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SIM and SIM · R mice are histoincompatible
VIROLOGY
86, 287-290
(1978)
SIM PHILIP
and SIM
FURMANSKI,
Department
of Biology,
. R Mice
Are Histoincompatible
MICHAEL DIETZ, JOHN MARCELLETTI Michigan Accepted
Cancer
DAVID
Foundation,
December
Detroit,
L. HINES,
Michigan
AND
48201
19, 1977
SIM and SIM. R mice were bred for congenicity at the Fv-I locus. difference in their permissiveness for infection by murk C-type viruses, to Fv-I, these mice differ in antigens determining bistocompatibility.
In addition presumably
to a due
The ability to infect mouse cells producsion of FV-induced leukemia, we found that tively with oncornaviruses is governed by a virus tropism and Fv-1 type are critical number of host genes. Preeminent among determinants in the outcome of the disease these is Fv-1, which was originally identi- (11). To analyze the effects further, we fied by its effect on Friend virus (FV) and initiated studies using these congenic mice. We found that in addition to a difference at is now known to be the host contribution to the phenomenon of virus tropism affect- Fv-1, the SIM @‘v-l, n/n) and SIM . R (Fving all of the murine leukemia viruses 1, b/b) mice are histoincompatible. SIM and SIM R mice were obtained (MuLVs) (1). Those MuLVs that are capable of replicating in mouse cells are iden- from colonies maintained at the Michigan tified as N-tropic or B-tropic depending on Cancer Foundation derived from breeding whether or not they can infect cells with stock originally provided by Dr. A. Axelrad. the Fv-1 genotypes n/n or b/b, respectively. Animals were also obtained directly from NB-tropic viruses replicate equally well in Dr. Axelrad and used for these experiments. either type of cell, as well as in cells with In accord with previous reports (4,5), we the n/b genotype, which are restrictive to found that the SIM mice were permissive both N- and B-tropic viruses. for N- and NB-tropic viruses and restricted Studies on the mechanism of the Fv-1 B-tropic viruses, while the SIM . R mice effect and virus tropism have generated were permissive for B- and NB-tropic viconsiderable interest as a model system for ruses and restricted N-tropic viruses. These the regulation of oncornavirus infection and results were obtained using a spleen focus expression. The data obtained to date in- assay with FVs of known tropism, as predicate that the effect is mediated at a step viously described (11, 12) and using the after virus penetration but prior to the in- same viruses in an in vitro XC plaque assay sertion of proviral DNA (2,3). on cells derived from SIM and SIM . R emA valuable resource in attempting to de- bryos or trypsinized adult kidneys ( II). The lineate the mechanism of gene effects is the Fv-1 n/n (SIM) restriction of the B-tropic availability of animals which differ only at virus and the restriction of the N-tropic the locus under study, that is, coisogenic, or virus by Fv-1 b/b (SIM . R) was greater more realistically, congenic animals. Axel- than lO&fold in each case in the animals rad and co-workers have developed strains used in these experiments. of mice which are congenic for Fv- 1 (4, 5) Histocompatibility was determined by and these animals, and cell lines derived skin graft reactions of the SIM and SIM R from them, have been used in several of the mice. Disks of full thickness skin 0.7 cm in studies of Fv-1 and virus tropism (2, 5, diameter were cut from the lateral thorax 6-10). of donor mice with a biopsy punch. After In our studies on the spontaneous regres- removal of blood vessels and subcutaneous 287 0042~6822/78/0861-0287$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved
288
SHORT
COMMUNICATIONS
