Generation of cytotoxic lymphocytes by SV40-induced antigens

In order to study the correlation of in vivo tumor transplantation immunity and in vitro immunologic assays, cell-mediated cytotoxicity against SV40-transformed cells was studied in AL/N strain mice by using 51Cr-release assay. Killing of SV40-transformed AL/N fibroblast cells was observed by spleen cells of AL/N mice immunized with syngeneic SV40-transformed cells. Immunization with the solubilized SV40 tumor-specific transplantation antigen (TSTA) that induced transplantation immunity in vivo did not elicit cytotoxic spleen cells in vitro. However, the spleen cells from mice immunized with solubilized TSTA and then sensitized in vitro with SV40-transformed cells became cytotoxic against SV40-transformed fibroblasts. Similarly, SV40 TSTA (T antigen) purified by immunoprecipitation was able to prime the lymphocytes in AL/N mice: the primed lymphocytes could differentiate into cytotoxic lymphocytes upon in vitro stimulation by SV40-transformed cells. These data indicate that SV40 TSTA (T antigen) plays a role in the induction of cytotoxic lymphocytes.     

Demonstration of multiple antigenic sites of the SV40 transplantation rejection antigen by using cytotoxic T lymphocyte clones

Multiple antigenic sites on the simian virus 40 (SV40) tumor-specific transplantation antigen (TSTA) were detected by the use of cytotoxic T lymphocyte (CTL) clones isolated from continuous cultures of SV40-specific CTL (H-2b). Two independently derived clones, K11 and K19, specific for the SV40 TSTA in association with H-2Db, each recognized a different antigenic determinant of the SV40 TSTA. This conclusion was based on the observation that a human papovavirus BK virus (BKV) transformed cell line, which possesses a T antigen serologically cross-reactive with that of SV40, was lysed by a heterogeneous population of SV40-immune lymphocytes and by clone K19 but not by K11. Therefore, these CTL clones must recognize two different antigenic determinants of the SV40 TSTA:K19 recognizes a cross-reactive determinant of the SV40 and BKV TSTA, whereas K11 is reactive against an SV40-specific determinant.     

Insensitivity of a ricin-resistant mutant of Chinese hamster ovary cells to fusion induced by Newcastle disease virus.

The simian virus 40 (sv40) tumor antigen (T-antigen) and tumor-specific transplantation antigen (TSTA) have been partially purified and studied to clarify their relationship. The T-antigen and the TSTA were partially purified from nuclei of SV AL/N cells, and SV40-transformed mouse embryo fibroblast line, by precipitation with ammonium sulfate and chromatography on DEAE- and DNA-cellulose. The T-antigen was assayed by complement fixation, and the TSTA was assayed by its ability to immunize mice against SV40-containing ascites tumor cells. When T-antigen- and TSTA-containing preparations were sedimented through sucrose gradients, each antigen had a major peak of activity at a sedimentation coefficient of 6.7 and minor peaks in other regions. Antiserum against T-antigen (from tumor-bearing hamsters) immunoprecipitated the TSTA activity. A preparation of T-antigen from human SV80 cells, which exhibited only one protein band after sodium dodecylsulfate-polyacrylamide gel electrophoresis, had TSTA activity when as little as 0.6 microgram of protein per mouse was used for immunization. These experiments demonstrate that the T-antigen, the product of the SV40 early A gene is capable of inducing specific immunity against transplantation of SV40-transformed tumor cells in mice.     

Cytotoxic T-Lymphocyte Epitope Immunodominance in the Control of Choroid Plexus Tumors in Simian Virus 40 Large T Antigen Transgenic Mice

The simian virus 40 (SV40) large tumor antigen (Tag) is a virus-encoded oncoprotein which is the target of a strong cytotoxic T-lymphocyte (CTL) response. Three immunodominant H-2(b)-restricted epitopes, designated epitopes I, II/III, and IV, have been defined. We investigated whether induction of CTLs directed against these Tag epitopes might control Tag-induced tumors in SV11(+) (H-2(b)) mice. SV11(+) mice develop spontaneous tumors of the choroid plexus due to expression of SV40 Tag as a transgene. We demonstrate that SV11(+) mice are functionally tolerant to the immunodominant Tag CTL epitopes. CTLs specific for the H-2Kb-restricted Tag epitope IV were induced in SV11(+) mice following adoptive transfer with unprimed C57BL/6 spleen cells and immunization with recombinant vaccinia viruses expressing either full-length Tag or the H-2Kb-restricted epitope IV as a minigene. In addition, irradiation of SV11(+) mice prior to adoptive transfer with unprimed C57BL/6 spleen cells led to the priming of epitope IV-specific CTLs by the endogenous Tag. Induction of epitope IV-specific CTLs in SV11(+) mice by either approach correlated with increased life span and control of the choroid plexus tumor progression, indicating that CTLs specific for the immunodominant Tag epitope IV control the progressive growth of spontaneous tumors induced by this DNA virus oncogene in transgenic mice.     

Ribosomal RNA synthesis in spermatogonia and Sertoli cells of the mouse testis

Simultaneously cloned monkey CV-1 cell sublines were found to differ in morphology, cloning efficiency, chromosome number, and sensitivity to SV40 virion productive infection. A fibroblast-like clone, FC7, when compared with an epithelioid clone, TC7, had a lower mean chromosome number and was resistant to SV40 virion infection. Virion adsorption and penetration were similar in both the FC7 and TC7 cells, and both cell types were equally sensitive to first cycle SV40 DNA infection. As the subdiploid mean chromosome number of the FC7 cells increased with passage time toward the TC7 subtetraploid number, the FC7 cells became more sensitive to virion infection. This host gene-dosage effect on SV40 productive infection suggests that a monkey cell function participates in the SV40 uncoating and/or viral genome activation process.     

Abrogation of simian virus 40 DNA-mediated transformation of primary C57BL/6 mouse embryo fibroblasts by exposure to a simian virus 40-specific cytotoxic T-lymphocyte clone.

Primary mouse embryo fibroblasts of C57BL/6 origin (B6/MEF) were transformed in vitro by transfection with simian virus 40 (SV40) DNA. The transformation frequency was markedly reduced if the SV40 DNA-transfected cultures were briefly exposed to K11 cells, an SV40-specific clone of cytotoxic T lymphocytes. This abrogation of SV40 transformation in vitro by cytotoxic T-lymphocyte clone K11 was specific, since transformation of B6/MEF cells by adenovirus type 5 DNA was not affected. The approach described here should serve as an ideal model of dissecting immunological events during in vivo tumorigenesis.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses VIII. Association of Simian Virus 40 Transplantation Antigen with a Specific Region of the Early Viral Genome

Two of the five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrids induce SV40 transplantation resistance in immunized hamsters. These two hybrids, Ad2(+)ND(2) and Ad2(+)ND(4), contain 32 and 43% of the SV40 genome, respectively. The pattern of induction of SV40 transplantation antigen (TSTA) by the various hybrids differentiates TSTA from both SV40 U and T antigens. Since the SV40 RNA induced by both these hybrids is early SV40 RNA, these findings confirm that TSTA is an early SV40 function. By combining available data on SV40 antigen induction by these hybrids with electron microscopy heteroduplex mapping studies, the DNA segment responsible for the induction of SV40 TSTA can be inferred to lie in the region between 0.17 and 0.43 SV40 units from the site on the SV40 chromosome cleaved by E. coli R(1) restriction endonuclease.     

Induction of SV40-related transplantation immunity in mice by BK virus-transformed cells.

The reactivity of tumor-specific transplantation antigens (TSTAs) induced by SV40 and BK virus (BKV) was studied in Balb/Ccr mice with SV40-transformed tumor cells (mKSA-4AC). The mice, which had received BKV-transformed mouse or hamster cells, were highly resistant to the challenge with mKSA-4AC. The mKSA-rejected animals, however, were as susceptible as non-immunized ones to the subsequent challenge with Moloney sarcoma virus (Kirsten)-transformed cells. The result shows that BKV-transformed cells have a TSTA immunologically related closely to that induced by SV40.     

Characterization of a progressive tumor from C3H fibroblasts transformed in vitro with SV40 virus. Immunoresistance in vivo correlates with phenotypic loss of H-2Kk

Cultured SV40-transformed fibroblasts from C3H mice (SV-C3H) were "adapted" to in vivo growth by serial passage through sublethally irradiated, syngeneic recipients. After four in vivo passages, a population of cells was obtained (V4) that was weakly oncogenic in nonirradiated mice. Cells isolated from large V4 tumors (V5) were found to be highly oncogenic, producing lethal tumors at doses of less than 10(3) cells. V5 is insensitive to SV40-specific transplantation immunity in syngeneic animals but can be rejected completely by H-2 allogeneic mice. In vitro studies revealed that although V4 and the parent SV-C3H cells can induce SV40-specific cytotoxic T cells (CTL) in vitro and are lysed by these CTL, V5 does neither. The failure of V5 to interact with CTL was traced to the loss of H-2Kk antigen expression on these cells. The correlation between H-2Kk loss and immunoresistance in vivo suggests a central role for the cytotoxic T cell in in vivo tumor elimination in this system.     

