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.     

SV40 tumor rejection induced by vesicular stomatitis virus bearing SV40 tumor-specific transplantation antigen (SV40-TSTA). I. Specificity of immunoprotection and effect of enzyme treatment on TSTA activity.

Highly purified vesicular stomatitis virus (VSV) was obtained from VSV-infected SV40-transformed hamster cell lines. Immunization with this virus protected hamsters against challenge with SV40-transformed cells (TSV5-cl2). This protection was obtained regardless of the source of the SV40-transformed cells (e.g. cat, rat, hamster) used to produce VSV, and was therefore associated with the SV40 tumor-specific transplantation antigen (SV40-TSTA). Furthermore, when grown on spontaneously transformed cell lines or on cells transformed by a different oncogenic DNA virus, such as polyoma virus, the VSV failed to protect against the SV40-induced tumor. It was concluded that the SV40-TSTA activity of purified VSV is due to the incorporation of SV40-TSTA within the viral envelope. When VSV was treated with proteolytic enzymes (bromelain, trypsin) no loss of TSTA-induced tumor rejection was observed, although VSV had lost its ability to induce virus-neutralizing antibody. This clearly demonstrates that the TSTA activity is not related to the viral spikes. Phospholipase C suppressed the TSTA activity but neutralizing activity was still detectable in the anti-VSV sera. The results presented here demonstrate that the protection afforded by VSV is highly specific. It is particularly interesting that SV40-TSTA activity may be conveyed by the lipid core of the viral envelope.     

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.     

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.     

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.     

Expression of fetal antigens and tumor-specific antigens in SV40-transformed cells. II. Tumor transplantation studies

The effect of immunization with fetal antigens and tumor-specific antigens on the transplantation of SVT2, a BALB/c mouse tumor induced by Simian virus 40 (SV40), has been studied. Hyperimmunization with 5,000 R X-irradiated, syngeneic fetuses of 1–2 weeks' gestation or syngeneic spleen cells gave no protection to SVT2 cell challenge. In contrast, immunization with SV40 or SV40-transformed cells gave a 100- to 1,000-fold protection. These results gave additional evidence that fetal antigens are different from tumor-specific transplantation antigen (TSTA).     

Virus-induced cellular immunity to simian virus 40 tumor-specific surface antigen(s) in adult LVG:LAK hamsters

Cell-mediated immunity directed against simian virus 40 (SV40) tumor-specific surface antigen(s) (TSSA) was detected in SV40-immunized noninbred LVG:LAK hamsters with the use of a lymphocyte transformation assay. Sensitized spleen cells underwent a proliferative response in the presence of cells of an SV40-induced hamster sarcoma cell line (WF5-1) or the plasma membranes from these cells. In addition, 3-M KCl extracts of the plasma membranes of WF5-1 cells elicited such a lymphocyte proliferative response. Neither the cells of a chemically induced sarcoma cell line (OBT), their plasma membranes, nor 3-M KCl extracts of these membranes caused a similar proliferative response. These results established that immunization of adult hamsters with SV40 resulted in a specific autochthonous cell-mediated immune response against SV40 TSSA. The in vitro demonstration of this virus-induced immunity provided a unique method for the investigation of the SV40 TSSA involved in the establishment or rejection of tumors. The described lymphocyte transformation assay was also used to follow the solubilization and purification of the SV40 TSSA.     

Antigenic similarity between simian virus 40-induced surface and fetal antigens in hamster.

The tumor-associated cell-surface antigen (TSSA) on simian virus 40 (SV40)-transformed hamster cells was studied serologically by a complement-dependent cytotoxicity test. An antiserum was obtained from guinea pigs inoculated with SV40-transformed hamster cells. The serum was cytotoxic to SV40-transformed hamster cells after absorption with 15-day hamster embryo cells, hamster cells transformed either by polyoma virus or adenovirus 12, various tissues of hamster origin (brainliver, spleen, and kidney), or sheep red blood cells. These results indicated that the major TSSA induced specifically by SV40 was similar or identical to the antigen present during early stages of embryogenesis.     

Assessment of host immune status during progressive growth and after excision of DNA virus (simian virus 40) tumors in hamsters: comparison of tumor-specific and tumor-unrelated parameters of immune responsiveness.

