role of the mammary tumor virus in the immunogenicity of ... · responsiveness of the hosts1...

[CANCER RESEARCH 28, 1759-1772,September 1968]

Role of the Mammary Tumor Virus in the Immunogenicity of

Spontaneous Mammary Carcinomas of BALB/c Mice and in theResponsiveness of the Hosts1

Manouchehr Dezfulian,2 Tina Zee,3 K. B. DeOme, Phyllis B. Blair, and David W. Weiss

Department of Bacteriology and Immunology, and Cancer Research Genetics Laboratory, University of California, Berkeley, Cali

fornia 94720

SUMMARY

Immunization of young adult BALB/c mice, mammary tumorvirus (MTV)-negative, with preparations of normal lactatingmammary tissue from young primaparous females of the iso-genie BALB/cfCSH subline (MTV-infected) conferred significantly heightened reactivity against challenge with isograftsof recently arisen spontaneous BALB/cfC3H mammary carcinomas. Whole blood from BALB/cfC3H animals also appearedto be immunogenic in BALB/c hosts.

Normal, untreated young adult BALB/c animals offered considerably greater resistance to first-set mammary carcinomaisografts of BALB/cfC3H origin than did the infectedBALB/cfC3H hosts. The greater refractoriness of BALB/cmice was abolished by foster-nursing on a BALB/cfC3H mother.

The results of experiments with splenectomized animals wereconsistent with an immunologie interpretation of these fiindings.It was also possible to confer on normal BALB/c mice enhancedsusceptibility to the tumor isografts with sera from BALB/cmice immunized with BALB/cfC3H, but not with BALB/c,milk.

Neonatal infection with MTV did not reduce the ability ofmice of the BALB/c genotype to react against unrelated antigens or against three spontaneous or methylcholanthrene-induced mammary carcinomas of BALB/c origin and hence freeof the MTV.

It is apparent from these observations that MTV-infectednormal mammary tissue and whole blood possess one or moreof the tumor-associated antigens which are expressed on neo-plastic cells and that infection with the MTV at or before birthlowers or abrogates immunologie capacity specifically towardssuch antigen(s). The present results thus provide additional

1 Supported by Research Grants CA-05388 from the NIH,USPHS ; E-344 and E-292 from the American Cancer Society ; andby the Cancer Research Funds of the University of California.

2 This work is in partial fulfillment of the requirements for the

Ph.D. degree in Bacteriology and Immunology. Present address:Medical School, Pahlavi University, Shiraz, Iran.

3 This work is in partial fulfillment of the M.A. degree inBacteriology and Immunology. Present address: The UpjohnCompany, Kalamazoo, Michigan.

Received October 16, 1967; accepted May 7, 1968.

evidence that at least part of the unique antigenicity of spontaneous mammary tumors of mice is determined or controlledby the MTV.

INTRODUCTION

Recently reported studies from this (3, 4, 9, 19, 20, 45-49)and from other (25-29, 39) laboratories have shown that spontaneous mammary carcinomas of mice are, in fact, specificallyimmunogenic in animals of the strain of origin and also in theprimary autochthonous hosts. States of considerably heightenedresistance or susceptibility to autografts and isografts of thetumor can be induced by appropriate immunization with livingor irradiated tumor tissue, cell membrane concentrates preparedfrom tumor cells, and cell preparations of organs rich in re-ticuloendothelial elements derived from animals carrying thetumors. Both the heightened resistance and the heightened susceptibility to tumor challenge appear to be based on immunologie mechanisms. Moreover, immunization with outgrowth linesdescendant from the preneoplastic hyperplastic alveolar nodules(HAN) of mammary parenchyma increases reactivity againstlater implants of frank carcinomas originating from the sameor from other HAN cell lines.

Consideration of the data already available also suggestsstrongly that the mammary tumor virus (MTV) controls themanifestation of at least one strong immunogen or category ofimmunogens which characterize mouse mammary carcinoma.This impression is based on several repeated observations inanimals of the C3H genotype : Mice not infected with the MTV(C3Hf and C3H/2) are consistently more reactive to implantsof MTV-infected preneoplastic and neoplastic mammary tissuesthan are animals of the same genotype but infected at birth(or perhaps already in utero) with the agent (19, 20, 48, 49),presumably because the latter are specifically unresponsive toantigens experienced early in life and maintained thereafterin the tissues. This interpretation is strengthened by the recentobservation that untreated, MTV-infected mice usually do notmake precipitating antibodies against the MTV virion, whereasMTV-free subjects of the same genotype (C3H) frequentlydo so following a first contact with the virus in adult life (7).Neoplastic and HAN tissues elicit strongly heightened reactivityin MTV-free hosts against subsequent challenge with MTV-infected tumors if the immunizing implants are also infected

SEPTEMBER 1968 1759

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Manouchehr Dezjulian, Tina Zee, K. B. DeOme, Phyllis B. Blair, and David W. Weiss

with, but not if they lack, the MTV (9, 19). Neonatal infectionwith MTV abolishes much of the ability of animals from C3Hsublines which are free of the virus to develop resistance againstinfected isogenic implants of mammary tumors and nodulartissue (20). And, there is a strong cross-immunogenicity ofspontaneous C3H mammary carcinomas in the MTV-free, isogenic C3Hf and C3H/2 sublines (9).

At the same time, evidence has developed which points towards the existence of specific antigens in spontaneous mammary carcinomas of mice which are independent of the presenceor activities of the MTV. Thus Strain A mice are capable ofspecific acquisition of heightened resistance or immunologieenhancement towards tumor isografts despite the similar viralstatus of the immunizing and challenge tissues and the testanimals (4). Animals of the C3Hf subline can also develop suchheightened reactivity, seemingly of immunologie nature, againsta second experience with tumors equally free of the MTV andequally infected with a variant agent termed nodule-inducingvirus (NIV) (46, 47). These findings, which show acquiredantitumor reactivity on the part of mice of at least some linesdespite a presumed immunologie tolerance vis-a-vis virus-con

trolled antigens, suggested the presence of immunologie determinants unrelated to the virion or its activities in infected cells.On the other hand, the findings could be explained as resultingfrom a "breaking" of specific immunologie tolerance towards

virus-determined antigens (4).Further experiments were therefore conducted to provide

additional evidence for the presence of antigen(s) dependenton the MTV and for the existence of another class of antigen (s)independent of the agent.

The results of one avenue of investigation support the suspected occurrence of antigens specific to the neoplastic stateand unrelated to the oncogenic virus. A considerable portionof a large number of newly arisen spontaneous mammarytumors of MTV-infected C3H mice were found to be immuno-genic both in C3H and in C3Hf (i.e., MTV-free) hosts but tobe cross-reactive only in the latter. The immunogenic behaviorof these neoplasms was not altered appreciably by serial passage in isogenic animals for up to eight transplant generations(J. Vaage and D. W. Weiss, submitted for publication; J.Vaage, submitted for publication).4

The findings coming from a second experimental approachhave corroborated the existence and strong immunogenicityof MTV-dependent antigens. This study was an exploration ofthe immunogenicity of normal tissues infected with the MTVagainst isogenic tumors similarly infected with the virus andis the subject of the present communication.

Animals of the BALB/c genotype were chosen for these experiments, in part because the parent strain bred in our laboratory is free of both the MTV and the NIV, thus facilitating

4 It is proposed in the present and subsequent communicationsto reserve the term "tumor-specific" for those antigens which are,

indeed, limited to the neoplastic condition and which are not expressed by nonneoplastic cells despite infection with an oncogenicagent. The broader term "tumor-associated" will be employed to

designate antigens held by both neoplastic and preneoplastic cells,including those antigens which are possessed or induced by anoncogenic virus and are already present in normal tissue.

an analysis of the influence of both agents on the antigenicityof tissues, including mammary parenchyma of all three majorstages in the progression from normal to neoplastic, and in partbecause it seemed desirable to extend investigations of mammary tumor immunology to still another strain.

