 days, 9.0_+ 1.32 days, 8.75 + 1.03 days and 9.0 _+1.32 days respectively. These values are not significantly different as compared to that of control mice (P > 0.05). Except for 0 and 28 days, all these mean values are significantly different (P < 0.05) as compared to the mean values of corresponding groups of mice inoculated with fetal cells under the kidney capsule. Effect of lymphoid cells from fetal cells immunized from multiparous mice Figure 6 compares the effect of lymphoid cells from fetal cells inoculated mice and from multiparous pregnant mice for their capacity to adoptively transfer resistance to tumor challenge. Mean time of appearance of tumors in control mice challenged with tumor cells only was 9.4 _+ 1.35 days. Normal lymphoid cells did not affect the time of appearance of tumors since the mean time of appearance of tumors in these mice was 9.6_+ 2.33 days. Mean time of tumor appearance in mice transferred with lymphoid cells from fetal cells immunized mice and from multiparous pregnant mice was 17.2 +2.4 days and 14.0_+ 1-76 respectively. These mean values are significantly different as compared to both the control groups (P < 0.05). Effect of serum The results of this experiment are presented in Fig. 7. Pre-treatment of tumor cells with serum from fetal cells injected mice did not delay the tumor appearance. O n the other hand, it shortened the time of appearance of tumors. The mean time of appearance of tumors in mice injected with tumor cells treated
75
with serum from fetal cells immunized mice was 14.0_+1-5 days and in mice injected with normal serum treated tumor cells and M E M medium treated tumor cells was 18.25_+ 1.7 days and 19.25+ 1.3 days respectively. These mean values are significantly different as compared to that of experimental animals (P < 0.05). DISCUSSION
There is considerable evidence to suggest the existence of a variety of fetal antigens in association with h u m a n tumors [3, 4, 20, 21]. Experimental studies have further proved the association of embryonic antigen with chemically induced tumors [6, 22, 23]. Tumors induced by viruses have also been shown to share antigens with embryos [24, 25]. Prehn [7] for the first time found suppression of transplanted MC induced tumor growth in mice pre-immunized with viable embryo cells. Blair [11], Ting et al. [26], Castro et al. [12], Baldwin et al. [13], Parmiani and Lembo [14] failed to find transplantation immunity against virus induced m a m m a r y tumors, SV40 induced tumors and chemically induced tumors after immunization with fetal cells. But studies of Buttle and Frayn [27], Coggin et al. [24], Girardi et al. [16] Lemevel and Wells [8], Bendich et al. [9] and Grant et al. [10] encouraged various workers to carry on further studies with other tumor types since these workers showed that preimmunization with fetal antigens produced significant resistance against development and suppression in growth of imferon induced sarcoma, SV40 induced tumors, adeno virus induced tumors, Rouscher leukemia virus injected cells, plasma cell tumors and MC induced tumors. In the present studies, inoculation of fetal cells followed by tumor challenge after 7, 14 and 21 days resulted in significant delay in tumor appearance as compared to the control mice. Tumor challenge on 0 day and after 28 days of fetal cells inoculation did not affect the time of appearance of tumors (Fig. 5). Failure of different investigators to induce immunity against various types of tumors following immunization with fetal cells may be attributed to several factors and different experimental conditions. First and most important may be the strain dependent sensitivity. Whether the host responds to these fetal antigens or not will determine their suitability for immunotherapy. Ting et al. [25] could produce anti fetal serum in C57 mice which was found to be highly reactive. Antisera
76
Savita Gautam and B. K. Aikat
produced by the same schedule in C3H mice was weakly reactive whereas repeated hyperimmunization of BALB/C or CBFI mice with X irradiated fetal cells failed to produce serum that would react with tumor antigen or fetal antigen. Several workers reported the induction of immunity against tumors in C57 mice by preimmunizing them with fetal cells [8, 9], while Blair [11], Ting et al. [26], Parmiani and Lembo [14] failed to induce such immunity in BALB/C mice. Similarly hamsters have been found to be good responders to fetal antigen as compared to BALB/C mice [25, 10]. 32 o
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Fig. 5. Figure showing the effect of fetal cells inoculation followed by tumor challenge at different intervals on time of appearance of tumors in mice.
