Tumor-Associated Transplantation Antigens in Immune Rejection of Mouse Malignant Cell Hybrids

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1 Proc. Nat. Acad. Sci. USA Vol. 72, No. 6, pp , June 1975 Tumor-Associated Transplantation Antigens in Immune Rejection of Mouse Malignant 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, Villejuif, France Communicated by Boris Ephrussi, March 14, 1976 ABSTRACT The inability of mouse cell hybrids derived from two malignant parental cells to produce tumors in syngeneic F, hosts was analyzed. YC hybrids, derived from the fusion of Cl. ID and L1210 cells, failed to induce any tumor in adult mice, while 91% of x-irradiated newborn mice developed tumors and died. Some telocentric chromosomes were lacking in hybrid tumors; however, none of the immunologically intact adult mice developed tumors when grafted with tumors grown in x-irradiated newborn mice. This indicates that histocompatibility factors interfered in the failure of YC tumors to grow in adult hosts. Syngeneic F, mice immunized with Y2C hybrid cells had cytotoxic antibodies against Y2C hybrids and Cl. ID and L1210 parental cells. Complete absorption of cytotoxic antibodies directed against hybrid cells by mixtures of both parental cell lines demonstrates the absence of any new antigen on hybrid cells. Hybrid cells had a higher density of Cl. 1D-tumor antigenic sites, as compared to Cl.1D parental cells. This possibly explains the higher antigenicity and/or the higher sensitivity to immune lysis of hybrid cells. In previous experiments, hybrids (YC) from two mouse malignant cells (L1210 and Cl.1D) failed to develop tumors in adult compatible hosts (1). However, it appeared doubtful that malignancy of both parental cells was suppressed by complementation (2), because YC hybrids in culture had characteristics of malignant cells (1), and expressed the tumor-associated transplantation antigens of L1210 parental cells (3). Further, hybrids between the same Cl.1D cell line and mouse teratocarcinoma cells were malignant (4). Experiments reported here demonstrate that the inability of YC hybrids to grow in vivo is due to histocompatibility factors. As Cl.lD and L1210 cells, either separately or mixed together, produced tumors in F, mice (1), an analysis of hybrid-tumor-associated antigens was performed. Three possible hypotheses were considered to explain the efficiency of immune reaction. First, hybrid cells might express parental-tumor-associated antigens in a greater amount than parental cells. Second, silent genes in parental cells might express themselves upon hybridization (5), resulting in new antigens on the surface of hybrids. Third, hybrid molecules on hybrid cell surface, having new antigenic determinants, might result from combination of molecule subunits coded by homologous parental genes in hybrids. The existence of hybrid molecules in somatic cell hybrids is well documented (6) Ṫhe origin of tumor-associated surface antigens of YC hybrids was analyzed by absorbing with parental and hybrid cells 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 antigenic determinants. MATERIALS AND METHODS The origin and description of Cl.lD fibroblastic parental cells, of L1210 leukemic parental cells, and of the four YC hybrid clones were reported elsewhere (1). Cl.lD cells were derived 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 (except L1210 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 e DBA/2)Fl (C3D2F1) mice within 48 hr after birth. All newborn mice were x-irradiated (400 R) prior to inoculation. Tumors were excised and divided into three parts. One part was grafted to groups of nonirradiated adult F1 mice. The second part was fixed in Bouin's solution for histology. Single-cell suspensions obtained from the third part were used to initiate cell cultures (7). Karyotypes were analyzed after staining with acetoorcein. Antisera against Y2C hybrid cells were prepared by hyperimmunizing 4-month-old syngeneic C3D2F1 males with Y2C cells harvested in Versene solution and suspended in growth medium (Table 1). Sera obtained by tail bleeding were heatinactivated at 560, pooled, and stored at -80 until tested. Assays for antibody-mediated cytotoxicity were made with Hanks' solution supplemented with 4% fetal calf serum free TABLE 1. Immunization schedule* Immunization Date Cell dose/mouse no. (weeks) (X 10-6) * All immunizations were performed ip. Mice were bled 9, 11, 13, 15, 17, and 19 weeks after initiation of immunization. 2130

