breakdown of friend virus-induced tolerance and...

[CANCER RESEARCH 34, 543 550, March 1974]

Breakdown of Friend Virus-induced Tolerance and Development ofRunting Syndrome in Rats1

Noritoshi Takeichi, Hiroshi Kaji, Takao Kodama, and Hiroshi Kobayashi

Cancer Insiliate, Hokkaido University School of Medicine, Sapporo, Japan

SUMMARY

The effects of the inoculation of lymphoid cells intosyngeneic rats made Friend virus (FV) tolerant by neonatalinjections of FV were studied. The inoculation of lymphoidcells from immune donors brought about the runtingsyndrome in five of seven recipients, while the inoculation oflymphoid cells from normal donors induced the runtingsyndrome in one of five recipients. The titer of FV in theblood decreased markedly by the inoculation of lymphoidcells, but immunological tolerance to Friend lymphomacells was not broken in the survivors. The repeated inoculation of lymphoid cells from immune rats induced the runtingsyndrome in all cases. The inoculation of immune lymphoidcells into young tolerant rats induced a low incidence of therunting syndrome and a high incidence of resistance toFriend lymphoma transplants. The major pathologicalfindings in the runted rats were atrophy of the thymus andenlargement of the spleen with a depletion of lymphocytes.The pathogenic mechanism of the runting syndrome in theFV-tolerant rats induced by inoculation of syngeneic lymphoid cells was discussed.

INTRODUCTION

Studies with murine leukemias induced by Gross (1),Moloney leukemia (8, 9), and Graffi (6) viruses havesuggested that newborn mice exposed to the leukemia viruswere more susceptible to a subsequent challenge withsyngeneic tumor transplants or were unable to producecirculating antibodies to the leukemia antigens. Therefore,they were considered to be immunologically specificallytolerant to the tumor-specific cellular and/or virion antigens.

Where immunological tolerance to FV2-induced trans

plantation antigen in rats is concerned, we have reportedthat neonatal injections of high doses of FV (IO4Â°to 10sÂ°MIDso/rat) always resulted in the development of a stateof complete tolerance in the rats, as judged by the persistence of a constant viremia and the lethal growth of Friend

1This work was supported in part by a research grant l'or cancer

research from Ministry of Education of Japan.2The abbreviations used are: FV. Friend virus; MIDSO, 50% minimal

infective doses; WFT-13, Friend virus-induced lymphoma in WKA/Mkrat.

Received August 14, 1973; accepted November 12, 1973.

lymphomas (10-14). These FV-tolerant rats developedsplenic lymphomas about 200 days after the neonatalinjection of FV (10).

The current study represents an attempt to determinewhether immunological tolerance in rats given neonatalinjections of FV can be broken by the inoculation ofimmunocompetent cells from syngeneic donors.

MATERIALS AND METHODS

Rats. An inbredstrain of male and female WKA/Mk ratswas used. These rats are the offspring of parents maintainedby consecutive brother-sister mating for more than 200generations at the Laboratory for the Breeding of Experimental Animals, Hokkaido University, Sapporo, Japan.

FV. The FV used has been serially maintained inDHS/Mk mice by the injection of spleen homogenates. TheFV was recovered from Friend-diseased spleens by themethod of Chenaille et ai. (5). Usually, 0.1 to 0.3 ml FVsuspension at a titer of IO5Â°to 10eÂ°MID50/ml was injectedi.p. and s.c. into newborn rats. The titer unit, expressed asMIDso/ml, is the dose at which 50% of the mice are positivefor the development of Friend disease as measured by thespleen focus assay method of Axelrad and Sleeves (2) and iscalculated according to the cumulative method of Reed andMuench.

Friend Lymphoma. Transplantable Friend lymphoma(WFT-13) was induced in WKA/Mk rats at 172 days afterthe neonatal injection of FV. This tumor grew well either inrats made FV tolerant by the neonatal injection of FV or inimmunologically depressed rats but failed to grow lethallyeven in syngeneic normal adult rats. Detailed characteristicsof Friend lymphomas have been reported elsewhere (10, 11,13, 14).

Serum. Serum was obtained from rats immunized repeatedly with WFT-13 cells by cardiac puncture under etheranesthesia. After the blood clotted at room temperature, theserum was separated by centrifugation at 3000 rpm for 30min.

