Tumor-specific Transplantation Antigens in Reticulum Cell … · [CANCER RESEARCH 33, 551-558, March 19731 Tumor-specific Transplantation Antigens in Reticulum Cell Sarcomas and Lymphomas

[CANCER RESEARCH 33, 551-558, March 19731

Tumor-specific Transplantation Antigens in Reticulum CellSarcomas and Lymphomas Induced by the Friend Virus Complex1

A. Howard Fieldsteel, Peter J. Dawson, and Carole Kurahara

Division of Life Sciences, Stanford Research Institute, Menlo Park, California 94025 (A. H. F., C. K.J, and Department of Pathology, University ofOregon Medical School, Portland, Oregon 97201 Â¡P.J. D./

SUMMARY

The reticulum cell sarcomas induced by Friend leukemiavirus (FLV) and the lymphomas induced by the Friendvirus-associated lymphatic leukemia virus (LLV) have beeninvestigated for both their antigenic relationships and thepresence in them of common tumor-specific transplantationantigens. Since noninfectious reticulum cell sarcomas containing the Friend virus genome were available, this was a uniqueopportunity to determine whether new tumor-specific transplantation antigens were present in these tumors in theabsence of virus.

All of the lymphomas induced by LLV in different strainsof mice contained LLV and each induced transplantationresistance against the other. The reticulum cell sarcomasinduced by FLV were of two kinds: (a) those that containedFLV and (Â¿>)those that apparently contained no FLV but didcontain the FLV genome, which could be rescued by LLV.Both the infectious and noninfectious reticulum cell sarcomasinduced transplantation resistance against each other. Furthermore, the lymphomas induced by LLV and the infectious andnoninfectious reticulum cell sarcomas induced by FLV alsoinduced transplantation resistance against each other. Finally,LLV itself induced transplantation resistance against not onlythe lymphomas, but also the virus-containing and noninfec

tious reticulum cell sarcomas.It is concluded that both the LLV-induced lymphomas and

the FLV-induced reticulum cell sarcomas probably containnew nonviral, cross-reacting, tumor-specific transplantationantigen(s).

INTRODUCTION

Antigenic studies on FLV,2 RLV, and MLV leukemia

viruses have been the subject of several reports (11, 15-17,19). Virus neutralization, cytotoxic and immunofluorescentantibody, and transplantation studies have demonstrated theirclose antigenic relationship to one another. However, there are

1This investigation was supported by USPHS Grant CA-07868 from

the National Cancer Institute.2The abbreviations used are: FLV, Friend leukemia virus; RLV,

Rauscher leukemia virus; MLV, Moloney leukemia virus; LLV,lymphatic leukemia virus; MSV, murine sarcoma virus; 1D50, 50%infective dose; BSS, balanced salt solution; TSTA, tumor-specifictransplantation antigens;Â£s,group-specific antigens.

Received October 18, 1972;accepted December?, 1972.

a number of unanswered questions with respect to the basis ofthis relationship, the chief one being whether new cellularantigens are formed in the tumors induced by these viruses,since all tumors studied thus far contained infectious virus.Only the lymphomas induced by FLV and RLV have beeninvestigated for their relationship to each other and tolymphomas induced by MLV (11). The interrelationshipamong the lymphomas and reticulum cell sarcomas induced byFLV has yet to be determined.

We have shown that FLV is invariably associated with avirus (LLV) that induces lymphatic leukemia and that thisvirus cannot be distinguished immunologically from FLV (3).It has also been demonstrated that LLV not only could beseparated from FLV, but also was able to induce transplanta-ble lymphocytic lymphomas. All attempts to obtain FLVpreparations free of LLV have failed. Subsequently, it wasfound that FLV was defective and required the presence ofLLV as a helper for its replication (7,10).

Rauscher (20) also showed that, after passage in BALB/cmice, the virus that he isolated induced lymphatic leukemia inC57BL mice and rats. He was unable to separate a LLV fromextracts that induced early splenic disease in BALB/c mice,although some of the mice in his study did develop lymphaticleukemia. However, we have shown (9) that a LLV can readilybe separated from early-passage RLV that induces splenicdisease. These preparations of LLV do not induce the earlysplenic disease but readily induce transplantable lymphocyticlymphomas. It is probable that these viruses bear the samerelationship to each other as does LLV to FLV.

