Department of Immunology, Imperial College School of Medicine (St Marys 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
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Abstract |
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Introduction |
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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-
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.
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Methods |
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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 Dulbeccos modified essential medium (DMEM). BHK-TK- cells were cultured in DMEM containing 25 µg/ml BrdU.
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.
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.
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).
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.
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.
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.
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.
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 TrisHCl 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 ASepharose (Sigma) overnight at 4 °C and incubated with anti-proteasome antiserum at 4 °C for 90 min. Protein ASepharose 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% SDSpolyacrylamide 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).
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-
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-
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.
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Results |
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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
-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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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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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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Coupled with the low frequencies of IFN--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.
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Discussion |
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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 3846, 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 5057 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
-type subunit X (
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 M15866 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-B precursor complex and subsequent activation of the NF-
B transcription factor. Both p105, the inactive precursor of the NF-
B subunit p50, and a cytoplasmic inhibitor of NF-
B, I
B
, are processed by the proteasome (Palombella et al., 1994
). Tax has been shown to associate physically with several members of the cytoplasmic NF-
B precursor complex, including p105 and I
B
. By associating with both the proteasome and the NF-
B precursor complex, Tax may be able to facilitate the processing of the NF-
B precursor complex and activation of NF-
B, which in turn activates the transcription of several genes, including IL-2R
, 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 Tax1119-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
).
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Acknowledgments |
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Received 1 August 2001;
accepted 2 November 2001.