Titration of FV in Blood. At different ages, 3 to 4 rats thatwere given neonatal injections of FV were bled by cardiacpuncture under ether anesthesia. The infectivity of the FVwas titrated by a focus spleen assay. Tenfold dilutions ofpooled plasma virus preparation in 0.5 M sodium citratebuffer, pH 6.75, were injected i.p. into groups of 5 mice.Seven days later, the mice were sacrificed, the spleens were

MARCH 1974 543

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N. Takeichi, H. Kaji, T. Kodama, and H. Kobayashi

fixed in Bouin's fluid, and the foci on the surface of each

spleen were counted with the naked eye. Virus titers wereexpressed as MID50/ml as mentioned above.

Cell Preparations. Donor rats for the injection of lymph-oid cells were killed by ether. Spleens and lymph nodes wereminced with fine scissors and gently homogenized with aloose-fitting glass TenBroeck homogenizes The crudespleen and lymph node cell suspensions were filteredthrough surgical gauze and were washed 3 times at 1000rpm for 5 min, after which 1 to 5 x IO8 cells/ml wereresuspended in Eagle's minimal essential medium. Cell

viability was determined by lack of permeability to trypanblue. All suspensions had 95% or more viable cells.

Autopsy and Microscopic Examination. For histolÃ³gica!examination, the spleen, thymus, lymph nodes, and othertissues were fixed in a 10% formalin solution and embeddedin paraffin, and 4-^m-thick sections were prepared. Hema-toxylin and eosin staining was used routinely for all sections.

RESULTS

Changes of Body Weight. Two months after neonatalinjections of high doses of FV, 43 FV-tolerant rats weredivided into 4 groups as follows. In Group 1, 7 rats wereinoculated twice at 5-day intervals with spleen and lymphnode cells (5 x IO7) obtained from rats preimmunizedrepeatedly with WFT-13 cells. In Group 2, 5 rats wereinoculated twice at 5-day intervals with spleen and lymphnode cells (5 x 10') obtained from normal rats. In Group 3,7 rats were inoculated 4 times at 4-day intervals with seraobtained from rats immunized with WFT-13 cells. In Group4, 7 rats were not inoculated as a control group.

Clinical symptoms and body weights were recorded inindividual rats of the 4 groups at different stages followingthe above inoculations. Six days after the 1st inoculation, allrats in the 1st group and 1 in the 2nd group showed acessation of body weight gain, diarrhea, ruffled hair,anemia, hunched posture, etc. Five of the 7 rats in the 1stgroup and 1of the 5 rats in the 2nd group died of the runtingsyndrome from 11 to 18days after the 1st inoculations. Thebody weight of the runted rats in the late stages was lessthan one-half of that of the control rats (Chart 1). Typicalrunting syndrome was not observed in the rats of the 3rdgroup although 2 of them showed a loss of body weight from12 to 18 days after the 1st inoculation with immune sera.

Titer of FV in the Blood. The induction of tolerance toFV-specific transplantation antigen in newborn rats mustdepend not only on the dose of the virus injected but alsoon the degree of virus multiplication. For determination ofthe titer of the virus in the rats of the 4 groups describedabove, all rats from each group were bled by cardiacpuncture at different ages, and the blood was pooledaccording to groups for the titration of the virus. Afterinitial inoculations, the virus titer curves in each group wererecorded in Chart 2. In the 1st group, the titer of the FVdecreased rapidly and could not be detected at 6 days. In the2nd group, the titer of the virus decreased from 6 days,reaching its minimal level with a titer of 10Â°5MID50/ml at

130- Immune lymphoid cells Normal lymphoid cells

I I

â€¢¿�M

Â«H

50-

0 6 12 18 24 0 6

Days after Inoculation

Non-treated

~i 1 r-12 18 24

I I I IÃ“ 6 12 Ã•8 24 ~0 6~


12 18 24

Chart I. Body weight curves of FV-tolerant rats receiving lymphoid cellsand sera.

24


Chart 2. The titer of plasma virus in FV-tolerant rats receivinglymphoid cells and sera.

12 days and thereafter gradually increased again. In the 3rdgroup, the titer of the virus decreased slowly and reached aplateau with a titer of IO17 MID50/ml at 6 days. In the

control group, the titer of the virus remained nearlyconstant at a titer of IO27 to IO29 MID50/ml.

