Expression of fetal antigens and tumor-specific antigens in SV40-transformed cells. I. Serological analysis of the antigenic specificities

The fetal antigens expressed in a variety of tumors have previously been shown to be different from tumor-specific antigens. The present study on the expression of fetal antigens in various SV40-transformed cells showed that more than one fetal antigen may be found in these cell lines, and that the expression of fetal antigens was not directly related to the function of the SV40 genome. These fetal antigens were not found in syngeneic spleen cells but were found in the normal spleens of some other strains of mice.     

Detection of tumor-specific cell surface antigen of simian virus 40-induced tumors by the isotopic antiglobulin technique

Simian virus 40 (SV40) tumor-specific cell surface antigen (TSSA) was assayed by the isotopic antiglobulin technique. Antisera were produced by immunization of A/LN mice with syngeneic SV40-induced tumor designated SV-A/LN. Positive reactions were only obtained with SV40-transformed mouse or hamster cells which possessed both SV40 TSTA (tumor-specific transplantation antigen) and T antigen. Negative reactions were obtained with untransformed cells or non-SV40 tumors. The T antigen-negative cells (“T- S+” by Tevethia et al., 1968), which lack TSTA and viral RNA or viral DNA, also gave negative reactions. Absorption experiments further confirmed the specificity. These results showed that the SV40 TSSA detected by the isotopic antiglobulin technique was virus-specific, and was correlated with the expression of TSTA.     

Quantitative in vivo studies of soluble simian virus 40 tumor-specific transplantation antigens of the mouse.

Quantitative studies have been performed on the immunogenicity of a membrane-bound antigen of a simian virus 40 (SV40) -induced sarcoma in syngeneic BALB/c mice and of subcellular fractions derived from this tumor. The objectives of the investigation were: a) to develop a quantitative in vivo assay of the tumor-specific transplantation antigen (TSTA) and b) to compare the distribution of histocompatibility antigens, H-2, with that of the SV40 TSTA during several fractionation steps. The immunogenicity of the TSTA-containing fractions was assessed from dose-response curves relating tumor size and the amount of protein used for immunization. After digestion of the tumor cell membranes with a limited amount of papain, H-2 as well as TSTA were present in a soluble form. A single immunization with only 2 microng of the solubilized TSTA reduced the tumor size by 70% compared to that in nonimmunized control animals. The results of several fractionation steps suggest that H-2 and the TSTA are not tightly associated in the solubilized immunogenic material.     

Differential extraction of tumor-specific transplantation antigen and embryonic antigen from simian virus 40- and adenovirus 7-induced sarcoma cells of hamsters with 1-butanol and 3 M potassium chloride

Simian virus 40 (SV40)-induced sarcomas and adenovirus 7-induced sarcomas (Adv-7) exhibit both specific tumor-specific transplantation antigens (TSTA) and cross-protective embryonic antigens at the cell surface in the LAK:LVG(SYR) strain of Syrian golden hamsters. Specific SV40 TSTA could be released from the surfaces of living hamster sarcoma cells in a 2.5% crude 1-butanol extract (CBE) and served as immunogen to protect syngeneic recipients against subsequent homologous but not heterologous tumor cell challenge. The CBE-extracted SV40-induced TSTA (tumor-specific) was observed to be free of detectable, cross-protective embryonic antigens (EA) by tumor transplantation assays. The induction of cytotoxic lymphocyte-mediated immunity with the CBE-released TSTA was dependent on the administration of a single sensitizing injection of 12-20 micrograms antigen protein. Higher concentrations (50-1,000 micrograms) of the CBE tumor cell extract, given in a single injection, enhanced tumor growth as did two injections of 12.5 micrograms CBE-extracted SV40-induced TSTA at 1-week intervals. A cross-protective antigen(s), not detected in the CBE tumor extracts, was retained in the intact, 1-butanol-extracted SV40 and Adv-7-induced tumor cell lines after completion of the CBE extraction procedure and in similarly extracted 10-day hamster fetal cells. Some alterations in the normal immunogenicity of EA extracted with CBE followed by KCI from SV40-induced sarcoma cells could be detected in the transplantation assays and lymphocyte transformation assays, whereas EA extracted from CBE-KCI-treated Adv-7 cells or 10-day hamster fetal cells retained normal immunogenicity in vivo and in vitro. These procedures provide a means for successful separation of immunogenic SV40- and Adv-7-induced TSTA from detectable, biologically active, cross-protective EA from the surfaces of these sarcoma cells.     