2 Proc. Nat. Acad. Sci. USA 72 (1975) Malignancy of Hybrid Cells 2131 of gammaglobulin (HIPT), measuring release of 5'Cr from labeled target cells. Cytotoxic titers of sera were determined by incubating 0.1 ml of serial 2-fold dilutions of serum with 0.05 ml of 5'Cr-labeled target cells (2.5 X 104 cells) and 0.1 ml of 1:10 dilution of rabbit serum as a source of complement, for 45 min at 37. Then 1.8 ml of cold HIPT was added to each tube, the cells were centrifugated at 800 X g for 10 min, and the radioactivity of 1 ml of supernatants and that remaining in the original tubes were counted in a well-type gamma counter to determine the percentage of 5"Cr released in 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 than with Cl.1D and L1210 cells. The cytotoxic index is expressed as cytotoxicity (%) in experimental tubes minus cytotoxicity (%) in complement control tubes, and is corrected for maximal 51Cr released by freezing and thawing cells three times. The titer of the serum is expressed as the reciprocal of the dilution that produced 50% of the maximum release above complement control. Sera from some nonimmunized F1 mice had complementdependent cytotoxic activity (25-30% 5'Cr release) only against Y2C target cells. The activity disappeared completely 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 additional period of 60 min at +4 with frequent shaking. The cells were centrifugated, and the absorbed sera were tested for their cytotoxic titers against the three cell lines. All tests were made in triplicate. All experiments were made at least twice. Y2C cells were tested for Sendai virus production with supernatants of cell cultures in agglutination tests of chicken erythrocytes. Sera of F1 mice immunized with Y2C cells were examined for the presence of antibodies against Sendai virus by measuring the inhibition of agglutination of chicken erythrocytes by Sendai virus. RESULTS Inoculation of YC Hybrids. Groups of x-irradiated newborn F1 mice were inoculated ip with 2.5 X 106 cells of each YC TABLE 3. TABLE 2. Inoculation of YC hybrids to F1 mice Cell culture grafts Secondary Hybrid Adult X-irradiated grafts to clone mice newborn mice adult mice Y1C 0/11 9/9 8/8 0/9 Y2C 0/32 9/11 5/5 Y4C 0/7 7/8 8/8 Y5C 6/8 0/19 Total 0/60 52/57 0/88 (91%) Numbers are mice developing tumors/total inoculated. hybrid clone. 52/57 newborn mice developed tumors within 2-4 weeks and died (91%). By contrast, none of the 60 adult F, mice developed tumors within 1 year (Table 2). Karyologic analyses were made on cell cultures initiated from eight tumors grown in newborn mice. Karyotypes of tumors resembled very closely those of the inoculated cell populations, but some telocentric chromosomes were missing. The modal number of chromosomes was slightly reduced (Table 3). Therefore, tumors might have developed as a consequence 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 unirradiated adult F1 mice. None of the 88 mice tested developed tumors within 9 months (Table 2). It was concluded that YC hybrids failed to grow in adult mice because of immune rejection, and grew in x-irradiated newborn because the hosts were immunologically incompetent. Histology of Hybrid Tumors. Three different histologic features were observed. (1) Fibrosarcoma (Fig. 1) similar to Cl.1D tumors. (2) Lymphoid tumor (Fig. 2), closely resembling those developed when parental L1210 leukemia cells were Karyotype of YC tumors Number of Number Of chromosomes Hybrid clone metaphases inoculated Tumor no. examined Total Telocentric Submetacentric "D" marker Y1C 25 89(81-91) 81(72-83) 9(7-10) 1 J (81-94) 76(73-85) 9(8-11) 1 J (74-88) 75(65-79) 9(7-12) 1 Y2C 25 93(54-108) 82(49-96) 9(5-12) 1 J (75-113) 78(64-103) 10(7-13) 1 Y4C 25 89(81-102) 77(71-92) 12(9-12) 1 J (71-94) 73(63-84) 11(8-13) 1 Y5C 25 90(82-121) 79(73-108) 9(7-15) 1 J (77-92) 75(67-82) 11(8-13) 1 J (76-100) 74(65-84) 10(9-14) 1 J (76-91) 75(66-81) 11(7-12) 1 J (65-94) 76(57-85) 9(6-13) 1 The modal number of chromosomes per metaphase is given, with the range in parentheses.