Studies are also reported herewith on the differential susceptibility to MTV-infected mammary tumor isografts of normal, unimmunized animals of the BALB/c strain and of miceof a reciprocally isogenic subline, BALB/cfC3H, infected withthe MTV as a result of foster nursing the female ancestor ona C3H mother.

MATERIALS AND METHODS

The general methodology of these experiments has been described in the series of preceding communications (3, 4, 19, 20,47).

Mice. Healthy male and female animals of the BALB/cCrgl(MTV-free, NIV-free) and BALB/cfC3HCrgl (MTV-infected,NIV-free (32)) sublines were employed. During the course ofthese experiments, the BALB/cfC3H subline had been removedfrom the parental BALB/c strain for 6-8 generations. Theisogenicity of each line and of the two lines vis-a-vis each otherwas demonstrated by the survival of second-set skin grafts performed between randomly chosen pairs of animals of the samesex throughout the course of the study.

In some experiments the animals were subjected to sple-nectomy. This was accomplished by exteriorizing the spleensof the nembutal-anesthetized subjects via a small lateral incision through the skin and abdominal wall and resecting theorgan after ligation of the blood vessels supplying it.

Tumors. Four spontaneous mammary carcinomas ofBALB/CÃ•C3H origin were employed, in most instances in the1st to 3rd transplant generations; in only two of the experiments reported here were tumors used after more than 3 isogenic passages. The tumors were always maintained in youngadult virgin females of the same line, with the one exceptionnoted in the text.

The BALB/cfC3H tumors, designated respectively BfSMT-1,BfSMT-2, BfSMT-3, and BfSMT-TZ, were all infected withthe MTV, and numerous virus-like particles usually identifiedas representing at least one form of this virus could be seen onelectron photomicrographs.

In addition, the following tumors were used: a mammarycarcinoma originating spontaneously in an old, multiparousBALB/c female, two mammary carcinomas which developedin transplanted outgrowth lines of hyperplastic alveolar nodulesarising in methylcholanthrene-treated BALB/c females, andtwo mammary carcinomas which appeared in BALB/c femalescarrying several implanted isogenic pituitaries. The incidenceof spontaneous mammary carcinomas in BALB/cCrgl mice isless than 0.1%. These MTV-negative tumors are described inmore detail in the text. The normal tissues of mice of theBALB/cCrgl line appear to be free of any virus-like particlescommonly associated with MTV or NIV, and such particlesare also not found in the spontaneous or in induced mammaryhyperplasias and neoplasias of these animals.

The carcinomatous nature of all the tumors was verified byhistologie examination.

1760 CANCER RESEARCH VOL. 28



Mouse Immunogenicity

Calculations of tumor volumes are derived from the equationdescribed in a preceding report (4).

Immunizing Tissue Preparations. The immunizing preparations of normal lactating mammary glands and of mixtures ofspleen and thymus tissue were made in an identical manner,as described fully in another communication from this laboratory (6). The tissues were minced finely with scissors and razorblade and homogenized with isotonic saline in a Waring or Vir-Tis blendor with a crushed ice jacket. The resulting homog-enates were centrifugecl at low speed and the precipitatesdiscarded. The supernates were now centrifuged at high speedin a Spinco centrifuge (at approx. 18,000 rpm) for 90 minutes;these supernates were discarded and the pellets resuspendedin saline. The pellet-suspensions were then centrifuged oncemore at low speed and the precipitates discarded. The super-nates of this last centrifugation constituted the desired materialand are referred to as "high-speed centrifugation pellet preparations." The preparations were kept frozen in the freezing

compartment of a commercial refrigerator.When made from lactating mammary glands of MTV-infected

mice, these preparations are rich in MTV activity and containthe B-particles believed to represent the mature virion.

The tissue donors were young (approx. 3 months old) BALB/cand BALB/cfCSH mice in lactation after first pregnancy;there are very few, if any, preneoplastic structures in the mammary glands of such animals, and it can be assumed that theproportion of any preneoplastic tissue components which mighthave been present in the high-speed pellet preparations of thesemammary glands was minute.

Statistical Methods. The tests for significance of the resultshave also been described previously (4). No finding will betermed "significant" unless the P value of the respective com

parison is 0.05 or less.

RESULTS

Titration of a BALB/cfC3H Mammary Carcinoma inBALB/c Mice. In most of the experiments conducted in theBALB/c-BALB/cfC3H system, tumor challenge was with suspensions of tumor cells obtained by mechanical disruption offreshly resected neoplasms. It is necessary in most immunologiestudies to guard against overwhelming challenges so as not toobscure a definite but limited response. Accordingly, many ofthe tumors employed here were subjected to titration (in the1st or 2nd transplant generation) to ascertain the relationshipsbetween number of living cells (i.e., cells alive at the time ofimplantation, as determined by vital staining) and appearanceof tumors at the sites of implantation in normal, young adulthost animals. The results of one such estimation of the response of BALB/c hosts to graded numbers of cells of aBALB/cfC3H spontaneous mammary tumor, representativeof the findings with most of the mammary neoplasms investigated in this strain, are shown in Table 1.

It is apparent from the results of this introductory experiment that there exists, in fact, a direct relationship betweenthe numbers of viable tumor cells in the implantation inoculumand the frequency and rapidity of appearance of palpable tumors in the hosts. [It is not unlikely that a larger number of

Table 1

No.oflivingtumorcells

ininoculum5X10*1X1055X10Â«IX

10Â«No.

ofanimals12121212No.

ofanimalswithtumors

attermination241112Mean

timeofappear

anceofpalpabletumors(days)33352716Moan

ofcalculatedtumorvolumes

attermination(cu

mm)9806601,3003,200

Relationship between numbers of living cells in a dispersed neo-plastic isograft and the development of challenge tumors innormal hosts. The suspension of neoplastic cells was preparedfrom a freshly-excised spontaneous BALB/cfC3H mammary carcinoma, designated BfSMT-TZ, in the 1st transplant generation.The cells were suspended in Morgan #199 tissue culture fluid;viable cell counts were made after trypan blue staining immediately before implantation. The inoculum dilutions were injected in 0.1-ml quantities subcutaneously into the lower rightquadrant. The host animals were young adult BALB/c males (3months old). The animals were observed for tumor development,and their tumors were measured for 10 weeks after challenge.

animals would have been found to have tumors had the periodof observation been continued for several months (L. J.Faulkin, Jr. and D. W. Weiss, unpublished observations)]. Ithas been reported elsewhere (46) that variations in the size ofsolid tumor fragments did not significantly affect the incidenceor rate of development of tumors appearing in isogenic StrainA/Crgl hosts. It seems likely that the dose effect observedhere reflects the much greater sensitivity of grafts of dispersedrather than of solid neoplastic tissues to host responses; and,it is also probable that the numbers of living cells containedin solid grafts vary considerably and not necessarily in directrelation to the mass of the graft.

Antitumor Immunogenicity of a Normal Mammary TissuePreparation Infected with MTV. Groups of young adultBALB/c virgin females (10-11 weeks old) were given seriesof injections of high-speed centrifugation pellet preparationsof normal, lactating mammary glands taken from BALB/c orfrom BALB/cfC3H females. The tissue donors were young(12-14 weeks old) primaparous animals. The tissue preparations were administered intraperitoneally in 0.1-ml volumes ina diluent of isotonic saline; the quantities of tissue concentrate in each aliquot are expressed as gram equivalents ofstarting mammary tissue (GEMT) and are indicated in Table2. Several weeks after the last treatment the animals werechallenged with an inoculum of living tumor cells and thenobserved for several weeks or months for the appearance oftumors. The challenge tumor was a mammary adenocarcinoma(designated BfSMT-1), which had arisen spontaneously in amultiparous BALB/cfC3H female, and was here employed inthe 1st and 2nd transplant generations.

