Presentation of a new H-2Dk-restricted epitope in the Tax protein of human T-lymphotropic virus type I is enhanced by the proteasome inhibitor lactacystin

Mehnaaz Lomas1, Emmanuel Hanon1, Yuetsu Tanaka2, Charles R. M. Bangham1 and Keith G. Gould1

Department of Immunology, Imperial College School of Medicine (St Mary’s Campus), Norfolk Place, London W2 1PG, UK1
Department of Infectious Disease and Immunology, Okinawa-Asia Research Centre of Medical Science, Faculty of Medicine, University of the Ryukyus, Uehara-cho 207, Nishihara, Okinawa 903-0215, Japan2

Author for correspondence: Keith Gould. Fax +44 20 7402 0653. e-mail k.gould{at}ic.ac.uk


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Tax, the trans-activator of human T-lymphotropic virus type I (HTLV-I), is the dominant target antigen for cytotoxic T lymphocytes (CTLs) in the majority of infected individuals, although the reason for this immunodominance is not clear. Tax has been shown to associate physically with the proteasome, a protease that is responsible for the generation of the majority of major histocompatibility complex (MHC) class I ligands recognized by CTLs. This association could lead to the preferential targeting of Tax to the MHC class I pathway and account for its high immunogenicity. Here, the CTL response to Tax was investigated in mice by priming with a Tax expression vector and boosting with a Tax recombinant vaccinia virus (modified vaccinia virus Ankara strain). This approach led to the identification of a new H-2Dk-restricted epitope in Tax, amino acid residues 38–46, sequence ARLHRHALL. Surprisingly, presentation of this epitope was found to be enhanced by the proteasome inhibitor lactacystin, although Tax was shown to associate with proteasomes in murine cells. The difficulties encountered in generating Tax-specific CTL responses and the results of enzyme-linked immunospot (ELISpot) analysis suggested that Tax is only poorly immunogenic for CTLs in mice. Therefore, the immunodominance of Tax in human CTL responses to HTLV-I is probably not due to an intrinsic property of the protein itself, such as an association with the proteasome, but instead may result from the fact that Tax is the predominant protein synthesized early after infection.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Human T-lymphotropic virus type I (HTLV-I) is a pathogenic retrovirus that causes two different types of disease (Cann & Chen, 1996 ). Although most infected individuals remain asymptomatic carriers of the virus, 2–3% develop one of a number of inflammatory diseases (most commonly HTLV-I-associated myelopathy/tropical spastic paraparesis) and another 2–3% develop adult T-cell leukaemia/lymphoma (reviewed by Bangham, 2000 ; Uchiyama, 1997 ). The role of the host immune response to the virus in these different outcomes of infection has been investigated extensively. Most people infected with HTLV-I mount a highly active cytotoxic T lymphocyte (CTL) response against the virus (Bangham et al., 1999 ; Kannagi et al., 1984 ), although whether this CTL response is beneficial or contributes to the inflammatory disease is controversial (Bangham et al., 1996 , 1999 ; Jacobson et al., 1990 ).

HTLV-I shares with other retroviruses the three main genomic regions of gag, pol and env but, in addition, encodes two transcriptional regulatory proteins, Tax and Rex (Cann & Chen, 1996 ; Ciminale et al., 1992 ). The majority of CTLs in infected individuals recognize the same antigen, the Tax protein, and these CTLs are able to lyse autologous Tax-expressing cells without the need for in vitro restimulation. Although CTL responses to Pol and Env have also been identified, Tax is clearly the immunodominant target antigen for CTLs freshly isolated from infected individuals (Jacobson et al., 1990 ; Kannagi et al., 1991 ; Parker et al., 1992 , 1994 ). The factors contributing to immunodominance in CD8+ CTL responses have been investigated in detail (Yewdell & Bennink, 1999 ). They include the efficiency with which a peptide epitope is generated in the cell, transport of the peptide into the lumen of the endoplasmic reticulum, the affinity with which a peptide binds to a particular major histocompatibility complex (MHC) class I molecule, egress to the cell surface and the quality of the responding CD8+ T-cell repertoire. It is important to understand why Tax is such an immunodominant target for CTLs, as recent evidence has suggested that the CTL response to Tax plays an important part in the outcome of an HTLV-I infection (Bangham et al., 1999 ).