Tumor-specific antigens on rat liver cells transformed in vitro by chemical carcinogens.

With the use of membrane immunofluorescence and xenogeneic antisera, tumor-specific membrane antigens were detected on rat epithelial-like liver cells transformed in vitro by chemical carcinogens. These antigens were not detected in 10-, 15-, and 19-day rat fetuses. Xenogeneic antisera were produced in rabbits by immunization of the rabbits with cultivated BD rat liver cells transformed by dimethylnitrosamine or N-methyl-N'-nitro-N-nitrosoguanidine. The specific antisera against tumor-associated antigen(s) were obtained by in vivo absorption in syngeneic male rats and by in vitro absorption with various cell lines. One tumor-specific individual antigen and two tumor-specific cross-reacting antigens were shown to be present on the surface of chemically and/or spontaneously transformed rat liver cell lines. They were not detected on liver and spleen cells of normal BD adult rats, on fetal liver cells, or on liver and intestinal carcinoma cells of Wistar rats. Sera from multiparous pregnant rats had no antibodies against these tumor antigens (although they reacted with fetal cells).     

In vitro induction of tumor-specific immunity by highly purified VSV grown in SV40-transformed cells and tumor glycolipids incorporated into liposomes

Cytotoxic effector lymphocytes (CL) were induced by in vitro immunization of spleen cells from normal Syrian hamsters to syngeneic tumor cells, either SV40-transformed (EH-SV) or spontaneously transformed (EH-N). The lymphocyte reactivity was measured in a direct 51Cr release cytotoxicity assay performed with EH-SV- and EH-N-labelled targets. A specific cytotoxic effect against tumor cells carrying the sensitizing antigens was observed. Cytotoxic effector lymphocytes were also induced by in vitro immunization of hamster spleen cells to highly purified vesicular stomatitis virus (VSV) grown either in syngeneic SV40-transformed fibroblasts or in "normal" fibroblasts. Purified virus possessing an intact envelope or virus subparticles devoid of their glycoprotein spikes stimulated the cellular immune responses against host tumor antigens present within the viral envelope. Cytotoxicity assays have revealed two tumor-specific antigens (TSA), one induced by SV40 and present in SV40-transformed cell lines and the other present in "normal" cells. CL were also induced by in vitro sensitization of spleen cells from normal hamsters to liposomes containing the polar glycolipid fraction from EH-SV and/or EH-N cells. A specific cytotoxic effect against tumor cells that have supplied the glycolipid extract was observed, suggesting specific recognition of glycolipid antigens characteristic for each tumor line. This study supports the view that surface glycolipids act as tumor-specific antigens implicated in the destruction of SV40-induced tumors in Syrian hamsters.     

In vivo induction of tumor-specific immunity by glycolipid extracts of SV40-transformed cells

Glycolipid extracts were prepared from various Syrian golden hamster cell lines, either SV40-transformed or spontaneously transformed. To detect possible SV40-TSTA activity of the glycolipid preparations, normal hamsters were inoculated with different glycolipid extracts and were subsequently challenged with an SV40 tumor-cell line. Significant immunoprotection against SV40 tumor challenge was induced with glycolipids obtained from SV40-transformed cell lines. This was expressed as complete tumor rejection or as a decrease in tumor growth rate, when compared to controls. No protective effects were induced with glycolipid extracts from spontaneously transformed cells. Results suggest that tumor-specific glycolipids synthesized in cells transformed by SV40 virus could act as tumor transplantation antigens responsible for specific tumor rejection in syngeneic hosts.     

Incorporation of SV40-tumor specific transplantation antigen (SV40-TSTA) in vesicular stomatitis virus grown in SV40 transformed hamster cells (author's transl)

Highly purified and inactivated VSV (VSV/TSV-5 C1.2) grown in SV40 transformed hamster cells, TSV-5 C1.2, induces in hamster a transplantation immunity against host-tumor cells. Immunization with VSV/TSV-5 C1.2 gave more than a 10-fold protection against TSV-5 C1.2 tumor cells. The transplantation immunity confered by the immunization is specific for tumor cells which possess SV40-TSTA. VSV/TSV-5 C1.2 protects hamsters against another SV40-transformed cell line, EHSVi-C1.1. In contrast, immunization with VSV/EHB, a purified preparation of VSV grown in spontaneously transformed hamster cells, EHB, gave no protection to TSV-5 C1.2 cell challenge. It was concluded that the presence of SV40-TSTA activity in VSV/TSV-5 C1.2 virions was due to incorporation of this tumor cell antigen in the viral envelope. The existence of cell antigens in VSV/TSV-5 C1.2 and VSV/EHB preparations was also shown in vitro by Cr⁵¹ release assay. The anti-VSV/TSV-5 C1.2 rabbit serum reacted specifically with SV40-transformed cells, TSV-5 C1.2 and EHSVi-C1.1.     

