Proc. Nati. Acad. Sci. USA
Vol. 75, No. 3, pp. 1519-1523, March 1978 Immunology
Teratocarcinoma cell variants rejected by syngeneic mice: Protection of mice immunized with these variants against other variants and against the original malignant cell line (tumor immunology)
THIERRY BOON AND ALINE VAN PEL Cellular Genetics Unit, International Institute of Cellular and Molecular Pathology, Avenue Hippocrate, 74, B-1200 Bruxelles, Belgium
Communicated by C. de Duve, December 27,1977
ABSTRACT We reported previously that, by mutagenesis of a malignant teratocarcinoma cell line, it is possible to obtain a number of variant clones that are incapable of forming progressive tumors. Each of these "tum" variants is rejected in syngeneic mice and stimulates the production of immune memor cells (self-protection). We show here that four different tum- clones confer an immune protection against each other although this cross-protection is invariably weaker than the self-protection. Moreover, mice immunized with living tumcells are partially protected against the original malignant teratocarcinoma cells, even though the latter cells are incapable of conferring any immune protection when injected after being killed by irradiation. These results indicate that each tumvariant carries at least one specific transplantation antigen that is absent from the original tumor cell line and from most other tum- variants. Other tumor-specific transplantation antigens are probably present on all the tum- variants and also on the malignant teratocarcinoma cell line. A large number of permanent clonal cell lines have been derived from the transplantable mouse teratocarcinoma tumors obtained in mouse strain 129/Sv (1-5). Many of these cell lines are malignant and pluripotent: syngeneic mice injected with these clonal cell lines form progressive tumors that contain the large variety of different tissues usually found in teratomas. Because of their ability to differentiate into many cell types, these teratocarcinoma cells are equivalent to the cells present in early embryos. The similarity between these two cell types extends to their surface antigens: both cells are devoid of H-2 antigens but carry the F9 antigen which is absent from any adult tissue with the exception of the germ line (6, 7). We reported previously that, by treatment of a malignant teratocarcinoma cell line with a mutagen, it is possible to obtain a number of variant clones that are incapable of forming progressive tumors. The cell line used was PCC4-azal, an azaguanine-resistant mutant derived from the malignant and pluripotent line PCC4. A population of PCCH-azal cells was treated with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine. Fifty-five clones were isolated from the surviving cells. Twelve of these clones (22%) were found to be unable to produce tumors in syngeneic mice upon injection of 1 X 106 cells, a dose that regularly generates a tumor with PCC4-azal. These variant clones were called tum- (nontumorigenic) as opposed to the tum+ (tumorigenic) initial PCC4-azal cells (8). The tum- phenotype appears to be linked to a phenomenon of immune rejection (8). Indeed, the tum- variants produce tumors as efficiently as the tum+ control in sublethally irradiated mice. Moreover, when mice injected with living tum- cells are irradiated 3 weeks later and are again injected with the same The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
tum- clone, few or no tumors appear. This demonstrates the existence of a radioresistant immune memory against tumcells. Finally, irradiated mice can be protected against a given tum- clone by adoptive transfer of spleen cells from mice immunized with living cells of the same clone. Representative results obtained with the tum+ line and the tum- clone 25 are summarized in Fig. 1. Similar results were obtained with tum- variants 20, 70, and 133 (8). The tum- phenotype could result from many different modifications of the teratocarcinoma cells, ranging from the creation of new surface antigens to the loss of immunosuppressive functions. To restrict the range of possible interpretations, we decided to investigate whether independently isolated tum- clones are immunologically identical or not. We took advantage of the fact that animals injected with a tumclone develop a radioresistant immune memory. Such mice were challenged with other tum- clones. We report here that the tum- clones that we have tested crossreact but are not immunologically identical. In the course of these experiments we observed that mice that have been immunized with tum- cells are partially protected against the tum+ control cells, even though these tum+ cells are almost completely unable to generate by themselves an immune protection in vvo. A description of this phenomenon is also given. MATERIALS AND METHODS Mice. Mice from the inbred 129/Sv line were obtained from J. L. Guenet (Institut Pasteur, Paris). The mice were more than 8 and less than 15 weeks old. Cell Lines. Line PCC4-azal is a clonal permanent cell line resistant to azaguanine at a concentration of 15 ,ug/ml (3). It was derived from line PCC4, which itself was derived from the transplantable teratocarcinoma OTT 6050 (9). PCC4-azal and the tum- clones derived from it were found to be free of my-
coplasma. The culture conditions have been described (3, 8). tum- Variants. These variant clones were obtained from PCC4-azal by a mutagen treatment and a cloning procedure as described (8). Clones 20, 25, 70, and 133 were isolated independently and have been described (8). The tum- clones used
in the experiments described below were maintained in permanent, exponentially growing cultures. New cultures were started from frozen stocks approximately every 3 months. Injection of Cells and Tumor Analysis. The cells are always injected both subcutaneously and intraperitoneally as described (8). This procedure yields about 90% subcutaneous tumors and 10% intraperitoneal solid tumors. A result is considered positive Abbreviations: tum-, not tumorigenic in syngeneic hosts; tum+, tumorigenic in syngeneic hosts.
