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.     

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.     

Stress protein (hsp73)-mediated,TAP-independent processing of endogenous,truncated SV 40 large T antigen for Db-restricted peptide presentation

Transporter associated with antigen processing (TAP)-competent and TAP-deficient cell lines were transfected with expression plasmids encoding either the wild-type (wt) large tumor antigen (T-Ag) of SV40, or a truncated cytoplasmic variant (cT-Ag) of this viral protein. Stable expression of comparable levels of both forms of the viral protein was observed in different transfectants. The truncated cT-Ag variant, but not the wtT-Ag was stably associated with the con-stitutively expressed, cytosolic heat shock protein (hsp)73 chaperone. Two D^b-binding peptides and one K^b-binding peptide of T-Ag were presented to cytotoxic T lymphocyte lines (CTLL) by TAP-competent transfectants expressing either wtT-Ag or cT-Ag. TAP-deficient transfectants expressing the wtT-Ag did not present any of these epitopes to CTLL. In contrast, TAP-deficient transfectants expressing the truncated hsp73-associated cT-Ag, presented the two D^b-binding epitopes, but not the K^b-binding T-Ag epitope to CTLL. Regurgitation of peptides by transfectants was not detectable. The described data indicate that a pool of post-Golgi D^b molecules is available for 2–3 h in TAP-deficient transfectants for loading with peptides released during endolysosomal processing of hsp73-associated, endogenous antigen.     

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.     

Differential effect of adenovirus 2 E3/19K glycoprotein on the expression of H-2Kb and H-2Db class I antigens and H-2Kb- and H-2Db-restricted SV40-specific CTL-mediated lysis

The E3/19-kDa glycoprotein (E3/19K) coded by adenovirus type 2 (Ad2) is known to inhibit the cell-surface expression of major histocompatibility complex (MHC) class I antigens by binding to the MHC antigens intracellularly, and thus reduces recognition of antigens by MHC-restricted cytotoxic T lymphocytes (CTL). We have studied the effect of the E3/19K expression in SV40-infected monkey cells, TC-7/H-2Kb and TC-7/H-2Db expressing transfected H-2Kb and H-2Db antigens, respectively, on the cell-surface H-2 class I antigens and on lysis of the cells by SV40 large tumor (T)-antigen-specific H-2Kb- and H-2Db-restricted CTL clones. H-2Db antigen expression on TC-7/H-2Db cells was drastically reduced by infection with Ad2 but not with an E3/19K-negative SV40-Ad2 hybrid virus, Ad2+ND1, as early as 12 hr postinfection. However, H-2Kb antigen expression on Ad2-infected TC7/H-2Kb cells remained unaltered, even at 24 hr postinfection. Specific lysis of SV40-infected TC-7/H-2Db cells by H-2Db-restricted SV40 T-antigen-specific CTL clones, Y-1 and Y-3, was strongly reduced by coinfection of the target cells with Ad2 but not with Ad2+ND1. Lysis of SV40-infected TC-7/H-2Kb cells by a H-2Kb-restricted SV40 T-antigen-specific CTL clone Y-4 was also reduced significantly by Ad2 infection, but not Ad2+ND1. These results indicate that the E3/19K protein affects cell-surface expression of H-2Db antigen but not H-2Kb antigen.     

Inhibition of glycosylation prevents H-2K and D antigen expression on SV40 virus-transformed cells

Tunicamycin, an inhibitor of glycosylation, was used to examine the role of carbohydrate moieties of glycoproteins involved in recognition of Simian virus 40 (SV40)-transformed cells by cytotoxic T lymphocytes (CTL). Tunicamycin treatment renders such cells refractory to lysis by H-2 restricted, SV40-specific CTL as well as by allogeneic CTL directed only against H-2 antigens. Treated cells are no longer susceptible to lysis by anti-H-2 antisera and complement, nor are they stained by monoclonal anti-H-2 antibodies in an indirect immunofluorescence assay. No accumulation of H-2 proteins was detected in immunoprecipitates from cells labeled with [35S]methionine in the presence of tunicamycin. It is concluded that tunicamycin prevents the expression of H-2 glycoproteins in these cells, perhaps due to accelerated degradation of the unglycosylated molecules.     

