Proc. Nat. Acad. Sci. USAVol. 72, No. 6, pp. 2130-2134, June 1975

Tumor-Associated Transplantation Antigens in Immune Rejection of MouseMalignant Cell Hybrids

(analysis of malignancy/cell surface antigens/cytotoxicity)

JACQUES JAMI AND EVELYNE RITZ

Institut de Recherches Scientifiques sur le Cancer, CNRS, Boite Postale No. 8, 94800 Villejuif, France

Communicated by Boris Ephrussi, March 14, 1976

ABSTRACT The inability of mouse cell hybrids de-rived from two malignant parental cells to produce tumorsin syngeneic F, hosts was analyzed. YC hybrids, derivedfrom the fusion of Cl. ID and L1210 cells, failed to induceany tumor in adult mice, while 91% of x-irradiated new-born mice developed tumors and died. Some telocentricchromosomes were lacking in hybrid tumors; however,none of the immunologically intact adult mice developedtumors when grafted with tumors grown in x-irradiatednewborn mice. This indicates that histocompatibilityfactors interfered in the failure of YC tumors to grow inadult hosts. Syngeneic F, mice immunized with Y2Chybrid cells had cytotoxic antibodies against Y2C hybridsand Cl. ID and L1210 parental cells. Complete absorptionof cytotoxic antibodies directed against hybrid cells bymixtures of both parental cell lines demonstrates the ab-sence of any new antigen on hybrid cells. Hybrid cellshad a higher density of Cl. 1D-tumor antigenic sites, ascompared to Cl.1D parental cells. This possibly explainsthe higher antigenicity and/or the higher sensitivity toimmune lysis of hybrid cells.

In previous experiments, hybrids (YC) from two mouse malig-nant cells (L1210 and Cl.1D) failed to develop tumors inadult compatible hosts (1). However, it appeared doubtfulthat malignancy of both parental cells was suppressed bycomplementation (2), because YC hybrids in culture hadcharacteristics of malignant cells (1), and expressed thetumor-associated transplantation antigens of L1210 parentalcells (3). Further, hybrids between the same Cl.1D cell lineand mouse teratocarcinoma cells were malignant (4).

Experiments reported here demonstrate that the inabilityof YC hybrids to grow in vivo is due to histocompatibilityfactors. As Cl.lD and L1210 cells, either separately or mixedtogether, produced tumors in F, mice (1), an analysis ofhybrid-tumor-associated antigens was performed. Threepossible hypotheses were considered to explain the efficiencyof immune reaction. First, hybrid cells might express par-ental-tumor-associated antigens in a greater amount thanparental cells. Second, silent genes in parental cells mightexpress themselves upon hybridization (5), resulting in newantigens on the surface of hybrids. Third, hybrid moleculeson hybrid cell surface, having new antigenic determinants,might result from combination of molecule subunits codedby homologous parental genes in hybrids. The existence ofhybrid molecules in somatic cell hybrids is well documented(6).The origin of tumor-associated surface antigens of YC

hybrids was analyzed by absorbing with parental and hybridcells antisera against hybrid cells prepared in syngeneic F,mice. It was found that YC hybrids had more Cl.lD-tumor-

Abbreviation: ip, intraperitoneal.

associated antigens than Cl.1D cells and no new antigenicdeterminants.

MATERIALS AND METHODS

The origin and description of Cl.lD fibroblastic parentalcells, of L1210 leukemic parental cells, and of the four YChybrid clones were reported elsewhere (1). Cl.lD cells werederived from a C3H mouse (histocompatibility factor H-2k),and L1210 cells from a DBA/2 mouse (H-2d).For inoculation and cytotoxicity experiments, cells were

grown in Dulbecco's modified Eagle's medium with 10%calf serum (growth medium), and were dispersed (exceptL1210 cells, which grew in suspension cultures) with 0.02%Versene in Dulbecco's phosphate-buffered saline.

