Previous Article | Next Article 
J Virol, February 1998, p. 1469-1481, Vol. 72, No. 2
Department of Microbiology and Immunology,
Pennsylvania State University College of Medicine, Hershey,
Pennsylvania 17033,1 and
Laboratory of
Viral Disease, National Institute of Allergy and Infectious
Diseases, Bethesda, Maryland 208922
Received 12 September 1997/Accepted 5 November 1997
An immunological hierarchy among three H-2Db-restricted
cytotoxic T lymphocyte (CTL) determinants in simian virus 40 (SV40) large T antigen (Tag) was described previously: determinants I and
II/III are immunodominant, whereas determinant V is immunorecessive. To
assess the immunogenicity of each determinant individually and define
mechanisms that contribute to the immunorecessive nature of determinant
V, we constructed a panel of recombinant vaccinia viruses (rVVs)
expressing minigenes encoding these determinants in various polypeptide
contexts. We found the following. (i) Immunization of mice with an rVV
encoding full-length SV40 Tag resulted in priming for CTL responses to
determinants I and II/III but not determinant V. (ii) rVVs encoding
peptide I or II/III in the cytosol or targeted to the endoplasmic
reticulum (ER) were highly antigenic and immunogenic. (iii) rVVs
encoding peptide V minigenes were antigenic and immunogenic if the
peptide was targeted to the ER, expressed in the cytosol with short
flanking sequences, or expressed from within a self-protein, murine
dihydrofolate reductase. (iv) Presentation of the nonflanked peptide V
(preceded by a Met codon only) could be enhanced by using a potent
inhibitor of the proteasome. (v) H-2Db-epitope V peptide
complexes decayed more rapidly than complexes containing epitope I or
II/III peptides. In brefeldin A blocking experiments, functional
epitope V complexes were detected longer on targets expressing
ER-targeted epitope V than on targets expressing forms of epitope V
dependent on the transporter associated with antigen processing.
Therefore, limited formation of relatively unstable cell surface
H-2Db complexes most likely contributes to the
immunorecessive nature of epitope V within SV40 Tag. Increasing the
delivery of epitope V peptide to the major histocompatibility complex
class I presentation pathway by ER targeting dramatically enhanced the
immunogenicity of epitope V.
Cells infected with viruses or
undergoing oncogenic transformation express new or altered
self-proteins that may trigger host immune responses. CD8+
cytotoxic T lymphocytes (CTL) recognize such proteins in the form of
small antigenic peptide fragments complexed with cell surface major
histocompatibility complex (MHC) class I molecules (32, 86).
Peptides presented by MHC class I complexes are generated largely from
endogenously synthesized proteins through proteolysis in the cytosol,
often by the action of proteasomes (22, 47, 63, 80), and are
delivered to the endoplasmic reticulum (ER) by the transporter
associated with antigen processing (TAP) (49, 83, 87), where
they are assembled with nascent class I molecules. The resulting
complex is then transported via the secretory pathway to the cell
surface (17, 55, 81, 85). Multiple factors contribute to the
ability of a given determinant in a protein to elicit a CTL response.
These include the efficiency of production of the peptide and transport
to the ER (7, 8, 20, 26, 54, 57), peptide affinity for class
I molecules (15, 82), and the frequency of potentially
reactive T cells in the repertoire (12, 18, 52, 56).
We have used simian virus 40 (SV40) large tumor, or T, antigen (SV40
Tag) to understand factors which govern the immunogenicity of CTL
determinants within a tumor antigen. SV40 Tag is a multifunctional 94-kDa nuclear oncoprotein which can initiate and maintain
transformation of a wide variety of cell types in vitro
(28). In vivo, the Tag can induce neoplasia with a
metastatic potential when expressed as a transgene under control of a
tissue-specific promoter or when expressed from the viral promoter
(10, 34, 37, 59). In the immunocompetent host, the Tag often
induces a vigorous cellular immune response which leads to the
rejection of transplanted Tag-induced tumors (reviewed in references
76 and 77). Although SV40 Tag has
long served as a model tumor inducer and immunogen in experimental
systems, more recent detection of SV40 virus in isolates derived from
human tumors has prompted renewed interest in the potential role of the
immune system in controlling tumor induction by SV40 (9, 13, 41,
46).
SV40 Tag contains four H-2b-restricted CTL determinants, of
which three are H-2Db restricted (19, 43, 51,
77). These include CTL determinants I (residues 206 to 215),
II/III (residues 223 to 231), and determinant V (residues 489 to 497).
Determinant V was previously characterized as immunorecessive within
the context of the full-length Tag (50, 69). That is,
determinant V-specific CTL responses were detected only (and then with
some difficulty) following immunization of mice with transformed cells
expressing a mutated Tag in which the three immunodominant determinants
(determinants I, II/III, and IV) were deleted or rendered nonantigenic
(50, 69).
To further characterize the immunological potential of the
H-2b-restricted SV40 Tag CTL epitopes, including the
immunorecessive determinant V, we have extended these findings by
examining the antigenicity and immunogenicity of SV40 Tag CTL
determinants expressed in various polypeptide contexts from recombinant
vaccinia viruses (rVVs). Our results show that the immunogenicity of
the immunorecessive epitope V is significantly enhanced by targeting it
to the ER.
Plasmid construction and manipulation.
Plasmid DNA used for
sequencing and transfection was routinely prepared by the QIAwell-8
procedure (QIAGEN Inc., Chatsworth, Calif.).
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
An Endoplasmic Reticulum-Targeting Signal Sequence
Enhances the Immunogenicity of an Immunorecessive Simian Virus 40 Large T Antigen Cytotoxic T-Lymphocyte Epitope
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Animals. Four- to six-week-old male C57BL/6 (B6/) mice were purchased from Jackson Laboratory, Bar Harbor, Maine, and routinely used between the ages of 5 and 12 weeks. All mice were maintained in the animal facility at Pennsylvania State University College of Medicine, Hershey.
Cell lines and viruses. B6/WT-19 cells have been described previously (50, 78). B6/K-1,4,5 (H-2b) cells, which were generated by sequential cocultivation of B6/K-0 cells (B6/K-0 cells express wild-type SV40 Tag) with SV40 Tag-specific CTL clones, have been described elsewhere (71, 72). B6/K-1,4,5 cells are resistant to lysis by SV40 Tag-specific CTL clones due to a deletion which removes sequences encoding determinants I and II/III and to point mutations which alter determinants IV and V (42, 51).
B6/T117A1 cells were derived by isolation of a Tag-expressing, immortalized focus following calcium phosphate-mediated transfection of mouse embryo fibroblasts with the plasmid pSLM361-13 as described elsewhere (50, 73). Plasmid pSLM361-13 was generated from plasmid pLM247 (50) by site-directed mutagenesis using the Altered Sites procedure. B6/T117A1 cells express a Tag derivative in which only determinants I (
207-215) and II/III (223-231) are
deleted and in which three residues within determinant IV (residues
406, 408, and 411) have been replaced by Ala residues.
Tag-transformed cell lines were routinely maintained in closed vessels
in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 or
5% (vol/vol) heat-inactivated fetal bovine serum (FBS) (HyClone,
Logan, Utah), 2 mM L-glutamine, 100 U of penicillin per ml,
100 µg of streptomycin per ml, 25 µg of kanamycin per ml, 20 mM
HEPES, and 0.0225% sodium bicarbonate.
HuTK
143 cells were derived from human osteosarcoma cells
(ATCC CRL-8303) (62). The CV-1 cell line is a continuous
cell line derived from African green monkey kidney cells (ATCC CCl-70)
(45). Ltk/Db cells have been described
previously (3); these cells were generated by transfecting
Ltk cells (H-2k) with the
H-2Db gene, along with the pSV2-neo plasmid as a
selectable marker.
T2/Db cells (2) were generously provided by P. Cresswell (Yale University, New Haven, Conn.). The cells were
maintained in Iscove's modified Dulbecco's medium (IMDM) supplemented
with 10% FBS, 0.0225% sodium bicarbonate, 25 mM HEPES, 2 mM
L-glutamine, 100 U of penicillin per ml, and 100 µg of
streptomycin per ml, as well as 5 × 105 M
-mercaptoethanol and 100 µg of G418 per ml to maintain selection for the transfected H-2Db gene. The RMA cell
line and the antigen presentation-defective, TAP2-deficient RMA/s cell
line have been described elsewhere (36, 44).