Three such experiments were conducted. The details of theexperimental protocols and the results are presented in Table2. It is apparent that immunization with the concentrate ofnormal mammary tissue derived from MTV-infected donorssignificantly reduced the incidence of tumors which arose fromthe challenge inocula; 15 of 42 mice treated with the MTV+

SEPTEMBER 1968 1761



Manouchehr Dezfulian, Tina Zee, K. B. DeOme, Phyllis B. Blair, and David W. Weiss

Table 2

ExperimentNo.1Immunization

with apreparaScheduleof

injections (i.p.) oftest animals

(10- to 11-week-oldBALB/cfemales)6

injections of 0.03 GEMT each, onfollowing days before tumor implantation: 131, 128, 125, 62,59,and

56.tion

of normal mammarytissue"Tissue

donor strain(12- to 14-week-old

primaparousfemales)BALB/cfC3H

(MTV+)

BALB/c (MTV-)No.

ofSRBC test

included in animalspreparation*(BALB/c)+

10

+ 10No.

ofanimalswith

palpabletumors

at termination"5

10calculated

tumorvolumes

at termination*1(cumm)480

480

Same as in Experiment 1

4 injections, on following days beforetumor challenge: 55, 0.012 GEMT;52, 0.025 GEMT; 49, 0.04 GEMT;and 21, 0.06 GEMT.

Total

BALB/cfC3H (MTV+)BALB/c (MTV-)

BALB/cfC3H (MTV+)

BALB/c (MTV-)

BALB/cfC3H (MTV+)BALB/c (MTV-)

1010

22

22

4242

58

5

16

15 (35.7%)34 (80.9%)

2,300630

1,520

320

Antitumor immunogenicity of a normal lactating mammary tissue preparation infected with the mammary tumor virus (MTV).SRBC, sheep red blood cells; GEMT, gm equivalent of mammary tissue.

Â«Seetext for details of the high-speed centrifugation pellet mammary tissue preparation.<>Each injection inoculum contained 20% recently drawn sheep RBC."Challenge was made with a spontaneous mammary carcinoma, BfSMT-1, obtained from a multiparous BALB/cfC3H female; the

1st and 2nd transplant generations were used. Challenge was with 4 X IO4 living tumor cells implanted subcutaneously into the leftinguinal area. The animals in Experiments 1 and 2 were observed for 78 days after challenge, those in Experiment 3 for 35 days.

tf In Experiments 1 and 2, the calculations of the tumor volumes given are based on measurements taken 63 days after challenge,the last time at which the tumors were measured; in Experiment 3, the calculations are based on values obtained 33 days afterchallenge.

tissue preparation developed progressively growing tumors,compared to 34 of 42 animals given the uninfectcd mammarypreparation (P < 0.01). It was also noted in 2 of the 3 experiments that those tumors which did develop in animals immunized with MTV+ tissue were considerably larger at theend of the observation period than were the neoplasms in thegroup treated with the MTV~ preparation. This observation

resembles previously recorded findings in C3Hf mice whichhad been immunized with living preneoplastic mammary tissue(19). In one of the present experiments in which tumors appearing in MTV-treated animals did not develop more rapidlythan those in parallel animals given the MTV-free concentrate,the tissue vaccines were administered together with a suspension of fresh sheep RBC. (The sheep RBC were introducedinto the vaccine in this experiment because of recent reportsthat the response to unrelated antigens can be potentiated ormodified by the simultaneous administration of red blood cells(13). The present observation thus supports a modulating effect by sheep RBC on the reactivity of animals challenged withimmunologically heterogeneous substances). Opposite effectsaccruing in different animals of the same inbred strain fromidentical treatment with tumor-specific or tumor-associatedantigens have been reported previously (46, 47), and it wassuggested that the immune response to such antigens couldresult in either heightened resistance or in immunologie enhancement, depending on whether cellular or certain humoralcomponents of the response predominate vis-a-vis a givennumber of challenge cells. It was also found that immunologieenhancement could be elicited predictably in mice by certain

schedules of tumor immunization (4). It thus appeared likelythat the more rapid development of challenge tumors seen herein some of the animals immunized with the MTV-containingnormal mammary tissue preparation resulted, in fact, from theevocation of a state of specific immunologie enhancement. Totest this possibility, a passive transfer experiment was performed employing MTV+ milk rather than a normal tissuepreparation for immunization so as to further reduce the likelihood of contamination of the vaccine by any virus-independentpreneoplastic or neoplastic cell antigens.

Enhancement of Isografts of a Spontaneous BALB/cfC3HMammary Carcinoma in BALB/c Hosts Pretreated with Immune Serum. Twelve-week-old BALB/c virgin females weregiven three intraperitoneal injections, 1 week apart, of 0.3-mlquantities each of pooled milk derived from lactating BALB/cor BALB/cfC3H donors. Four weeks after the last immunizinginjection, the animals were bled and the sera from each groupwere pooled. Normal, virgin BALB/c females, 4 or 10 weeksold, were administered a single intraperitoneal injection of oneor the other of the pooled sera; the younger animals received0.1-ml quantities, the older ones 0.15-ml amounts. Eight hourslater, the mice were challenged subcutaneously in the left inguinal area with 1 X IO5 or 4 X IO5 living tumor cells; thechallenge tumor, designated BfSMT-3, was in the 2nd transplant generation. The test animals were observed for tumordevelopment for 4 weeks after challenge.

Mice of both age groups developed larger tumors within theobservation period of 4 weeks if they were given serum fromisogenic donors immunized with MTV-infected milk prior to




Mouse Immuno genicity

tumor challenge (Table 3). The difference in tumor size between these animals and the mice pretreated with serum fromdonors immunized with MTV-negative milk was statisticallysignificant (P < 0.05). The ability of immune serum to conferheightened tumor susceptibility (i.e., to further the development of the neoplastic challenge cells) presumably places thisphenomenon into the category of immunologie enhancement. Itcannot be concluded, however, that the responsible serum fac-tor(s) were immunoglobulins directed only at virion antigensin the immunizing milk. It is not impossible that the milk suspension also contained immunogenically significant quantitiesof tissue fragments, carrying antigenic determinants coded forby the virion, or that living MTV in the milk actively inducedsuch antigens in the serum donors. [It has already been learnedfrom other experiments, however, that formalinin-activated,purified MTV B-particle preparations can evoke immune responses against tumor isografts (D. Burton, P. B. Blair, andD. W. Weiss, to be published.)]

The results shown in Table 3 also point to the much greaterability of the tumor implants to grow in the 4-week-old thanin the 10-week-old isogenic hosts, despite the larger tumorchallenge given the latter (P < 0.05). This observation is inaccord with previous findings pointing to host age as a significant determinant of tumor growth rate (3, 46) and may wellreflect the immunologie immaturity of mice aged only 3-5 weeks(2, 21, 43).

An experiment was now designed to determine whether othernormal tissues from MTV-infected mice of the BALB/c geno

type can immunize against subsequent challenge with anMTV+ mammary tumor.

Determination of Antitumor Immunogenicity of Preparations of Red Blood Cells, Spleen and Thymus Tissue, andLactating Mammary Gland from Normal Animals Infectedand Uninfected with MTV. Groups of young adult BALB/cmales (9-11 weeks old) were given a series of intraperitonealinjections of 0.1-ml quantities of one of the following: highspeed centrifugation pellet preparations made either from lac-tating glands derived from young, primaparous donors or froma mixture of splenic and thymic tissue obtained from the sameanimals; or, whole, undiluted blood drawn from the tail veinsof 7- to 8-month-oId females and collected in heparin to prevent coagulation. The spleen-thymus preparation consisted of 1

part thymus to 8 parts spleen in the case of BALB/c tissues,and 1 part thymus to 3 parts spleen for the BALB/cfC3Hpreparations. The following schedule of injections was employed: 1st injection, 0.012 gram equivalents of tissue (GET) ;2nd injection, 3 days later, 0.025 GET; 3rd injection, 3 daysthereafter, 0.04 GET; and 4th injection, 30 days after the 3rd,0.06 GET. All the treated animals, plus a group of normal,untreated BALB/c males of the same age were challenged 30days later with a single subcutaneous injection, in the leftinguinal area, of 4 X 10Â°living tumor cells. The tumor designated BfSMT-TZ was a spontaneous BALB/cfC3H mammarycarcinoma obtained from a multiparous female; the 1st and2nd transplant generations were used. The animals were observed for 76 days after challenge, and the incidence of developing tumors was recorded (Table 4).