We have induced transplantable lymphocytic lymphomasfrom LLV and the LLV associated with RLV (2,9) in severalstrains of mice. We have also induced transplantable reticulumcell sarcomas from FLV in BALB/c and C57BL X DBA/2 F,(hereafter called BDFj) mice (6). Both types of tumorscontain varying quantities of infectious virus. In addition, thereticulum cell sarcomas have been grown in vitro as suspendedcell cultures (5, 8). These cultures do not contain detectableinfectious virus by any of the following criteria, (a) Innewborn BALB/c mice, an extensive bioassay of lysed cells,irradiated cells, and tissue culture fluids (concentrated 100times) from these cultures has failed to reveal the presence ofany infectious agent, (ft) Immunization of BALB/c mice withtissue culture fluid concentrated 100 times or with cell lysatesfrom these cultures has failed to protect the mice againstchallenge with as few as 20 ID50's of FLV. (c) Mice exhibiting

complete transplantation resistance after repeated inoculationwith IO7 tissue culture cells had no detectable neutralizing

MARCH 1973 551

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A. H. Fieldsteel, P. J. Dawson, and C. Kurahara

antibodies against FLV in their serum, (d) Extensive examination of these tissue culture cells by electron microscopy failedto reveal the presence of type C particles, although occasionalcells contained several type A particles (8).

In a recent study intended to determine whether the cells ofthese cultures release noninfectious virus particles, a largenumber of cells (3 X IO8) were cultured in the presence ofuridine-3H. After 24 hours of incubation, the cell culture

medium was concentrated and examined for the presence ofRNA tumor virus-like particles in a sucrose density gradient.No zone of radioactivity occurred at a buoyant density of 1.14to 1.16 g/cu cm, at which viruses of this type might beexpected to band (D. R. Black and A. H. Fieldsteel,unpublished observations).

These noninfectious tumors, when inoculated back intosyngeneic mice, induce typical reticulum cell sarcomasindistinguishable from the original virus-containing tumors.They have also been shown, by in vivo and in vitro methods,to contain the FLV genome (7, 10).

The studies reported here were carried out to determine theantigenic relationship between the FLV-induced reticulum cellsarcomas, both virus-containing and virus free, and theLLV-induced lymphomas.

MATERIALS AND METHODS

Mice. The BALB/c mice used in transplantation experimentswere 6- to 10-week-old females from our own inbred colony,which readily accepted skin grafts from one another. All otherBALB/c mice were obtained from Simonsen Laboratories,Gilroy, Calif. Each subline appeared to be equally susceptibleto all tumors and viruses. The RF, C57BL/6, and BDFj hybridmice were 6 to 8 weeks old, and they also were obtained fromSimonsen Laboratories.

Tumors. The FLV-induced, transplantable reticulum cellsarcomas in BALB/c and BDP! mice, the noninfectious tissueculture tumors derived from each of these, and the methodused in cultivating them have been described (1,6,8).

The lymphomas induced by LLV were initiated by theinoculation of newborn BALB/c, C57BL/6, RF, and BDF,mice with a 20% cell-free extract of a mixture of mesentericnode, thymus, and spleen from mice with lymphatic leukemia.When these mice developed lymphatic leukemia (45 to 90 dayslater), a cell suspension of the greatly enlarged mesentericnode was inoculated into the same strain of mouse, producingrapidly fatal, transplantable lymphomas.

Two spontaneous tumors have been used as controls. One ofthese, a lymphocytic lymphoma, occurred in an RF mouse andwas subsequently carried as a transplantable tumor. The othertumor was derived from the CL-1 cell line (12), a continuousculture of BALB/c mouse embryo cells. After 59 passages, itwas found to have transformed spontaneously, producingreticulum cell sarcomas when transplanted into BALB/c mice.

