- [Show abstract] [Hide abstract] ABSTRACT: Va14Ja18 natural T (iNKT) cells rapidly elicit a robust effector response to different glycolipid Ags, with distinct functional outcomes. Biochemical parameters controlling iNKT cell function are partly defined. However, the impact of iNKT cell receptor beta-chain repertoire and how alpha-galactosylceramide (alpha-GalCer) analogues induce distinct functional responses have remained elusive. Using altered glycolipid ligands, we discovered that the Vb repertoire of iNKT cells impacts recognition and Ag avidity, and that stimulation with suboptimal avidity Ag results in preferential expansion of high-affinity iNKT cells. iNKT cell proliferation and cytokine secretion, which correlate with iNKT cell receptor down-regulation, are induced within narrow biochemical thresholds. Multimers of CD1d1-alphaGalCer- and alphaGalCer analogue-loaded complexes demonstrate cooperative engagement of the Va14Ja18 iNKT cell receptor whose structure and/or organization appear distinct from conventional alphabeta TCR. Our findings demonstrate that iNKT cell functions are controlled by affinity thresholds for glycolipid Ags and reveal a novel property of their Ag receptor apparatus that may have an important role in iNKT cell activation.
- [Show abstract] [Hide abstract] ABSTRACT: Mouse models in which tumors arise spontaneously due to the transgenic expression of an oncoprotein provide an opportunity to test approaches that target the immune-mediated control of tumor progression. In this report we investigated the role of SV40 Tag-specific CD8(+) T cells in the control of advanced choroid plexus tumor progression using large tumor Ag (Tag) transgenic mice. Since mice of the SV11 line are tolerant to the immunodominant SV40 Tag-derived CTL epitopes, mice with advanced stage tumors were reconstituted with naive C57BL/6 spleen cells following a low dose of gamma-irradiation. This led to the priming of CTLs specific for the H2-K(b)-restricted epitope IV by the endogenous Tag and a significant increase in the life span of Tag transgenic mice. Epitope IV-specific CD8(+) T cells accumulated and persisted in the brains and tumors of SV11 mice, as determined by analysis with epitope-specific MHC class I tetramers. Brain-infiltrating epitope IV-specific T cells were capable of producing IFN-gamma as well as lysing syngeneic Tag-transformed cells in vitro. In addition, the adoptive transfer of spleen cells from Tag-immune C57BL/6 mice resulted in a dramatic increase in the control of tumor progression in SV11 mice and was associated with the accumulation of CD8(+) T cells specific for multiple Tag epitopes in the brain. These results indicate that the control of advanced stage spontaneous choroid plexus tumors is associated with the induction of a strong and persistent CD8(+) T cell response to Tag.
- [Show abstract] [Hide abstract] ABSTRACT: Recent reports have documented the presence of SV40 large T antigen (T ag) sequences in a number of human tumors and raised the question of whether cellular immunity to T ag is elicited in such individuals. We used HLA-A2.1 transgenic C57BL/6 mice to identify an epitope from T ag recognized by CD8+ CTLs when presented by this human MHC class I molecule. Immunization of HLA-A2.1 transgenic mice with syngeneic T ag-transformed cells resulted in the induction of HLA-A2.1-restricted, T ag-specific CTLs. The target epitope, residues 281-289 (KCDDVLLLL) of T ag, was identified using both cell lines expressing T ag variants and synthetic T ag peptides. Peptide 281-289 bound stably to HLA-A2.1 molecules, effectively sensitized target cells for CTL lysis, and was efficiently processed from endogenous T ag in cells of both mouse and human origin. CTLs were not cross-reactive on the human BK or JC virus T ags. Thus, SV40 T ag 281-289 represents a potential specific CTL recognition epitope for humans.
- [Show abstract] [Hide abstract] ABSTRACT: Viral oncogenes, mutated cellular oncogenes, or other adventitious agents that might contaminate vaccine preparations on inoculation of the host will encounter a T cell-mediated immune response which will play a determining role in the progression of neoplastic events or replication of contaminating viral agents. Using SV40 T antigen tumour systems as a model we discuss the regions of the oncoprotein that have an impact on tumourigenicity and the role of CD8 T lymphocyte immune responses in eliminating potential tumour cells. In addition, we discuss measures that counteract T cell immune responses to abrogate T cell-mediated immunosurveillance.