All but one of the 12 untreated control animals developedprogressively growing tumors from the challenge inocula within6 weeks after implantation. None of the groups treated withany of the tissue concentrates, regardless of the viral status ofthe donor animals, showed quite as high a tumor incidence. [Asmall "nonspecific" protective effect against tumor challenge

has also been observed by other investigators for mammaliantissue preparations (18)]. Infection with the MTV was againfound to confer a high degree of antitumor immunogenicity onnormal lactating gland. Only 2 of 24 animals immunized withthe MTV+ mammary preparation developed challenge tumors within the period of observation, compared to 8 of 12animals which had received the MTV~ concentrate (P < 0.01).

In contrast, the spleen and thymus mixture from MTV-infecteddonors was no more effective than the similar tissue from micefree of the virus.

A significant degree of immunogenicity was also manifestedby the whole-blood vaccine obtained from MTV+ hosts. Tumors in animals so immunized arose somewhat later and developed more slowly than in hosts injected with blood fromMTV- donors. It has been demonstrated that the red cell

fraction of the blood of MTV-infected mice carries MTV biologic activity (33), but it is not yet clear whether this activityreflects the presence of an infective MTV entity distinct fromthe B-particle commonly identified as the complete MTV virion,as postulated by Nandi (31), or whether it is caused by thepresence of very small numbers of highly infective B-particles.

Table 3

Source of milkforimmunizationoftheBALB/c

serumdonorsBALB/cfCSH

(MTV+)"BALB/c(MTV-)BALB/cfCSH(MTV+)BALB/c

(MTV-)Age

ofBALB/ctestanimals(weeks)441010No.

oflivingtumorcellsinchallengeinoculum0IX

IO5IX1054X1054X

105No.

oftestanimals5555No.

ofanimalswithpalpabletumors


ofevenly

bisectingtumordiameters

attermination5(mm)19x2513x206x

95x5Mean

ofcalculatedtumorvolumes

attermination(cu

mm)3,6001,30013050

Development of isografts of a spontaneous BALB/cfCSH mammary carcinoma in BALB/c hosts pretreated with sera from BALB/cdonors immunized with BALB/c and BALB/cfCSH milk. See text for experimental details.

a Tumor BfSMT-3 in the 2nd transplant generation.6 Experiment terminated 4 weeks after challenge.c MTV, mammary tumor virus.

SEPTEMBER 1968 1763



Manouchehr Dezfulian, Tina Zee, K. B. DeOme, Phyllis B. Blair, and David W. TFeiss

Table 4

Immunization of BALB/cmale test animals with

preparations of the followingtissues derived from

BALB/cfCSH or BALB/cdunorsDonor

tissue:BALB/cfC3HWholebloodSpleen

+thymusLactatingmammaryglandDonor

tissue :BALB/cWholebloodSpleen

-j-thymusLactatingmammaryglandUntreated

controlsNo.

oftest

animals12242412121212Cumulative

No. of palpable tumorsin test animals at following times

after challenge implantation of4 X 105 living cells of a BALB/cfC3H

spontaneous mammary carcinoma0(days)1702000002013003212325013252739034283131015548353101755938310285510423102858114931128681156311286811633112868116951328681176513286811%

withtumors at

termination41.654.28.375.050.075.091.6Mean

time ofappearanceof palpable

tumors(days)422634283324Mean

ofcalculated

tumorvolumes

at termination (cumm)3501,7001,4001,9001,1001,700

Antitumor immunogenicity of preparations of several normal tissues derived from normal BALB/cfC3H donors (MTV + ). MTV,mammary tumor virus. See text for experimental details.

0 Tumor BfSMT-TZ, in the 2nd transplant generation. In this instance, the tumor was passaged once in BALB/c males.

In either event a degree of immunogenicity in the blood of infected mice is not an unexpected finding, nor is the much lowerefficacy of such preparations than the one displayed by lactat-ing mammary glands, which are known to contain substantialnumbers of discrete viral particles and which constitute thespecific target tissue of this agent.

Experiments were conducted to determine whether normalmice of BALB/c genotype behave as those of the C3H varietydo (19, 20), i.e., whether animals infected with MTV frombirth (BALB/cfCSH) are less able to offer resistance to thedevelopment of MTV-infected neoplastic isografts than MTV-free BALB/c's.

Development of Isografts of Spontaneous BALB/cfC3HMammary Carcinomas in BALB/c and BALB/cfC3H Hosts.Groups of 16-week-old BALB/c and BALB/cfC3H virgin females were immunized intraperitoneally with 0.1-ml quantitiesof a high-speed centrifugation pellet preparation of lactatingmammary glands taken from young, primaparous BALB/cfC3Hdonors. The following schedule of injections was employed: 1st

injection, 0.015 GEMT; 2nd injection, 2 days later, 0.02GEMT; 3rd injection, 4 days after the 2nd, 0.03 GEMT; andthe 4th injection, 2 weeks after the 3rd, 0.04 GEMT. Groupsof other animals of the same age remained untreated. All micewere challenged 5 weeks after the last immunization of thetest animals with either 2 X IO4 or 7 X IO4 living cells derived

from a spontaneous BALB/cfC3H mammary carcinoma, designated BfSMT-3, in the 1st transplant generation; the challengeinoculum was placed subcutaneously in the left inguinal region.The animals were observed periodically for 12 weeks afterchallenge for tumor development at the sites of implantation(Table 5).

A categoric difference in the responsiveness to isografts ofthe same MTV-containing mammary tumor was evident between young adult BALB/c and BALB/cfC3H hosts. Eight of16 of the pretreated BALB/cfC3H mice developed progressively growing tumors within the 12-week observation period,while only 1 of 21 similarly pretreated BALB/c animals did(P < 0.01). The difference in tumor incidence between the un-

Table 5

Tumorhosts(24-week-oldfemales)BALB/cfCSH

(MTV+)BALB/c(MTV-)BALB/cfCSH(MTV+)BALB/c

(MTV-)ImmunizationwithMTV+mammarytissuepreparation(BALB/cfCSH)+-fâ€”â€”No.


ofanimalswithpalpabletumorsat

termination08190Mean

ofevenlybisectingtumordiameters6(mm)10x126x

7Mean

ofcalculatedtumorvolumes6(cu

mm)480100

Development of isografts of a spontaneous BALB/cfCSH mammary carcinoma in immunized and normal, young adult BALB/c and BALB/cfC3H hosts. See text for experimentaldetails. MTV, mammary tumor virus.

Â«Twelve weeks after challenge implantation. Half of the animals in each group werechallenged with 2 X IO4, the other half with 7 X IO4 living tumor cells derived from mammarycarcinoma BfSMT-3 in the 1st transplant generation; the data presented are pooled for thetwo challenge-size subgroups in each instance.

6Determination 10 weeks after challenge implantation.




treated BALB/cfC3H and the BALB/c hosts was of the samemagnitudo: 9 of 17 of the former, compared with none of 20of t lie latter, supported tumor growth (P < 0.01). An immunizing effect of pretreatment with the MTV-infected mammarytissue preparation could not be detected in this experiment because the challenge inoculum proved insufficient to initiate progressively growing tumors in even the untreated BALB/c animals. Immunization was ineffective in reducing tumor incidencein the BALB/cfC3H mice, but it did lead to a significan* acceleration of tumor development in these animals (P < 0.05).Whether this enhancement of tumor growth arose from immunologie events, such as the activation of a predominantlyhumoral antibody response against MTV-associated antigensdespite the neo- or prenatal infection of the animals with thevirus, was not determined.