Data on all tumors in this study are summarized in Table 1.Preparation of Cell Suspensions. Solid tumors and normal

tissues were finely minced, suspended in Hanks' BSS, taken up

in a 10-ml syringe, and expelled through a graduated series ofneedles ranging from 18 to 23 gauge. A single-cell suspension

was finally obtained by differential centrifugation.Ascites tumors were centrifuged at 200 X g for 5 min, and

the supernatant fluid was discarded. The cells were re-suspended in Hanks' BSS and differentially centrifuged to

remove red blood cells.Since the cells of the noninfectious reticulum cell sarcomas

in tissue culture remained discrete and did not attach to theglass, it was necessary only to shake the culture vessel toobtain a uniform suspension. Prior to use, the cells wereresuspended in Hanks' BSS.

Viability of all cell suspensions was determined by thefailure of cells to take up trypan blue at a final concentrationof 0.05%.

Viruses. LLV was derived from preparations of FLV, passedserially through Scott-Russ rats, purified by ether treatment,and finally passed in newborn BALB/c mice as describedpreviously (3). The virus induced the typical gross andhistological changes of lymphatic leukemia, with a latentperiod of at least 45 days. It did not induce Friend disease.FLV was from the 8th and 9th passages in BALB/c mice andwas also prepared as described previously (4). MLV, from Dr.J. B. Moloney, National Cancer Institute, Bethesda, Md., wasused as a 20% cell-free extract of spleen, thymus, and lymphnodes from infected BALB/c mice.

Immunization. In all of the transplantation studies, immunization with tumor cells was carried out with allogeneic cells,and the mice were challenged with syngeneic tumor cells. Micewere given 2 doses of viable allogeneic cells s.c. at 14-dayintervals. Initially, they received IO6 cells; the 2nd injectionaveraged approximately 5 X IO7 cells. Included in each

experiment were control groups of mice; these were inoculatedwith equivalent numbers of viable liver cells from normal miceof the same strain as that from which the tumor used forimmunization originated. This experiment was done to test forany nonspecific reactions that might occur simply as the resultof inoculating allogeneic tissues. The mice were then challenged 2 weeks later with graded doses of syngeneic tumorcells. Challenge with the reticulum cell sarcomas was s.c. onthe side opposite to that used for immunization. Thelymphomas were inoculated i.p.

The FLV preparation was a 20% cell-free extract of infectedspleen that contained IO6'2 IDso's of FLV per ml. The LLV

was a 20% extract of infected spleen, thymus, and lymphnodes with a titer of >107 ID50's per ml. For immunization,

the mice received 2 i.p. doses of 0.2 ml of the 20% suspension,14 days apart. They were challenged s.c. with a syngeneictumor 14 days after the 2nd injection.

RESULTS

Relationship among Lymphomas Induced by LLV. LLV,upon passage into C57BL/6, RF, and BDF! mice, inducedlymphatic leukemia and readily transplantable lymphomas.Cell-free extracts of each of these lymphomas, when inoculated into newborn BALB/c mice at various passage levels,were found to contain at least 10s 50% lethal doses of LLV

per ml. In no instance did these extracts induce Friend diseasewhen inoculated into newborn mice.

Experiments were carried out to determine whether these

552 CANCER RESEARCH VOL. 33



TSTA in Tumors Induced by FL V Complex

Table 1Description of transplantable tumors utilized in these experiments

Tumordesignation"FLV-RCS-BALB(V+)FLV-RCS-BDF,(V+)FLV-RCS-BALB(V-)FLV-RCS-BDF,(V-)LLV-L-BALB(V+)LLV-L-BDF,(V+)LLV-L-BL(V+)LLV-L-RF(V+)SL-RFeCL-1-RCS-BALB(V-/VirusetiologyFriendFriendFriendFriendLymphaticleukemiaLymphaticleukemiaLymphaticleukemiaLymphaticleukemiaNone(spontaneous)NoneTumortypeReticulum

cellsarcomaReticulum



cellsarcomaLymphocyticlymphomaLymphocyticlymphomaLymphocyticlymphomaLymphocyticlymphomaLymphocyticlymphomaReticulum

cellsarcomaTumor

formAscitesAscitesAscites0Ascites0SolidSolidSolidSolidAscitesSolidMouseof

originBALB/cBDF,BALB/cBALB/cBALB/cBDF,C57BL/6RIRFBALB/cPassageno/586447d32d5812610811

a Tumors still containing the virus that induced them are designated (V+). At the time of this study,extensive testing of the other tumors no longer revealed the viruses that induced them; these tumors aredesignated (V-).