fat, donor skin was placed on prepared graft beds over the lateral thorax of the recipients. A graft was considered rejected when at least 50% of it had become necrotic (13). Each grafting experiment included control isografts, and alI grafts were exchanged with individuals of the same sex. SIM and SIM . R mice rejected all skin grafts made between them. Rejection occurred within 14 days after grafting. Isografts were uniformly accepted among both the SIM and SIM . R mice. The magnitude of the response was similar to that for transplants across a major histocompatibility barrier (13). Further analysis of histocompatibility differences was made in cytotoxicity tests using H-2 typing alloantisera (obtained from the Jackson Laboratory through the courtesy of the Transplantation Immunology Branch, National Institute of Allergy and Infectious Diseases). 51Cr-labeled target cells (5 x lo4 spleen or lymph node cells) were incubated with the antiserum and guinea pig complement (final dilution, 1:20) for 2 hr. Radioactivity released by the cells was determined following centrifugation and the specific cytotoxicity was calculated as
specific cytotoxicity was obtained with SIM . R cells at a 1:lOOO dilution of antiserum, while SIM cells did not react even at 1:lOO (not shown). The results of the cytotoxicity tests were confirmed by adsorption. All the cytotoxic activity in D-13 serum directed against SIM . R cells was removed by 10” homologous cells, while as many as 10’ SIM cells or 5 x lo6 F6 cells removed no activity. F6 is an oncornavirus-producing erythroblastic cell line derived in our laboratory from FVinfected SIM mice. This test was included because of previous reports demonstrating anomalous results obtained with anti-H-2 alloantisera due to anti-C-type virus activity in the sera (14). SIM mice which had been grafted with and rejected SIM ’ R skin possessed humoral cytotoxic activity against SIM . R cells. But in contrast to the D-13 serum, this SIM-anti-SIM . R antiserum had no activity against Balb/c cells. Furthermore, while Balb/c cells removed all D-13 activity against both SIM . R and Balb/c targets, adsorption with SIM . R cells removed only a maximum of 40% of the D-13 anti-Balb/c activity. These results indicate that all of the antigenic specificities recognized by D-13 serum on SIM . R cells are also present on Balb/c cells but that the converse is not true. Moreover, there are differences between SIM and SIM . R cells other than those detected by the D-13 serum. To determine the extent of the difference between SIM and SIM . R cells, we tested them in the two-way mixed lymphocyte reaction. In the mouse, lymphocytes from animals that differ in the I region of the major histocompatibility complex will react in this assay; differences in the D or K ends of the complex contribute to only a minor
a-bXlOO,
c-b
where a is the number of counts released in the presence of the test serum and complement, b is the number of counts released by complement alone, and c is the number of counts released by an unadsorbed rabbit anti-mouse thymocyte serum and complement (taken as maximum release). The results are shown in Table 1. We found that SIM and SIM. R mice differ markedly in their reaction with the D-13 serum. In titrations of D-13 activity, TABLE REACTIVITY
Antiserum
a Specifx
cytotoxicity
SIM
AND
SIM
recognized
Dilution
2, 56 13, 4, 41, 42, 43, 44 28, 36, 42
1:25 1:250 1:25
Specificities
D-2 D-13 D-28
OF
(percent).
R
1 CELLS
WITH
H-2
--hPINC
Mouse
SERA
strain
Balb/c
C57B1/6
SIM
S1M.R
8” 98 85
100 5 83
0 0 99
7 88 95
SHORT
289
COMMUNICATIONS
extent ( 15). Mixed lymphocyte cultures were prepared in microtiter plates using 2 x lo5 spleen cells of each type. Following incubation for 3 days, 1 PCi of [3H]thymidine was added to each well and the uptake of label was determined 18 hr later. We found that while there was a strong reaction in cultures of SIM or SIM . R cells with allogeneic Balb/c or C57B1/6 lymphocytes, there was no significant reaction in the mixtures of SIM and SIM R cells (data not shown). These results indicate that while the SIM and SIM R mice differ in antigens specified by the D end of the major histocompatibility complex (at least, as determined by the D-13 serum), they are the same at the closely linked I locus. We have considered three categories of explanations for these results: (i) SIM and SIM R mice are not coisogenic or congenic and differ at several (or many) genes including Fv-1 and H-2, which are in different linkage groups. This may be a trivial consequence of an insufficient number of backcrosses in generating the SIM R line, or it may