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.      

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.     

MHC-restricted cytotoxic T-lymphocyte assay: an improved method based on normal and SV40-immortalized rabbit epidermal target cells

Although several techniques are available to evaluate cell-mediated immunity, numerous difficulties have prevented their use in rabbits. Cytotoxic T-lymphocyte (CTL) assays have been used to determine the ex vivo cytolytic activity of CD8+ T-lymphocytes in immunization protocols. However, this assay cannot be performed with rabbit peripheral blood mononuclear cell (PBMC) targets because of their high spontaneous (51)Cr release. To overcome this intrinsic difficulty shown by rabbit cells, syngeneic normal and SV40-immortalized cells were prepared from skin biopsies. The results show that: (i) skin-derived rabbit fibroblasts can be used as target cells after infection with a fowlpox virus recombinant; (ii) SV40-immortalized skin fibroblasts appear to be more appropriate for repeated assays; (iii) antigen-expanded T-cells and fresh PBMCs can be used as effectors with a similar efficiency; and (iv) dissociation of adherent skin fibroblast target cells with EDTA is to be preferred over TrypLE enzymatic treatment.     

Biochemical properties of a transforming nonkaryophilic T antigen of SV40

We reconstructed into wt SV40 DNA a previously described deletion of the A gene, eliminating amino acids 110 through 152 of the large T (L. Fischer-Fantuzzi and C. Vesco (1985) Proc. Natl. Acad. Sci. USA 82, 1891-1895); the gene product of the new recombinant pACTSV2, like the previous product, has a cytoplasmic instead of a nuclear localization and efficiently transforms NIH3T3 cells. Three main functions of this nonkaryophilic large T (NKLT) were examined, and the results obtained were the following: the NKLT does not bind to the SV40 origin DNA under conditions where the normal large T shows specific binding; the NKLT has conserved the ability to form high molecular weight aggregates; the NKLT becomes phosphorylated in vivo at only two residues: serine 639 and threonine 701. This indicates that the NH2-terminal phosphorylation of the large T is unnecessary for established-cell transformation. In addition, this and previous evidence (K. H. Scheidtmann et al. (1984) J. Virol. 50, 636-640) suggest that the lack of phosphorylation in serines 106, 676, 677, and 679 may constitute a characteristic of the large T molecules with extranuclear localization.     

The induction of respiratory syncytial virus-specific cytotoxic T-cell responses following immunization with a synthetic peptide containing a fusion peptide linked to a cytotoxic T lymphocyte epitope.

Previously published work has shown that a cytotoxic T-cell epitope (CTL) representing residues 82-90 of the M2 protein of respiratory syncytial virus (RSV) is the target for a protective response against the virus. In this report, we demonstrate that a synthetic peptide representing residues 81-95, when covalently linked to a fusion peptide derived from the conserved N-terminal 19 residues of the F1 protein of measles virus efficiently primes RSV-specific CTLs in vivo following immunization without adjuvant.     

Localization of two cytotoxic T lymphocyte epitopes and three anchoring residues on a single nonameric peptide that binds to H-2Ld and is recognized by cytotoxic T lymphocytes against mouse tumor P815

