Int. J. Cancer: 23, 514-518 (1979)
EXPRESSION OF Fv-4' ALLELE IN HEMATOPOIETIC CELLS FROM G MICE RESISTANT TO FRIEND LEUKEMIA VIRUS Hidetoshi IKEDAand Takeshi ODAKA Department of Genetics, The Institute of Medical Science, The University of Tokyo, P . 0. Takanawa, Tokyo 108, Japan
G mice carrying the Fv-4 resistant allele supported virus growth neither a t an early nor a later (Kai et ol., 1976) stage of infection with NB-tropic FLV. This resistance could not be abolished by treatment of G mice with cyclophosphamide or cortisone acetate. By bone-marrow or spleen-cell transplantation into irradiated mice, the resistance of G mice could be transferred t o Fv-Csusceptible mice. Conversely, transfer of bone-marrow o r spleen cells of Fv-4-susceptible mice rendered G mice susceptible. It could be concluded that, as assessed by the virus content in the spleen, helper LLV grows mainly in radiosensitive, bone-marrow-derived cells, and the Fv-4 gene is expressed in these cells.
Friend leukemia virus (FLV) is composed of defective spleen focus-forming virus (SFFV) and its helper lymphatic leukemia virus (LLV). Several host genes affect leukemogenesis by FLV (Lilly and Pincus, 1973). The Fv-I gene (Lilly, 1970) controls the replication of N- or B-tropic LLV, whereas the Fv-2 gene (Odaka and Yamamoto, 1962; Odaka, 1973) controls the replication of SFFV and spleen focus formation and splenomegaly induction by the FLV complex. Recently, Suzuki (1975) identified the Fv-4 gene which is epistatic for Fv-I and Fv-2 genes (Odaka and Ikeda, 1977). G mice carrying Fv-4" neither support the growth of N-tropic and NB-tropic FLV nor develop splenomegaly (Kai et a/., 1976). More recently, a gene identical or similar to the Fv-4' was found in Japanese wild mice (Odaka et al., 1978). Mechanisms of Fv-4 restriction in vivo have not yet been characterized. In this report, we investigated susceptibility to FLV infection of radiation chimeras between G and Fv-4-susceptible D D D or DDD-Fv'. The results indicated that the Fv-4 gene is expressed in heniatopoietic cells. The results further suggested that radioresistant non-hematopoietic cells do not participate in supporting viral growth in vivo. MATERIAL A N D METHODS
Mice Strains DDD (Fv-I"", Fv-~", F v - ~ " ) ,DDD-Fv' (Fv-I"", Fv-2", F V - ~ and ~ ~ ) G (Fv-I"", Fv-ZSs, Fv+frT)were described previously (Odaka, 1970; Suzuki, 1975; Odaka and Ikeda, 1977; Yoshikura and Odaka, 1978; Odaka, unpublished data). Mice of these strains and (G x DDD)F, hybrids were produced in our laboratory. Male and female mice (4-16 weeks) were used. Random-bred female ddYS mice, purchased from the Shizuoka Agricultural Cooperative Association for Laboratory Animals, Hamamatsu, Japan, were used at 4-5 weeks of age for SFFV assay.
