The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mylin, L. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mylin, L. M.
The Journal of Immunology, 1999, 162: 2171-2179.
Copyright © 1999 by The American Association of Immunologists

Context-Dependent Immunogenicity of an S206G-Substituted H-2Db-Restricted Simian Virus 40 Large T Antigen Epitope I Variant1

Lawrence M. Mylin2

Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, PA 17033


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
SV40 large tumor Ag (Tag) contains four H-2b-restricted (I, II/III, IV, and V) CTL epitopes. A hierarchy exists among these CTL epitopes. CTL directed against epitopes I, II/III, and IV are readily detected following immunization of H-2b mice with SV40, Tag-transformed syngeneic cells, or a vaccinia recombinant that expresses full-length Tag, while epitope V-specific CTL are not. The mechanisms that define this hierarchy remain unknown. Initial studies have shown that the locations of epitopes I and V within SV40 Tag do not determine the immunological potencies of these epitopes. Like the wild-type Tag, derivatives in which the locations of epitopes I and V were precisely reversed within Tag failed to induce epitope V-specific CTL, but did induce epitope I-specific CTL. The use of an S206G-substituted epitope I variant (GAINNYAQKL) revealed that the S206G variant sequence induced CTL when located within the native epitope I context, but failed to do so when located within the epitope V context of Tag. Mutagenesis of residues adjacent to the S206G-substituted epitope I variant revealed that the identity of the residue flanking the amino terminus of the S206G variant was critical when it resided within the epitope V location, but not within the epitope I location. These results demonstrate that effects imposed by both regional context and adjacent residues can modulate immunogenicity, but that the relative importance of such effects varies in an epitope-dependent manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD8+ cytotoxic T lymphocytes (CTL)3 recognize and lyse target cells that display cell surface MHC I molecule complexes containing appropriate short epitope peptides (8–10 residues) derived from viral or oncogene-encoded protein products 1, 2 . Peptides presented by MHC class I molecules are largely derived by proteolysis from proteins that gain access to the cytoplasmic compartment of the APC. The proteasome, a large cytosolic multicatalytic proteinase complex, has been implicated in generating a majority of peptides that are presented by MHC class I complexes 3, 4 . Peptides liberated from antigenic proteins in the cytosol are transported into the lumen of the endoplasmic reticulum by TAP, where they can be assembled into MHC class I complexes and are exported to the cell surface 5 .

The factors that regulate the efficiency with which epitope peptides are liberated from antigenic proteins, traverse the MHC class I Ag presentation pathway, and remain on display at the cell surface for T lymphocyte recognition are still only poorly understood. Efficient processing and presentation of MHC class I-restricted epitopes appear to be largely independent of the native amino acid flanking sequence context. CTL epitopes have been efficiently processed and presented from antigenic proteins (or segments thereof) in which the flanking sequences have been altered 6, 7 or from within heterologous proteins or short polyepitope minigenes where sequences surrounding the epitope are completely foreign 8, 9, 10, 11, 12, 13, 14, 15 . Results from several studies, however, indicate that amino acid sequences that surround an epitope within a protein (or short polypeptides encoded by minigene constructs) can profoundly influence MHC class I-restricted Ag processing and presentation 12, 16, 17, 18, 19 . For example, proline substitutions at amino acid residue positions immediately flanking the amino or carboxyl termini of the H-2Kd-restricted influenza virus nucleoprotein (NP) epitope (NP147–155) dramatically reduced presentation from within full-length NP to CTL in vitro and reduced immunogenicity in vivo 16 . However, proline residue substitutions at the flanks of two other epitopes within NP resulted in only minor or no noticeable change in the processing and presentation of those determinants 16 . Therefore, it is likely that properties determined by the primary sequence of a test epitope influence the magnitude of effects imposed on processing and presentation by the amino acid context surrounding that epitope within a polypeptide. It is equally possible, however, that effects imposed by nonadjacent flanking sequences (regional effects) contributed to the differential effects observed in those studies because three different test epitopes (at three different locations within NP) were examined.

We have used the SV40 large tumor Ag (SV40 Tag) to study factors that control the processing, presentation, and immunogenicity of CTL epitopes 6, 9, 15, 20 . Tag induces oncogenic transformation in a wide variety of cell types in vitro and in vivo; in the immunocompetent host, Tag also induces a vigorous cellular immune response that leads to rejection of developing Tag-induced tumors (reviewed in Refs. 21 and 22). SV40 Tag contains three H-2Db-restricted (I, II/III, and V) and one H-2Kb-restricted (IV) CTL epitopes, among which an immunological hierarchy has been defined 15, 21, 23, 24, 25 . Epitopes I, II/III, and IV are immunologically dominant, while epitope V has been characterized as weak or immunorecessive 15, 23 . CTL directed against epitopes I, II/III, and IV are readily detected following immunization of H-2b mice with SV40, Tag-transformed syngeneic cells, or a vaccinia recombinant that expresses full-length Tag 9, 15 . By contrast, Tag-induced epitope V-specific CTL were detected only following immunization with Tag derivatives that lacked the immunodominant CTL epitopes 9, 15 . Nonetheless, CTL clones specific for each of the Tag epitopes (I, II/III, IV, and V) readily lyse Tag-transformed cells, indicating that each of these epitopes (including epitope V) is readily processed and presented in cells of the H-2b haplotype 15 .

In a previous study we reported that relocation of the immunorecessive epitope V within SV40 Tag to the site (Tag protein context) normally occupied by the immunodominant epitope I did not lead to an observable increase in the immunogenicity of epitope V 15 . In this report we extend these studies by using epitope I (SAINNYAQKL; Tag residues 206–215) and an S206G-substituted epitope I variant (GAINNYAQKL) to examine effects on processing, presentation, and immunogenicity contributed by adjacent and nonadjacent sequences that flank epitopes I and V in SV40 Tag. Our results indicate that the magnitude of inhibitory effects imposed by adjacent residues is influenced by the region of Tag examined (regional context effects), and that the identity of the test epitope determines whether regional or adjacent residue effects will noticeably compromise MHC class I-restricted processing and presentation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Four- to six-week-old male C57BL/6 (H-2b) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and routinely used between the ages of 5–12 wk. All mice were maintained in the animal facility at Pennsylvania State University College of Medicine (Hershey, PA).