Mouse mastocytoma P815 expresses tumor antigens P815A and P815B encoded by a single gene called P1A and carried by a single peptide named P1A 35–43 (NH₂-Leu-Pro-Tyr-Leu-Gly-Trp-Leu-Val-Phe-COOH). P1A 35–43 is presented to anti-P815A and anti-P815B cytotoxic T lymphocytes (CTL) by major histo-compatibility complex (MHC) H-2L^d molecules. In order to determine the individual role played by each amino acid residue of P1A 35-43 in binding to H-2L^d and in recognition by anti-A and anti-B T cell receptors (TcR), a series of P1A 35-43 peptides substituted by alanine at single positions was synthesized and tested for binding to H-2L^d and for CTL recognition. Binding to H-2L^d was estimated by measuring the ability of the peptide to up-regulate cell surface expression of H-2L^d. We found that three residues were important for interaction of P1A 35-43 with H-2L^d. Two of them, Pro at position 2 and Phe at position 9 were consistent with the described H-2L^d binding motif. A third residue, Trp at position 6, was also required for effective MHC binding of the tumor antigen. CTL sensitization assays showed that alanine substitution at position 7 (Leu) or at position 8 (Val) dramatically affected peptide recognition by anti-A CTL while positions 3 (Tyr) and 4 (Leu) were critical for recognition by anti-B CTL. We conclude that Pro², Trp⁶ and Phe⁹ constitute the anchor residues of P1A 35-43 to H-2L^d, whereas the dipeptidyl sequences Tyr³-Leu⁴ and Leu⁷-Val⁸ form the core epitopes recognized by the TcR of anti-P815B and anti-P815A CTL, respectively.     

Expression of SV40 T antigen in finite life-span hybrids of normal and SV40-transformed fibroblasts

The fusion of normal human fibroblasts with SV40-transformed human fibroblasts resulted in hybrid clones, 85% of which exhibited a finite in vitro life-span. Foci of rapidly dividing cells appeared in 15% of the hybrid clones. The cells within these foci repopulated the culture and could then be subcultured through more than 100 population doublings. One or two foci of dividing cells occurred per culture of 10⁵ or more cells. The change to an indefinite life-span was, therefore, a rare event. All hybrid clones, including those that exhibited a finite in vitro life-span, expressed viral T antigen. Thus, even though viral DNA was present and being expressed in all hybrid clones, the senescent phenotype was dominant in these hybrids.This work is in partial fulfillment of requirements for a doctoral degree.     

Promiscuous T cell recognition of an H-2 IA-presented mycobacterial epitope

Genetically permissive T cell epitopes are an important prerequisite for the development of peptide-based vaccines or immunodiagnostic reagents. We have investigated the structural requirements of permissive T cell recognition of peptide p350—369 from the 38-kDa antigen of Mycobacterium tuberculosis. This peptide was found to be immunogenic in mice of the H-2^{b, bm12, d. s and k}, but not of the H-2^f genotype. T cell responses were restricted by I-A class II molecules. The same epitope core was recognized in the H-2^{b, d and k} genotypes. T cell hybrids from BALB/c and C57BL/10 mice were used to determine: (i) the critical residues using substituted peptide derivatives and (ii) the degree of T cell promiscuity. Two out of five BALB (H-2^d)-derived hybridomas tested displayed promiscuous peptide recognition in the context of H-2^b and H-2^bm12 antigen-presenting cells. The recognition of critical residues was found to be uniform for all five hybridomas when tested with syngeneic antigen-presenting cells; additional critical residues were identified when the peptide was recognized in the context of allogeneic antigen-presenting cells. Only one of the four tested C57BL/10 (H-2^b) hybridomas showed promiscuity in the context of H-2^bm12. Each of these C57BL/10-derived clones had a distinct response profile toward the critical residues. We propose that the demonstrated T cell promiscuity involves peptide interaction with polymorphic H-2 I-A residues.     

Effect of inhibitors on induction of SV40 tumor antigen by an adenovirus-SV40 hybrid