Tax has been shown to associate physically with two subunits of the human proteasome (Beraud & Greene, 1996 ; Rousset et al., 1996 ), the multi-subunit proteolytic system responsible for generating the majority of peptides presented by MHC class I molecules (Rock et al., 1994 ). Proteins from a variety of other viruses have also been reported to interact with proteasomes (Berezutskaya & Bagchi, 1997 ; Huang et al., 1996 ; Turnell et al., 2000 ) and, although the primary function of the association between Tax and the proteasome may be to affect processing of the transcription factor NF-{kappa}B (Palombella et al., 1994 ), we hypothesized that this interaction may also facilitate the intracellular degradation of Tax in the MHC class I pathway. If correct, this could help to explain the immunodominance of Tax in the CTL response to HTLV-I.

In this study, we have investigated the CTL response to Tax in mice as a model system amenable to manipulation, with a view to testing the hypothesis that the association of Tax with proteasomes contributes to its immunogenicity for CTLs. Although Tax associates with proteasomes in murine cells and contains at least one murine CTL epitope, Tax was found to be only poorly immunogenic for CTLs in mice, suggesting that its association with proteasomes is not the explanation for the immunodominance of Tax in human CTL responses.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Antibodies.
Lt4, a mouse monoclonal anti-Tax antibody, was obtained from Yuetsu Tanaka (University of the Ryukyus, Okinawa, Japan). Anti-B5R, a rat monoclonal anti-vaccinia virus antibody, was obtained from Geoff Smith (Imperial College, London, UK). Two polyclonal rabbit anti-vaccinia virus antisera were obtained from John Williamson (Imperial College, London, UK). pAb186, a rabbit polyclonal anti-rat proteasome antibody, which cross-reacts with mouse proteasomes, was obtained from Jennifer Rivett (University of Bristol, Bristol, UK).

{blacksquare} Cell lines.
BHK and BHK-TK- cells were obtained from Sarah Gilbert (University of Oxford, Oxford, UK). L-Db cells were obtained from Alain Townsend (University of Oxford, Oxford, UK). BHK and L-Db cells were cultured in Dulbecco’s modified essential medium (DMEM). BHK-TK- cells were cultured in DMEM containing 25 µg/ml BrdU.

{blacksquare} Preparation of Tax modified vaccinia virus Ankara (MVA).
Wild-type (MVA) was obtained from Geoff Smith (Imperial College, London, UK). The tax gene was cloned into the SmaI site of pSC11 (Chakrabarti et al., 1985 ) and the recombinant plasmid was transfected into BHK cells infected 90 min earlier with wild-type MVA at 0·05 p.f.u. per cell. Virus was harvested 2 days later and used to re-infect a monolayer of BHK-TK- cells. TK- recombinant viruses were amplified in three re-infection cycles by BrdU selection in BHK-TK- cells. Recombinant virus was purified by five successive rounds of plaque purification. Bulk stocks were purified by centrifugation of cytoplasmic extracts of infected cells through a 36% (w/v) sucrose cushion in a Sorvall HB-4 rotor at 13000 r.p.m. for 60 min.

{blacksquare} Staining for wild-type and recombinant vaccinia virus.
BHK cell monolayers were infected with Tax MVA at various m.o.i. After 2 days, cells were either overlaid with agarose containing 0·25 mg/ml X-Gal or fixed in 50% acetone and 50% methanol for immunostaining. Recombinant MVA stained blue in the X-Gal overlay and vaccinia virus-positive plaques were stained brown using a polyclonal rabbit anti-vaccinia virus antiserum, followed by a peroxidase-conjugated secondary antibody and DAB substrate.

{blacksquare} Flow cytometry.
Intracellular staining of Tax was as described previously (Hanon et al., 2000 ). BHK or L-Db cells were infected overnight with vaccinia virus using 10 p.f.u. per cell. Single-staining for Tax used Lt4 alone, whereas double-staining experiments also incorporated the anti-vaccinia virus antibody B5R. Samples were analysed on an EPICS XL flow cytometer using Expo 2 software (Beckman Coulter).

{blacksquare} Tax peptides.
A total of 86 unpurified 13-mer Tax peptides was generated on a 5 µmol synthesis scale (Genosys). The peptides, which overlapped by nine amino acids, were dissolved in RPMI 1640 (Gibco) at 1 mg/ml and used either separately or in four pools of 22 (except for pool 1, which contained 20 peptides) at various dilutions. The peptides were used to restimulate spleen effector cells in vitro and were incubated with target cells in chromium-release assays. Several shorter peptides were also made on a 5 µmol synthesis scale (Research Genetics) and used as described above for the 13-mer peptides.

{blacksquare} Animals and immunizations.
Female CBA mice, 12 weeks old, were immunized in groups of two by Ivor Brown (Imperial College, London, UK). Mice were injected with 100 µg pJFE-Tax plasmid DNA in 50 µl PBS into a hind leg muscle and boosted 3 weeks later with 106 p.f.u. Tax MVA in 100 µl PBS injected into the tail vein. The spleens of the mice were removed 2 weeks later as a source of effector cells.

