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.     

Localization of an immunorecessive epitope on SV40 T antigen by H-2Db-restricted cytotoxic T-lymphocyte clones and a synthetic peptide

SV40 tumor (T) antigen possesses four distinct antigenic determinants, sites I, II, III, and IV, recognized by SV40-specific H-2b-restricted cytotoxic T-lymphocytes (CTL) clones. SV40-transformed C57BL/6 (B6) mouse kidney cells, designated K-3, 1, 4, K-1, 4, and K-4, 1, have been isolated by immunological selection with SV40 T antigen site-specific CTL clones in vitro. The cells have lost the expression of all four antigenic sites and cannot be lysed by the CTL clones specific for antigenic sites I, II, III, and IV. To search for additional SV40-specific antigenic sites on SV40 T antigen, B6 mice were immunized with K-3,1,4 cells and stimulated spleen cells with K-3,1,4 cells in vitro. Repeated stimulation of the spleen cell culture with gamma-irradiated K-3,1,4 cells in the presence of IL-2 was necessary to generate CTL activity against K-3,1,4 cells. A new group of H-2Db-restricted CTL clones designated as Y-5 was isolated which were cytotoxic to K-3,1,4 cells. The antigenic site recognized by CTL clone Y-5, site V, was localized in the carboxy terminal half of the SV40 T antigen. By the use of a synthetic peptide corresponding to SV40 T antigen in the carboxy region, the antigenic site V was localized between amino acids 489 and 503.     

Demonstration of unique and common antigenic sites located on the SV40 large T and small t antigens

Two distinct antigenic sites were demonstrated on SV40 small t antigen using sera from hamsters bearing tumors induced by SV40-transformed cells. One of the antigenic sites on small t antigen cross-reacts with antigenic sites on large T antigen as rabbit antisera prepared against denatured large T antigen immunoprecipitated both proteins and its activity could be absorbed with extracts prepared from cells infected with the $\text{0.54}\text{0.59}$ deletion mutant dl2005 which synthesized large T antigen but not small t antigen. The second antigenic site was found to be unique for small t antigen and was identified using serum from a hamster bearing an SV40 lymphoma which preferentially immunoprecipitated small t antigen. Its activity to small t antigen could not be removed by absorption with dl 2005-infected cell extracts. Activity against unique antigenic sites on small t antigen was also found in sera from animals bearing another SV40-induced tumor (TSV5). The unique antigenic sites on small t antigen were located on the C-terminal half of the protein since shortened small t polypeptide induced by d1891 (which lacks C-terminal sequences) was not immunoprecipitated by the lymphoma serum. Unique antigenic sites on large T antigen were also demonstrated as certain sera from tumor-bearing hamsters immunoprecipitated only the large T antigen. These results indicate that animals bearing SV40 tumors immunologically distinguish the large T and small t antigens and produce antibodies specific for the common and unique antigenic sites on the two proteins.     

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.     

Cell surface binding affinity of Simian virus 40 T-antigen

Simian virus 40-transformed cells are characterized by a virus-induced tumor transplantation antigen (SV40 TSTA) defined in vivo by the rejection of tumorigenic SV40-transformed cells in SV40-immunized mice and in vitro by SV40 tumor cell-specific cytotoxic T cells. Several experimental findings support the notion that SV40-infected and -transformed cells express SV40 large tumor antigen (TAg) or closely related antigens on the cell surface (surface T). In this report, evidence is presented for a cell surface binding affinity of SV40 TAg solubilized and extracted by disruption of SV40-transformed and SV40-infected cells in growth medium. Incubation of various transformed and nontransformed living monolayer cells in situ with these extracts led to a significant uptake of TAg to the cell surface (called “externally bound TAg”) up to two to five times higher amounts in comparison to native surface T on SV40-transformed cells. This was demonstrated by the highly sensitive ¹²⁵I-protein A assay using rabbit antisera directed against purified SV40 TAg. Serological analysis of TAg in cellular extracts and of externally bound TAg revealed no apparent differences suggesting the cell surface binding affinity as a new property of SV40 TAg. We interpret our results as an indication that this property enables purified TAg to initiate the cellular immune response necessary for the SV40-tumor rejection in mice.     