Plasmid construction and manipulation

Plasmid DNA used for sequencing and transfection was routinely prepared by the QIAwell-8 procedure (Qiagen, Chatsworth, CA). The plasmid pSelectESV-1 15 was constructed by inserting a 3.0-kb KpnI-BamHI fragment representing VA45–54 SV40 early region DNA (encodes large T Ag under control of the SV40 early promoter) into the phagemid pSelect (Promega, Madison, WI). Substitution and deletion derivatives of T Ag (Table IGo) were generated by site-directed mutagenesis (using pSelectESV-1 or subsequent Tetr Amps derivatives encoding mutant T Ag proteins) according to the Altered Sites Mutagenesis procedure (Promega) as previously described 15 . Oligonucleotides used for mutagenesis and DNA sequencing were synthesized by a MilliGen/Biosearch 7500 DNA synthesizer in the Macromolecular Core Facility at the Pennsylvania State University College of Medicine (Hershey, PA). Codons used to construct T Ag CTL epitope exchange derivatives were identical with those found in the wild-type VA45–54 coding sequence. The nucleotide sequences of VA45–54 regions that encompass the H-2b-restricted T Ag CTL epitopes are identical with that which has been reported previously 26 (data not shown). Alterations to T Ag were verified by DNA sequence analysis.


View this table:
[in this window]
[in a new window]
  		Table I. Cell lines used in this study

 
Plasmid pLM299 (S206G in Tag) was derived from pSelect-ESV-1 15 by a TCT->GGT alteration affecting codon 206 and were subsequently used to construct plasmids pRLM340, pRLM343, pRLM344, and pRLM345. A deletion of the epitope V coding sequence in plasmid pLM254 15 was filled with sequences encoding the S206G-215 epitope I variant to generate plasmid pLM332. Plasmid pLM332 was used to construct plasmids pRLM346, pRLM347, pRLM348, and pRLM349 by alteration of codons surrounding the S206G-215 sequence located within the epitope V context of Tag.

Cell lines

The cell lines used in this study are listed in Table IGo. B6/WT-19 cells have been described previously 27 . B6/K-1,4,5 (H-2b) cells, which were generated by sequential cocultivation of cells expressing wild-type SV40 Tag with SV40 Tag-specific CTL clones, have been previously described 28, 29 . B6/K-1,4,5 cells are resistant to lysis by SV40 Tag-specific CTL clones due to a deletion that removes sequences encoding determinants I and II/III, and point mutations that alter determinants IV and V 30, 31 .

Tag-transformed cell lines were derived by isolation of Tag-expressing, immortalized foci following calcium phosphate-mediated transfection of C57BL/6-derived embryonic fibroblasts with plasmid DNA (see Table IGo) as previously described 15, 32 . Tag-transformed cell lines were routinely maintained in closed vessels in DMEM supplemented with 7.5% (v/v) heat-inactivated FBS (HyClone, Logan, UT), 2 mM L-glutamine, 100 U of penicillin/ml, 100 µg of streptomycin/ml, 25 µg of kanamycin/ml, 20 mM HEPES, and 0.15% (w/v) sodium bicarbonate 15 . RMA and RMA/s cells were maintained in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 5 x 10-5 M 2-ME, 25 µg of pyruvic acid/ml, 100 U of penicillin/ml, 100 µg of streptomycin/ml, and 5 mM HEPES (CTL medium) 15 .

SV40-specific CTL clones

SV40-specific CTL clones Y-1 or K-11, K-19, Y-4 and Y-5, or H-1 recognize, respectively, SV40 Tag CTL determinants I (Tag amino acid residues 206–215), II/III (223–231), IV (404–411), and V (489–497) 6, 15, 23, 24, 25, 31, 33 . The CTL clones were maintained in vitro by restimulation twice weekly with gamma-irradiated B6/WT-19 or B6/K-3,1,4 cells in CTL medium containing Human rIL-2 (AmGen, Thousand Oaks, CA) or T-Stim culture supplement (Collaborative Biomedical Products, Bedford, MA) and methyl-{alpha}-D-manno-pyranoside (Sigma, St. Louis, MO) as previously described 15, 25 .

Generation of bulk culture CTL for cytotoxicity assays

Bulk culture CTL were prepared from splenic lymphocytes obtained from C57BL/6 mice immunized with 2 x 107 Tag-transformed cells as previously described 15 . Red cell-depleted splenic lymphocytes were cultured in 12-well plates (Costar, Cambridge, MA). Each well was seeded with 1 x 107 splenocytes and 5 x 105 gamma-irradiated (10,000 rad) stimulator cells in 4 ml of CTL medium, incubated at 37°C in 5% CO2 for 6 days, and used as effectors in 51Cr release assays.

Preparation of target cells for use in cytotoxicity assays

Procedures for the preparation and use of peptide-pulsed target cells in cytotoxicity assays have been previously described 9, 15, 25, 34 . Peptides corresponding to sequences within T Ag were synthesized in the Macromolecular Core Facility of Pennsylvania State University College of Medicine by 9-fluoronylmethoxycarbonyl chemistry (9050 MilliGen PepSynthesizer; Millipore, Framingham, MA). Peptides were used for cytotoxicity assays without further purification. 51Cr-labeled target cells were incubated in CTL medium containing 1 µM synthetic peptide for at least 1 h at 37°C in 5% CO2. Peptide-pulsed cells were washed with CTL medium three times before use in the cytotoxicity assay. Tag peptides are designated LT followed by the numbers of Tag residues that correspond to the first and last residues of the synthetic peptide.

Cytotoxicity assay

Lysis of Tag-transformed or peptide-pulsed target cells by C57BL/6-derived, in vitro restimulated splenic lymphocytes or CTL clones was measured using a standard 51Cr release assay as previously described 9, 15 . Tag transformed fibroblast cell lines were routinely cultured in the presence of 40 U of {gamma} IFN (provided by H. M. Shepard, Genentech, San Francisco, CA)/ml for 2 days before use as 51Cr-labeled targets in cytotoxicity assays. The percentage of specific CTL-mediated 51Cr release was calculated using the formula: % specific release = [(E - S)/(M - S)] x 100, where E indicates the radioactivity released from targets incubated in the presence of effector cells, S indicates the spontaneous release of radioactivity by target cells incubated in the presence of medium alone, and M indicates the quantity of radioactivity released into the supernatant by target cells incubated in the presence of 2.5% (w/v) SDS. All determinations were made in triplicate.