1519
Proc. Natl. Acad. Sci. USA 75 (1978)
Immunology: Boon and Van Pel
1520
Table 1. Crossreactions among tum- clones % injected mice with tumors (no. mice with tumors/no. mice injected)
% mice with tumors
(no.' with tumors/no. injected)
PCC4.azal :tum'
F)
2-106
100% (81/81)
8% (5/65)
600 rads ()2-1 06
clone 20 25
100% (82/82)
70
600
a2y1O6
rads
2-116
12% (3 /24)
Day 21 Day 0 FIG. 1. The tum- phenotype. Result of injection of 129/Sv mice with 2 X 106 living tum+ cells or the same number of cells from tumclone 25. For clone 25 are also shown the results of the injection of animals given 600 rads of gamma radiation a few hours earlier and of animals immunized 3 weeks earlier. Data pooled from a number of independent experiments.
if the animal acquires a large tumor (about 1 within 2 months of the injection day.
cm
diameter)
RESULTS Existence of an Immune Cross-Protection between Different tum- Clones. A crossreaction experiment performed with tum- clones 20 and 25 is described in Fig. 2. One group of mice was injected with living cells from clone 20 and another with cells from clone 25. As expected, no tumor appeared. Three weeks later, the mice were given 600 rads (6J/kg) of gamma radiation. Each group was then divided into two subgroups that were challenged with either clone 20 or clone 25. Nonimmunized controls were also irradiated and injected with clone 20 or 25. In agreement with previous results, all the irradiated nonimmunized mice formed tumors, whereas none of the mice immunized and challenged with the same tum% mice with tumors (no. with tumors/no. injected)
600
t2-1 6
2.106
rids
Challenging clone
Immunizing
2-106
25:tum-
133
Controls
70
25
20
133
17
71
79
70
(1/6)
(15/21)
(19/24)
(16/23)
52
33
81
76
(11/21)
(3/9)
(17/21)
(16/21)
42
62
30
62
(5/12)
(8/13)
(3/10)
(8/13)
29
58
76
41
(7/24)
(14/24)
(19/25)
(7/17)
63
84
94
85
(23/27) (15/16) (21/25) (15/24) Mice were injected on the left side of the abdomen with 2 X 106 living cells of tum- clone 20, 25, 70, or 133 ("immunizing clone"). Control mice were injected with the same amount of culture medium. Three weeks later, the mice were given 600 rads of gamma radiation and were injected on the right side of the abdomen with 1 X 106 living cells from clone 20, 25, 70 or 133 ("challenging clone"). These results were obtained in a single experiment with a homogeneous group of 129/Sv mice.
clone did. In the subgroups immunized with one clone and challenged with the other, a significant number of mice failed to form tumors. In these groups, the number of mice with tumors was intermediate between that obtained with the control mice and that obtained with mice challenged and immunized with the same clone. A second experiment was performed to examine the crossreaction patterns existing among tum- clones 20, 25, 70, and 133. The results, shown in Table 1, are less clearcut than those of the first experiment. However, taken altogether, they confirm that, for a given challenging clone, immunization with a different tum- clone confers some degree of protection ("cross-protection"), even though this protection is invariably weaker than that conferred by an immunization with the same clone ("self-protection"). Similar results obtained in a third experiment are summarized in Table 2.
0% (0/15 ) 600 rads
2-1 06
53% (9/17) 600
(82- 106
2.106
rads
-
20
600 rads 1
2.106
i
78% (14/18 ) 2-106
rads
,
100% (12/12) 600 rads (8) 2-106
I
1
100%
(9/9)
Day 25 2. FIG. Cross-protection between tum- clones 20 and 25 in 129/Sv mice. Mice were immunized with 2 X 106 living cells of clones 20 or 25. Controls were not immunized. Twenty-five days later, all the mice were given 600 rads of gamma radiation and injected with 2 X 106 living cells from clone 20 or clone 25. Data are pooled from two inde-
Day 0
pendent experiments.
25
133
600
-(Controls)
0% (0/19 )
Table 2. Crossreactions among tum- clones % injected mice with tumors (no. mice with tumors/no. mice injected) Challenging clone Immunizing 133 25 20 clone
Controls
7
50
52
(2/27)
(12/24)
(14/27)
52
4
39
(14/27)
(1/28)
(11/28)
46
64
8
(12/26)
(16/25)
(2/26)
74
77
92
(20/27)
(20/26)
(24/26)
Mice were injected on the left side of the abdomen with 1 X 106 living cells of tum- clone 20,25, or 133 ("immunizing clone") on day 0. On day 9, these mice were boosted with 1 X 107 living cells of the same clone. Control mice were injected with the same amount of culture medium. On day 30, the mice were given 600 rads of gamma radiation and were injected on the right side of the abdomen with 1 x 106 living cells from clone 20,25, or 133 ("challenging clone"). These results were obtained in a single experiment with a homogeneous group of 129/Sv mice.
Immunology:
Boon and Van Pel % mice with tumors (no. with tumors/no. injected)
-(Control)
tumr 5-10S
Proc. Nati. Acad. Sci. USA 75 (1978)
% mice with tumors (no. with tumors/no. injected)
Experiment 1
3*10'
-(Control)
*100% (9/9)
93% (14/15)
9 3.106
5*105 55% (6/11)
3.106
5.1e Ij~fjeIrradiated
3.106
Iu i
($10i°;g l ivi ng
FU5M10s 33% (4/12)
5 -105 71% (5/7)
1521
Day 0
3 10S *100% (12/12)
3-105 30 Day 22
* 55%Y (5/9)
Experiment 2 -(Control)
[X
106 95% (19/20)
~i107
120a2
1.5-106
E
tu3 5 105
irradiated
~E11106
50% (5/10)
1207011.5.106 v257
1.5.106
107
()irradiated
5105 56% (5/9)
[1
-(Control)
5i10s
210770~
iIrradiated
5.105 25% (3/12)
Day 21 Cross-protection against tum+ cells after immunization with living tum- cells. 129/Sv mice were injected with living tumn cells on their left side. Some groups were injected with a mixture of cells from different clones; the amounts indicated correspond to the amount of each clone. Control mice were not injected. Twenty-one days later, all the mice were injected on the right side with 5 X 105 living tum+ cells. Mice were scored for tumors on the right side as well as for intraperitoneal tumors. These results were obtained in a single experiment. Day 0
FIG. 3.