Whether sex plays any significant role in response to fetal antigens is again a controversial question. Some workers found that males responded better to fetal antigen immunization [24, 16]. However, Grant et al. [10] did not find any such sex difference in case of M C induced tumors. The degree of immunity also depends upon the immunization schedule, latent period etc. In the present study, failure of viable fetal cells to induce resistance in mice challenged with tumor cells after 28 days may be explained on the basis that fetal tissues become mature in adult hormonal environment as has been discussed by Coggin et al. [24]. This is confirmed by the histological study of fetal cell growth below the kidney capsule in mice sacrificed after 28 days of fetal cell injection which constituted almost all mature structures like bone with bone marrow, cartilage, cavities lined by columnar epithelium (Fig. 4). However, it is difficult to suggest whether the tumor associated fetal antigens were present at this stage or not. Appearance of blocking antibodies by this time may be another factor. Buttle and Frayn [27] found that degree of tumor inhibition was highest if the interval between fetal cells injec-
tion and tumor challenge was 7 days and the effect gradually reduced as this interval was increased. Under natural conditions only females are exposed to fetal antigens during pregnancy. Blair et al. [28] reported that spontaneous tumor development in the virgin females occurs at a later time than in the parous females. On the other hand, Fraumeni et al. [29] after studying a large group of patients concluded that unmarried females are more susceptible to tumors of the colon, breast, corpus uteri and ovary. Apart from hormonal influences, the cause of this difference may be that during pregnancy females are exposed to fetal antigens which sensitize the lymphoid cells. Baldwin et al. [5, 6], Steele and Sjogren [23], Hellstrom and Hellstrom [17, 18] reported that multiparous female animals possess cytotoxic lymph node cells that destroy several viral and MC induced tumors in vitro. Very few investigators have tried to demonstrate the in vivo role of fetal antigen sensitized lymphocytes in tumor rejection response. Only few contradictory reports are available. In the present investigation studies were undertaken to examine the capacity of lymphoid cells from fetal cells injected mice and from muhiparous pregnant mice to transfer adoptively the resistance to tumor challenge. It was found that lymphoid cells from fetal cells immunized mice and from multiparous pregnant mice significantly delayed the time of appearance of tumors as compared to the control mice whereas the normal lymphoid cells did not (Fig. 6). 28 24
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cells from cells from fetol cells mulqiporous injected mice preqnon~r mice
Fig. 6. This figure shows the effect of lymphoid cell transfer from mice immunized with fetal cells and from multiparous pregnant mice on appearance of tumors.
Hanna et al. [30] reported that transfer of spleen cells from multiparous mice protected the normal mice from infection with Rouse leukemia virus. Girardi et al. [16] showed that
Role o f Fetal Antigens in Tumor Immunity
lymphoid cells from primiparous and multiparous hamsters protected normal male hamsters against SV40 induced tumor challenge. O n the other hand, studies of Baldwin et al. [13], Parmiani and Lembo [14] showed that lymphoid cells from multiparous rats and mice were ineffective in adoptively transferring tumor immunity to normal animals. In the next set of experiments, three groups of normal mice received tumor cells incubated previously with M E M , with serum from normal virgin female mice, with serum from fetal cells immunized mice respectively. Inoculation of mice with tumor cells treated with serum from fetal cells immunized mice did not protect them against tumor formation (Fig. 7). Surprisingly, the tumor appearance time was shortened in these mice as compared to the control groups.
77
studies, Baldwin et al. [6, 13] have shown that lymph node cells from multiparous pregnant mice were cytotoxic in vitro for cultured tumor cells and fetal cells. But this cytotoxicity for tumor cells and fetal cells was blocked ill presence of serum from multiparous mice (Fig. 8). Similar findings have been given by Hellstrom and Hellstrom [17, 18] who ascribed the inability of fetal antigens to induce tumor resistance in body to these blocking antibodies.