MHC class I molecule stabilization assay

The relative binding efficiency of synthetic peptides for MHC class I molecules was determined by measuring the level of MHC class I molecules on the surface of RMA/s cells. The RMA/s cell line is a Rauscher virus-induced T cell lymphoma that has a low surface expression of MHC class I molecules 35, 36 . Increased expression of surface MHC class I molecules can be induced by incubation of RMA/s cells in the presence of haplotype-specific peptide or at low temperature 37, 38, 39 . RMA/s cells (3 x 105 to 6 x 105) were incubated in 1 ml of supplemented RPMI medium containing varying concentrations of peptides for 14–24 h as previously described 6, 15 . Expression of H-2Db molecules was determined by FACS analysis using an EPICS V flow cytometer/sorter (Coulter, Hialeah, FL) set at an excitation fluorescence of 500 mW at 488 nm. Cells were washed twice in PBS containing 2% FCS and 0.1% sodium azide (FACS buffer), then incubated for 45 min on ice with 0.1 ml of the appropriately diluted anti-H-2Db mAb 28-14-8 40 . After washing with FACS buffer, the cells were incubated with 0.1 ml FITC-conjugated goat anti-mouse Ab for 45 min on ice. Cells were washed, fixed in 1% paraformaldehyde in PBS, and analyzed by flow cytometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of relocation of epitope I within SV40 Tag on processing, presentation, and immunogenicity

We reported previously that relocation within SV40 Tag did not alter the immunorecessive nature of SV40 Tag CTL epitope V 15 . To extend those results, Tag derivatives in which epitope I was relocated into the epitope V Tag context were generated. Exchange derivatives were generated in which 10 (206–215, SAINNYAQKL) or nine (207–215, AINNYAQKL) epitope I residues were transferred into the epitope V location (Fig. 1Go; 206–215 {iff} 489–497 or 207–215 {iff} 489–497, respectively). The results of cytotoxicity assays using SV40-specific CTL clones indicated that Tag derivatives in which 10 epitope I residues were transferred into the epitope V location were recognized efficiently by epitope I-specific CTL clones (Fig. 1Go; 206–215 {iff} 489–497; B6/T69A1, B6/T70A1), while derivatives in which only nine epitope I residues were transferred were recognized poorly by the same CTL clones (Fig. 1Go; 207–215 {iff} 489–497; B6/T20Aa, B6/T20Ba) 15 . Immunization experiments using the same cell lines confirmed that the 10-mer epitope I was immunogenic when located within the epitope V location of Tag (Table IIGo, B6/T69A1). Cells that expressed the Tag derivative in which only nine epitope I residues replaced epitope V did not induce detectable levels of epitope I-specific CTL in the same experiments (Table IIGo, B6/T20Ba). These results support our previous conclusion that epitope I corresponds to a 10-mer peptide that includes Tag residues 206–215 (SAINNYAQKL) 6 . Furthermore, the processing, presentation, and immunogenicity of epitope I (10 mer) do not appear to be limited by the protein context that surrounds epitope V within Tag.



View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 1. Lysis of SV40 Tag-transformed cell lines by SV40 Tag-specific CTL clones. Cells expressing Tag derivatives deleted of CTL epitopes or in which 10 (206–215 {iff} 489–497) or 9 (207–215 {iff} 489–497) epitope I residues replace epitope V were combined in a standard cytotoxicity assay with SV40-specific CTL clones. A, Tag derivatives. The positions of epitopes I, II/III, IV, and V are indicated for the wild-type Tag (wt Tag). Epitope I is represented by two hatched boxes; the letter S appears below the smaller box, which represents residue Ser206, while the larger box represents residues 207–215. The numbers in square brackets at the left of some Tag diagrams indicate the corresponding epitope I and flanking residue amino acid sequences shown in Fig. 2Go. Tag and cell line descriptions appear in Table IGo. B, Lysis of cells expressing Tag derivatives shown in A by the CTL clones Y-1 (epitope I-specific) and K-19 (epitope II/III-specific) used at an E:T cell ratio of 10:1. Cell lines represented in the figure are: wt Tag, B6/T5Aa; {Delta} I, B6/T7Ba; {Delta} II/III, B6/T8Ab; {Delta} V, B6/T11Bb; 207–215 {iff} 489–497, B6/T20Aa and B6/T20Ba; 206–215 {iff} 489–497, B6/T69A1 and B6/T70A1.

 

View this table:
[in this window]
[in a new window]
  		Table II. CTL induction by B6 cell lines immortalized by SV40 T Ag CTL epitope deletion or relocation derivatives

 
Effects of an S206G substitution on H-2Db stabilization and recognition in vitro by epitope I-specific CTL clones

Inspection of the predicted amino acid sequence of the Tag derivative constructed by relocating nine epitope I residues into the epitope V context (Fig. 2GoB, number 7) revealed the presence of a 10 mer sequence corresponding to GAINNYAQKL. We have previously reported an analysis of the effects of substitutions within and surrounding epitope I on its processing and presentation from within Tag to epitope I-specific CTL clones 6 . The results of that study indicated that Pro or Thr substitutions at the P1 position of epitope I (residue 206) could abrogate recognition of Tag-transformed cells by epitope I-specific CTL clones; the effect of a glycine substitution at the 206 position was not analyzed in that study. Accordingly, a synthetic peptide corresponding to the sequence GAINNYAQKL was synthesized and analyzed for MHC class I stabilization (Fig. 3GoA) and recognition by epitope I-specific CTL clones (Fig. 3Go, B and C). The results indicated that the peptide GAINNYAQKL was recognized efficiently by two epitope I-specific CTL clones, Y-1 (Fig. 3GoB) and K-11 (Fig. 3GoC). The S206G-substituted epitope I peptide also stabilized H-2Db molecules on the surface of RMA/s cells with an efficiency similar to that of a 10 mer peptide corresponding to the native epitope I sequence, SAINNYAQKL (Fig. 3GoA). Peptides bearing Pro (P), Met (M), or Thr (T) substitutions affecting the P1 position (residue 206) were also analyzed (Fig. 3Go). Both CTL recognition and H-2Db stabilization by peptides corresponding to only nine epitope I residues (LT207–215) or bearing an S206P substitution were reduced relative to both the wild-type (SAINNYAQKL) and S206G-substituted (GAINNYAQKL) peptides. Recognition of the Thr- and Met-substituted epitope I peptides was reduced only for CTL clone K-11 (Fig. 1GoC). These results support an important role for both the presence and the identity of residue position 206 in the epitope I peptide and suggest that an S206G-substituted epitope I peptide should be recognized by epitope I-specific CTL if it is efficiently processed and presented from within Tag.