The fact that self-protection is invariably stronger than cross-protection indicates that the four tum- clones tested here have specific individual antigens. The existence of cross-protection suggests that these tum- clones may also have crossreacting antigens. Rejection of tum+ Cells by Mice Immunized with Living tum- Cells. A cross-protection experiment was performed with PCC4-azal challenging cells. Mice were injected with 3 X 106 living cells from tum- clone 20, 25, or 70. No tumor appeared. Three weeks later, these mice were challenged with 5 X 105
living tum+ cells, injected on the other side of the abdomen. The number of tumors produced by the challenging tum+ cells is shown in the upper part of Fig. 3. Clearly, the animals immunized with tum- cells formed significantly fewer tumors than did the controls. Similar results were obtained in a large number of independent experiments (see below). The protection against tum+ cells was not abolished by irradiation (600 rads) given just before the injection of the challenging cells (data not shown). The protection obtained in mice challenged 45 days after immunization was as good as that obtained after 21 days (data not shown). These results suggest that the tum+ cells and the three tumclones tested have crossreacting antigens. Absence of Protection by Irradiated tum+ Cells. The results described in the preceding section would not be particularly worthy of notice were it not for the fact that the tum+ PCC4-azal teratocarcinoma cells have little ability, if any, to
106 95% (19/20)
670 rads (4)106 100% (10/10)
60% (6/10)
J20,25,701 1.1 06
EfD0
100%(21/21)
Day 0
670 rads (i106 Day 21
72% (13 /18)
FIG. 4. Immunization with dead tum+ or tum- cells. Exp. 1. 129/Sv mice were inoculated on the left side with 106 living cells of clone 20 or 5 X 106 tum+ cells killed with 4000 rads of gamma radiation. Controls were not injected. Twenty-two days later, the mice were challenged with 3 X 105 living tum+ cells injected on the right side. Exp. 2. 129/Sv mice were immunized on the left with cells killed with 2500 rads. Controls were injected with medium. Twenty-one days later, the mice were challenged on the other side with either 106 tum+ cells or 106 cells of clone 25. Mice were scored for tumors on the right side and for intraperitoneal tumors.
induce a rejection response in the syngeneic 129/Sv mice. Animals immunized with a large number of tum+ cells killed by irradiation failed to show any protection when they were challenged with tum+ cells (Fig. 4). Even multiple immunizations with killed tum+ cells failed to induce a significant protection (data not shown). Preliminary experiments with mice whose PCC4*azal tumor had been surgically removed suggest that the protection of these mice against a challenge with tum+ cells is at best very weak. Factors Influencing the Protection Against tum+ Cells. The degree of protection observed in animals that had rejected tum- cells decreased rapidly when the number of challenging tum+ cells was increased. In the experiment reported in Table 3, the protection broke down coppletely when the number of challenging cells exceeded 10 times the minimal number of tum+ cells required to obtain a tumor in the majority of the control mice (about 2 X 105). Repeating the injections of tum- cells three times at 15 day intervals did not markedly improve the protection (data not shown). However, an increase in the number of immunizing tum- cells well above that needed to produce a tumor in irradiated mice improved the protection considerably (Table 4). Immunizations with mixtures of clones 20, 25, and 70 did not result in better protections than immunizations with single clones (Fig. 3 lower). However, in this experiment, the total number of cells injected in the mixture was equal to the number
1522
Proc. Natl. Acad. Sci. USA 75 (1978)
Immunology: Boon and Van Pel
Table 3. Influence of dose of tum+ challenging cell % with tumors (no. tumors/ Challenging cell Immunizing cell no. mice injected Dose Dose Type Type 2.5 X 106 (control)
tum+
5 X 105
-
41 (11/27) 93 (27/29)
20
2.5 X 106
tum+
1 X 106
-
(control) 2.5 X 106 (control) 2.5 X 106 (control)
76 (22/29) 100 (28/28)
tum+
2.5 X 106
93 (28/30) 97 (29/30)
tum+
6 X 106
93 (28/30) 100 (29/29)
20
20 -
20
Mice were immunized on the left side with living cells of tumr clone 20 at the dose indicated. The controls were injected with an equal amount of medium. Three weeks later, the mice were challenged with living tum+ cells at the dose indicated. Data are pooled from two separate experiments.
of cells injected in the immunizations performed with a single clone. In view of increased protection obtained with large numbers of immunizing tum- cells, the possibility remains that mixtures of tum- clones may improve the protections obtained with saturating numbers of tum- cells. Finally, injections of tum- cells killed by irradiation failed to induce any significant protection against tum+ cells (Fig. 4 lower). Irradiated tum- cells nevertheless could induce a partial self-protection. To summarize, we found that the only practicable way to obtain a significant protection against tum+ cells was immunization with a large number of living tum- cells.
DISCUSSION The initial purpose of the immunization experiments described here was to find a difference between the tum+ and the tumcells that could account for the rejection of the latter. Our main relevant observation is that the self-protection obtained against a tum- variant after immunization with the same variant is always better than the cross-protection obtained after immunization with another tum- variant. This result, obtained with four tum- clones, is clearly compatible with the hypothesis that the mutagenic treatment resulted in the stable acquisition of new antigens specific for each tum- clone. Other explanations of the tum- phenotype were considered. Some of them do not postulate the presence of new antigens on the tum- variants. For instance, these variants could carry increased amounts of a weak tumor-specific antigen already present on the tum+ cell. Alternatively, they could have lost an immunosuppressive function. This loss would allow for an increased effectiveness of either the afferent or the efferent branch of the immune rejection process. Similar effects could also result from a decrease in antigen shedding. In our opinion, such hypotheses can be reduced either to an increase in the immunogenicity of the teratocarcinoma cells or to an increase in their sensitivity to immune effector cells. They all imply that the same set of antigenic specificities is present on all the tumvariants. None of these hypotheses predicts that the self-protection will invariably be superior to the cross-protection. On the contrary, they predict that, if a difference is to be found, the cross-protection found against tum- variants ought in some cases to be stronger than the corresponding self-protection. It is therefore difficult to reconcile any of these explanations with our observations, and we are led to postulate that the tum-
Table 4. Influence of number of immunizing tum- cells on protection against the tum+ cells % with tumors Immunizing cell Challenging cell Dose Type Dose (no. tumors/no. mice injected) 59 (10/17) 4 X 105 tum+ 5 X 105 20 35 (6/17) 2 X 106 20 35 (6/17) 1 X 107 20 4 X 105 tum+ 5 X 105 61 (11/18) 25 2X 106 50(8/16) 25 1 x 107 11 (2/18) 25
Type
(control) tum+ 5 X 105
100 (19/19)
Mice were injected with various numbers of tum- cells on their left side. Controls were injected with an equal amount of medium. Three weeks later, the mice were injected on the right with living tum+ cells. Mice were scored for tumors on the right side and for intraperitoneal tumors.
