Dissection of H-2Db-restricted cytotoxic T-lymphocyte epitopes on simian virus 40 T antigen by the use of synthetic peptides and H-2Dbm mutants

Tevethia, Satvir S.; Lewis, Marc S.; Tanaka, Yuetsu; Milici, Janice; Knowles, Barbara B.; Maloy, W L; Anderson, Robert

doi:10.1128/jvi.64.3.1192-1200.1990

Cited by 30 publications

(25 citation statements)

References 59 publications

(41 reference statements)

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“…Cytotoxicity assays. Susceptibility of B6 cell lines to lysis by SV40 T-antigenspecific CTL clones was measured in 51 Cr release assays as described previously (72,79). Similar results were obtained whether target cells were labeled with 51 Cr before (as a monolayer) or after (in suspension) trypsinization.…”

Section: Methodsmentioning

confidence: 76%

Hierarchy among multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian virus 40 T antigen

Mylin

Bonneau

Lippolis

et al. 1995

J Virol

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Simian virus 40 large tumor (T) antigen contains three H-2D b -restricted (I, II/III, and V) and one H-2K brestricted (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-2 b mice with syngeneic, T-antigen-expressing cells. By contrast, a weak CTL response against the H-2D b -restricted epitope V was detected only following immunization of H-2 b 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-2D b -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 specifies an optimized H-2D b binding motif and was found to induce CTL in H-2 b 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-2D b 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-2D b 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.

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Section: Methodsmentioning

confidence: 76%

Hierarchy among multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian virus 40 T antigen

Mylin

Bonneau

Lippolis

et al. 1995

J Virol

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“…la, b). This can [21] this study [22]; this study [211 [12]; this study [19] [12]; this study this study this study…”

Section: Identification Of Empty Mhc Class-i Binding Peptidesmentioning

confidence: 88%

“…Peptides. Nine amino-acid long synthetic peptides (Table I) corresponding to sequence residues in SV40 T-antigen epitopes I. II-III, and V [19][20][21] were synthesized by the solid-phase method described by Merrifield [31] with an applied Biosystems Model 430 A automated peptide synthesizer (Foster City, CA, USA). The peptides were purified by reverse-phase HPLC (Waters) and the purity ofthe peptides was analysed by mass spectrometry (VG ZAB-VSE).…”

Section: Methodsmentioning

confidence: 99%

“…Synthetic peptides corresponditig to residues in the H-2Drestricted epitopes I, II-III, and V of SV40 T antigen ( [21]; Table I) were tested in an immunoprecipitation assay [34,35] to thermostabilize empty MHC class-1 molecules in iysates of the mutant murine cell line RMA/S [29,30]. This cell line is deficient in the expression of the transporter associated with antigen presentation (TAP2, [36,37]) and accumulates thermolabile empty class-I molecules in the endoplasmic reticulum (ER; [34,38]).…”

Section: Identification Of Empty Mhc Class-i Binding Peptidesmentioning

confidence: 99%

“…Fourofthem(epitopesl, II-lII, and V)are H-2D^estricted. whereas T-cell epitope IV is H-2K'' restricted [21]. Sites II and III share overlapping amino-acid residues [21].…”

Section: Introductionmentioning

confidence: 99%

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Interaction of In Vitro‐ and In Vivo‐Generated Cytotoxic T Cells with SV40 T Antigen: Analysis with Synthetic Peptides

Alsheikhly¹

1994

Scand J Immunol

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Virus-specific cytotoxic T cells recognize antigens in the form of peptides (8 or 9 amino acids long) bound to MHC class-I molecules. Exposure of unprimed murine splenocytes to synthetic peptides of viral antigens elicits primary CTL in vitro. The fine specificity of such CTL as well as the correlation between binding affinity of peptides to class-I molecules and CTL induction was analysed using synthetic peptides corresponding to overlapping and distinct amino-acid residues in SV40 T antigen (Tag) Db-restricted T-cell epitopes I, II-III, and V. The peptides induced cross-reactive CD8+ primary CTL in splenocytes of naive C57 BL/6 mice. This reactivity was seen regardless of the peptides allelic anchor motifs or their abilities to stabilize empty class-I molecules. However, none of the primary CTL and CTL lines lysed Tag-expressing cells. In contrast, CTL generated in vivo by immunizing mice with Tag-expressing cells recognized endogenously processed Tag as well as synthetic peptides. The peptides recognized by these CTL depended on the intracellular concentration of Tag antigen in the immunizing cells. The reactivity of these CTL was peptide specific as shown by a functional peptide competition assay. Moreover, three peptides bound to and were recognized in the context of both Kb and Db molecules. These results have revealed a flexible disposition of MHC class-I molecules with regard to peptide binding and also reflected lack of correlation between binding affinity to class-I molecules and the capacity of peptides to induce primary CTL or to serve as potential targets. The significance of these findings in relation to identifying major T-cell epitopes using allele specific peptide motif and in vitro maintained CTL clones is discussed.

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Peptide transporter‐independent, stress protein‐mediated endosomal processing of endogenous protein antigens for major histocompatibility complex class I presentation

Schirmbeck

Reimann

1994

Eur J Immunol

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The peptide transporter-defective cell line RMA-S expressing the wild-type simian virus 40 large T antigen (wtT-Ag) from a transfected gene did not present two well-defined, H-2 class I (Db)-restricted epitopes of T-Ag to cytotoxic T lymphocytes (CTL). Hence, "endogenous" processing and presentation of the wtT-Ag depended on a functional peptide transporter heterodimer. In contrast, both T-Ag epitopes were efficiently presented to CTL by transfected RMA-S cells expressing a truncated, cytoplasmic T-Ag variant (cT-Ag) or a karyophilic, amino-terminal 272-amino acid T-Ag fragment. Transporter-independent "endogenous" processing of mutant T-Ag molecules correlated with their association with the constitutively expressed heat shock protein 73 (hsp73). Class I-restricted presentation of both epitopes processed from these hsp73-associated protein antigens was sensitive to NH4Cl and chloroquine. These data indicate that selected intracellular proteins access an alternative, hsp73-mediated pathway for class I-restricted presentation that operates independent of peptide transporters in an endosomal compartment.

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Dissection of H-2Db-restricted cytotoxic T-lymphocyte epitopes on simian virus 40 T antigen by the use of synthetic peptides and H-2Dbm mutants

Cited by 30 publications

References 59 publications

Hierarchy among multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian virus 40 T antigen

Hierarchy among multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian virus 40 T antigen

Interaction of In Vitro‐ and In Vivo‐Generated Cytotoxic T Cells with SV40 T Antigen: Analysis with Synthetic Peptides

Peptide transporter‐independent, stress protein‐mediated endosomal processing of endogenous protein antigens for major histocompatibility complex class I presentation

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