{blacksquare} Restimulation of spleen effector cells in vitro.
Spleens from each group of two mice were homogenized, washed twice and resuspended in 15 ml RPMI 1640 supplemented with 10% heat-inactivated foetal calf serum, penicillin, streptomycin, 10 mM HEPES, 15 µM 2-mercaptoethanol (Sigma) and, for the third and subsequent restimulations, 10 U/ml human IL-2 (Cetus). Effector cells were restimulated with spleen feeder cells from uninfected mice that were incubated with peptide for 90 min and then irradiated. Peptides were used either in pools (20 µM of each individual peptide) or alone (100 µM). Effector cells were restimulated at weekly intervals and maintained in humidified incubators in 5% CO2 at 37 °C.

{blacksquare} Cytotoxic assay.
On day 4 or 5 after restimulation, effector cells were either diluted in U-bottom wells (96-well plate) to give the indicated effector:target ratios or used at a single ratio (specified in results section). Approximately 10000 51Cr-labelled L-Db target cells and either peptide pools (10 µM of each individual peptide) or individual peptides (33 µM) were added to the effector cells and incubated at 37 °C for 5 h (Townsend et al., 1985 ). For assays in which the whole Tax protein was processed in the cell, the target cells were infected with MVA for 90 min (2 p.f.u. per cell) in suspension. The cells were then washed, incubated in suspension overnight and labelled the following day. For the proteasome inhibitor experiments, target cells were pre-treated for 2 h in medium containing 10 µM lactacystin, which was then maintained in the medium for the duration of the infection and overnight incubation. Spontaneous and total chromium release values were estimated from wells in which the target cells were kept in medium alone or with 5% Triton X-100, respectively. The percentage of specific lysis was calculated using the formula (sample release-spontaneous release/total release-spontaneous release)x100.

{blacksquare} Immunoprecipitation and Western blot analysis.
L-Db cells (8x106 cells) were infected overnight with vaccinia viruses using 10 p.f.u. per cell. The cells were then harvested, lysed in 150 mM NaCl, 50 mM Tris–HCl pH 7·5, 0·5 % Nonidet P-40, 0·5% Triton X-100, and a mammalian cell protease inhibitor cocktail (Sigma), incubated on ice for 40 min and clarified by centrifugation. A sample of the whole cell lysate was removed to confirm Tax expression by Western blotting. The lysate was pre-cleared with protein A–Sepharose (Sigma) overnight at 4 °C and incubated with anti-proteasome antiserum at 4 °C for 90 min. Protein A–Sepharose was then added and the mixture incubated for a further 90 min at 4 °C. The Sepharose beads were then washed and resuspended in 2xSDS sample buffer.

Duplicate immunoprecipitation and whole cell lysate samples were fractionated on two 10% SDS–polyacrylamide gels. Proteins were transferred to nitrocellulose using a semi-dry electroblotter (25 V constant for 20 min). The filters were blocked in TBS containing 0·1% Tween (v/v) and 5% (w/v) milk powder overnight at 4 °C. One of the filters was probed with anti-Tax antibody and the other was probed with the anti-mouse proteasome antiserum. Bound antibodies were detected using horseradish peroxidase-linked secondary antibodies (anti-rabbit IgG and anti-mouse IgG) (New England Biolabs) and chemiluminescence reagents (New England Biolabs).

{blacksquare} ELISpot assay.
ELISpot analysis was carried out as described previously (Power et al., 1999 ). Nitrocellulose-bottomed 96-well plates (Millipore) were coated for 2 h at 37 °C followed by overnight incubation at 4 °C with rat anti-mouse IFN-{gamma} antibody (clone R4-6A2) (Pharmingen). Dilutions of responder spleen cells from immunized mice were cultured in complete medium with or without peptide epitope for 48 h. Plates were then washed and incubated with biotinylated IFN-{gamma} antibody (clone XMG1.2) (Pharmingen) followed by streptavidin conjugated to alkaline phosphatase (Boehringer Mannheim). Spots were visualized using BCIP/NBT substrate (Promega) and counted using an automated ELISpot plate counter (Autoimmun Diagnostika). Test wells were assayed in triplicate and the frequency of peptide-specific T-cells present was calculated by subtracting the mean number of spots obtained in the absence of peptide from the mean number of spots obtained in the presence of peptide.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Generation and characterization of Tax MVA
The initial aim of this study was to establish whether there are any murine CTL epitopes in the HTLV-I Tax protein. A method of immunizing mice with Tax that would be optimal for generating CTL responses was required and the well-characterized approach of priming with a DNA expression plasmid and boosting with a recombinant MVA was chosen (Hanke et al., 1998 ; Schneider et al., 1998 ).