Limitations and utility of a cytolytic assay for measuring simian virus 40-induced cell surface antigens.

Antisera were produced against an SV40-transformed cell line in the syngeneic AL/N mouse. With a microcytolytic assay, the specificity of antisera produced by various immunization schedules and their ability to lyse numerous SV40-transformed cell lines were determined. Various AL/N mouse cell lines, newly transformed by SV40 and cloned, were found to be lysed by the antisera. When tumors were induced by SV40-transformed cells in the syngeneic mouse and cell lines were reestablished from tumors and such procedures were repeated, the susceptibility to serum-mediated cytolysis of the sublines was the same as that of the original SV40-transformed cell line, in spite of differences in tumorigenicity. Polyoma virus-transformed AL/N cell lines were also lysed while AL/N embryo cells, untransformed by SV40 or by polyoma, were not. SV40-transformed T-antigen-positive BALB/c mouse or hamster cell lines or a T-antigen-positive tissue cultured human cell were also resistant to lysis. A competition type of microassay demonstrated specific inhibition of the serum-mediated cytolysis by all of the SV40 T-antigen-positive cell lines tested. Thus, the lack of lysis of cells did not necessarily indicate the absence of SV40-induced surface antigens. The polyoma-transformed AL/N cell line also inhibited the antisera, but to a lesser extent, suggesting the possibility that SV40 and polyoma virus transformation may result in the appearance of partially common cell surface antigens.     

Induction of simian virus 40 (SV40) transplantation immunity in mice by SV40-transformed cells of various species.

Specific tumor rejection was obtained with the use of simian virus 40 (SV40)-transformed cells from several species including man, rat, ape, sheep, and hamster. Growth of the syngeneic sarcoma mKSA in BALB/c mice was strikingly inhibited following a single immunization with as few as 10(3) intact, viable cells. Non-SV40-transformed cells did not induce tumor rejection activity nor did SV40-transformed lines induce immunity against the 3-methylcholanthrene-induced sarcoma Meth A, syngeneic with BALB/c mice. A close relationship existed between the tumor rejection antigen, the tumor-specific transplantation antigen (TSTA) located on the plasma membrane, and the intranuclear tumor antigen (T-ag). Both were associated with the DNA sequence of the early region of the SV40 genome, and TSTA activity was found in the nucleus. However, we did not observe a close parallelism between T-ag activity and TSTA. Neverthesless, the results strongly suggested that TSTA, like T-ag, was encoded by the virus.     

Restoration of specific immunity against SV40 tumor-specific transplantation antigen to lymphoid cells from tumor-bearing mice.

Specific cell-mediated immunity to SV40 tumor-specific transplantation antigen (TSTA) in BALB/c mice undergoing progressive tumorigenesis by syngeneic SV40-transformed cells (VLM) was investigated in vivo using a tumor-cell neutralization test. Specific cellular reactivity to SV40 TSTA was not detected in BALB/c mice bearing large tumors (10-15 mm mean diameter) but was demonstrable after tumor excision. Specific cytotoxic reactivity against syngeneic SV40-transformed cells in vivo could be restored to lymphoid cells from VLM tumor-bearing mice either by culturing the lymphoid cells in vitro or by treating them with papain or trypsin. Enzyme-treated lymphoid cells from MCA tumor-bearing BALB/c mice had no cytotoxic reactivity against VLM cells. These studies suggest that tumor-bearing hosts possess lymphocytes which are sensitized to the TSTA of the tumor but that the reactivity of these lymphocytes is blocked.     

Differences in the capacity of simian virus 40 (SV40) tumor antigens on cells, membranes and in soluble form to induce transplantation immunity in hamsters and mice.

The immunogenicity of the SV40 tumor-specific transplantation antigen (TSTA) on cells, cell particulates and solubilized membranes was studied in mice and in Syrian hamsters. Immunizations were done with various concentrations of tissue-culture-passaged, non-virus-releasing transformed cells, purified cell membranes and in some cases purified nuclei and papain-solubilized membranes obtained from several species, including the mouse, hamster, man, and sheep. All transformed cell lines were T-antigen-positive. The immunosensitive mKSA line of BALB/c mice and the immunosensitive SV34 cell line of the hamster were used for tumor challenge. All materials, regardless of source and of type of preparation, were strikingly immunogenic in the mouse but only SV40 virus and SV34 (hamster) cells provided protection against tumor cell challenge in the hamster. Also, in a limited study, BKV-transformed hamster cells and purified cell membranes and JCV-transformed hamster cells were found to be immunogenic by the tumor rejection assay in the mouse but not in the hamster. SV40 immunization did not protect the hamster against BKV- and JCV-transformed hamster cells. These results are discussed in terms of possible different specificities resident on the TSTA molecule.     