variants carry a new antigen not present on the tum+ cells. A priori, this antigen could be the same for all the tum- variants. For instance, the mutagen could induce the same viral antigen in all the tum- variants. However, this would make all tumclones immunologically identical and this is clearly incompatible with the advantage of self-protection over cross-protection. We therefore conclude that it is most likely that every tum- variant is endowed with at least one new antigenic specificity that is absent from most of the other tum- variants and from the tum+ teratocarcinoma cells. In this respect, these antigens would be analogous to the individual tumor-specific transplantation antigens found on tumors induced by methylcholanthrene (10). A second observation that requires an interpretation is the occurrence of cross-protection not only between different tumvariants but also between tum- and the original tum+ teratocarcinoma cells. Our results suggest that this cross-protection is not due to a nonspecific stimulation of the immune system consequent to the rejection of the tum- cells. Indeed, the cross-protection against tum- as well as that against tum+ cells can occur after irradiation of the immunized animals. Because first-set rejections are sensitive to irradiation and second-set rejections are rather resistant (11, 12), this suggests that these cross-protections are secondary reactions. This, in turn, implies that they act upon an antigenic specificity common to the immunizing cell and the challenging cell. It appears therefore that, besides the rejection antigens that are specific for each tumclone, there must be one or more common antigenic specificities shared by all the tum- variants as well as by the tum+ cells. These common antigenic specificities may be completely independent of the individual tum- antigens. Immunization with living tum- cells would then simply allow for the presentation of this common antigen to the immune system with an efficiency that cannot be achieved by injecting tum+ or tum- cells previously killed by irradiation. Alternatively, this common antigen may function on the tum+ cells like a hapten, not immunogenic but able to serve as a target for immune effector cells. The new antigenic specificity present on each tumvariant would be part of a complex functioning as a carrier with respect to this common hapten, rendering it immunogenic. Hence, the ability of the tum- variant to raise an immune response against the tum+ teratocarcinoma cells. Further experiments will be required to decide between these two models. Unlike many virally or chemically induced tumors, most spontaneous tumors found in mice and rats have proved to be
Immunology: Boon and Van Pel unable to immunize syngeneic hosts (13-15). This is likely to apply also to human tumors. Consequently, it may be of great benefit for cancer therapy to obtain modified tumor cells capable of provoking the rejection of the tumor from which they are derived (16). The results described here indicate that tumvariants may represent such a possibility. The first question that needs to be answered is whether the possibility of obtaining tum- variants at high frequencies will be restricted to certain tumors or whether it will prove to be generally applicable to all cancer cells. We gratefully acknowledge the excellent assistance of G. Warnier, J. C. Gaudin, and Paul Wauters. We thank M. Kumps for her help in preparing the manuscript. We are particularly grateful to P. Medawar, C. de Duve, J. Van Snick, and P. Masson for stimulating discussions. This work was supported by the Fonds cancerologique de la Caisse Generale d'Epargne et de Retraite, Brussels, Belgium. 1. Rosenthal, M. D., Wishnow, R. M. & Sato, G. H. (1970) J. Natl. Cancer Inst. 44, 1001-1009. 2. Kahan, B. W. & Ephrussi, B. (1970) J. Natl. Cancer Inst. 44, 1015-1029.
Proc. Nati. Acad. Sci. USA 75 (1978)
1523
3. Jakob, H., Boon, T., Gaillard, J., Nicolas, J. F. & Jacob, F. (1973) Ann. Microbiol. (Paris) 124B, 269-282. 4. Evans, M. J. (1972) J. Embryol. Exp. Morphol. 28, 163-176. 5. Lehman, J. M., Speers, W. C., Swartzendruber, D. E. & Pierce, G. B. (1974) J. Cell. Physiol. 84, 13-28. 6. Artzt, K., Dubois, P., Bennett, D., Condamine, H. & Jacob, F. (1973) Proc. Nati. Acad. Sci. USA 70,2988-2992. 7. Artzt, K. & Jacob, F. (1974) Transplantation 17,632-634. 8. Boon, T. & Kellermann, 0. (1977) Proc. Natl. Acad. Sd. USA 74, 272-275. 9. Stevens, L. C. (1970) Dev. Biol. 21, 364-382. 10. Main, J. M. & Prehn, R. T. (1957) J. Natl. Cancer Inst. 19, 1053-1064. 11. Tyan, M. L. & Cole, L. J. (1963) Transplantation 1, 546. 12. Dempster, W. J., Lennox, B. & Boag, J. W. (1950) Br. J. Exp. Pathol. 31, 670. 13. Baldwin, R. W. (1966) J. Natl. Cancer Institute 1, 257-264. 14. Prehn, R. T. (1976) Adv. Cancer Res. 23, 203-236. 15. Hewitt, H. B., Blake, E. R. & Walder, A. S. (1976) Br. J. Cancer 33,241-259. 16. Klein, G. & Klein, E. (1977) Proc. Natl. Acad. Sci. USA 74, 2121-2125.