Virus NB-tropic FLV that had been serially passaged in BALB/c mice was used. The methods of preparation and storage were described previously (Odaka, 1973). In this experiment, all mice were inoculated with FLV of 104.2or lo4.*ID,, (splenomegaly induction in 4 weeks). Virus assay
Spleens were individually homogenized in 5 ml of cold phosphate-buffered saline containing 0.25 gelatin, and the homogenates were assayed on C-182 (S+L-) cells (Bassin et al., 1971) by the uv-XC assay (Rowe et a/., 1970). Titers were expressed as PFU per spleen. SFFV titer was estimated by the spleen focus assay (Axelrad and Steeves, 1964). The ddYS mice were injected IP with 0.2 ml of appropriately diluted sample, and killed 10 days later. The spleens were removed, fixed in Bouin's solution and examined for superficial macroscopic foci. Titers were expressed as FFU. Radiation chimera
Mice were irradiated with 900-1,100 rads from a 6oCo source. Between 5 and 28 h after radiation, the mice were injected IV with 0.5 rnl suspension of bone-marrow or spleen cells suspended in Eagle's minimum essential medium containing 10 % bovine serum. Unless otherwise stated, one mouse received 3-15 x lo6 bone-marrow cells or 7-80 x lo6 spleen cells. RESULTS
LLVgrowth at early stage of infection
In order to compare the growth of LLV in G and DDD mice, espxially in the early stage of infection, the mice were infected with the FLV complex. Three mice of each group were killed daily. Spleens were extracted and assayed for LLV by the uv-XC assay (Fig. 1). N o trace amount of LLV was detected in G mice throughout the observation period, whereas LLV began lo increase in titer in DDD mice from day 2 on. Therefore, LLV did not replicate in G mice in early (Fig. 1) and later (Kai et al., 1976) stages of infection, or if it did replicate, it was very rapidly eliminated in G mice. Treating G mice twice (3 days before and on the day of infection) with cyclophosphamide (0.2 g/kg), Received: December 11, 1978.
RESISTANCE TO FRIEND LEUKEMIA VIRUS
or three times (3 days before, on the day of, and 3 days after infection) with cortisone acetate (83 mg/ kg) did not affect the resistance of G mice; no virus growth was detected in the spleens 10 days after infection (data not shown).
104 c-
Z
w W -I
%
103
\
3
U
a
3 -I
515
102
2.5 X I0
L 1 3 0 2 DAY AFTER I NFECTION FIGURE1 - LLV growth in spleens of G ( 0 ) and DDD (0)mice. Mice were injected IP with NB-tropic FLV, and their spleens were homogenized and assayed for LLV by uv-XC assay.
Virus growth in chimeric mice If the Fv-4 gene is expressed in hematopoietic cells, and non-hematopoietic cells do not contribute to virus growth, the resistance could be transferred by bone-marrow transplantation, as in the case of the Fv-2 controlled resistance (Odaka and Matsukura, 1969). Though G , DDD and DDD-Fv' strains are all derived from a random-bred closed colony dd, the H-2 haplotype of G differs from that of DDD (and hence DDD-Fvr; Ikeda, unpublished data). Therefore, before proceeding to bone-marrow transplantation experiments, it was important to see whether hematopoietic stem cells can settle efficiently in allogeneic hosts or not. Reciprocal transfer of bone-marrow cells was done between G and DDD mice. The transfer of lo5 nucleatcd G bone-marrow cells formed 9.3 & 1.6 (SD) spleen colonies in G mice and 9.0&2.7 in DDD mice, while the same number of DDD bone-marrow cells formed 6.3*1.2 spleen colonies in G mice and 11.3kO.6 in DDD mice. The results indicate that stem cells are similar in number in G and DDD mice and can settle in allogeneic recipients almost as well as in syngeneic hosts.
1oL
z W
1 o3 0
2
v)
0
z
i
0
0 0
102
0
2.5
xl0
---
0
c--------
DDD
MD-Fv'
G
OONDR BONE MARROW CELLS
DDD
DDO-Fv'
G
WNDR SPLEEN CELLS
FIGURE2 - LLV growth in chimeric mice. Recipient mice ( 0 , G, 0,DDD) were irradiated with 900-1,100 rads and reconstituted with 3-15 x loR bone-marrow cells (a) or 7-80 x lofi spleen cells (b). Chimeric mice were intraperitoneally inoculated with NB-tropic FLV, and 7 days later their spleens were homogenized and assayed for LLV by uv-XC assay.
516
IKEDA AND ODAKA TABLE I
SFFV RECOVERY FROM SPLEENS OF MICE WHICH WERE IRRADIATED, RECONSTITUTED WITH BONE-MARROW CELLS AND INOCULATED WITH NB-TROPIC FLV.
