Establishment of transformed swine fibroblast cell lines using SV40 large T antigen

Summary Swine testicle cell lines were established by transformation of primary swine testicle (PST) cells with an SV40 plasmid (pSV3-neo), which contains genes conferring resistance to neomycin and expressing SV40 large T antigen. Plasmid DNA was transfected into PST cells using a lipofection system. Two related plasmids, pSV2-neo and pSV5-neo, failed to induce transformed cells. Cells transformed with pSV3-neo formed single colonies that were resistant to the antibiotic, G418, and expressed large T antigen. Upon two cycles of cloning by endpoint dilution method, three transformed clones, designated transformed swine testicle (tST)-3, tST-14 and tST-18, were selected and characterized in regards to cell replication and susceptibility to swine viruses. The resultant clones were compared with a counterpart non-transformed ST cell line (ATCC-ST). The three tST cell lines showed longer or the same doubling times and higher saturation densities compared to ATCC-ST cells. These cells were free from a range of adventitious agents and supported the replication of porcine parvovirus (PPV), pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV), comparable to ATCC-ST cells. All three cell lines have been maintained in continuous cultures for over 60 passages with no changes in growth characteristics. These findings indicate that lipofection with pSV3-neo is an efficient means for the introduction of exogenous DNA into porcine cells and for establishment of transformed immortalized cell lines.     

Diversity of escape variant mutations in Simian virus 40 large tumor antigen (SV40 Tag) epitopes selected by cytotoxic T lymphocyte (CTL) clones

To better understand the relationship between epitope variation and tumor escape from immune surveillance, SV40 T antigen-transformed B6/K-0 cells were subjected to selection with individual CTL clones specific for the SV40 T antigen H-2D(b)-restricted epitopes I or V. CTL-resistant populations were isolated from a majority of the selection cultures and substituted epitope sequences were identified within most of the resistant populations. Tag sequences deleted of all or portions of the selection-targeted epitope were identified, but in lower numbers compared to epitope sequences bearing single residue substitutions. Relatively few flanking residue substitutions were identified, and only in epitope I-targeted selections. The diversity (numbers and epitope residue locations) of substituted epitope residue positions varied between selections. These findings suggest that the scope of spontaneously occurring mutations that could allow for escape from individual CD8+ T cell clones is large.     

Phosphorylation of SV40 large T antigen in SV40 nucleoprotein complexes

Plaques produced by our P^? mutants of vesicular stomatitis virus (VSV), which are defective in the inhibition of total protein synthesis in infected cells, stop increasing in size after several days of incubation under conditions where those produced by P⁺ mutants increase linearly in size. The basis for the arrest in size of P^? plaques has been shown to be due to the induction of interferon, and the phenotype is termed PIF⁺ for “plaque interferon positive.” Thus P^? plaques can inhibit the increase in size of adjacent P⁺ plaques and the factor responsible has the biological and physical properties of interferon. Also P^? mutants, when plaqued on VERO cells which cannot be induced to produce interferon, produce plaques which increase linearly in size like P⁺ plaques. Finally, the inclusion of anti-interferon antibody in the overlay medium also causes P^? mutants to produce plaques like P⁺ mutants. VERO cells were found to be useful to separate the effects of is mutations on plaque size from the interferon effect. Using other cell types the latter effect (PIF assay) can be used as an assay for the ability of viruses to induce interferon, for the isolation of PIF⁺ mutants from PIF^? virus, and as a test for the ability of cells to respond to interferon induction by PIF⁺ viruses. The assay can be increased in sensitivity through the use of specific cell types and of cell cultures preincubated for several days in the stationary phase of growth. In its most sensitive form, the assay could detect PIF⁺ behavior in certain ts mutants of VSV at permissive temperatures and in VSV mutants emerging from persistent infection. The assay has also been used to isolate novel mutants of VSV which show alterations in the viral P function.     

