Loss of anti-tumour immunogenicity of a somatic cell hybrid line with increasing subculture.

Good immunoprotection was afforded by A9/SEWA somatic hybrid cells in the C3H mouse/C3H Py tumour system, confirming results previously obtained in the A.SW mouse/SEWA tumour system. However, in this study the immunogenicity decreased with increasing serial subculture of the hybrid line and concomitant chromosome loss.     

Novel antigen expression on syngeneic somatic cell hybrids of murine spontaneous mammary tumor cells

Spontaneous mammary carcinoma cells of C3H/He mice were fused with syngeneic L-cells, and two types of hybrid cell clones were obtained. In group A, hybrid cells showed contact inhibition, and parental H-2k and L-antigens were well expressed. Metacentric chromosomes of L-cell origin and estrogen dependency were also well preserved in this group. However, in group B, hybrid cells proliferated to pile up, and the expression of parental antigens, H-2 and L-cell antigens, was strongly suppressed. But, mouse mammary tumor antigens (MM-antigens), which were expressed on ascites mammary tumor cells with hypotetraploidy of C3H/He origin and which were not expressed on both parent cells, were newly expressed. The number of metacentric chromosomes was decreased, and estrogen dependency was lost in this group. The relationship between the expression of MM-antigens and that of H-2 antigens was reciprocal, and tumorigenicity was independent of cellular behavior in vitro and of MM-antigen expression. MM-antigen-positive hybrid cell clones were frequently obtained when tumor cells were fused with metaphase-rich L-cells.     

Humoral antibody response and tumor transplantation resistance elicited by fetal tissues in mice.

Immune responses to fetal antigen immunization were studied in C57BL/6 (B6), C3H/HeN (C3H), and BALB/c (BALB) mice. In tests by the isotopic antiglobulin technique, the mice could be grouped in three classes according to their antibody responses: good responders (B6), poor responders (C3H), and nonresponders (BALB). In tumor transplantation experiments, protection against syngeneic tumor-cell challenge, after fetal tissue immunization, was observed only with B6 mice. In addition, experiments on B6 mice showed that resistance to tumor challenge after fetal tissue immunization depended on the quantitative expression of fetal antigens by the tumor cells. Our results indicated that, in addition to other well-known factors, the effectiveness of fetal tissue immunization in tumor-challenge resistance depended on both host responsiveness and the amount of fetal antigen expressed by the tumor cells.     

Characterization of "H-2K,D-like structures" on MM2 x mouse L cell hybrids. II. An "H-2Kd-like" structure on C3H-derived tumor

Anti-MM2 serum, which had been prepared by immunizing C3H/He mice with syngeneic MM2 mouse mammary ascites tumor, immunoprecipitated "H-2K,D-like" molecules on DBA/2 and C3H.H-2 degrees lymph node cells as well as on somatic cell hybrids between MM2 tumor and mouse L cells. Preclearing of lysates from C3H.H-2 degrees lymph node cells with anti-H-2.31 serum removed all "H-2K,D-like" molecules reactive with anti-MM2 serum, indicating that the molecules detected by anti-MM2 serum are H-2Kd antigens. The anti-H-2.31 serum detected an "H-2K,D-like structure" on the hybrid cells and absorption of the anti-H-2.31 serum with the hybrid cells deprived the serum of anti-H-2Kd reactivity. The hybrid cells could induce antibodies against the H-2Kd antigen in C3H/He mice. These results indicate that on the hybrid cells, whose parental cells were both derived from C3H mice, there is an "H-2K,D-like structure" that has the H-2Kd private specificity. Absorption of anti-MM2 serum with EL4 cells did not affect the capacity of the serum to detect the H-2Kd antigen on C3H.H-2 degrees lymph node cells, indicating that the "H-2Kd-like structure" is distinct from "H-2K,D-like structure A" which was previously reported. Nine isozymes were examined and MM2 cells, mouse L cells, and the hybrids were found to have the same isozyme markers as those of the C3H/He mouse.     