Transplantation of WFT-13 Cells. For a determination ofwhether immunological tolerance to FV-specific transplan-

544 CANCER RESEARCH VOL. 34



Friend Tolerance and Runt ing Syndrome in Rats

tation antigen was abolished, the surviving rats from eachgroup received s.c. transplants of WFT-13 cells. The resultsare presented in Table 1. The 2 survivors in the 1st grouprejected the WFT-13 transplants but then died of the runtingsyndrome. Two of the 4 survivors in the 2nd group diedfrom the growth of WFT-13 cells, while the other 2 ratsresisted the WFT-13 challenge but thereafter succumbed tothe runting syndrome. All rats in the 3rd and 4th groupsdied from the growth of the WFT-13 transplants.

Effects of Repeated Inoculations of Lymphoid Cells. Inthe experiment described above, most of the FV-tolerantrats developed the runting syndrome after the inoculation oflymphoid cells from rats preimmunized with WFT-13 cells.Some of them resisted the WFT-13 challenge but subsequently developed the runting syndrome. Experiments werethen carried out to investigate the still remaining possibilitythat the repeated inoculation of lymphoid cells might completely break a tolerant state, thereby inhibiting the development of the runting syndrome. Nine FV-tolerant rats at 2months of age were inoculated i.p. 5 times at 3-day intervalswith 5 x IO7lymphoid cells per rat. As the result, all (9 of 9)

tolerant rats developed severe runting syndrome and died ata mean survival period of 20.0 days (Table 2).

Inoculation of Lymphoid Cells into Young FV-tolerantRats. Spleen and lymph node cells from syngeneic ratsimmunized with or without WFT-13 cells were inoculatedinto young FV-tolerant rats at 21 days after neonatalinjections of FV. The results are shown in Table 3. In theyoung tolerant rats inoculated with immune lymphoid cells(5 x IO7)twice at 5-day intervals, 3 of the 13 rats (23.1%)

died of the runting syndrome from 14 to 21 days after the1st inoculation. Nine of the 10 remaining rats rejected theWFT-13 transplants and survived, while 1 rat succumbed tothe runting syndrome after rejecting the WFT-13 transplants. The 9 survivors did not develop primary lymphomasuntil more than 300 days. One of the 8 (12.5%) youngtolerant rats inoculated with normal lymphoid cells developed the runting syndrome. Of the remaining 7 rats, 5 diedfrom the growth of WFT-13 cells and 2 developed therunting syndrome after rejecting WFT-13 transplants. Noneof the 7 young tolerant rats not inoculated developed the

Table 1The effect of inoculation of syngeneic lymphoid cells into FV-wleranl rats

Table 2The effects of repeated inoculation of syngeneic lymphoid cells into

F V-tolerant rats

SubstancesinoculatedImmune

lymphoid cells"

Normal lymphoid cellsImmune seraNoneIncidence

of runtingsyndrome5/7

(7I.OK1/5 (20.0)0/7 (0)0/7 (0)Transplantation

of WFT- 13cellsinsurvivors"0/2"

(2/2)'

2/4 (2/2)7/77/7

Â°WFT-13 cells (5 x 10') were transplanted s.c."Spleen and lymph node cells (5 x 10') were injected i.p. twice at an

interval of 5 days.' Numbers in parentheses, percentage of incidence." Number of rats that died of tumor growth/number of rats challenged

with WFT-13 cells.' Number of rats that developed the runting syndrome/number of rats

that rejected tumor.

Cells inoculated/No. ofcells5

x 10' (5 times)Routei.p.Incidence

of runtingsyndrome9/9Mean

survivaltime(days)20.0

Table 3Inoculation of syngeneic lymphoid cells from rats immunized with

WFT-13 into young FV-toleranl rats

Incidence of Transplantation Incidencethe runting of WFT-130 in of primary

Cells inoculated syndrome survivors tumor

Immunelymphoidcells"Normal

lymphoidcellsNone3/13

(23.1)<1/8

(12.5)0/7

(0)0/10"

(1/10)'5/7

(2/7)7/7

(0/7)0/9'

" WFT-13 cells (5 x IO7)were transplanted s.c. interval of 5 days." Spleen and lymph node cells (5 x 10') were injected Â¡.p.twice at an

interval of 5 days.' Numbers in parentheses, percentage of incidence." Number of rats that died of tumor growth/number of rats challenged

with WFT-13 cells.' Number of rats that developed the runting syndrome/number of rats

that rejected tumor.' Rats observed more than 300 days after infection.

runting syndrome, and all of them died from the growth ofWFT-13 transplants.

Pathological Findings in the Runted Rats. By macroscopicexamination of the runted rats, the most common findingswere atrophy of the thymus and enlargement of the spleen.However, the spleen appeared atrophie in severely runtedrats or in rats that survived for a relatively long period afterfirst exhibiting clinical symptoms. The degree of thymic atrophy varied individually, but many of the thymuses wereone-half normal size or less. In some of the severely runtedrats inoculated repeatedly with immune lymphoid cells, itwas not possible to identify any surviving thymic tissue atnecropsy. The size of lymph nodes varied from normal inmost rats to atrophie in some extremely runted rats. Theliver was anemic but normal in size. Under microscopic examination, the type of lesions observed in the thymus depended on the degree of the runting syndrome and thelength of survival after the initial inoculation. In general,the histological changes in the atrophie thymuses involvedboth their lymphoid component and epithelial framework.In the relatively long-surviving rats, which displayed mildsymptoms of wasting, thymic lesions accompanied by a depletion of lymphocytes were located at cortical areas; but inthe severely runted rats, individual lobules of the thymuswere markedly small and corticomedullary differentiationdisappeared (Figs. 2 and 3). In the cortex, thymocytes disappeared completely and the reticulum cells were altered byvacuolar degeneration (Fig. 4). In the medulla of thethymus, lymphocytes decreased and reticulum cells proliferated. The histological change of the spleen consisted of

MARCH 1974 545




atrophy of the white pulp with a marked depletion oflymphocytes and lymphoblasts in follicular areas. In mostof the runted rats, the follicles were replaced by proliferating histiocytes and reticulum cells. Megakaryocytes increased relative to other cell components in the red pulp(Fig. 5). The lymph nodes displayed a loss of lymphocytes,and germinal centers varied in size from extremely small toprominent (Fig. 6). The bone marrow of the runted ratsshowed a decrease of erythroid cells and a relative increaseof metamyelocytes. In other organs such as the kidney,lung, parotid gland, and testes, we could not discernhistological changes except for cell-inflammatory infiltrations in the lungs of some severely runted rats. Thesehistological changes of the lymphoid organs were verysimilar to the runting syndrome in the rats induced byneonatal injections of low doses of FV as reported previously (18).

DISCUSSION

Previously, we have reported that rats given neonatalinjections of low doses of FV showed a high incidence of therunting syndrome, while rats given neonatal injections ofhigh doses of FV grew normally as FV-tolerant rats (18).Histological and immunological studies of these runted ratshave suggested that the pathogenesis of the runting syndrome induced by neonatal injections of low doses of FVmight be ascribed to the specific immune reaction betweenimmunocompetent cells of the host and their own tissueswhich had acquired FV-specific transplantation antigen(17). The pathogenic mechanism might be considered to bethe same as that for rats rendered tolerant by the inoculation of syngeneic lymphoid cells. The inoculation of immunelymphoid cells into FV-tolerant rats brought about a highincidence of the runting syndrome, but the inoculation ofimmune sera did not induce the runting syndrome. This factindicates that the development of this syndrome is not basedon humoral immunity but on cell-mediated immunity in thehost. The incidence of the runting syndrome in tolerant ratsby the inoculation of specific immune lymphoid cells washigher than that in tolerant rats by the inoculation ofnormal lymphoid cells. Repeated inoculations of immunelymphoid cells brought about the runting syndrome in alltolerant rats. Furthermore, the clinical and histologicalfindings in these runted rats were almost identical to thoseof classical runt disease. The evidence of this experimentsuggests that an immune reaction similar to graft-veww.v-host reaction occurs between tolerant rats and syngeneiclymphoid cells. Graft-verms-host reaction is known to havedeveloped in rodents given neonatal injections of allogeneiclymphoid cells or in FI hybrid rodents receiving lymphoidcells of parental origin (4). In these models, the runt diseaseis believed to be produced by a specific reaction ofimmunocompetent donor cells recognizing the transplantation antigen of the host as foreign. The same is considered tohold true in tolerant rats receiving syngeneic lymphoid cells.In our experiments, inoculated lymphoid cells recognizedthe FV-specific transplantation antigen in the virus-infected

cells as foreign, and the consequent specific immunereaction results in the development of the runting syndrome.

A few reports have described the breakdown of immunological tolerance induced by viral infection. Volkert andHannover Larsen (19, 20) have shown that a viremic state inmice infected neonatally with lymphocytic choriomeningitisvirus could be terminated by the adoptive inoculation ofsyngeneic immune lymphoid cells. SpÃ¤rck and Volkert (16)have also demonstrated that the adoptive inoculation ofsyngeneic immune lymphoid cells could eliminate an onco-genic viremia and prevent the development of primarylymphomas in mice. In our studies, the inoculation ofsyngeneic immune lymphoid cells into tolerant rats couldterminate a viremia but could not break immunologicaltolerance to WFT-13 cells. Most of the rats developed therunting syndrome and died. On the other hand, the inoculation of immune lymphoid cells into young tolerant ratscould terminate a viremia and also abrogate immunologicaltolerance to WFT-13 cells. However, there were a fewyoung tolerant rats that produced FV-specific cytotoxicantibody and were resistant to WFT-13 transplants (17).Almost all of these rats developed the runting syndrome anddied within 2 months. This fact indicates that even rats givenhigh doses of FV neonatally do not develop completetolerance to WFT-13 cells in the early stages of infection.Therefore, the immunological tolerance in young tolerantrats might be broken easily by the inoculation of syngeneiclymphoid cells.

Finally, the cause of the runting syndrome in tolerant ratsby the inoculation of lymphoid cells may be due to extensivedamage to immature hematopoietic cells, which are considered to be the target cells of FV. If this is true, the additionalinoculation of syngeneic bone marrow cells may prevent thedevelopment of the runting syndrome and abolish completely the immunologically tolerant state.

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1. Axelrad, A. A. Antigenic Behavior of Lymphoma Cell Populations inMice to the Gross Leukemia Virus. Progr. Exptl. Tumor Res., 6:30 83, 1965.

2. Axelrad, A. A., and Sleeves, R. A. Assay for Friend Leukemia Virus:Rapid Quantitative Method Based on Enumeration of MacroscopicSpleen Foci in Mice. Virology, 24: 513 518. 1964.

3. Bianco, A. R., Glynn, J. P., and Goldin. A. Induction of Resistanceagainst the Transplantation of Leukemias Induced by Rauscher Virus.Cancer Res., 26: 1722 1728. 1966.

4. Billingham, R. E., Defendi, V., Silvers, W. K., and Steinmiller, D.Quantitative Studies on the Induction of Tolerance of Skin Homo-

grafts and Runt Disease in Neonatal Rats. J. Nati. Cancer Inst., 28:365-435, 1962.

5. Chenaille, D., Davy, J. P., Tavitian, A., and Bairon. M. RoutineMethod for Concentration and Partial Purification of Murine Leukemia Virus (Rauscher). Nature, 213 (5071): 107, 1967.

6. Chicco-Bianchi. L.. Fiore-Donati. L.. Tridente, G., and Pennelli, N.

Graffi Virus and Immunological Tolerance in Leukemogenesis. Nature, 214: 1227 1228, 1967.

7. Glynn, J. P., Bianco, A. R., and Goldin. A. Studies on InducedResistance against Isotransplants of Virus-induced Leukemias. Cancer

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8. Klein, E., and Klein, G. Antibody Response and Leukemia Development in Mice Inoculated Neonatally with Moloney Virus. Cancer

Res., 25: 851 854, 1965.9. Klein, E., and Klein, G. Immunological Tolerance of Neonatally

Infected Mice to the Moloney Leukemia Virus. Nature, 209: 163 165,

1966.10. Kobayashi, H., Hosokawa, M., Takeichi, N., Sendo, F., and Kodama,

T. Transplantable Friend Virus-induced Tumors in Rats. Cancer Res.,29: 1385-1392, 1969.

11. Kobayashi, H., Kuzumaki, N., Gotohda, E., Takeichi, N., Sendo, F.,Hosokawa, M., and Kodama, T. Specific Antigenicity of Tumors andImmunological Tolerance in the Rat Induced by Friend, Gross andRauscher Virus. Cancer Res., 33: 1598 1603, 1973.