View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 2. Predicted amino acid sequences of the epitope I or epitope V region Tag derivatives bearing epitope I or the S206G-substituted epitope I variant sequence GAINNYAQKL. Wild-type epitope I (solid line) and epitope V (dashed line) sequences are underlined. Brackets group derivatives in A and B that bear reciprocal flanking residue substitutions. Flanking residues occupying positions 205 and 216, and 488 and 498 are indicated for the sequences shown in A and B, respectively.A, Native epitope I amino acid sequence and amino acid context within SV40 Tag (wild-type Tag) is given above sequences showing substitutions (outline font) affecting the flanks of the S206G epitope I variant located within the epitope I context of SV40 Tag. B, Epitope V amino acid sequence and amino acid context within SV40 Tag (wild-type Tag) are shown above sequences corresponding to the S206G epitope I variant located within the epitope V context of SV40 Tag. Substitutions affecting the flanks of the S206G epitope I variant located within the native epitope V context of SV40 Tag are indicated in bold.

 


View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 3. H-2Db stabilization and CTL clone recognition for epitope I synthetic peptide variants. A, RMA/s cells were incubated overnight in the presence of synthetic peptides and analyzed for cell surface H-2Db expression. B and C, Radiolabeled B6/K-1,4,5 cells were pulsed with synthetic peptides corresponding to epitope I (LT206-215) or position 206-substituted epitope I variants, washed, and combined with the epitope I-specific CTL clones Y-1 (B) and K-11 (C) in a standard 51Cr release assay.

 
Context-dependent immunogenicity of the S206G-substituted epitope I variant

To examine whether the S206G-substituted epitope I variant sequence GAINNYAQKL could be processed and presented from within SV40 Tag, an S206G-substituted derivative of Tag was generated (Fig. 2GoA, number 2). Cells transformed by this Tag derivative were lysed efficiently by the epitope I-specific CTL clone Y-1 (Fig. 4GoB). Immunization experiments using multiple S206G cell lines were performed. The results shown in Table IIIGo illustrate that CTL induced by wild-type Tag (B6/T5Aa) and the S206G variant (B6/T58A2) efficiently lysed target cells pulsed with synthetic peptides corresponding to either epitope I (SAINNYAQKL) or the S206G-substituted epitope I variant (GAINNYAQKL). Further, the results in Table IIIGo show that cells expressing either the wild-type (B6/WT-19) or the S206G-substituted epitope I sequence (B6/T58A2) provided efficient in vitro restimulation for epitope I-specific CTL obtained from mice immunized with cells expressing either the wild-type or S206G-substituted epitope I sequence. These results suggest that the sequence GAINNYAQKL can be efficiently processed and presented from and is immunogenic when located within the epitope I context of SV40 Tag.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 4. CTL clone lysis of cell lines expressing Tag derivatives bearing epitope I or the S206G-substituted epitope I variant in either the epitope I or epitope V location. CTL clones Y-1 and K-19 were combined with target cells at the E:T cell ratios indicated in a standard 4.5-h cytotoxicity assay in which nonspecific lysis was <10%. S or G indicates the residue occupying residue position P-1 of an epitope I 10 mer in the relevant Tag derivative (position 206 in wild-type Tag). Numbers in square brackets indicate the epitope I and flanking residue amino acid sequences shown in Fig. 2Go. The cell lines used in the assay were B6/T5Aa (A), B6/T58A2 (B), B6/T69A1 (C), and B6/T85A1 (D).

 

View this table:
[in this window]
[in a new window]
  		Table III. Induction of epitope I-specific CTL by SV40 T Ag, an S206G-substituted T Ag, or T Ag epitope relocation/substitution derivatives

 
In light of these results, it was of interest to determine why the sequence GAINNYAQKL was apparently inefficiently processed and presented (Fig. 1Go, cell lines B6/T20Aa and B6/T20Ba) and was not immunogenic when located within the epitope V region of Tag (Table IIGo, cell line B6/T20Ba; Table IIIGo, cell line B6/T20Aa). Efficient processing of a peptide corresponding to the 10 mer sequence GAINNYAQKL from the Tag derivative expressed by B6/T20Ba or B6/T20Aa cells would require that residue 488 Gly, which normally flanks the amino terminus of the epitope V peptide, serve as the amino terminal Gly residue of a 10-mer GAINNYAQKL peptide. To allow for a more direct comparison of processing constraints that might affect epitope V and the epitope V context-located GAINNYAQKL sequence, a Tag derivative was constructed in which the full 10-mer sequence GAINNYAQKL was surrounded by native epitope V flanking sequences (Fig. 2Go, number 9). Cell lines generated using such Tag derivatives (206G-215{iff}489–497) were specifically lysed by the epitope I-specific CTL clone Y-1 (Fig. 4GoD, B6/T85A1; Fig. 5GoE, B6/T105B1). In cytotoxicity assays where they were directly compared, target cells expressing Tag derivatives corresponding to that expressed by B6/T85A1 cells (206G-215{iff}489–497; Fig. 2GoB, number 9) were lysed more efficiently than B6/T20Ba or B6/T20Aa cells (207–215{iff}489–497; Fig. 2GoB, number 7; data not shown). Therefore, restoration of the native epitope V flanking context by (re)insertion of a glycine residue, which could then serve as an NH2-terminal flanking residue at Tag position 488, appeared to improve processing and presentation of the GAINNYAQKL epitope I variant from within the epitope V location of Tag.



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 5. CTL clone lysis of cell lines expressing Tag derivatives bearing the S206G epitope I variant and flanking residue substitutions in either the epitope I or epitope V location. CTL clones Y-1 and K-19 were combined with target cells at the E:T cell ratios indicated in a standard 4.5-h cytotoxicity assay in which nonspecific lysis was <10%. Numbers in square brackets indicate amino acid sequences for the S206G-substituted epitope I variant and flanking residues as shown in Fig. 2Go. Amino acid sequences are given above A and E, which correspond to the S206G-substituted epitope I variant and native Tag flanking sequences found in those derivatives. Adjacent flanking residues are indicated for all derivatives by the diagrams above the panels. The cell lines used in the assay were B6/T101B1 (A), B6/T102B2 (B), B6/T103B2 (C), B6/T104B2 (D), B6/T105B1 (E), B6/T106B1 (F), B6/T107A2 (G), and B6/T108A2 (H).

 
Tag derivatives bearing the GAINNYAQKL sequence as well as the Gly residue (re)inserted at position 488 (Fig. 2GoB, number 9) failed to induce epitope I-specific CTL in C57BL/6 mice (Table IIIGo, B6/T85A1 cells; Table VGo, B6/T105A4 cells). The CTL obtained from mice immunized with B6/T85A1 or B6/T105A4 cells failed to lyse target cells pulsed with 10 mer synthetic peptides corresponding to either the SAINNYAQKL or GAINNYAQKL sequences following in vitro restimulation with the wild-type Tag (Table IIIGo, B6/WT-19, and data not shown). In vitro restimulation of splenocytes obtained from mice immunized with B6/T105A4 cells with Tag derivatives that bore the GAINNYAQKL sequence in either the immunogenic epitope I location (Table VGo, B6/T58A2) or the nonimmunogenic epitope V location (Table VGo, B6/T86B1) yielded similar results. These results characterize GAINNYAQKL as a determinant that displays context-dependent immunogenicity within SV40 Tag.