Adoptive transfer of immunity induced by semi-allogeneic hybrid cells,against a murine fibrosarcoma

Semi-allogeneic somatic hybrid cells derived from the fusion of a C57BL/6 fibrosarcoma (MCB6–1) and A9 cells (C3H origin) were used to immunize C57BL/6 mice against the parental tumor cells. These hybrid cells expressed H-2 histocompatibility antigens of both parental cells (H-2_b and H-2^k), and failed to produce tumors in normal C57BL/6 mice. A single i.p. injection of hybrid cells induced anti-tumor immunity which could be transferred to normal C57BL/6 recipient mice by immune spleen or peritoneal cells; the efficient cells were T cells, as this activity was completely abrogated by treatment with anti-Thy-1–2 antiserum and complement. Among immune splenic T cells, only the light-density T cells, obtained after fractionation on Percoll gradient, were effective in the transfer of immunity. Immunity induced by the hybrid cells was specific for MCB6-1 parental tumor cells. This immunity could be transferred during two brief periods, 7 to 12 days, and 40 to 50 days, after hybrid cell injection; there appeared to be an intermediate period, 12 to 40 days after immunization, during which no immunity could be transferred. These results suggest a suppressive mechanism implicated during hybrid cell immunization and interacting with the anti-tumor immune response.     

Therapeutic tumor immunity induced by polyimmunization with melanoma antigens gp100 and TRP-2

To improve the immunogenicity of melanoma self-antigens, we used a novel strategy of nonviral genetic vaccination coupled with muscle electroporation. Electroporation-enhanced immunization with plasmids encoding either human gp100 or mouse TRP-2 antigens induced only partial rejection of B16 melanoma challenge. However, immunization with a combination of these two antigens caused tumor rejection in 100% of the immunized mice. Splenocytes from combination-immunized animals killed syngeneic targets loaded with peptides derived from both gp100 and TRP-2. Immune cell depletion experiments identified CD8+ T lymphocytes as the primary effectors of antitumor immunity. Most importantly, polyimmunization led to the generation of a therapeutic immune response that significantly improved the mean survival time of mice bearing established lung metastases. These results validated the usefulness of electroporation-enhanced, nonviral genetic immunization for the active immunotherapy of cancer and indicated that using a combination of different tumor antigens may be a decisive strategy for a successful therapeutic vaccination.     

Comparison between different techniques of immuno-protection in the A-SW mouse-SEWA tumour system

Various procedures for immunoprotection in the A-SW mouse SEWA tumour system were compared. Four types of protector agents were used: polyoma virus, allogeneic S724 cells, A9/SEWA hybrid cells and xenogeneic ST_py cells. Several injection schedules were also employed. On the whole, reasonable immunoprotection was obtained. The results, when allogeneic hybrid or xenogeneic cells were used, were comparable with those obtained for polyoma virus. This indicates that the T.A.T.A. of the SEWA tumour had been well conserved. When polyoma virus alone was used as the protector agent, injection 7 days before the tumour challenge, or iterative injection, afforded better protection than a single injection on the day of challenge.     

Expression of alien minor histocompatibility antigens distinct from tumor-specific transplantation antigen on a murine fibrosarcoma

The fibrosarcoma ST2, induced by 3-methylcholan-threne in BALB/c (H-2^d) mice, also expressed alien histocompatibility antigens of the C3Hf and B10 background not encoded by the MHC. To examine the relationship between these alien, minor antigens and the tumor-specific transplantation antigen (TSTA) of the tumor, in vivo immunogenicity test were performed in BALB/c mice and in hybrids between BALB/c and C3Hf (H-2^k), C3H.OH (H-2⁰²), C3H.SW (H-2^b), BALB.K (H-2^k), B10.BR (H-2^k), and B10.D2 (H-2^d) mice. A significant loss of TSTA immunogenicity was found in (BALB/c X C3Hf) and in (BALB/c X C3H.OH)F₁ animals and, to a lesser extent, in (BALB/c X C3H.SW)F₁ mice as compared to the immunogenicity of the tumor in BALB/c mice. Immunogenicity tests with ST2 in BALB/c X (BALB/c X C3Hf) or in BALB/c X (BALB/c X B10.D2) backcross mice, respectively, revealed that half of the BALB/c X (BALB/c X C3Hf) and 97% of the BALB/c X (BALB/c X B10.D2) animals were able to mount an immune response to ST2. To see whether the loss of TSTA immunogenicity in (BALB/c X C3Hf) was due to common determinants shared between TSTA and alien non-H-2 C3Hf antigens or to a genetically linked low responsiveness to TSTA introduced by C3Hf and C3H.OH strains, BALB/c mice were immunized with normal normal tissues of some BALB/c X (BALB/c X C3Hf) back-cross, anti-ST2 resistant mice. Normal tissues of anti-ST2 resistant, dd and dk typed backcrosses were able to immunize BALB/c mice against a challenge of an otherwise lethal dose of ST2 cells. Some but not all BALB/c X (BALB/c X B10.D2) anti-ST2 resistant donors had tissues able to immunize BALB/c hosts against the ST2 growth. Since resistance to tumor growth and expression of minor “alien” antigens shared with the tumor segregate independently, we concluded that alien, minor C3Hf and B10 antigens of the BALB/c sarcoma ST2 are distinct from the TSTA of this tumor.     

Immune response to somatic cell hybrids between ultraviolet radiation-induced regressor and spontaneous progressor C3H mouse tumor cells

We used somatic cell hybridization to determine whether the regressor phenotype exhibited by UV-induced murine tumors was dominant or recessive and whether this technique could confer immunogenic properties on nonimmunogenic syngeneic tumors. We transfected a highly antigenic UV-induced C3H mouse tumor cell line (UV-2240) with the plasmid pSV2-neo and selected G418-resistant clones. The resulting cell line was fused with a spontaneously transformed nonimmunogenic C3H progressor tumor cell line (SF-2T) that had been selected previously for resistance to 3.0 mM ouabain. These two cell lines were fused by a brief exposure to polyethylene glycol and heterokaryons isolated by growth in medium containing both G418 and ouabain. Hybrid cell lines established from individual colonies and from pools of colonies were tested for tumorigenicity in normal C3H and athymic nude mice. The results indicated that all the hybrid cell lines tested were highly antigenic in that they were completely rejected when transplanted into normal syngeneic mice but grew progressively in nude mice. Furthermore, immunization of C3H mice with the hybrid cell lines induced protective immunity against challenge with the immunizing tumor and generated cross-protective immunity against challenge with the regressor parental cell line but not against challenge with the progressor parental cell line. These results demonstrate that the regressor phenotype of the UV-2240 tumor is dominant in nature and that the immune response induced by somatic cell hybrids is uniquely directed against the dominant tumor-specific transplantation antigens expressed on the regressor tumor. This implies that introduction of tumor-specific transplantation antigens from an immunogenic tumor into a nonimmunogenic tumor, although sufficient to confer immunogenic properties to the hybrid, is insufficient to induce cross-protective transplantation immunity against the nonimmunogenic tumor.     

Redirecting adaptive immunity against foreign antigens to tumors for cancer therapy

Immunotherapy for cancer is often limited by weak immunogenicity of tumor antigens. However, immune systems are usually strong and effective against foreign invading antigens. To test whether the destructive effect of adaptive immunity against foreign antigens can be redirected to tumors for cancer therapy, we immunized mice with adenovector expressing LacZ (Ad/CMV-LacZ). Subcutaneous syngeneic tumors were then established in the immunized animals or in na?ve animals. The immune response against adenovirus or LacZ was redirected to tumors by intratumoral injection of Ad/CMV-LacZ. We found that immunization and treatment with the adenovector dramatically reduced the tumor growth rate compared with intratumoral administration of adenovector in na?ve mice. Complete tumor regression was observed in about 50% of the immunized animals but not in the na?ve animals. Similar effects were observed when oncolytic vaccinia virus was used to immunize and treat tumors. Lymphocyte infiltration in tumors was dramatically increased in the immunized group when compared with other groups. Moreover, immunity against parental tumor cells was induced in the animals cured with immunization and treatment with Ad/CMV-LacZ, as evidenced by the lack of tumor growth when the mice were challenged with parental tumor cells. Taken together, these results suggest that redirecting adaptive immunity against foreign antigens is a potential approach for anticancer therapy and that pre-existing immunity could enhance virotherapy against cancers.     

Quantitation of viral antigens released into plasma and culture fluids by murine mammary tumor cells

Radioimmunoassay capable of measuring mouse mammary tumor virus (MMTV) 52,000 M.W. envelope glycoprotein (gp52) and 27,000 M.W. protein (p27) have been used to quantitatively compare plasma concentrations of these viral antigens in mice bearing spontaneous mammary tumors. Although gp52 was detected in the plasma of all tumor-bearing mice tested, p27 was detected in only a portion of tumor-bearing animals. In p27-positive animals, gp52 was detected in higher concentrations than p27. These findings demonstrate that gp52 has preferential utility as a plasma marker for the presence of mammary tumors in MMTV-infected hybrid (BALB/c x DBA/8 F1), Paris RIII, and C3H/HeJ mice. In addition, cultures of MMTV-producing cells [GR-MMTV and MMTV(C3H)Fel I] were used as models to study the release of viral antigens in the absence of serum antibody or additional host factors. Comparisons of extracellular soluble and particulate antigen concentrations demonstrated that gp52 and p27 were present in substantially higher concentrations as soluble than virion-associated antigens. The mean ratio of non-virion-associated gp52 to virion-associated gp52 was 12.5:1 for GR-MMTV cells and 37.3:1 for MMTV(C3H)Fel I cells. The marked stability of MMTV in culture fluids suggested that virion breakdown was not responsible for the accumulation of soluble viral antigens in culture. The information obtained suggests that abundant virus-free antigens may be of greater use than virion-associated antigens as a source of viral antigen to evaluate mammary tumor status.     

Immunological studies on mouse mammary tumors. IV. Extraction and solubilization of transplantation antigen of mouse mammary tumor

Antigens present in mouse mammary tumor MM2 reacting with tumor-specific antibodies were extracted with 0.2% deoxycholate. After the removal of deoxycholate by dialysis, most antigens were precipitated by centrifugation at 105,000 × g for 60 min. Hyperimmunization of C3H/He mice with these antigens induced heightened resistance against MM2. Antibodies appeared in the serum after a booster injection of deoxycholate extract of tumor cells was given. Antigens were solubilized from deoxycholate extract with distilled water. Purified soluble antigen inhibited the neutralization activity of the tumor-specific antibody to MM2 tumor. From these observations, it is concluded that transplantation antigens of isografted mammary tumors in C3H/He mice are present in deoxycholate extracts of MM2 tumor.     

Down-regulation of MHC class I antigens is not a general mechanism for the increased tumorigenicity caused by c-myc amplification

Previous studies have shown that increased expression of oncogenes from the myc-family can down-regulate the level of MHC class I antigens in tumor cells. This has suggested a mechanism by which amplification/overexpression of myc-genes may contribute to the malignancy development of certain tumors. Earlier published data from the murine SEWA tumor, a polyomavirus-induced osteosarcoma, have correlated the degree of tumorigenicity of different sublines to their level of c-myc amplification. Here I present a quantitative and qualitative analysis of MHC class I antigens from five sublines of this tumor system. No differences could be found, between sublines with different degrees of tumorigenicity, regarding MHC class I antigen expression. Thus, in the SEWA tumor, the enhancement of the tumorigenicity caused by c-myc amplification is not mediated through down-regulation of MHC class I antigens.     

Characterization of "H-2K,D-like structures" on MM2 X mouse L cell hybrids. I. "H-2K,D-like" alloantigen encoded by the D region and/or to the right of the D region of the H-2 gene complex

It was found that anti-MM2 serum, which had been prepared by immunizing C3H/He mice with syngeneic MM2 mouse mammary ascites tumor, detected molecules of 44-46,000 and 12,000 daltons on EL4 leukemic cells and on C57BL/6 lymph node cells, as well as on somatic cell hybrids between the MM2 and mouse L cells. Experiments with a known rabbit anti-mouse beta2-microglobulin serum showed that the two molecules detected by anti-MM2 serum were hydrophobically associated with each other in membrane extracts; thus, the antigen detected by anti-MM2 serum was structurally similar to H-2K and D antigens. Preclearing of lysates from C57BL6 lymph node cells with a mixture of anti-H-22, anti-H-2.33, and anti-H-2.28 sera did not remove the "H-2-like" antigen, indicating that the antigen was distinct from the H-2Kb and Db molecules. Neither C3H/He nor B10.BR lymph node cells expressed the "H-2-like" antigen, but B10.A(4R) and B10.AM lymph node cells did possess the antigen. Absorption of anti-MM2 serum with EL4 cells abolished the capacity of the absorbed serum to precipitate the "H-2-like" antigen activity on C57BL/6 lymph node cell extracts and reduced the "H-2-like" radioactive peaks of the hybrid cells. These results indicate that there are at least two components being recognized by the anti-MM2 serum in hybrid cells between MM2 tumor and mouse L cells, both of which had originated in the C3H/He mouse (H-2k). One is the same as or cross-reactive with an "H-2-like" alloantigen of normal C57BL/6 lymph node cells (H-2d) and the other is another "H-2-like" antigen. Experiments with recombinant mice show that the "H-2-like" alloantigen on lymph node cells is coded for by the D region and/or to the right of the D region of the major histocompatibility complex (MHC).     

CD80 CD86和CD137L基因联合表达对小鼠肝癌种植模型肿瘤免疫原性的影响

目的探讨CD80、CD86和CD137L基因联合表达对肿瘤免疫原性的影响。方法按接种变异瘤株不同,将BALB／C小鼠随机分成A组（H22．Wt细胞）、B组（H22-neo细胞）、C组（H22-CD80／CD86^＋细胞）、D组（H22．CD137L^＋细胞）、E组（H22-CD80／CD86／CD137L^＋细胞）5组,建立H22．BALB／C小鼠荷肝癌模型,A、B组为对照组。观察小鼠成瘤率、成瘤潜伏期、荷瘤鼠存活率及肿瘤增殖情况。通过复种试验观察转基因对H22变异株免疫原性和机体免疫保护作用的影响。结果E组首次接种成瘤率仅有50．0％,显著低于其余4组（P〈0．01）。首次接种后,C组荷瘤鼠肿瘤生长受到明显抑制,有2只荷瘤鼠肿瘤完全消退。E组肿瘤生长所受抑制较C组更为明显,肿瘤峰值体积显著小于C组,且有3只荷瘤鼠肿瘤完全消退。其余3组荷瘤鼠未见肿瘤完全消退。与A、B、D组相比,C、E组荷瘤鼠生存率显著改善（P〈0．01）,而C、E两组荷瘤鼠生存率差异无统计学意义（P〉0．05）。复种试验表明,C、E组荷瘤鼠再次成瘤率低于对照组,E组与C组差异也有统计学意义（P〈0．01）;第3次接种后,E组成瘤率显著低于C组（P〈0．01）。E组中5只首次接种未成瘤的小鼠,于第21天重复接种H22-Wt细胞,小鼠100％排斥肿瘤,于第56天第3次接种H22／Wt细胞,小鼠仍然100％排斥肿瘤。结论CD80＋CD86和CD137L单独或者联合表达均可显著降低野生型H22细胞株致瘤性,CD80、CD86和CD137L基因联合表达显著改善了野生型H22细胞的免疫原性。     

Structure of tumor antigen on hybrid cells between mouse mammary ascites tumor and mouse fibroblast L cells.