Cells were injected intraperitoneally (ip) into newborn(9 DBA/2 X d C3H)F1 (D2C3F,) and (9 C3H X eDBA/2)Fl (C3D2F1) mice within 48 hr after birth. Allnewborn mice were x-irradiated (400 R) prior to inoculation.Tumors were excised and divided into three parts. One partwas grafted to groups of nonirradiated adult F1 mice. Thesecond part was fixed in Bouin's solution for histology.Single-cell suspensions obtained from the third part wereused to initiate cell cultures (7).

Karyotypes were analyzed after staining with aceto-orcein.

Antisera against Y2C hybrid cells were prepared by hyper-immunizing 4-month-old syngeneic C3D2F1 males with Y2Ccells harvested in Versene solution and suspended in growthmedium (Table 1). Sera obtained by tail bleeding were heat-inactivated at 560, pooled, and stored at -80° until tested.

Assays for antibody-mediated cytotoxicity were made withHanks' solution supplemented with 4% fetal calf serum free

TABLE 1. Immunization schedule*

Immunization Date Cell dose/mouseno. (weeks) (X 10-6)

1 0 0.12 5 0.23 6 0.54 7 15 8 26 10 27 12 48 14 109 16 1510 18 20

* All immunizations were performed ip. Mice were bled 9, 11,13, 15, 17, and 19 weeks after initiation of immunization.

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of gammaglobulin (HIPT), measuring release of 5'Cr fromlabeled target cells. Cytotoxic titers of sera were determinedby incubating 0.1 ml of serial 2-fold dilutions of serum with0.05 ml of 5'Cr-labeled target cells (2.5 X 104 cells) and 0.1ml of 1:10 dilution of rabbit serum as a source of comple-ment, for 45 min at 37°. Then 1.8 ml of cold HIPT was addedto each tube, the cells were centrifugated at 800 X g for 10min, and the radioactivity of 1 ml of supernatants and thatremaining in the original tubes were counted in a well-typegamma counter to determine the percentage of 5"Cr releasedin each tube. Complement controls were made in triplicate.Percentages of 5"Cr released in complement controls were

9-17%. 51Cr release background was higher with Y2C thanwith Cl.1D and L1210 cells. The cytotoxic index is expressedas cytotoxicity (%) in experimental tubes minus cytotoxicity(%) in complement control tubes, and is corrected for maxi-mal 51Cr released by freezing and thawing cells three times.The titer of the serum is expressed as the reciprocal of thedilution that produced 50% of the maximum release abovecomplement control.

Sera from some nonimmunized F1 mice had complement-dependent cytotoxic activity (25-30% 5'Cr release) onlyagainst Y2C target cells. The activity disappeared com-

pletely when sera were diluted 1:4-1:8.Absorption experiments were performed by incubating 1

ml of 1:10 dilutions of serum with various numbers of L1210,Cl.1D, and Y2C cells at 370 for 20 min and then for an addi-tional period of 60 min at +4° with frequent shaking. Thecells were centrifugated, and the absorbed sera were testedfor their cytotoxic titers against the three cell lines. All testswere made in triplicate. All experiments were made at leasttwice.Y2C cells were tested for Sendai virus production with

supernatants of cell cultures in agglutination tests of chickenerythrocytes. Sera of F1 mice immunized with Y2C cells were

examined for the presence of antibodies against Sendai virusby measuring the inhibition of agglutination of chickenerythrocytes by Sendai virus.

RESULTS

Inoculation of YC Hybrids. Groups of x-irradiated newbornF1 mice were inoculated ip with 2.5 X 106 cells of each YC

TABLE 2. Inoculation of YC hybrids to F1 mice

Cell culture grafts SecondaryHybrid Adult X-irradiated grafts toclone mice newborn mice adult mice

Y1C 0/11 9/9 0/108/8 0/9

Y2C 0/32 9/11 0/105/5

Y4C 0/7 7/8 0/108/8

Y5C 0/10 6/8 0/190/100/100/10

Total 0/60 52/57 0/88(91%)

Numbers are mice developing tumors/total inoculated.

hybrid clone. 52/57 newborn mice developed tumors within2-4 weeks and died (91%). By contrast, none of the 60 adultF, mice developed tumors within 1 year (Table 2).