be the result of phenomena such as quasi-linkage ( 16, 17) as has been reported for the association between the Gix antigen and H-2 and Fv-1 (although a specific association between H-2 and Fv-1 was not observed). It is of interest, however, that the added specificity detected on SIM R cells by the D-13 serum is not present on the cells of either parent strain (SIM and C57Bl/6); the introduction of genes from another strain of mice or mutation (18) may thus also be a necessary concurrence. (ii) SIM and SIM R mice may be congenic and differ only at the Fv-1 locus. The fact that SIM R also differs from SIM by possession of the C57B1/6 allele of Gpd-1 (19), which is closely linked to Fv-1, would support this possibility. An Fv-1 locus gene product (or a gene closely linked to Fv-1) could itself be responsible for the observed histoincompatibility. Alternatively, an H-2 allele causing the histoincompatibility may have become closely linked to Fv-1 in these mice as a result of translocation or mutation. (iii) Finally, it is possible that SIM and SIM R mice are congenic but that the difference in the two strains lies in a locus
other than that of Fv-1, which includes an Fv-l-like restriction effect on virus replication as well as a histocompatibility difference. The major histocompatibility complex of the mouse is known to possess these properties (20). Further, we have found that inoculation of SIM mice with the RFV strain of FV causes an erythroleukemia that spontaneously regresses, as we have reported for a number of other mouse strains ( 11, 21). But inoculation of the same virus into SIM R mice causes a chronic, lethal erythroleukemia. Studies of similar phenomena in other laboratories have implicated several genes as regulators of regression or recovery from FV-induced erythroleukemia, including H-2 (2U), Rfv-1 (22), and Rgv-1 (23), all of which map within the major histocompatibility complex of the mouse. The determination of the actual difference(s) between the SIM and SIM R strains awaits further genetic analysis. ACKNOWLEDGMENTS The authors thank Dr. A. Axelrad for providing the mice used in these experiments and helpful comments and Dr. M. A. Rich for his support and encouragement, This work was supported by Grant CA-14100 from the National Cancer Institute, an institutional grant to the Michigan Cancer Foundation from the United Foundation of Greater Detroit, and the Suzanne Korman Morton Cancer Research Laboratory. REFERENCES 1. LII,I.Y, F., and PINCXIS, T., Aduan. Cancer Res. 17, 231-277 (1973). 2. JOLICO~JR, P., and BALTIMOHE, D., Proc. Nat. Acad. Sci. USA 73, 22362240 (1976). 3. SVF,DA, M. M., and SOEIKO, R., Proc. Nat. Acad. Sci. USA 73,2356-2360 (1976). 4. AX~LHAI), A. A., WAHE, M., and VAN DEB GAAC:, H. C., in “RNA Viruses and Host Genomes in Oncogenesis” (P. Emmelot and P. Bentvelzen, eds.), pp. 239-254. North-Holland, Amsterdam. 1972. 5. WAHE, L. M., and AXF.I,I~AI), A.. Virology 50, 339-348 (1972). 6. Jor.~com:n, P., and BAI,TIMOHF:, D.. J. Viral. 16, 1593-1598 (1975). 7. SC~~IH, V., BLACKST~IN, M. E., and AXFX
290
SHORT
COMMUNICATIONS
ogy 74,252-255 (1976). M. A., BLACKSTEIN, M. E., and MCCARTER, J. A., Virology 79, 302-311 (1977). DIETZ, M., FOUCHEY, S. P., LANGLEY, C., RICH, M. A., and FURMANSKI, P., J. Exptl. Med. 145, 594406 ( 1977). AXELRAD, A., and STEEVES, R. A., Virology 24, 513-518 (1964). LEVEY, R. M., and MEDAWAR, P. B., Ann. N.Y. Acad. Sci. 129,164-177 (1966). KLEIN, P. A., J. Immunol. 115,1254-1260 (1975). SHREFFLER, D. C., and DAVID, C. S., Aduun. Zmmunol. 20,125-195 (1975). STOCKERT, E., BOYSE, E. A., SATO, H., and ITAK.
10. KOCHMAN, 11. 12. 13. 14. 15. 16.
17.
18. 19. 20.
21. 22. 23.
URA, K., Proc. Nat. Acad. Sci. USA 73, 2077-2081 (1976). BOYSE, E. A., Immunol. Rev. 33,125-145 (1977). KLEIN, J., and EGOROV, I. K., J. Immunol. 111, 976-979 (1973). ROWE, W. P., HUMPHREY, J. B., and LILY, F., J. Exptl. Med. 137,850-853 (1973). LILLY, F., J. Exptl. Med. 127.465-473 (1968). DIETZ, M., and RICH, M. A., Znt. J. Cancer 10, 99-104 (1972). CHESEBRO, B., WEHRLY, K., and STIMPFLINC, J., J. Exptl. Med. 140,1457-1467 (1974). DAWSON, P. J., and FIELDSTEEL, A. K., Cancer Res. 33,2456-2458 (1973).