SV40 strain VA45-54 (74) was prepared as described
previously (75). The wild-type vaccinia virus strain
(Western Reserve isolate [WR]) was propagated in
HuTK143 cells. The vaccinia virus recombinant TM-1
(referred to in this study as rVV-Dhfr/) has been described elsewhere
(29).
SV40-specific CTL clones. SV40-specific CTL clones Y-1 or K-11, Y-2 or K-19, and Y-5 or H-1 recognize, respectively, SV40 Tag CTL determinants I (Tag amino acid residues 206 to 215), II/III (residues 223 to 231), and V (residues 489 to 497) (11, 19, 43, 50, 69, 70). The CTL clones were maintained in vitro as described previously (19, 50).
Generation of rVVs.
The procedure for generating rVVs was
modified from a protocol described elsewhere (24). CV-1
cells in 25-cm2 cell culture flasks were infected with
wild-type vaccinia virus (WR strain; multiplicity of infection [MOI]
of 0.1) in 1 ml of PBS/BSA (phosphate-buffered saline [PBS] [pH
7.4] supplemented with 0.1% [wt/vol] bovine serum albumin [BSA]).
After 2 h, the virus solution was aspirated and replaced with 5 ml
of complete DMEM and calcium phosphate-DNA precipitates, which
contained 10 µl of pSC-derivative recombinant plasmid DNA (20 µg)
and were prepared by using a CellPhect transfection kit (Pharmacia LKB Biotechnology Inc., Uppsala, Sweden). The cultures were incubated at
37°C in 5% CO2 for 2 to 3 days, a time at which
cytopathic effects were evident. The cells were harvested by
trypsinization, lysed in 1 ml of PBS/BSA by three cycles of
freeze-thawing, and sonicated for 1 min. The cell lysate was diluted
and used to infect HuTK-143 cells in a six-well plate in the presence
of 25 µg of 5-bromodeoxyuridine (to select thymidine kinase-negative
[TK] progeny viruses) per ml in plaquing medium
(equal-volume mixture of 1.8% Noble agar and 2× Eagle's medium
supplemented with 10% FBS) at 37°C in 5% CO2. Potential
recombinants were further screened for -galactosidase expression.
After 48 to 72 h, the wells were overlaid with 2 ml of plaquing
medium supplemented with 0.025% 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal)
and incubated until blue plaques appeared (usually overnight). Blue
plaques were picked and subjected to three rounds of plaque
purification. Viral stocks were prepared by reinfection of
HuTK143 cells.
Cytotoxicity assay. Cytotoxicity assays were performed as described previously (19, 50, 70). Vaccinia virus-infected targets were prepared by one of two procedures for use in CTL assays. Fibroblasts were harvested by trypsinization and washed with media and PBS/BSA, and 5 × 105 cells were infected in 15-ml conical tubes at an MOI of 10 in a final volume of 0.5 ml of PBS/BSA for 1 h at 37°C. Infection suspensions were then diluted with 5 ml of complete DMEM and rocked at 37°C for 3 h. The infected cells were then labeled with Na51CrO4 for an additional 30 min, washed three times, and combined with effectors. Alternatively, fibroblast monolayers were labeled overnight in culture with Na51CrO4, harvested by trypsinization, and infected as described above. For some experiments, infections were performed in T/B, which is PBS/BSA supplemented with an equal volume of incomplete DMEM prepared as described previously (50) but without FBS and sodium bicarbonate.
Targets were also prepared for CTL assays by incubation of fibroblasts (B6/K-1,4,5) or nonadherent cells (RMA) in media containing synthetic peptides corresponding to CTL determinants. Either the targets were incubated in suspension with both peptide and Na51CrO4, or peptide was added after the cells had been labeled and washed free of unincorporated 51Cr. Unbound peptides and unincorporated Na51CrO4 were subsequently removed by a minimum of two washes. In experiments involving proteasome inhibitors, 51Cr-labeled targets were incubated in T/B containing inhibitor for at least 30 min prior to addition of virus. Targets were maintained in the presence of inhibitor throughout the infection period but were washed free of inhibitor before being combined with effectors in the cytotoxicity assay. 51Cr-labeled targets infected with vaccinia virus recombinants were incubated in the presence of 4 mg of brefeldin A (BFA) per ml for various times to monitor the relative rates of H-2Db-peptide complex decay (50, 85). Briefly, Ltk/Db cells were labeled in culture overnight with 51Cr, harvested by trypsinization, washed in media and T/B, combined with virus at an MOI of 10 in 1 ml of T/B, and incubated at 37°C. After 4 h, infected cells were diluted with T/B containing BFA for a final concentration of 4 mg/ml, and incubation was continued at 37°C. Targets were washed in T/B containing 2 mg of BFA per ml, resuspended in RPMI containing 2 mg of BFA per ml, combined with equal volumes of effector suspension in 96-well plates, centrifuged, and incubated for 4 h at 37°C in 5% CO2.Generation of bulk culture vaccinia virus-specific CTL for in vitro cytotoxicity assays. Bulk culture vaccinia virus-specific secondary in vitro-stimulated CTL (Vac-CTL) were generated by a procedure modified from that described by Eisenlohr and coworkers (25). Briefly, a single-cell suspension was prepared from the spleens of two mice which had been immunized intravenously (i.v.) with wild-type vaccinia virus (rVV-WR; 2 × 106 PFU/mouse) for at least 2 weeks prior to sacrifice. One-third of the splenocytes were infected with 108 PFU of rVV-WR for 1 h at 37°C in 1 ml of PBS/BSA. After three washes with complete IMDM, the infected splenocytes were mixed with the remaining splenocytes and seeded into 12-well plates in complete IMDM. The cultures were maintained at 37°C in 5% CO2 for 5 to 7 days before being used in cytotoxicity assays.
Generation of bulk culture SV40 Tag-specific CTL from mice immunized with vaccinia virus recombinants. Bulk culture SV40 Tag-specific CTL were generated from splenic lymphocytes isolated at least 3 weeks following immunization of groups of four C57BL/6 mice with 0.5 × 107 to 1 × 107 PFU of rVV (i.v.). Splenic lymphocytes were prepared and restimulated in vitro with gamma-irradiated B6/WT-19 cells as described elsewhere (50). To verify vaccinia virus infection, 2 × 107 splenocytes from the same groups were restimulated for 6 days in loosely capped, upright T25 flasks containing 12 ml of RPMI medium supplemented with 1 × 106 naive C57BL/6 splenocytes which had been infected for 4 h with rVV-WR (MOI, 10), washed free of virus, and gamma irradiated on ice (60,000 rads).
Peptide transport assays utilizing SLO-permeabilized Ltk/Db cells. The capacity of TAP to transport peptides was determined as described by Androlewicz et al. (4). Briefly, TYNRTRALV synthetic peptide (20 µg) was iodinated with 1 mCi of Na125I by using chloramine T to a specific activity of 6 × 104 to 9 × 104 cpm/pmol (125I-TYN). Ltk/Db cells were washed once in serum-free medium and incubated for 10 min on ice with streptolysin O (SLO) (Murex, Norcross, Ga.) at 1 U/ml. The cells were then washed three times in serum free-medium and resuspended in intracellular transport buffer (ICT) (50 mM HEPES [pH 7.0], 78 mM KCl, 4 mM MgCl2, 8.37 mM CaCl2, 10 mM EGTA, 1 mM dithiothreitol, 10 mM ATP) containing BSA (4 mg/ml). SLO-permeabilized cells (106/sample) were incubated with 125I-TYN (125 ng) mixed with different amounts of unlabeled peptides at 37°C for 30 min. The cells were then lysed with 2% Triton X-100-0.15 M NaCl-10 mM Tris (pH 7.4)-5 mM phenylmethylsulfonyl fluoride, and the lysate was incubated with 50 µl of packed concanavalin A (ConA)-Sepharose to recover glycosylated 125I-TYN. The amount of bound peptide was quantitated by gamma counting.