b Number of passages in mice at time of initial experiments unless otherwise indicated.0 These tumors are carried in tissue culture but when inoculated i.p. in syngeneic mice they form

ascites tumors.d Number of passages in tissue culture at time of experiments.e Spontaneous lymphocytic lymphoma in RF mice.' Spontaneous reticulum cell sarcoma derived from "normal" BALB/c embryonic tissue culture.

lymphomas were capable of inducing transplantation resistance against each other. The results of several experiments(Table 2) show that all of the lymphomas induced by LLVinduced transplantation resistance against the lymphoma fromBALB/c mice, and that the lymphoma from the latter alsoinduced transplantation resistance against the BDF! lymphoma. In this and other experiments, some transplantationresistance occasionally appeared to be induced by preparationsof normal liver cells. This phenomenon was described by Kleinand Klein (14), who were able to demonstrate a slightlyincreased transplantation resistance to methylcholanthrene-induced sarcomas in mice pretreated with irradiated normalcells as compared with untreated normal controls. Thisphenomenon was ascribed "to a nonspecific bolstering effect

on the immunological reactivity against small, near-thresholdinocula." Whole-body irradiation was found to abolish these

nonspecific effects of pretreatment, leaving specific transplantation immunity intact. Since, basically, we were comparingdifferences in immune responses of animals pretreated withtumor cells as compared with normal cells derived from thesame strain of mice, the significance of the results wasconsidered to be unaltered by the occasional occurrence ofnonspecific reactions. Additionally, the spontaneous lymphoma from RF mice did not induce resistance against theBALB/c lymphoma in BALB/c mice.

Relationship between the Virus-containing and Noninfec-tious Reticulum Cell Sarcomas Induced by FLY. Since thelymphomas induced by LLV all contained virus, it was not

unexpected that they would show a relationship to each other.However, since the reticulum cell sarcomas that had beengrown in tissue culture did not contain infectious virus (5, 8),it was possible to determine not only whether a relationshipexisted between these tumors but also whether this relationship was dependent on the presence of virus. BALB/c or BDFjmice were immunized with either the virus-containing ornoninfectious reticulum cell sarcomas from BDF! or BALB/cmice, respectively. Each group was then challenged with eitherthe virus-containing or noninfectious reticulum cell sarcoma ofsyngeneic origin. It is apparent (Table 3) that both thevirus-containing and noninfectious reticulum cell sarcomasfrom BDFi mice protected BALB/c mice against thecorresponding syngeneic tumors. However, in the reversesituation, only the virus-containing reticulum cell sarcomafrom BALB/c mice appeared to induce protection, but thiswas against the syngeneic noninfectious reticulum cell sarcomafrom BDF[ mice. The failure of both the virus-containing andnoninfectious BALB/c reticulum cell sarcomas to protectagainst the virus-containing BDF, reticulum cell sarcoma canprobably be explained by the fact that the latter was highlymalignant, requiring as few as 10 cells to induce a tumor. Thesmallest quantity used for challenge in these experiments was250 cells.

Because the noninfectious BALB/c reticulum cell sarcomaalso failed to protect against the noninfectious BDFjreticulum cell sarcoma, we tested the ability of the former toprotect against itself. During the course of passage in tissue

MARCH 1973 553




Table 2Interrelationship among Â¡ymphomasinduced by LL V

Immunization wasbyImmunizing

tumorLLV-L-BL(V+)Â°LLV-L-RF(V+)aLLV-L-BALB(V+)bLLV-L-BDF,(V+)aSL-RF(V-)a2

s.c. doses ofviableChallenge

tumorLLV-L-BALBCV+)LLV-L-BALB(V+)LLV-L-BDF,(V+)LLV-L-BALB(V+)LLV-L-BALB(V+)cells

administeredChallenge

~

dose(no. ofcells)5X

IO31 X 10Â«

Total5X

IO31 X 10"