Cytotoxic T lymphocyte escape variants, induced mutations, and synthetic peptides define a dominant H-2Kb-restricted determinant in simian virus 40 tumor antigen

Immunization of C57BL/6 mice with syngeneic cells transformed by simian virus 40 large T antigen (SV40 T ag) induces the generation of T antigen-specific cytotoxic T lymphocytes (CTL) which are restricted by the major histocompatibility class I antigens H-2Db and H-2Kb. Previous studies have shown that the H-2Db-restricted CTL response is directed to at least three distinct epitopes (I, II/III, and V) in the SV40 T antigen which have been precisely mapped using deletion mutagenesis and overlapping synthetic peptides. Although in vivo the CTL response to SV40 T antigen is dominated by the H-2Kb class I antigen, the precise location of the H-2Kb-restricted epitope(s) was not known, and whether there was multiplicity of H-2Kb-restricted epitopes remained unclear. In this study, we have defined the minimal recognition epitope for the SV40-specific H-2Kb-restricted CTL clone Y-4 as T antigen residues 404-411 by using T antigen deletion and point mutants and synthetic peptides. DNA sequence analysis of the region encoding residues 404-411 from the T antigens expressed in three independently isolated CTL clone Y-4 escape variants identified inactivating mutations capable of abrogating CTL recognition. Estimation of CTL precursor (CTLp) frequencies by limiting dilution analysis revealed that CTLp specific for epitope IV represent a large percentage of the total CTL response elicited by the intact T antigen in H-2b mice. Immunization of B6 mice with cells expressing a T antigen derivative deleted of residues 404-411 revealed that site IV represents the only immunodominant H-2Kb-restricted epitope within T antigen.     

SV40-transformed cells express SV40 T antigen-related antigens on the cell surface

SV40 tumor antigen (T-Ag)-related antigens were detected serologically on the surface (surface T) of living SV40-transformed human and mouse monolayer cells by an ¹²⁵I-protein A binding assay. In immunofluorescence analysis, these cells were negative for surface T. However, on mKSA, a SV40-transformed mouse cell line grown in suspension or on SV40-transformed human and mouse monolayer cells put into suspension, surface T could be visualized by immunofluorescence microscopy. The antisera used in these experiments were raised in rabbits with purified, SDS-denatured SV40 T-Ag or came from hamsters bearing SV40 tumors. Both types of antisera had in common high titers against SV40 T-Ag (?1:1000). All these antisera were negative on normal cells or on polyoma virus-transformed cells. The specificity of both antisera for SV40 T-Ag-related binding sites on the surface of SV40-transformed cells were demonstrated by an ¹²⁵I-IgG blocking assay in which preincubation of the cells with rabbit anti-T-Ag serum inhibited the binding of hamster SV40 tumor serum to the cell surface by about 85%. These results demonstrate the expression of T-Ag-related antigens on the surface of living cells and, therefore, support the hypothesis that SV40 T-Ag-related antigens participate in the formation of the SV40-specific tumor transplantation antigen (TSTA).     

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.     

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.     

Differential presentation of endogenously processed cytotoxic T lymphocyte epitopes by mouse hepatocarcinoma cell lines induced by SV40 large T antigen

Tumor cells can have different morphologic or metabolic phenotypes and display genetic instability. Thus they could also vary in their ability to present epitopes to the immune system. We have analyzed the presentation of H-2 Kb- and Db-restricted cytotoxic T lymphocyte (CTL) epitopes of a tumor-associated antigen by three cell lines derived from hepatocarcinomas developed in vivo by mice transgenic for SV40 T targeted to the liver. SV40 T is the obvious tumor-specific antigen and epitopes derived from this antigen were therefore studied. The study included four already known epitopes that can be presented by SV40- transformed kidney cells and two new CTL epitopes that were identified in the present work. CTL lines specific for each epitope were obtained from C57BL/6 mice and were used to map the presentation of SV40 T peptides by the hepatocarcinoma cells. These tumor cells were derived from the same tissue, induced by the same agent and all naturally presented peptide p232-240 from p53. Despite these common features, they all had different patterns of spontaneous presentation of SV40 T CTL epitopes. The mechanisms underlying this disparity are discussed, together with the possible consequences for establishing immunotherapeutic strategies.      