The kinetics of cell-mediated immunity to simian virus 40 (SV40) tumor-specific transplantation antigen (TSTA) were compared to the kinetics of tumor-unrelated parameters of immune responsiveness in the assessment of the immune statuses of inbred MHA/SsLAK hamsters during the course of progressive syngeneic SV40 tumor growth and after tumor excision. With the use of the tumor cell neutralization test in vivo and the macrophage migration inhibition assay in vitro, specific cellular immunity to SV40 TSTA was detected by 4 days after tumor cell inoculation, when the tumor was small. This tumor-specific immune response was no longer detected at 7 days after tumor cell inoculation, when the tumor had reached a mean diameter of 12.5 mm, but it returned by 14 days after surgical excision of the tumor. The patterns of host responsiveness to mitogens in spleen cells derived from tumor-bearing animals or from tumor-excised animals generally showed little or no correlation with the kinetics of tumor-specific cellular immunity. The kinetics of the humoral immune response to murine erythrocytes, as determined by hemagglutination assays, correlated much more closely with the kinetics of tumor-specific immunity than did the responses to mitogens. IgG antibody (T-dependent) responses were more affected by progressive tumor growth than were IgM antibody (T-independent) responses. The data suggest that results of tests with the use of tumor-unrelated parameters of immune responsiveness for the assessment of the immune status of cancer patients should be interpreted with caution.     

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.     

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.     

Tumor-specific chemoimmunotherapy of murine fibrosarcoma using tumor-specific transplantation antigen, cyclophosphamide, and interleukin-2.

The anti-tumor effect of active specific chemoimmunotherapy, using butanol-extracted tumor-specific transplantation antigen (TSTA), cyclophosphamide (CY), and continuous intrasplenic infusion of interleukin-2 (IS-IL-2), was assessed in a C3H/HeJ murine methylcholanthrene (MCA)-induced fibrosarcoma model. Sole administration of TSTA induced tumor-specific, suppressor T cells in the spleens of mice bearing 3-day established tumors. Concomitant low-dose (20 mg/kg) CY treatment not only inhibited TSTA-mediated suppressor cell induction, but also evoked splenic lymphocytes of tumor-bearing mice to display tumor-specific cytotoxic activity. High-dose (200 mg/kg) CY abrogated the immunotherapeutic benefit. The immune effectors generated by TSTA plus CY bear the Thy 1, L3T4, Lyt 2 phenotype. Continuous IS-IL-2 infusion in combination with TSTA and CY induced tumor-specific Lyt 2+ cytolytic T cells, as well as the activation of L3T4+ cytostatic T cells. Thus, a triple regimen using TSTA, CY, and IS-IL-2 appears to augment CTL induction in tumor-bearing hosts undergoing stimulation of helper elements by TSTA and inhibition of suppressor cells by CY.     

Rauscher leukemia virus-induced tumor antigens: complete separation from gp70, p30 and H-2.

The possibility that some or all of the viral proteins, gp70, p30, and the histocompatibility antigen, H-2, function as the tumor-specific transplantation antigen (TSTA) of the R-MuLV-induced leukemia, RBL-5, and also in the secondary in vitro induction of cytotoxic T lymphocytes (CTL), was investigated. The antigen was obtained by isolating the plasma membranes of RBL-5 cells and solubilizing with sodium deoxycholate (DOC) followed by gel filtration chromatography. A fraction containing excellent tumor-rejection activity but low amounts of gp70, p30 and H-2 was chromatographed on goat anti-gp 70 goat anti-p 30 and sheep anti-H-2b immunoaffinity columns. The data obtained indicate that gp 70, p 30 or H-2 do not function as TSTA of RBL-5 leukemia, individually or as a complex. Similarly, the antigen responsible for the specific secondary induction of CTL in vitro is distinct from these three proteins.     

Subcellular distribution of the tumor-specific transplantation antigen of simian virus 40-transformed cells.

TU-5, a simian virus 40 (SV40)-transformed cell line of BALB/c origin, expressed the SV40-specific T-antigen and a transplantation antigen (TSTA). Nuclei and plasma membranes were prepared from these cells and shown on the basis of the distribution of T-antigen and histocompatibility (H-2) antigens to be relatively free of cross contamination. Most of the TSTA, estimated by tumor rejection, was associated with the nuclear fraction.     

In vivo and in vitro TSTA-inducing activity of temperature-sensitive (ts) mutants of SV40.

In vivo TSTA induction in Syrian hamsters was studied with the use of SV40 ts mutants (A, B, C, BC and D). The ts A30, TS A239 and possibly also the ts BC210 mutants were defective in resistance-inducing activity in hamsters in contrast to wild type SV40 and other ts mutnats. At the permissive temperature ts A30 and ts A239 did not induce TSTA in hamster cells during abortive infection in vitro, while they did so in green monkey cells at both permissive and non-permissive temperatures. In hamster cells transformed by ts A30, ts A239 and ts A209 mutants none at all or very little TSTA was detected by in vivo transplantation immunological tests. Thus, expression of TSTA induced by these three SV40 ts A mutants was found to be dependent from the species of infected cells and was being a temperature independent viral function.     