86, 287-290
(1978)
SIM PHILIP
and SIM
FURMANSKI,
Department
of Biology,
. R Mice
Are Histoincompatible
MICHAEL DIETZ, JOHN MARCELLETTI Michigan Accepted
Cancer
DAVID
Foundation,
December
Detroit,
L. HINES,
Michigan
AND
48201
19, 1977
SIM and SIM. R mice were bred for congenicity at the Fv-I locus. difference in their permissiveness for infection by murk C-type viruses, to Fv-I, these mice differ in antigens determining bistocompatibility.
In addition presumably
to a due
The ability to infect mouse cells producsion of FV-induced leukemia, we found that tively with oncornaviruses is governed by a virus tropism and Fv-1 type are critical number of host genes. Preeminent among determinants in the outcome of the disease these is Fv-1, which was originally identi- (11). To analyze the effects further, we fied by its effect on Friend virus (FV) and initiated studies using these congenic mice. We found that in addition to a difference at is now known to be the host contribution to the phenomenon of virus tropism affect- Fv-1, the SIM @‘v-l, n/n) and SIM . R (Fving all of the murine leukemia viruses 1, b/b) mice are histoincompatible. SIM and SIM R mice were obtained (MuLVs) (1). Those MuLVs that are capable of replicating in mouse cells are iden- from colonies maintained at the Michigan tified as N-tropic or B-tropic depending on Cancer Foundation derived from breeding whether or not they can infect cells with stock originally provided by Dr. A. Axelrad. the Fv-1 genotypes n/n or b/b, respectively. Animals were also obtained directly from NB-tropic viruses replicate equally well in Dr. Axelrad and used for these experiments. either type of cell, as well as in cells with In accord with previous reports (4,5), we the n/b genotype, which are restrictive to found that the SIM mice were permissive both N- and B-tropic viruses. for N- and NB-tropic viruses and restricted Studies on the mechanism of the Fv-1 B-tropic viruses, while the SIM . R mice effect and virus tropism have generated were permissive for B- and NB-tropic viconsiderable interest as a model system for ruses and restricted N-tropic viruses. These the regulation of oncornavirus infection and results were obtained using a spleen focus expression. The data obtained to date in- assay with FVs of known tropism, as predicate that the effect is mediated at a step viously described (11, 12) and using the after virus penetration but prior to the in- same viruses in an in vitro XC plaque assay sertion of proviral DNA (2,3). on cells derived from SIM and SIM . R emA valuable resource in attempting to de- bryos or trypsinized adult kidneys ( II). The lineate the mechanism of gene effects is the Fv-1 n/n (SIM) restriction of the B-tropic availability of animals which differ only at virus and the restriction of the N-tropic the locus under study, that is, coisogenic, or virus by Fv-1 b/b (SIM . R) was greater more realistically, congenic animals. Axel- than lO&fold in each case in the animals rad and co-workers have developed strains used in these experiments. of mice which are congenic for Fv- 1 (4, 5) Histocompatibility was determined by and these animals, and cell lines derived skin graft reactions of the SIM and SIM R from them, have been used in several of the mice. Disks of full thickness skin 0.7 cm in studies of Fv-1 and virus tropism (2, 5, diameter were cut from the lateral thorax 6-10). of donor mice with a biopsy punch. After In our studies on the spontaneous regres- removal of blood vessels and subcutaneous 287 0042~6822/78/0861-0287$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved
288
SHORT
COMMUNICATIONS