'
Recipient
Donor
No. of mice tested
LLV (log,,PFU/spleen)
(log,,FFU/spleen)
DDD DDD DDD
DDD DDD-Fv" G
5 5 6
a11>4 a1b4 a115.4, >5.4, 4.4 4.7, 4.3, 2.4, 10' PFU (LLV) per spleen.
in the reconstituted G mice was partially inhibited by graft-versus-host reaction or resistance to In the present study, we tested the possibility allogeneic marrow graft, because G mice have a that NR-tropic FLV grows well at early stage of H-2 haplotype (Ikeda, unpublished data) and infection and is rapidly eliminated in G mice. probably also an Hh system (Cudkowicz, 1971) We assayed LLV in the spleens for 4 days after different from those of DDD mice. In the experinfection, but found no virus. Treatment of G mice iments of allogeneic spleen cell transfer, an alternawith cyclophosphaniide or cortisone acetate did tive explanation is that target cells for FLV growth not enhance the virus growth. are fewer in spleen-derived cells than in boneMice with anemia gene, W, SI or f, are resistant marrow-derived cells. However, the exact reason to spleen focus-formation by FLV (Steeves et a/., for these partial resistances in irradiated G mice is 1968; Bennett et al., 1968; Axelrad, 1968). G mice not understood. had heniatocrit values (47.5-fO.60 %) comparable It seems unlikely that bone-marrow-derived cells to those of DDD (49.01 1.99 %). Hematopoietic of G mice produce some factors suppressing virus stem cells of G and DDD mice. as assayed by growth or are cytotoxic for FLV-infected cells, colony formation in the spleen (CFU,), were also since the mixtures of bone-marrow cells of G similar in number. and DDD did not render the recipients resistant (Table 11). Reciprocal bone-marrow or spleen-cell transplanReciprocal bone-marrow transplantation contation between G and DDD or DDD-Fvr converted the susceptibility of the recipients to that expxted verted the susceptibility of the irradiated mice to from the genotypes of the donors (Fig. 2). The that of rhe donor. This finding indicates that the DDD mice which had been lethally irradiated and Fv-4 gene is expressed in bone-marrow-derived reconstituted with bone-marrow cells of G mice cells. The finding further suggests that non-hematoneither supported virus growth nor developed poietic cells do not support LLV replication, splenomegaly. This response of the chimeric DDD although LLV can efficiently replicate in fibroblasts mice was completely comparable with that of in vitro. Though expressed in bone-marrow-derived untreated G mice. Conversely, the chimeric G mice cells, the Fv-2 gene affects SFFV growth and splenowith bone-marrow cells of DDD origin responded megaly induction (Odaka and Matsukura, 1969), but not LL.V growth (Fig. 2). In contrast, the Fv-4 to FLV as untreated DDD mice. gene primarily affects LLV growth, and consequently However, G mice reconstituted with DDD or SFFV growth and splenomegaly induction. The DDD-Fvr cells were not as susceptible as DDD mice Fv-lr allele may determine either non-permissiveness reconstituted with DDD or DDD-Fv' cells. G mice of hematopoietic cells to LLV infection or deletion reconstituted with spleen cells of DDD or DDD-Fvr of a subpopulation of hematopoietic cells required yielded lower LLV titers than did DDD recipients for FLV replication. reconstituted with DDD or DDD-Fv' spleens (Fig. 2b). Similarly, G mice reconstituted with ACKNOWLEDGEMENTS DDD or DDD-Fv' bone-marrow cells yielded We thank Miss M. Yamashita and Mrs. S. lower SFFV titers than did DDD mice reconstituted with DDD or DDD-Fb'bone-marrow cells (Table I). Enokida for excellent technical assistance. We are Furthermore, G mice reconstituted with DDD also indebted to Dr. H. Yoshikura for reading this bone-marrow cells developed splenomegaly after paper and making invaluable comments. This a longer latent period than did DDD mice recon-. work was supported in part by a Grant-in-Aid stituted with DDD bone-marrow cells (Table 111). for Cancer Research from the Ministry of Education, These observations may indicate that viral growth Science, and Culture, Japan. DISCUSSION