View this table:
[in this window]
[in a new window]
  		Table V. Induction of epitope I-specific CTL by SV40 T Ag derivatives in which the 206G–215 sequence replaces epitope V and that bear additional substitutions at adjacent flanking residue positions

 
Analysis of the effects of substitutions at flanking residue positions on the processing, presentation, and immunogenicity of the GAINNYAQKL epitope I variant from two locations within SV40 Tag

The immunogenicity of the GAINNYAQKL sequence appeared to be controlled differentially by native Tag sequences flanking dominant (I) and recessive (V) CTL determinants. This difference provided us with a model system with which to specifically address the roles of individual flanking residues in controlling immunological potency. Importantly, the influence(s) of both regional context and adjacent residues could be assessed by determining whether substitutions at adjacent flanking residue positions would have similar consequences on the immunological potency of one determinant examined within different locations of Tag.

To investigate the role of adjacent residues in controlling the immunogenicity of the GAINNYAQKL determinant, Tag derivatives were generated in which residues immediately flanking the amino and/or carboxyl termini of the GAINNYAQKL determinant within the epitope I or epitope V locations were reversed (Fig. 2GoA, numbers 3–5; Fig. 2GoB, numbers 10–12). Derivatives bearing substitutions that flanked the amino, carboxyl, or both amino and carboxyl termini of the GAINNYAQKL sequence were generated. Standard cytotoxicity assays (Fig. 5Go) employing the SV40-specific CTL clones Y-1 (epitope I specific) and K-19 (epitope II/III specific) revealed little (or no) difference between multiple cell lines expressing the Tag derivatives that bore flanking residue substitutions around the GAINNYAQKL sequence relative to the cell lines that expressed the derivatives that represented, respectively, the corresponding native epitope I and V flanking contexts.

The impact of flanking residue substitutions on CTL induction by the GAINNYAQKL sequence in vivo was determined by immunization of C57BL/6 mice using cell lines expressing Tag derivatives bearing substitutions affecting the epitope I and epitope V flanking contexts. The results shown in Table IVGo reveal that the identity of the residues that immediately flanked the GAINNYAQKL determinant (flanking residues 205 and 216) was not critical when it was located within the epitope I context of Tag (Table IVGo). By contrast, the identity of the residue that immediately flanked the amino terminus of the GAINNYAQKL determinant when it was located within the epitope V context of Tag was important (position 488; Table VGo). Derivatives bearing a G488V substitution were able to induce epitope I-specific CTL (Table VGo, B6/T106B1 and B6/T107A2). In repeated experiments, however, lysis of target cells pulsed with either epitope I peptide (SAINNYAQKL or GAINNYAQKL) was lower by CTL obtained following immunization with G488V substitution derivatives (B6/T106A1 and B6/T107A2) than by CTL obtained following immunization with S206G cell lines such as B6/T58A2 (Table VGo and data not shown). Note that the GAINNYAQKL sequence encoded by B6/T58A2 cells resides within the epitope I context of Tag where it also is flanked by an amino terminal Val residue. An R498C substitution affecting the carboxyl-terminal flanking residue appeared to have little affect on CTL induction by the GAINNYAQKL determinant when located within the epitope V context (Table VGo, B6/T108A2 and B6/T107A2).


View this table:
[in this window]
[in a new window]
  		Table IV. Induction of epitope I-specific CTL by SV40 Tag S206G derivatives bearing additional substitutions at adjacent flanking residue positions

 
These results suggest that a Gly residue immediately flanking the amino terminus of the GAINNYAQKL sequence dramatically reduced the immunogenicity of that sequence when located within the epitope V, but not the epitope I, context of Tag. Even so, induction of epitope I-specific CTL by the G488V derivative (or the G488V, R498C double substitution derivative) was not as efficient as induction of epitope I-specific CTL by derivatives bearing the GAINNYAQKL sequence in the epitope I location (flanked at the amino terminus by Val205). Therefore, our results support the idea that influences imposed by sequences that surround the epitope V location (regional effects) modify limitations imposed by an adjacent amino-terminal flanking Gly residue (Gly488) on CTL induction by the GAINNYAQKL sequence.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results from this study demonstrate that native CTL epitope-flanking sequences can differentially affect the immunogenicity of a test epitope. The Tag epitope I variant sequence, GAINNYAQKL, was characterized as a CTL recognition epitope that displayed protein context-dependent immunogenicity within SV40 T Ag. The immunogenic and nonimmunogenic contexts analyzed using the GAINNYAQKL sequence are of interest because they correspond precisely to native amino acid sequences that flank two H-2Db-restricted SV40 Tag CTL recognition epitopes, epitopes I and V. Tag epitopes I and V display differing immunological potencies; epitope I is immunodominant, while epitope V has been characterized as immunorecessive within SV40 Tag. Our results show that the GAINNYAQKL sequence was immunogenic when located within the epitope I context, but not when located within the epitope V context of Tag.

The key finding of this study is that epitope identity, adjacent flanking residues, and a broader region of Tag surrounding the epitope together determined whether flanking sequences negatively influenced the immunogenicity of the GAINNYAQKL epitope. SV40 Tag provides an ideal model for the study of MHC class I-restricted Ag processing and immunological hierarchy because it contains multiple H-2b-restricted epitopes of differing immunological potencies. Analysis of a test epitope within different Tag epitope locations allows for dissection of effects that are context dependent and/or epitope specific. From the results presented here, it is clear that the residue that flanked the amino terminus of the GAINNYAQKL sequence could dramatically regulate its immunogenicity when the GAINNYAQKL sequence was located precisely within the epitope V location of Tag. A Gly residue at position 488 (native residue) prevented induction of GAINNYAQKL-specific CTL, while a Val residue at position 488 allowed for induction of GAINNYAQKL-specific CTL. By contrast, either a Gly or a Val (native) residue at the amino-terminal flanking position (residue 205) allowed for efficient induction of GAINNYAQKL-specific CTL when the GAINNYAQKL sequence was located within the epitope I context. These results clearly indicate that the effects imposed by an adjacent flanking residue(s) can be influenced by other, more regional protein context influences.