- [Show abstract] [Hide abstract] ABSTRACT: The role of CTL tolerance in tumor immunity to SV40 large T antigen (T ag)-induced tumors was studied using T ag transgenic mice of the line 501 (H2b). 501 mice express SV40 T ag under the influence of the alpha-amylase promoter, which leads to the development of osteogenic osteosarcomas late in life and eventual death between 12 and 17 months of age. We determined the ability of 501 mice to respond to the four H2b-restricted T ag CTL epitopes, which include epitope I (T ag 206-215), epitope II/III (T ag 223-231), the immunorecessive epitope V (T ag 489-497), restricted by H2-Db, and epitope IV (T ag 404-411), restricted by H2-Kb. We demonstrate that 501 mice are partially tolerant to the H2b-restricted T ag epitopes. Immunization of 4-month-old 501 mice with T ag-transformed syngeneic cell lines or a recombinant vaccinia virus expressing full-length T ag elicited CTL responses against the H2-Kb-restricted T ag epitope IV only. In contrast, immunization of 4-month-old 501 mice with recombinant vaccinia viruses expressing individual T ag epitopes as minigenes elicited CTLs against epitopes I, IV, and V, but not against epitope II/III. Complete tolerance to epitopes I, IV, and V developed in 501 mice, but the age when tolerance was detected varied for each epitope. Tolerance to epitope I occurred by 6 months of age and was accelerated in the absence of CD4+ T cells. Tolerance to the immunorecessive epitope V was observed in 12-month-old 501 mice but was independent of the presence of osteosarcomas. In contrast, CTLs specific for epitope IV were detected in mice from 3 to 14 months of age but not in mice that had developed osteosarcomas. Analysis of epitope IV-specific CD8+ cells derived from 3-month-old 501 mice with H2-Kb/epitope IV tetramers revealed decreased numbers of epitope IV-specific CD8+ cells in 501 mice relative to C57BL/6 mice, with a further decrease in older 501 mice. Tumor progression resulted in loss of H2-Kb/epitope IV tetramer staining CD8+ cells. Thus, progression to tolerance to individual T ag CTL epitopes in 501 mice is epitope dependent.
- [Show abstract] [Hide abstract] ABSTRACT: The role of CTL tolerance in tumor immunity to SV40 large T antigen (T ag)-induced tumors was studied using T ag transgenic mice of the line 501 (H2(b)). 501 mice express SV40 T ag under the influence of the alpha-amylase promoter, which leads to the development of osteogenic osteosarcomas late in life and eventual death between 12 and 17 months of age. We determined the ability of 501 mice to respond to the four H2(b)-restricted T ag CTL epitopes, which include epitope I (T ag 206-215), epitope II/III (T ag 223-231), the immunorecessive epitope V (T ag 489-497), restricted by H2-D-b, and epitope TV (T ag 404-411), restricted by H2-K-b. We demonstrate that 501 mice are partially tolerant to the H2(b)-restricted T ag epitopes, Immunization of 4-month-old 501 mice with T ag-transformed syngeneic cell lines or a recombinant vaccinia virus expressing full-length T ag elicited CTL responses against the H2-K-b-restricted T ag epitope IV only. In contrast, immunization of 4-month-old 501 mice with recombinant vaccinia viruses expressing individual T ag epitopes as minigenes elicited CTLs against epitopes I, IV, and V, but not against epitope II/III. Complete tolerance to epitopes I, IV, and V developed in 501 mice, but the age when tolerance was detected varied for each epitope. Tolerance to epitope I occurred by 6 months of age and was accelerated in the absence of CD4(+) T cells. Tolerance to the immunorecessive epitope V was observed in 12-month-old 501 mice but was independent of the presence of osteosarcomas. In contrast, CTLs specific for epitope IV mere detected in mice from 3 to 14 months of age but not in mice that had developed osteosarcomas. Analysis of epitope IV-specific CD8(+) cells derived from 3-month-old 501 mice with H2-K-b/epitope IV tetramers revealed decreased numbers of epitope IV-specific CD8(+) cells in 501 mice relative to C57BL/6 mice, with a further decrease in older 501 mice. Tumor progression resulted in loss of H2-K-b/epitope IV tetramer staining CD8(+) cells, Thus, progression to tolerance to individual T ag CTL epitopes in 501 mice is epitope dependent.