518
IKEDA AND ODAKA
EXPRESSION DE L’ALLELE Fv-4‘ DANS LES CELLULES HeMATOPOYETIQUES DE SOURIS G RESISTANTES AU VIRUS DE LA LEUCEMIE DE FRIEND La replication virale ne s’est produite, chez des souris G portant I’allble Fv-4 rbsistant, ni a u premier stade de I’infection par le FLV A tropisme NB ni B un stade ulttrieur (Kai et a / . 1976). Cette resistance ne pouvait pas ttre supprimte par un traitenient A la cyclophosphamide ou 8 I’acetate de cortisone. La rtsistance des souris G a pu Ztre transftrke B des souris portant I’all&le Fv-4 sensible par transplantation d e cellules de moelle osseuse ou de rate B des souris irradiks. A I’inverse, si les cellules de moelle osseuse ou de rate provenaient de souris B Fv-4 sensible, leur transfert rendait les souris G sensibles. On peut en conclure que, d’aprbs la teneur de la rate en virus, le LLV auxiliaire se rtplique surtout dans les cellules radiosensibles provenant de la moelle osseuse, et que le gene Fv-4 est exprim6 dans ces cellules.
REFERENCES
AXELRAD, A. A., Genetic and cellular basis of susceptibility or resistance to Friend leukemia virus infection in mice. Can. Cancer Conf., 8, 313-343 (1968). AXELRAD, A. A., and STEEVES, R. A., Assay for Friend leukemia virus: rapid quantitative method based on enumeration of macroscopic spleen foci in mice. Virology, 24, 513-518 (1964).
BASSIN,R. H., TUTTLE,N., and FISCHINGER, P. J., Rapid cell culture assay technique for murine leukemia viruses. Nature (Lond.), 229, 564-566 (1971). BENNETT,M., STEEVES,R. A., CUDKOWICZ, G., MIRAND, E. A,, and RUSSEL,L. B., Mutant S1 alleles of mice affect susceptibility to Friend spleen focus-forming virus. Science, 162, 564-565 (1968).
CUDKOWICZ, G., Genetic control of bone marrow graft rejection. I. Determinant specific difference of reactivity in two pairs of inbred mouse strains. J . exp. Med., 134, 281-293
in Friend virus preparation. l n t . J. Cancer, 11, 567-574 (1973).
ODAKA,T., and IKEDA,H., Genetic resistance to Friend leukemia virus in mice: masking of Fv-2 phenotype by an epistatic gene, Fv-4‘. Jap. J . exp. Med., 47, 515-521 (1977). ODAKA,T., IREDA,H., MORIWAKI,K., MATSUZAWA,A., MIZUNO,M., and KONDO,K., Genetic resistance in Japanese wild mice (Mus musculus molossinus) t o an NB-tropic Friend murine leukemia virus. J . nat. Cancer Inst., 61, 1301-1306 (1978).
ODAKA,T., and MATSUKURA, M., Inheritance of susceptibility t o Friend mouse leukemia virus. VI. Reciprocal alteration of innate resistance o r susceptibility by bone marrow transplantation between congeneic strains. J . Virol., 4, 403-415 (1969). ODAKA,T., and YAMAMOTO, T.. Inheritance of susceptibility to Friend mouse leukemia virus. Jap. J . exp. Med., 32,
(1971).
405-413 (1962).
KAI, K., IKEDA,H., YUASA,Y., SUZUKI,S., and ODAKAT., Mouse strain resistant t o N-, B-, and NB-tropic murine leukemia viruses. J . Virol., 20, 436-440 (1976). LILLY,F., Fv-2: identification and location of a second gene governing the spleen focus response to Friend leukemia virus in mice. J. nat. Cancer Inst., 45, 163-169 (1970). LILLY,F., and PINCUS,T., Genetic control of murine viral leukemogenesis. Advanc. Cancer Res., 17, 231-277 (1973).
