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Induction of transplantation resistance to Rous sarcoma isograft by avian leukosis virus
VIROLOGY
39,
482--190
Induction
(1969)
of Transplantation
Resistance
by Avian HEINZ
BAUER,
Max-Planck-Institut
JAN
BUBENEK,’
Leukosis THOMAS
jiiv Virusjorschung,
to Rous
July
GRAF,
Most of the virus tumors examined thus far contain tumor-specific transplantation antigens (TSTA) which are common for the neoplasmsinduced by the same virus within a given animal species(Old and Boyse, 1964; SjGgren, 1965; Klein, 1966). Reports on immunological cross resistance between polyoma tumors from different animal species (Hable, 1963; Irlin, 1967), mouse and rat Rous sarcomas (Jonsson, 1966), SV40-transformed human and hamster cells (Girardi, 1965; Kluchareva et al., 1967), and adeno-12 virus-induced hamster and mouse tumors (HellstrGm and Sjijgren, 1967) suggest that even tumors of the same virus origin in different speciescarry identical antigens. Whether or not the TSTA play an essential role in the transformation process has, as yet, not been resolved. A polyoma virus mutant was described that induced tumors withalmost UndetectableTSTA (Hare, 1967). Two serotypes of adenovirus with serologitally different T-antigens and different grades of oncogenicity in mammals induced com1 On leave of absence from the Institute of Ex. perimental Biology and Genetics, Czechoslovak Academy of Sciences, Prague, Czechoslovakia. 482
,4ND
CHRISTEL
Abteilung,
Ttibingen,
ALLGAIER Germany
8, 1969
Avian leukosis virus (ALV) particles were shown to production of transplantation antigens(s) that cross Rous sarcoma virus (RSV) specific TSTA. Mice injected leukosis virus or ALV-transformed chicken myeloblasts tumors induced by Rous sarcoma virus. It was found that newborn mice was sufficient to induce transplantation particles themselves seemed not to be capable of transferring These results suggested that infection of mouse cells by the induction of tumor-specific transplantation antigens to the ROW sarcoma isografts. INTRODUCTION
lsograft
Virus
Biologisch-medizinische
Accepted
Sarcoma
be capable of inducing the react immunologically with with high doses of avian were resistant to isografts of a single ALV-injection into resistance whereas the ALV transplantation antigen. ALV may be a requisite for and subsequent immunity
mon specific transplantation antigens, whereas a nononcogenic adenovirus did not induce immunity against adenovirus tumors (Hellstriim and SjGgren, 1967). In the case of avian tumor viruses tumorspecific transplantation antigens were detected in mouse sarcomata induced by Rous sarcoma virus (RSV) (Sjijgren, 1965). At present it is not known whether avian leukosis viruses (ALV), which are morphologically similar and serologically related to RSV, also induce TSTA. ALV cause tumors in chickens and are able to transform certain types of chicken cells in vitro (Baluda and Goetz, 1961), but these target cells are different from those transformed by RSV. We undertook this study to determine whether ALV are able to induce TSTA and, if so, whether these TSTA are immunologically related to those induced by RSV. Inbred mice seemedto be the most appropriate animal system to perform such studies. Since we were unable to induce tumors in mice by ALV, the immunity to ALV-injected mice could not be tested with syngeneic ALV-tumor cells. However, in preliminary experiments we found that mice which were immunized with ALV or
COMMON
ALV-producing came resistant Therefore in all mice injected were tested for syngeneic Rous MATERIAL
TRANSPLANTATION
chicken myeloblasts beto a Rous sarcoma isograft. experiments to be described, with ALV or myeloblasts immunity by challenge with sarcoma cells. AND
METHODS
Virus Strains RSV. The Schmidt-Ruppin (SR) strain of RSV originally obtained from Dr. Ahlstrom (Lund, Sweden), was used to induce the SR-A-tumor. Some properties of the cloned SR-RSV-H strain which was used for the primary induction of the SR-D-tumors, were described previously. This virus was shown to be free of a contaminating leukosis virus and belongs to avian tumor virus subgroup D (Bauer and Graf, 1969). BH-RSV (RAV-l), a variant of the Bryan “high titer” RSV, and SR-RSV-1 were kindly supplied by Dr. P. M. Biggs (Houghton, Huntingdon, England) and Dr. P. K. Vogt (Seattle, Washington), respectively. The first virus passage on C/B chick fibroblasts was used as a stock for both strains. ALV. The BAI strain A of avian myeloblastosis virus, originally obtained from Dr. J. W. Beard (Durham, North Carolina), was used as a model virus for ALV. It was passaged about 20 times in chickens. Antisera produced in rabbits against this virus strain cross-reacted with viruses of avian tumor virus subgroups A and B (Bauer, 1969). These results are in accordance with Vogt’s finding (196513) that the BAI strain A contains viruses belonging to subgroup A and BI now called MAV-1 and MAV-2, respectively (Moscovici and Vogt, 1968). No focus formation was observed after plating the BAI-A virus on chick fibroblast cultures of different genotypes, and after subcutaneous injection of virus or myeloblast cells no fibrosarcomas appeared in chickens. These observations indicate that our BAI-A strain is free of RSV. An antigenically homogeneous stock of subgroup B virus was isolated from the BAI-A strain by the end-point dilution technique on C/A chick fibroblasts. It failed to induce myeloblastosis after injection of lo* TCID 60into l-day-old chicks and was there-
ANTIGENS
OF
ALV
AND
RSV
483
fore called myeloblastosis associated virus B (MAV-B) . All virus stocks were stored at -70” and centrifuged for 15 min at 2000 g before injection into mice. Virus assay. The RSV-focus test was performed according to the method described by Temin and Rubin (1958). TCIDr,,, was estimated as described earlier (Bauer et al., 1965) : the end point of infectivity in chicken fibroblast cultures was determined by using group-specific (gs) complement-fixing (CF) rabbit antiserum (Bauer and Schafer, 1965) to detect gs-antigen in the virus particles. Animals Mice. The inbred strain STU2 was used. Antigenic homogeneity of these mice was tested by skin grafting. No spontaneous mammary tumor has been observed during a period of several years, and no indication for latent leukemia virus infection was obtained by electronmicroscopical observation (Gelderblom et al., in preparation) or serological methods (Schafer and Seifert, 1968). Chickens. For the preparation of tissue cultures, chick embryos from the line 15 (originally obtained from Dr. Burmester, East Lansing, Michigan) and the Spafas line were used. The latter chicken line was originally obtained from Dr. Luginbuhl (Storrs, Connecticut) and maintained by Lohmann & Co. (Cuxhaven). Normal chicken plasma and cells for the immunization of mice were taken from either of these chicken lines. Embryo fibroblast cultures from both lines were routinely tested for latent leukosis virus infection by gs-CF-tests (Bauer et al., 1965) and interference tests (Rubin, 1961) with RSV-strains of subgroups A and B, and recently also of subgroups C and D. No latent infection could be detected by these methods. For the production of ALV (BAI strain A) and myeloblasts (ALV-producing chicken myeloblastic leukemia cells) chickens from the Shaver line were used. This flock was not tested for latent leukosis viruses, but 2 Committee on standardized nomenclature for inbred strains of mice (1968). Standardized nomenclature for inbred strains of mice: fourth listing. Cancer Res. 28, 391-420.
484
BAUER
these birds were highly susceptible to induction of myeloblastosis by BAI strain A. Tumors Chicken myeloblastosis was produced by intraperitoneal injection of 10’ TCID,, of BAI strain A into 2-day-old Shaver chickens. Thirty to fifty percent of the chickens developed myeloblastosis within 2-4 weeks after infection. Tumors SR-A, SR-Dl, SR-D2, SR-D4, and SR-D5 were induced by subcutaneous administration of approximately 5 X lo6 RSV-transformed chick embryo cells into newborn mice of the STU strain. The minimal cell dose which gave rise to a tumor was 1 X 106. SR-A, SR-D2, and SR-D4 tumors were tested by complement fixation and found to contain group-specific antigen of the avian sarcoma leukosis virus type. The injection of cells from each of the five tumor lines caused fibrosarcomas in chickens. All mouse tumors were serially transplanted. Cells used for challenge were derived from the second to the eleventh passage of these tumors. The minimal number of tumor cells which produced tumors in 100% of the animals (DiLlloo) was lo2 cells for each of the different tumor lines. Tumors induced primarily in males (SR-A, SR-Dl, SR-D2) or females (SR-D4, SR-D5) were used as challenge for immunized males and females, respectively. Design of Experiments 1. Immunization with cells. Newborn mice were inoculated subcutaneously with 2 to 5 X lo6 living myeloblasts or control cells suspended in 0.1 ml PBS (0.1 M phosphatebuffered saline, pH 7.2). The injection was repeated at 2-4-week intervals (Table 1, Expt. 2). When adult mice were immunized, lOa myeloblasts or control cells suspended in 0.3 ml of PBS were inoculated subcutaneously. The injection was repeated at intervals of 14-28 days (Table 1, Expt. 1). 2. Immunization with ALV. Unless otherwise indicated, mice were injected subcutaneously with 0.1 ml of plasma from leukemic chickens containing 10’ to lo* TCIDso of ALV. A control group was either treated with the same volume of normal chicken
E?’