Multiparous pregnant mice
Cytotoxic
28
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4 Tumor cells
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Multiporous pregnant mice
Fig. 7. This figure shows the effect of in vitro treatment of tumor cells with serum from normal mice and from mice immunized with fetal cells on time of appearance of tumors.
Coggin and Anderson [31] demonstrated that females splenectomized to reduce the antibody forming capacity react protectively to fetal vaccine. Antibody response against fetal antigens is more because these antigens contain more antigenic sites as compared to the tumor antigen. Recently in a series of their in vitro
Fig. 8. Diagrammaticpresentation of ln vitro cytotoxicity of lymphoid cells from multiparous mice against tumor cells, fetal cells and blocking of this cytotoxicity by serum from multiparous mice.
Recently, Tamerius et al. [32] described that fetal antigens have a special property to form blocking factors. These factors prevent either the development of cell mediated immunity or act against the expression of an already developed cell mediated immunity.
REFERENCES
1. 2. 3. 4. 5.
Y. H. PILCI-I and R. S. RlOOlNS, Antibodies to spontaneous and methylcholanthrene induced tumors in inbred mice. Cancer Res. 26, 871 (1966). J . E . DELORME and P. ALEXANDER,Treatment of primary fibrosarcoma in the rat with immune lymphocytes. Lancet ii, 117 (1964). G . I . ABELEV, Production of embryonal serum alpha-globulin by hepatomas. Review of experimental and clinical data. Cancer Res. 28, 1344 (1968). E. ~/[. EDYNAK, L. J. O L D , ~/~. VRANA and M. P. LARDIS, A fetal antigen associated with human neoplasia. New Engl. J. Med. 286, 1178 (1972). R . W . BALDWIN, D. GLAVES, l~. V. PIMM and B. M. VasE, Tumor specific and embryonic antigen expression on chemically induced rat tumors. Ann. Inst. Pasteur 122, 715 (1972).
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Savita Gautam and B. K. Aikat
6.
7.
8. 9.
10.
I 1.
12. 13. 14.
15.
16.
17.
18.
19. 20.
21. 22.
23. 24.
25. 26. 27.
R . W . BALDWIN, D. GLAVES and B. M. VOSE, Differentiation between the embryonic and tumour specific antigens of chemically induced rat tumours. Int. d. Cancer 29, 1 (1974). R . T . PREHN, The significance of tumor distinctive histo-compatibility antigens. In: Cross Reacting Antigens and Neoantigens. (Edited by J. J. TRENTIN), p. 105. Williams Wilkins Co., Baltimore (1967). B . P . LEMEVEL and S. A. WELLS, Foetal antigens cross reactive with tumour specific transplantation antigens. Nature New Biol. 244, 183 (1973). A. BENDICH, E. BOREN FREUND and E. H. STONEHILL,Protection of adult mice against tumor challenge by immunization with irradiated adult skin or embryo cells, d. Immunol. 111, 284 (1973). J . P . GRANT, S. LADISCHand S. A. WELLS, Immunologic similarities between fetal cell antigens and tumor cell antigens in guinea pigs. Cancer (Philad.) 33, 376 (1974). P . B . BLAIR, Search for cross reacting antigenicity between m a m m a r y tumor virus induced m a m m a r y tumors and embryonic antigens: Effect of immunization on development of spontaneous m a m m a r y tumors. Cancer Res. 30, 1199 (1970). J. E. CASTRO, E. M. LANCE, P. B. MEDAWAR, J. ZANELLI and R. HUNT, Foetal antigens and cancer. Nature (Lond.) 243, 225 (1973). R . W . BALDWIN, D. GLAVES and B. M. VOSE, Immunogenicity of embryonic antigens associated with chemically induced rat tumors. Int. d. Cancer 13, 135 (1974). G. PARMIANI and R. LEMBO, Effect of antiembryo immunization on Methylcholanthrene induced sarcoma growth in BALB/C mice. Int. J. Cancer 