ROWE, W. P., PUGH,W. E., and HARTLEY, J. W., Plaque assay techniques for murine leukemia viruses. Virology,
ODAKA,T., Inheritance of susceptibility to Friend leukemia virus. VII. Establishment of a resistant Int. J . Cancer, 6, 18-23 (1970). ODAKA,T., Inheritance of susceptibility to Friend leukemia virus. X. Separate genetic control of two
mouse strain. mouse viruses
42, 1136-1139 (1970).
STEEVES,R. A., BENNETT,M., MIRAND, E. A., and CUDKOWICZ, G., Genetic control by the W locus of susceptibility t o (Friend) spleen focus-forming virus. Nature (Land.), 218, 372-374 (1968). SUZUKI,S., Fv-4: A new gene affecting the splenomegaly induction by Friend leukemia virus. Jap. J . exp. Med., 45, 473-47%(1 975).
YOSHIKURA, H., and ODAKA,T., Resistance of G mice t o murine leukemia virus infection : apparent disparity in in vivo and in vitro resistances. J . nai. Cancer -Inst.: 61, 461-463 (1978).
EXPRESSION OF Fv-4' ALLELE IN HEMATOPOIETIC CELLS FROM G MICE RESISTANT TO FRIEND LEUKEMIA VIRUS Hidetoshi IKEDAand Takeshi ODAKA Department of Genetics, The Institute of Medical Science, The University of Tokyo, P . 0. Takanawa, Tokyo 108, Japan
G mice carrying the Fv-4 resistant allele supported virus growth neither a t an early nor a later (Kai et ol., 1976) stage of infection with NB-tropic FLV. This resistance could not be abolished by treatment of G mice with cyclophosphamide or cortisone acetate. By bone-marrow or spleen-cell transplantation into irradiated mice, the resistance of G mice could be transferred t o Fv-Csusceptible mice. Conversely, transfer of bone-marrow o r spleen cells of Fv-4-susceptible mice rendered G mice susceptible. It could be concluded that, as assessed by the virus content in the spleen, helper LLV grows mainly in radiosensitive, bone-marrow-derived cells, and the Fv-4 gene is expressed in these cells.
Friend leukemia virus (FLV) is composed of defective spleen focus-forming virus (SFFV) and its helper lymphatic leukemia virus (LLV). Several host genes affect leukemogenesis by FLV (Lilly and Pincus, 1973). The Fv-I gene (Lilly, 1970) controls the replication of N- or B-tropic LLV, whereas the Fv-2 gene (Odaka and Yamamoto, 1962; Odaka, 1973) controls the replication of SFFV and spleen focus formation and splenomegaly induction by the FLV complex. Recently, Suzuki (1975) identified the Fv-4 gene which is epistatic for Fv-I and Fv-2 genes (Odaka and Ikeda, 1977). G mice carrying Fv-4" neither support the growth of N-tropic and NB-tropic FLV nor develop splenomegaly (Kai et a/., 1976). More recently, a gene identical or similar to the Fv-4' was found in Japanese wild mice (Odaka et al., 1978). Mechanisms of Fv-4 restriction in vivo have not yet been characterized. In this report, we investigated susceptibility to FLV infection of radiation chimeras between G and Fv-4-susceptible D D D or DDD-Fv'. The results indicated that the Fv-4 gene is expressed in heniatopoietic cells. The results further suggested that radioresistant non-hematopoietic cells do not participate in supporting viral growth in vivo. MATERIAL A N D METHODS
Mice Strains DDD (Fv-I"", Fv-~", F v - ~ " ) ,DDD-Fv' (Fv-I"", Fv-2", F V - ~ and ~ ~ ) G (Fv-I"", Fv-ZSs, Fv+frT)were described previously (Odaka, 1970; Suzuki, 1975; Odaka and Ikeda, 1977; Yoshikura and Odaka, 1978; Odaka, unpublished data). Mice of these strains and (G x DDD)F, hybrids were produced in our laboratory. Male and female mice (4-16 weeks) were used. Random-bred female ddYS mice, purchased from the Shizuoka Agricultural Cooperative Association for Laboratory Animals, Hamamatsu, Japan, were used at 4-5 weeks of age for SFFV assay.