Somatic cell hybrids between mouse fibroblast L cells and MM2 mouse mammary ascites tumor grown in BALB/c mice were isolated and the structures of tumor-associated surface antigen of the hybrid cells, and parental MM2 and mouse L cells were investigated by the methods of radioiodination of membrane proteins, immunoprecipitation with a specific antiserum against tumor-associated surface antigens of MM2 tumor (anti-MM2 serum), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two molecules of 105,000 and 76,000 daltons were detected on the MM2 cell surface, but no MM2 tumor antigen was detected on the mouse L cells. On the hybrids between these two kinds of cells, in addition to the two MM2 tumor antigens, molecules of 48,000-51,000 and 12,000 daltons were observed. On Sendai-virus-infected mouse L cells only a molecule of 68,000 daltons was detected by the anti-MM2 serum, and furthermore this molecule was also detected by normal mouse serum, indicating that antibodies against Sendai virus were contaminating in both the anti-MM2 and normal mouse sera used, and thus the molecules detected on the hybrid cells were distinguishable from possible viral components of Sendai virus on the hybrid cells. The results indicate that somatic cell hybrids between mouse L cells and MM2 tumor grown in BALB/c mice expressed on their cell surface the molecules that were not exposed on either parent cell. The experiments comparing newly detected molecules with the H-2 antigen suggested that they were similar to H-2 in their electrophoretic pattern.     

Enhancement of graft-versus-tumor activity and graft-versus-host disease by pretransplant immunization of allogeneic bone marrow donors with a recipient-derived tumor cell vaccine

Allogeneic bone marrow transplantation (BMT) can be accompanied by a beneficial T cell-mediated antitumor immune response known as graft-versus-tumor (GVT) activity. However, BMT donor T cells are not exposed to target antigens of GVT activity until transfer to the host, where tumor antigen presentation may be suboptimal. This study tested in a murine model the hypothesis that immunization of MHC-matched allogeneic donors with a recipient-derived tumor cell vaccine would substantially increase GVT activity and extend survival of BMT recipients with preexisting micrometastatic tumor. C3H.SW and C57BL/10 mice were immunized against a C57BL/6-derived fibrosarcoma or leukemia, and they were used as BMT donors. Recipients were H-2-matched, minor histocompatibility antigen-mismatched C57BL/6 mice with previously established micrometastatic tumors. Donor immunization led to a significant increase in GVT activity that was T cell dependent and cell dose dependent. In some settings, donor immunization also prolonged survival of recipients with preexisting micrometastatic tumors. However, donor immunization significantly increased the incidence of fatal graft-versus-host disease such that long-term survival was uncommon. In vitro cytotoxicity assays indicated that donor immunization induced both tumor-selective and alloreactive cytolytic T-cell populations. In vivo cross-protection assays showed that a substantial portion of the GVT effect was mediated by alloreactive cells not specific for the immunizing tumor. In conclusion, immunization of allogeneic BMT donors with a recipient-derived whole tumor cell vaccine substantially increases GVT activity but also exacerbates graft-versus-host disease.     

The semistability of centric chromatin bodies during long-term passage of multidrug resistant mouse-Chinese hamster cell hybrids

In a cell fusion experiment with multidrug resistant (MDR) mouse SEWA tumor cells and sensitive Chinese Hamster CHO cells, the resistant hybrid cells were completely without recognizable mouse chromosomes. Instead, numerous chromatin bodies (CB) were found that contained copies of a high molecular weight P-glycoprotein gene (PGY1) associated with the MDR condition. The present paper reports on the CB under long-term selective and nonselective growth. The CB were of a stability intermediate between that of double minutes (DM) and homogeneously staining regions (HSR). The stability of the CB was different in the two hybrid lines studied.     