The results presented in this study do not yet allow us to define the boundaries of Tag sequences surrounding the epitope I or V locations that contribute to the regional context effects observed. For example, Pro, Ser, and Gly residues that flank the amino terminus of epitope V (residues 486–488, PSG; see Fig. 2GoA, numbers 6 and 9) could reduce the efficiency of proteolytic events that may be important for the release of the amino terminus of the adjacent epitope. Peptide bonds involving glycine residues in small m.w. substrates or proteins appear to be cleaved efficiently by proteasomes in vitro 41, 42 . Nonetheless, several examples have been reported where neighboring glycine residues have appeared to negatively influence processing and presentation of MHC class I-restricted determinants in vivo 7, 12, 16, 43, 44 . It should be noted that the GAINNYAQKL sequence was also nonimmunogenic when located within the epitope V region and flanked at the amino terminus by residue Ser487 (Fig. 2Go, number 7). The extent to which regional influences contributed to this effect, however, cannot be addressed by the results of this study because a corresponding V205S substitution derivative was not analyzed.

The results presented here illustrate that the identity of the test epitope determines whether flanking effects will be important in regulating immunogenicity. The native epitope I sequence, SAINNYAQKL, was insensitive to a flanking residue context that dramatically reduced the immunogenicity of the variant sequence GAINNYAQKL. This apparent differential sensitivity could be accounted for by two models. First, the intrinsic immunogenicity of the test epitope could determine whether its ability to maximally induce CTL in vivo will be noticeably affected by the flanking sequences. Nonpermissive flanking sequences could reduce the amount of the epitope peptide productively liberated during Ag processing. If partially limiting production of the epitope peptide does not prevent saturating immune stimulation, no effect of flanking sequences would be observed. In this regard, the epitope V context-located GAINNYAQKL sequence behaves like Tag epitopes V (H-2Db restricted) 15, 23, 25 and T499–507 (H-2Kd restricted) 45 ; sufficient levels of these epitopes are generated from Tag-transformed cells to trigger lysis by CTL clones in vitro, but these levels are apparently insufficient to readily induce CTL induction in vivo. Increased production of these peptides from short minigenes encoded on vaccinia virus recombinants allows for efficient CTL induction by epitopes V or T499–507 9, 45 . Alternately, enhanced proteolytic cleavage could occur within a sensitive epitope if the combination of flanking sequences and the test epitope sequence generate protease-sensitive sites within the epitope 16, 46, 47 . The same flanking sequences may have no effect on a second epitope (that has a different primary sequence) because the same protease-sensitive sites would not be generated. The H-2Kd-restricted NP147–155 epitope appears to be sensitive to proteolytic destruction during Ag processing; flanking Pro substitutions that reduce processing, presentation, and immunogenicity also appear to enhance destruction of the NP147–155 peptide by proteasomes in vitro 16 . Our limited knowledge of the sequence preferences of proteases that participate in Ag processing in vivo makes it difficult to predict how flanking sequences will influence immunogenicity. It will be of interest to determine whether similar N and/or C terminal flanking residue substitutions that control the immunogenicity of the GAINNYAQKL epitope will also influence the immunogenicity of epitope V from within a Tag derivative that lacks the immunodominant CTL epitopes I, II/III, and IV.

Studies of the hierarchy of CTL epitopes within SV40 Tag have characterized three types of determinants. The wild-type epitope I (SAINNYAQKL) was immunogenic from within both epitope I and epitope V Tag contexts. On the other hand, epitope V failed to induce CTL when located within either the epitope I or V locations of full-length Tag derivatives that retained expression of an immunodominant CTL epitope (I, II/III, or IV) 9, 15 . Finally, the epitope I variant sequence GAINNYAQKL displayed context-dependent immunogenicity; it was immunogenic from within the epitope I location, but not from within the epitope V location. It will be of interest to determine whether, like epitope V, immunogenicity of the GAINNYAQKL sequence will be unmasked within the epitope V location by inactivation of dominant Tag CTL epitopes.

Induction of SV40 Tag-specific CTL following immunization of C57BL/6 mice with syngeneic Tag-transformed cells appears to rely on cross-priming 48, 49, 50 . Professional host APCs present the relevant Tag determinants to prime Tag-reactive T lymphocytes in vivo, since the injected tumor cells do not express costimulatory molecules. Therefore, developing an understanding of factors that control the processing/reprocessing of Tag protein fragments in professional host APCs will be important to understanding how the immunogenicity of tumor cell epitopes is regulated in vivo 51 . The results of a recent study that showed that apoptotic rather than necrotic cell residues are efficiently used by dendritic cells to efficiently stimulate T cell responses in vitro 52 imply that the ability of Tag (or Tag fragments) to be preserved during apoptosis may be important in regulating the efficiency (and perhaps hierarchy) of cross-priming following immunization with Tag-transformed cells.

In summary, the results presented in this study demonstrate that effects imposed by both regional context and adjacent residues can modulate immunogenicity for MHC class I-restricted CTL epitopes. The effects imposed by adjacent flanking residues appeared to be tempered by influences contributed by other neighboring sequences. The relative importance of such effects, however, varied in an epitope-dependent manner.


   Acknowledgments
 
I thank Melanie Epler and Rebecca Pound for excellent technical assistance, S. Joyce and M. J. Tevethia for critical reading of the manuscript, and Satvir Tevethia for his advice and support during this study.


   Footnotes
 
1 This work was supported by Grant CA25000 (to Satvir S. Tevethia) from the National Cancer Institute, National Institutes of Health (Bethesda, MD). Back

2 Address correspondence and reprint requests to Dr. Lawrence M. Mylin, Department of Microbiology and Immunology H107, Pennsylvania State University College of Medicine, P.O. Box 850, 500 University Drive, Hershey, PA 17033. E-mail: Back

3 Abbreviations used in this paper: CTL, CD8+ cytotoxic T lymphocyte; NP, influenza virus nucleoprotein; SV40 Tag, simian virus 40 large tumor antigen; I, II/III, IV, and V, cytotoxic T lymphocyte epitopes in simian virus 40 Tag; LT, synthetic peptide corresponding to amino acid residues in simian virus 40 Tag. Back