The tax gene was cloned into the pSC11 vaccinia virus shuttle plasmid (Chakrabarti et al., 1985 ), which was then used to generate a recombinant MVA vaccinia virus expressing Tax. MVA is a highly attenuated strain of vaccinia virus that can be used as a gene delivery system to immunize mice. After plaque purification and before bulk stocks of Tax MVA were purified, crude preparations were tested for contaminating wild-type virus by staining duplicate plates of cells infected with different dilutions of virus stock with X-Gal or an anti-vaccinia virus antiserum. Roughly the same number of vaccinia virus-positive plaques and blue {beta}-galactosidase-positive plaques (indicating recombinant virus) were obtained at each dilution with both methods of staining, showing that the virus stocks were not contaminated significantly with wild-type virus. After purification, the titres of Tax MVA stocks were found to be in the region of 109 p.f.u./ml.

Flow cytometry showed that approximately 80% of Tax MVA-infected BHK cells stained positively with both the anti-Tax and the anti-B5R vaccinia virus-specific antibodies after overnight infection (Fig. 1a), indicating that the recombinant virus expressed Tax protein and was not contaminated significantly with wild-type virus. Uninfected cells were negative for both these proteins in the immunofluorescence assay (Fig. 1a). Tax expression by Tax MVA was also tested in L-Db mouse fibroblast cells, as these cells were to be used as target cells in cytotoxicity assays. Although L-Db cells are non-permissive for MVA replication, Tax expression was detected in Tax MVA-infected cells by intracellular Tax staining and flow cytometry (Fig. 1b). Tax expression was not detected in L-Db cells infected with wild-type MVA (Fig. 1b).



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Fig. 1. Determination of purity of Tax MVA by intracellular staining of uninfected BHK cells and BHK cells infected with Tax MVA. (a) BHK cells were infected overnight at an m.o.i. of 10 p.f.u. per cell. Tax expression in L-Db cells infected overnight with 10 p.f.u. per cell of wild-type MVA and (b) Tax MVA was also detected. Cells were stained with either Lt4 (anti-Tax) and anti-B5R (anti-vaccinia virus) antibodies (a) or Lt4 alone (b) followed by secondary fluorochrome-conjugated antibodies.

 
Recognition of Tax peptides by H-2k CTLs
Tax MVA was used to boost CBA mice (H-2k haplotype) that had been primed with pJFE-Tax DNA (see Methods). pJFE is an expression plasmid in which cloned genes are under the control of the SR-{alpha} promoter (Elliott et al., 1990 ). Spleen effector cells were restimulated in vitro with one of the four pools of the 13-mer Tax peptides (see Methods) and subsequently tested in a standard 5 h chromium-release assay for lysis of L-Db cells incubated with the same pool of Tax peptides (Fig. 2). Only Tax peptide pool 1 gave specific lysis above background levels in the absence of peptide and there was no recognition of peptides in pools 2, 3 and 4.



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Fig. 2. Recognition of L-Db target cells incubated with (solid lines) and without (dashed lines) Tax peptide pools 1 (a), 2 (b), 3 (c) and 4 (d) (10 µM final concentration of each peptide) by Tax-specific CBA effector cells after one in vitro restimulation with the corresponding Tax peptide pool (20 µM final concentration of each peptide). The percentage of spontaneous lysis was 19%.

 
Tax peptide pool 1 is made up of 20 overlapping 13-mer peptides, numbered 1–20. Spleen effector cells that had been restimulated with peptide pool 1 were then tested for lysis of target cells incubated with the individual peptides comprising pool 1. There was specific lysis of target cells incubated with the individual 13-mer peptides 1, 9 and 10 at an effector:target ratio of 10:1 (Fig. 3). Peptides 9 and 10 overlap by nine amino acid residues and so probably contain a single CTL epitope.



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Fig. 3. Recognition of L-Db target cells incubated with individual peptide pool 1 13-mer Tax peptides (33 µM) by Tax-specific CBA effector cells after four in vitro restimulations with Tax peptide pool 1 (20 µM final concentration of each peptide). Target cells were incubated with no peptide as a negative control and pool 1 (10 µM final concentration of each peptide) as a positive control. The percentage of spontaneous lysis was 13·3% at an effector:target ratio of 20:1.