Clustering of antigenic sites recognized by cytotoxic T lymphocyte clones in the amino terminal half of SV40 T antigen

The distribution of antigenic sites recognized by cytotoxic T lymphocytes (CTL) in the amino terminal half of SV40 T antigen was studied using SV40-specific CTL clones. Spleen cells of C57BL/6 (B6) mice immunized with B6/pSV3T3-20GV cells, which synthesize a truncated SV40 T antigen of amino acids 1-368, were restimulated in vitro with B6/pPVU-5-70K cells expressing SV40 T antigen of amino acids 109-708 and then cloned. The recognition sequence for all 10 CTL clones established mapped in the amino terminal half of SV40 T antigen between amino acids 109 and 271. Fine mapping of these 10 CTL clones defined three distinct antigenic sites. These three sites were abolished by the deletion of SV40 T antigen amino acids 193-211, 220-223, and 220-228, respectively. Additional CTL clones were established from spleen cells of B6 mice immunized with B6-K/S11-S24 cells, which synthesize a SV40 T antigen missing amino acids 127-250. None of these CTL clones reacted with B6/pSV3T3-20GV cells. These CTL clones recognized an antigenic site(s) which mapped in the carboxy terminal half of SV40 T antigen. Our results indicate that the antigenic sites in the amino terminal half of SV40 T antigen are tightly clustered between amino acids 193 and 271 and most probably between 193 and 228.     

In vitro phosphorylation of SV40 large T antigen

Phosphorylation of simian virus 40 large T antigen (large T) was investigated in vitro. "Autophosphorylation" of large T resulted in the modification of Ser106, Ser112, Ser123, Thr124, either Ser676, Ser677, or Ser679, and Thr701. All of these residues were also found to be phosphorylated in vivo. Reaction of large T with purified casein kinase I resulted in phosphorylation of Ser123, possibly Thr124, and either Ser676, Ser677, or Ser679, while purified casein kinase II phosphorylated Ser106 and possibly Ser112. Submolar amounts of phosphate were transferred to large T indicating that only a fraction of large T served as substrate for the casein kinases. Removal of serine-bound phosphate did not affect the subsequent autophosphorylation or phosphorylation by casein kinase I and II. No phosphorylation at in vivo sites was observed with the cAMP-, cGMP-, or Ca2+/phospholipid-dependent protein kinases, or with the protease-activated kinase I and II.     

Analysis of the nonviral antigens immunoprecipitable by SV40 T antibody from SV40-transformed human/mouse hybrid cell lines

A temperature-sensitive (ts) mutant of herpes simplex virus type 1 (HSV-1), tsJ12, is able to undergo one cycle of replication at the nonpermissive temperature (39°) yielding wild-type quantities of enveloped virus particles. These particles contain viral DNA which is as infectious as wild-type viral DNA; however, they are not infectious. Analysis of [¹⁴C]glucosamine-labeled mutant-infected cell extracts by one- and two-dimensional polyacrylamide gel electrophoresis demonstrated that at 39° tsJ12 fails to induce the synthesis of both the mature gB glycoprotein and its dimeric form which are normal constituents of the virion envelope. Polyethylene glycol, an agent which promotes membrane fusion, enhances the infectivity of tsJ12 virions by greater than 1000-fold following adsorption of virus to susceptible cells demonstrating that mutant virions are able to attach to cells but not penetrate. Consistent with a defect in the virion envelope, tsJ12 is able to interfere with the production of infectious wild-type virus, presumably by the formation of pseudotypic virions composed of wild-type viral genomes in gB-deficient envelopes. Physical mapping of the is defect in this mutant demonstrates that it lies within the limits of the DNA sequence which specifies gB on the physical map of the genome. A ts⁺ revertant of tsJ12 is as infectious as wild-type virus and synthesizes a gB glycoprotein which is indistinguishable from that of wild-type virus. Thus, biological and biochemical studies of tsJ12 and of a ts+ revertant of this mutant (1) demonstrate that glycoprotein gB is essential for infectivity at the level of penetration and (2) further define the physical map location of the gene for this glycoprotein.     

Frequency of SV40-specific cytotoxic T-lymphocyte precursors in two SV40 T-antigen transgenic mouse lines.

Cytotoxic T-lymphocyte precursors (CTL-P) from control C57BL/6 mice and from mice of two simian virus 40 (SV40) T-antigen transgenic lines, 427 and 419, specifically nonresponsive and responsive, respectively, to SV40 T antigen, were quantitated by limiting dilution (LD) after immunization with SV40. CTL-P frequencies for the SV40 T antigen-responsive 419 line transgenic mice were within the range established in C57BL/6 mice, whereas no CTL-P could be demonstrated for the SV40 T antigen-tolerant 427 line mice. These results suggest that deletion or anergy of SV40 T antigen-responsive clones underlies the specific profound tolerance of 427 line mice.     