Karyologic analyses were made on cell cultures initiatedfrom eight tumors grown in newborn mice. Karyotypes oftumors resembled very closely those of the inoculated cellpopulations, but some telocentric chromosomes were missing.The modal number of chromosomes was slightly reduced(Table 3). Therefore, tumors might have developed as aconsequence of some specific chromosome loss (2).To test this hypothesis, we grafted fragments from the

tumors examined for their karyotypes (or, in some instances,2.5 X 108 cells explanted from the same tumors) to unir-radiated adult F1 mice. None of the 88 mice tested developedtumors within 9 months (Table 2). It was concluded that YChybrids failed to grow in adult mice because of immune re-jection, and grew in x-irradiated newborn because the hostswere immunologically incompetent.

Histology of Hybrid Tumors. Three different histologicfeatures were observed. (1) Fibrosarcoma (Fig. 1) similar toCl.1D tumors. (2) Lymphoid tumor (Fig. 2), closely resem-bling those developed when parental L1210 leukemia cells were

TABLE 3. Karyotype of YC tumors

Number of Number Of chromosomesHybrid clone metaphasesinoculated Tumor no. examined Total Telocentric Submetacentric "D" marker

Y1C 25 89(81-91) 81(72-83) 9(7-10) 1J159 25 85(81-94) 76(73-85) 9(8-11) 1J160 70 84(74-88) 75(65-79) 9(7-12) 1

Y2C 25 93(54-108) 82(49-96) 9(5-12) 1J162 50 90(75-113) 78(64-103) 10(7-13) 1

Y4C 25 89(81-102) 77(71-92) 12(9-12) 1J166 50 85(71-94) 73(63-84) 11(8-13) 1

Y5C 25 90(82-121) 79(73-108) 9(7-15) 1J158 54 86(77-92) 75(67-82) 11(8-13) 1J164 50 83(76-100) 74(65-84) 10(9-14) 1J167 40 85(76-91) 75(66-81) 11(7-12) 1J168 80 86(65-94) 76(57-85) 9(6-13) 1

The modal number of chromosomes per metaphase is given, with the range in parentheses.

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2132 Cell Biology: Jami and Ritz

FIG. 1. Histologic section of YC fibrosarcoma. Hematoxylinand eosin X 250.

injected subcutaneously. (3) Morphology intermediate be-tween those of the two parental tumors (Fig. 3): cell mor-

phology ranged from spindle-shaped with elongated nuclei toround with thin rims of cytoplasm. Tumoral ascites similarto those obtained in all mice inoculated with L1210 leukemiacells was never observed upon ip injections of YC hybrids.

Distribution of these features among the eight tumorsexamined appeared to be independent of the clone of origin.Two tumors were only fibrosarcoma, and two had onlyintermediate morphology. The other tumors were hetero-geneous, either mainly intermediate with areas of fibrosarcoma(2) or of lymphoid tumor (1), or mainly fibrosarcoma withareas of lymphoid tumor (1).

Syngeneic Antisera Against Y2C Hybrids. Sera of F1 miceimmunized with Y2C hybrids were pooled, and two poolswere tested for cytotoxic antibodies, using parental and hy-brid cells as target cells. Fig. 4 shows titration curves of thepool of sera used in the absorption experiments describedbelow. Titers for Y2C, Cl.lD, and L1210 target cells were

160, 80, and 15, respectively. Similar relative titers were

found with the other pool of sera for the three target cells(110, 40, and 5, respectively). Thus, Y2C hybrids were more

sensitive to antibodies than either of the parental cells.

Absorption Experiments. Cytotoxic antibodies against allthree target cell lines were completely absorbed by incubat-ing sera with Y2C cells (Fig. 5).

Aliquots of antisera were absorbed with 1: 1 mixtures ofCl.lD and L1210 parental cells. Antibodies active against

FIG. 2. Section of YC lymphoid tumor area. Hematoxylinand eosin X250.