fat, donor skin was placed on prepared graft beds over the lateral thorax of the recipients. A graft was considered rejected when at least 50% of it had become necrotic (13). Each grafting experiment included control isografts, and alI grafts were exchanged with individuals of the same sex. SIM and SIM . R mice rejected all skin grafts made between them. Rejection occurred within 14 days after grafting. Isografts were uniformly accepted among both the SIM and SIM . R mice. The magnitude of the response was similar to that for transplants across a major histocompatibility barrier (13). Further analysis of histocompatibility differences was made in cytotoxicity tests using H-2 typing alloantisera (obtained from the Jackson Laboratory through the courtesy of the Transplantation Immunology Branch, National Institute of Allergy and Infectious Diseases). 51Cr-labeled target cells (5 x lo4 spleen or lymph node cells) were incubated with the antiserum and guinea pig complement (final dilution, 1:20) for 2 hr. Radioactivity released by the cells was determined following centrifugation and the specific cytotoxicity was calculated as
specific cytotoxicity was obtained with SIM . R cells at a 1:lOOO dilution of antiserum, while SIM cells did not react even at 1:lOO (not shown). The results of the cytotoxicity tests were confirmed by adsorption. All the cytotoxic activity in D-13 serum directed against SIM . R cells was removed by 10” homologous cells, while as many as 10’ SIM cells or 5 x lo6 F6 cells removed no activity. F6 is an oncornavirus-producing erythroblastic cell line derived in our laboratory from FVinfected SIM mice. This test was included because of previous reports demonstrating anomalous results obtained with anti-H-2 alloantisera due to anti-C-type virus activity in the sera (14). SIM mice which had been grafted with and rejected SIM ’ R skin possessed humoral cytotoxic activity against SIM . R cells. But in contrast to the D-13 serum, this SIM-anti-SIM . R antiserum had no activity against Balb/c cells. Furthermore, while Balb/c cells removed all D-13 activity against both SIM . R and Balb/c targets, adsorption with SIM . R cells removed only a maximum of 40% of the D-13 anti-Balb/c activity. These results indicate that all of the antigenic specificities recognized by D-13 serum on SIM . R cells are also present on Balb/c cells but that the converse is not true. Moreover, there are differences between SIM and SIM . R cells other than those detected by the D-13 serum. To determine the extent of the difference between SIM and SIM . R cells, we tested them in the two-way mixed lymphocyte reaction. In the mouse, lymphocytes from animals that differ in the I region of the major histocompatibility complex will react in this assay; differences in the D or K ends of the complex contribute to only a minor
a-bXlOO,
c-b
where a is the number of counts released in the presence of the test serum and complement, b is the number of counts released by complement alone, and c is the number of counts released by an unadsorbed rabbit anti-mouse thymocyte serum and complement (taken as maximum release). The results are shown in Table 1. We found that SIM and SIM. R mice differ markedly in their reaction with the D-13 serum. In titrations of D-13 activity, TABLE REACTIVITY
Antiserum
a Specifx
cytotoxicity
SIM
AND
SIM
recognized
Dilution
2, 56 13, 4, 41, 42, 43, 44 28, 36, 42
1:25 1:250 1:25
Specificities
D-2 D-13 D-28
OF
(percent).
R
1 CELLS
WITH
H-2
--hPINC
Mouse
SERA
strain
Balb/c
C57B1/6
SIM
S1M.R
8” 98 85
100 5 83
0 0 99
7 88 95
SHORT
289
COMMUNICATIONS
extent ( 15). Mixed lymphocyte cultures were prepared in microtiter plates using 2 x lo5 spleen cells of each type. Following incubation for 3 days, 1 PCi of [3H]thymidine was added to each well and the uptake of label was determined 18 hr later. We found that while there was a strong reaction in cultures of SIM or SIM . R cells with allogeneic Balb/c or C57B1/6 lymphocytes, there was no significant reaction in the mixtures of SIM and SIM R cells (data not shown). These results indicate that while the SIM and SIM R mice differ in antigens specified by the D end of the major histocompatibility complex (at least, as determined by the D-13 serum), they are the same at the closely linked I locus. We have considered three categories of explanations for these results: (i) SIM and SIM R mice are not coisogenic or congenic and differ at several (or many) genes including Fv-1 and H-2, which are in different linkage groups. This may be a trivial consequence of an insufficient number of backcrosses in generating the SIM R line, or it may be the result of phenomena such