Relative decay rates of H-2Db complexes stabilized on RMA/s cells by synthetic peptides corresponding to SV40 Tag CTL epitopes. Relative rates of H-2Db-peptide complex decay were monitored by flow cytometric analysis of H-2Db complexes remaining on the surface of RMA/s cells following incubation at 37°C. Briefly, RMA/s cells were incubated overnight at 29°C (5% CO2) in the presence of synthetic peptides corresponding to SV40 Tag CTL epitopes as described previously (43, 50). The cells were washed free of peptide and resuspended in complete RPMI medium lacking peptide, and incubation was continued at 37°C for up to 6 h. Aliquots were removed at appropriate intervals and stored on ice to stabilize the remaining complexes. Samples were prepared for flow cytometric analysis of H-2Db molecules using the monoclonal antibody 28-14-8 (60) as described elsewhere (43, 50).
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
CTL recognition of SV40 Tag determinants expressed from rVVs. To study the immunogenicity and antigenicity of three H-2Db-restricted SV40 Tag determinants, rVVs (Fig. 1A) that express each determinant as a cytosolic peptide with an NH2-terminal Met to enable efficient translation initiation (25) or with an NH2-terminal 16-amino-acid ER insertion sequence from the adenovirus type 5 E3/19K glycoprotein (plus an additional Ala) to allow for TAP-independent presentation were constructed (1, 26, 67). We also constructed rVVs expressing full-length Tag or a polydeterminant minigene consisting of determinants I, V, and II/III fused without intervening residues (again with an initiating NH2-terminal Met) to examine CTL recognition and immunogenicity for these epitopes when processed and presented from within more complex substrates.
|
|
), or bearing no
Tag insert (rVV-SC). ES, the 16-residue E3/19K signal sequence derived
from adenovirus (Fig.
|
CTL recognition of determinant V is influenced by short flanking sequences. A trivial explanation for the failure of rVV-V to sensitize target cells for lysis by determinant V-specific CTL was that this recombinant failed to produce the proper peptide. Since the only means of identifying such minigene products is by CTL recognition, it was not possible to completely eliminate this possibility. Three additional rVVs independently produced from the original shuttle plasmid similarly failed to sensitize infected targets for lysis by determinant V-specific CTL clones (data not shown).
Studies conducted with Saccharomyces cerevisiae suggested that the initiating Met was likely to be retained by the determinant V peptide expressed from rVV-V (48). It was unclear whether the Met extension would interfere with presentation of the determinant V peptide. To explore this possibility, we synthesized peptide derivatives of determinant V extended at the amino terminus with a single Met and/or Ala residue and examined these peptides for Db binding and recognition by determinant V-specific CTL clones. Our results indicated that single Met or Ala NH2-terminal extensions reduced the ability of determinant V synthetic peptides to sensitize target cells for lysis by Y-5 by at least 10- to 100-fold (in assays performed in the presence or absence of FBS, respectively). We also compared H-2Db binding of the Met- or Ala-extended 10-mer peptides and the determinant V 9-mer. Consistent with results obtained from the CTL lysis assays, the ability of the NH2-extended peptides to stabilize H-2Db molecules on tap2-deficient RMA/s cells was also reduced greater than 10-fold (data not shown). These results implied that extension of the determinant V peptide by a single residue (Ala or Met) might significantly reduce peptide presentation in vivo. To determine whether an NH2-terminal Met would always interfere with presentation of determinant V, derivatives of mouse DHFR into which the sequence MQGINNLDNL (rVV-Dhfr/M-V) or AQGINNLDNL (rVV-Dhfr/A-V) was inserted were generated (Fig. 1B). The DHFR derivative utilized serves as an efficient carrier for heterologous CTL determinants in vitro and in vivo (29). Ltk/Db cells infected with rVV-Dhfr/M-V or rVV-Dhfr/A-V were recognized by the determinant V-specific CTL clone H-1 (Table 1); in fact, the Met-flanked determinant V peptide appeared to be presented better than the Ala-flanked peptide. These results demonstrate that processing and presentation of determinant V are not prevented by the presence of a Met residue immediately adjacent to the NH2 terminus of determinant V when the determinant is located in the context of a larger polypeptide.
|
|
|
A proteasome inhibitor allows limited recognition of rVV-V by a determinant V-specific CTL clone. Our results indicate that a COOH (Ala)2 extension apparently overcomes a defect which blocks presentation of the cytosolic rVV-V determinant V peptide. Since the NH2 coding sequences of the two minigenes (rVV-V and rVV-V-A2) are identical (Fig. 1A and C), it is unclear how the COOH extension could increase the efficiency with which the initiating Met residue is removed. TAP does not appear to represent a pertinent limitation, as the (Ala)2-extended determinant V synthetic peptides are transported with 100-fold-decreased efficiency (Fig. 5). Alternately, addition of two COOH-terminal Ala residues may simply stabilize the Met-extended determinant V peptide against rapid destruction by cytosolic proteases.
To examine the stability of the rVV-V-encoded product, we utilized the peptide aldehyde proteasome inhibitor cbz-LLL-CHO (MG132), which has been shown to reduce protein degradation within intact cells and to interfere with the generation of antigenic peptides from proteins (33, 58, 88). Incubation with cbz-LLL-CHO did indeed enhance presentation of the determinant V minigene product to CTL clone H-1 (Fig. 6). This effect was specific, as H-1 recognition of targets infected with an irrelevant vaccinia virus recombinant (rVV-ES-I) was not induced; inappropriate recognition of rVV-V by a determinant I-specific CTL clone was not induced under the same conditions (data not shown).
|
, rVV-ES-V; 
, rVV-V;
, rVV-ES-I; 
, no rVV; 
, LT489-497.
Immunogenicity of rVVs expressing Tag determinants. To correlate the findings from these in vitro assays to antigen presentation in vivo, we determined the capacity of the rVVs to prime splenocytes for secondary in vitro restimulation by Tag-expressing tissue culture cells. The lytic activity of restimulated splenocyte cultures was determined by using target cells pulsed with appropriate synthetic peptides (Fig. 7).
|
|
|
H-2Db complexes containing epitope V peptides are relatively unstable. A simple explanation for the increased potency of rVV-ES-V may be related to increased production of the determinant V peptide by this recombinant (5). It has previously been reported that incubation of SV40 Tag-transformed cells in the presence of BFA leads to a rapid reduction in the susceptibility of targets to lysis by epitope V-specific CTL clones (50). These results were taken to indicate that the abundance of epitope V complexes was low and/or that functional cell surface H-2Db-epitope V complexes were relatively unstable. Both intrinsic instability of H-2Db-epitope V complexes and low abundance of available epitope V peptide could contribute to low cell surface densities of H-2Db-epitope V complexes on Tag-transformed cells in vitro or on professional antigen-presenting cells in vivo. Limited production of the determinant V peptide in vivo and/or instability of H-2Db-epitope V complexes could influence the immunogenicity of epitope V.
To determine whether H-2Db-epitope V complexes are intrinsically unstable, peptide-pulsed RMA/s cells were used to examine the relative stability of H-2Db complexes containing the SV40 Tag epitopes I, II/III, and V. Incubation of RMA/s cells overnight in the presence of each of these peptides (10 mM each) induced stabilization of similar levels of H-2Db complexes on the cell surface as determined by flow cytometry (Fig. 8) (50). To evaluate the stability of such complexes, RMA/s cells incubated overnight in the presence of epitope peptides were washed free of peptide, and the decay of cell surface H-2Db expression was analyzed by flow cytometry (60). The results presented in Fig. 8 reveal that complexes formed with the synthetic epitope V peptide (LT489-497) decayed more rapidly at 37°C than complexes formed with peptides corresponding to epitope I (LT206-215) or II/III (LT223-231). These results suggest that H-2Db-epitope V complexes formed by exogenously added synthetic peptide with cell surface H-2Db molecules are relatively unstable.
|
),
II/III (LT223-231) (
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
A principal factor which regulates CTL responses is the quantity of peptide-MHC complexes displayed on the cell surface (16, 86). This could be limited at any of the steps in the antigen processing pathway. Recent findings with rVVs expressing cytosolic or ER-targeted peptides suggest that proteolysis is commonly the major limiting factor in peptide production. Expression of CTL determinants as rVV-encoded cytosolic or ER-targeted peptides results in the expression of enormous amounts of peptide class I complexes at the cell surface, with more than 50,000 complexes being expressed within the first 6 h of infection. In general, this represents a 10- to 100-fold increase in the amount of peptide relative to that recovered from cells expressing the corresponding full-length proteins (5).