Y2C target cells were completely absorbed, indicating thatthere was no new surface antigen on Y2C hybrid cells, i.e.,antigen absent on parental cells (Fig. 6).50% of antibodies cytotoxic for Cl.1D target cells were

absorbed by half as many Y2C cells (Fig. 7), indicatingthat Y2C cells had two times more "Cl.1D" antigenic sitesthan Cl.1D cells. 50% of antibodies cytotoxic for L1210target cells were absorbed by the same number of Y2C asL1210 cells (Fig. 8), showing that Y2C and L1210 cells hadapproximately the same number of "L1210" antigenic sites.

Diameters of 100 cells of each line were measured with anocular micrometer, and mean surface areas were calculated.Since the ratios of Y2C:Cl.lD and Y2C:L1210 cell surfaceswere 5:4 and 2:1, respectively, "Cl.1D" and "L1210"antigenic site densities on Y2C cells were estimated to beabout 1.6 and 0.5 those on Cl.1D and L1210 cells, respectively.

Test for Sendai Virus. Y2C cell cultures were found to befree of Sendai virus production, and sera of F1 mice hyperim-munized with Y2C cells had no antibody inhibiting chickenerythrocyte agglutination by Sendai virus.

DISCUSSION

The inability of mouse cell hybrids derived from two malig-nant parental cells to produce tumors in syngeneic hostswas analyzed.

Production of Sendai virus by hybrid cells, as a result ofincomplete ultraviolet-inactivation of the stock virus usedfor fusion of parental cells, was ruled out by absence of Sendaivirus in cell cultures and absence of hemagglutinating anti-

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Proc. Nat. Acad. Sci. USA 72 (1975)

FIG. 3. Section of YC tumor with intermediate morphology.Hematoxylin and eosin X 250.

bodies in mice immunized with Y2C hybrids. This eliminatesa viral oncolysis (8) to explain the apparent nonmalignancyof YC hybrid cells.

Rejection by adult F1 mice of all secondary grafts of YCtumors developed in x-irradiated newborn demonstratesthat, even if some telocentric chromosomes were lacking inhybrid tumors, specific chromosome losses are not responsiblefor YC growth in newborn mice, and that suppression ofmalignancy (2) did not occur in YC hybrid cell populations.In this respect, YC hybrids are comparable to hybrids derivedfrom highly malignant A9HT and mouse tumor cells (9).Hybrids between two malignant cells always appear malig-nant (4, 9-15), i.e., the characters determining malignancy donot show complementation (15).

Graft experiments reported here demonstrate that YChybrids fail to grow because they are rejected by adult hosts.Analysis of an antiserum prepared by injecting Y2C hybridsinto syngeneic F1 mice did not reveal the presence of new anti-gen on hybrid cells, as indicated by complete absorption bymixtures of both parental cell lines of cytotoxic antibodiesdirected against hybrid cells.The absolute number and the density of "Cl.1D" antigenic

sites on Y2C hybrids were found to be approximately 2 timesand 1.6 times, respectively, that of Cl.lD cells. This possiblyexplains the higher antigenicity and/or the higher sensitivityto immune lysis of hybrid cells. Concerning the question ofsensitivity, this explanation appears insufficient, becausecytotoxic activity of antisera absorbed by Y2C cells dis-appears first for Y2C target cells, and then for Cl.lD target

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5 20 80 160 350 1,300 5,000 20.000Serum Dilution

FIa. 4. Humoral complement-dependent cytotoxic anti-bodies of C3D2F1 mice immunized with Y2C hybrids. Titrationcurves were determined with 0, Y2C hybrids; 0, Cl.1D; and0, L1210 parental cells as target cells. Complement controlvalues were subtracted.

cells (Fig. 5). Since absorption by Y2C cells removes bothanti-"Cl.1D" and anti-"L1210" antibodies present in theantiserum, it is possible that anti-"Cl.1D" and anti-"L1210"antibodies attached to their respective antigenic sites onhybrid cell surface cooperate in binding complement, asintermixing of antigenic sites of both parental cell origins(16, 17) makes it possible (18). It is also possible that thedifference is due to intrinsic sensitivity to lysis of Y2C cells,as indicated by higher 5'Cr release background with Y2Cthan with Cl.1D cells in all experiments.