Vol. 75, No. 3, pp. 1519-1523, March 1978 Immunology
Teratocarcinoma cell variants rejected by syngeneic mice: Protection of mice immunized with these variants against other variants and against the original malignant cell line (tumor immunology)
THIERRY BOON AND ALINE VAN PEL Cellular Genetics Unit, International Institute of Cellular and Molecular Pathology, Avenue Hippocrate, 74, B-1200 Bruxelles, Belgium
Communicated by C. de Duve, December 27,1977
ABSTRACT We reported previously that, by mutagenesis of a malignant teratocarcinoma cell line, it is possible to obtain a number of variant clones that are incapable of forming progressive tumors. Each of these "tum" variants is rejected in syngeneic mice and stimulates the production of immune memor cells (self-protection). We show here that four different tum- clones confer an immune protection against each other although this cross-protection is invariably weaker than the self-protection. Moreover, mice immunized with living tumcells are partially protected against the original malignant teratocarcinoma cells, even though the latter cells are incapable of conferring any immune protection when injected after being killed by irradiation. These results indicate that each tumvariant carries at least one specific transplantation antigen that is absent from the original tumor cell line and from most other tum- variants. Other tumor-specific transplantation antigens are probably present on all the tum- variants and also on the malignant teratocarcinoma cell line. A large number of permanent clonal cell lines have been derived from the transplantable mouse teratocarcinoma tumors obtained in mouse strain 129/Sv (1-5). Many of these cell lines are malignant and pluripotent: syngeneic mice injected with these clonal cell lines form progressive tumors that contain the large variety of different tissues usually found in teratomas. Because of their ability to differentiate into many cell types, these teratocarcinoma cells are equivalent to the cells present in early embryos. The similarity between these two cell types extends to their surface antigens: both cells are devoid of H-2 antigens but carry the F9 antigen which is absent from any adult tissue with the exception of the germ line (6, 7). We reported previously that, by treatment of a malignant teratocarcinoma cell line with a mutagen, it is possible to obtain a number of variant clones that are incapable of forming progressive tumors. The cell line used was PCC4-azal, an azaguanine-resistant mutant derived from the malignant and pluripotent line PCC4. A population of PCCH-azal cells was treated with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine. Fifty-five clones were isolated from the surviving cells. Twelve of these clones (22%) were found to be unable to produce tumors in syngeneic mice upon injection of 1 X 106 cells, a dose that regularly generates a tumor with PCC4-azal. These variant clones were called tum- (nontumorigenic) as opposed to the tum+ (tumorigenic) initial PCC4-azal cells (8). The tum- phenotype appears to be linked to a phenomenon of immune rejection (8). Indeed, the tum- variants produce tumors as efficiently as the tum+ control in sublethally irradiated mice. Moreover, when mice injected with living tum- cells are irradiated 3 weeks later and are again injected with the same The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
tum- clone, few or no tumors appear. This demonstrates the existence of a radioresistant immune memory against tumcells. Finally, irradiated mice can be protected against a given tum- clone by adoptive transfer of spleen cells from mice immunized with living cells of the same clone. Representative results obtained with the tum+ line and the tum- clone 25 are summarized in Fig. 1. Similar results were obtained with tum- variants 20, 70, and 133 (8). The tum- phenotype could result from many different modifications of the teratocarcinoma cells, ranging from the creation of new surface antigens to the loss of immunosuppressive functions. To restrict the range of possible interpretations, we decided to investigate whether independently isolated tum- clones are immunologically identical or not. We took advantage of the fact that animals injected with a tumclone develop a radioresistant immune memory. Such mice were challenged with other tum- clones. We report here that the tum- clones that we have tested crossreact but are not immunologically identical. In the course of these experiments we observed that mice that have been immunized with tum- cells are partially protected against the tum+ control cells, even though these tum+ cells are almost completely unable to generate by themselves an immune protection in vvo. A description of this phenomenon is also given. MATERIALS AND METHODS Mice. Mice from the inbred 129/Sv line were obtained from J. L. Guenet (Institut Pasteur, Paris). The mice were more than 8 and less than 15 weeks old. Cell Lines. Line PCC4-azal is a clonal permanent cell line resistant to azaguanine at a concentration of 15 ,ug/ml (3). It was derived from line PCC4, which itself was derived from the transplantable teratocarcinoma OTT 6050 (9). PCC4-azal and the tum- clones derived from it were found to be free of my-
coplasma. The culture conditions have been described (3, 8). tum- Variants. These variant clones were obtained from PCC4-azal by a mutagen treatment and a cloning procedure as described (8). Clones 20, 25, 70, and 133 were isolated independently and have been described (8). The tum- clones used
in the experiments described below were maintained in permanent, exponentially growing cultures. New cultures were started from frozen stocks approximately every 3 months. Injection of Cells and Tumor Analysis. The cells are always injected both subcutaneously and intraperitoneally as described (8). This procedure yields about 90% subcutaneous tumors and 10% intraperitoneal solid tumors. A result is considered positive Abbreviations: tum-, not tumorigenic in syngeneic hosts; tum+, tumorigenic in syngeneic hosts.