 
CBA mice express the Kk and Dk MHC class I molecules and to define the precise epitopes recognized by CTLs, the sequences of peptides 1, 9 and 10 were compared with the known Kk peptide-binding motif and the sequences of known Dk epitopes (de Bergeyck et al., 1994 ; Gould et al., 1991 ; Lukacher & Wilson, 1998 ; Rammensee et al., 1999 ; Tourdot et al., 2001 ; Wilson et al., 1999 ) (Table 1). Although a Dk peptide-binding motif derived by sequencing eluted peptides has not been reported, the alignment of known Dk epitopes suggests a well-conserved binding motif for Dk (Table 1). The peptide-binding motifs were used to predict the sequences of possible epitopes within peptides 1, 9 and 10, giving the sequences 1.1, 1.2, 10.1 and 10.2 (Table 2), which were subsequently synthesized. The epitope within peptides 9 and 10 was predicted to be presented by the Dk class I molecule (Table 1). Although peptide 1 does not contain anchor residues in the correct positions for either the Dk or the Kk motifs, peptides 1.1 and 1.2 were the sequences most likely to be epitopes, as they both have a carboxy-terminal leucine residue: a common carboxy-terminal anchor residue. Not all epitopes conform to a consensus binding motif (Apostolopoulos et al., 1997 ; Dethlefs et al., 1997 ; Mata et al., 1998 ).


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Table 1. Alignment of known Dk-restricted epitopes and the Kk-peptide-binding motif

 

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Table 2. Amino acid sequences of synthetic Tax peptides

 
Additional chromium-release assays using CBA spleen effector cells showed that lysis of L-Db target cells incubated with the individual peptide 1 diminished after four restimulations in vitro with peptide pool 1, although there was still efficient lysis of target cells incubated with pool 1 itself (data not shown). Furthermore, target cells incubated with the shorter peptides 1.1 and 1.2 (Table 2) did not show significant specific lysis when incubated with pool 1-restimulated effector cells (data not shown). The combined evidence suggested that peptide 1 contains, at best, a subdominant CTL epitope. Experiments with serial dilutions of the peptides 10, 10.1 and 10.2 (Table 2) in chromium-release assays using pool 1-restimulated effector and L-Db target cells showed that peptide 10.2, amino acid sequence ARLHRHALL, gave efficient lysis at the lowest peptide concentrations, with half-maximal lysis at a concentration of approximately 2 nM (data not shown). The MHC restriction element of this peptide was determined using P1-Dk and P815-Kk cells (H-2d haplotype background, transfected with the Dk or Kk class I molecule, respectively) as target cells in chromium-release assays. As predicted by comparison with the binding motifs (Table 2), the epitope is Dk-restricted, as it was presented by P1-Dk but not P815-Kk cells (data not shown).

Presentation of a Dk-restricted epitope in Tax is enhanced by lactacystin
The CTLs described above were tested for their ability to recognize L-Db and P1-Dk target cells infected with Tax MVA in order to ascertain whether the determinant corresponding to peptide 10.2 is processed from full-length protein and, therefore, represents an authentic CTL epitope. Chromium-release assays demonstrated that there was specific lysis of Tax MVA-infected cells but not of target cells infected with wild-type MVA using both L-Db (Fig. 4) and P1-Dk cells (data not shown), showing that the Dk-restricted epitope is presented in Tax MVA-infected cells.



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Fig. 4. Lysis of L-Db target cells infected overnight in suspension at an m.o.i. of 2 p.f.u. per cell of wild-type MVA ({circ}), Tax MVA ({square}) or Tax MVA+10 µM lactacystin, including pre-treatment with 10 µM lactacystin for 2 h prior to infection ({blacksquare}), by Tax-specific CBA effector cells after seven in vitro restimulations with Tax peptide pool 1 (20 µM final concentration of each peptide). Uninfected target cells were incubated with Tax peptide 12 (33 µM) as a positive control ({bullet}). The percentages of spontaneous lysis were 28·6% for uninfected target cells, 33·5% for wild-type MVA-infected target cells, 30·9% for Tax MVA-infected target cells and 32·3% for Tax MVA+lactacystin-infected target cells. Similar results were seen in six separate assays.

 
The involvement of the proteasome in the processing and presentation of the Dk-restricted Tax epitope was investigated using the proteasome inhibitor lactacystin. Lactacystin is a covalent inhibitor of the proteasome, which has been shown to inhibit the chymotrypsin-like and trypsin-like peptidase activities of the proteasome by modifying the amino-terminal threonine residue of catalytically active {beta}-subunits (Lee & Goldberg, 1998 ). L-Db target cells were incubated with lactacystin at a concentration of 10 µM for 2 h prior to infection with Tax MVA and then during the overnight infection. Surprisingly, presentation of the Dk-restricted epitope was enhanced reproducibly by the lactacystin treatment, as seen from the increased lysis of Tax MVA-infected cells that were treated with lactacystin compared with Tax MVA-infected cells (Fig. 4). There was an approximate doubling of the level of specific lysis of the lactacystin-treated cells at an effector:target ratio of 20:1. Intracellular staining for Tax and flow cytometry showed that this enhanced presentation of the Dk-restricted Tax epitope was not due to an increase in Tax protein expression in lactacystin-treated cells (data not shown). Nor was it due to a non-specific effect of lactacystin, as wild-type MVA-infected target cells treated with 10 µM lactacystin were not lysed by the CTLs (data not shown).