Received for publication September 11, 1998. Accepted for publication November 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Yewdell, J. W., J. R. Bennink. 1992. Cell biology of antigen processing and presentation to major histocompatibility complex class I molecule-restricted T lymphocytes. Adv. Immunol. 52:1.[Medline]
  2. Germain, R. N., D. H. Margulies. 1993. The biochemistry and cell biology of antigen processing and presentation. Annu. Rev. Immunol. 11:403.[Medline]
  3. Vinitsky, A., L. C. Anton, H. L. Snyder, M. Orlowski, J. R. Bennink, J. W. Yewdell. 1997. The generation of MHC class I-associated peptides is only partially inhibited by proteasome inhibitors. J. Immunol. 159:554.[Abstract]
  4. York, I. A., K. L. Rock. 1996. Antigen processing and presentation by the class I major histocompatibility complex. Annu. Rev. Immunol. 14:369.[Medline]
  5. Heemels, M. T., H. Ploegh. 1995. Generation, translocation, and presentation of MHC class I-restricted peptides. Annu. Rev. Biochem. 64:463.[Medline]
  6. Lippolis, J. D., L. M. Mylin, D. T. Simmons, S. S. Tevethia. 1995. Functional analysis of amino acid residues encompassing and surrounding two neighboring H-2Db-restricted cytotoxic T-lymphocyte epitopes in simian virus 40 tumor antigen. J. Virol. 69:3134.[Abstract]
  7. Hahn, Y. S., C. S. Hahn, V. L. Braciale, T. J. Braciale, C. M. Rice. 1992. CD8+ T cell recognition of an endogenously processed epitope is regulated primarily by residues within the epitope. J. Exp. Med. 176:1335.[Abstract/Free Full Text]
  8. Hahn, Y. S., V. L. Braciale, T. J. Braciale. 1991. Presentation of viral antigen to class I major histocompatibility complex-restricted cytotoxic T lymphocyte: recognition of an immunodominant influenza hemagglutinin site by cytotoxic T lymphocyte is independent of the position of the site in the hemagglutinin translation product. J. Exp. Med. 174:733.[Abstract/Free Full Text]
  9. Fu, T.-M., L. M. Mylin, T. D. Schell, I. Bacik, G. Russ, J. W. Yewdell, J. R. Bennink, S. S. Tevethia. 1998. An endoplasmic reticulum targeting signal sequence enhances the immunogenicity of an immunorecessive simian virus 40 T antigen cytotoxic T lymphocyte epitope. J. Virol. 72:1469.[Abstract/Free Full Text]
  10. Fu, T.-M., R. H. Bonneau, M. J. Tevethia, S. S. Tevethia. 1993. Simian virus 40 T antigen as a carrier for the expression of cytotoxic T-lymphocyte recognition epitopes. J. Virol. 67:6866.[Abstract/Free Full Text]
  11. Chimini, G., P. Pala, J. Sire, J. B. R., and J. L. Maryanski. 1989. Recognition of oligonucleotide-encoded T cell epitopes introduced into a gene unrelated to the original antigen. J. Exp. Med. 169:297.
  12. Del Val, M., H. J. Schlicht, T. Ruppert, M. J. Reddehase, U. H. Koszinowski. 1991. Efficient processing of an antigenic sequence for presentation by MHC class I molecules depends on its neighboring residues in the protein. Cell 66:1145.[Medline]
  13. Bergmann, C. C., L. Tong, R. Cua, J. Sensintaffar, S. Stohlman. 1994. Differential effects of flanking residues on presentation of epitopes from chimeric peptides. J. Virol. 68:5306.[Abstract/Free Full Text]
  14. Bergmann, C. C., Q. Yao, C.-K. Ho, S. L. Buckwold. 1996. Flanking residues alter antigenicity and immunogenicity of multi-unit CTL epitopes. J. Immunol. 157:3242.[Abstract]
  15. Mylin, L. M., R. H. Bonneau, J. D. Lippolis, S. S. Tevethia. 1995. Hierarchy among multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian virus 40 T antigen. J. Virol. 69:6665.[Abstract]
  16. Yellen-Shaw, A. J., E. J. Wherry, G. C. Dubois, L. C. Eisenlohr. 1997. Point mutation flanking a CTL epitope ablates in vitro and in vivo recognition of a full-length viral protein. J. Immunol. 158:3227.[Abstract]
  17. Yellen-Shaw, A. J., L. C. Eisenlohr. 1997. Regulation of class I-restricted epitope processing by local or distal flanking residues. J. Immunol. 158:1727.[Abstract]
  18. Couillin, I., B. Culmann-Penciolelli, E. Gomard, J. Choppin, J.-P. Levy, J.-G. Guillet, S. Saragosti. 1994. Impaired cytotoxic T lymphocyte recognition due to genetic variations in the main immunogenic region of the human immunodeficiency virus 1 NEF protein. J. Exp. Med. 180:1129.[Abstract/Free Full Text]
  19. Shastri, N., T. Serwold, F. Gonzalez. 1995. Presentation of endogenous peptide/MHC class I complexes is profoundly influenced by specific C-terminal flanking residues. J. Immunol. 155:4339.[Abstract]
  20. Tevethia, S. S., M. Lewis, Y. Tanaka, J. Milici, B. B. Knowles, W. L. Maloy, R. Anderson. 1990. 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. J. Virol. 64:1192.[Abstract/Free Full Text]
  21. Tevethia, S. S.. 1990. Recognition of simian virus 40 T antigen by cytotoxic T lymphocytes. Mol. Biol. Med. 7:83.[Medline]
  22. Tevethia, S. S.. 1980. Immunology of simian virus 40. G. Klein, ed. Viral Oncology 581. Raven Press, New York.
  23. Tanaka, Y., R. W. Anderson, W. L. Maloy, S. S. Tevethia. 1989. Localization of an immunorecessive epitope on SV40 T antigen by H-2Db-restricted cytotoxic T-lymphocyte clones and a synthetic peptide. Virology 171:205.[Medline]
  24. Tanaka, Y., M. J. Tevethia, D. Kalderon, A. E. Smith, S. S. Tevethia. 1988. Clustering of antigenic sites recognized by cytotoxic T lymphocyte clones in the amino terminal half of SV40 T antigen. Virology 162:427.[Medline]
  25. Deckhut, A. M., J. D. Lippolis, S. S. Tevethia. 1992. Comparative analysis of core amino acid residues of H-2Db-restricted cytotoxic T-lymphocyte recognition epitopes in simian virus 40 T antigen. J. Virol. 66:440.[Abstract/Free Full Text]
  26. Tooze, J.. 1980. DNA Tumor Viruses: Molecular Biology of Tumor Viruses Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
  27. Tevethia, S. S., R. S. Greenfield, D. C. Flyer, M. J. Tevethia. 1980. SV40 transplantation antigen: relationship to SV40-specific proteins. Cold Spring Harbor Symp. Quant. Biol. 1:235.