Detergent solubilization and partial purification of tumor specific surface and transplantation antigens from SV40-virus-transformed mouse cells.

A solubilization technique employing 0.5% Triton X-100 was developed to obtain both SV40 virus (SV40)-induced tumor-specific surface antigen(s) (TSSA) from SV40-transformed mouse cells, as determined by a serum-mediated microcytolytic assay, and tumor-specific transplantation antigen(s) (TSTA), as determined by in invivo experiments. High yields (approximately 50%) of TSSA were obtained in whole-cell extracts and also after ammonium sulfate fractionation. Additional fractionation of a 30-50% ammonium sulfate fraction by gel exclusion chromatography on Sephadex G-150 resulted in two pooled fractions which contained TSSA activity. The first eluted close to the void volume, and the second in the 45,000 molecular weight region. The various TSSA active fractions were also active in vivo TSTA tests. Detergent solubilization provides a suitable technique to recover the SV40-induced antigens in good yield, and apparently in intact form.     

In vitro induction of tumor-specific immunity. II. Activation of cytotoxic lymphocytes to murine oncofetal antigens.

The presence of oncofetal antigens (OFA) on a wide variety of murine tumor cells was demonstrated to a totally in vitro system of cellular immunity. Nonimmune spleen lymphocytes were cocultivated with irradiated syngeneic fetal liver cells and, at various times after initiation of culture, were tested for the presence of cytotoxic lymphocytes (CL) by 51Cr-release assay with labeled tumor target cells. Significant cytotoxic activity was regularly detected after such culture, whereas only minor levels appeared in control cultures of spleen lymphocytes with irradiated syngeneic spleen cells. Specificity of the reaction was assessed by inhibition tests in which nonlabeled cells were admixed to the CL and 51Cr-labeled tumor targets. Fetal liver cells gave significant inhibition; however, no inhibition was found with adult spleen cells. Various tumor types gave inhibition, and fibrosarcomas were more effective than plasmacytomas or lymphomas. The results suggested that all tumor types tested possess such OFA, as well as their unique or virus-associated, tumor-associated transplantation antigens, and that the in vitro system permits a more active response to the tumor-associated OFA than that observed in in vivo studies.     

Propionibacterium acnes-mediated humoral immune responses to tumor-specific antigens on rat liver cells transformed in vitro by chemical carcinogens.

Effects of Propionibacterium acnes on production of antibodies against tumor-specific membrane antigens were investigated in syngeneic inbred BD IV and BD VI rats. BD rat liver cell lines transformed in vitro by chemical carcinogens were used as target cells for tumor-specific antigens. By membrane immunofluorescence, antibodies against these rat liver cell lines were detected in syngeneic BD rat sera. Antibodies were produced in syngeneic rats under the adjuvant effect of heat-killed P. acnes only. In assays with various target cells and absorption experiments, the antibodies reacted with a tumor-specific individual antigen or tumor-specific cross-reacting antigen on the surfaces of transformed BD rat liver cells. No antibodies against these antigens were found in the sera obtained from syngeneic rats immunized with either the transformed cell lines or P. acnes but not with both. Freund's complete adjuvant did not induce antibodies against these tumor-specific antigens.     

Comparative behavior of simian virus 40 T-antigen and of tumor-specific surface and transplantation antigens during partial purification.

The simian virus 40-specific T-antigen has been extracted from SV AL/N mouse embryo tissue culture cells by treatment with Triton X-100 detergent. The extracts contained tumor-specific transplantation antigen (TSTA) and tumor-specific surface antigen. These extracts were purified by ammonium sulfate precipitation and diethyl-aminoethyl cellulose and phosphocellulose column chromatography and were assayed for the three antigens. We found that T-antigen, TSTA, and much of the tumor-specific surface antigen copurified through all purification steps. This finding is consistent with previous suggestions of the close degree of homology that must exist between the protein species carrying these three antigenic determinants. The antibody-mediated cytolytic assay appears to detect a new type of antigen on the cell surface, different from T-antigen and TSTA; two antigenic fractions were obtained from the phosphocellulose column that had tumor-specific surface antigen activity, but one of these did not have T-antigen or TSTA activities.     