3 2132 Cell Biology: Jami and Ritz Proc. Nat. Acad. Sci. USA 72 (1975) FIG. 1. Histologic section of YC fibrosarcoma. Hematoxylin and eosin X 250. injected subcutaneously. (3) Morphology intermediate between those of the two parental tumors (Fig. 3): cell morphology ranged from spindle-shaped with elongated nuclei to round with thin rims of cytoplasm. Tumoral ascites similar to those obtained in all mice inoculated with L1210 leukemia cells was never observed upon ip injections of YC hybrids. Distribution of these features among the eight tumors examined appeared to be independent of the clone of origin. Two tumors were only fibrosarcoma, and two had only intermediate morphology. The other tumors were heterogeneous, either mainly intermediate with areas of fibrosarcoma (2) or of lymphoid tumor (1), or mainly fibrosarcoma with areas of lymphoid tumor (1). Syngeneic Antisera Against Y2C Hybrids. Sera of F1 mice immunized with Y2C hybrids were pooled, and two pools were tested for cytotoxic antibodies, using parental and hybrid cells as target cells. Fig. 4 shows titration curves of the pool of sera used in the absorption experiments described below. 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 all three target cell lines were completely absorbed by incubating sera with Y2C cells (Fig. 5). Aliquots of antisera were absorbed with 1: 1 mixtures of Cl.lD and L1210 parental cells. Antibodies active against FIG. 2. Section of YC lymphoid tumor area. Hematoxylin and eosin X250. Y2C target cells were completely absorbed, indicating that there 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), indicating that Y2C cells had two times more "Cl.1D" antigenic sites than Cl.1D cells. 50% of antibodies cytotoxic for L1210 target cells were absorbed by the same number of Y2C as L1210 cells (Fig. 8), showing that Y2C and L1210 cells had approximately the same number of "L1210" antigenic sites. Diameters of 100 cells of each line were measured with an ocular micrometer, and mean surface areas were calculated. Since the ratios of Y2C:Cl.lD and Y2C:L1210 cell surfaces were 5:4 and 2:1, respectively, "Cl.1D" and "L1210" antigenic site densities on Y2C cells were estimated to be about 1.6 and 0.5 those on Cl.1D and L1210 cells, respectively. Test for Sendai Virus. Y2C cell cultures were found to be free of Sendai virus production, and sera of F1 mice hyperimmunized with Y2C cells had no antibody inhibiting chicken erythrocyte agglutination by Sendai virus. DISCUSSION The inability of mouse cell hybrids derived from two malignant parental cells to produce tumors in syngeneic hosts was analyzed. Production of Sendai virus by hybrid cells, as a result of incomplete ultraviolet-inactivation of the stock virus used for fusion of parental cells, was ruled out by absence of Sendai virus in cell cultures and absence of hemagglutinating anti-

4 Proc. Nat. Acad. Sci. USA 72 (1975) Malignancy of Hybrid Cells C.) 250 0~~~~~~~~~~~~~~~ ,300 5, Serum Dilution FIa. 4. Humoral complement-dependent cytotoxic antibodies of C3D2F1 mice immunized with Y2C hybrids. Titration curves were determined with 0, Y2C hybrids; 0, Cl.1D; and 0, L1210 parental cells as target cells. Complement control values were subtracted. FIG. 3. Section of YC tumor with intermediate morphology. Hematoxylin and eosin X 250. bodies in mice immunized with Y2C hybrids. This eliminates a viral oncolysis (8) to explain the apparent nonmalignancy of YC hybrid cells. Rejection by adult F1 mice of all secondary grafts of YC tumors developed in x-irradiated newborn demonstrates that, even if some telocentric chromosomes were lacking in hybrid tumors, specific chromosome losses are not responsible for YC growth in newborn mice, and that suppression of malignancy (2) did not occur in YC hybrid cell populations. In this respect, YC hybrids are comparable to hybrids derived from highly malignant A9HT and mouse tumor cells (9). Hybrids between two malignant cells always appear malignant (4, 9-15), i.e., the characters determining malignancy do not show complementation (15). Graft experiments reported here demonstrate that YC hybrids fail to grow because they are rejected by adult hosts. Analysis of an antiserum prepared by injecting Y2C hybrids into syngeneic F1 mice did not reveal the presence of new antigen on hybrid cells, as indicated by complete absorption by mixtures of both parental cell lines of cytotoxic antibodies directed against hybrid cells. The absolute number and the density of "Cl.1D" antigenic sites on Y2C hybrids were found to be approximately 2 times and 1.6 times, respectively, that of Cl.lD cells. This possibly explains the higher antigenicity and/or the higher sensitivity to immune lysis of hybrid cells. Concerning the question of sensitivity, this explanation appears insufficient, because cytotoxic activity of antisera absorbed by Y2C cells disappears first for Y2C target cells, and then for Cl.lD target cells (Fig. 5). Since absorption by Y2C cells removes both anti-"cl.1d" and anti-"l1210" antibodies present in the antiserum, it is possible that anti-"cl.1d" and anti-"l1210" antibodies attached to their respective antigenic sites on hybrid cell surface cooperate in binding complement, as intermixing of antigenic sites of both parental cell origins (16, 17) makes it possible (18). It is also possible that the difference is due to intrinsic sensitivity to lysis of Y2C cells, as indicated by higher 5'Cr release background with Y2C than with Cl.1D cells in all experiments. Speculations concerning quantitative antigenic variations in parental and hybrid cells requires more information about %~~~~ ( 75\ CL Number of Absorbing Y2C Cells x 10-6 FIG. 5. Absorption of antibodies by Y2C hybrids. Each sample was titrated in triplicate with each cell line: 0, Y2C hybrids; 0, Cl.1D; and 0, L1210 parental cells.