Total5

X IO31 X 10"

Total5X

IO31 X IO4

Total5X

IO31 X IO4

Total14

days apart; challenge wason Day28.No.

of mice with tumors/total no. ofmiceImmunized

with tumorcells1/15

9/3510/502/30

0/292/593/14

2/155/290/15

0/150/3013/15

14/1527/30Immunized

with normallivercells8/15

12/1520/3014/15

14/1528/3015/15

15/1530/309/15

7/1516/30Untreated15/1534/35

49/5030/30

34/3464/6415/15

15/1530/3015/15

15/1530/3015/1S

15/1530/30

" Immunization was in BALB/c mice.b Immunization was in BDF, mice.

culture, 1 passage line of the noninfectious BALB/c reticulumcell sarcoma lost much of its ability to induce tumors,although it still contained the FLV genome (A. H. Fieldsteel,P. J. Dawson, and C. Kurahara, unpublished observations).When 16 BALB/c mice were inoculated s.c. with between IO6and IO7 cells of the 256th tissue culture passage of the

noninfectious reticulum cell sarcoma, only 3 of themsubsequently developed progressive lethal tumors. Fifty-fivedays after the initial inoculation, the 13 survivors werechallenged s.c. with 10" cells of the 100th tissue culture

passage; none of these developed tumors. However, when 10previously uninoculated BALB/c mice were inoculated simultaneously with the same dose of cells, 9 of them developedprogressive, fatal tumors. In addition, when the 13 survivors ofthe 2 previous inocula of the noninfectious BALB/c reticulumcell sarcoma were challenged i.p. with IO4 cells of the BALB/c

lymphoma [LLV-L-BALB (V+)], only 1 developed a tumor.This was a very slow-growing lymphocytic lymphoma whichappeared 28 days after inoculation. Ten previously uninoculated BALB/c mice that were inoculated simultaneously withthe same number of LLV-L-BALB(V+) cells died 15 to 18days later with lymphocytic lymphomas. Thus, it was apparentthat the noninfectious BALB/c reticulum cell sarcoma wassufficiently antigenic to confer solid immunity not onlyagainst itself but also, under appropriate circumstances, againstthe lymphomas induced by LLV.

Relationship between Lymphomas Induced by LLV and theInfectious and Noninfectious Reticulum Cell Sarcomas Induced by FLV. We had previously established a relationshipbetween LLV and FLV (3) and have shown here that not onlyare the LLV lymphomas related to each other but thevirus-containing and noninfectious reticulum cell sarcomas

induced by Friend virus are also related to each other. Todevelop further the relationship among these tumors and therole played by virus in this relationship, we conductedtransplantation studies on the lymphomas and the reticulumcell sarcomas. The results (Table 4) indicate that, althoughcomplete cross-protection did not occur among all the tumors,1 of the lymphomas [LLV-L-BALB(V+)] protected against 1of the noninfectious reticulum cell sarcomas[FV-RCS-BDF,(V-)], and the other [LLV-L-BDF,(V+)]induced strong protection against a virus-containing reticulumcell sarcoma [FV-RCS-BALB(V+)]. Conversely, the virus-containing reticulum cell sarcoma from BALB/c mice and thenoninfectious reticulum cell sarcoma from BDF, mice eachinduced strong protection against syngeneic lymphomas inBDF] and BALB/c mice, respectively. Again, the noninfectious reticulum cell sarcoma from BALB/c mice failed toinduce protection, this time against the lymphoma from BDF!mice.

Induction of Transplantation Resistance to SyngeneicNoninfectious FLV-induced Reticulum Cell Sarcomas andLLV-induced Lymphomas by Pretreatment with LLV. McCoyet al. (16) showed that infectious FLV was essential for theinduction of transplantation resistance against a FLV-inducedlymphoma in C57BL/6 mice, and they concluded that thoseresults were consistent with the theory that transplantationresistance is mediated by cellular antigens induced byinfectious virus. However, since the lymphomas used by themalso contained infectious virus, the transplantation resistancecould have been the result of antibody directed against thevirus itself rather than against virus-induced antigens. If itcould be shown that the virus that induced these lymphomas(LLV) could also induce transplantation resistance against the




TSTA in Tumors Induced by FLV Complex

Table 3Relationship between the virus-containing and noninfeclious Friend virus-induced reticulum cell sarcomas

Immunization was by 2 s.c. doses of viable cells administered 14 days apart; challenge was on Day 28.

No. of mice with tumors/total no. of mice

ImmunizingtumorFLV-RCS-BDF,(V+)Â°FLV-RCS-BDF,(V-)Â°FLV-RCS-BALB(V+)bFLV-RCS-BALB(V-)bChallengetumorFLV-RCS-BALB(V+)FLV-RCS-BALB(V-)FLV-RCS-BALB(V+)FLV-RCS-BALB(V-)FLV-RCS-BDF,(V+)FLV-RCS-BDF.(V-)FLV-RCS-BDF,(V+)FLV-RCS-BDF,(V-)Challengedose1

XIO35XIO31

X10*Total1

XIO35XIO31XIO4Total1

XIO35XIO3Total5X

IO31XIO4Total2.5

XIO21XIO35XIO3Total5X

IO31X10"Total2.5

XIO21XIO35XIO3Total5

XIO31X10"Totalimmunized

with tumorcells6/1415/3010/1531/591/156/309/1516/603/152/155/305/154/159/3011/1515/1515/1541/453/152/155/3014/1413/1515/1542/4413/1513/1526/30Immunized

with normallivercells15/1526/3014/1555/604/1517/309/1530/6013/1511/1524/309/1510/1519/3015/1515/1515/1545/4515/1515/1530/3015/1515/1515/1545/4515/1515/1530/30Untreated13/1428/3012/1553/596/1521/3012/1539/6010/1512/1522/3010/159/1519/3015/1515/1515/1545/4514/1514/1528/3015/1515/1515/1545/4514/1514/1528/30

" Immunization was in BALB/c mice.b Immunization was in BDF, mice.

noninfectious reticulum cell sarcomas, the evidence that thisresistance was truly mediated by cellular antigens againstvirus-induced but nonvirion antigens in the tumors would bemore convincing. Therefore, a series of experiments wascarried out to determine whether LLV could induce transplantation resistance against not only the virus-containing lym-phomas but also the noninfectious reticulum cell sarcomas.The data summarized in Table 5 show that LLV inducedstrong transplantation resistance not only against the BALB/clymphoma but also against both of the noninfectious,FLV-induced reticulum cell sarcomas. FLV itself also inducedprotection against the noninfectious FLV-induced reticulumcell sarcoma (p < 0.01), but at a level lower than that inducedby LLV.

In an additional experiment, a preliminary attempt wasmade to determine whether MLV would protect BDF, miceagainst the noninfectious reticulum cell sarcoma of BDF,mice. Eight newborn BDF, mice were inoculated i.p. with 0.1ml of a 20% cell-free extract of MLV-infected tissue.Fifty-three days later, these mice, plus 8 BDF, mice of thesame age, were inoculated s.c. between the scapulae with 10s

cells of the noninfectious BDF, tumor. By Day 22 afterinoculation, all of the latter group had developed progressivereticulum cell sarcomas. None of the MLV-inoculated micedeveloped tumors, although all of them eventually developedlymphatic leukemia.

DISCUSSION

Noninfectious tumors induced by MSV, another of theRNA murine tumor viruses, have been available since 1966(13), although only recently have they been investigated forthe presence of new cell surface antigens. Stephenson andAaronson (21) failed to demonstrate transplantation antigenson nonproducer MSV-transformed cells. Strouk et al. (22) alsowere unable to detect new surface antigens in MSV-transformed cells known to contain the viral genome. On the otherhand, McCoy et al. (18) obtained results that suggested that anonproducing, MSV-induced tumor possessed a new, tumor-associated transplantation antigen that is not a virion antigen.