Localization of common cytotoxic T lymphocyte recognition epitopes on simian papovavirus SV40 and human papovavirus JC virus T antigens.

Human papovavirus JC virus (JCV) and Simian virus 40 (SV40) tumor or T antigens demonstrate considerable sequence homology which is reflected by antibody cross-reactivity. This similarity raised the possibility that JCV and SV40 T antigen also might contain common cytotoxic T lymphocyte (CTL) recognition epitopes. In this study we identified and mapped such sites on the JCV T antigen. C57Bl/6 cell lines transformed by JCV/SV40 T antigen chimeras were generated and tested for susceptibility to lysis by five H-2b restricted SV40-specific CTL clones: Y-1, Y-2, Y-3, Y-4, and Y-5. These CTL clones recognize specific epitopes within amino acids 205-219 (site I), 220-233 (sites II and III), 369-511 (site IV), and 489-503 (site V) on SV40 T Ag, respectively. The results show that sites I, II, III, and IV (recognized by CTL clones Y-1, Y-2, Y-3, and Y-4, respectively) represent common epitopes on SV40 and JCV T antigens. CTL clone Y-5 failed to recognize JCV T antigen indicating that CTL can discriminate between the two antigenically related T antigens.     

An immunoaffinity purification procedure for SV40 large T antigen

A rapid purification procedure for SV40 large T antigen has been developed which combines the use of an adenovirus-SV40 hybrid virus which overproduces large T antigen, and immunoaffinity chromatography on an anti-large T monoclonal antibody coupled to protein A Sepharose. The protein exhibits the p53-binding, ATPase, and sequence-specific DNA-binding activities of T antigen. The purification procedure can be completed in 1 day and allows the isolation of milligram amounts of large T in excellent yield. The pure protein is extremely antigenic and is tolerant of iodination to high specific activity, permitting the development of a competition radioimmunoassay for large T that reliably detects nanogram amounts of the protein.     

Cross-reaction of BK virus large T antigen with monoclonal antibodies directed against SV40 large T antigen

Seventy-five percent of the amino acid sequence of simian virus 40 (SV40) large T antigen is identical to that of the large T antigen of the human papovavirus, BK virus (BKV). Cross-reactivity between BKV T antigen and monoclonal antibodies directed against SV40 T antigen was studied by immunofluorescence, an enzyme immunoassay, immunoprecipitation of radiolabeled extracts, and Western blotting. BKV T antigen was found to be recognized by two monoclonal antibodies, PAb 416 and 430, which react with two distinct sites toward the amino terminus of SV40 large T antigen. These two sites may correspond to two hydrophilic regions of shared amino acid sequence which exist toward the amino termini of the T antigens.     

SV40 large T antigen-specific human T cell memory responses

The continued presence of simian virus 40 (SV40), a monkey polyomavirus, in man is confirmed by the regular detection of SV40-specific antibodies in 5-10% of children who are unlikely to have received contaminated polio-vaccines. The aim of our experiments was to find cellular immunological evidence of SV40 infection in humans by testing memory T cell responses to SV40 large T antigen (Tag). As there is some indication that the virus may be present in malignant pleural mesothelioma (MPM) cells, we analyzed T cell responses in MPM patients and in healthy donors. The frequencies of responding T cells to overlapping Tag peptides were tested by cytokine flow cytometry. CD8+ T cells from 4 of 32 MPM patients responded (above twofold of control) to SV40 Tag peptides, while no positive responses were detected in 12 healthy donors. Within SV40 Tag we identified three 15 amino acid-long immunogenic sequences and one 9 amino acid-long T cell epitope (p138) (138FPSELLSFL146), the latter including a HLA-B7-restriction motif. T cell responses to p138 were SV40-specific as T cells stimulated with p138 did not cross-react with the corresponding sequences of Tag of human polyomaviruses BKV and JCV. Similarly, the relevant BKV and JCV Tag peptides did not generate T cell responses against SV40 TAg p138. Peptide-stimulated T cells also killed SV40 Tag-transfected target cells. This article demonstrates the presence, and provides a detailed analysis, of SV40-specific T cell memory in man.     