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.     

Common antigens found on fetal cells and viral transformed adult prostatic tissue.

In vivo and in vitro immunologic cross reactivity between SV40 virus transformed prostatic tissue and fetal antigens of LVG/LAK strain hamsters has been studied. The hamsters immunized with fetal antigens demonstrated significant resistance to tumor growth. Complement-dependent humoral cytotoxicity of target SV40 tumor cells in microtest plates was used to demonstrate the presence of significant antibody titer in fetal-immunized hamsters. When the immune sera were absorbed with SV40 transformed prostatic tissue, cytotoxicity indices were markedly lower than with the unabsorbed immune sera. The transformed prostatic tissue was found to have an elevated tartrate inhibited acid phosphatase indicative of the presence of epithelial components in the transformed tissue. These experiments demonstrate in vivo and in vitro cross reactviity between fetal antigens and tumor associated antigens of transformed prostatic tissue. This study suggests a similarity between fetal antigens and antigens of a specific organ tissue (prostate) transformed by a DNA oncogenic virus.     

Biochemical characterization of 1-butanol-extracted murine tumor-specific transplantation antigens

This investigation sought to characterize biochemically the tumor-specific transplantation antigens (TSTA) expressed on the cell surface of a panel of chemically induced fibrosarcomas of C3H/HeJ mice. Results suggest a uniform antigenic framework upon which individual specificities are superimposed. The antigens expressed by the 3-methylcholanthrene-induced fibrosarcomas MCA-D, MCA-F, and MCA-2A fulfill the requirements of a TSTA; namely, immunization of syngeneic hosts with irradiated cells or soluble extracts engenders a tumor-specific immune response such that animals resist challenge with the same, but not another, tumor. Brief incubation of intact tumor cells in single-phase aqueous solutions of 2.5% (v/v) 1-butanol extracts an immunoprotective TSTA, but not alloantigenic activity, from MCA-F cells. This extraction protocol was extended to the two other MCA-induced neoplasms. The butanol-extracted TSTA from the three tumors displayed isoelectric pHs of 6.4 to 6.6 following preparative isoelectric focusing. The tumor-specific immunoprotective activity from all three tumors displayed an apparent molecular weight of 150,000 (150 kDa) during high-performance gel permeation chromatography. The chromatographic properties of the 150 kDa antigens were unaffected by reduction using dithiothreitol, but incubation in acetate buffer, pH 3.0, dissociated the 150 kDa complex into at least two components with molecular weights of 70 to 100 kDa and 20 to 40 kDa. Only the smaller component displayed TSTA activity. The presence of two major components in the 150-kDa antigen was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. TSTA activity was sensitive to digestion with pronase, papain, chymotrypsin, and alpha-mannosidase, but resistant to DNase, RNase, neuraminidase, trypsin, endoglycosidase H, and a mixed-function glycosidase. In addition, the TSTA activity was unaffected by heating. These data demonstrate that MCA carcinogenesis results in the expression of immunologically unique epitopes on biochemically related glycoproteins and suggest a unified mechanism for the generation of TSTA polymorphism.     

Effect of Propionibacterium acnes on the cellular immune responses to tumor-specific antigens on malignant rat liver cells.

The effect of Propionibacterium acnes on the cellular immune responses to tumor-specific membrane antigens was investigated by microcytotoxicity assays (MA) and 51Cr release assays (CRA) with use of mesenteric lymph node cells (LNC) of syngeneic BD IV and BD VI rats. BD rat liver cell lines transformed in vitro by chemical carcinogens were used as target cells with tumor-specific antigens. By MA, the LNC from rats that were inoculated with malignant liver cells under the adjuvant effect of heat-killed P. acnes showed significant cytotoxic response to the target cells but not to nonmalignant liver cells. By CRA, these LNC did not show specific cytolysis to the malignant liver cells. Assays with various target cells derived from BD rat liver and inhibition tests with syngeneic and xenogeneic antisera against tumor-specific antigens on the malignant liver cells proved that LNC reacted with tumor-specific individual or tumor-specific cross-reacting antigens on the malignant liver cells. Cytotoxic responses against the malignant liver cells were not demonstrated even by MA with use of the LNC from rats inoculated with either the malignant liver cells or P. acnes alone. LNC from the rats inoculated with both nonmalignant liver cells and P. acnes were not cytotoxic to malignant or nonmalignant liver cell lines.