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: We have evaluated the potential of conferring protective immunity to herpes simplex virus type 2 (HSV-2) by selectively inducing an HSV-specific CD8(+) cytotoxic T-lymphocyte (CTL) response directed against a single major histocompatibility complex class I-restricted CTL recognition epitope. We generated a recombinant vaccinia virus (rVV-ES-gB498-505) which expresses the H-2Kb-restricted, HSV-1/2-cross-reactive CTL recognition epitope, HSV glycoprotein B residues 498 to 505 (SSIEFARL) (gB498-505), fused to the adenovirus type 5 E3/19K endoplasmic reticulum insertion sequence (ES). Mucosal immunization of C57BL/6 mice with this recombinant vaccinia virus induced both a primary CTL response in the draining lymph nodes and a splenic memory CTL response directed against HSV gB498-505. To determine the ability of the gB498-505-specific memory CTL response to provide protection from HSV infection, immunized mice were challenged with a lethal dose of HSV-2 strain 186 by the intranasal (i.n.) route. Development of the gB498-505-specific CTL response conferred resistance in 60 to 75% of mice challenged with a lethal dose of HSV-2 and significantly reduced the levels of infectious virus in the brains and trigeminal ganglia of challenged mice. Finally, i.n. immunization of C57BL/6 mice with either a recombinant influenza virus or a recombinant vaccinia virus expressing HSV gB498-505 without the ES was also demonstrated to induce an HSV-specific CTL response and provide protection from HSV infection. This finding confirms that the induction of an HSV-specific CTL response directed against a single epitope is sufficient for conferring protective immunity to HSV. Our findings support the role of CD8(+) T cells in the control of HSV infection of the central nervous system and suggest the potential importance of eliciting HSV-specific mucosal CD8(+) CTL in HSV vaccine design.
- [Show abstract] [Hide abstract] ABSTRACT: CD8+ T cells respond to Ags when their clonotypic receptor, the TCR, recognizes nonself peptides displayed by MHC class I molecules. The TCR/ligand interactions are degenerate because, in its life time, the TCR interacts with self MHC class I-self peptide complexes during ontogeny and with self class I complexed with nonself peptides to initiate Ag-specific responses. Additionally, the same TCR has the potential to interact with nonself class I complexed with nonself peptides. How a single TCR interfaces multiple ligands remains unclear. Combinatorial synthetic peptide libraries provide a powerful tool to elucidate the rules that dictate how a single TCR engages multiple ligands. Such libraries were used to probe the requirements for TCR recognition by cloned CD8+ T cells directed against Ags presented by H-2Kb class I molecules. When H-2Kb contact residues were examined, position 3 of the peptides proved more critical than the dominant carboxyl-terminal anchor residue. Thus, secondary anchor residues can play a dominant role in determining the antigenicity of the epitope presented by class I molecules. When the four solvent-exposed potential TCR contact residues were examined, only one or two of these positions required structurally similar residues. Considerable structural variability was tolerated at the remaining two or three solvent-exposed residues of the Kb-binding peptides. The TCR, therefore, requires close physico-chemical complementarity with only a few amino acid residues, thus explaining why TCR/MHC interactions are of low affinity and degenerate.
- [Show abstract] [Hide abstract] ABSTRACT: Simian virus 40 (SV40) has been shown to be associated with a number of human tumours. Two other human papova viruses, BKV and JCV, infect humans at a relatively high frequency and are activated upon immune suppression. The T antigens of both of these viruses share considerable homologies with the transforming protein T antigen of SV40. We have used SV40 T antigen specific cytotoxic T lymphocyte (CTL) clones to discriminate among the T antigens of SV40, BKV and JCV. These CTL clones directed to four distinct CTL epitopes serve as specific probes and can differentiate subtle alterations or deletions in the CTL epitopes relative to SV40 T antigen. Using this strategy, we have been able to authenticate three SV40 viruses isolated from humans as all four distinct CTL epitopes in the T antigens encoded by these three SV40 human isolates (SVCPC, SVMEN, and SVPML-1) were found to be identical to prototype SV40. We have further identified a 198 amino acid deletion T antigen variant of SVCPC. The finding of a deletion mutant in the SVCPC virus population suggests that the cellular immune response may play a role in the selection of antigenic loss variants.