as quasi-linkage ( 16, 17) as has been reported for the association between the Gix antigen and H-2 and Fv-1 (although a specific association between H-2 and Fv-1 was not observed). It is of interest, however, that the added specificity detected on SIM R cells by the D-13 serum is not present on the cells of either parent strain (SIM and C57Bl/6); the introduction of genes from another strain of mice or mutation (18) may thus also be a necessary concurrence. (ii) SIM and SIM R mice may be congenic and differ only at the Fv-1 locus. The fact that SIM R also differs from SIM by possession of the C57B1/6 allele of Gpd-1 (19), which is closely linked to Fv-1, would support this possibility. An Fv-1 locus gene product (or a gene closely linked to Fv-1) could itself be responsible for the observed histoincompatibility. Alternatively, an H-2 allele causing the histoincompatibility may have become closely linked to Fv-1 in these mice as a result of translocation or mutation. (iii) Finally, it is possible that SIM and SIM R mice are congenic but that the difference in the two strains lies in a locus
other than that of Fv-1, which includes an Fv-l-like restriction effect on virus replication as well as a histocompatibility difference. The major histocompatibility complex of the mouse is known to possess these properties (20). Further, we have found that inoculation of SIM mice with the RFV strain of FV causes an erythroleukemia that spontaneously regresses, as we have reported for a number of other mouse strains ( 11, 21). But inoculation of the same virus into SIM R mice causes a chronic, lethal erythroleukemia. Studies of similar phenomena in other laboratories have implicated several genes as regulators of regression or recovery from FV-induced erythroleukemia, including H-2 (2U), Rfv-1 (22), and Rgv-1 (23), all of which map within the major histocompatibility complex of the mouse. The determination of the actual difference(s) between the SIM and SIM R strains awaits further genetic analysis. ACKNOWLEDGMENTS The authors thank Dr. A. Axelrad for providing the mice used in these experiments and helpful comments and Dr. M. A. Rich for his support and encouragement, This work was supported by Grant CA-14100 from the National Cancer Institute, an institutional grant to the Michigan Cancer Foundation from the United Foundation of Greater Detroit, and the Suzanne Korman Morton Cancer Research Laboratory. REFERENCES 1. LII,I.Y, F., and PINCXIS, T., Aduan. Cancer Res. 17, 231-277 (1973). 2. JOLICO~JR, P., and BALTIMOHE, D., Proc. Nat. Acad. Sci. USA 73, 22362240 (1976). 3. SVF,DA, M. M., and SOEIKO, R., Proc. Nat. Acad. Sci. USA 73,2356-2360 (1976). 4. AX~LHAI), A. A., WAHE, M., and VAN DEB GAAC:, H. C., in “RNA Viruses and Host Genomes in Oncogenesis” (P. Emmelot and P. Bentvelzen, eds.), pp. 239-254. North-Holland, Amsterdam. 1972. 5. WAHE, L. M., and AXF.I,I~AI), A.. Virology 50, 339-348 (1972). 6. Jor.~com:n, P., and BAI,TIMOHF:, D.. J. Viral. 16, 1593-1598 (1975). 7. SC~~IH, V., BLACKST~IN, M. E., and AXFX
290
SHORT
COMMUNICATIONS
ogy 74,252-255 (1976). M. A., BLACKSTEIN, M. E., and MCCARTER, J. A., Virology 79, 302-311 (1977). DIETZ, M., FOUCHEY, S. P., LANGLEY, C., RICH, M. A., and FURMANSKI, P., J. Exptl. Med. 145, 594406 ( 1977). AXELRAD, A., and STEEVES, R. A., Virology 24, 513-518 (1964). LEVEY, R. M., and MEDAWAR, P. B., Ann. N.Y. Acad. Sci. 129,164-177 (1966). KLEIN, P. A., J. Immunol. 115,1254-1260 (1975). SHREFFLER, D. C., and DAVID, C. S., Aduun. Zmmunol. 20,125-195 (1975). STOCKERT, E., BOYSE, E. A., SATO, H., and ITAK.
10. KOCHMAN, 11. 12. 13. 14. 15. 16.
17.
18. 19. 20.
21. 22. 23.
URA, K., Proc. Nat. Acad. Sci. USA 73, 2077-2081 (1976). BOYSE, E. A., Immunol. Rev. 33,125-145 (1977). KLEIN, J., and EGOROV, I. K., J. Immunol. 111, 976-979 (1973). ROWE, W. P., HUMPHREY, J. B., and LILY, F., J. Exptl. Med. 137,850-853 (1973). LILLY, F., J. Exptl. Med. 127.465-473 (1968). DIETZ, M., and RICH, M. A., Znt. J. Cancer 10, 99-104 (1972). CHESEBRO, B., WEHRLY, K., and STIMPFLINC, J., J. Exptl. Med. 140,1457-1467 (1974). DAWSON, P. J., and FIELDSTEEL, A. K., Cancer Res. 33,2456-2458 (1973).