Induction of simian virus 40 transplantation immunity in hamsters by gel-purified SV40 large T antigen

Simian virus 40 (SV40) large T antigen and p53 cellular protein were isolated from an SV40-transformed hamster cell line by immunoprecipitation with anti-T sera and purified by sodium dodecyl sulfate-gel electrophoresis. These two protein were tested in hamsters for the presence of SV40 transplantation rejection antigenic sites by in vivo transplantation rejection assay. The large T antigen immunized the hamsters against a challenge of SV40 tumor cells and the protected animals generated cytotoxic spleen cells. Hamsters immunized with the p53 cellular protein were not protected against SV40-induced tumor but there was some delay in the appearance of tumor.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). IX. Analysis of TrAg in mouse cells synthesizing truncated SV40 large T antigen

Mouse LTK- cells (H-2k) were transfected with a series of recombinant plasmids consisting of the herpes simplex virus type 1 thymidine kinase (TK) gene linked to fragments of SV40 DNA coding for portions of SV40 T antigen in pBR322, and TK+ transformants (LTK+) were selected in HAT medium. The TK+ transformants were analyzed for SV40 transplantation rejection antigen (TrAg) at the cell surface by reacting them with cytotoxic lymphocytes (CTL) generated to SV40 TrAg in C3H/HeJ (H-2k) mice. The results indicated that the cells transformed by pVBETK-1 and synthesizing full size SV40 large T antigen were efficiently lysed by SV40 CTL. In addition, cells transformed by the plasmid pVBt1TK-1 and synthesizing a truncated 33 K T antigen were also found to be susceptible to lysis by the CTL. However, LTK+ cells that were transformed with the plasmid pVBt2TK-1 and which synthesized a truncated T antigen of 12.3 K did not provide a target for SV40 CTL nor did pVBETK-1-transformed cells that did not express any of the SV40 tumor antigens. Only the pVBETK-1-transformed cells that express 94 K T antigen were able to immunize mice against a challenge of syngeneic SV40-transformed cells. These results suggest that the TrAg expression at the cell membranes of transformed cells may be associated with the proximal half of SV40 T antigen.     

Decidualization and maintenance of a functional prostaglandin system in human endometrial cell lines following transformation with SV40 large T antigen

Prostaglandins (PGs) are key regulators of reproductive function and associated pathologies. We have established stable endometrial stromal and epithelial cell lines with SV40 large T antigen (TAG) as a model to study PG action in the human endometrium. Two clones for each cell type were selected for rapid growth, PG production and response to interleukin-1beta (IL-1beta). The resulting stromal (HIESC) and epithelial (HIEEC) cells retain their characteristics for at least 40 population doublings (PDs). The selected clones express progesterone (PR) and estrogen receptor-alpha (ER-alpha) at both mRNA and protein levels. By contrast, with the existing known human endometrial cell lines Ishikawa and KLE, HIESC and HIEEC increase their production of PGF2alpha and PGE2 and cyclooxygenase (COX)-2 protein expression in response to IL-1beta. The latter cells also express the main biosynthetic enzymes involved in PG production, cytosolic phospholipase A2 (PLA2), COX-1 and COX-2, PGF synthase and PGE synthase and the corresponding EP2, EP3, EP4 and FP receptors. The selective COX-2 inhibitor NS-398 completely inhibits the increased production of PGs induced by IL-1beta in both cell types, whereas dexamethasone (DEX) exerts a stronger inhibition in HIESC than in HIEEC. The latter observation may be related to the higher expression of COX-1 measured in HIEEC. On the basis of the present characterization and previous determination of corresponding primary cell cultures, HIESC and HIEEC appear appropriate to study the contribution of PGs in the regulation of human endometrium function and associated pathologies.     

Specific DNA binding activity of T antigen subclasses varies among different SV40-transformed cell lines

Large tumor antigen (T antigen) occurs in at least three different oligomeric subclasses in cells infected or transformed by simian virus 40 (SV40): 5-7 S, 14-16 S, and 23-25 S. The 23-25 S form is complexed with a host phosphoprotein (p53). The DNA binding properties of these three subclasses of T antigen from nine different cell lines and free p53 protein were compared using an immunoprecipitation assay. All three subclasses of T antigen bound specifically to SV40 DNA sequences near the origin of replication. However, the DNA binding activity varied between different cell lines over a 40- to 50-fold range. The 23-25 S and 14-16 S forms from most of the cell lines tested bound much less SV40 origin DNA than 5-7 S T antigen. The free p53 phosphoprotein did not bind specifically to any SV40 DNA sequences.     