1519
Proc. Natl. Acad. Sci. USA 75 (1978)
Immunology: Boon and Van Pel
1520
Table 1. Crossreactions among tum- clones % injected mice with tumors (no. mice with tumors/no. mice injected)
% mice with tumors
(no.' with tumors/no. injected)
PCC4.azal :tum'
F)
2-106
100% (81/81)
8% (5/65)
600 rads ()2-1 06
clone 20 25
100% (82/82)
70
600
a2y1O6
rads
2-116
12% (3 /24)
Day 21 Day 0 FIG. 1. The tum- phenotype. Result of injection of 129/Sv mice with 2 X 106 living tum+ cells or the same number of cells from tumclone 25. For clone 25 are also shown the results of the injection of animals given 600 rads of gamma radiation a few hours earlier and of animals immunized 3 weeks earlier. Data pooled from a number of independent experiments.
if the animal acquires a large tumor (about 1 within 2 months of the injection day.
cm
diameter)
RESULTS Existence of an Immune Cross-Protection between Different tum- Clones. A crossreaction experiment performed with tum- clones 20 and 25 is described in Fig. 2. One group of mice was injected with living cells from clone 20 and another with cells from clone 25. As expected, no tumor appeared. Three weeks later, the mice were given 600 rads (6J/kg) of gamma radiation. Each group was then divided into two subgroups that were challenged with either clone 20 or clone 25. Nonimmunized controls were also irradiated and injected with clone 20 or 25. In agreement with previous results, all the irradiated nonimmunized mice formed tumors, whereas none of the mice immunized and challenged with the same tum% mice with tumors (no. with tumors/no. injected)
600
t2-1 6
2.106
rids
Challenging clone
Immunizing
2-106
25:tum-
133
Controls
70
25
20
133
17
71
79
70
(1/6)
(15/21)
(19/24)
(16/23)
52
33
81
76
(11/21)
(3/9)
(17/21)
(16/21)
42
62
30
62
(5/12)
(8/13)
(3/10)
(8/13)
29
58
76
41
(7/24)
(14/24)
(19/25)
(7/17)
63
84
94
85
(23/27) (15/16) (21/25) (15/24) Mice were injected on the left side of the abdomen with 2 X 106 living cells of tum- clone 20, 25, 70, or 133 ("immunizing clone"). Control mice were injected with the same amount of culture medium. Three weeks later, the mice were given 600 rads of gamma radiation and were injected on the right side of the abdomen with 1 X 106 living cells from clone 20, 25, 70 or 133 ("challenging clone"). These results were obtained in a single experiment with a homogeneous group of 129/Sv mice.
clone did. In the subgroups immunized with one clone and challenged with the other, a significant number of mice failed to form tumors. In these groups, the number of mice with tumors was intermediate between that obtained with the control mice and that obtained with mice challenged and immunized with the same clone. A second experiment was performed to examine the crossreaction patterns existing among tum- clones 20, 25, 70, and 133. The results, shown in Table 1, are less clearcut than those of the first experiment. However, taken altogether, they confirm that, for a given challenging clone, immunization with a different tum- clone confers some degree of protection ("cross-protection"), even though this protection is invariably weaker than that conferred by an immunization with the same clone ("self-protection"). Similar results obtained in a third experiment are summarized in Table 2.
0% (0/15 ) 600 rads
2-1 06
53% (9/17) 600
(82- 106
2.106
rads
-
20
600 rads 1
2.106
i
78% (14/18 ) 2-106
rads
,
100% (12/12) 600 rads (8) 2-106
I
1
100%
(9/9)
Day 25 2. FIG. Cross-protection between tum- clones 20 and 25 in 129/Sv mice. Mice were immunized with 2 X 106 living cells of clones 20 or 25. Controls were not immunized. Twenty-five days later, all the mice were given 600 rads of gamma radiation and injected with 2 X 106 living cells from clone 20 or clone 25. Data are pooled from two inde-
Day 0
pendent experiments.
25
133
600
-(Controls)
0% (0/19 )
Table 2. Crossreactions among tum- clones % injected mice with tumors (no. mice with tumors/no. mice injected) Challenging clone Immunizing 133 25 20 clone
Controls
7
50
52
(2/27)
(12/24)
(14/27)
52
4
39
(14/27)
(1/28)
(11/28)
46
64
8
(12/26)
(16/25)
(2/26)
74
77
92
(20/27)
(20/26)
(24/26)
Mice were injected on the left side of the abdomen with 1 X 106 living cells of tum- clone 20,25, or 133 ("immunizing clone") on day 0. On day 9, these mice were boosted with 1 X 107 living cells of the same clone. Control mice were injected with the same amount of culture medium. On day 30, the mice were given 600 rads of gamma radiation and were injected on the right side of the abdomen with 1 x 106 living cells from clone 20,25, or 133 ("challenging clone"). These results were obtained in a single experiment with a homogeneous group of 129/Sv mice.
Immunology:
Boon and Van Pel % mice with tumors (no. with tumors/no. injected)
-(Control)
tumr 5-10S
Proc. Nati. Acad. Sci. USA 75 (1978)
% mice with tumors (no. with tumors/no. injected)
Experiment 1
3*10'
-(Control)
*100% (9/9)
93% (14/15)
9 3.106
5*105 55% (6/11)
3.106
5.1e Ij~fjeIrradiated
3.106
Iu i
($10i°;g l ivi ng
FU5M10s 33% (4/12)
5 -105 71% (5/7)
1521
Day 0
3 10S *100% (12/12)
3-105 30 Day 22
* 55%Y (5/9)
Experiment 2 -(Control)
[X
106 95% (19/20)
~i107
120a2
1.5-106
E
tu3 5 105
irradiated
~E11106
50% (5/10)
1207011.5.106 v257
1.5.106
107
()irradiated
5105 56% (5/9)
[1
-(Control)
5i10s
210770~
iIrradiated
5.105 25% (3/12)
Day 21 Cross-protection against tum+ cells after immunization with living tum- cells. 129/Sv mice were injected with living tumn cells on their left side. Some groups were injected with a mixture of cells from different clones; the amounts indicated correspond to the amount of each clone. Control mice were not injected. Twenty-one days later, all the mice were injected on the right side with 5 X 105 living tum+ cells. Mice were scored for tumors on the right side as well as for intraperitoneal tumors. These results were obtained in a single experiment. Day 0
FIG. 3.