Virus NB-tropic FLV that had been serially passaged in BALB/c mice was used. The methods of preparation and storage were described previously (Odaka, 1973). In this experiment, all mice were inoculated with FLV of 104.2or lo4.*ID,, (splenomegaly induction in 4 weeks). Virus assay
Spleens were individually homogenized in 5 ml of cold phosphate-buffered saline containing 0.25 gelatin, and the homogenates were assayed on C-182 (S+L-) cells (Bassin et al., 1971) by the uv-XC assay (Rowe et a/., 1970). Titers were expressed as PFU per spleen. SFFV titer was estimated by the spleen focus assay (Axelrad and Steeves, 1964). The ddYS mice were injected IP with 0.2 ml of appropriately diluted sample, and killed 10 days later. The spleens were removed, fixed in Bouin's solution and examined for superficial macroscopic foci. Titers were expressed as FFU. Radiation chimera
Mice were irradiated with 900-1,100 rads from a 6oCo source. Between 5 and 28 h after radiation, the mice were injected IV with 0.5 rnl suspension of bone-marrow or spleen cells suspended in Eagle's minimum essential medium containing 10 % bovine serum. Unless otherwise stated, one mouse received 3-15 x lo6 bone-marrow cells or 7-80 x lo6 spleen cells. RESULTS
LLVgrowth at early stage of infection
In order to compare the growth of LLV in G and DDD mice, espxially in the early stage of infection, the mice were infected with the FLV complex. Three mice of each group were killed daily. Spleens were extracted and assayed for LLV by the uv-XC assay (Fig. 1). N o trace amount of LLV was detected in G mice throughout the observation period, whereas LLV began lo increase in titer in DDD mice from day 2 on. Therefore, LLV did not replicate in G mice in early (Fig. 1) and later (Kai et al., 1976) stages of infection, or if it did replicate, it was very rapidly eliminated in G mice. Treating G mice twice (3 days before and on the day of infection) with cyclophosphamide (0.2 g/kg), Received: December 11, 1978.
RESISTANCE TO FRIEND LEUKEMIA VIRUS
or three times (3 days before, on the day of, and 3 days after infection) with cortisone acetate (83 mg/ kg) did not affect the resistance of G mice; no virus growth was detected in the spleens 10 days after infection (data not shown).
104 c-
Z
w W -I
%
103
\
3
U
a
3 -I
515
102
2.5 X I0
L 1 3 0 2 DAY AFTER I NFECTION FIGURE1 - LLV growth in spleens of G ( 0 ) and DDD (0)mice. Mice were injected IP with NB-tropic FLV, and their spleens were homogenized and assayed for LLV by uv-XC assay.
Virus growth in chimeric mice If the Fv-4 gene is expressed in hematopoietic cells, and non-hematopoietic cells do not contribute to virus growth, the resistance could be transferred by bone-marrow transplantation, as in the case of the Fv-2 controlled resistance (Odaka and Matsukura, 1969). Though G , DDD and DDD-Fv' strains are all derived from a random-bred closed colony dd, the H-2 haplotype of G differs from that of DDD (and hence DDD-Fvr; Ikeda, unpublished data). Therefore, before proceeding to bone-marrow transplantation experiments, it was important to see whether hematopoietic stem cells can settle efficiently in allogeneic hosts or not. Reciprocal transfer of bone-marrow cells was done between G and DDD mice. The transfer of lo5 nucleatcd G bone-marrow cells formed 9.3 & 1.6 (SD) spleen colonies in G mice and 9.0&2.7 in DDD mice, while the same number of DDD bone-marrow cells formed 6.3*1.2 spleen colonies in G mice and 11.3kO.6 in DDD mice. The results indicate that stem cells are similar in number in G and DDD mice and can settle in allogeneic recipients almost as well as in syngeneic hosts.