Further experiments were conducted to explore the differential ability of normal, untreated BALB/c and BALB/cfC3Hhosts to support the development of isografts of spontaneousBALB/cfC3H mammary carcinomas.

Groups of BALB/c and BALB/cfC3H normal, virgin femaleswere given implants of varying numbers (ranging from 5 X IO3


to 2.5 X IO5) of living cells derived from either of two spontaneous BALB/cfC3H mammary carcinomas, BfSMT-1 orBfSMT-2. The tumor inocula were placed subcutaneously into

the left inguinal area. Mice ranging in age from 3 to 25 weekswere employed. The animals were observed for from severalweeks to several months after challenge for the developmentof tumors at the sites of implantation. Four distinct experiments were conducted; the results are presented together inTable 6.

In all but one of the eight comparisons made of the relativeabilities of normal, unimmunized BALB/c and BALB/cfC3Hhosts to support the growth of the neoplastic isografts, theBALB/cfC3H animals provided a better milieu for tumor development, as indicated by the considerably larger size of theneoplasms at termination, and, occasionally, by a higher incidence of successful takes of the grafts. The one exception inwhich BALB/cfC3H and BALB/c mice offered equal supportto the neoplastic isografts was seen in very young (3-week-old) hosts (Experiment 4). In that experiment, 11-week-oldBALB/c's offered very strong resistance to the tumor cells. This

finding points again to age maturity as a requisite condition for

Table 6

ExperimentNo.i234Tumor hostsBALB/cfC3HBALB/cBALB/cfC3HBALB/cBALB/cfCSHBALB/eBALB/cfCSHBALB/cBALB/cfCSHBALB/cBALB/cfCSHBALB/cBALB/cfCSHBALB/cBALB/cfC3HBALB/cBALB/c(MTV+)(MTV-)(MTV+)(MTV-)(MTV+)(MTV-)(MTV+)(MTV-)(MTV+)(MTV-)(MTV+)(MTV-)(MTV+)(MTV-)(MTV+)(MTV-)(MTV-)Age

ofhosts(weeks)191977141425251515151515153311No.

ofchallenge

tumorcellsÂ«2.52.5111111551.5IM1J1.5555XXXXXXXXXXXXXXXXXIOÂ»10Â»10Â«10Â»IOÂ«10510Â»10Â«10Â«10810Â«10*10Â»10Â»IOÂ«IOS10Â»No.

ofanimals91012121212149666666181818No.

ofanimals

withpalpabletumors at

termination9Â»1012Â«1211121394Â»1426614"134Mean

of calculated tumorvolumes at intervals after

challenge implantation (cumm)1,700Â»40030a101553015400Â«101,200100800/40<y2,600Â»3,200250440Â«16030070500100

Differential susceptibility of normal BALB/c and BALB/cfCSH mice of different ages to isografts of spontaneous BALB/cfCSHmammary carcinomas. MTV, mammary tumor virus.

Â«The following spontaneous BALB/cfCSH mammary carcinomas were employed in these studies: Experiment 1: BfSMT-1, 2ndtransplant generation; Experiment 2: BfSMT-1, 8th transplant generation; Experiment 3: BfSMT-2, 1st transplant generation;Experiment 4: BfSMT-1, 16th transplant generation.

6 Five weeks after challenge implantation.Â«Nineteen days after challenge implantation.d Twelve days after challenge implantation, i.e., in this experiment, tumors were measured shortly after appearance as well as at

termination.e Eight weeks after challenge implantation./ Five weeJcs after implantation ; tumors could not be measured accurately after the 5th week because they were very large,

necrotic, and ulcerated."Throe months after challenge implantation.* Nine weeks after challenge; tumors could not be measured accurately thereafter because of their large size, necrosis, and ulcÃ©ration.

SEPTEMBER 1968 1765




the effective development of specific tumor resistance. The relatively small tumor inocula which were used in this experiment,5 X 10s living cells, gave rise to very rapidly growing neo-plastic masses in most of the 3-week-old hosts. Physiologic factors peculiar to very young mice as well as their immunologieimmaturity could account for this event, but it might also havebeen the consequence of employing a tumor which had beenpassaged repeatedly (16th transplant generation): Selection ofimmunoresistant variants is a possible consequence of repeatedsojourns of populations of cells in immunologically hostileenvironments.

In Experiment 3 in which animals were given graded numbers of tumor cells, it was evident that a state of appreciableresistance can be masked to a considerable extent by a one-logincrease in challenge dose; this is a commonly observed eventin tissue transplantation experiments, especially where the tissues are neoplastic in nature (18).

These findings thus confirm in mice of BALB/c genotypethe observations made previously with C3H animals, that infection with MTV at or before birth markedly reduces theability of the animal to impede the development of isogenicneoplasms in adult life. The question still remained, however,whether this reduced resistance to tumor isografts accrues froma specific immunologie unrcsponsiveness or whether it arisesfrom a general impairment of immunologie function causedsomehow by the viral infection or from some physiologic alteration in host-tissue environment favoring later neoplasticgrowth. In one of the latter events, the greater susceptibilityto MTV-infected tumor isografts of mice exposed early in lifeto MTV could obviously not be taken as evidence in itself forthe MTV-associated antigenicity of the protective antigen(s).Experiments were therefore conducted to explore the relativeimmunologie capacity of BALB/c and BALB/cfC3H animalsto respond immunologically to unrelated antigens and to mammary tumors which appear to have arisen without the agencyof the MTV or any other known viral agent.

Four-month-old BALB/c and BALB/cfC3H virgin femaleswere given a series of immunizing injections of a mixture ofsheep red blood cells and bovine serum albumin intraperito-neally. No differences were seen in the titers of circulating antibodies measured by direct and indirect hemagglutination testsat intervals after the termination of immunization; both groupsof animals responded well to the antigens.

Experiments were initiated to ascertain the relative abilityof several MTV-free mammary carcinomas of BALB/c mice todevelop in these and in BALB/cfC3H hosts.

Differential Development of Isografts of Mammary Carcinomas of BALB/c Mice Free of the MTV in BALB/c andBALB/cfC3H Hosts. The following mammary tumors wereemployed in these experiments: two tumors which had arisen intransplanted nodular outgrowths derived from female BALB/cmice fed methylcholanthrene and designated MIMT-1 andMIMT-2 respectively (these tumors were kindly made available by Dr. L. J. Faulkin, Jr., of the University of Californiain Davis) and a mammary carcinoma designated BSMT-

MD16D which appeared spontaneously in an old, multiparousBALB/c female. The tumors were employed in the 1st and 2ndtransplant generations; their carcinomatous nature was con

firmed by histologie examination. Electron microscopic examination carried out by Dr. Dorothy Pitelka of our laboratoryfailed to reveal structures resembling viral particles associatedwith the MTV ; it has also been found that several other spontaneous BALB/c mammary carcinomas fail to induce in rabbitsantibodies detectable in the immunodiffusion test for MTV (P.B. Blair, unpublished observations).

Groups of BALB/c and BALB/CÃ•C3Hnormal, virgin femaleswere given single implants of suspensions of living cells derivedfrom these tumors subcutaneously in the left inguinal area. Theanimals were observed periodically after implantation for thedevelopment of tumors at the challenge sites.