AT,.
plasma or remained untreated. The immunizat’ion schedule and challenge time used for the different experiments is described in the legend of the corresponding tables. RESULTS
Immunization and ALV
oj
Mice
with
Myeloblasts
Groups of adult mice were preimmunized repeatedly with chicken myeloblasts. Control mice either remained untreated or were injected with similar amounts of normal chicken muscle cells. As a positive control, mice were immunized with syngeneic Rous sarcoma cells. Two weeks after the last injection all groups of mice were challenged with syngeneic cells from the SR-A-tumor. It is shown in Table 1 (Expt. 1) that chicken myeloblasts were able to induce a resistance in mice against the isograft of RSV-induced mouse sarcoma cells. Although the number of animals treated with myeloblasts was low, 3 resistant mice out of 7 seems to be significant, since none of 41 control mice was resistant. TABLE IMMUNIZaTION
OF MICE
ALV
AGAINST ISOGRAFT INDUCED BYANUNCLONED
1 WITH
MYELOBLASTS
Rous SR-RSV
OF A
Tumor Treatment
of micea
None Syngeneic
Rous
sarcoma
cells Chicken myeloblasts Chicken spleen Chicken muscle Chick embryo fibroblasts Normal chicken plasma ALV-containing plasma
OR
SARCOMA STRSIN challenne
Expt 1 50 X DMIOO
dose*
Expt. 2 5 X DMlao
21/21” 6/13
19/19 -
4/7 20/20 -
27/36 34/35 30/30 7/7 7/12
a Mice were immunized 3 (Expt. 1) or 6 times (Expt. 2) with chicken material. Immunizat,ion with syngeneic mouse tumor SR-A was accomplished by temporary growth and ligature of the syngeneic tumor (Bubenik and Bauer, 1967). * Challenge was performed 10 days (Expt. 2) and 2 weeks (Expt. 1) after the last immunization with SR-A-tumor cells. c Number of mice with tumor per total number of mice. The observation period aft,er challenge was 2 months (Expt. 1) and 5 weeks (Expt. 2).
COMMON
TRANSPLANTATION
ANTIGENS
OF
ALV
AND
RSV
485
In a second experiment, where the immunization was begun in newborn mice, a similar resistance of the myeloblast-treated animals against the Rous sarcoma isograft was observed (Table 1, Expt. 2). Noninfected chick embryo fibroblasts or spleen cells from normal chickens of the line 15 did not induce a significant resistance against transplantation of 5 DMloo of SR-A tumor cells (Table 1). Since the myeloblasts contain large amounts of ALV particles, the question arose whether the virus alone is able to induce a transplantation resistance. This was tested in the same experiment by immunizing mice repeatedly with 10’ to lo8 TCIDso of leukosis virus. Control mice were injected with virus-free chicken plasma. A marked immunizing effect was observed in ALV-injected mice, whereas none of the seven control mice were found to be resistant (Table 1).
is only inactivated by 0.5 loglo. Virus infectivity (TCIDGO), however, is inactivated about 8 loglo. If a virus infection of mouse cells is necessary for the induction of TSTA, HA-treated virus should not confer immunity. On the other hand, if the virus is a carrier of TSTA, HA-treated virus may still be immunizing. The possibility of soluble TSTA in the ALV chicken plasma was tested by infection of mice with plasma from myeloblastosis chickens from which virus was removed by ultracentrifugation at a performance index of 10 (Bauer and Schafer, (1966a). Three weeks after the last immunizing injection, mice were challenged with either 5 or 25 X DMloo of syngeneic Rous sarcoma cells. As a result, neither ALV-deprived plasma nor HA-treated ALV induced transplantation resistance, whereas an immune effect was observed after injection of untreated ALV (Table 2).
Attempts to Immunize ALV Particles
Single ALV Injection of Mice When Newborn and Challenge with a Rous Sarcoma Induced by a Cloned RSV Strain
with
Noninfectious
Different mechanisms may be considered in order to explain the immunity against subsequent isografting of Rous tumor cells observed after pretreatment of mice with ALV or chicken myeloblasts. The ALV-induced immmunity could be caused either by TSTA synthesized in ALVinfected mouse cells or by TSTA carried by the inoculated virus itself. In the latter case the TSTA may be on the surface of virus particles similarly to other cellular components (for a review, see Vogt, 1965a; Bauer and Schafer, 196613) and thus immunogenic in mice. Since the ALV used for immunization was usually suspended in the original chicken plasma, it seemed also possible that a soluble TSTA as contained in the plasma is responsible for the immunity. Both of the last-mentioned possibilities were tested in the following experiment. A group of mice were injected with hydroxylamine (HA)-treated ALV. Studies in this laboratory (Graf and Bauer, 1969) indicate that, after 15 hours of HA treatment, the antigenicity of group-specific internal antigen (Bauer and Schafer, 1966a) is not affected, while that of the typespecific antigen (Ishizaki and Vogt, 1966)
If the immunity depends on an infection of mouse cells by ALV, one might expect that a single virus injection into newborn mice would be sufficient for the induction of immunity. To determine this, mice were injected only once with ALV when newborn and challenged 4% weeks thereafter with syngeneic tumor cells. To study the degree of immunity conferred by ALV, experimental and control mice were divided into three groups, and each group was challenged with a different dose of syngeneic Rous sarcoma cells. Since in the previous experiments the immunity induced by ALV was tested by challenge with a Rous sarcoma isograft, the question arose whether the immunity was due to a common leukosis virus present in the BAI-A and RSV stocks or whether it represented real cross reaction between leukosis and sarcoma viruses. Therefore, in the present experiment SR-RSV-H induced Rous sarcomata were used for challenge. For this purpose the SR-RSV-H was cloned three times prior to tumor induction and shown to be free of a contaminating leukosis virus (Bauer and Graf, 1969). Since we also
BAUER zY:Td1,.
456 TABLE 2
IMMUNIZATION AGAINST SR-A TUMOI~ ISOGUFT OF STU-MICE WITH HYDROXYL~MINE TREAY~EDALV AND ALV-FREE CHICKEN I'L.~SMA
Treatment’
Number of mice with turnorb totalnumber of mice Challenge
dose of cells
5 X DMlon
25 X DMloa
None
g/g
g/g
Normal chicken plasma ALV-free plasma
4/4 5/5
4/4 414
ALV 50-hr HA-treated” ALV 15-hrHA-treated ALV 0-hr HA-treated
v3
6/f3
4/4 4/7
4/4 5/7
0 Mice were 1 day old at the time of their first immunizing injection. The injection was repeated 6 times at 2-4-week intervals. The challenge was applied 3 weeks after the last immunis zation. b The observation period w&s 2 months after the challenge. c Hydroxylamine (HA) treatment was performed as follows: Virus suspended in chicken plasma was mixed with an equal amount of 2 M hydroxylamine, pH 7.5, and incubated for the indicated times at 20”. Thereafter the hydroxylamine was removed by dialysis at 4”.