Immunologic selection of simian virus 40 (SV40) T-antigen-negative tumor cells which arise by excision of early SV40 DNA.

A clonal line of highly oncogenic spontaneously transformed mouse cells (104C) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned (106CSC). This 106CSC cell line expressed T antigen and transplantation antigen but was about 100 times less tumorigenic than the 104C parent. When 10(5) 106CSC cells were injected into immunocompetent syngeneic mice, tumors were produced. From such tumors, cell lines were established in culture, all of which were consistently negative for T antigen. We found previously by solution DNA hybridization methods that the tumor cells were depleted in the early region of SV40 DNA which codes for the T antigen. We postulated that this loss occurs through a DNA rearrangement of unknown mechanism in one or a few 106CSC cells and that the tumors are then produced from such a cell or cells, whereas all the T-antigen-positive 106CSC cells are rejected by immunologic means. In this investigation we showed by the DNA transfer method using appropriately selected SV40 DNA probes that indeed the tumor cell clone (130CSCT) we selected to investigate came from one 106CSC cell in which the T-antigen-coding SV40 DNA sequences (but not all the early SV40 DNA sequences) were lost by an excision and recombination mechanism. We also showed that the 130CSCT cells, which are highly tumorigenic, could again be transformed by SV40 and that the resulting T-antigen-positive cloned derivative cells became much less tumorigenic (approximately 10(5)-fold), apparently again because of immunologic recognition and rejection. Indeed, when 10(7) T-antigen-positive cloned cells were injected, all the T-antigen-positive cells were rejected and the tumor was produced again from one or more T-antigen-negative cells. Thus, a one-step in vivo transplantation experiment allowed a selection (for tumorigenicity and against the SV40 T antigen) of a mutant mammalian cell with a DNA deletion at a definable site.     

Transformation of human fibroblasts by ionizing radiation, a chemical carcinogen, or simian virus 40 correlates with an increase in susceptibility to the autonomous parvoviruses H-1 virus and minute virus of mice.

Morphologically altered and established human fibroblasts, obtained either by 60Co gamma irradiation, treatment with the carcinogen 4-nitroquinoline 1-oxide, or simian virus 40 (SV40) infection, were compared with their normal finite-life parental strains for susceptibility to the autonomous parvoviruses H-1 virus and the prototype strain of minute virus of mice (MVMp). All transformed cells suffered greater virus-induced killing than their untransformed progenitors. The cytotoxic effect of H-1 virus was more severe than that of MVMp. Moreover, the level of viral DNA replication was much (10- to 85-fold) enhanced in the transformants compared with their untransformed parent cells. Thus, in this system, cell transformation appears to correlate with an increase in both DNA amplification and cytotoxicity of the parvoviruses. However, the accumulation of parvovirus DNA in the transformants was not always accompanied by the production of infectious virus. Like in vitro-transformed fibroblasts, a fibrosarcoma-derived cell line was sensitive to the killing effect of both H-1 virus and MVMp and amplified viral DNA to high extents. The results indicate that oncogenic transformation can be included among cellular states which modulate permissiveness to parvoviruses under defined growth conditions.     

Transformation of bone marrow cells in rodents by recombinant plasmid pBRSV

Bone marrow cells (mouse strain CBA/Ca and Syrian hamster cells) were transformed with pBRSV DNA containing T-antigen of the SV40 virus. The SV40 T-antigen in transformed cell was detected in 0.5% cases by immunofluorescence with specific antibodies. Extrachromosomal localization of recombinant DNA was shown by means of retransformation of E. coli cells with cytoplasmic spleen DNA from mice previously injected intravenously the transformed bone marrow cells.     

Cell surface antigens coded for by the human chromosome 7

Human-mouse somatic cell hybrids, containing chromosome 7 from an SV40-transformed human cell line as the only human chromosome, were injected into the same inbred strain of mouse as the mouse parental cell, and the humoral immune response assayed. A cell-surface antigen(s) coded for by the chromosome 7 common to all human fibroblastic cell lines tested and also found on African green monkey kidney cell lines was demonstrated. No reactivity to SV40-induced TSTA was detected.Abbreviations used in this paper are as follows SV40 simian virus 40 - MEM minimal essential medium - FBS fetal bovine serum - FITC fluorescein isothiocyanate - C121 53-87 (1) clone 21 - Cl36 53-87-3 clone 36 - 52-62 52-62 (1) clone 5 subclone 9 - MSV murine sarcoma virus - T tumor - TSTA tumor-specific transplantation antigen - RIA radioimmunoassay - PBS phosphate-buffered saline - PBS⁺ PBS with sodium azide and FBS - IgG immunoglobulin - cpm counts per minute