The results of these experiments (Table 7) show that theyoung adult BALB/c and BALB/cfC3H hosts behaved verysimilarly towards isografts of the three BALB/c mammary carcinomas apparently free of the MTV; in no instance was therea significant difference in the incidence or rapidity of development of the tumors. The two neoplasms which arose in hyper-plastic alveolar mammary tissue from animals treated withmethylcholanthrene can be presumed to have been specificallyantigenic in view of the tumor-specific antigenicity commonlyfound to distinguish neoplasms induced by this carcinogen (17,35, 37). It is not yet known whether the third tumor, whicharose spontaneously in a BALB/c female, possessed specificantigens; experiments are in progress along this line. Recentfindings in our laboratory already suggest, however, that spontaneous mammary carcinomas of mice do possess antigens notheld by normal mammary tissue and which are not determinedby the presence or activities of the MTV (J. Vaage and D. W.Weiss, to be published).

Two further experiments were initiated for assaying the relative susceptibility of BALB/c and BALB/cfC3H animals toisografts of two mammary carcinomas (in the 2nd transplantgeneration) which had arisen in MTV-free BALB/c femalescarrying several whole isogenic pituitary implants. In one instance, the tumor incidences were again identical in the hostsof both BALB/c lines, but the neoplastic implants developedin the MTV-infected BALB/cfC3H animals at a slightly butsignificantly more rapid rate. In the other experiment, therewere no differences in the rates of tumor development when theneoplastic implants were able to initiate growth, but a significantly larger proportion of young adult (4-month-old) BALB/cmice than of the BALB/cfC3H animals prevented entirelythe appearance of palpable tumors from the implanted inocula. These findings cannot, however, be taken to suggestthat the seemingly greater susceptibility of the MTV-infectedBALB/cfC3H hosts to some pituitary-''induced" mammary

carcinomas reflects a more general depression of immunologiecapacity by the MTV. At least one of these tumors (the otherwas lost before testing was completed) failed entirely to initiategrowth in 90% of immunologically immature 3-week-oldBALB/c animals, a behavior opposite to that expected of antigenic implants if immunologie considerations are the only, orthe predominant, ones governing the fate of the grafts. As hasbeen shown in this and in preceding communications, spontaneous MTV-infected mammary carcinomas commonly developmore rapidly in 3-week-old than in young adult, immunologically mature hosts. It may well be that the anomalous behavior





Table 7

Experi- Tumor employedment No. (of BALB/corigin)123MIMT-1,

from nodular outgrowthofanimal

fed3-meth-ylcholanthrene.MIMT-2,

from nodular outgrowthofanimal

fed3-meth-ylcholanthrene.BSMT-MD16D,arisen

spontaneously.Tumor

hostsBALB/cfC3HBALB/cBALB/cfCSHBALB/cBALB/cfC3HBALB/cBALB/cfCSHBALB/c(MTV+)(MTV-)(MTV+)(MTV-)(MTV+)(MTV-)(MTV+)(MTV-)Age

ofhosts

(weeks)18181818991212No.

ofchallenge

tumorcells2.52.52.52.55522XXXXXXXX10Â»10Â»10Â«10Â«10*10*10Â»105No.


ofanimals

withpalpabletumors


of calculatedtumor volumes at

intervals afterchallenge implantation

(cumm)90"7050Â«50SO/60300"350190*220115"200400Â»480

Differential susceptibility of BALB/c and BALB/cfCSH mice to isografts of mammary carcinomas arisen in BALB/c females free ofthe MTV, mammary tumor virus.

Â»Sixteen weeks after challenge implantation.6 Eighteen weeks after challenge implantation. The tumor incidence data cited in the table are also based on tumor incidence at this

time.rSix weeks after challenge implantation.d Eleven weeks after challenge implantation. Tumor measurements were taken at shorter intervals after challenge in the animals

implanted with 2.5 X 10Â°tumor cells because of the more rapid development of the isografts. The tumor incidence data for theseanimals are based on the 11-week findings.

e Five weeks after challenge. The tumor incidence data, however, are based on the numbers of tumors seen 8 weeks after challenge./Fourteen weeks after challenge."Eighteen weeks after challenge. The tumor incidence figures are based on the 18-week interval.

of the tumors from pituitary-stimulated animals accrued froma special susceptibility to hormonal stimuli, as is often the casewith neoplasms induced by hormonal manipulations of theprimary autochthonous host (1, 34). Infection with MTV hasbeen shown to affect the hormonal reactivity of mammarytissue of mice (5, 8, 14, 41) and perhaps also other nonimmu-nologic functions of the host (11, 15, 16, 38, 40, 44), and thegreater susceptibility of BALB/cfC3H animals to implants ofpituitary-induced mammary tumors may thus be based onalterations in hormonal or other physiologic, rather than inimmunologie, parameters.

The Effect of Splenectomy on the Resistance of BALB/cMice to BALB/cfC3H Mammary Carcinomas. Two other experiments were conducted to provide additional support for theimmunologie nature of the heightened resistance conferred onBALB/c mice by immunization with normal mammary tissueinfected with the MTV, and for the greater refractoriness ofnormal, unimmunized animals of the BALB/c than of theBALB/cfCSH line to a first experience with MTV-infectedmammary tumor cells. In these experiments, the response ofmice of both sublines to infected tumor cells, with and withouta previous immunizing experience with infected normal mammary tissue, was tested in animals previously subjected to sple-nectomy. An immunologie basis for the different behavior ofnormal MTV-infected and MTV-free animals and for the immunizing potential of infected normal tissues would be in keeping with either an abrogation of the resistance effects followingsplenectomy or with an increased tumor resistance. The former

eventuality is frequently seen in resistance states based on immunologie mechanisms and would be the less surprising event.It has also been demonstrated, however, that splenectomy sometimes heightens resistance to foreign tissues, presumably because the reduction in the levels of circulating antibodies whichoften accompanies removal of the spleen might vitiate or lessenan active immunologie enhancement of the antigenic challengecells (23, 36).

In the first of these experiments, 15-week-old virgin BALB/cfemales were subjected to splenectomy. Two weeks later, whenthe animals appeared to have recovered fully from the effectsof the surgery, they were given single subcutaneous implants,in the left inguinal area, of 2 X IO4living cells derived from thespontaneous BALB/cfC3H mammary carcinoma BfSMT-3, inthe 2nd transplant generation. This number of tumor cells waschosen as representing a challenge dose sufficient to initiateprogressively growing tumors in approximately half the normalBALB/c hosts, i.e., a circumstance favoring the detection ofan appreciable lowering of active immunologie enhancement.For control purposes, groups of normal BALB/c females andnormal and splenectomized BALB/cfC3H females were challenged at the same time.

In the second experiment, groups of virgin BALB/c femaleswere immunized with a high-speed centrifugation pellet preparation of either BALB/cfCSH or BALB/c lactating mammarytissue. Immunization was begun 2 weeks after splenectomy, whenthe animals were 10 weeks old; the vaccination schedule andthe tissue preparation used were identical to those described

SEPTEMBER 1968 1767




for Experiment 3 presented in Table 2. The animals werechallenged at 19 weeks of age with a subcutaneous implant inthe left inguinal region oÃ4 X 10* living cells derived fromBALB/cfC3H spontaneous mammary carcinoma BfSMT-1, inthe 3rd transplant generation.

The animals in both experiments were observed periodicallyfor the development of tumors at the sites of implantation. Itis seen from the results of Experiment 1 (Table 8) that a largerproportion of nonimmunized BALB/cfC3H than BALB/c hostspermitted progressive growth of the implanted tumor cells, thusindicating once more the greater refractoriness of the latteranimals to MTV-infected neoplastic isografts. There was inthis experiment no appreciable difference, however, in the meansize of the tumors which did develop from the implants in thenonsplenectomized mice of the two sublines. In contrast, sple-nectomy appeared to reduce the size of the tumors which developed in the BALB/c animals; in the BALB/cfC3H subline,removal of the spleen appeared to have no significant effect ontumor development. This observation is thus compatible withthe suggestion that some degree of active enhancement normallytakes place in BALB/c animals against isogenic tumors anti-genie by reason of their MTV status.