1oL
z W
1 o3 0
2
v)
0
z
i
0
0 0
102
0
2.5
xl0
---
0
c--------
DDD
MD-Fv'
G
OONDR BONE MARROW CELLS
DDD
DDO-Fv'
G
WNDR SPLEEN CELLS
FIGURE2 - LLV growth in chimeric mice. Recipient mice ( 0 , G, 0,DDD) were irradiated with 900-1,100 rads and reconstituted with 3-15 x loR bone-marrow cells (a) or 7-80 x lofi spleen cells (b). Chimeric mice were intraperitoneally inoculated with NB-tropic FLV, and 7 days later their spleens were homogenized and assayed for LLV by uv-XC assay.
516
IKEDA AND ODAKA TABLE I
SFFV RECOVERY FROM SPLEENS OF MICE WHICH WERE IRRADIATED, RECONSTITUTED WITH BONE-MARROW CELLS AND INOCULATED WITH NB-TROPIC FLV.
'
Recipient
Donor
No. of mice tested
LLV (log,,PFU/spleen)
(log,,FFU/spleen)
DDD DDD DDD
DDD DDD-Fv" G
5 5 6
a11>4 a1b4 a115.4, >5.4, 4.4 4.7, 4.3, 2.4, 10' PFU (LLV) per spleen.
in the reconstituted G mice was partially inhibited by graft-versus-host reaction or resistance to In the present study, we tested the possibility allogeneic marrow graft, because G mice have a that NR-tropic FLV grows well at early stage of H-2 haplotype (Ikeda, unpublished data) and infection and is rapidly eliminated in G mice. probably also an Hh system (Cudkowicz, 1971) We assayed LLV in the spleens for 4 days after different from those of DDD mice. In the experinfection, but found no virus. Treatment of G mice iments of allogeneic spleen cell transfer, an alternawith cyclophosphaniide or cortisone acetate did tive explanation is that target cells for FLV growth not enhance the virus growth. are fewer in spleen-derived cells than in boneMice with anemia gene, W, SI or f, are resistant marrow-derived cells. However, the exact reason to spleen focus-formation by FLV (Steeves et a/., for these partial resistances in irradiated G mice is 1968; Bennett et al., 1968; Axelrad, 1968). G mice not understood. had heniatocrit values (47.5-fO.60 %) comparable It seems unlikely that bone-marrow-derived cells to those of DDD (49.01 1.99 %). Hematopoietic of G mice produce some factors suppressing virus stem cells of G and DDD mice. as assayed by growth or are cytotoxic for FLV-infected cells, colony formation in the spleen (CFU,), were also since the mixtures of bone-marrow cells of G similar in number. and DDD did not render the recipients resistant (Table 11). Reciprocal bone-marrow or spleen-cell transplanReciprocal bone-marrow transplantation contation between G and DDD or DDD-Fvr converted the susceptibility of the recipients to that expxted verted the susceptibility of the irradiated mice to from the genotypes of the donors (Fig. 2). The that of rhe donor. This finding indicates that the DDD mice which had been lethally irradiated and Fv-4 gene is expressed in bone-marrow-derived reconstituted with bone-marrow cells of G mice cells. The finding further suggests that non-hematoneither supported virus growth nor developed poietic cells do not support LLV replication, splenomegaly. This response of the chimeric DDD although LLV can efficiently replicate in fibroblasts mice was completely comparable with that of in vitro. Though expressed in bone-marrow-derived untreated G mice. Conversely, the chimeric G mice cells, the Fv-2 gene affects SFFV growth and splenowith bone-marrow cells of DDD origin responded megaly induction (Odaka and Matsukura, 1969), but not LL.V growth (Fig. 2). In contrast, the Fv-4 to FLV as untreated DDD mice. gene primarily affects LLV growth, and consequently However, G mice reconstituted with DDD or SFFV growth and splenomegaly induction. The DDD-Fvr cells were not as susceptible as DDD mice Fv-lr allele may determine either non-permissiveness reconstituted with DDD or DDD-Fv' cells. G mice of hematopoietic cells to LLV infection or deletion reconstituted with spleen cells of DDD or DDD-Fvr of a subpopulation of hematopoietic cells required yielded lower LLV titers than did DDD recipients for FLV replication. reconstituted with DDD or DDD-Fv' spleens (Fig. 2b). Similarly, G mice reconstituted with ACKNOWLEDGEMENTS DDD or DDD-Fv' bone-marrow cells yielded We thank Miss M. Yamashita and Mrs. S. lower SFFV titers than did DDD mice reconstituted with DDD or DDD-Fb'bone-marrow cells (Table I). Enokida for excellent technical assistance. We are Furthermore, G mice reconstituted with DDD also indebted to Dr. H. Yoshikura for reading this bone-marrow cells developed splenomegaly after paper and making invaluable comments. This a longer latent period than did DDD mice recon-. work was supported in part by a Grant-in-Aid stituted with DDD bone-marrow cells (Table 111). for Cancer Research from the Ministry of Education, These observations may indicate that viral growth Science, and Culture, Japan. DISCUSSION
518
IKEDA AND ODAKA
EXPRESSION DE L’ALLELE Fv-4‘ DANS LES CELLULES HeMATOPOYETIQUES DE SOURIS G RESISTANTES AU VIRUS DE LA LEUCEMIE DE FRIEND La replication virale ne s’est produite, chez des souris G portant I’allble Fv-4 rbsistant, ni a u premier stade de I’infection par le FLV A tropisme NB ni B un stade ulttrieur (Kai et a / . 1976). Cette resistance ne pouvait pas ttre supprimte par un traitenient A la cyclophosphamide ou 8 I’acetate de cortisone. La rtsistance des souris G a pu Ztre transftrke B des souris portant I’all&le Fv-4 sensible par transplantation d e cellules de moelle osseuse ou de rate B des souris irradiks. A I’inverse, si les cellules de moelle osseuse ou de rate provenaient de souris B Fv-4 sensible, leur transfert rendait les souris G sensibles. On peut en conclure que, d’aprbs la teneur de la rate en virus, le LLV auxiliaire se rtplique surtout dans les cellules radiosensibles provenant de la moelle osseuse, et que le gene Fv-4 est exprim6 dans ces cellules.
REFERENCES
AXELRAD, A. A., Genetic and cellular basis of susceptibility or resistance to Friend leukemia virus infection in mice. Can. Cancer Conf., 8, 313-343 (1968). AXELRAD, A. A., and STEEVES, R. A., Assay for Friend leukemia virus: rapid quantitative method based on enumeration of macroscopic spleen foci in mice. Virology, 24, 513-518 (1964).
BASSIN,R. H., TUTTLE,N., and FISCHINGER, P. J., Rapid cell culture assay technique for murine leukemia viruses. Nature (Lond.), 229, 564-566 (1971). BENNETT,M., STEEVES,R. A., CUDKOWICZ, G., MIRAND, E. A,, and RUSSEL,L. B., Mutant S1 alleles of mice affect susceptibility to Friend spleen focus-forming virus. Science, 162, 564-565 (1968).
CUDKOWICZ, G., Genetic control of bone marrow graft rejection. I. Determinant specific difference of reactivity in two pairs of inbred mouse strains. J . exp. Med., 134, 281-293
in Friend virus preparation. l n t . J. Cancer, 11, 567-574 (1973).
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