14, 555 (1974). l~. J. BRAWN, Possible association of embryonal antigen(s) with several primary 3-methyl cholanthrene induced murine sarcomas. Int. J. Cancer 6, 245 (1970). A . J . GIRARDI, P. PEPPUCCI, P. DIERLAM, W. RUTALA and J. H. COGOlN, Prevention of Simian Virus 40 tumors by hamster fetal tissue, influence of parity status of donor females on immunogenicity of fetal tissue and on immune cell cytotoxicity. Proc. nat. Acad. Sci. (Wash.) 70, 183 (1973). I. HELLSTROMand K. E. HELLSTROM, Cytotoxic effect of lymphocytes from pregnant mice on cultivated tumor cells. II. Blocking and unblocking of cytotoxicity. Int. J. Cancer 15, 30 (1975). I. I-IELLSTROMand K. E. HELLSTROM, Cytotoxic effect of lymphocytes from pregnant mice on cultivated tumor cells. I. Specificity, nature of effector cells and blocking by serum. Int. d. Cancer 15, 1 (1975). J. REINER and C. M. SOUTHAM,Evidence of common antigenic properties in chemically induced sarcomas of mice. Cancer Res. 27, 1243 (1967). P. GOLDMANand S. O. FREEDMAN,Specific carcino embryonic antigens of the human digestive system, d. exp. Med. 122, 467 (1965). D. BUFFE, C. RIMBAUT, J. LEMERLE, O. SCHWEISGUTCHand P. BURTIN, Presence of a ferroprotein of tissular origin, the .. 2H globulin in sera of children with tumours. Int. J. Cancer 5, 85 (1970). D . M . P . THOMSON and P. ALEXANDER, A cross reacting embryonic antigen in the membrane of rat sarcoma cells which is immunogenic in the syngeneic host. Brit. J. Cancer 27, 35 (1973). G. STEELEand H. O. SJOOREN, Embryonic antigens associated with chemically induced colon carcinoma in rats. Int. J. Cancer 14, 435 (1974). J . H . COGOIN, K. R. AMBROSE,B. B. BELLAMYand N. G. ANDERSON, T u m o r immunity in hamsters immunized with fetal tissues. J. Immunol. 107, 526 (1971). C.C. TING,J. R. ORTALDOand R. B. HERBERMAN,Expression of fetal antigens and tumor specific antigens in SV40-transformed cells. I. Serological analysis of the antigenic specificities. Int. J. Cancer 12, 511 (1973). C.C. TING, D. RODRIGUESand K. B. HERBERMAN,Expression of fetal antigens and tumor specific antigens in SV40-transformed cells. II. T u m o r transplantation studies. Int. J. Cancer 12, 519 (1973). G . A . H . BUTTLE and A. FRAYN, Effect of previous injection of homologous embryonic tissue on the growth of certain transplantable mouse tumours. Nature (Lond.) 215, 1495 (1967).
Role of Fetal Antigens in Tumor Immunity 28.
29. 30.
31.
39.
P . B . BLAIR, S. M. BLAIR, W. R. LYONS, H. A. BERN and C. H. LI, Effect of hormones and of parity on the occurrence of hyperplastic alveolar nodules and tumors in m a m m a r y glands of female A/Crgl mice. Cancer Res. 20, 1640 (1960). J . F . FRAUMENI,J. W. LLOYD, E. M. SMITH and J. K. WAGONER, Cancer mortality among nuns: Role of marital status in etiology of neoplastic disease in women. J. nat. Cancer Inst. 42, 455 (1969). !k/[. G. HANNA, R. W. TENNANT and J. H. COGOIN, Suppressive effect of immunization with mouse fetal antigens on growth of cells infected with Rauscher leukemia virus and on plasma cell tumors. Proc. nat. Acad. Sci. (Wash.) 68, 1748 (1971). J. H. COGGIN and N. G. ANDERSON, Phase specific autoantigens (fetal) in model tumor systems. In: Embryonic and Fetal Antigens in Cancer. (Edited by N. G. ANDERSON,J. H. COGGIN,E. COLE a n d J . W. HOLLEMAN)Vo1. II, p. 91. U.S. Department of Commerce, Springfield. J. TAMERIUS, I. HELLSTROMand K. E. HELLSTROM,Evidence that blocking factors in the sera of multiparous mice are associated with immuno-globulins. Int. d. Cancer20, 456 (1975).
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