Simian virus 40 specific cytotoxic lymphocyte clones localize two distinct TSTA sites on cells synthesizing a 48 kD SV40 T antigen

Simian virus 40 specific antigenic sites (TSTA) which react with SV40 specific cytotoxic lymphocytes (CTL) were localized on the surface of mouse embryo fibroblasts (H-2b) transformed with a recombinant plasmid which contain SV40 large T antigen coding DNA sequences (0.65-0.37 map units). These cells (B6/pSV-20-GV) synthesize a large T antigen polypeptide of 48 kD and could be lysed with two independently isolated CTL clones which recognize distinct antigenic sites on SV40 transformed cell surface. These results suggest that at least two distinct TSTA sites are present in cells synthesizing only the amino terminal half of SV40 T antigen.     

Analysis of mechanisms controlling the interactions of SV40 large T antigen with the SV40 ORI region

We have characterized the interactions of simian virus 40 (SV40) large tumor antigen (large T) with the control region of the SV40 genome, the SV40 ORI, by analyzing the specific binding of large T antigen to SV40 wild-type origin DNA and to isolated binding sites I and II, respectively. DNA binding affinities of large T antigen were determined under standardized conditions and DNA excess, using a target-bound DNA binding assay (M. Hinzpeter, E. Fanning, and W. Deppert, 1986, Virology 148, 159-167). Our results show that large T antigen exhibits similar affinities for isolated binding sites I and II and for combined sites I and II on wild-type ORI DNA. When the fraction of large T antigen molecules (calculated per large T antigen monomers) able to bind specifically to these sites was determined (DNA binding activity of large T antigen) we found that only 2% of large T antigen molecules present in extracts of lytically infected cells were able to bind to isolated site II, whereas about 50% bound to isolated site I. However, only about 10% of large T antigen molecules bound to the complete wild-type ORI, containing combined binding sites I and II. Thus, a much larger proportion of large T antigen molecules is capable of binding specifically to site I as is suggested by analysis of large T antigen binding to combined sites I and II on the SV40 wild-type ORI. These findings indicate that the interaction of large T antigen with the SV40 wild-type ORI is restricted on one hand by the ability of large T antigen to bind to site II, and on the other hand by the spatial arrangement of binding sites I and II on the SV40 wild-type ORI.     

Limitations of HLA-transgenic mice in presentation of HLA-restricted cytotoxic T-cell epitopes from endogenously processed human papillomavirus type 16 E7 protein

We investigated the use of mice transgenic for human leucocyte antigen (HLA) A*0201 antigen-binding domains to test vaccines composed of defined HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitopes of human papillomavirus (HPV) type 16 E7 oncoprotein. HPV is detected in >90% of cervical carcinomas. HPV16 E7 oncoprotein transforms cells of the uterine cervix and functions as a tumour-associated antigen to which immunotherapeutic strategies may be directed. We report that although the HLA A*0201 E7 epitope peptides function both to prime for E7 CTL responses, and to sensitize target cells for E7-directed CTL killing in situations where antigen processing is not required, the epitopes are not processed out of either endogenously expressed or immunization-introduced E7, by the mouse antigen-processing and presentation machinery. Thus (1) CTL induced by HLA A*0201 peptide immunization killed E7 peptide-pulsed target cells, but did not kill target cells expressing whole E7; (2) immunization with whole E7 protein did not elicit CTL directed to HLA A*0201-restricted E7 CTL epitopes; (3) HLA A*0201-restricted CTL epitopes expressed in the context of a DNA polytope vaccine did not activate E7-specific T cells either in 'conventional' HLA A*0201 transgenic (A2.1Kb) mice, or in HHD transgenic mice in which expression of endogenous H-2 class 1 is precluded; and (4) HLA A*0201 E7 peptide epitope immunization was incapable of preventing the growth of an HLA A*0201- and E7-expressing tumour. There are generic implications for the universal applicability of HLA-class 1 transgenic mice for studies of human CTL epitope presentation in murine models of human infectious disease where recognition of endogenously processed antigen is necessary. There are also specific implications for the use of HLA A2 transgenic mice for the development of E7-based therapeutic vaccines for cervical cancer.