This improvement in generating functional peptide-class I complexes makes the use of peptide-encoding vaccinia virus vectors an appealing strategy for enhancing the immunogenicity of weak CTL determinants. We tested this vaccine strategy by using three H-2Db-restricted CTL determinants from SV40 Tag, among which an immunological hierarchy has been demonstrated (50, 77). Our results demonstrate that rVVs expressing the immunodominant CTL determinants I and II/III as minigenes (rVV-I and rVV-II/III) were recognized by the respective determinant-specific CTL clones in vitro and were effective immunogens.
The most important finding of this study is that the immunogenicity of determinant V was dramatically improved by fusion to the ES sequence. Targeting determinant I and II/III peptides to the ER provided little, if any, enhancement of immunogenicity or antigenicity, suggesting that peptide translocation into the ER for these immunodominant determinants is not a rate-limiting step in antigen presentation. Similar results have been obtained by others using immunologically potent CTL epitopes (6, 40, 61). On the other hand, the rVV-ES-V recombinant consistently induced more-potent epitope V-specific CTL responses than any of the other epitope V-containing derivatives tested in this study. While other determinant V-containing vaccinia virus recombinants were immunogenic, the responses induced were weaker, and reliable detection of those responses required the use of peptide-pulsed targets which allowed for increased sensitivity in the cytotoxicity assay.
ER targeting contributed to enhanced antigenicity and immunogenicity of determinant V in TAP-expressing cells. It is unlikely that the ES sequence improved the immunogenicity of epitope V by functioning simply as an amino-terminal flanking sequence, for the following reasons: (i) the ES sequence did allow presentation of epitope V from within TAP-deficient T2/Db cells, while, as expected, none of the other epitope V-containing vaccinia virus recombinants were recognized under similar conditions (data not shown); and (ii) the ES sequence did increase the quantity of functional H-2Db-epitope V complexes on the surface of TAP1/2+ cells, as indicated in BFA blocking experiments (Fig. 9). These results demonstrate that the ES sequence can enhance the immunogenicity of a weak CTL epitope, such as epitope V, most likely because increasing the delivery of the appropriately preprocessed peptide directly into the ER enhances MHC class I-restricted antigen presentation. However, other factors, such as T-cell repertoire, will determine whether enhanced delivery of weak epitopes in general will induce an efficient CTL response.
Consistent with our previous results obtained from the analysis of the immunogenicity of SV40 Tag-transformed cells, expression of the full-length SV40 Tag from a vaccinia virus recombinant produced targets which could be recognized by determinant V-specific CTL clones in vitro but did not induce detectable levels of determinant V-specific CTL in vivo, even when cytotoxicity assays with improved sensitivity were used. The poor immunogenicity of rVV-941 Tag may result from relatively low levels of determinant V produced from the Tag protein. It is also likely that T cells responding to determinants I, II/III, and IV suppress determinant V-specific CTLs. Such immunodomination has been observed repeatedly in immunization experiments which utilize cells expressing full-length Tag or SV40 virus (50). Such a domination model is consistent with our demonstration that immunization with vaccinia virus recombinants which express derivatives of a self-protein (murine DHFR) bearing an epitope V insert do induce epitope V-specific CTL. Similar results have been obtained with influenza virus NP expressed by rVV (21) or from plasmid DNA (30); CTL specific for weak determinants in NP were induced only when the immunodominant determinant was destroyed by mutation of a crucial class I binding residue(s). If so, results presented here, namely, the induction of determinant V-specific CTL by rVV-I-V-II/III, imply that efficient suppression may require that the immunorecessive determinant be expressed in limiting amounts. Perhaps differences in the amount of epitope V peptide generated by differing immunization procedures account for the ability of others to detect epitope V-specific CTL in immunization experiments involving purified, full-length SV40 Tag protein or SV40 Tag expressed following immunization with plasmid DNA (65). Immunization with SV40 Tag-transformed cells consistently fails to result in induction of detectable levels of epitope V-specific CTL (50).
A second important finding resulting from this study is that the antigenicity and immunogenicity of determinant V, when expressed as a minigene product, are dramatically influenced by the presence of short flanking sequences. The rVV-V recombinant (encoding MQGINNLDNL) failed to produce sufficient amounts of the appropriate determinant V peptide to sensitize infected targets for lysis by two independently derived determinant V-specific CTL clones. This failure could imply that the minimal determinant V peptide is unstable in cells or that a property intrinsic to the determinant V peptide may prevent efficient presentation of the Met-extended form of the determinant V peptide expressed by rVV-V. Although our observations imply that the determinant V peptide is intrinsically unstable in cells, the defect in rVV-V recognition may also result from an artifact imposed by the construction of the epitope V-encoding minigene.
Findings for yeast have shown that an N-terminal Met is efficiently removed by Met aminopeptidase only if the radius of gyration of the penultimate residue is less than 1.29 Å (48). If similar constraints apply to murine cells, the initiating Met preceding determinant V is likely to be retained since the penultimate residue in the rVV-V-encoded minigene peptide is a Gln (1.75 Å). As addition of an NH2-terminal Met to the corresponding nonamer determinant synthetic peptide reduces its antigenicity by 100-fold, the inability to remove Met may contribute to this poor antigenicity. Insertion of an (Ala)2 spacer between the initiating Met and determinant V (MAAQGINNLDNL as in rVV-A2-V) would be expected to, and did, overcome such a defect. It is clear that amino acid sequences predicted to result in inefficient removal of an NH2 Met extension do not necessarily prevent efficient recognition of minigene products. It was previously found that an H-2Kb-binding peptide from vesicular stomatitis virus nucleocapsid is efficiently presented as a cytosolic minigene, despite possessing Arg (the residue with the largest radius of gyration) as the penultimate NH2-terminal residue (67). These apparently contradictory results may suggest that the yeast and mammalian cells differ in their ability to remove initiating Met, that small peptides are processed by different proteases than proteins (proteins were used to establish the rules for yeast), or that factors intrinsic to the specific determinant peptide determine the extent to which a residual NH2 Met residue will interfere with accumulation of determinant-MHC class I molecules at the cell surface.
The antigenicity of the determinant V peptide expressed from rVV-V was enhanced by treating cells with the protease inhibitor cbz-LLL-CHO, a potent inhibitor of proteasomes that also inhibits other cellular proteases. The simplest explanation for the cbz-LLL-CHO-mediated rescue of determinant V is that cbz-LLL-CHO prevents its destruction by cellular proteases. For example, cbz-LLL-CHO-mediated inhibition of cellular proteolysis may allow extra time for removal of the NH2 Met extension. Alternatively, it is possible that the enhanced presentation of determinant V mediated by cbz-LLL-CHO is an indirect effect of reducing the amount of competing peptides and/or prolonging TAP interactions with peptide-receptive H-2Db molecules (68). Finally, if the relevant form of the protected determinant V peptide is the nonamer, our results could imply that the nonamer is not a cytosolic intermediate in the presentation of determinant V from T antigen (or the other constructs). This raises the specter of TAP-mediated transport of longer determinant V-containing peptides that are subsequently trimmed in the ER (27, 64, 67).
An intriguing finding is that the antigenicity and immunogenicity of the rVV-V construct were restored by addition of two Ala residues to the carboxy terminus of the determinant V sequence (rVV-V-A2 [Fig. 4 and Table 2]) to encode a primary translation product corresponding to MQGINNLDNLAA. The amino terminus of this (Ala)2-extended peptide is identical to the determinant V peptide encoded by the rVV-V minigene (MQGINNLDNL) which was only poorly antigenic and not immunogenic. Recognition of the Ala-extended peptide most likely does not result from enhanced import into the ER by TAP1/2 (Fig. 5) or improved MHC class I molecule binding (data not shown). Instead, these results suggest that longer peptides containing determinant V may be more resistant to cytosolic proteases which destroy a shorter determinant V peptide. This would be consistent with the enhancing effect of cbz-LLL-CHO on the presentation of MQGINNLDNL.
On the basis of the data at hand, we propose a simple model to unify the results of our analyses involving the recombinants which express cytosolic forms of determinant V. Inefficient removal of the amino-terminal initiating Met residue could limit the pool of determinant V peptide nonamer to levels insufficient to trigger CTL lysis. Since H-2Db complexes containing determinant V peptide are relatively unstable, the few which could be loaded from a limited pool of epitope V 9-mer may dissociate too rapidly for sufficient numbers to accumulate at the cell surface to trigger CTL lysis; alternately, if loaded, the Met-extended 10-mer determinant V peptide would be expected to form 10- to 100-fold-fewer complexes, and this level might also be below the threshold required to trigger CTL clone lysis. We hypothesize that stabilization of the core determinant V peptide (by treatment of rVV-V-infected targets with the protease inhibitor cbz-LLL-CHO or by extension of the minimal peptide) favors increasing the pool of available nonamer determinant V peptide.