- [Show abstract] [Hide abstract] ABSTRACT: Replication-deficient viruses provide an attractive alternative to conventional approaches used in the induction of antiviral immunity. We have quantitatively evaluated both the primary and memory cytotoxic T-lymphocyte (CTL) responses elicited by immunization with a replication-deficient mutant of herpes simplex virus type 1 (HSV-1). In addition, we have examined the potential role of these CTL in protection against HSV infection. Using bulk culture analysis and limiting-dilution analysis, we have shown that a replication-deficient virus, d301, generates a strong primary CTL response that is comparable to the response induced by the wild type-strain, KOS1.1. Furthermore, the CTL induced by d301 immunization recognized the immunodominant, H-2Kb-restricted, CTL recognition epitope gB498-505 to a level similar to that for CTL from KOS1.1-immunized mice. The memory CTL response evoked by d301 was strong and persistent, even though the frequencies of CTL were slightly lower than the frequencies of CTL induced by KOS1.1. Adoptive transfer studies indicated that both the CD8+ and the CD4+ T-cell responses generated by immunization with d301 and KOS1.1 were able to limit the extent of a cutaneous HSV infection to comparable levels. Overall, these results indicate that viral replication is not necessary to elicit a potent and durable HSV-specific immune response and suggest that replication-deficient viruses may be effective in eliciting protection against viral pathogens.
- [Show abstract] [Hide abstract] ABSTRACT: Simian virus 40 large tumor (T) antigen contains three H-2Db-restricted (I, II/III, and V) and one H-2Kb-restricted (IV) cytotoxic T lymphocyte (CTL) epitopes. We demonstrate that a hierarchy exists among these CTL epitopes, since vigorous CTL responses against epitopes I, II/III, and IV are detected following immunization of H-2b mice with syngeneic, T-antigen-expressing cells. By contrast, a weak CTL response against the H-2Db-restricted epitope V was detected only following immunization of H-2b mice with epitope loss variant B6/K-3,1,4 cells, which have lost expression of CTL epitopes I, II/III, and IV. Limiting-dilution analysis confirmed that the lack of epitope V-specific CTL activity in bulk culture splenocytes correlated with inefficient expansion and priming of epitope V-specific CTL precursors in vivo. We examined whether defined genetic alterations of T antigen might improve processing and presentation of epitope V to the epitope V-specific CTL clone Y-5 in vitro and/or overcome the recessive nature of epitope V in vivo. Deletion of the H-2Db-restricted epitopes I and II/III from T antigen did not increase target cell lysis by epitope V-specific CTL clones in vitro. The amino acid sequence SMIKNLEYM, which species an optimized H-2Db binding motif and was found to induce CTL in H-2b mice, did not further reduce epitope V presentation in vitro when inserted within T antigen. Epitope V-containing T-antigen derivatives which retained epitopes I and II/III or epitope IV did not induce epitope V-specific CTL in vivo: T-antigen derivatives in which epitope V replaced epitope I failed to induce epitope V-specific CTL. Recognition of epitope V-H-2Db complexes by multiple independently derived epitope V-specific CTL clones was rapidly and dramatically reduced by incubation of target cells in the presence of brefeldin A compared with the recognition of the other T-antigen CTL epitopes by epitope specific CTL, suggesting that the epitope V-H-2Db complexes either are labile or are present at the cell surface at reduced levels. Our results suggest that processing and presentation of epitope V is not dramatically altered (reduced) by the presence of immunodominant CTL epitopes in T antigen and that the immunorecessive nature of epitope V is not determined by amino acids which flank its native location within simian virus 40 T antigen.
- [Show abstract] [Hide abstract] ABSTRACT: Simian virus 40 tumor (T) antigen contains three H-2Db-and one H-2Kb-restricted cytotoxic T lymphocyte (CTL) epitopes (sites). Two of the H-2Db-restricted CTL epitopes, I and II/III, are separated by 7 amino acids in the amino-terminal one third of T antigen. In this study, we determine if the amino acids separating these two H-2Db-restricted CTL epitopes are dispensable for efficient processing and presentation. In addition, the importance of amino acid residues lying within and flanking the H-2Db-restricted epitopes I and II/III for efficient processing, presentation, and recognition by site-specific CTL clones was determined by using T-antigen mutants containing single-amino-acid substitutions between residues 200 and 239. Using synthetic peptides in CTL lysis and major histocompatibility complex class I stabilization assays, CTL recognition site I has been redefined to include residues 206 to 215. Substitutions in amino acids flanking either site I or site II/III did not affect recognition by any of the T-antigen-specific CTL clones. Additionally, the removal of the 7 residues separating site I and site II/III did not affect CTL recognition, thus demonstrating that these two epitopes when arranged in tandem in the native T antigen can be efficiently processed and presented to CTL clones. Differences in fine specificities of two CTL clones which recognize the same epitope (Y-1 and K-11 for site I and Y-2 and Y-3 for site II/III) have been used in conjunction with synthetic peptide variants to assign roles for residues within epitopes I and II/III with respect to TCR recognition and/or peptide-major histocompatibility complex association.