Tax associates with the mouse proteasome
The physical association reported between Tax and the proteasome was demonstrated with human proteasome subunits by yeast two-hybrid screening (Beraud & Greene, 1996 ; Rousset et al., 1996 ). In view of the unexpected effects of the proteasome inhibitor lactacystin, described above, immunoprecipitation assays were carried out to determine whether Tax also interacts physically with the murine proteasome. Immunoprecipitation of lysates of Tax MVA-infected L-Db cells with an anti-proteasome antiserum, followed by Western blotting for Tax (developed with the Lt4 anti-Tax antibody), showed the presence of a band of approximately 40 kDa (Fig. 5, lane 4), which was absent in immunoprecipitates of lysates of wild-type MVA-infected cells (Fig. 5, lane 3). The 40 kDa band corresponds to the size of band obtained in a Western blot of whole cell lysate of Tax MVA-infected cells (Fig. 5, lane 2). Therefore, Tax associates physically with the murine, as well as the human, proteasome, although lactacystin treatment does not inhibit the presentation of Tax to murine CTLs.



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Fig. 5. Immunoprecipitation of Tax and the murine proteasome. Tax MVA-infected cells were lysed and the whole cell lysates or samples immunoprecipitated with an anti-proteasome antibody were fractionated on a 12% polyacrylamide gel and then transferred to a nitrocellulose membrane. The membrane was probed with Lt4 (anti-Tax antibody) (1/10000 dilution of ascites fluid) followed by a secondary horseradish peroxidase-conjugated antibody. Lanes 1 and 2 show whole cell lysate samples, confirming the detection of a 40 kDa Tax band in Tax MVA-infected cells (lane 2) but not in wild-type MVA-infected negative control cells (lane 1). A band of identical size was found in Tax MVA-infected cells immunoprecipitated with the anti-proteasome antibody (lane 4) but not wild-type MVA-infected cells (lane 3).

 
Tax is poorly immunogenic for CTLs in CBA mice
In order to quantify the CTL response to Tax in immunized CBA mice, the ELISpot assay was used to measure the frequencies of spleen effector cells that release IFN-{gamma} upon specific peptide stimulation. CBA mice were primed with pJFE-Tax and boosted with Tax MVA according to the usual immunization regime (see Methods). The frequencies of IFN-{gamma}-secreting cells in two experiments in response to stimulation by peptide 1 were no higher than background (no stimulating peptide) and in response to peptides 10 and 10.2 were only marginally higher than background (Fig. 6).



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Fig. 6. Quantification of the specific CD8+ T-cell response to Tax in CBA mice by IFN-{gamma} ELISpot assay. Freshly isolated spleen cells from Tax-immunized mice were incubated with 2 µM peptide and IFN-{gamma}-secreting T-cells were enumerated. Tax-specific T-cell frequencies were measured in two separate assays (experiments 1 and 2), denoted by filled and open symbols, respectively. Circles represent frequencies measured in response to peptide 1.2 and squares represent frequencies measured in response to peptide 10 in experiment 1 and peptide 10.2 in experiment 2.

 
The highest frequency of IFN-{gamma}-secreting cells measured corresponded to a frequency of no more than 120 per 106 spleen cells. The cells used for experiment 2 (Fig. 6, open symbols) gave measurable specific lysis of L-Db target cells incubated with peptides 10, 10.1 and 10.2 in chromium-release assays (data not shown) and the results shown in Fig. 3 were, in fact, obtained using these effector cells. The cells used for experiment 1 (Fig. 6, filled symbols) and two other sets of effector cells obtained from immunized mice did not lyse target cells incubated with Tax peptides in chromium-release assays (data not shown) and these two sets of effector cells did not secrete IFN-{gamma} in response to stimulation with Tax peptides.

Coupled with the low frequencies of IFN-{gamma}-producing Tax-specific CD8+ T-cells was the poor reproducibility of the generation of Tax-specific CTL responses. CBA mice were immunized in groups of two, three or four animals on 11 separate occasions and only four of these groups of mice produced Tax-specific CTLs that were detectable by chromium-release or ELISpot assays. These observations suggest that, contrary to the case seen in HTLV-I infection of humans, Tax is not very immunogenic for CTLs in mice.