5 2134 Cell Biology: Jami and Ritz Proc. Nat. Acad. Sci. USA 72 (1975) 100-0% el (A U ( % %% a 7' In a u 54 LC) 25_.0 Cn 2' 6 i i Number of Absorbing C11D and L1210 Cells x 10-6 FIG. 6. Absorption of antibodies with 1: 1 mixtures of Cl.1D and L1210 parental cells. Each sample was titrated in triplicate with each cell line: 0, Y2C hybrids; 0, Cl.1D; and 0, L1210 parental cells. the chromosome constitution of YC hybrids, which are derived from two aneuploid cells. However, H-2k antigenic density of Y2C cells is 0.55 of that on Cl.1D cells (19). This fact, taken together with the present results, suggests an inverse relationship between the quantitative expression of H-2 and tumor-associated antigens (20, 21). Histologic features of hybrid tumors did not provide clearcut information about regulation of phenotypic expression. The intermediate morphology of five of eight tumors suggests quantitative variations in expression of differentiated traits. Greater frequency of fibrosarcoma than lymphoid morphology indicates the possible role of gene dosage in phenotypic expression in hybrid cells (4, 22), since parental fibroblasts were hypotetraploid and parental leukemic cells were only pseudo-diploid. Lymphoid morphology, even limited to 0 cn , u) 5 0 en %% Number of Absorbing Cells x 10-6 FIG. 7. Absorption of antibodies with *, Y2C; or 0, Cl.1D cells. Each sample was titrated in triplicate, using Cl.1D as target cells Number of Absorbing Cells x 10-6 FIG. 8. Absorption of antibodies with *, Y2C; or 0, L1210 cells. Each sample was titrated in triplicate, using L1210 cells as target cells. small area in two of eight tumors, suggests that information for lymphoid morphology is carried cryptically for many generations in hybrid cells (23). J.J. is charg6 de recherche A l'institut National de la SantA et de la Recherche M6dicale. This work was conducted with the aid of a grant from the Institut National de la Sant, et de la Recherche M6dicale. 1. Jami, J. & Ritz, E. (1973) J. Nat. Cancer Inst. 51, Harris, H., Miller, 0. J., Klein, G., Worst, P. & Tachibana, T. (1969) Nature 223, Jami, J. & Ritz, E. (1973) Cancer Res. 33, Jami, J., Failly, C. & Ritz, E. (1973) Exp. Cell Res. 76, Peterson, J. & Weiss, M. C. (1972) Proc. Nat. Acad. Sci. USA 69, Ephrussi, B. (1972) Hybridization of Somatic Cells (Princeton IJniv. Press, Princeton, N.J.). 7. Jami, J. & Ritz, E. (1975) J. Nat. Cancer Inst. 54, Lindenmann, J. (1964) J. Immunol. 92, Wiener, F., Klein, G. & Harris, H. (1973) J. Cell Sci. 12, Defendi, V., Ephrussi, B., Koprowski, H. & Yoshida, M. C. (1967) Proc. Nat. Acad. Sci. USA 57, Scaletta, L. & Ephrussi, B. (1965) Nature 207, Silagi, S. (1967) Cancer Res. 27, Belehradek, J., Jr. & Barski, G. (1971) imt. J. Cancer 8, Blanchard, M. G., Barski, G., IAon, B. & H6mon, D. (1973) Int. J. Cancer 11, Wiener, F., Klein, G. & Harris, H. (1974) J. Cell Sci. 16, Watkins, J. F. & Grace, D. M. (1967) J. Cell Sci. 2, Frye, L. D. & Edidin, M. (1970) J. Cell Sci. 7, Lengerova, A. & Peknicova, 'J. (1973) Eur. J. Cancer 9, Rubio, N. (1974) Nature 249, Haywood, G. R. & McKhann, C. F. (1971) J. Exp. Med. 133, Ting, C. C. & Herberman, R. B. (1971) Nature New Biol. 232, Fougbre, C., Ruiz, F. & Ephrussi, B. (1972) Proc. Nat. Acad. Sci. USA 69, Wiener, F., Cochran, A., Klein, G. & Harris, H. (1972) J. Nat. Cancer Inst. 48,