Pretransplant tumor antigen-specific immunization of allogeneic bone marrow transplant donors enhances graft-versus-tumor activity without exacerbation of graft-versus-host disease

Allogeneic bone marrow transplantation (BMT) causes a beneficial graft-versus-tumor (GVT) immune response that is often associated with graft-versus-host disease (GVHD). There is substantial interest in developing therapeutic strategies that augment GVT without GVHD. We have demonstrated recently that immunization of BMT donors with cellular tumor vaccines leads to curative GVT but induces unacceptable GVHD because of the presence of recipient minor histocompatibility antigens (mHAgs) in whole-cell tumor vaccines. This study tested the hypothesis that immunization of BMT donors against a defined tumor-specific antigen with a vaccine not containing recipient mHAgs would help to separate the two responses by enhancing GVT activity without exacerbating GVHD, even when cellular vaccines were used after BMT. Recipient strain C57BL/6 fibrosarcoma cells engineered to express the well-characterized model tumor antigen, influenza nucleoprotein (NP), were used in these studies. C3H.SW donors were immunized against NP prior to BMT, and cytolytic T cells were transferred along with bone marrow into irradiated H-2-matched, mHAg-mismatched C57BL/6 recipients with established micrometastatic 205-NP tumors. Donor immunization led to a significant increase in GVT activity, as measured by reduction in tumor growth and enhanced survival. However, deaths in recipients of tumor antigen-specific immune BMT ultimately occurred because of the growth of antigen-loss variants; such tumor growth did not occur in animals receiving BMT from donors treated with whole-cell vaccines. Donor immunization did not lead to an exacerbation of GVHD, even when BMT recipients received additional immunization after BMT with a 205-NP "whole" tumor cell vaccine (which was shown to induce fatal GVHD when used for donor immunization). In conclusion, immunization of allogeneic BMT donors against a tumor-specific antigen significantly enhances GVT activity without an associated exacerbation of GVHD.     

Murine tumor cell lysis by antibody-dependent macrophage-mediated cytotoxicity using syngeneic monoclonal antibodies

Four monoclonal antibodies to MH134 murine syngeneic hepatoma cells, 3H1, 7C2, 11G2, and 12A2, were produced by hybridomas constructed by fusing P3-X63-Ag8-U1 murine myeloma cells with spleen cells of a C3H/HeN mouse immunized with the syngeneic tumor cells. Immunodiffusion analysis with rabbit anti-mouse immunoglobulin antisera showed that 3H1, 7C2, 11G2, and 12A2 are IgG2a, IgM, IgG1, and IgG2a, respectively. Enzyme-linked immunosorbent assay using cells of five syngeneic tumor lines, MH134, MM102, MM46, MM48, and X5563, and lymph node cells of C3H/HeN and C57BL/6 mice showed that 3H1 specifically bound to MH134 tumor cells, whereas 7C2, 11G2, and 12A2 reacted not only with MH134 but also with MM102 and MM46 tumor cells. None of these monoclonal antibodies bound either to cells of MM48 or X5563 tumor lines or to normal lymph node cells. These results strongly suggest that MH134 tumor cells display at least two kinds of tumor-associated antigens on their cell surfaces: one is expressed uniquely by MH134 tumor cells, which are recognized by 3H1; the other is commonly shared by MH134, MM102, and MM46 tumor cells, which are determined by the other three antibodies. 3H1, 11G2, and 12A2 but not 7C2 were found to be able to induce antibody-dependent cellular cytotoxicity (ADCC) against MH134 tumor cells. Target specificity of ADCC induced by these monoclonal antibodies was identical with that seen in enzyme-linked immunosorbent assay. 3H1, 7C2, and 12A2 but not 11G2 exhibited complement-dependent cytotoxicity, showing the same specificity in target cell lysis as that seen in enzyme-linked immunosorbent assay or ADCC. Pretreatment of MH134 tumor cells with 7C2 inhibited ADCC of both 11G2 and 12A2. Pretreatment of the tumor cells with 11G2 inhibited complement-dependent cytotoxicity of both 7C2 and 12A2. Neither ADCC nor complement-dependent cytotoxicity of 3H1 was inhibited by the pretreatment of the cells with 7C2 or 11G2. These results strongly suggest that tumor-associated antigens recognized by 3H1 are located apart from that recognized by 7C2, 11G2, and 12A2 and that the binding sites of the latter three antibodies are closely associated with, or identical with, each other in the tumor-associated antigen. The ability of 12A2 to induce ADCC against MH134 tumor cells was significantly stronger than that of 3H1 or 11G2.(ABSTRACT TRUNCATED AT 400 WORDS)