Speculations concerning quantitative antigenic variationsin parental and hybrid cells requires more information about

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( 75\

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0 1 2.5 10 20 30Number of Absorbing Y2C Cells x 10-6

FIG. 5. Absorption of antibodies by Y2C hybrids. Eachsample was titrated in triplicate with each cell line: 0, Y2Chybrids; 0, Cl.1D; and 0, L1210 parental cells.

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2134 Cell Biology: Jami and Ritz

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6 i i.5 5 10 20 40 60Number of Absorbing C11D and L1210 Cells x 10-6

FIG. 6. Absorption of antibodies with 1: 1 mixtures of Cl.1Dand L1210 parental cells. Each sample was titrated in triplicatewith each cell line: 0, Y2C hybrids; 0, Cl.1D; and 0, L1210parental cells.

the chromosome constitution of YC hybrids, which are de-rived from two aneuploid cells. However, H-2k antigenicdensity of Y2C cells is 0.55 of that on Cl.1D cells (19). Thisfact, taken together with the present results, suggests an

inverse relationship between the quantitative expression ofH-2 and tumor-associated antigens (20, 21).

Histologic features of hybrid tumors did not provide clear-cut information about regulation of phenotypic expression.The intermediate morphology of five of eight tumors suggestsquantitative variations in expression of differentiated traits.Greater frequency of fibrosarcoma than lymphoid morphol-ogy indicates the possible role of gene dosage in phenotypicexpression in hybrid cells (4, 22), since parental fibroblastswere hypotetraploid and parental leukemic cells were onlypseudo-diploid. Lymphoid morphology, even limited to

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FIG. 7. Absorption of antibodies with *, Y2C; or 0, Cl.1Dcells. Each sample was titrated in triplicate, using Cl.1D as

target cells.

a 7'Inaa'

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FIG. 8. Absorption of antibodies with *, Y2C; or 0, L1210cells. Each sample was titrated in triplicate, using L1210 cells astarget cells.

small area in two of eight tumors, suggests that informationfor lymphoid morphology is carried cryptically for manygenerations in hybrid cells (23).

J.J. is charg6 de recherche A l'Institut National de la SantAet de la Recherche M6dicale. This work was conducted with theaid of a grant from the Institut National de la Sant, et de laRecherche M6dicale.

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T. (1969) Nature 223, 363-368.3. Jami, J. & Ritz, E. (1973) Cancer Res. 33, 2524-2528.4. Jami, J., Failly, C. & Ritz, E. (1973) Exp. Cell Res. 76,

191-199.5. Peterson, J. & Weiss, M. C. (1972) Proc. Nat. Acad. Sci.

USA 69, 571-575.6. Ephrussi, B. (1972) Hybridization of Somatic Cells (Prince-

ton IJniv. Press, Princeton, N.J.).7. Jami, J. & Ritz, E. (1975) J. Nat. Cancer Inst. 54, 117-122.8. Lindenmann, J. (1964) J. Immunol. 92, 912-919.9. Wiener, F., Klein, G. & Harris, H. (1973) J. Cell Sci. 12,

253-261.10. Defendi, V., Ephrussi, B., Koprowski, H. & Yoshida, M. C.

(1967) Proc. Nat. Acad. Sci. USA 57, 299-305.11. Scaletta, L. & Ephrussi, B. (1965) Nature 207, 1169-1171.12. Silagi, S. (1967) Cancer Res. 27, 1953-1960.13. Belehradek, J., Jr. & Barski, G. (1971) imt. J. Cancer 8,

1-9.14. Blanchard, M. G., Barski, G., IAon, B. & H6mon, D. (1973)

Int. J. Cancer 11, 178-185.15. Wiener, F., Klein, G. & Harris, H. (1974) J. Cell Sci. 16,

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471-475.19. Rubio, N. (1974) Nature 249, 491-463.20. Haywood, G. R. & McKhann, C. F. (1971) J. Exp. Med.

133, 1171-1187.21. Ting, C. C. & Herberman, R. B. (1971) Nature New Biol.

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