In summary, we have shown the following. (i) The immunogenicity of an immunorecessive (or weak) CTL determinant was dramatically enhanced by ER targeting. Similar improvement was not observed for the dominant Tag CTL epitope I or II/III. (ii) The immunorecessive determinant V peptide was immunogenic without ER targeting, but only if short flanking residues were provided. (iii) Determinant V was antigenic and immunogenic when located within the context of an immunologically neutral (self-protein) context, namely, murine DHFR. (iv) The determinant V peptide forms relatively unstable complexes with H-2Db. These findings illustrate that appropriate immunization strategies can be used to effectively induce CTL specific for an immunorecessive epitope. We are currently using SV40 Tag transgenic mice to investigate the therapeutic potential of individual dominant or recessive Tag CTL epitopes in the rejection of SV40 Tag-induced tumors in vivo.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by grant CA25000 from the National Cancer Institute. T.D.S. is currently supported by a fellowship from the Cancer Research Institute of New York.
We are grateful for the pSC11 plasmid provided by B. Moss (National Institutes of Health) and T2/Db cells provided by P. Cresswell (Yale University). We thank Melanie Epler for excellent technical assistance.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Microbiology and Immunology H107, Pennsylvania State University College of Medicine, P.O. Box 850, 500 University Dr., Hershey, PA 17033. Phone: (717) 531-8872. Fax: (717) 531-5578. E-mail: sst1{at}psu.edu.
Present address: Department of Virus and Cell Biology, Merck
Research Laboratories, West Point, PA 19486.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
| 1. |
Anderson, K.,
P. Cresswell,
M. Gammon,
J. Hermes,
A. Williamson, and H. Zweerink.
1991.
Endogenously synthesized peptide with an endoplasmic reticulum signal sequence sensitizes antigen processing mutant cells to class I-restricted cell-mediated lysis.
J. Exp. Med.
174:489-492 |
| 2. | Anderson, K. S., J. Alexander, M. Wei, and P. Cresswell. 1993. Intracellular transport of class I MHC molecules in antigen processing mutant cell lines. J. Immunol. 151:3407-3419[Abstract]. |
| 3. |
Anderson, R. W.,
M. J. Tevethia,
D. Kalderon,
A. E. Smith, and S. S. Tevethia.
1988.
Fine mapping two distinct antigenic sites on simian virus 40 (SV40) T antigen reactive with SV40-specific cytotoxic T-cell clones by using SV40 deletion mutants.
J. Virol.
62:285-296 |
| 4. |
Androlewicz, M. J.,
K. S. Anderson, and P. Cresswell.
1993.
Evidence that transporters associated with antigen processing translocate a major histocompatibility complex class I-binding peptide into the endoplasmic reticulum in an ATP-dependent manner.
Proc. Natl. Acad. Sci. USA
90:9130-9134 |
| 5. | Anton, L. C., J. W. Yewdell, and J. R. Bennink. 1997. MHC class I-associated peptides produced from endogenous gene products with vastly different efficiencies. J. Immunol. 158:2535-2542[Abstract]. |
| 6. | Bacik, I., J. H. Cox, R. Anderson, J. W. Yewdell, and J. R. Bennink. 1994. TAP (transporter associated with antigen processing)-independent presentation of endogenously synthesized peptides is enhanced by endoplasmic reticulum insertion sequences located at the amino- but not carboxyl-terminus of the peptide. J. Immunol. 152:381-387[Abstract]. |
| 7. |
Bergmann, C. C.,
L. Tong,
R. Cua,
J. Sensintaffar, and S. Stohlman.
1994.
Differential effects of flanking residues on presentation of epitopes from chimeric peptides.
J. Virol.
68:5306-5310 |
| 8. | Bergmann, C. C., Q. Yao, C.-K. Ho, and S. L. Buckwold. 1996. Flanking residues alter antigenicity and immunogenicity of multi-unit CTL epitopes. J. Immunol. 157:3242-3249[Abstract]. |
| 9. | Bergsagel, D. J., M. J. Finegold, J. S. Butel, W. J. Kupsky, and R. L. Garcea. 1992. DNA sequences similar to those of simian virus 40 in ependymomas and choroid plexus tumors of childhood. N. Engl. J. Med. 326:988-993[Abstract]. |
| 10. | Brinster, R. L., H. Y. Chen, A. Messing, T. van Dyke, A. J. Levine, and R. D. Palmiter. 1984. Transgenic mice harboring SV40 T-antigen genes develop characteristic brain tumors. Cell 37:367-379[Medline]. |
| 11. | Campbell, A. E., F. L. Foley, and S. S. Tevethia. 1983. Demonstration of multiple antigenic sites of the SV40 transplantation rejection antigen by using cytotoxic T lymphocyte clones. J. Immunol. 130:490-492[Abstract]. |
| 12. | Cao, W., B. A. Myers-Powell, and T. J. Braciale. 1996. The weak CD8+ CTL response to an influenza hemagglutinin epitope reflects limited T cell availability. J. Immunol. 157:505-511[Abstract]. |
| 13. | Carbone, M., H. I. Pass, P. Rizzo, M. Marinetti, M. Di Muzio, D. J. Y. Mew, A. S. Levine, and A. Procopio. 1994. Simian virus 40-like DNA sequences in human pleural mesothelioma. Oncogene 9:1781-1790[Medline]. |
| 14. |
Chakrabarti, S.,
K. Brechling, and B. Moss.
1985.
Vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques.
Mol. Cell. Biol.
5:3403-3409 |
| 15. |
Chen, W.,
S. Khilko,
J. Fecondo,
D. Margulies, and J. McCluskey.
1994.
Determinant selection of major histocompatibility complex class I-restricted antigenic peptides is explained by class I-peptide affinity and is strongly influenced by nondominant anchor residues.
J. Exp. Med.
180:1471-1483 |
| 16. | Christinck, E. R., M. A. Luscher, B. H. Barber, and D. B. Williams. 1991. Peptide binding to class I MHC on living cells and quantitation of complexes required for CTL lysis. Nature (London) 352:67-70[Medline]. |
| 17. |
Cox, J. H.,
J. R. Bennink, and J. W. Yewdell.
1991.
Retention of adenovirus E19 glycoprotein in the endoplasmic reticulum is essential to its ability to block antigen presentation.
J. Exp. Med.
174:1629-1637 |
| 18. | Daly, K., P. Nguyen, D. L. Woodland, and M. A. Blackman. 1995. Immunodominance of major histocompatibility complex class I-restricted influenza virus epitopes can be influenced by the T-cell receptor repertoire. J. Virol. 69:7416-7422[Abstract]. |
| 19. |
Deckhut, A. M.,
J. D. Lippolis, and 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.
66:440-447 |
| 20. | Del Val, M., H. J. Schlicht, T. Ruppert, M. J. Reddehase, and 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 66:1145-1153[Medline]. |
| 21. | Deng, Y., J. W. Yewdell, L. C. Eisenlohr, and J. R. Bennink. 1997. MHC affinity, peptide liberation, T cell repertiore, and immunodominance all contribute to the paucity of MHC class I-restricted peptides recognized by anti-viral cytotoxic T lymphocytes. J. Immunol. 158:1507-1515[Abstract]. |
| 22. |
Dick, L. R.,
C. Aldrich,
S. C. Jameson,
C. R. Moomaw,
B. C. Pramanik,
C. K. Doyle,
G. N. DeMartino,
M. J. Bevan,
J. M. Forman, and C. A. Slaughter.
1994.
Proteolytic processing of ovalbumin and -galactosidase by the proteasome to yield antigenic peptides.
J. Immunol.
152:3884-3894[Abstract].
|
| 23. |
Earl, P. L.,
A. W. Hügin, and B. Moss.
1990.
Removal of cryptic poxvirus transcription termination signals from the human immunodeficiency virus type 1 envelope gene enhances expression and immunogenicity of a recombinant vaccinia virus.