In Experiment 2 (Table 8), splenectomized BALB/c micewere found to be highly susceptible to the larger challenge(4 X IO4 living cells of spontaneous BALB/cfC3H mammarycarcinoma BfSMT-1), and equally so whether they had beenimmunized with BALB/cfC3H or with BALB/c mammarypreparations. This finding is in marked contrast to the high degree of protection which was elicited in nonsplenectomizedyoung adult BALB/c females by identical immunization withan MTV-infected mammary preparation against an equal num

ber of living cells of the same tumor (Experiment 3, Table 2).It is very improbable that the failure of the MTV-infected normal tissue vaccine in the present instance can be ascribed to thefact that tumor BfSMT-1 was here used in the 3rd transplantgeneration, whereas it was employed in the 2nd generation inthe earlier experiment. Investigations of the effects of serialtransplantation on both the tumor-specific and tumor-associatedantigenicity of spontaneous mammary carcinomas failed to provide any evidence for the loss of immunogenicity for at least8 transplant generations (J. Vaage and D. W. Weiss, to bepublished) [a finding in support of the belief that tumor-specific antigens may represent entities indispensable to theexistence of neoplastic cells (42)]. The more conservative interpretation of the present observations is that splenectomyabolished the immunologie resistance which is normally conferred by specific immunization.

Obviously conclusions cannot be drawn from these preliminary splenectomy experiments, and further studies with splenectomized mice are in progress. The findings so far are inagreement with an immunologie basis for the higher resistanceof normal BALB/c than of BALB/cfC3H mice to MTV-infectedmammary tumor isografts and for the heightened reactivity ofBALB/c hosts following immunization with MTV-infected tissuepreparations.

The Effect of Neonatal Infection with MTV on the Responsiveness of BALB/c Mice to Mammary Tumor Isograftsof BALB/cfC3H Origin. Routine skin-graft tests of the reciprocal isogenicity of mice of the BALB/c and BALB/cfC3H lines,carried out throughout the course of these experiments amongrandomly chosen pairs of animals, failed to reveal any operativelack of isogenicity within or between the lines. Nonetheless,

Table 8

TumorhostsExperiment

1BALB/cBALB/cBALB/cfC3HBALB/cfC3HExperiment

ZBALB/c(MTV-)(MTV-)(MTV+)(MTV+)(MTV-)ImmunizationNoneNoneNoneNoneHigh-speedcentrifugationSplenectomy

statusSplenectomizedNormalSplenectomizedNormalSplenectomizedNo.

oflivingtumorcells in

challengeinoculum2

X10<Â°2X10*2X10*2X10*4X104Â«No.

ofanimals8961116No.

ofanimals

withpalpabletumors at

termination445915Mean

of evenly bisectingtumor diameters at

intervals after challengeimplantation(mm)2x26

2x3Â°7x77x97x8

10x125x68x98x9>

BALB/c (MTV-)

pellet preparation ofBALB/cfC3H lactatingmammary tissue*

Same with tissue ofBALB/c origin

Splenectomized 4 X IO4 16 14 7x8

Effect of splenectomy on the acceptance of isografts of BALB/cf3CH spontaneous mammary carcinomas by BALB/c and BALB/cfCSHmice. MTV, mammary tumor virus.

a BfSMT-3 tumor in the 2nd transplant generation.6 Thirty-nine days after challenge implantation.c Forty-six days after challenge implantation. The tumor incidence data cited in the table are, however, based on tumor incidence

59 days after challenge, a time at which some of the neoplastic masses had become too large to permit measurement.d The immunization schedule was the same as shown for Experiment 3 in Table 2.e BfSMT-1 tumor in the 3rd transplant generation./ Twenty-one days after challenge. The tumor incidence data are based on the numbers of tumors seen 28 days after challenge.





there remained the possibility, albeit remote, that the greaterresistance of BALB/c than of BALB/cfC3H mice to tumorisografts of BALB/cfC3H origin arose from the presence of atransplantation antigen in the latter animals not shared by theformer, expressed sufficiently to trigger effective homograftresistance in BALB/cfC3H lactating and neoplastic mammarytissue but not in skin. In order to rule out this possibility, anexperiment was performed in which the development of implants of a BALB/cfCSH spontaneous mammary carcinomawas followed comparatively in mice of the BALB/cfC3H sub-line and in mice of the BALB/c strain infected at birth withMTV by foster nursing on a BALB/cfC3H mother. Abrogation by MTV infection of the greater impediment which normalBALB/c hosts manifest against such implants would arguestrongly against the artifact of a residual heterozygosis of iso-antigenic characteristics as an explanation of the findings.

Groups of 10- to 16-week-old normal, virgin females of theBALB/cfC3H line and of BALB/c mice which were fosternursed on a BALB/cfC3H mother were given single subcutaneous implants, in the left inguinal region, of 2 X 10Blivingcells derived from spontaneous BALB/cfC3H mammary tumorBfSMT-3, in the 3rd transplant generation. The developmentof the tumor implants was followed for 5 weeks after challenge.[Although this challenge inoculum is rather large, the resultsof the preceding experiments show that heightened reactivityof BALB/c animals can be manifested against BALB/cfC3Hmammary tumors even when challenge is of the same largeorder, i.e., 1 to 4 X IO5living cells (Table 3, Table 4, Table 6)].

As is evident from Table 9, BALB/cfC3H mice and BALB/canimals foster nursed on a BALB/cfC3H mother and therebyinfected with the MTV at birth supported equally well thedevelopment of isografts of a BALB/cfC3H mammary tumoralready shown to be immunosensitive in MTV-free BALB/chosts. Neonatal introduction of MTV thus obliterated thegreater resistance normally offered by BALB/c mice againstBALB/cfC3H mammary carcinomaisografts. This observationagain indicates that infection of host animals with MTV is amajor determinant of the specific ability of mice to resist infected isografts of mammary cancers and supports the analogous findings of Morton et al. (27) and of Lavrin et al. (19, 20)with mice of C3H genotype.

Table 9

No.ofTumorhosts animalsNo.

ofanimalswithpalpabletumors

atterminationMean

ofevenlybisectingtumordiametersat

termination(mm)

BALB/cfCSH (MTV+) 12 11 11x16BALB/c fosternursed

on a BALB/cfC3Hfemale (MTV+) 17 17 12x18

Differential susceptibility to isografts of a BALB/cfCSH mammary carcinoma of mice of the BALB/cfCSH subline and ofBALB/c mice infected with the mammary tumor virus MTV atbirth by foster nursing on a BALB/cfCSH mother. The tumorhosts were 10- to 16-week-old normal, virgin females. Tumorchallenge was with 2 X IO5 living cells derived from spontaneousmammary carcinoma BfSMT-3 in the 3rd transplant generation.The experiment was terminated 5 weeks after challenge.

DISCUSSION

The results presented here provide strong additional evidencefor a causal role of the MTV in the specific immunogenicityof spontaneous mammary carcinomas of inbred mice. Immunization with preparations of normal mammary tissue or wholeblood derived from mice infected with MTV elicited heightenedreactivity in uninfected, isogenic BALB/c hosts against laterimplantation with isografts of MTV+ spontaneous mammarycarcinomas. Untreated mice of the same genotype were foundto be less resistant in adult life to the development of first-setMTV+ mammary tumor isografts if they were infected withMTV at birth than if they had not previously come into contactwith the agent.

The postulation of a specific immunologie tolerance towardsMTV-dependent antigens in MTV-infected adult mice does notrequire the assumption that these antigens are already expressed shortly after neonatal infection; specific immunologieunresponsiveness can also be induced in adulthood under avariety of circumstances (10, 22). MTV-associated antigens ofpreneoplastic and carcinomatous mammary tissue could conceivably come into being only some time after the virus reachesthe mammary glands and after the B-particles accepted bymost investigators as representing the mature MTV virion (24,30, 31) can be detected there, and perhaps even only concomitant with the preneoplastic transformation. Failure to find thesame antigens in MTV-infected young normal mammary glandsand other normal organs on the one hand, and in preneoplasticand tumorous mammary tissue on the other, would thereforehave said little. Conversely, the demonstration of a common,MTV-associated immunogenicity of normal tissues of infectedyoung adults and of HAN and mammary carcinoma cellspoints strongly to the viral etiology of at least one strongtransplantation antigen in MTV-containing mouse mammarytumors and preneoplastic nodules.