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
Proteasomes are multicatalytic enzyme complexes that are responsible for the turnover of most cellular proteins and also for the generation of most of the peptides presented at the cell surface by MHC class I molecules for recognition by CTLs. An increasing number of viral proteins have been found to interact physically with proteasome components and HTLV-I Tax is one such protein (Rousset et al., 1996 ). Others include the E7 protein of human papillomavirus (Berezutskaya & Bagchi, 1997 ), hepatitis B virus X protein (Huang et al., 1996 ; Zhang et al., 2000 ), human immunodeficiency virus type 1 Nef (Rossi et al., 1997 ) and Tat proteins (Seeger et al., 1997 ) and the E1A protein of adenovirus (Turnell et al., 2000 ). Many of these viral proteins are transcriptional regulators and/or oncoproteins and they may target the proteasome because of its central role in cell-cycle regulation. However, some of these proteins, including Tax, are also major target antigens for CTLs. It is not known whether physical association of a viral protein with proteasomes affects its immunogenicity for CTLs. On the one hand, targeting of a protein to proteasomes may increase its turnover and presentation to CTLs, but, on the other hand, binding of a protein to the outside of proteasomes could protect it from proteasome-mediated degradation.

We wished to investigate the effects of association of HTLV-I Tax with the proteasome on its immunogenicity for CTLs using a mouse model. In this report, we have identified a murine CTL epitope in Tax, quantified the response specific for this epitope, demonstrated that Tax does associate physically with proteasomes in murine cells and tested the effects of the proteasome inhibitor lactacystin on the presentation of the Tax CTL epitope. Although it is well established that Tax is highly immunogenic for CTLs in humans (Bangham et al., 1999 ; Kannagi et al., 1991 ; Jacobson et al., 1990 ; Kannagi et al., 1984 ), there have been no reports of CTL responses to Tax in mice; a single report suggested that Tax was not immunogenic for CTLs in a rat system (Tanaka et al., 1991 ).

Using a DNA-prime, MVA-boost immunization protocol, we have defined a new H-2Dk-restricted epitope in Tax, amino acid residues 38–46, sequence ARLHRHALL. The amino acid sequence of this epitope shows similarity to other reported Dk-restricted epitopes (Table 1) and contains the Dk-binding motif, in this case consisting of arginine residues at positions 2 and 5 and leucine at the carboxy-terminal end of a 9-mer peptide. We reasoned that if physical association of Tax with the proteasome enhances its degradation and presentation to CTL, proteasome inhibitors would be expected to reduce presentation of the Dk-restricted epitope. However, presentation of this epitope by Tax MVA-infected cells was found to be enhanced markedly by the proteasome inhibitor lactacystin, suggesting that normal proteasome activity is not required for the generation of this epitope. A similar enhancement of presentation of certain CTL epitopes by proteasome inhibitors has been reported previously (Anton et al., 1998 ; Luckey et al., 1998 ; Schwarz et al., 2000 ) and is dependent not only on the epitope studied but also on the concentration of inhibitor used (Anton et al., 1998 ; Schwarz et al., 2000 ). Schwarz et al. (2000) reported that presentation of the H-2Db-restricted lymphocytic choriomeningitis virus-derived epitope GP276 was enhanced at low concentrations of the proteasome inhibitors lactacystin or epoxomicin but abrogated at high concentrations of these inhibitors. A low concentration of lactacystin was defined to be between 0·5 and 1 µM. Anton et al. (1998) found that presentation of the Kk-restricted influenza virus nucleoprotein 50–57 epitope, which was barely affected at 10 µM lactacystin, was reduced at a concentration of 100 µM lactacystin. However, they also found that cell surface expression of recombinant vaccinia virus-encoded genes was reduced at 100 µM but not at 10 µM lactacystin, suggesting that high concentrations of lactacystin may have non-specific effects. In our experiments, 10 µM lactacystin was used, as this concentration has been shown to inhibit the presentation of several different CTL epitopes (Anton et al., 1998 ; Cerundolo et al., 1997 ) while having minimal secondary effects. Proteasomes may still be responsible for the proteolytic processing of the Dk-restricted Tax epitope because lactacystin inhibits the chymotrypsin-like activity of the proteasome more strongly than other catalytic activities and modifies preferentially the amino-terminal threonine residue of the {beta}-type subunit X ({beta}5) (Dick et al., 1996 ; Fenteany et al., 1995 ; Groll et al., 1997 ). Therefore, cells treated with lactacystin would be expected to retain some proteasome activity.