J. Virol.
64:2448-2451 |
| 24. | Earl, P. L., and B. Moss. Generation of recombinant vaccinia viruses, p. 16.17.1-16.17.16. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecular biology. John Wiley & Sons, Inc., New York, N.Y. |
| 25. | Eisenlohr, L. C., I. Bacik, J. R. Bennink, K. Bernstein, and J. W. Yewdell. 1992. Expression of a membrane protease enhances presentation of endogenous antigens to MHC class I-restricted T lymphocytes. Cell 71:963-972[Medline]. |
| 26. |
Eisenlohr, L. C.,
J. W. Yewdell, and J. R. Bennink.
1992.
Flanking sequences influence the presentation of an endogenously synthesized peptide to cytotoxic T lymphocytes.
J. Exp. Med.
175:481-487 |
| 27. |
Elliott, T.,
A. Willis,
V. Cerundolo, and A. Townsend.
1995.
Processing of major histocompatibility class I-restricted antigens in the endoplasmic reticulum.
J. Exp. Med.
181:1481-1491 |
| 28. | Fanning, E. 1992. Structure and function of simian virus 40 large tumor antigen. Annu. Rev. Biochem. 61:55-85[Medline]. |
| 29. | Fu, T.-M., R. H. Bonneau, M. Epler, M. J. Tevethia, S. Alam, K. Verner, and S. S. Tevethia. 1996. Induction and persistence of a cytotoxic T lymphocyte (CTL) response against a herpes simplex virus-specific CTL epitope expressed in a cellular protein. Virology 222:269-274[Medline]. |
| 30. | Fu, T.-M., A. Friedman, J. B. Ulmer, M. A. Liu, and J. J. Donnelly. 1997. Protective cellular immunity: cytotoxic T-lymphocyte responses against dominant and recessive epitopes of influenza virus nucleoprotein induced by DNA immunization. J. Virol. 71:2715-2721[Abstract]. |
| 31. |
Gairin, J. E., and M. B. A. Oldstone.
1992.
Design of high-affinity major histocompatibility complex-specific antagonist peptides that inhibit cytotoxic T-lymphocyte activity: implications for control of viral disease.
J. Virol.
66:6755-6762 |
| 32. | Germain, R. N., and D. H. Margulies. 1993. The biochemistry and cell biology of antigen processing and presentation. Annu. Rev. Immunol. 11:403-450[Medline]. |
| 33. | Hahn, Y. S., B. Yang, and T. J. Braciale. 1996. Regulation of antigen processing and presentation to class I MHC restricted CD8+ T lymphocytes. Immunol. Rev. 151:31-49[Medline]. |
| 34. |
Hanahan, D.
1985.
Heritable formation of pancreatic -cell tumors in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes.
Nature (London)
315:115-122[Medline].
|
| 35. |
Jennings, S. R.,
K. L. Fresa,
P. A. Lippe,
J. E. Milici, and S. S. Tevethia.
1988.
Frequency analysis of simian virus 40-specific cytotoxic T lymphocyte precursors in the high responder C57BL/6 mouse strain.
J. Gen. Virol.
69:2493-2503 |
| 36. | Kärre, K., H. G. Ljunggren, G. Piontek, and R. Kiessling. 1986. Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature (London) 319:675-678[Medline]. |
| 37. |
Knowles, B. B.,
J. McCarrick,
N. Fox,
D. Solter, and I. Damjanov.
1990.
Osteosarcomas in transgenic mice expressing an  -amylase-SV40 T-antigen hybrid gene.
Am. J. Pathol.
137:259-262[Abstract].
|
| 38. |
Kozak, M.
1989.
The scanning model for translation: an update.
J. Cell Biol.
108:229-240 |
| 39. | Lanford, R. E. 1988. Expression of simian virus 40 T antigen in insect cells using a baculovirus expression vector. Virology 167:72-81[Medline]. |
| 40. |
Lawson, C. M.,
J. R. Bennink,
N. P. Restifo,
J. W. Yewdell, and B. R. Murphy.
1994.
Primary pulmonary cytotoxic T lymphocytes induced by immunization with a vaccinia virus recombinant expressing influenza A virus nucleoprotein peptide do not protect mice against challenge.
J. Virol.
68:3505-3511 |
| 41. | Lednicky, J. A., R. L. Garcea, D. J. Bergsagel, and J. S. Butel. 1995. Natural simian virus 40 strains are present in human choroid plexus and ependymoma tumors. Virology 212:710-717[Medline]. |
| 42. |
Lill, N. L.,
M. J. Tevethia,
W. G. Hendrickson, and 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.
176:449-457 |
| 43. | Lippolis, J. D., L. M. Mylin, D. T. Simmons, and 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. 69:3134-3146[Abstract]. |
| 44. |
Ljunggren, H. G., and K. Kärre.
1985.
Host resistance directed selectively against H-2-deficient lymphoma variants. Analysis of the mechanism.
J. Exp. Med.
162:1745-1759 |
| 45. |
Mackett, M.,
G. L. Smith, and B. Moss.
1984.
General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes.
J. Virol.
49:857-864 |
| 46. |
Martini, F.,
L. Iaccheri,
L. Lazzarin,
P. Carinci,
A. Corallini,
M. Gerosa,
P. Iuzzolino,
G. Barbanti-Brodano, and M. Tognon.
1996.
SV40 early region and large T antigen in human brain tumors, peripheral blood cells, and sperm fluids from healthy individuals.
Cancer Res.
56:4820-4825 |
| 47. | Michalek, M. T., E. P. Grant, C. Gramm, A. L. Goldberg, and K. L. Rock. 1992. A role for the ubiquitin-dependent proteolytic pathway in MHC class I-restricted antigen presentation. Nature (London) 363:552-554. |
| 48. |
Moerschell, R. P.,
Y. Hosokawa,
S. Tsunasawa, and F. Sherman.
1992.
The specificities of yeast methionine aminopeptidase and acetylation of amino-terminal methionine in vivo.
J. Biol. Chem.
265:19638-19643 |
| 49. | Monaco, J. J. 1992. A molecular model of MHC class-I-restricted antigen processing. Immunol. Today 13:173-179[Medline]. |
| 50. | Mylin, L. M., R. H. Bonneau, J. D. Lippolis, and S. S. Tevethia. 1995. Hierarchy among multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian virus 40 T antigen. J. Virol. 69:6665-6677[Abstract]. |
| 51. | Mylin, L. M., A. M. Deckhut, R. H. Bonneau, T. D. Kierstead, M. J. Tevethia, D. T. Simmons, and S. S. Tevethia. 1995. Cytotoxic T lymphocyte escape variants, induced mutations and synthetic peptides define a dominant H-2Kd-restricted determinant in simian virus 40 tumor antigen. Virology 208:159-172[Medline]. |
| 51a. | Mylin, L. M., and S. S. Tevethia. Unpublished results. |
| 52. |
Nanda, N. K., and E. Sercarz.
1996.
A truncated T cell receptor repertoire reveals underlying immunogenicity of an antigenic determinant.
J. Exp. Med.
184:1037-1043 |
| 53. | Neisig, A., J. Roelse, A. J. Sijts, F. Ossendorp, M. C. Feltkamp, W. M. Kast, C. J. M. Melief, and J. J. Neefjes. 1995. Major differences in transporter associated with antigen processing (TAP)-dependent translocation of MHC class I-presentable peptides and the effect of flanking sequences. J. Immunol. 154:1273-1279[Abstract]. |
| 54. | Niedermann, G., S. Butz, H. G. Ihlenfeldt, R. Grimm, M. Lucchiari, H. Hoschützky, G. Jung, B. Maier, and K. Eichmann. 1995. Contribution of proteosome-mediated proteolysis to the hierarchy of epitopes presented by major histocompatibility complex class I molecules. Immunity 2:289-299[Medline]. |
| 55. | Nuchtern, J. G., J. S. Bonifacino, W. E. Biddison, and R. D. Klausner. 1989. Brefeldin A implicates egress from endoplasmic reticulum in class I restricted antigen presentation. Nature (London) 339:223-226[Medline]. |
| 56. | Oldstone, M. B. A., H. Lewicki, P. Borrow, D. Hudrisier, and J. E. Gairin. 1995. Discriminated selection among viral peptides with the appropriate anchor residues: implications for the size of the cytotoxic T-lymphocyte repertoire and control of viral infection. J. Virol. 69:7423-7429[Abstract]. |
| 57. | Ossendorp, F., M. Eggers, A. Neisig, T. Ruppert, M. Groettrup, A. Sijts, E. Mengedé, P.-M. Kloetzel, J. Neefjes, U. Koszinowski, and C. Melief. 1996. A single residue exchange within a viral CTL epitope alters proteasome-mediated degradation resulting in lack of antigen presentation. Immunity 5:115-124[Medline]. |
| 58. |
Palombella, V. J.,
O. J. Rando,
A. L. Goldberg, and T. Maniatis.
1994.
The ubiquitin-proteasome pathway is required for processing the NF- B1 precursor protein and the activation of NF-B.
Cell
78:773-785[Medline].
|
| 59. | Paltimer, R. D., H. Y. Chen, A. Messing, and R. L. Brinster. 1985. SV40 enhancer and large T antigen are instrumental in development of choroid plexus tumors in transgenic mice. Nature (London) 316:457-460[Medline]. |
| 60. |
Potter, T. A.,
C. Boyer,
A. Verhuslt,
P. Goldstein, and T. V. Rajan.
1984.
Expression of H-2Db on the cell surface in the absence of detectable 2 microglobulin.
J. Exp. Med.
160:317-322 |
| 61. | Restifo, N. P., I. Bacik, K. R. Irvine, J. W. Yewdell, B. J. McCabe, R. W. Anderson, L. C. Eisenlohr, S. A. Rosenberg, and J. R. Bennink. 1995. Antigen processing in vivo and the elicitation of primary CTL responses. J. Immunol. 154:4414-4422[Abstract]. |
| 62. | Rhim, J. S., H. Y. Cho, and R. J. Huebner. 1975. Non-producer human cells induced by murine sarcoma virus. Int. J. Cancer 15:23-29[Medline]. |
| 63. | Rock, K. L., G. Gramm, L. Rothstein, K. Clark, R. Stein, L. Dick, D. Hwang, and A. L. Goldberg. 1994. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78:761-771[Medline]. |
| 64. |
Roelse, J.,
M. Grommé,
F. Momberg,
G. Hämmerling, and J. Neefjes.
1994.
Trimming of TAP-translocated peptides in the endoplasmic reticulum and in the cytosol during recycling.
J. Exp. Med.
180:1591-1597 |
| 65. | Schirmbeck, R., W. Böhm, and J. Reimann. 1996. DNA vaccination primes MHC class I-restricted, simian virus 40 large tumor antigen-specific CTL in H-2d mice that reject syngeneic tumors. J. Immunol. 157:3550-3558[Abstract]. |
| 66. |
Schumacher, T. N. M.,
D. V. Kantesaria,
M.-T. Heemels,
P. G. Ashton-Rickardt,
J. C. Shepherd,
K. Fruh,
Y. Yang,
P. A. Peterson,
S. Tonegawa, and H. L. Ploegh.
1994.
Peptide length and sequence specificity of the mouse TAP1/TAP2 translocator.
J. Exp. Med.
179:533-540 |
| 67. |
Snyder, H. L.,
J. W. Yewdell, and J. R. Bennink.
1994.
Trimming of antigenic peptides in an early secretory compartment.
J. Exp. Med.
180:2389-2394 |
| 68. |
Suh, W.-K.,
E. K. Mitchell,
Y. Yang,
P. A. Peterson,
G. L. Waneck, and D. B. Williams.
1996.
MHC class I molecules form ternary complexes with calnexin and TAP and undergo peptide-regulated interaction with TAP via their extracellular domains.
J. Exp. Med.
184:337-348 |
| 69. | Tanaka, Y., R. W. Anderson, W. L. Maloy, and 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 171:205-213[Medline]. |
| 70. | Tanaka, Y., M. J. Tevethia, D. Kalderon, A. E. Smith, and S. S. Tevethia. 1988. Clustering of antigenic sites recognized by cytotoxic T lymphocyte clones in the amino terminal half of SV40 T antigen. Virology 162:427-436[Medline]. |
| 71. | Tanaka, Y., and S. S. Tevethia. 1988. In vitro selection of SV40 T antigen epitope loss variants by site-specific cytotoxic T lymphocyte clones. J. Immunol. 140:4348-4354[Abstract]. |
| 72. | Tanaka, Y., and 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 31:197-202[Medline]. |
| 73. | 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 137:414-421[Medline]. |
| 74. | Tevethia, M. J., and L. W. Ripper. 1977. Biology of SV40 transplantation antigen (TrAg). II. Isolation and characterization of additional temperature sensitive mutants of SV40. Virology 81:192-211[Medline]. |
| 75. | Tevethia, M. J., L. W. Ripper, and S. S. Tevethia. 1974. A simple qualitative spot complementation test for temperature sensitive mutants of SV40. Intervirology 3:245-255[Medline]. |
| 76. | Tevethia, S. S. 1980. Immunology of simian virus 40, p. 581-601. In G. Klein (ed.), Viral oncology. Raven Press, New York, N.Y. |
| 77. | Tevethia, S. S. 1990. Recognition of simian virus 40 T antigen by cytotoxic T lymphocytes. Mol. Biol. Med. 7:83-96[Medline]. |
| 78. | Tevethia, S. S., R. S. Greenfield, D. C. Flyer, and M. J. Tevethia. 1980. SV40 transplantation antigen: relationship to SV40-specific proteins. Cold Spring Harbor Symp. Quant. Biol. 1:235-242. |
| 79. | Tooze, J. 1980. DNA tumor viruses, p. 799-829. In J. Tooze (ed.), Molecular biology of tumor viruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 80. |
Townsend, A.,
J. Bastin,
K. Gould,
G. Brownlee,
M. Andrew,
B. Coupar,
D. Boyle,
S. Chan, and G. Smith.
1988.
Defective presentation to class I-restricted cytotoxic T lymphocytes in vaccinia-infected cells is overcome by enhanced degradation of antigen.
J. Exp. Med.
168:1211-1224 |
| 81. | Townsend, A., C. Ohlen, J. Bastin, H. G. Ljunggren, L. Foster, and K. Karre. 1989. Association of class I major histocompatibility heavy and light chains induced by viral peptides. Nature (London) 340:443-448[Medline]. |
| 82. | van der Burg, S. H., M. J. W. Visseren, R. M. P. Brandt, W. M. Kast, and C. J. M. Melief. 1996. Immunogenicity of peptides bound to MHC class I molecules depends on the MHC-peptide complex stability. J. Immunol. 156:3308-3314[Abstract]. |
| 83. |
Van Kaer, L.,
P. G. Ashton-Rickardt,
H. L. Ploegh, and S. Tonegawa.
1992.
TAP1 mutant mice are deficient in antigen presentation, surface class I molecules, and CD48+ T cells.
Cell
71:1205-1214[Medline].
|
| 84. | Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119[Medline]. |
| 85. |
Yewdell, J. W., and J. R. Bennink.
1989.
Brefeldin A specifically inhibits presentation of protein antigens to cytotoxic T lymphocytes.
Science
244:1072-1075 |
| 86. | Yewdell, J. W., and J. R. Bennink. 1992. Cell biology of antigen processing and presentation to major histocompatibility complex class I molecule-restricted T lymphocytes. Adv. Immunol. 52:1-123[Medline]. |
| 87. |
Yewdell, J. W.,
F. Esquivel,
D. Arnold,
T. Spies,
L. C. Eisenlohr, and J. R. Bennink.
1993.
Presentation of numerous viral peptides to mouse major histocompatibility complex (MHC) class I-restricted T lymphocytes is mediated by the human MHC-encoded transporter or by a hybrid mouse-human transporter.
J. Exp. Med.
177:1785-1790 |
| 88. | York, I. A., and K. L. Rock. 1996. Antigen processing and presentation by the class I major histocompatibility complex. Annu. Rev. Immunol. 14:369-396[Medline]. |
J Virol, February 1998, p. 1469-1481, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Brosi, H., Reiser, M., Rajasalu, T., Spyrantis, A., Oswald, F., Boehm, B. O., Schirmbeck, R.
(2009). Processing in the Endoplasmic Reticulum Generates an Epitope on the Insulin A Chain that Stimulates Diabetogenic CD8 T Cell Responses. J. Immunol.
183: 7187-7195
[Abstract] [Full Text] -
Grebe, K. M., Hickman, H. D., Irvine, K. R., Takeda, K., Bennink, J. R., Yewdell, J. W.
(2009). Sympathetic nervous system control of anti-influenza CD8+ T cell responses. Proc. Natl. Acad. Sci. USA
106: 5300-5305
[Abstract] [Full Text] -
Otahal, P., Schell, T. D., Hutchinson, S. C., Knowles, B. B., Tevethia, S. S.
(2006). Early Immunization Induces Persistent Tumor-Infiltrating CD8+ T Cells against an Immunodominant Epitope and Promotes Lifelong Control of Pancreatic Tumor Progression in SV40 Tumor Antigen Transgenic Mice.. J. Immunol.
177: 3089-3099
[Abstract] [Full Text] -
Ryan, C. M., Schell, T. D.
(2006). Accumulation of CD8+ T Cells in Advanced-Stage Tumors and Delay of Disease Progression following Secondary Immunization against an Immunorecessive Epitope. J. Immunol.
177: 255-267
[Abstract] [Full Text] -
Otahal, P., Hutchinson, S. C., Mylin, L. M., Tevethia, M. J., Tevethia, S. S., Schell, T. D.
(2005). Inefficient Cross-Presentation Limits the CD8+ T Cell Response to a Subdominant Tumor Antigen Epitope. J. Immunol.
175: 700-712
[Abstract] [Full Text] -
Yoshimura, Y., Yadav, R., Christianson, G. J., Ajayi, W. U., Roopenian, D. C., Joyce, S.
(2004). Duration of Alloantigen Presentation and Avidity of T Cell Antigen Recognition Correlate with Immunodominance of CTL Response to Minor Histocompatibility Antigens. J. Immunol.
172: 6666-6674
[Abstract] [Full Text] -
Barton, L. F., Runnels, H. A., Schell, T. D., Cho, Y., Gibbons, R., Tevethia, S. S., Deepe, G. S. Jr., Monaco, J. J.
(2004). Immune Defects in 28-kDa Proteasome Activator {gamma}-Deficient Mice. J. Immunol.
172: 3948-3954
[Abstract] [Full Text] -
Schell, T. D.
(2004). In Vivo Expansion of the Residual Tumor Antigen-Specific CD8+ T Lymphocytes That Survive Negative Selection in Simian Virus 40 T-Antigen-Transgenic Mice. J. Virol.
78: 1751-1762
[Abstract] [Full Text] -
Paster, W., Kalat, M., Zehetner, M., Schweighoffer, T.
(2002). Structural Elements of a Protein Antigen Determine Immunogenicity of the Embedded MHC Class I-Restricted T Cell Epitope. J. Immunol.
169: 2937-2946
[Abstract] [Full Text] -
Schell, T. D., Mylin, L. M., Tevethia, S. S., Joyce, S.
(2002). The assembly of functional {beta}2-microglobulin-free MHC class I molecules that interact with peptides and CD8+ T lymphocytes. Int Immunol
14: 775-782
[Abstract] [Full Text] -
Casimiro, D. R., Tang, A., Perry, H. C., Long, R. S., Chen, M., Heidecker, G. J., Davies, M.-E., Freed, D. C., Persaud, N. V., Dubey, S., Smith, J. G., Havlir, D., Richman, D., Chastain, M. A., Simon, A. J., Fu, T.-M., Emini, E. A., Shiver, J. W.
(2002). Vaccine-Induced Immune Responses in Rodents and Nonhuman Primates by Use of a Humanized Human Immunodeficiency Virus Type 1 pol Gene. J. Virol.
76: 185-194
[Abstract] [Full Text] -
Schell, T. D., Tevethia, S. S.
(2001). Control of Advanced Choroid Plexus Tumors in SV40 T Antigen Transgenic Mice Following Priming of Donor CD8+ T Lymphocytes by the Endogenous Tumor Antigen. J. Immunol.
167: 6947-6956
[Abstract] [Full Text] -
Chikh, G. G., Kong, S., Bally, M. B., Meunier, J.-C., Schutze-Redelmeier, M.-P. M.
(2001). Efficient Delivery of Antennapedia Homeodomain Fused to CTL Epitope with Liposomes into Dendritic Cells Results in the Activation of CD8+ T Cells. J. Immunol.
167: 6462-6470
[Abstract] [Full Text] -
Herberts, C. A., Stittelaar, K. J., van der Heeft, E., van Gaans-van den Brink, J., Poelen, M. C. M., Roholl, P. J. M., van Alphen, L. J. W., Melief, C. J. M., de Jong, A. P. J. M., van Els, C. A. C. M.
(2001). A measles virus glycoprotein-derived human CTL epitope is abundantly presented via the proteasomal-dependent MHC class I processing pathway. J. Gen. Virol.
82: 2131-2142
[Abstract] [Full Text] -
Norbury, C. C., Princiotta, M. F., Bacik, I., Brutkiewicz, R. R., Wood, P., Elliott, T., Bennink, J. R., Yewdell, J. W.
(2001). Multiple Antigen-Specific Processing Pathways for Activating Naive CD8+ T Cells In Vivo. J. Immunol.
166: 4355-4362
[Abstract] [Full Text] -
Schell, T. D., Lippolis, J. D., Tevethia, S. S.
(2001). Cytotoxic T Lymphocytes from HLA-A2.1 Transgenic Mice Define a Potential Human Epitope from Simian Virus 40 Large T Antigen. Cancer Res.
61: 873-879
[Abstract] [Full Text] -
Fu, T.-M., Freed, D. C., Trigona, W. L., Guan, L., Zhu, L., Long, R., Persaud, N. V., Manson, K., Dubey, S., Shiver, J. W.
(2001). Evaluation of Cytotoxic T-Lymphocyte Responses in Human and Nonhuman Primate Subjects Infected with Human Immunodeficiency Virus Type 1 or Simian/Human Immunodeficiency Virus. J. Virol.
75: 73-82
[Abstract] [Full Text] -
Mylin, L. M., Schell, T. D., Roberts, D., Epler, M., Boesteanu, A., Collins, E. J., Frelinger, J. A., Joyce, S., Tevethia, S. S.
(2000). 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.
74: 6922-6934
[Abstract] [Full Text] -
Schell, T. D., Knowles, B. B., Tevethia, S. S.
(2000). Sequential Loss of Cytotoxic T Lymphocyte Responses to Simian Virus 40 Large T Antigen Epitopes in T Antigen Transgenic Mice Developing Osteosarcomas. Cancer Res.
60: 3002-3012
[Abstract] [Full Text] -
Schell, T. D., Mylin, L. M., Georgoff, I., Teresky, A. K., Levine, A. J., Tevethia, S. S.
(1999). Cytotoxic T-Lymphocyte Epitope Immunodominance in the Control of Choroid Plexus Tumors in Simian Virus 40 Large T Antigen Transgenic Mice. J. Virol.
73: 5981-5993
[Abstract] [Full Text] -
Mylin, L. M.
(1999). Context-Dependent Immunogenicity of an S206G-Substituted H-2Db-Restricted Simian Virus 40 Large T Antigen Epitope I Variant. J. Immunol.
162: 2171-2179
[Abstract] [Full Text] -
Xie, Y. C., Hwang, C., Overwijk, W., Zeng, Z., Eng, M. H., Mule, J. J., Imperiale, M. J., Restifo, N. P., Sanda, M. G.
(1999). Induction of Tumor Antigen-Specific Immunity In Vivo by a Novel Vaccinia Vector Encoding Safety-Modified Simian Virus 40 T Antigen. JNCI J Natl Cancer Inst
91: 169-175
[Abstract] [Full Text] -
Blaney, J. E. Jr., Nobusawa, E., Brehm, M. A., Bonneau, R. H., Mylin, L. M., Fu, T.-M., Kawaoka, Y., Tevethia, S. S.
(1998). Immunization with a Single Major Histocompatibility Complex Class I-Restricted Cytotoxic T-Lymphocyte Recognition Epitope of Herpes Simplex Virus Type 2 Confers Protective Immunity. J. Virol.
72: 9567-9574
[Abstract] [Full Text] -
Anton, L. C., Snyder, H. L., Bennink, J. R., Vinitsky, A., Orlowski, M., Porgador, A., Yewdell, J. W.
(1998). Dissociation of Proteasomal Degradation of Biosynthesized Viral Proteins from Generation of MHC Class I-Associated Antigenic Peptides. J. Immunol.
160: 4859-4868
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»