  28. Tanaka, Y., S. S. Tevethia. 1990. Loss of immunorecessive cytotoxic T lymphocyte determinant V on SV40 T antigen following cocultivation with site-specific cytotoxic T lymphocyte clone Y-5. Intervirology 31:197.[Medline]
  29. Tanaka, Y., S. S. Tevethia. 1988. In vitro selection of SV40 T antigen epitope loss variants by site-specific cytotoxic T lymphocyte clones. J. Immunol. 140:4348.[Abstract]
  30. Lill, N. L., M. J. Tevethia, W.G. Hendrickson, S. S. Tevethia. 1992. Cytotoxic T lymphocytes (CTL) against a transforming gene product select for transformed cells with point mutations within sequences encoding CTL recognition epitopes. J. Exp. Med. 176:449.[Abstract/Free Full Text]
  31. Mylin, L. M., A. M. Deckhut, R. H. Bonneau, T. D. Kierstead, M. J. Tevethia, D. T. Simmons, S. S. Tevethia. 1995. Cytotoxic T lymphocyte escape variants, induced mutations, and synthetic peptides define a dominant H-2Kb-restricted determinant in simian virus 40 tumor antigen. Virology 208:159.[Medline]
  32. Tevethia, M. J.. 1984. Immortalization of primary mouse embryo fibroblasts with SV40 virions, viral DNA, and a subgenomic DNA fragment in a quantitative assay. Virology 137:414.[Medline]
  33. Campbell, A. E., F. L. Foley, S. S. Tevethia. 1983. Demonstration of multiple antigenic sites of the SV40 transplantation rejection antigen by using cytotoxic T lymphocyte clones. J. Immunol. 130:490.[Abstract]
  34. Deckhut, A. M., S. S. Tevethia. 1992. Effect of point mutations in the native simian virus 40 tumor antigen, and in synthetic peptides corresponding to the H-2Db-restricted epitopes, on antigen presentation and recognition by cytotoxic T lymphocyte clones. J. Immunol. 148:3012.[Abstract]
  35. Ljunggren, H. G., K. Kärre. 1985. Host resistance directed selectively against H-2-deficient lymphoma variants: analysis of the mechanism. J. Exp. Med. 162:1745.[Abstract/Free Full Text]
  36. Kärre, K., H. G. Ljunggren, G. Piontek, R. Kiessling. 1986. Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature 319:675.[Medline]
  37. Ljunggren, H. G., N. J. Stam, C. Ohlen, J. J. Neefjes, P. Hoglund, M. T. Heemels, J. Bastin, T. N. Schumacher, A. Townsend, K. Karre. 1990. Empty MHC class I molecules come out in the cold. Nature 346:476.[Medline]
  38. Townsend, A., C. Ohlen, J. Bastin, H. G. Ljunggren, L. Foster, K. Karre. 1989. Association of class I major histocompatibility heavy and light chains induced by viral peptides. Nature 340:443.[Medline]
  39. Townsend, A., T. Elliott, V. Cerundolo, L. Foster, B. Barber, A. Tse. 1990. Assembly of MHC class I molecules analyzed in vitro. Cell 62:285.[Medline]
  40. Potter, T. A., C. Boyer, A. Verhuslt, P. Goldstein, T. V. Rajan. 1984. Expression of H-2Db on the cell surface in the absence of detectable ß2 microglobulin. J. Exp. Med. 160:317.[Abstract/Free Full Text]
  41. Driscoll, J., M. G. Brown, D. Finley, J. J. Monaco. 1993. MHC-linked LMP gene products specifically alter peptidase activities of the proteasome. Nature 365:262.[Medline]
  42. Dick, L. R., C. Adlrich, S. C. Jameson, C. R. Moomaw, B. C. Pramanik, C. K. Doyle, G. N. DeMartino, M. J. Bevan, J. M. Forman, C. A. Slaughter. 1994. Proteolytic processing of ovalbumin and ß-galactosidase by the proteasome to yield antigenic peptides. J. Immunol. 152:3884.[Abstract]
  43. Eggers, M., B. Boes-Fabian, T. Ruppert, P.-M. Kloetzel, U. H. Koszinowski. 1995. The cleavage preference of the proteasome governs the yield of antigenic peptides. J. Exp. Med. 182:1865.[Abstract/Free Full Text]
  44. Eisenlohr, L. C., J. W. Yewdell, J. R. Bennink. 1992. Flanking sequences influence the presentation of an endogenously synthesized peptide to cytotoxic T lymphocytes. J. Exp. Med. 175:481.[Abstract/Free Full Text]
  45. Newmaster, R. S., L. M. Mylin, T.-M. Fu, S. S. Tevethia. 1998. Role of a subdominant H-2Kd-restricted SV40 tumor antigen cytotoxic T lymphocyte epitope in tumor rejection. Virology 244:427.[Medline]
  46. Ossendorp, F., M. Eggers, A. Neisig, T. Ruppert, M. Groettrup, A. Sijts, E. Mengedé, P.-M. Kloetzel, J. Neefjes, U. Koszinowski, et al 1996. A single residue exchange within a viral CTL epitope alters proteasome-mediated degradation resulting in lack of antigen presentation. Immunity 5:115.[Medline]
  47. Niedermann, G., S. Butz, H. G. Ihlenfeldt, R. Grimm, M. Lucchiari, H. Hoschutzky, G. Jung, B. Maier, K. Eichmann. 1995. Contribution of proteasome-mediated proteolysis to the hierarchy of epitopes presented by major histocompatibility complex class I molecules. Immunity 2:289.[Medline]
  48. Gooding, L. R., C. B. Edwards. 1980. H-2 antigen requirements in the in vitro induction of SV40-specific cytotoxic T lymphocytes. J. Immunol. 124:1258.[Abstract]
  49. Bevan, M. J.. 1976. Cross-priming for a secondary cytotoxic response to minor H-antigens with H-2 congenic cells which do not cross-react in the cytotoxic assay. J. Exp. Med. 143:1283.[Abstract/Free Full Text]
  50. Bevan, M. J.. 1995. Antigen presentation to cytotoxic T lymphocytes in vivo. J. Exp. Med. 182:639.[Free Full Text]
  51. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245.[Medline]
  52. Albert, M. L., B. Sauter, N. Bhardwaj. 1998. Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 392:86.[Medline]



This article has been cited by other articles:


Home page
 J. Virol.Home page
L. M. Mylin, T. D. Schell, D. Roberts, M. Epler, A. Boesteanu, E. J. Collins, J. A. Frelinger, S. Joyce, and S. S. Tevethia
Quantitation of CD8+ T-Lymphocyte Responses to Multiple Epitopes from Simian Virus 40 (SV40) Large T Antigen in C57BL/6 Mice Immunized with SV40, SV40 T-Antigen-Transformed Cells, or Vaccinia Virus Recombinants Expressing Full-Length T Antigen or Epitope Minigenes
J. Virol., August 1, 2000; 74(15): 6922 - 6934.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mylin, L. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mylin, L. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS