induction of resistance against the transplantation of ...induction of resistance against the...

[CANCER RESEARCH 26 Part 1, 1722-1728, August 1966]

Induction of Resistance against the Transplantation of LeukemiasInduced by Rauscher Virus1

A. R. BIANCO,' J. P. GLYNN, AND A. GOLDIN

Cancer Chemotherapy National Service Center, National Cancer Institute, Bethesda, Maryland

Summary

A state of relative resistance against the transplantation ofisografts of leukemias induced by the Rauscher virus (RV) wasdeveloped in HALB/*' an(l CDFi mice by pretreatment withX-irradiated tumor cells. In mice pretreated with cells from1 tumor, significant cross-protection was obtained for all otherRV-induced tumors tested and for some tumors induced byMoloney virus, but not for leukemia LI 210. Although marginalresistance was observed on a few occasions, the inoculation ofinfectious RV was not an efficient method of inducing transplantation resistance in BALB/c and CDFi mice. In CDFi miceresistance to grafts of RV-induced tumors was accompanied byresistance to challenge with RV and the presence of circulatingRV neutralizing antibodies.

Introduction

Tumors induced by most of the known murine leukemogenicviruses have been reported to have components which areantigenic for syngeneic mice. One general feature of this anti-genicity which has been detected by a variety of technics is thatall or most of the tumors induced by a single virus seem topossess the same or antigenically related components.

The development of transplantation resistance and circulatingantibody demonstrable by either cytotoxicity or immuno-fluorescence, or both, have teen used to study these relationshipswith tumors induced by Graffi (12, 13), Gross (1, 9, 17), Moloney(6, 8, 15), Rauscher (5, 11) and Friend (10, 18) viruses. In thecurrent study, data are presented on the development of transplantation resistance to tumors induced by the Rauscher virus(RV) in mice highly susceptible to the oncogenic potential ofRV.

Materials and Methods

MICE.BALB/c and (BALB/c x DBA/2) FI (CD) male mice,8-12 weeks old, were obtained from the production colonies ofthe NIH.

TUMORS.The basic data on the transplantable neoplasms are

1Presented in part at the 55th Annual Meeting, American Association for Cancer Kesearch, Chicago, Illinois, April 9-11, 19(>4.

2Visiting Associate from Institute of Medical Pathology, University of Naples, Naples, Italy.

Received for publication November 8, 1965; revised February28, 19(i(i.

presented in Table 1. With the exception of the L1210 sublim-(19), the tumors were established from primary lymphaticneoplasms of animals that had been inoculated with eitherMoloney or Rauscher virus when less than 72 hr old. All thetumor stock are maintained by serial transplantation at weeklyor bi-weekly intervals.

TRANSPLANTATION.For purposes of quantitating transplantation resistance, counted numbers of tumor cells were injected s.c.in the right inguinal region. Usually 3-5 serial dilutions of tumorcells were used with each mouse receiving the appropriate cellconcentration in 0.1 ml. All of the transplants grew as localtumors at the site of inoculation and frequently metastasized toother regions. The median survival time of animals inoculatedwith the minimum cell concentration required to cause 100%death with tumor varied between 20-30 days. Spontaneousregression of the tumors in syngenic mice have not been observed.

IMMUNIZATION.Unless indicated, mice were given 2 injectionsi.p. of X-irradiated (6000-10,000 r) tumor cells 2 weeks apart.In individual experiments, the numbers of irradiated cellsinjected ranged from 2 to 7 X 10'/mouse. The challenge withviable tumor cells usually was given 2 weeks after the lastimmunizing injection.

VIRUS.The virus (14) was obtained from Dr. F. J. Rauscher.Four stocks, designated P-237, P-327, P-387, and P-641, wereused. They were all 10<;0extracts of infected BALB/c splenic

material. The titers expressed as IDM/ml of the individualpreparations were: P-237, IO4-4;P-327, IO5; P-387, IO6-2;andP-641, IO6-8.All virus dilutions were made in phosphate-buffered

saline (pH 7.2).X-IRRADIATION.Tumor cells were irradiated with 2 X-ray

tubes placed opposite each other, operated at 200 kvp and 15 ma.Filtration was 0.25 mm Cu and 0.55 mm Al. The target distancewas 25 cm and the dose rate was 520 r/min.

SKIN GRAFTING.Technic used was that of Billingham andMedawar (3). Circular disks of skin (9-10 mm in diameter) werecut from the dorsal region of donor and placed on a prepared bedon the lateral aspect of the thorax of recipients. Bandages wereremoved on the 9th day and the grafts were inspected at regularintervals.

Results

Although many of the transplantation experiments were clonein CDFi mice to provide some flexibility in the use of the availablevirus-induced tumors, transplantation resistance with RV-induced leukemias was demonstrable in wholly syngeneic sys-

1722 CANCER RESEARCH VOL. 26

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Resistance against Induced-Rauscher Virus

TABLE 1BASICDATAON THE TKANSPLANTABLENEOPLASMSUSED IN THEPKESENTSTUDY

DesignationMCDV-12RCL-1RCL-2MCDV-10LSTR-AMNACL-1MNACL-5MCAS-10L1210/C95Â«EtiologyÂ«RVRVRVRVMVMVMV6MVMCGenotypeBALB/cBALB/cBALB/c(BALB/c

xDBA/2)FiBALB/cBALB/cBALB/c(BALB/c

xDBA/2)FiDBA/2FormAsci

tesAscitesAscitesAscitesAscitesSolidSolidAscitesAscitesXo.

of transplantgenerations prior

touse3225275015222101195

" RV, Rauscher virus; MV, Moloney virus; MC, methylcholanthrene.6 Nucleic acid from MV.c Subline of L1210 resistant to therapy. The generation number refers to the number of passages

of this subline since its establishment; however, the parent tumor has been maintained by transplantation at weekly intervals for several years.

TABLE 2GROWTH OF MCDV-12 IN SYNGENEICMICE PRETREATED

WITH X-IRRADIATED MCDV-12 CELLS"

CHALLENGEINOCULUM(CEILS/MOUSE)1

XIO43.3XIO31.1XIO33.6XIO21.2X IO2NORMAL

MICETakes/total10/1010/108/102/108/10MST61416.51818PRETREATEDMICETakes/total1/100/100/101/100/10

" Three i.p. injections (2 weeks apart) of X-irradiated (10,000r) MCDV-12 cells (6.89 X 10', 5.86 X 10', and 5.35 X 10' cells/mouse, respectively). The mice were challenged with MCDV-121 week after the last pretreatment.

6MST, median survival time (days).

tems. The data from a typical experiment with MCDV-12 inBALB/c mice are presented in Table 2.

In several experiments, groups of CDFi mice immunize toMCDV-12 were challenged with a number of histocompatibleneoplasms. Summary data are presented in Table 3. Significanttransplantation resistance was obtained for the 4 RV-inducedtumors and for 1 (MCAS-10) of the 2 MV-induced tumors bypretreatment of CDFi mice with X-irradiated MCDV-12 cells.LSTRA and L1210 grew equally well in both control andMCDV-12 immunized animals.

The results of 2 transplantation experiments in which irradiated RCL-1 cells were used to immunize BALB/c (top portion)and CDFi (bottom portion) are shown in Table 4. In both experiments, marked inhibition of tumor growth was observed with theRV-induced tumors and, to a lesser extent, with the MV-inducedLSTRA. No transplantation resistance for the MV-inducedMCAS-10 was seen in CDFi mice pretreated with RCL-1 cells.

Some additional data on cross-protection are presented inTable 5. The growth of 2 MV-induced tumors was not inhibitedin animals pretreated with MCDV-12 cells, although in other

experiments these tumors were shown to contain the MV-inducedtransplantation antigens.

The possibility that the cross-protection observed in CDFianimals was a result of FI anti-parent effects is unlikely. CDFianimals pretreated with a concentration of X-irradiated BALB/cspleen cells approximately equivalent to the concentration ofX-irradiated MCDV-12 required for immunization failed toprotect the animals to subsequent inoculation with viableMCDV-12 cells (Chart 1). In addition, the results of BALB/cskin grafting and tumor transplantation in CDFi mice pretreated with X-irradiated MCDV-12 cells (Table 6) show that,although pretreatment conferred resistance against tumorchallenge, it did not influence the survival of BALB/c skingrafts. An additional injection of X-rayed MCDV-12 cells(5.44 X IO7cells/mouse) given at 73 days after skin grafting,was without effect. The skin grafts did not show any signs ofincompatibility (i.e., loss of hair or shrinkage) when observed formore than 150 days.

Although animals immunized with X-irradiated RV-inducedtumor cells did not develop the splenomegalic response usuallyassociated with RV infection, small amounts of infectious RVwere detectable in the various tumor lines either by extraction(F. J. Rauscher, personal communication) or by inoculation ofX-irradiated cell suspensions into newborn BALB/c mice. Thus,animals immunized with X-irradiated tumor cells are concomi-tantly infected with low concentrations of RV.

Several attempts were made to determine if infectious virusper se would elicit transplantation resistance in BALB/c orCDFi mice, particularly since in studies (6) with the Moloneyvirus in these strains of mice, transplantation resistance wasproduced regularly by X-irradiated tumor cells, but only sporadically by infectious virus.

In 1 experiment, groups of adult BALB/c mice inoculated i.p.with 1CT2dilution of RV Stock P-237, were challenged at either

2 or 4 weeks postvirus inoculation with 1 of several dilutions ofMCDV-12 cells (Table 7). As an indication of the infectivity of

the virus preparation, 2 weeks after virus inoculation the spleensof the infected mice were slightly but consistently enlarged, and

AUGUST 1966 1723



A. R. Bianco, J. P. Glynn, and A. Goldin

TABLE 3TRANSPLANTABILITYOF PARENTALANDFi NEOPLASMSIN (BALB/c x DBA/2)Fi HYBRIDMICE PRETKEATEDWITH

X-lRRADIATED MCDV-12 CELLS

NEOPLASMMCDV-12RCL-1RCL-2MCDV-1O-LSTRA-AMCAS-10L1210/C95TRANSPLANTGENERA

TION647983252755152171101195TUMOR

TAKES IX NORMAL MICEATCHALLENGEDOSE (CELLS XIO1/MOUSE)I.I10/108/104/100/103.39/102/108/109/109/105/108/109/107/87/81010/1010/108/1010/109/1010/1010/1010/108/88/83010/1010/1010/1010/1010/1010/1010/1010/108/88/89010/1010/1010/1010/108/8TUMOR

TAKES INPRETREATEDMICEAT CHALLENGEDOSE(CELLSXIO'/MOUSE)1.10/102/101/100/103.31/100/101/101/106/100/109/107/94/86/8102/100/100/108/104/100/109/109/102/87/8305/101/104/109/106/104/1010/1010/106/88/8901/107/109/109/107/8SUMTAKES/TOTAL(%)Untreated39/40

(97.5)32/40(80)36/40(90)37/40(92.5)32/40(80)35/40(87.5)28/40(70)39/40(97.5)31/32(97)23/24

(95.8)Pretreated8/40

(20)2/40(5)12/40(30)20/40(50)17/40(42.5)13/40(32.5)28/40(70)35/39(89.7)19/32(59)21/24

(87.5)X0'46.4243.0227.5515.6211.8522.970.0560.9111.060.27P<0.001<0.001<0.001<0.001<0.001<0.0010.70

< P <0.900.30< P <0.50<0.0010.50

< P < 0.70

Â«Cell levels used for this neoplasm were 5.5 to 16.5-50 to 150X IO3instead of 3.3 to 10-30 to 90 X IO3,respectively.

TABLE 4TRANSPLANTABILITYOF SEVERALRAUCHERVIRUS(RV) ANDMOLONEYVIRUS(MV) INDUCEDNEOPLASMS

IN HlSTOCOMPATIBLE MlCE PRETREATED WITH RCL-1 CELLS

NEOPLASMRCL-1MCDV-12LSTRARCL-1MCDV-10LSTRAMCAS-10TRANSPLANTGENERA

TION491031954792193106TUMOR

TAKES IN NORMALMICE(CELLSXIO'/MOUSE)0.125/85/80.367/88/80/71.18/87/84/810/103.38/88/88/810/108/108/10108,88/88/810/108/1010/108/10308/810/1010/1010/1010/109010/1010/1010/1027010/10TUMOR

TAKES IN PRETREATEDMICE(CELLSXIO'/MOUSE)0.121/82/80.361/82/81/81.10/84/80/84/103.31/86/84/82/102/105/10101/87/88/81/101/1010/109/10303/84/104/1010/108/10907/107/109/1027010/10SUMTAKES/TOTAL(%)Untreated39/40

(97.5)36/40(90)25/39(64.1)40/40

(100)36/40(90)38/40(95)38/40

(95)Pretreated6/40

(14)20/40(50)15/40(37.5)14/40

(35)H/40(35)29/40(72.5)36/40

(90)Xc-59.9513.394.5628.924.97.00.1P<0.001<0.0010.02

<P <0.05<0.001<0.001<0.01>0.1

TABLE 5TRANSPLANTABILITYOF 2 MV-INDUCEDTUMORSIN CDFi MICE PRETREATEDWITH

X-IRRADIATED MCDV-12 CELLS"

TUMORUSEDFORCHALLENGEMNACL-1MNACL-5TUMOR

TAKES IN CONTROL MICE AT CHALLENGE LEVEL (CELLS

XlO'/MOUSE)18/2060/10196/10Cells

XlO'/mouse0.44/826/8107/8TUMOR

TAKESINPRETREATEDMICEAT

CHALLENGELEVEL(CELLSXIO'/MOUSE)10/2064/10194/10Cells

XlO'/mouse0.42/827/8108/8SUM

TAKES/TOTAL(%)14/40

(35)8/40(20)17/24

(71)17/24 (71)xÂ»!1.510.1P0.20

< P <0.300.70

< P < 0.90

Â«Significant levels of rejection (P < 0.001) of MCDV-12 cells were obtained in each of the experiments.

at 4 weeks the spleens of the infected mice were conspicuouslyenlarged. No transplantation resistance was observed in theanimals challenged 2 weeks after virus inoculation, while marginalresistance was observed in mice challenged at 4 weeks after

infection with RV. In a more extensive experiment, no trans-plantation resistance was observed in adult BALB/c miceinoculated with 1 of 2 dilutions (10-1 or 10~4)of RV stock P-387.

The mice were infected at weekly intervals from 1 week to 10




Resistance against Induced-Rauscher Virus

EFFECT OF PRETREATMENT WITH X-IRRADIATED BALB/c SPLEEN CELLS OR X-IRRADIATED

TUMOR CELLS ON THE TRANSPLANTABILITY OF THE MCDV-12 LYMPHOMA IN

(BALB/c X D8A/21F. MICE (CDBA)

100

90

80

70

60

50

40

30

20

10

0

(DAYShMEDIAN SURVIVAL TIME

D Normal CDBA MiceM CDBA Mice Pretreoted With X-Irradiated BALB/c Spleen Cells

UCD8A Mice Pretrealeit With X-lrradioled MCDV-12 Cells

II3IH3ÃŒ 116X16.5)m

118.51

(22}

1201 (19.51

(Â¿U

I90 30 IO

MCDV-12 CELLS X IO33.3

CHART!. Mice immunized with X-irradiated (10,000 r) MCDV-12 received 6.9 X 10' and 7.2 X IO7 cells i.p. at 2-week intervals.Groups receiving X-irradiated (10,000 r) BALB/c spleen were given 1.3 X 10" and 6.6 X IO7cells at the same intervals. Mice in allgroups received viable MCDV-12 cells s.c. in the right inguinal region 2 weeks after the last pretreatment.

TABLE 6TRANSPLANTABILITYOF MCDV-12 AND OF BALB/c SKIN

(BALB/c x DBA/2)Fi HYBRID MICE PRETREATEDWITHX-IRRADIATED MCDV-12 CELLS

IN

TABLE 7TRANSPLANTABILITYOF MCDV-12 IN RAUSCHERVIRUS

(RV)-lNFECTED0 ISOLOGOUS MlCE

Kind ofgraftMCDV-12Â»

BALB/c skinTakes

in untreatedmice(%)38/40

(95)14/14 (100)Â»Takes

in pretreatedmice(%)12/40

(30)16/16 (100)6

Â«Inoculum varied from 3.3 X IO3to 9 X IO4cells/mouse.6 Day of last reading = 150 days after grafting.

weeks prior to tumor challenge, and all groups designated toreceive a certain level of tumor were inoculated with cells fromthe same suspension. At the time of tumor challenge, 4 mice fromeach of the infected groups were sacrificed and the spleen weightsrecorded. The degree of splenomegaly ranged from an averageweight of 1.9 gm in those infected 10 weeks prior to tumorchallenge to an average of 0.27 gm for those animals infected 1week prior to tumor challenge. The average spleen weight ofuninfected control mice was 0.15 gm.

The results of another attempt to demonstrate transplantationresistance with infectious RV is shown in Table 8. Groups ofCDFi mice infected with different dilutions of RV stock P-641(10~" through 10~9) were challenged 4 weeks later with 1 of 5

dilutions of an MCDV-10 tumor suspension. At the time of

tumor challenge appreciable splenic enlargement was observedonly in those animals infected with the IO"1 virus dilution.

Equivocal transplantation resistance was obtained only in thosegroups infected with the 10~3 dilution of RV; the LDÂ»expressed

CELLS/MOUSE4

XIO42

XIO41

XIO45

XIO32.5

X 10"KIND

OF PRE-TBEATMEXTRVNoneRVNoneRVNoneRVNoneRVNoneWH.

FOLLOWING VIÃ•USINOCULATION2Takes/total10/1010/106/109/100/100/100/100/10Msr151521.519.54Â»Takes/total10/1010/103/1010/104/96/100/103/104/104/10MST15141822

0 Each mouse was injected Â¡.p.with 0.2 ml of a 10~2dilution of

RV (P-237).6 For the 4-week set the x* = 6.37 and P < 0.025.c MST, median survival time (days).

as MCDV-10 cells/mouse was increased approximately 3-fold

over that of the controls.Since transplantation resistance was observed consistently

when animals were immunized with X-irradiated tumor cells,this type of system was used to check for cross-protection to aninoculation of infectious RV. CDFi mice immunized with X-irradiated MCDV-12 cells were challenged with RV and, at bi-weekly

AUGUST 1966 1725




intervals, groups of 7 animals were sacrificed for spleen weightassay. In a separate group of immunized mice challenged with2.4 X IO4MCDV-12 cells, only 1 of 10 (1/10) mice developed a

tumor. In contrast, all the control mice (10/10) developedtumors. The spleen weight of the RV-infected control animalsincreased progressively to an average of approximately 1.5 gm at10 weeks (Chart 2). The average spleen weight of RV-infectedimmunized mice did not increase progressively, but remainedonly slightly greater than the average spleen weights of thenormal and uninfeeted immunized mice during the entire experi-

TABLE 8TRANSPLANTABILITYOF MCDV-10 IN ISOLOGOUS(BALB/c x

DBA/2)F] HYBRIDMICE PRETKEATEDWITHDIFFERENTDOSESOF RAUSCHERVIRUS (RV)

TUMORCHAL-LENGE{CELLS/MOUSE)9

XIO43XIO41XIO13.3XIO31.1XIO3Sum%

positiveTUMOR

TAKESINUNTREATEDMICE8/88/87/82/105/1030/44G8LDsoÂ»

2640TUMOR

TAKES IX MICE PRETREATED WITH RVDOSE"18/88/85/83/90/1024/435659(5036/86/86/83/100/821/4250980058/88/87/86/91/1030/4370284078/88/86/84/101/927/4363420098/88/87/85/103/1031/447ÃŒ2530

0 Negative log of virus dilution.6MCDV-10 cells/mouse.

ment, indicating a strong and persistent inhibition of the earlysplenomegalic phase of the Rauscher disease.

The plasma collected from animals sacrificed for spleen weightassay in the above experiment (Chart 2) was bioassayed forinfectious RV in young BALB/c mice, using the splenomegalicresponse of the recipients as the indicator of virus activity. Theplasma from each group was pooled, diluted in buffered saline(pH 7.2), and each dilution was injected into 5 recipients whichwere sacrificed for spleen weight assay 28 days later. The resultsfrom an assay of the groups sacrificed 6 weeks after infection withRV (Chart 2) are shown in Chart 3. RV was not detectable in theplasma from the immunized animals. Similar assays at 8 and 10weeks after infection with RV (Chart 2) failed to detect circulating infectious virus.

The sera of CDFi mice immunized with X-irradiatedMCDV-12 cells were tested for their capacity to neutralize RV.Ten-fold dilutions of RV, stock P-641 were mixed with equalvolumes either of undiluted pooled immune sera or pooled normalsera, and the mixtures were kept at room temperature for 1 hrwith occasional shaking. A group of 5 BALB/c mice were inoculated i.p. with the serum-virus mixtures and sacrificed 4 weekslater for spleen weight assay. The results (Chart 4) showed thatserum from mice pretreated with MCDV-12 neutralized asignificant portion of the early splenomegalic response associatedwith RV infection.

Discussion

The results show that resistance can be developed against thetransplantation of RV-induced tumors in BALB/c and CDF

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CHART2. Inhibition of RV-induced splenomegaly in CDFi mice. Pretreated mice received 2 i.p. injections of X-irradiated MCDV-12cells, 2.4 X 10' and 7.2 X IO7cells/mouse, respectively, 2 weeks apart. Two weeks after the last injection, each mouse was inoculatedi.p. with 0.2 ml of a 10~' dilution of RV stock P-641. Appropriate groups of 7 mice each were sacrificed at the intervals indicated and

their spleen weights recorded.




Resistance against Induced-Rauschcr Virus

BALB/C mice inoculated with plasma from:

O Horma/ mice challenged with RVâ€¢Prelreated mice challenged with RVA Pretreated controls

Normal controls

1234NEGATIVE LOG OF PLASMA DILUTION

CHART3. Assay for infectious RV in plasma from animals sacrificed in experiment shown in Chart 2. Five 6-week-old BALB/c malemice were inoculated 0.1 ml/mouse i.p. with each of the dilutions of the pooled plasma. All recipients were sacrificed 4 weeks after inoculation and their spleen weights recorded.

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1234NEGATIVE LOG OF VIRUS DILUTION

mice by pretreatment with X-irradiated tumor cells. For thedemonstration of antigenicity it was necessary to graft withrather closely spaced dilutions of tumor cells. However, significant levels of cross-resistance were obtained for all the RV-induced tumors. Any attempt to relate the strength of theresponse obtained in this system with what has been observedwith tumors induced by either RV (11) or other leukemogenicagents (1, 6, 8, 9, 13), is compromised to a large extent bydifferences in the strains of mice in which the tumors wereinduced and the resistance developed. In our experience, RV- andMV-induced (6) tumors in BALB/c and CDFi mice are lessantigenic than tumors induced by these viruses in C57BL/6mice. With certain MV- and RV-induced tumors in C57BL/6, wehave observed regressions of subcutaneous transplants fromearly transplant generations similar to what has been reportedfor MV- and RV-induced tumors in C57BL/6 mice by Old et al.(11) and by the Kleins (8) for MV-induced tumors in theirstrains of mice. In our experience such regressions do not occurwith MV- and RV-induced tumors of BALB/c or CDFt mice.

RCL-2, a BALB/c tumor induced with RV which had beenpassaged twice in the rat, was indistinguishable with respect totransplantation resistance from tumors induced with RV ofmouse origin. Recently, Hartley et al. (7) have shown that sera

CHART 4. Neutralization of RV splenomegalic response. Serumwas collected 2 weeks after the last immunization from mice whichhad received 7.4 X 10' and 0.1 X 1C7irradiated (8000 r) MCDV-12cells 2 weeks apart. Each group of recipients contained five G-week-old BALB/c male mice, each of which was inoculated i.p. with0.2 ml of the appropriate mixture.

AUGUST 1966 1727




from rats bearing a transplantable RV-induced leukemia wouldfix complement with extracts from both rat and mouse tumorsinduced by RV.

Reproducible levels of transplantation resistance to RV-induced tumors were not obtained in BALB/c and CDFi miceby infection with RV. On a few occasions in an extensive series ofexperiments, marginal resistance was observed to isografts ofRV-induced tumors, in contrast to the regular appearance ofresistance following pretreatment with inactivated tumor cells.This apparent difference in the immunizing capacity of infectiousvirus vs. inactivated tumor cells need not necessarily be taken assupport for the existence of transplantation antigens on thetumor cells distinct from those of the virus. The ratio of virusantigens to infectious virus might be rather high in tumor cellsas compared to infectious virus preparations, and in strains ofmice as susceptible as BALB/c and CDFi are to the RV, immunizing concentrations of virus antigen may be achieved withinfectious RV only in the presence of a severe viremia and thusbe ineffective.

Transplantation resistance produced by pretreatment withX-irradiated cells was accompanied by resistance to challengewith infectious RV and the presence of circulating antibodiescapable of neutralizing RV similar to what had been reported byFink and Rauscher (5) using a virus vaccine. The resistance toRV occurred in animals pretreated with cells from MCDV-12,a poor source of infectious vims, suggesting that the immuneresponse was evoked primarily by the cells rather than by virusshedding from the cells. Barski and Youn (2) found that cellsfrom long term spleen-thymus monolayers chronically infected

with RV immunized BALB/c mice against challenge withinfectious RV. However, none of the data really preclude theexistence of tumor-specific cross-protective transplantationantigens induced by RV. If nonvirus-producing transplantablelines of virus-induced leukemias which did not elicit the production of antiviral antibody could be identified, then transplantation studies such as has been done with polyoma tumors (16)could provide some data as to whether the presence of virusantigen is a necessary prerequisite for the demonstration oftransplantation resistance.

In some but not all instances, significant cross-resistance forMV-induced tumors was observed in animals pretreated withRV-induced tumors. Whether the differences observed are due toqualitative or quantitative differences is not known. Old et al.(11), based on serologie studies, have postulated the existence ofrelated cellular antigens (FMR) in leukemias induced by Friend,Moloney, and Rauscher viruses. In a different system, Fink (4)found that BALB/c animals actively immunized with formalin-

ized RV vaccine were protected against challenge with Rauscherand Friend virus, but not against challenge with MV. Withrespect to transplantation resistance, it would seem that thequestion of cross-resistance should be studied by obtaining aseries of tumors in strains such as C57BL/6 where the tumorsseem to be highly antigenic.

References

1. Axelrad, A. A. Changes in Resistance to the Proliferation ofIsotransplanted Gross Virus-Induced Lymphoma Cells, asMeasured with a Spleen Colony Assay. Nature, 199: 80-83,1963.

2. Barski, G., and Youn, J. K. Immunization against RauscherMouse Leukemia with Tissue Culture Material. Science, 149:751-52, 1905.

3. Billingham, 11.S., and Medawar, P. B. The Technique of FreeSkin Grafting in Mammals. J. Exptl. Biol., 28: 385-402, 1951.

4. Fink, M. A. Some Immunological Cross-Reactions among theMurine Leukemia Viruses. Proc. Am. Assoc. Cancer Res., 5:18, 1964.

5. Fink, M. A., and Rauscher, F. J. Immune Reactions to a Murine Leukemia Virus. I Induction of Immunity to Infectionwith Virus in the Natural Host. J. Nati. Cancer Inst., 32:1075-82, 1964.

6. Glynn, J. P., Blanco, A. R., and Goldin, A. Studies on InducedResistance against Isotransplants of Virus-induced Leukemia.Cancer Res., %: 502-8, 1964.

7. Hartley, J. W., Rowe, W. P., Capps, W. I., and Heubner, R. J.Complement Fixation and Tissue Culture Assays for MouseLeukemia Viruses. Proc. Nati. Acad. Sci. U.S., 53:931-38, 1965.

8. Klein, E., and Klein, G. Antigenic Properties of LymphomasInduced by the Moloney Agent. J. Nati. Cancer Inst., SÃ©:547-68, 1964.

9. Klein, G., Sjogren, II. O., andKlein, E. Demonstration of HostResistance against Isotransplantation of Lymphomas Inducedby the Gross Agent. Cancer Res., 82: 955-61, 1962.

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15. Sachs, L. Transplantability of an X-ray-induced and a Yirus-induccd Leukemia in Isologous Mice Inoculated with a Leukemia Virus. Ibid., 29: 758-64, 1962.

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19. Venditti, J. M., Goldin, A., and Kline, I. Studies on the Effectiveness of 4',4"-Di-2-imidazolin-2-yl-terephalanilides againstMouse Leukemia L1210and Resistant Variants. Cancer Chem-otherap. Rept., 19: 5-11, 1962.

172S CANCER RESEARCH VOL. 26



1966;26:1722-1728. Cancer Res A. R. Bianco, J. P. Glynn and A. Goldin Leukemias Induced by Rauscher VirusInduction of Resistance against the Transplantation of

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