- [Show abstract] [Hide abstract] ABSTRACT: A panel of herpes simplex virus type 1 (HSV-1)-specific, CD8+, major histocompatibility complex class I (H-2Kb)-restricted cytotoxic T-lymphocyte (CTL) clones was derived from HSV-1-immunized C57BL/6 (H-2b) mice in order to identify the HSV-1 CTL recognition epitope(s) which confers type specificity. HSV-1 x HSV-2 intertypic recombinants were used to narrow the region encoding potential CTL recognition epitopes to within 0.51 to 0.58 map units of the HSV-1 genome. Using an inhibitor of viral DNA synthesis and an ICP6 deletion mutant, the large subunit of ribonucleotide reductase (ICP6, RR1) was identified as a target protein for these type-specific CTL. Potential CTL recognition epitopes within RR1 were located on the basis of the peptide motif predicted to bind to the MHC class I H-2Kb molecule. A peptide corresponding to residues 822 to 829 of RR1 was shown to confer susceptibility on H-2Kb-expressing target cells to lysis by the type 1-specific CTL. On the basis of a comparison of the HSV-1 RR1 epitope (residues 822 to 829) with the homologous sequence of HSV-2 RR1 (residues 828 to 836) and by the use of amino acid substitutions within synthetic peptides, we identified HSV-1 residue 828 as being largely responsible for the type specificity exhibited by HSV-1-specific CTL. This HSV-1 RR1 epitope, when expressed in recombinant simian virus 40 large T antigen in primary C57BL/6 cells, was recognized by the HSV-1 RR1-specific CTL clones. These results indicate that an early HSV protein with enzymatic activity provides a target for HSV-specific CTL and that type specificity is dictated largely by a single amino acid.
- [Show abstract] [Hide abstract] ABSTRACT: Simian virus 40 (SV40) large T antigen can immortalize a wide variety of mammalian cells in culture. We have taken advantage of this property of T antigen to use it as a carrier for the expression of cytotoxic T-lymphocyte (CTL) recognition epitopes. DNA sequences corresponding to an H-2Db-restricted SV40 T-antigen site I (amino acids 205 to 215) were translocated into SV40 T-antigen DNA at codon positions 350 and 650 containing EcoRI linkers. An H-2Kb-restricted herpes simplex virus glycoprotein B epitope (amino acids 498 to 505) was also expressed in SV40 T antigen at positions 350 and 650. Primary C57BL/6 mouse kidney cells were immortalized by transfection with the recombinant and wild-type T-antigen DNA. Clonal isolates of cells expressing chimeric T antigens were shown to be specifically susceptible to lysis by CTL clones directed to SV40 T-antigen site I and herpes simplex virus glycoprotein B epitopes, indicating that CTL epitopes restricted by two different elements can be processed, presented, and recognized by the epitope-specific CTL clones. Our results suggest that SV40 T antigen can be used as a carrier protein to express a wide variety of CTL epitopes.
- [Show abstract] [Hide abstract] ABSTRACT: The 94-kD large tumor (T) antigen specified by simian virus 40 (SV40) is sufficient to induce cell transformation. T antigen contains four H-2Db-restricted cytotoxic T lymphocyte (CTL) recognition epitopes that are targets for CTL clones Y-1, Y-2, Y-3, and Y-5. These epitopes have been mapped to T antigen amino acids 207-215 (site I), 223-231 (sites II and III), and 489-497 (site V), respectively. Antigenic site loss variant cells that had lost one or more CTL recognition epitopes were previously selected by coculturing SV40-transformed H-2Db cells with the site-specific Db-restricted CTL clones. The genetic bases for T antigen CTL recognition epitope loss from the variant cells were identified by DNA amplification and direct sequencing of epitope-coding regions from variant cell DNAs. Cells selected for resistance to CTL clone Y-1 (K-1; K-1,4,5; K-3,1) carry deleted SV40 genomes lacking site I, II, and III coding sequences. Point mutations present within the site II/III coding region of Y-2-/Y-3-resistant cell lines specify the substitution of asparagine for lysine as T antigen amino acid 228 (K-2) or phenylalanine for tyrosine at position 230 (K-3). Point mutations identified within independently selected Y-5 resistant populations (K-5 and K-1,4,5) direct the substitution of isoleucine for asparagine at position 496 (K-5) or the substitution of phenylalanine for isoleucine at position 491 (K-1,4,5) of T antigen. Each substitution causes loss of the relevant CTL recognition epitope, apparently by compromising CTL T cell receptor recognition. These experiments identify specific amino acid changes within a transforming protein that facilitate transformed cell escape from site-specific CTL clones while allowing maintenance of cellular transformation. This experimental model system provides unique opportunities for studying mechanisms of transformed cell escape from active immunosurveillance in vivo, and for analysis of differential host immune responses to wild-type and mutant cell-transforming proteins.