Summary A type 7 adenovirus-SV40 hybrid induces the synthesis of SV40 tumor or T antigen in hamster, rabbit, human, and monkey cells. Actinomycin D inhibits synthesis of the antigen suggesting that development of the antigen requires DNA-dependent RNA. Puromycin allows synthesis of T antigen which is therefore, apparently a low molecular weight protein or polypeptide. Synthesis of the antigen is not inhibited by p-fluorophenylalanine. Neither mitomycin C, fluorouracil, iododeoxyuridine, nor cytosine arabinoside prevent development of T antigen. It therefore appears probable that for synthesis of T antigen to proceed the synthesis of new virus DNA is not required regardless of whether this is virus DNA of SV40 or of adenovirus.Antiserum prepared against type 7 adenovirus neutralizes the ability of the hybrid to induce SV40 tumor antigen. Antibody against the SV40 tumor antigen or against whole SV40 virus failed to do so. The evidence therefore suggests that the adenovirus hybrid is carrying a piece of SV40 tumor-inducing genome which replicates with the adenovirus. Adenovirus-SV40 hybrids have been found not only with type 7 but also with many other types (24, 25).Dedicated to the Honor of the 60th birthday of ProfessorSven Gard.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). VI. Mechanism of induction of SV40 transplantation immunity in mice by purified SV40 T antigen (D2 protein)

According to the base sequence homology of the gene coding for the nonstructural (NS) protein the influenza A viruses can be divided into at least three groups. Within each group the homology is about 90% or higher. The avian influenza A viruses fall at least into two groups between which the homology is about 40%. All human strains tested belong to another group. Influenza viruses of other species might comprise their own group(s). The related regions of the NS gene among the two groups of avian influenza viruses overlap completely and they are highly conserved. The results are discussed in terms of a selection pressure concerning the function exerted by the host during the evolution of the different NS genes, which also might explain a certain species specificity concerning the NS gene of influenza A viruses.     

Identification of an HLA-DQ6-derived peptide recognized by mouse MHC class I H-2Db-restricted CD8+ T cells in HLA-DQ6 transgenic mice

Summary CD8⁺ T cells from C57BL/6(B6) mice show cytotoxicity to B cell blasts prepared from syngeneic transgenic mice expressing HLA-DQ6 molecules in a mouse MHC class I H-2D^b restricted manner. Although these results suggest that CD8⁺ T cells recognize peptides derived from DQ6 molecule bound to H-2D^b on target cells, no direct evidence so far has been obtained. To clarify this, we synthesized 23 peptides corresponding to DQ6α orβ chain and carrying the motifs of D^b-binding peptides, and examined their capacity to induce cytotoxicity in the CD8⁺ T cell line. We show here that DQA1-2, one of these peptides, induced cytotoxicity of the CD8⁺ T cells when this peptide was pulsed to H-2D^b expressing target cells, as efficiently as HLA-DQ6 expressing target cells did. Thus, our results suggest that DQA1-2 can be naturally processed from DQ6 molecules and recognized by the CD8⁺ T cells in the context of H-2D^b molecules. These results suggest that allogeneic HLA class II molecules are involved in the rejection not only as the ligand for T cell receptor of alloreactive CD4⁺ T cells but also as self-peptides bound to HLA class I molecules recognized by CD8⁺ T cells.     

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.     

Preferential usage of Vα4 and Vβ10 T cell receptor genes by lymphocytic choriomeningitis virus glycoprotein-specific H-2Db-restricted cytotoxic T cells

Correlations between the T cell receptor (TcR) V gene usage and the specificity of T cells have been primarily described for major histocompatibility complex (MHC) class II-restricted helper T cell responses. In the present study the TcR genes expressed by MHC class I-restricted murine cytotoxic T cells (CTL) specific for a major epitope of the lymphocytic choriomeningitis virus (LCMV), LCMV-GP2_275–289, were investigated. The TcR primary structure of an LCMV-GP2_275–289 specific H-2D^b-restricted CTL clone has been determined. It uses a member of the V_α4 family joined to J_αAN14.4 for the α chain and V_β10 rearranged to D_β2.1 and J_β2.4 for its β chain. Four other independent LCMV-GP2_275–289 specific H-2D^b-restricted CTL clones also expressed V_α4 and V_β10 gene elements. Furthermore, V_α4 and V_β10 were preferentially expressed by polyclonal CTL of C57BL/6 origin specific for LCMV. These results suggest that both TcR V_α and V_β regions are important for the recognition of the LCMV-GP2_275-289 epitope on H-2D^b molecules.