An immunochemical investigation of SV40 T antigens. 1. Production properties and specificity of rabbit antibody to purified simian virus 40 large-T antigen.

Large quantities of a species of T antigen with an apparent molecular weight of 84,000 have been isolated from monkey kidney cells infected with SV40 by using the protein A Antibody Adsorbent (P.A.A.) technique and preparative SDS-polyacrylamide gel electrophoresis. The purified polypeptide was found to be immunogenic, inducing a specific antibody response in a rabbit. The resulting antiserum was 10 times as potent as a hamster anti-tumor serum and reacted with native as well as SDS- and DTT-treated T antigens from SV40-transformed or lytically infected cells. It failed to show any reaction with T antigen from polyoma-infected cells and showed similar specificity to antitumor serum obtained from hamsters which had been inoculated with cells of an SV40-transformed, virus-free cell line. In both cases two distinct polypeptides, large T (84,000 and 94,000) and small t (19,000) were precipitated from extracts of SV40-transformed or lytically infected cells. The rabbit antiserum was shown to be capable of specifically precipitating small-t antigen in the absence of large-T antigen and therefore these two polypeptides must share common antigenic determinants. A radioimmunoassay showed large-T antigen to be very heat stable in direct contrast to earlier results obtained using the complement fixation test. The reasons for this discrepancy and its functional significance are discussed.     

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.     

Comparison of SV40 induced antigens on the surface of cultivated cells by a cytolytic microassay

By means of a direct cytolytic assay, SV40 specific antisera were shown to kill SV40 T antigen positive AL/N mouse cell lines, including lines which were repeatedly passed through the syngeneic mouse as tumors. The sera also killed a polyoma transformed SV40 T antigen negative AL/N cell line, but not untransformed or spontaneously transformed AL/N embryo cell lines. With the antisera tested there was also no lysis observed on SV40 T antigen positive Balb/c mouse, hamster or human cell lines, although by use of a competition assay SV40 specific common antigens were detectable on the surface of these cells.     

Antigenic relationship of SV40 early proteins to purified large T polypeptide.

Rabbit antiserum was produced against SV40 large T antigen purified by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. This antiserum immunoprecipitated both large T and small t antigens, and reacted with SV40 T, U, and S antigens by immunofluorescence. these data establish the antigenic relatedness of all the known SV40 early gene products, with the exception of transplantation antigen activity, and confirm the virus-specific nature of each. The reactivity of the anti-T polypeptide serum was compared with the specificities of T-reactive antisera produced by different methods, including conventional tumor-bearing hamster sera, rabbit antiserum directed against whole-cell SDS-lysates of SV40-transformed rabbit kidney cells, and high-titer ascites fluid from hamsters in which ascites was induced by injection of SV40-transformed hamster ascites cells. Each of the antisera was reactive in all of the tests for SV40-induced early antigens, but the relative reactivity toward each protein varied considerably. It is postulated that the differences in reactivity to small t antigen and U antigen represent differences in the immune response of individual animals to the amino and carboxyl termini of the large T antigen polypeptide, respectively. Antiserum produced against the SDS-denatured large T polypeptide exhibited the highest reactivity to both small t and U antigenic sites relative to its reactivity against intranuclear large T antigen.     

Rescue of defective SV40 from a transformed mouse 3T3 cell line: Selection of a specific defective

Small-plaque type SV40 yields various defectives containing deletions, insertions, and substitutions. To select a specific defective lacking a part of the late genes, we attempted to rescue a defective mutant from a transformed mouse cell line. A triple heterokaryon culture was prepared which consisted of monkey cells, 3T3 cells transformed by defective SV40, and 3T3 cells transformed by large-plaque type SV40 that yields no defective virus having the capacity to direct T antigen synthesis. Virus recovered from the heterokaryon culture was propagated in monkey cells, and defective light virions were concentrated by CsCl equilibrium centrifugation. The virions and their DNA thus obtained were found to direct T antigen synthesis but not synthesis of virion antigen. Heteroduplex DNA molecules presumably consisting of a complete and a defective strand had mostly two single-stranded loops. The rescued defective SV40 genome appeared to have a deletion and an insertion at two separate and fixed sites.     

Antibody response to human papovavirus JC (JCV) and simian virus 40 (SV40) T antigens in SV40 T antigen-transgenic mice.