could not detect any transforming virus in our ALV stock, it appeared likely that our ALV preparation was free of viruses in common with the SR-RSV-H. The results of this experiment are shown in Table 3. It can be seen that a single injection of ALV in newborn mice induces a clearcut immunity against isograft with a Rous sarcoma which was primarily induced by a cloned RSV strain. This immunity is as strong as that observed in the experiments in which adult mice were repeatedly immunized. It can be seen in this experiment that the immunity is not overcome by a challenge dose of 25 X DM100, although it is overcome by higher doses, such as 125 X
DMm. Dependenceof Transplantation Resistanceon the Dose of Injected ALV With the finding that a single ALVinjection into newborn mice causes immunity against Rous sarcoma isograft, it was then possible to study the effect of virus dose on
this resistance. For this purpose l-day-old mice were injected with different dilutions of ALV. At the age of S weeks the mice were challenged with either 5 or 25 X DMloo of SR-D tumor cells and checked weekly for appearance of tumors. It can be seen from the results in Table 4 that the degree of transplantation resistance is dependent on the dose of the immunizing virus. As in the experiments described above, the absence of immunity in normal plasma injetted mice is evidence for the specificity of the transplantation resistance. Immunization with Avian Tumor Virus Strains Other Than the BAI-A Strain of ALV Since the BAI strain A used throughout the experiments represents a mixture of myeloblastosis virus and myeloblastosisassociated viruses (Moscovici and Vogt, 1968), it remained unclear which virus is responsible for the observed immunity.d One approach to study this question was to immunize mice with other avian tumor virus strains. For this purpose we injected mice with the BAI-A derived MAV-B strain, SR-RSV-1, BH-RSV (RAV-l), and SR-RSV-H. The first three strains are not oncogenic in mice as far as could be deTABLE 3 TRANSPLANTATION RESISTANCE AGAINST A Rous SARC~M.~ ISOGRAFT AFTER A SINGLE INJECTION OF NEWBORN MICE WITH ALV Number of mice with tumorsb total number of mice Treatment
of mice”
Challengec
25 x DMm
4/4 2/5
8/8 4/10
DMm
Normal
chicken plasma
ALV containing plasma
dose of cells
5X
125 x DMm
g/g 12/12
a Before injection the ALV was sedimented from the original chicken plasma by ultracentrifugation and resuspended in the same volume of normal chicken plasma as was used for injection of control mice. b The observation period was 6 weeks after the
chal1enge,
c Challenge tumors were SR-D5 0 for females and SR-Dl c? for males. The challenge was performed when mice were 4% weeks old.
COMMON
TRANSPLANTATION
ANTIGENS
termined from repeated experiments. For immunization, the respective virus was concentrated loo-fold by ultracentrifugation of medium from virus-infected chick TABLE DOSE-DEPENDENT
4
TRANSPLANTATION
ANCE TO Rows SARCOMA INJECTED ONCE WITH
I I Chd
TreatmaP
lenge dose (DMm)
RESIST-
ISOGRAFT IN MICE ALV AT BIRTH
Number of mice with tumors total number of mice Weeks
after
None Normal chicken plasma ALV 1OWc ALV
10-r
ALV
10”
4
5
6
5 25 5 25
6/9 2/5 3/7 5/8
9/9 5/5 7/7 8/8
9/9 5/5 7/7 8/8
5 25 5 25 5 25
2/5 l/3 2/10 5/10 l/10 3/11
3/5 3/3 7/11 7/10 3/10 S/11
5/5 3/3 S/11 7/10 4/10 7/10
755
-
-
15/15
8/8 8/118/11 15/21 7/10 7/10 5/10 5/10 12/20 7/10 - i
TABLE RESISTANCE DIFFERENT
AGAINST A Rous VIRUS STRAINS
Normal chicken plasma SR-RSV-1 BH-RSV (RAV-1) MAV-B SR-RSV-H
Injection
dose
107 107 10’ 5 x
FFU FFUd TCIDW 106 FFU
Antibody
5
SARCOMA ISOGRAFT AFTER INJECTION OF THE AVIAN SARCOMA LEUKOSIS
OF NEW-BORN
COMPLEX
of mice with
total of mice
487
VSV
embryo fibroblast cultures, titered, and thereafter injected into mice (Table 5). It can be seen in Table 5 that neither SRRSV-1, BH-RSV (RAV-1)) nor MAV-B induced any significant transplantation immunity, in contrast to the results obtained with BAI-A virus of a similar infectivity titer (Tables 3 and 4). A clearcut resistance was only obtained with SRRSV-H, despite its low infectivity titer.
Number Treatmenta
AND
SR-RSV-H shares common neutralizing envelope (V-) antigen(s) with the subgroup B-viruses present in the BAI-A strain of ALV which was used for immunization of mice. If these common V-antigens are responsible for the transplantation cross immunity, ALV-injected mice could cotktain neutralizing antibody against SR-RSV-H at the time of challenge. To prove this, l-day-old mice were injected with the same dose of ALV as in the experiments described before. Mice were tested 5 weeks and 6 weeks, respectively, later for neutralizing serum antibody. Sera were used in a dilution of 1:2 and 1: 10 after heat inactivation and tested against 100 FFU of SR-RSV-H as described elsewhere (Bauer and Graf, 1969). None out of 18 sera tested showed any significant virus-neutralizing capacity. The sensitivity of the test was such that
14/14
a Before injection ALV was removed from the original chicken plasma by ultracentrifugation and resuspended in the same normal chicken plasma that has been used for injection of mice. The dilutions were also performed in the same normal plasma. b Challenge was performed with SR-D t,umor cells when mice were 8 weeks old. c Dilution of ALV.
TRANSPLANTATION MICE WITH
ALV
Test for SR-RSV-H Neutralizing in Mice Immunized with ALV
challenge z
3
OF
number
ChallengeC
tumors* of mice
dose of cells
5 X DMm
25 X DMm
125 X DMloo
12/12
13/13 13/13 15/15 14/14 5/12
12/12 16/16 14/14 3/5
W6 14/16 11/12 -
a Mice were injected once when newborn. b The observation period was 5 weeks after challenge. c Challenge tumors were SR-D4 0 for females and SR-D2 when mice were 5% weeks old. d The TCIDso titer of RAV-1 in this stock is approximately
CT for 10 times
625 X DMmo
6/6
males. higher
Challenge than
4/4
6/6 -
w l/3
was the FFU
performed titer.
4ss
BAUER
approximately 5 % neutralization could not be excluded. DISCUSSION
The experiments reported here present evidence for avian leukosis virus-induced transplantation antigens. Adult mice which were repeatedly preimmunized with chicken myeloblasts were resistant against a Rous sarcoma isograft. Resistance was also obtained when mice were repeatedly immunized with high doses of ALV-containing chicken plasma. It has been found that only a single injection of ALV was sufficient to induce a clear transplantation resistance in newborn mice. The fact that mice treated with normal chicken tissue or plasma were not more resistant than untreated control mice indicates that normal chicken antigen is of no importance for the induction of the transplantation resistance obtained after injection of ALV or myeloblasts. In the first set of experiments it was not possible to determine whether the observed resistance was due to a common virus in the immunizing material and in the graft, or if it reflected a true cross reaction between the TSTA of ALV and RSV. For this reason immunization experiments were repeated using Rous sarcomas induced by a cloned and characterized strain of RSV, SR-RSV-H. This virus strain has been described earlier to be free of contaminating leukosis virus (Bauer and Graf, 1969). Likewise the BAI strain A has been shown to be free of Rous sarcoma virus. As was the case in the first experiments, a clearcut resistance of ALV-infected mice against these new challenge tumors was observed. It is therefore concluded that leukosis and sarcoma viruses, i.e., viruses with different target cells for oncogenicity in chickens, share common TSTA. Concerning the nature of immunization by ALV, two possibilities come to mind. 1. A structural component of the ALVparticles present in the immunizing material could act as TSTA. So far, two types of virus specific structural antigens, namely internal and envelope antigens, are known. The internal gs-antigen is group specific for all avian leukosis sarcoma viruses so far tested (Bauer and Schafer, 1965; Bauer,
ET z4L
1969), whereas the envelope (V-) antigens are subgroup- and type-specific (Vogt et al., 1966). The HA experiment speaks against the internal gs-antigen of ALV as being capable of inducing immunity against a Rous sarcoma isograft. The same experiment also tends to exclude, that the V-antigen(s) of the immunizing virus acts as transplantation antigen. If V-antigen(s) were identical with TSTA, one might also expect the formation of virus neutralizing antibodies after immunization of mice with ALV. Our negative results in this respect are in accordance with the findings of other authors (Jonsson and Sjogren, 1966; Koldovsky et al., 1966) that no virus neutralizing antibodies are present in sera of mice resistant to transplantation of syngeneic Rous sarcoma cells. However, the possibility of either cell-bound immunity against V-antigen or the presence of a low titer of neutralizing antibody cannot be excluded. It is also conceivable that ALV particles are carrying TSTA that are not identical to any one of the two aforementioned antigens. If ALV-induced chicken leukemic cells contain the same or similar TSTA as RSVinduced mouse sarcomas, this immunogenic TSTA could also be present on the surface of ALV particles released by such cells, as is the casewith different cellular components present in ALV and related virus particles (for review see Vogt, 1965a; Bauer and Schafer, 1966b). Such a phenomenon was suggested by Lindenmann and Klein (1967) for influenza virus produced by Ehrlich ascites cells. At present this possibility cannot be excluded, in spite of the fact that HA-treated ALV does not induce any immunity. It could still be that TSTA on the virus surface is inactivated by HA despite our finding that the antigenicity of gs- and V-antigens are almost unaltered by HA (Graf and Bauer, 1969). 2. The alternative is that virus of the BAI-strain A used in the present studies infects mouse cells and induces the synthesis of new cellular TSTA. Whether or not this TSTA is identical with a virus structural antigen, it may be similar to or identical with the TSTA of RSV-induced
COMMON
TRANSPLANTATION
ANTIGENS
OF
ALV
AND
RSV
489
mouse sarcomas. In favor of this explanareaction between TSTA of ALV and RSV tion is the finding that the immunity ob- should also be considered. served after a single ALV injection into ACKNOWLEDGMENT newborn mice is as strong as that obtained after repeated injection of adult mice. If The competenttechnical assistance of Miss A. the TSTA were present in the inoculated Seydel is gratefully acknowledged.We wish to material, adult mice, because they are fully thank ProfessorW. SchSiferfor his interest and immune competent, would be expected to support of this work. This investigation wassupported by the DeutscheForschungsgemeinschaft. show a stronger immunity effect. It thus seems likely that certain leukosis viruses REFERENCES induce the synthesis of TSTA in mouse BALUDA, M. A., and GOETZ, I. E. (1961). Morphocells. Such cells with foreign antigen may logical conversion of cell cultures by avian survive and mult,iply longer in newborn. myeloblastosis virus. Virology 15, 185-199. immunologically immature mice than in BAUER, H. (1969).Untersuchungen iiber Struktur, adults. Therefore a single injection in newVermehrung und onkogene Wirkung der born mice results in an equally strong imHtihner-Leukose-Sarkomatose-Viren.Habilitamunizing effect as that obtained by retions-Schrift, Tiibingen. peated injection of adult mice. The fact BAUER, H., and GRAF, T. (1969). Evidence for the that no tumors were observed after ALV possible existence of two envelope antigenic infection in mice is no real contradiction determinants and corresponding cell receptors of this hypothesis. It is shown in Table 5 for avian t,umor viruses. Virology 37, 157-161. BAUER, H., and SCHAFER, W. (1965). Isolierung that a SR-RSV-H dose insufficient to induce eines gruppenspezifischen Antigens aus dem a tumor is still able to immunize mice. Hiihner-Myeloblast,ose-Virus (BAI Stamm A). Such an explanation was also given by 2. Naturjorsch. 20b, 815-817. Habel (1961) and Sjiigren et al. (1961) for BAUER, H., and SCHAFER, W. (1966a). Origin of the finding, that polyoma infection of newgroup-specific antigen of chicken leukosis viborn or adult mice produces resistance to ruses. virology 29,494-496. polyoma tumor isografts. They suggested BAUER, H., and SCHAFER, W. (1966b). Studies on that a new cellular TSTA was induced by physical, chemical and antigenic properties of a the virus in infected and/or transformed chicken leukosis virus. In “Subviral Carcinogenesis” (Y. Ito, ed.), pp. 337-352. Aichi Center, mouse cells. This elicits an immunological Nagoya, Japan. response which leads to a subsequent reH., and SCH;~FER, W. jection of the transformed challenge cells. BAUER, H., BAHNEMANN, (1965). Untersuchungen iiber das MyeloblastoseAt present it is not known whether the Virus des Huhnes (BAI Stamm A). I. Nachweis immunity is caused by the myeloblastosisund Vermehrungskinetik des Virus in Htihnerinducing virus or by any one of the myelofibroblasten-Kulturen. 2. Naturjorsch. 20b, 959blastosis-associated viruses. It seems re965. markable that injection of 5 X lo5 FFU BuBENfK, J., and BAUER, H. (1967). Antigenic of SR-RSV-H induces a stronger immunity characteristics of the interaction between Rous than 10’ to lo8 TCIDSO of ALV. On the sarcoma virus and mammalian cells. Complement-fixing and transplantation other hand, similar amounts of other avian antigens. Virology 31,489497. tumor viruses [MAV-B, SR-RSV-1, and A. J. (1965). Prevention of SV40 virus BH-RSV (RAV-l)] did not induce a clear GIRARDI, oncogenesis in hamsters. I. Tumor resistance resistance. This may be explained by the induced by human cells transformed by SV40. finding of Hanafusa and Hanafusa (1966) Proc. Natl. Acad. Sci. U.S. 54, 445-451. that only virus strains with specific enGRAF, T. and BAUER, H. (1969). Studies on the velope antigens are able to infect and transrelative target size of various functions in the form mammalian cells. A virus of low titer genome of avian tumor viruses. In II. Int. Meetmay be present in our ALV stock which ing on Tumor Viruses, Royaumont, in press. possessesthe necessary envelope antigens HABEL, K. (1961). Resistance of polyoma virus immune animals to transplanted polyoma tuto infect mouse cells and thereafter to mors. Proc. Sot. Exptl. Biol. Med. 106,722-725. induce TSTA. However, a partial cross
490
RAUER
HA~I~L, K. (1963). Common antigen in polyoma t,umors. Federation Proc. 22, 438. (Abstract..) HANAFUSA, H., and HANAFUSA, T. (1966). Determining factor in the capacity of RSV to induce tumors in mammals. Proc. Natl. Acad. Sci. U.S. 55, 538-552. HARE, J. D. (1967). Transplant immunity to polyoma virus-induced tumor cells. IV. A polyoma strain defective in transplant antigen induction. Virology 31, 62&632. HELLSTR~M, I., and SJ~GREN, H. 0. (1967). In vitro demonstration of humoral and cell-bound immunity against common specific transplantation antigen(s) of adenovirus la-induced mouse and hamster tumors. J. Ezptl. Med. 125, 11051118. IRLIN, I. S. (1967). Immunofluorescent demonstration of a specific surface antigen in cells infected or transformed by polyoma virus. Virilogy 32,725-728. ISHIZUI, R., and VOGT, P. K. (1966). Immunological relationships among envelope antigens of avian tumor viruses. Virology 30, 375-387. JONSSON, N. (1966). Studies on the occurrence of common specific transplantation antigens in Rous tumors of various mammalian species. Acta Pathol. Microbial. &and. 67, 339-353. JONSSON, N., and SJ~GREN, H. 0. (1966). Specific transplantation immunity in relation to Rous sarcoma virus tumorigenesis in mice. J. Exptl. Med. 123,487+X3. KLEIN, G. (1966). Tumor antigens. Ann. Rev. ikficrobiol. 20, 223-252. KLUCHAREVA, T. E., SHACHARINA, K. L., BELOVA, S., CHILISOVA, V., and DEICHMAN, G. I. (1967). Use of immunofluorescence for detection of specific membrane antigen in simian virus ~CIinfected nontransformed cells. J. Natl. Cancer Inst. 39, 825-832. KOLDOVSK+, P., SVOBODA, J., and BUBEN~K, J. (1966). Further studies on the immunobiology of
ET
AL
tumor RVAI induced by RSV in C57Bl strain mice. FoEia l3iol. (Prague) 12, l-10. LINDENMANN, J., and KLEIN, P. A. (1967). Viral analysis: increased immunogenicity of host cell antigen associated with influenza virus. J. Exptl. Med. 126,93-108. MOSCOVICI, C., and VOGT, P. K. (1968). Effects of genetic cellular resistance on cell transformation and virus replication in chicken hematopoietic cell cultures infected with avian myeloblastosis virus (BAI-A) . Virology 35,487497. OLD, L. J., and BOYSE, E. A. (1964). Immunology of experimental tumors. Ann. Rev. Med. 15, 167-186. RUBIN, H. (1961). The nature of a virus-induced cellular resistance to Rous sarcoma virus. Virology 13, 200-206. SCHBFER, W., and SEIFERT, E. (1968). Production of a potent complement-fixing murine leukemia virus-antiserum from the rabbit and its reactions with various types of tissue culture cells. Virology 35, 323328. SJ~~GREN, H. 0. (1965). Transplantation methods as a tool for detection of tumor-specific antigens. Progr. Exptl. Tumor Res. 6, 289-322. SJ~QREN, H. O., HELLSTR~M, J., and KLEIN, G. (1961). Resistance of polyoma virus immunized mice against transplantation of established polyoma tumors. Exptl. GelE Res. 23, 204-208. TEMIN, H., and RUBIN, N. (1958). Characteristics of an assay for Rous sarcoma virus and Rous sarcoma cells in tissue culture. Virology 6, 669688. VOGT, P. K. (1965a). Avian tumor viruses. Advun. Virus Res. 11, 293-385. VOGT, P. K. (196513). A heterogeneity of Rous sarcoma virus released by selectively resistant chick embryo cells. Virology 25, 237-247. VOGT, P. K., ISHIZAKI, R., and DUFF, R. (1966). Studies on the relationships among avian tumor viruses. In “Subviral Carcinogenesis” Cy. Ito, ed.), pp. 297-310. Aichi Center, Nagoya, Japan.
39,
482--190
Induction
(1969)
of Transplantation
Resistance
by Avian HEINZ
BAUER,
Max-Planck-Institut
JAN
BUBENEK,’
Leukosis THOMAS
jiiv Virusjorschung,
to Rous
July
GRAF,
Most of the virus tumors examined thus far contain tumor-specific transplantation antigens (TSTA) which are common for the neoplasmsinduced by the same virus within a given animal species(Old and Boyse, 1964; SjGgren, 1965; Klein, 1966). Reports on immunological cross resistance between polyoma tumors from different animal species (Hable, 1963; Irlin, 1967), mouse and rat Rous sarcomas (Jonsson, 1966), SV40-transformed human and hamster cells (Girardi, 1965; Kluchareva et al., 1967), and adeno-12 virus-induced hamster and mouse tumors (HellstrGm and Sjijgren, 1967) suggest that even tumors of the same virus origin in different speciescarry identical antigens. Whether or not the TSTA play an essential role in the transformation process has, as yet, not been resolved. A polyoma virus mutant was described that induced tumors withalmost UndetectableTSTA (Hare, 1967). Two serotypes of adenovirus with serologitally different T-antigens and different grades of oncogenicity in mammals induced com1 On leave of absence from the Institute of Ex. perimental Biology and Genetics, Czechoslovak Academy of Sciences, Prague, Czechoslovakia. 482
,4ND
CHRISTEL
Abteilung,
Ttibingen,
ALLGAIER Germany
8, 1969
Avian leukosis virus (ALV) particles were shown to production of transplantation antigens(s) that cross Rous sarcoma virus (RSV) specific TSTA. Mice injected leukosis virus or ALV-transformed chicken myeloblasts tumors induced by Rous sarcoma virus. It was found that newborn mice was sufficient to induce transplantation particles themselves seemed not to be capable of transferring These results suggested that infection of mouse cells by the induction of tumor-specific transplantation antigens to the ROW sarcoma isografts. INTRODUCTION
lsograft
Virus
Biologisch-medizinische
Accepted
Sarcoma
be capable of inducing the react immunologically with with high doses of avian were resistant to isografts of a single ALV-injection into resistance whereas the ALV transplantation antigen. ALV may be a requisite for and subsequent immunity
mon specific transplantation antigens, whereas a nononcogenic adenovirus did not induce immunity against adenovirus tumors (Hellstriim and SjGgren, 1967). In the case of avian tumor viruses tumorspecific transplantation antigens were detected in mouse sarcomata induced by Rous sarcoma virus (RSV) (Sjijgren, 1965). At present it is not known whether avian leukosis viruses (ALV), which are morphologically similar and serologically related to RSV, also induce TSTA. ALV cause tumors in chickens and are able to transform certain types of chicken cells in vitro (Baluda and Goetz, 1961), but these target cells are different from those transformed by RSV. We undertook this study to determine whether ALV are able to induce TSTA and, if so, whether these TSTA are immunologically related to those induced by RSV. Inbred mice seemedto be the most appropriate animal system to perform such studies. Since we were unable to induce tumors in mice by ALV, the immunity to ALV-injected mice could not be tested with syngeneic ALV-tumor cells. However, in preliminary experiments we found that mice which were immunized with ALV or
COMMON
ALV-producing came resistant Therefore in all mice injected were tested for syngeneic Rous MATERIAL
TRANSPLANTATION
chicken myeloblasts beto a Rous sarcoma isograft. experiments to be described, with ALV or myeloblasts immunity by challenge with sarcoma cells. AND
METHODS
Virus Strains RSV. The Schmidt-Ruppin (SR) strain of RSV originally obtained from Dr. Ahlstrom (Lund, Sweden), was used to induce the SR-A-tumor. Some properties of the cloned SR-RSV-H strain which was used for the primary induction of the SR-D-tumors, were described previously. This virus was shown to be free of a contaminating leukosis virus and belongs to avian tumor virus subgroup D (Bauer and Graf, 1969). BH-RSV (RAV-l), a variant of the Bryan “high titer” RSV, and SR-RSV-1 were kindly supplied by Dr. P. M. Biggs (Houghton, Huntingdon, England) and Dr. P. K. Vogt (Seattle, Washington), respectively. The first virus passage on C/B chick fibroblasts was used as a stock for both strains. ALV. The BAI strain A of avian myeloblastosis virus, originally obtained from Dr. J. W. Beard (Durham, North Carolina), was used as a model virus for ALV. It was passaged about 20 times in chickens. Antisera produced in rabbits against this virus strain cross-reacted with viruses of avian tumor virus subgroups A and B (Bauer, 1969). These results are in accordance with Vogt’s finding (196513) that the BAI strain A contains viruses belonging to subgroup A and BI now called MAV-1 and MAV-2, respectively (Moscovici and Vogt, 1968). No focus formation was observed after plating the BAI-A virus on chick fibroblast cultures of different genotypes, and after subcutaneous injection of virus or myeloblast cells no fibrosarcomas appeared in chickens. These observations indicate that our BAI-A strain is free of RSV. An antigenically homogeneous stock of subgroup B virus was isolated from the BAI-A strain by the end-point dilution technique on C/A chick fibroblasts. It failed to induce myeloblastosis after injection of lo* TCID 60into l-day-old chicks and was there-
ANTIGENS
OF
ALV
AND
RSV
483
fore called myeloblastosis associated virus B (MAV-B) . All virus stocks were stored at -70” and centrifuged for 15 min at 2000 g before injection into mice. Virus assay. The RSV-focus test was performed according to the method described by Temin and Rubin (1958). TCIDr,,, was estimated as described earlier (Bauer et al., 1965) : the end point of infectivity in chicken fibroblast cultures was determined by using group-specific (gs) complement-fixing (CF) rabbit antiserum (Bauer and Schafer, 1965) to detect gs-antigen in the virus particles. Animals Mice. The inbred strain STU2 was used. Antigenic homogeneity of these mice was tested by skin grafting. No spontaneous mammary tumor has been observed during a period of several years, and no indication for latent leukemia virus infection was obtained by electronmicroscopical observation (Gelderblom et al., in preparation) or serological methods (Schafer and Seifert, 1968). Chickens. For the preparation of tissue cultures, chick embryos from the line 15 (originally obtained from Dr. Burmester, East Lansing, Michigan) and the Spafas line were used. The latter chicken line was originally obtained from Dr. Luginbuhl (Storrs, Connecticut) and maintained by Lohmann & Co. (Cuxhaven). Normal chicken plasma and cells for the immunization of mice were taken from either of these chicken lines. Embryo fibroblast cultures from both lines were routinely tested for latent leukosis virus infection by gs-CF-tests (Bauer et al., 1965) and interference tests (Rubin, 1961) with RSV-strains of subgroups A and B, and recently also of subgroups C and D. No latent infection could be detected by these methods. For the production of ALV (BAI strain A) and myeloblasts (ALV-producing chicken myeloblastic leukemia cells) chickens from the Shaver line were used. This flock was not tested for latent leukosis viruses, but 2 Committee on standardized nomenclature for inbred strains of mice (1968). Standardized nomenclature for inbred strains of mice: fourth listing. Cancer Res. 28, 391-420.
484
BAUER
these birds were highly susceptible to induction of myeloblastosis by BAI strain A. Tumors Chicken myeloblastosis was produced by intraperitoneal injection of 10’ TCID,, of BAI strain A into 2-day-old Shaver chickens. Thirty to fifty percent of the chickens developed myeloblastosis within 2-4 weeks after infection. Tumors SR-A, SR-Dl, SR-D2, SR-D4, and SR-D5 were induced by subcutaneous administration of approximately 5 X lo6 RSV-transformed chick embryo cells into newborn mice of the STU strain. The minimal cell dose which gave rise to a tumor was 1 X 106. SR-A, SR-D2, and SR-D4 tumors were tested by complement fixation and found to contain group-specific antigen of the avian sarcoma leukosis virus type. The injection of cells from each of the five tumor lines caused fibrosarcomas in chickens. All mouse tumors were serially transplanted. Cells used for challenge were derived from the second to the eleventh passage of these tumors. The minimal number of tumor cells which produced tumors in 100% of the animals (DiLlloo) was lo2 cells for each of the different tumor lines. Tumors induced primarily in males (SR-A, SR-Dl, SR-D2) or females (SR-D4, SR-D5) were used as challenge for immunized males and females, respectively. Design of Experiments 1. Immunization with cells. Newborn mice were inoculated subcutaneously with 2 to 5 X lo6 living myeloblasts or control cells suspended in 0.1 ml PBS (0.1 M phosphatebuffered saline, pH 7.2). The injection was repeated at 2-4-week intervals (Table 1, Expt. 2). When adult mice were immunized, lOa myeloblasts or control cells suspended in 0.3 ml of PBS were inoculated subcutaneously. The injection was repeated at intervals of 14-28 days (Table 1, Expt. 1). 2. Immunization with ALV. Unless otherwise indicated, mice were injected subcutaneously with 0.1 ml of plasma from leukemic chickens containing 10’ to lo* TCIDso of ALV. A control group was either treated with the same volume of normal chicken
E?’
AT,.
plasma or remained untreated. The immunizat’ion schedule and challenge time used for the different experiments is described in the legend of the corresponding tables. RESULTS
Immunization and ALV
oj
Mice
with
Myeloblasts
Groups of adult mice were preimmunized repeatedly with chicken myeloblasts. Control mice either remained untreated or were injected with similar amounts of normal chicken muscle cells. As a positive control, mice were immunized with syngeneic Rous sarcoma cells. Two weeks after the last injection all groups of mice were challenged with syngeneic cells from the SR-A-tumor. It is shown in Table 1 (Expt. 1) that chicken myeloblasts were able to induce a resistance in mice against the isograft of RSV-induced mouse sarcoma cells. Although the number of animals treated with myeloblasts was low, 3 resistant mice out of 7 seems to be significant, since none of 41 control mice was resistant. TABLE IMMUNIZaTION
OF MICE
ALV
AGAINST ISOGRAFT INDUCED BYANUNCLONED
1 WITH
MYELOBLASTS
Rous SR-RSV
OF A
Tumor Treatment
of micea
None Syngeneic
Rous
sarcoma
cells Chicken myeloblasts Chicken spleen Chicken muscle Chick embryo fibroblasts Normal chicken plasma ALV-containing plasma
OR
SARCOMA STRSIN challenne
Expt 1 50 X DMIOO
dose*
Expt. 2 5 X DMlao
21/21” 6/13
19/19 -
4/7 20/20 -
27/36 34/35 30/30 7/7 7/12
a Mice were immunized 3 (Expt. 1) or 6 times (Expt. 2) with chicken material. Immunizat,ion with syngeneic mouse tumor SR-A was accomplished by temporary growth and ligature of the syngeneic tumor (Bubenik and Bauer, 1967). * Challenge was performed 10 days (Expt. 2) and 2 weeks (Expt. 1) after the last immunization with SR-A-tumor cells. c Number of mice with tumor per total number of mice. The observation period aft,er challenge was 2 months (Expt. 1) and 5 weeks (Expt. 2).
COMMON
TRANSPLANTATION
ANTIGENS
OF
ALV
AND
RSV
485
In a second experiment, where the immunization was begun in newborn mice, a similar resistance of the myeloblast-treated animals against the Rous sarcoma isograft was observed (Table 1, Expt. 2). Noninfected chick embryo fibroblasts or spleen cells from normal chickens of the line 15 did not induce a significant resistance against transplantation of 5 DMloo of SR-A tumor cells (Table 1). Since the myeloblasts contain large amounts of ALV particles, the question arose whether the virus alone is able to induce a transplantation resistance. This was tested in the same experiment by immunizing mice repeatedly with 10’ to lo8 TCIDso of leukosis virus. Control mice were injected with virus-free chicken plasma. A marked immunizing effect was observed in ALV-injected mice, whereas none of the seven control mice were found to be resistant (Table 1).
is only inactivated by 0.5 loglo. Virus infectivity (TCIDGO), however, is inactivated about 8 loglo. If a virus infection of mouse cells is necessary for the induction of TSTA, HA-treated virus should not confer immunity. On the other hand, if the virus is a carrier of TSTA, HA-treated virus may still be immunizing. The possibility of soluble TSTA in the ALV chicken plasma was tested by infection of mice with plasma from myeloblastosis chickens from which virus was removed by ultracentrifugation at a performance index of 10 (Bauer and Schafer, (1966a). Three weeks after the last immunizing injection, mice were challenged with either 5 or 25 X DMloo of syngeneic Rous sarcoma cells. As a result, neither ALV-deprived plasma nor HA-treated ALV induced transplantation resistance, whereas an immune effect was observed after injection of untreated ALV (Table 2).
Attempts to Immunize ALV Particles
Single ALV Injection of Mice When Newborn and Challenge with a Rous Sarcoma Induced by a Cloned RSV Strain
with
Noninfectious
Different mechanisms may be considered in order to explain the immunity against subsequent isografting of Rous tumor cells observed after pretreatment of mice with ALV or chicken myeloblasts. The ALV-induced immmunity could be caused either by TSTA synthesized in ALVinfected mouse cells or by TSTA carried by the inoculated virus itself. In the latter case the TSTA may be on the surface of virus particles similarly to other cellular components (for a review, see Vogt, 1965a; Bauer and Schafer, 196613) and thus immunogenic in mice. Since the ALV used for immunization was usually suspended in the original chicken plasma, it seemed also possible that a soluble TSTA as contained in the plasma is responsible for the immunity. Both of the last-mentioned possibilities were tested in the following experiment. A group of mice were injected with hydroxylamine (HA)-treated ALV. Studies in this laboratory (Graf and Bauer, 1969) indicate that, after 15 hours of HA treatment, the antigenicity of group-specific internal antigen (Bauer and Schafer, 1966a) is not affected, while that of the typespecific antigen (Ishizaki and Vogt, 1966)
If the immunity depends on an infection of mouse cells by ALV, one might expect that a single virus injection into newborn mice would be sufficient for the induction of immunity. To determine this, mice were injected only once with ALV when newborn and challenged 4% weeks thereafter with syngeneic tumor cells. To study the degree of immunity conferred by ALV, experimental and control mice were divided into three groups, and each group was challenged with a different dose of syngeneic Rous sarcoma cells. Since in the previous experiments the immunity induced by ALV was tested by challenge with a Rous sarcoma isograft, the question arose whether the immunity was due to a common leukosis virus present in the BAI-A and RSV stocks or whether it represented real cross reaction between leukosis and sarcoma viruses. Therefore, in the present experiment SR-RSV-H induced Rous sarcomata were used for challenge. For this purpose the SR-RSV-H was cloned three times prior to tumor induction and shown to be free of a contaminating leukosis virus (Bauer and Graf, 1969). Since we also
BAUER zY:Td1,.
456 TABLE 2
IMMUNIZATION AGAINST SR-A TUMOI~ ISOGUFT OF STU-MICE WITH HYDROXYL~MINE TREAY~EDALV AND ALV-FREE CHICKEN I'L.~SMA
Treatment’
Number of mice with turnorb totalnumber of mice Challenge
dose of cells
5 X DMlon
25 X DMloa
None
g/g
g/g
Normal chicken plasma ALV-free plasma
4/4 5/5
4/4 414
ALV 50-hr HA-treated” ALV 15-hrHA-treated ALV 0-hr HA-treated
v3
6/f3
4/4 4/7
4/4 5/7
0 Mice were 1 day old at the time of their first immunizing injection. The injection was repeated 6 times at 2-4-week intervals. The challenge was applied 3 weeks after the last immunis zation. b The observation period w&s 2 months after the challenge. c Hydroxylamine (HA) treatment was performed as follows: Virus suspended in chicken plasma was mixed with an equal amount of 2 M hydroxylamine, pH 7.5, and incubated for the indicated times at 20”. Thereafter the hydroxylamine was removed by dialysis at 4”.
could not detect any transforming virus in our ALV stock, it appeared likely that our ALV preparation was free of viruses in common with the SR-RSV-H. The results of this experiment are shown in Table 3. It can be seen that a single injection of ALV in newborn mice induces a clearcut immunity against isograft with a Rous sarcoma which was primarily induced by a cloned RSV strain. This immunity is as strong as that observed in the experiments in which adult mice were repeatedly immunized. It can be seen in this experiment that the immunity is not overcome by a challenge dose of 25 X DM100, although it is overcome by higher doses, such as 125 X
DMm. Dependenceof Transplantation Resistanceon the Dose of Injected ALV With the finding that a single ALVinjection into newborn mice causes immunity against Rous sarcoma isograft, it was then possible to study the effect of virus dose on
this resistance. For this purpose l-day-old mice were injected with different dilutions of ALV. At the age of S weeks the mice were challenged with either 5 or 25 X DMloo of SR-D tumor cells and checked weekly for appearance of tumors. It can be seen from the results in Table 4 that the degree of transplantation resistance is dependent on the dose of the immunizing virus. As in the experiments described above, the absence of immunity in normal plasma injetted mice is evidence for the specificity of the transplantation resistance. Immunization with Avian Tumor Virus Strains Other Than the BAI-A Strain of ALV Since the BAI strain A used throughout the experiments represents a mixture of myeloblastosis virus and myeloblastosisassociated viruses (Moscovici and Vogt, 1968), it remained unclear which virus is responsible for the observed immunity.d One approach to study this question was to immunize mice with other avian tumor virus strains. For this purpose we injected mice with the BAI-A derived MAV-B strain, SR-RSV-1, BH-RSV (RAV-l), and SR-RSV-H. The first three strains are not oncogenic in mice as far as could be deTABLE 3 TRANSPLANTATION RESISTANCE AGAINST A Rous SARC~M.~ ISOGRAFT AFTER A SINGLE INJECTION OF NEWBORN MICE WITH ALV Number of mice with tumorsb total number of mice Treatment
of mice”
Challengec
25 x DMm
4/4 2/5
8/8 4/10
DMm
Normal
chicken plasma
ALV containing plasma
dose of cells
5X
125 x DMm
g/g 12/12
a Before injection the ALV was sedimented from the original chicken plasma by ultracentrifugation and resuspended in the same volume of normal chicken plasma as was used for injection of control mice. b The observation period was 6 weeks after the
chal1enge,
c Challenge tumors were SR-D5 0 for females and SR-Dl c? for males. The challenge was performed when mice were 4% weeks old.
COMMON
TRANSPLANTATION
ANTIGENS
termined from repeated experiments. For immunization, the respective virus was concentrated loo-fold by ultracentrifugation of medium from virus-infected chick TABLE DOSE-DEPENDENT
4
TRANSPLANTATION
ANCE TO Rows SARCOMA INJECTED ONCE WITH
I I Chd
TreatmaP
lenge dose (DMm)
RESIST-
ISOGRAFT IN MICE ALV AT BIRTH
Number of mice with tumors total number of mice Weeks
after
None Normal chicken plasma ALV 1OWc ALV
10-r
ALV
10”
4
5
6
5 25 5 25
6/9 2/5 3/7 5/8
9/9 5/5 7/7 8/8
9/9 5/5 7/7 8/8
5 25 5 25 5 25
2/5 l/3 2/10 5/10 l/10 3/11
3/5 3/3 7/11 7/10 3/10 S/11
5/5 3/3 S/11 7/10 4/10 7/10
755
-
-
15/15
8/8 8/118/11 15/21 7/10 7/10 5/10 5/10 12/20 7/10 - i
TABLE RESISTANCE DIFFERENT
AGAINST A Rous VIRUS STRAINS
Normal chicken plasma SR-RSV-1 BH-RSV (RAV-1) MAV-B SR-RSV-H
Injection
dose
107 107 10’ 5 x
FFU FFUd TCIDW 106 FFU
Antibody
5
SARCOMA ISOGRAFT AFTER INJECTION OF THE AVIAN SARCOMA LEUKOSIS
OF NEW-BORN
COMPLEX
of mice with
total of mice
487
VSV
embryo fibroblast cultures, titered, and thereafter injected into mice (Table 5). It can be seen in Table 5 that neither SRRSV-1, BH-RSV (RAV-1)) nor MAV-B induced any significant transplantation immunity, in contrast to the results obtained with BAI-A virus of a similar infectivity titer (Tables 3 and 4). A clearcut resistance was only obtained with SRRSV-H, despite its low infectivity titer.
Number Treatmenta
AND
SR-RSV-H shares common neutralizing envelope (V-) antigen(s) with the subgroup B-viruses present in the BAI-A strain of ALV which was used for immunization of mice. If these common V-antigens are responsible for the transplantation cross immunity, ALV-injected mice could cotktain neutralizing antibody against SR-RSV-H at the time of challenge. To prove this, l-day-old mice were injected with the same dose of ALV as in the experiments described before. Mice were tested 5 weeks and 6 weeks, respectively, later for neutralizing serum antibody. Sera were used in a dilution of 1:2 and 1: 10 after heat inactivation and tested against 100 FFU of SR-RSV-H as described elsewhere (Bauer and Graf, 1969). None out of 18 sera tested showed any significant virus-neutralizing capacity. The sensitivity of the test was such that
14/14
a Before injection ALV was removed from the original chicken plasma by ultracentrifugation and resuspended in the same normal chicken plasma that has been used for injection of mice. The dilutions were also performed in the same normal plasma. b Challenge was performed with SR-D t,umor cells when mice were 8 weeks old. c Dilution of ALV.
TRANSPLANTATION MICE WITH
ALV
Test for SR-RSV-H Neutralizing in Mice Immunized with ALV
challenge z
3
OF
number
ChallengeC
tumors* of mice
dose of cells
5 X DMm
25 X DMm
125 X DMloo
12/12
13/13 13/13 15/15 14/14 5/12
12/12 16/16 14/14 3/5
W6 14/16 11/12 -
a Mice were injected once when newborn. b The observation period was 5 weeks after challenge. c Challenge tumors were SR-D4 0 for females and SR-D2 when mice were 5% weeks old. d The TCIDso titer of RAV-1 in this stock is approximately
CT for 10 times
625 X DMmo
6/6
males. higher
Challenge than
4/4
6/6 -
w l/3
was the FFU
performed titer.
4ss
BAUER
approximately 5 % neutralization could not be excluded. DISCUSSION
The experiments reported here present evidence for avian leukosis virus-induced transplantation antigens. Adult mice which were repeatedly preimmunized with chicken myeloblasts were resistant against a Rous sarcoma isograft. Resistance was also obtained when mice were repeatedly immunized with high doses of ALV-containing chicken plasma. It has been found that only a single injection of ALV was sufficient to induce a clear transplantation resistance in newborn mice. The fact that mice treated with normal chicken tissue or plasma were not more resistant than untreated control mice indicates that normal chicken antigen is of no importance for the induction of the transplantation resistance obtained after injection of ALV or myeloblasts. In the first set of experiments it was not possible to determine whether the observed resistance was due to a common virus in the immunizing material and in the graft, or if it reflected a true cross reaction between the TSTA of ALV and RSV. For this reason immunization experiments were repeated using Rous sarcomas induced by a cloned and characterized strain of RSV, SR-RSV-H. This virus strain has been described earlier to be free of contaminating leukosis virus (Bauer and Graf, 1969). Likewise the BAI strain A has been shown to be free of Rous sarcoma virus. As was the case in the first experiments, a clearcut resistance of ALV-infected mice against these new challenge tumors was observed. It is therefore concluded that leukosis and sarcoma viruses, i.e., viruses with different target cells for oncogenicity in chickens, share common TSTA. Concerning the nature of immunization by ALV, two possibilities come to mind. 1. A structural component of the ALVparticles present in the immunizing material could act as TSTA. So far, two types of virus specific structural antigens, namely internal and envelope antigens, are known. The internal gs-antigen is group specific for all avian leukosis sarcoma viruses so far tested (Bauer and Schafer, 1965; Bauer,
ET z4L
1969), whereas the envelope (V-) antigens are subgroup- and type-specific (Vogt et al., 1966). The HA experiment speaks against the internal gs-antigen of ALV as being capable of inducing immunity against a Rous sarcoma isograft. The same experiment also tends to exclude, that the V-antigen(s) of the immunizing virus acts as transplantation antigen. If V-antigen(s) were identical with TSTA, one might also expect the formation of virus neutralizing antibodies after immunization of mice with ALV. Our negative results in this respect are in accordance with the findings of other authors (Jonsson and Sjogren, 1966; Koldovsky et al., 1966) that no virus neutralizing antibodies are present in sera of mice resistant to transplantation of syngeneic Rous sarcoma cells. However, the possibility of either cell-bound immunity against V-antigen or the presence of a low titer of neutralizing antibody cannot be excluded. It is also conceivable that ALV particles are carrying TSTA that are not identical to any one of the two aforementioned antigens. If ALV-induced chicken leukemic cells contain the same or similar TSTA as RSVinduced mouse sarcomas, this immunogenic TSTA could also be present on the surface of ALV particles released by such cells, as is the casewith different cellular components present in ALV and related virus particles (for review see Vogt, 1965a; Bauer and Schafer, 1966b). Such a phenomenon was suggested by Lindenmann and Klein (1967) for influenza virus produced by Ehrlich ascites cells. At present this possibility cannot be excluded, in spite of the fact that HA-treated ALV does not induce any immunity. It could still be that TSTA on the virus surface is inactivated by HA despite our finding that the antigenicity of gs- and V-antigens are almost unaltered by HA (Graf and Bauer, 1969). 2. The alternative is that virus of the BAI-strain A used in the present studies infects mouse cells and induces the synthesis of new cellular TSTA. Whether or not this TSTA is identical with a virus structural antigen, it may be similar to or identical with the TSTA of RSV-induced
COMMON
TRANSPLANTATION
ANTIGENS
OF
ALV
AND
RSV
489
mouse sarcomas. In favor of this explanareaction between TSTA of ALV and RSV tion is the finding that the immunity ob- should also be considered. served after a single ALV injection into ACKNOWLEDGMENT newborn mice is as strong as that obtained after repeated injection of adult mice. If The competenttechnical assistance of Miss A. the TSTA were present in the inoculated Seydel is gratefully acknowledged.We wish to material, adult mice, because they are fully thank ProfessorW. SchSiferfor his interest and immune competent, would be expected to support of this work. This investigation wassupported by the DeutscheForschungsgemeinschaft. show a stronger immunity effect. It thus seems likely that certain leukosis viruses REFERENCES induce the synthesis of TSTA in mouse BALUDA, M. A., and GOETZ, I. E. (1961). Morphocells. Such cells with foreign antigen may logical conversion of cell cultures by avian survive and mult,iply longer in newborn. myeloblastosis virus. Virology 15, 185-199. immunologically immature mice than in BAUER, H. (1969).Untersuchungen iiber Struktur, adults. Therefore a single injection in newVermehrung und onkogene Wirkung der born mice results in an equally strong imHtihner-Leukose-Sarkomatose-Viren.Habilitamunizing effect as that obtained by retions-Schrift, Tiibingen. peated injection of adult mice. The fact BAUER, H., and GRAF, T. (1969). Evidence for the that no tumors were observed after ALV possible existence of two envelope antigenic infection in mice is no real contradiction determinants and corresponding cell receptors of this hypothesis. It is shown in Table 5 for avian t,umor viruses. Virology 37, 157-161. BAUER, H., and SCHAFER, W. (1965). Isolierung that a SR-RSV-H dose insufficient to induce eines gruppenspezifischen Antigens aus dem a tumor is still able to immunize mice. Hiihner-Myeloblast,ose-Virus (BAI Stamm A). Such an explanation was also given by 2. Naturjorsch. 20b, 815-817. Habel (1961) and Sjiigren et al. (1961) for BAUER, H., and SCHAFER, W. (1966a). Origin of the finding, that polyoma infection of newgroup-specific antigen of chicken leukosis viborn or adult mice produces resistance to ruses. virology 29,494-496. polyoma tumor isografts. They suggested BAUER, H., and SCHAFER, W. (1966b). Studies on that a new cellular TSTA was induced by physical, chemical and antigenic properties of a the virus in infected and/or transformed chicken leukosis virus. In “Subviral Carcinogenesis” (Y. Ito, ed.), pp. 337-352. Aichi Center, mouse cells. This elicits an immunological Nagoya, Japan. response which leads to a subsequent reH., and SCH;~FER, W. jection of the transformed challenge cells. BAUER, H., BAHNEMANN, (1965). Untersuchungen iiber das MyeloblastoseAt present it is not known whether the Virus des Huhnes (BAI Stamm A). I. Nachweis immunity is caused by the myeloblastosisund Vermehrungskinetik des Virus in Htihnerinducing virus or by any one of the myelofibroblasten-Kulturen. 2. Naturjorsch. 20b, 959blastosis-associated viruses. It seems re965. markable that injection of 5 X lo5 FFU BuBENfK, J., and BAUER, H. (1967). Antigenic of SR-RSV-H induces a stronger immunity characteristics of the interaction between Rous than 10’ to lo8 TCIDSO of ALV. On the sarcoma virus and mammalian cells. Complement-fixing and transplantation other hand, similar amounts of other avian antigens. Virology 31,489497. tumor viruses [MAV-B, SR-RSV-1, and A. J. (1965). Prevention of SV40 virus BH-RSV (RAV-l)] did not induce a clear GIRARDI, oncogenesis in hamsters. I. Tumor resistance resistance. This may be explained by the induced by human cells transformed by SV40. finding of Hanafusa and Hanafusa (1966) Proc. Natl. Acad. Sci. U.S. 54, 445-451. that only virus strains with specific enGRAF, T. and BAUER, H. (1969). Studies on the velope antigens are able to infect and transrelative target size of various functions in the form mammalian cells. A virus of low titer genome of avian tumor viruses. In II. Int. Meetmay be present in our ALV stock which ing on Tumor Viruses, Royaumont, in press. possessesthe necessary envelope antigens HABEL, K. (1961). Resistance of polyoma virus immune animals to transplanted polyoma tuto infect mouse cells and thereafter to mors. Proc. Sot. Exptl. Biol. Med. 106,722-725. induce TSTA. However, a partial cross
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HA~I~L, K. (1963). Common antigen in polyoma t,umors. Federation Proc. 22, 438. (Abstract..) HANAFUSA, H., and HANAFUSA, T. (1966). Determining factor in the capacity of RSV to induce tumors in mammals. Proc. Natl. Acad. Sci. U.S. 55, 538-552. HARE, J. D. (1967). Transplant immunity to polyoma virus-induced tumor cells. IV. A polyoma strain defective in transplant antigen induction. Virology 31, 62&632. HELLSTR~M, I., and SJ~GREN, H. 0. (1967). In vitro demonstration of humoral and cell-bound immunity against common specific transplantation antigen(s) of adenovirus la-induced mouse and hamster tumors. J. Ezptl. Med. 125, 11051118. IRLIN, I. S. (1967). Immunofluorescent demonstration of a specific surface antigen in cells infected or transformed by polyoma virus. Virilogy 32,725-728. ISHIZUI, R., and VOGT, P. K. (1966). Immunological relationships among envelope antigens of avian tumor viruses. Virology 30, 375-387. JONSSON, N. (1966). Studies on the occurrence of common specific transplantation antigens in Rous tumors of various mammalian species. Acta Pathol. Microbial. &and. 67, 339-353. JONSSON, N., and SJ~GREN, H. 0. (1966). Specific transplantation immunity in relation to Rous sarcoma virus tumorigenesis in mice. J. Exptl. Med. 123,487+X3. KLEIN, G. (1966). Tumor antigens. Ann. Rev. ikficrobiol. 20, 223-252. KLUCHAREVA, T. E., SHACHARINA, K. L., BELOVA, S., CHILISOVA, V., and DEICHMAN, G. I. (1967). Use of immunofluorescence for detection of specific membrane antigen in simian virus ~CIinfected nontransformed cells. J. Natl. Cancer Inst. 39, 825-832. KOLDOVSK+, P., SVOBODA, J., and BUBEN~K, J. (1966). Further studies on the immunobiology of
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tumor RVAI induced by RSV in C57Bl strain mice. FoEia l3iol. (Prague) 12, l-10. LINDENMANN, J., and KLEIN, P. A. (1967). Viral analysis: increased immunogenicity of host cell antigen associated with influenza virus. J. Exptl. Med. 126,93-108. MOSCOVICI, C., and VOGT, P. K. (1968). Effects of genetic cellular resistance on cell transformation and virus replication in chicken hematopoietic cell cultures infected with avian myeloblastosis virus (BAI-A) . Virology 35,487497. OLD, L. J., and BOYSE, E. A. (1964). Immunology of experimental tumors. Ann. Rev. Med. 15, 167-186. RUBIN, H. (1961). The nature of a virus-induced cellular resistance to Rous sarcoma virus. Virology 13, 200-206. SCHBFER, W., and SEIFERT, E. (1968). Production of a potent complement-fixing murine leukemia virus-antiserum from the rabbit and its reactions with various types of tissue culture cells. Virology 35, 323328. SJ~~GREN, H. 0. (1965). Transplantation methods as a tool for detection of tumor-specific antigens. Progr. Exptl. Tumor Res. 6, 289-322. SJ~QREN, H. O., HELLSTR~M, J., and KLEIN, G. (1961). Resistance of polyoma virus immunized mice against transplantation of established polyoma tumors. Exptl. GelE Res. 23, 204-208. TEMIN, H., and RUBIN, N. (1958). Characteristics of an assay for Rous sarcoma virus and Rous sarcoma cells in tissue culture. Virology 6, 669688. VOGT, P. K. (1965a). Avian tumor viruses. Advun. Virus Res. 11, 293-385. VOGT, P. K. (196513). A heterogeneity of Rous sarcoma virus released by selectively resistant chick embryo cells. Virology 25, 237-247. VOGT, P. K., ISHIZAKI, R., and DUFF, R. (1966). Studies on the relationships among avian tumor viruses. In “Subviral Carcinogenesis” Cy. Ito, ed.), pp. 297-310. Aichi Center, Nagoya, Japan.