Characterization of the surface proteins of SV40-transformed mouse and human cells: absence of SV40-specific proteins

The proteins of a number of SV40- and spontaneously transformed mouse and human cell lines were compared in an effort to identify a surface protein which would correspond to the SV40 tumor-specific transplantation antigen (TSTA). Analysis of the one- and two-dimensional electrophoretic patterns of 35S-methionine-labelled total proteins and 125I-labelled surface proteins of several of these cell lines failed to reveal the presence of proteins specific to transformation by SV40. Antisera were prepared against SV40- and spontaneously transformed mouse cells in syngeneic mice. In serological assays, these antisera reacted with surface antigens common to both SV40- and spontaneously transformed mouse cell lines. Electrophoretic analysis of the 125I-surface-labelled proteins which these antisera immunoprecipitated from extracts of SV40- and spontaneously transformed mouse and human cells identified a set of common surface proteins with apparent molecular weights of 15, 46, 50, 72, 77, 105, 150 and 230kdal. No SV40-specific surface proteins were detected. Two of the transformed cell surface proteins (105 and 150kdal) were present as well in membrane fractions of 35S-methionine-labelled primary mouse kidney cultures. The proteins of the primary cultures could not be iodinated by lactoperoxidase suggesting that these proteins were present at a "cryptic" location at the surface of normal cells. We were not able to obtain serological or immunochemical evidence for the presence of SV40 large T-antigen at the surface of any of the SV40-transformed cell lines tested using either hamster anti-SV40 tumor sera, a rabbit antiserum against SDS-denatured gel-purified large T-antigen or antisera against SV40-transformed mouse cells. In conjunction with the report that large T-antigen released from disrupted SV40-transformed cells will bind to cell surfaces (Lange-Mutschler and Henning, 1982), we consider the possibility that the specific rejection of SV40-induced tumors by sensitized animals is the result of immunological reactions against both common transformation-related surface antigens and SV40 T-antigen from disrupted cells that has bound to the surface of other tumor cells.     

Properties of transformed hamster cells containing SV40 tumor antigen in the cytoplasm

The properties of hamster cells containing SV40 tumor (T) antigen in the cytoplasm, rather than the nucleus, were determined. Eight cell lines were established from eight tumors induced by hamster embryo fibroblasts transformed in vitro by PARA (2cT)-adenovirus 7. Six cell lines contained only cytoplasmic SV40 T-positive cells while two were a mixture of nuclear T-positive and cytoplasmic T-positive cells. All the cell lines contained SV40 S antigen, all caused the production of SV40 T antibody in vivo, and four elicited the production of adenovirus T antibody. The cell lines seem to have acquired an infinite life span in vitro. The localization of T antigen apparently can be a stable phenomenon because five of the cell lines have retained T antigen exclusively in the cytoplasm for over 40 passages in tissue culture. The cytoplasmic T antigen could be detected by complement fixation in addition to immunofluorescence. Cytoplasmic T-positive cells were readily transplantable in vivo and contained SV40 TSTA demonstrable by both immunogenicity and immunosensitivity procedures. Two of the three cytoplasmic variants of PARA appear to be weakly oncogenic in newborn hamsters.     

Study by the isotopic antiglobulin technique of a cross-reacting murine antibody produced by immunization with a syngeneic SV40 tumor

Tumor line SV-AL/N, derived from SV40 transformation of an embryonic cell line, was employed to immunize syngeneic mice, and the resultant antisera were investigated with the modified isotopic antiglobulin assay. A single specificity was detected in the antiserum, which reacted strongly with an antigen present on SV40-transformed lines. However, the antigen was also present on cells derived from the same embryonic line as SV-AL/N but transformed instead either spontaneously or by polyoma virus. The antigen was present on one non-transformed syngeneic embryonic line and may have been present in low concentration in a non-transformed syngeneic fibroblast line; it was not detected in several methylcholanthrene-induced lines. Antiserum derived from multiple immunizations with irradiated syngeneic fetuses reacted weakly in a similar pattern. The results suggest that the antigen detected is present in the host genome in occult form, and is derepressed by several processes, perhaps including embryogenesis.     

Immunization against the polyoma tumor-specific transplantation antigen (TSTA) with polyoma T-antigens

The relationship between the polyoma virus tumor-specific transplantation antigen (TSTA) and 2 of the virus proteins coded from the early region of polyomavirus was investigated. Mice were immunized with small T antigen and a truncated mutant of middle T antigen, both purified from genetically engineered Escherichia coli. The 2 proteins induced protective immunity against polyomavirus-induced tumors, but not against non-polyoma tumors, indicating that one or more of the polyoma T antigens are directly involved in a TSTA function.