There are several different possible explanations for the enhancement of CTL epitope processing and presentation by proteasome inhibitors. As the majority of MHC class I ligands are generated by proteasomes, epitopes that are processed in a proteasome-independent manner would experience reduced competition for assembly with MHC class I molecules in the presence of proteasome inhibitors (Anton et al., 1998 ). This could be a particularly important factor in the presentation of peptides with a low affinity for MHC class I molecules. However, as yet there is no good evidence for completely proteasome-independent epitope processing of TAP-dependent epitopes. A more likely explanation is that one or more of the proteolytic activities of the proteasome actually destroys certain CTL epitopes. Luckey et al. (1998) have obtained some evidence for proteasomal destruction of the influenza A virus HLA-A*0201-restricted M158–66 epitope, an epitope whose presentation to CTLs was enhanced by 10 µM lactacystin. Interestingly, the level of cell surface expression of the Dk class I molecule on mouse L cells has been reported to increase by 50% after incubation with 10 µM lactacystin for 13 h (Vinitsky et al., 1997 ). This effect is apparently not due to inhibition of Dk turnover (Vinitsky et al., 1997 ) and so may help to account for the increased presentation of the Dk-restricted Tax epitope after lactacystin treatment. However, it is not the case that all Dk-restricted CTL epitopes show enhanced presentation after lactacystin treatment because presentation of the Dk-restricted epitope in the influenza virus PB1 protein is inhibited by lactacystin (Vinitsky et al., 1997 ; K. Gould, unpublished).

The evidence for Tax association with human proteasome subunits was obtained by yeast two-hybrid screening (Rousset et al., 1996 ) and we wished to confirm this association under more physiological conditions and using murine proteasomes. Immunoprecipitation of proteasomes from cells expressing Tax, followed by Western blotting for Tax, showed clearly that a significant proportion of Tax protein associates physically with proteasomes in mouse cells (Fig. 6). The results of the lactacystin experiments suggest that this association is not important for Tax CTL epitope generation but experiments with mutant forms of Tax that do not associate with the proteasome will be required to give a definitive answer to this question.

The probable function of Tax association with proteasomes is to influence the processing of the cytoplasmic NF-{kappa}B precursor complex and subsequent activation of the NF-{kappa}B transcription factor. Both p105, the inactive precursor of the NF-{kappa}B subunit p50, and a cytoplasmic inhibitor of NF-{kappa}B, I{kappa}B{alpha}, are processed by the proteasome (Palombella et al., 1994 ). Tax has been shown to associate physically with several members of the cytoplasmic NF-{kappa}B precursor complex, including p105 and I{kappa}B{alpha}. By associating with both the proteasome and the NF-{kappa}B precursor complex, Tax may be able to facilitate the processing of the NF-{kappa}B precursor complex and activation of NF-{kappa}B, which in turn activates the transcription of several genes, including IL-2R{alpha}, which causes proliferation of the infected cell.

The ELISpot assay results reported in this study suggest that Tax is not very immunogenic for CTLs in mice. Other reports using the same DNA-prime, MVA-boost immunization protocol have detected far greater CTL responses to different antigens (Schneider et al., 1998 ). The particular DNA expression plasmid used may have influenced the immunogenicity of Tax and this could be tested by direct comparison with a known dominant antigen for murine CTLs, such as the influenza virus nucleoprotein, using exactly the same plasmid vector and immunization protocol. Although Tax-specific CTLs were generated in CBA mice, roughly half of the immunizations failed to raise detectable anti-Tax CTLs (data not shown). Overall, our results suggest that the immunodominance of Tax in human CTL responses is not due to an inherent property of the protein itself, such as enhanced processing and presentation, because of targeting to proteasomes. It is possible that processing of Tax generates peptides that bind with high affinity to human MHC class I molecules and lead to immunodominant CTL responses, whereas the processed Tax peptide repertoire includes only sequences that bind to murine MHC class I molecules with low affinity and lead to poor CTL responses. This could be investigated by priming HLA-A2 transgenic mice and measuring the efficiency of the HLA-A2-restricted Tax11–19-specific CTL response, which is immunodominant in humans. Alternatively, the immunodominance of Tax in humans may be due to the temporal sequence of viral protein expression in natural HTLV-I infection because Tax is probably the first protein to be expressed (Hidaka et al., 1988 ).


   Acknowledgments
 
The authors would like to thank Geoff Smith for the anti-B5R antibody and wild-type MVA, John Williamson for the anti-vaccinia virus antisera, Jennifer Rivett for the pAb186 antibody, Sarah Gilbert for the BHK and BHK-TK- cell lines, Alain Townsend for the L-Db cell line, Cetus Corp for the generous gift of recombinant IL-2 and the Wellcome Trust for support.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 1 August 2001; accepted 2 November 2001.