It is very unlikely that the antitumor immunogenicity ofMTV-infected normal mammary glands and whole blood accrued from a contamination of the preparations with virus-independent tumor- or nodule-specific antigens, contributedby occult preneoplastic or neoplastic cells in the starting tissues.Young primaparous donors were used as the source of the normal, lactating mammae ; frank carcinomas were not seen in suchglands, and the quantity of any preneoplastic nodular structureswithin the total lobular tissue would be exceedingly small, ifat all present. It would also have to be assumed that any contaminating, virus-independent antigens are cross-reactive, evenacross strain boundaries (9), and that they are present in wholeblood as well as in mammary elements. It has already beenfound, moreover, that heightened antitumor resistance can beinduced by implantation of living grafts of normal, ductilemammary tissue, patently free of any frank nodular components (9).

The present studies indicate that infection with MTV inearly life does not generally reduce immunologie competence.Infected mice of the reciprocally isogenic BALB/cfC3H sub-line was as reactive to immunization with unrelated solubleand participate antigenic entities (bovine serum albumin andsheep red blood cells) and to isografts of spontaneous and

SEPTEMBER 1968 1769




methylcholanthrene-induced mammary carcinomas free of the

virus, as were animals of the BALB/c parental strain. Thegreater susceptibility of the neonatally infected BALB/cfC3Hmice towards MTV-infected mammary carcinoma isografts isthus most probably the consequence of a state of specific immunologie disability vis-a-vis replicating, virus-controlled antigens already experienced at birth or shortly thereafter. Thisinterpretation has also been given by Morton et al. (27) andby Lavrin et al. (19, 20) to similar findings in mice of C3Hgenotype, and other investigators have suggested similar conclusions in analogous investigations (H. Suit, to be published;D. Oth, personal communication).

The hypothesis that specific immunologie unresponsivenessagainst MTV-associated antigens, developing in consequenceof early and continued exposure, underlies the considerablygreater susceptibility of neonatally infected mice to infectedtumor isografts was further supported by another of the present experiments. Infection of mice of the BALB/c line withMTV by foster nursing on a BALB/cfC3H mother broughtabout the disappearance of the greater tumor resistance otherwise exhibited by these animals. This experiment also arguesagainst any undetected, residual isoantigenic heterozygosity asan explanation of the behavior of BALB/c mice towardsBALB/cfC3H neoplastic implants.

In one experiment reported, sera from mice immunized withMTV-infected, but not with MTV-free, milk of isogenic originconferred heightened susceptibility on isogenic animals againstlater challenge with mammary carcinoma isografts infectedwith the virus. This observation points once more (4, 7) tothe development of specific humoral antibodies as one not uncommon manifestation of the reaction of mice to contact inadult life with MTV-infected tissues or tissue derivatives.

The effects of splenectomy on the ability of MTV-freeBALB/c mice to react against isografts of MTV-infected mammary carcinomas upon first exposure and after immunizationwith an infected normal mammary tissue preparation were alsoexamined. The preliminary findings are compatible with animmunologie explanation of the behavior of BALB/c animalsagainst BALB/cfC3H tumors. Prior removal of the spleenabrogated the heightened reactivity otherwise conferred byimmunization with MTV-infected mammary tissue but increased the resistance of unimmunized mice; it is suggestedthat the latter effect may be based on a splenectomy-inducedreduction of the levels of autochthonous enhancing antibodiesagainst tumor-associated antigens.

The incrimination of the MTV in the tumor-associated an-tigenicity of the mammary tissues of infected mice is supportedstill further by experiments, to be published shortly, whichpoint to the MTV virion itself as at least one of the MTV-dependent antigens (D. Burton, P. B. Blair, and D. W. Weiss,to be published).

These findings in no way preclude the existence, on the surface of MTV-infected neoplastic mammary cells, of other tumor-specific antigens entirely independent of the presence andactivities of the virus; the expression of biologically andchemically different categories of antigens on the surface of acell is certainly a common phenomenon. In the spontaneousmammary carcinoma system, previously reported findings have

provided circumstantial evidence for the existence of specific,virus-unrelated antigenic entities, and stronger evidence willbe brought forward shortly (J. Vaage and D. W. Weiss, to bepublished). It is quite possible, however, that antigens possessedby the MTV virion itself, coded for by the viral genome, orderepressed in the host-cell genome by action of the virus,can mask or suppress the expression of other antigens; suggestive evidence for a marked reduction of the quantities ofsome antigens on the surface of neoplastic cells concomitantwith the expression of others has already been reported (12).The relationships of different categories of tumor-specific andtumor-associated antigens of spontaneously arising neoplasticcells is of considerable intrinsic interest and may also havesignificant clinical implications. Studies are currently under wayto detect and analyze such relationships in mice of the BALB/cgenotype where host animals as well as preneoplastic and neoplastic tissues infected with either MTV or NIV, both agents,or free of both are readily available.

ACKNOWLEDGMENTS

The authors express their appreciation to Mrs. Patricia Wood,Mrs. Rose S. Bonhag, and Miss Ulla Gars for skillful assistance inthese experiments, and to Miss Linda Gaede for aid in the preparation of the manuscript.

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2. Argyris, B. F. Acquired Tolerance to Skin Homografts in Mice.III. Role of Immune Status of Host in Induction and Maintenance of Tolerance. J. Immunol., 93: 114-121, 1964.

3. Attia, M. A., DeOme, K. B., and Weiss, D. W. Immunology ofSpontaneous Mammary Carcinomas in Mice. II. Resistance toa Rapidly and a Slowly Developing Tumor. Cancer Res., S5:451-457, 1965.

4. Attia, M. A. M., and Weiss, D. W. Immunology of Spontaneous Mammary Carcinomas in Mice. V. Acquired Tumor Resistance and Enhancement in Strain A Mice Infected withMammary Tumor Virus. Cancer Res., 26: 1787-1800, 1966.

5. Bern, H. A., and Nandi, S. Recent Studies of the HormonalInfluence in Mouse Mammary Tumorigenesis. Progr. Exptl.Tumor Res., 2: 90-144, 1961.

6. Blair, P. B. Immunology of the Mouse Mammary TumourVirus (MTV) : A Qualitative in Vitro Assay for MTV. Nature,908: 165-167, 1965.

7. Blair, P. B., Lavrin, D. H., Dezfulian, M., and Weiss, D. W.Immunology of the Mouse Mammary Tumor Virus (MTV) :Identification in Vitro of Mouse Antibodies Against MTV.Cancer Res., 26: 647-651, 1966.

8. Blair, S. M., Blair, P. B., and Daane, T. A. Differences in theMammary Response to Estrone and Progesterone in CastrateMale Mice of Several Strains and Hybrids. Endocrinology,61: 643-651, 1957.

9. Dezfulian, M., Lavrin, D. H., Shen, A., Blair, P. B., and Weiss,D. W. Immunology of Spontaneous Mammary Carcinomas inMice: Studies on the Nature of the Protective Antigens. In:Carcinogenesis: A Broad Critique, pp. 365-388. Baltimore:Williams & Wilkins Co., 1967.




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1968;28:1759-1772. Cancer Res Manouchehr Dezfulian, Tina Zee, K. B. DeOme, et al. Responsiveness of the HostsSpontaneous Mammary Carcinomas of BALB/c Mice and in the Role of the Mammary Tumor Virus in the Immunogenicity of

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