- [Show abstract] [Hide abstract] ABSTRACT: The effects on CTL recognition of individual amino acid substitutions within epitopes I, II, and III of SV40 tumor Ag (T Ag) were examined. Epitope I spans amino acids 207 to 215, and epitope II/III is within residues 223 to 231 of SV40 T Ag. An amino acid substitution at position 207 (Ala----Val) or 214 (Lys----Glu) of SV40 T Ag expressed in transformed cells resulted in loss of epitope I, recognized by CTL clone Y-1. The amino acid substitution at residue 214 in the corresponding synthetic peptide, LT207-215(214-Lys----Glu), also led to loss of recognition by CTL clone Y-1. The recognition, by CTL clone Y-1, of peptides LT207-215 and LT207-217 with an Ala----Val substitution at position 207 was severely affected. Peptides LT205-215 and LT205-219 with the Ala----Val substitution at residue 207 were, however, recognized by CTL clone Y-1, suggesting that residues 205 and 206 may be involved in presentation of site I. Alteration of residue 224 (Lys----Glu) in the native T Ag resulted in loss of recognition by both CTL clones Y-2 and Y-3. However, a peptide corresponding to epitope II/III with an identical amino acid substitution at residue 224 provided a target for CTL clone Y-3 but not clone Y-2. A change of Lys----Gln at residue 224 in both the native protein and a synthetic peptide caused loss of recognition by CTL clone Y-2 but not CTL clone Y-3. Further, an amino acid substitution of Lys----Arg at position 224 of the native T Ag decreased recognition of epitope II/III by CTL clones Y-2 and Y-3 but had no effect on recognition of a synthetic peptide bearing the same substitution. These results indicate that the mutagenesis approach, resulting in identical amino acid substitutions in the native protein and in the synthetic peptides, may provide insight into the role of individual residues in the processing, presentation, and recognition of CTL recognition epitopes.
- [Show abstract] [Hide abstract] ABSTRACT: Simian virus 40 (SV40) tumor (T) antigen expressed in H-2b SV40-transformed cells induces the generation of Lyt-2+ (CD8+) cytotoxic T lymphocytes (CTL), which are involved in tumor rejection, in syngeneic mice. Five CTL recognition sites on T antigen have been described by using mutant T antigens. Four of the sites (I, II, III, and V) are H-2Db restricted and have been broadly mapped with synthetic peptides of 15 amino acids in length overlapping by 5 residues at the amino and carboxy termini. The goal of this study was to define the minimal and optimal amino acid sequences of T antigen which would serve as recognition elements for the H-2Db-restricted CTL clones Y-1, Y-2, Y-3, and Y-5, which recognizes sites I, II, III, and V, respectively. The minimal and optimal residues of T antigen recognized by the four CTL clones were determined by using synthetic peptides truncated at the amino or carboxy terminus and an H-2Db peptide-binding motif. The minimal site recognized by CTL clone Y-1 was defined as amino acids 207 to 215 of SV40 T antigen. However, the optimal sequence recognized by CTL clone Y-1 spanned T-antigen amino acids 205 to 215. The T-antigen peptide sequence LT223-231 was the optimal and minimal sequence recognized by both CTL clones Y-2 and Y-3. Site V was determined to be contained within amino acids 489 to 497 of T antigen. The lytic activities of CTL clones Y-2 and Y-3, which recognize a single nonamer peptide, LT223-231, were affected differently by anti-Lyt-2 antibody, suggesting that the T-cell receptors of these two CTL clones differ in their avidities. As the minimal and optimal H-2Db-restricted CTL recognition sites have been defined by nonamer synthetic peptides, it is now possible to search for naturally processed H-2Db-restricted epitopes of T antigen and identify critical residues involved in processing, presentation, and recognition by SV40-specific CTL.
Penn State Hershey Medical Center and Penn State College of Medicine
هرشي، بنسيلفانيا, Pennsylvania, United States
- Microbiology and Immunology
Baylor College of MedicineHouston, Texas, United States