12. Kobayashi, H., Sendo, F., Shirai, T., Kaji. H., Kodama. T.. and Saito,H. Modification in Growth of Transplantable Rat Tumors Exposed toFriend Virus. J. Nati. Cancer Inst., 42: 413 419, 1969.

13. Kobayashi. H., Shirai, T., Takeichi, N., Hosokawa, M., Sendo, F.,and Kodama, T. Antigenic Variant (WFT-2N) of a TransplantableRat Tumor Induced by Friend Virus. Rev. Europeene d'Etudes Clin.

Biol., 15: 426 428, 1970.14. Kuzumaki, N., Takeichi. N., Sendo, F., Kodama, T., and Kabayashi,

H. Correlation between Various Cell-Surface Antigens Induced by

Friend Tolerance and Runt ing Syndrome in Rats

Murine Leukemia Virus in the Rat. Intern. J. Cancer, //: 575 585,1973.

15. McCoy, J. L., Fefer, A., Ting, R. C., and Glynn. J. P. TheDevelopment of Specific Cellular and Humoral Immunity in MiceInfected with Rauscher Leukemia Virus as Neonates or Adults.Cancer Res., 32. 1671 1678,1972.

16. SpÃ¤rck, J. K., and VÃ¶lker!, M. Effect of Adoptive Immunity onExperimentally Induced Leukemia in Mice. Nature, 206: 578 579,1965.

17. Takeichi, N., Kuzumaki, N., and Kobayashi, H. ImmunologicalStudies of Runting Syndrome in Rats Inoculated with Friend Virus.Cancer Res., 33: 3096-3102, 1973.

18. Takeichi, N., Kuzumaki. N.. Kodama, T., Sendo, F., Hosokawa, M.,and Kobayashi. H. Runting Syndrome in Rats Inoculated with FriendVirus. Cancer Res., 32: 445 449, 1972.

19. Volkert, M. Studies on Immunological Tolerance to LCM Virus. II.Treatment of Virus Carrier Mice by Adoptive Immunization. ActaPathol. Microbiol. Scand., 57: 465 487, 1963.

20. Volkert, M., and Hannover Larsen, J. Studies on ImmunologicalTolerance to LCM Virus. III. Duration and Maximal Effect toAdoptive Immunization of Virus Carriers. Acta Pathol. Microbiol.Scand., 60: 577 587, 1964.

Fig. 1. Portion of the thymus from a 4-week-old FV-tolerant rat. Note the distinct arrangement of cortex which consists of numerous dark-staining

lymphocytes. The medulla is pale and shows loosely arranged reticuloendothelial cells and large lymphocytes. H & E, x 150.Fig. 2. Thymus from a runted rat at 8 days after the 1st transfer of immune lymphoid cells. Individual lobules of the thymus are markedly small, and

corticomedullary differentiation is lost. H & E, x 150.Fig. 3. High-power magnification of the thymus from the runted rat in Fig. 2. Note acute necrosis of thymic cortex with karyorrhexis and pyknosis of

lymphocytes. H & E, x 600.Fig. 4. Thymus from a runted rat at 18 days after the 1st inoculation of immune lymphoid cells. Thymic lymphocytes disappeared completely from the

cortex, and the reticulum cells in the cortex converted to vacuolar degeneration. H & E, x 150.Fig. 5. Spleen from the runted rat in Fig. 4. Note the follicles replaced by proliferating reticulum cells. Megakaryocytes are seen surrounding the

follicles. H & E, x 300.Fig. 6. Parathymic lymph node from the runted rat in Fig. 2. Note the marked depletion of lymphocytes in the cortical areas. H & E, x 300.

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Friend Tolerance and Runting Syndrome in Rats

MARCH 1974 549



1974;34:543-550. Cancer Res Noritoshi Takeichi, Hiroshi Kaji, Takao Kodama, et al. of Runting Syndrome in RatsBreakdown of Friend Virus-induced Tolerance and Development

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