Human papovavirus JC (JCV) and simian virus 40 (SV40) genomes share approximately 69% homology; and there is antigenic cross-reactivity between JCV and SV40 tumor or T antigens. In order to determine whether a selective immune response to JCV T antigen could be demonstrated, transgenic mice (SV11+) that express SV40 T antigen in the choroid plexus and are partially tolerant to antigenic determinants on SV40 T antigen were immunized with SV40 or JCV T antigens and their antibody responses were analyzed. The results show that SV11+ mice responded as well as their nontransgenic litter mates to JCV T antigen. Monoclonal antibodies were derived from hybridomas generated from immunized mice which reacted specifically with epitopes in the amino and carboxy terminal halves on JCV T antigen. These studies show that transgenic mice expressing SV40 T antigen are capable of responding to determinants not shared between JCV and SV40 T antigen.     

Immunological properties of murine thymus-dependent lymphocyte surface glycoproteins.

The immunological properties of two murine thymus-dependent (T) lymphocyte surface glycoproteins, T200 and T25, were investigated. T200 is a lymphocyte-specific antigen with a high degree of species specificity. It shares antigenic determinants with molecules present on thymus-independent (B) lymphocytes. T25 has antigenic determinants which cross-react with antigens on mouse brain, rat thymocytes and rat brain. An antiserum against a purified rat brain glycoprotein which carries Thy-1.1 reacts with T25. Absorption of this antiserum with BALB/c thymocytes or brain homogenate produces a Thy-1.1 specific serum which reacts with T25 from AKR/J thymocytes but not with T25 from AKR/Cum thymocytes. These results confirm that T25 is the molecule on the surface of mouse T cells which carries the Thy-1 antigen. T25 also carries antigenic determinants, recognized by anti-thymocyte serum (ATS), which were found on secondary mouse embryo fibroblasts and untransformed fibroblast cell lines but which were not detected on fibroblast cell lines transformed with murine sarcoma virus (MSV) or with Simian virus 40 (SV40).     

High-affinity SV40 T-antigen binding sites in the human genome

We describe two different approaches to isolate human genomic sequences possessing high-affinity binding sites for the simian virus 40 (SV40) large T antigen. First, SV40 T antigen was added to Sau3A-restricted human DNA; the resulting T-antigen-DNA complexes were collected after repeated passages through nitrocellulose filters. The second approach involves the specific immunoprecipitation of chromatin fragments, generated by Sau3A treatment of nuclear chromatin from SV40-transformed human cells. The DNA fragments obtained were cloned in plasmid vectors for further investigation. Using the filter binding approach we isolated four different fragments with high-affinity binding sites. The binding site in one fragment was related to the strong T-antigen binding site I in the SV40 genome. The other three fragments contained multiple recognition pentamers, GA(G)GGC. Only one fragment with a high-affinity binding site was identified among the immunoprecipitable chromatin fragments. This DNA fragment belongs to the L1 family of human repetitive DNA. We present evidence suggesting that a significant fraction of human L1 elements possesses T-antigen binding sites. L1-related sequences appear as extrachromosomal elements in an SV40-transformed human cell line, and the amount of extrachromosomal L1 DNA was found to increase after fusion of transformed cells to permissive monkey cells.     

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.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). V In vitro demonstration of SV40 TrAg in SV40 infected nonpermissive mouse cells by the lymphocyte mediated cytotoxicity assay.

The development of SV40-specific transplantation antigen (TrAg) on the surface of nonpermissive mouse cells infected with SV40 was demonstrated using a sensitive in vitro lymphocyte mediated cytotoxicity assay. The in vitro lymphocyte mediated ⁵¹Cr release assay was shown to be specific for the detection of SV40 TrAg. SV40 TrAg was detected 24 hr after infection of mouse cells with SV40 and high levels of TrAg expression persisted for as long as 96 to 120 hr after virus infection. The development of TrAg on the surface of SV40-infected mouse cells correlated with the synthesis of tumor or T antigen in the nucleus of infected cells. The synthesis and the expression of TrAg at the surface of SV40-infected mouse cells may be an important step in the development of immunological resistance of the cell mediated type in an SV40-inoculated host leading to the elimination of stably transformed cells.