Immunological studies of tumors induced by murineleukemia viruses have been hampered by the previous

MARCH 1973 555




Table 4Relationship between lymphomas induced by lymphatic leukemia virus (LL V) and virus-containing and noninfectious reticuhtm cell sarcomas

induced by Friend virusImmunization was by 2 s.c. doses of viable cells administered 14 days apart; challenge was on Day 28.


ImmunizingtumorLLV-L-BALB(V+)Â°LLV-L-BDF,(V+)bFLV-RCS-BALB(V+)Â°FLV-RCS-BALB(V-)aFLV-RCS-BDF,

(V-)6Challenge

tumorFLV-RCS-BDF,(V+)FLV-RCS-BDF,(V-)FLV-RCS-BALB(V+)FLV-RCS-BALB(V-)LLV-L-BDF,(V+)LLV-L-BDF,

(V+)LLV-L-BALB(V+)Challenge

dose2.5

XIO21XIO35XIO3Total5X

IO31XIO4TotalIX

IO35XIO3Total5

XIO31X10*Total5

XIO31X10"Total5

X10'1X10'Total5X

IO31X10'TotalImmunized

with tumorcells13/1515/1515/1543/452/1410/1512/293/155/158/307/157/1514/302/141/153/2915/1515/1530/302/153/155/30Immunized

with normallivercells15/1515/1515/1545/4515/1515/1530/3013/1511/1524/309/1510/1519/3015/1515/1530/3015/1515/1530/3013/1512/1525/30Untreated15/1515/1515/1545/4514/1514/1528/3010/1512/1522/3010/159/1519/3015/1515/1530/3015/1515/1530/3014/1515/1529/30

" Immunization was in BDF, mice.b Immunization was in BALB/c mice.

unavailability of tumors free from infectious virus. Thesituation with FLV and RLV has been further complicated byfailure to distinguish between the viral component responsible for the early reticulum cell proliferation in the spleen andthat causing late onset of lymphatic leukemia. In addition,earlier investigators (11, 15-17) did not differentiate betweenthe reticulum cell sarcomas induced by the former componentand the lymphoid tumors induced by the latter. The recentdiscovery of the defectiveness of the component inducingearly splenomegaly (7, 10) adds a further dimension to theinterpretation of data obtained with the FLV system.

Theoretically, it seems that tumors induced by FLV mightbe expected to contain TSTA and, if the tumors containedvirus, viral antigens as well. Thus, the following 4 classes oftumors are theoretically possible: (a) virus-containing lymphoid tumors. Such tumors would contain TSTA, as well asthe internal gs and envelope antigens of LLV; (b) virus-freelymphoid tumors containing TSTA, but not viral antigens;(c)virus-containing reticulum cell sarcomas possessing viral antigens and TSTA, possibly differing from those in the 2preceding tumor classes. The viral antigens should include thegs and envelope antigens of LLV, as well as antigens associatedwith the genome of the viral component that induces earlyreticulum cell proliferation in the spleen (FLV). Because ofthe defectiveness of the latter component (7, 10), it might be

expected to share capsular antigens with its helper, LLV. (d)virus-free reticulum cell sarcomas containing only TSTA,which may or may not be the FLV genome.

Virus-free lymphoid tumors (Class 2) are not yet available,but examples of each of the other 3 classes of tumor have beeninvestigated.

As might be expected, all of the LLV-induced lymphomas(Class 1) in BALB/c, BDF,, RF, and C57BL/6 mice wereantigenic. The LLV-induced lymphomas all cross-reacted butdid not cross-react with a spontaneous lymphoma originatingin RF mice. Furthermore, mice immunized against LLVdisplayed transplantation resistance to these lymphomas. Also,immunization with these lymphomas protected against challenge with both the virus-containing and noninfectiousreticulum cell sarcomas, suggesting that either the viralantigens or lymphoma TSTA cross-reacted with the TSTA ofthe reticulum cell sarcomas.

The virus-containing reticulum cell sarcomas (Class 3) areclearly antigenic. Immunization of BALB/c mice with thesarcoma from BDF, mice [FLV-RCS-BDFi(V+)] protectedagainst the syngeneic virus-containing reticulum cell sarcomaas well as an LLV-induced lymphoma. It was not possible toprotect BDF, mice against the syngeneic virus-containingreticulum cell sarcoma by immunization with the LLV-induced lymphoma. This failure was considered to be due not




TSTA in Tumors Induced by FL V Complex

Table 5Induction of transplantation resistance to syngeneic noninfectious FL V-induced reticulum cell

sarcomas and LL V-induced lymphomas by pretreatment with LL V


Immunization"LLVFLVChallengetumorLLV-L-BALB

(V+)FLV-RCS-BALB(V-)FLV-RCS-BDF,

(V-)CL-1-RCS-BALB(V-)FLV-RCS-BDF,

(V-)Challenge

dose5X

IO31X10"Total5

XIO31X10"Total5

XIO31X10*Total5X

IO31X10"Total5

XIO31XIO4TotalMice

immunizedwithvirus7/154/1511/306/153/149/297/2916/2923/586/155/1511/3010/1412/1522/29Untreatedmice12/1512/1524/3011/1510/1521/3027/2930/3057/595/158/1513/3014/1415/1529/29

a Both virus pools were prepared from extracts of tissues from BALB/c mice that were given2 i.p. doses at 14-day intervals, followed 14 days later by s.c. inoculation of the tumor. Spleenextracts from normal BALB/c mice failed to influence the incidence of lymphomas or virus-freereticulum cell sarcomas.

6 p < 0.01.

to lack of cross-reactivity, since the same lymphoma gave goodprotection against the noninfectious reticulum cell sarcoma,but rather to the high virulence of the virus-containing tumorfrom BDP! mice. The 50% tumor end point of these tumors isfewer than 10 cells. It seems probable that challenge withnumbers of tumor cells of this order, rather than with theminimum of 5 X IO2 cells used in our study, would have

resulted in the demonstration of immunity.Nevertheless, it is obvious from these data that there is no

way to determine whether the cross-reaction between thevirus-containing reticulum cell sarcomas and the lymphomas isdue to cross-reacting TSTA or merely to LLV associated withthese tumors.

The Class 4 tumors (noninfectious reticulum cell sarcomas)are of the greatest interest. We demonstrated the presence ofTSTA in both syngeneic and allogeneic systems. In the lattersystem, a high degree of protection was demonstrated whenmice immunized with the noninfectious BDFj reticulum cellsarcoma were challenged with either the virus-containing ornoninfectious reticulum cell sarcomas from BALB/c mice.Immunization with the noninfectious BALB/c tumor failed toconfer similar protection but, significantly, it protected againstchallenge not only with itself but also with the LLV-inducedlymphoma.

Since exhaustive testing has demonstrated that both ofthese tumors [F LV-RCS-B ALB( V-) andFLV-RCS-BDF,(V-)] are free from both infectious FLVand LLV (5, 8), these data provide strong evidence of theexistence of nonviral TSTA within these tumors. Caution isneeded in accepting the theory that these antigens areunrelated to the virion, since we have shown that both of thesereticulum cell sarcomas contain the FLV genome, which may

be rescued by LLV (or other lymphatic leukemia viruses)under the appropriate experimental conditions (7, 10).Although these TSTA's still cannot be completely separatedfrom virion-associated antigens, data reported previously (8)appear to exclude the presence of both viral envelope and gsantigens in these tumors. Of special interest is the fact thatimmunization with LLV and MLV will protect against thenoninfectious reticulum cell sarcomas. Infection with both ofthese viruses results in the appearance of transplantablelymphomas. Since these lymphomas cross-react with thenoninfectious reticulum cell sarcomas, it is not unreasonableto assume that both classes of tumor contain a new nonviral,cross-reacting transplantation antigen.

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1973;33:551-558. Cancer Res A. Howard Fieldsteel, Peter J. Dawson and Carole Kurahara ComplexSarcomas and Lymphomas Induced by the Friend Virus Tumor-specific Transplantation Antigens in Reticulum Cell

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Tumor-specific Transplantation Antigens in Reticulum Cell … · [CANCER RESEARCH 33, 551-558, March 19731 Tumor-specific Transplantation Antigens in Reticulum Cell Sarcomas and Lymphomas