The fact that self-protection is invariably stronger than cross-protection indicates that the four tum- clones tested here have specific individual antigens. The existence of cross-protection suggests that these tum- clones may also have crossreacting antigens. Rejection of tum+ Cells by Mice Immunized with Living tum- Cells. A cross-protection experiment was performed with PCC4-azal challenging cells. Mice were injected with 3 X 106 living cells from tum- clone 20, 25, or 70. No tumor appeared. Three weeks later, these mice were challenged with 5 X 105
living tum+ cells, injected on the other side of the abdomen. The number of tumors produced by the challenging tum+ cells is shown in the upper part of Fig. 3. Clearly, the animals immunized with tum- cells formed significantly fewer tumors than did the controls. Similar results were obtained in a large number of independent experiments (see below). The protection against tum+ cells was not abolished by irradiation (600 rads) given just before the injection of the challenging cells (data not shown). The protection obtained in mice challenged 45 days after immunization was as good as that obtained after 21 days (data not shown). These results suggest that the tum+ cells and the three tumclones tested have crossreacting antigens. Absence of Protection by Irradiated tum+ Cells. The results described in the preceding section would not be particularly worthy of notice were it not for the fact that the tum+ PCC4-azal teratocarcinoma cells have little ability, if any, to
106 95% (19/20)
670 rads (4)106 100% (10/10)
60% (6/10)
J20,25,701 1.1 06
EfD0
100%(21/21)
Day 0
670 rads (i106 Day 21
72% (13 /18)
FIG. 4. Immunization with dead tum+ or tum- cells. Exp. 1. 129/Sv mice were inoculated on the left side with 106 living cells of clone 20 or 5 X 106 tum+ cells killed with 4000 rads of gamma radiation. Controls were not injected. Twenty-two days later, the mice were challenged with 3 X 105 living tum+ cells injected on the right side. Exp. 2. 129/Sv mice were immunized on the left with cells killed with 2500 rads. Controls were injected with medium. Twenty-one days later, the mice were challenged on the other side with either 106 tum+ cells or 106 cells of clone 25. Mice were scored for tumors on the right side and for intraperitoneal tumors.
induce a rejection response in the syngeneic 129/Sv mice. Animals immunized with a large number of tum+ cells killed by irradiation failed to show any protection when they were challenged with tum+ cells (Fig. 4). Even multiple immunizations with killed tum+ cells failed to induce a significant protection (data not shown). Preliminary experiments with mice whose PCC4*azal tumor had been surgically removed suggest that the protection of these mice against a challenge with tum+ cells is at best very weak. Factors Influencing the Protection Against tum+ Cells. The degree of protection observed in animals that had rejected tum- cells decreased rapidly when the number of challenging tum+ cells was increased. In the experiment reported in Table 3, the protection broke down coppletely when the number of challenging cells exceeded 10 times the minimal number of tum+ cells required to obtain a tumor in the majority of the control mice (about 2 X 105). Repeating the injections of tum- cells three times at 15 day intervals did not markedly improve the protection (data not shown). However, an increase in the number of immunizing tum- cells well above that needed to produce a tumor in irradiated mice improved the protection considerably (Table 4). Immunizations with mixtures of clones 20, 25, and 70 did not result in better protections than immunizations with single clones (Fig. 3 lower). However, in this experiment, the total number of cells injected in the mixture was equal to the number
1522
Proc. Natl. Acad. Sci. USA 75 (1978)
Immunology: Boon and Van Pel
Table 3. Influence of dose of tum+ challenging cell % with tumors (no. tumors/ Challenging cell Immunizing cell no. mice injected Dose Dose Type Type 2.5 X 106 (control)
tum+
5 X 105
-
41 (11/27) 93 (27/29)
20
2.5 X 106
tum+
1 X 106
-
(control) 2.5 X 106 (control) 2.5 X 106 (control)
76 (22/29) 100 (28/28)
tum+
2.5 X 106
93 (28/30) 97 (29/30)
tum+
6 X 106
93 (28/30) 100 (29/29)
20
20 -
20
Mice were immunized on the left side with living cells of tumr clone 20 at the dose indicated. The controls were injected with an equal amount of medium. Three weeks later, the mice were challenged with living tum+ cells at the dose indicated. Data are pooled from two separate experiments.
of cells injected in the immunizations performed with a single clone. In view of increased protection obtained with large numbers of immunizing tum- cells, the possibility remains that mixtures of tum- clones may improve the protections obtained with saturating numbers of tum- cells. Finally, injections of tum- cells killed by irradiation failed to induce any significant protection against tum+ cells (Fig. 4 lower). Irradiated tum- cells nevertheless could induce a partial self-protection. To summarize, we found that the only practicable way to obtain a significant protection against tum+ cells was immunization with a large number of living tum- cells.
DISCUSSION The initial purpose of the immunization experiments described here was to find a difference between the tum+ and the tumcells that could account for the rejection of the latter. Our main relevant observation is that the self-protection obtained against a tum- variant after immunization with the same variant is always better than the cross-protection obtained after immunization with another tum- variant. This result, obtained with four tum- clones, is clearly compatible with the hypothesis that the mutagenic treatment resulted in the stable acquisition of new antigens specific for each tum- clone. Other explanations of the tum- phenotype were considered. Some of them do not postulate the presence of new antigens on the tum- variants. For instance, these variants could carry increased amounts of a weak tumor-specific antigen already present on the tum+ cell. Alternatively, they could have lost an immunosuppressive function. This loss would allow for an increased effectiveness of either the afferent or the efferent branch of the immune rejection process. Similar effects could also result from a decrease in antigen shedding. In our opinion, such hypotheses can be reduced either to an increase in the immunogenicity of the teratocarcinoma cells or to an increase in their sensitivity to immune effector cells. They all imply that the same set of antigenic specificities is present on all the tumvariants. None of these hypotheses predicts that the self-protection will invariably be superior to the cross-protection. On the contrary, they predict that, if a difference is to be found, the cross-protection found against tum- variants ought in some cases to be stronger than the corresponding self-protection. It is therefore difficult to reconcile any of these explanations with our observations, and we are led to postulate that the tum-
Table 4. Influence of number of immunizing tum- cells on protection against the tum+ cells % with tumors Immunizing cell Challenging cell Dose Type Dose (no. tumors/no. mice injected) 59 (10/17) 4 X 105 tum+ 5 X 105 20 35 (6/17) 2 X 106 20 35 (6/17) 1 X 107 20 4 X 105 tum+ 5 X 105 61 (11/18) 25 2X 106 50(8/16) 25 1 x 107 11 (2/18) 25
Type
(control) tum+ 5 X 105
100 (19/19)
Mice were injected with various numbers of tum- cells on their left side. Controls were injected with an equal amount of medium. Three weeks later, the mice were injected on the right with living tum+ cells. Mice were scored for tumors on the right side and for intraperitoneal tumors.
variants carry a new antigen not present on the tum+ cells. A priori, this antigen could be the same for all the tum- variants. For instance, the mutagen could induce the same viral antigen in all the tum- variants. However, this would make all tumclones immunologically identical and this is clearly incompatible with the advantage of self-protection over cross-protection. We therefore conclude that it is most likely that every tum- variant is endowed with at least one new antigenic specificity that is absent from most of the other tum- variants and from the tum+ teratocarcinoma cells. In this respect, these antigens would be analogous to the individual tumor-specific transplantation antigens found on tumors induced by methylcholanthrene (10). A second observation that requires an interpretation is the occurrence of cross-protection not only between different tumvariants but also between tum- and the original tum+ teratocarcinoma cells. Our results suggest that this cross-protection is not due to a nonspecific stimulation of the immune system consequent to the rejection of the tum- cells. Indeed, the cross-protection against tum- as well as that against tum+ cells can occur after irradiation of the immunized animals. Because first-set rejections are sensitive to irradiation and second-set rejections are rather resistant (11, 12), this suggests that these cross-protections are secondary reactions. This, in turn, implies that they act upon an antigenic specificity common to the immunizing cell and the challenging cell. It appears therefore that, besides the rejection antigens that are specific for each tumclone, there must be one or more common antigenic specificities shared by all the tum- variants as well as by the tum+ cells. These common antigenic specificities may be completely independent of the individual tum- antigens. Immunization with living tum- cells would then simply allow for the presentation of this common antigen to the immune system with an efficiency that cannot be achieved by injecting tum+ or tum- cells previously killed by irradiation. Alternatively, this common antigen may function on the tum+ cells like a hapten, not immunogenic but able to serve as a target for immune effector cells. The new antigenic specificity present on each tumvariant would be part of a complex functioning as a carrier with respect to this common hapten, rendering it immunogenic. Hence, the ability of the tum- variant to raise an immune response against the tum+ teratocarcinoma cells. Further experiments will be required to decide between these two models. Unlike many virally or chemically induced tumors, most spontaneous tumors found in mice and rats have proved to be
Immunology: Boon and Van Pel unable to immunize syngeneic hosts (13-15). This is likely to apply also to human tumors. Consequently, it may be of great benefit for cancer therapy to obtain modified tumor cells capable of provoking the rejection of the tumor from which they are derived (16). The results described here indicate that tumvariants may represent such a possibility. The first question that needs to be answered is whether the possibility of obtaining tum- variants at high frequencies will be restricted to certain tumors or whether it will prove to be generally applicable to all cancer cells. We gratefully acknowledge the excellent assistance of G. Warnier, J. C. Gaudin, and Paul Wauters. We thank M. Kumps for her help in preparing the manuscript. We are particularly grateful to P. Medawar, C. de Duve, J. Van Snick, and P. Masson for stimulating discussions. This work was supported by the Fonds cancerologique de la Caisse Generale d'Epargne et de Retraite, Brussels, Belgium. 1. Rosenthal, M. D., Wishnow, R. M. & Sato, G. H. (1970) J. Natl. Cancer Inst. 44, 1001-1009. 2. Kahan, B. W. & Ephrussi, B. (1970) J. Natl. Cancer Inst. 44, 1015-1029.
Proc. Nati. Acad. Sci. USA 75 (1978)
1523
3. Jakob, H., Boon, T., Gaillard, J., Nicolas, J. F. & Jacob, F. (1973) Ann. Microbiol. (Paris) 124B, 269-282. 4. Evans, M. J. (1972) J. Embryol. Exp. Morphol. 28, 163-176. 5. Lehman, J. M., Speers, W. C., Swartzendruber, D. E. & Pierce, G. B. (1974) J. Cell. Physiol. 84, 13-28. 6. Artzt, K., Dubois, P., Bennett, D., Condamine, H. & Jacob, F. (1973) Proc. Nati. Acad. Sci. USA 70,2988-2992. 7. Artzt, K. & Jacob, F. (1974) Transplantation 17,632-634. 8. Boon, T. & Kellermann, 0. (1977) Proc. Natl. Acad. Sd. USA 74, 272-275. 9. Stevens, L. C. (1970) Dev. Biol. 21, 364-382. 10. Main, J. M. & Prehn, R. T. (1957) J. Natl. Cancer Inst. 19, 1053-1064. 11. Tyan, M. L. & Cole, L. J. (1963) Transplantation 1, 546. 12. Dempster, W. J., Lennox, B. & Boag, J. W. (1950) Br. J. Exp. Pathol. 31, 670. 13. Baldwin, R. W. (1966) J. Natl. Cancer Institute 1, 257-264. 14. Prehn, R. T. (1976) Adv. Cancer Res. 23, 203-236. 15. Hewitt, H. B., Blake, E. R. & Walder, A. S. (1976) Br. J. Cancer 33,241-259. 16. Klein, G. & Klein, E. (1977) Proc. Natl. Acad. Sci. USA 74, 2121-2125.
