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1 Institute of Virology, Johannes Gutenberg-Universität, Mainz, Germany
2 Department of Hematology and Oncology, Johannes Gutenberg-Universität, Mainz, Germany
3 Max von Pettenkofer Institut, Department of Virology, Ludwig-Maximilians-Universität München, Germany
Correspondence
Bodo Plachter
plachter{at}uni-mainz.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Novartis Pharma AG, Werk Klybeck, Klybeckerstrasse 141, CH-4057 Basel, Switzerland. 
Present address: Fred Hutchinson Cancer Research, 1100 Fairview Avenue N., Seattle, WA 98109-1024, USA.
Present address: Bioscientia Hamburg, Papenreye 63, D-22453 Hamburg, Germany.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), and in particular by HMCV-specific CD8+ cytoxic T lymphocytes (CTL) (Reddehase et al., 1985; Reusser et al., 1991). The CTL response is primed against short peptides that are proteolytically processed from intracellular viral proteins and presented on the cell surface by major histocompatibility complex (MHC) class I molecules (Kloetzel, 2004). For this, proteins are labelled by ubiquitination to be targeted to proteasomes for degradation. Peptides of appropriate length are released from proteasomes and processed further at their amino terminus by cytosolic aminopeptidases. Ultimately they are translocated to the endoplasmic reticulum (ER) by the transporter associated with antigen presentation (TAP), where they associate with MHC class I. In some instances, ER-resident proteases contribute to the trimming of antigenic peptides (reviewed by Kloetzel, 2004).
The HCMV genome encodes over 150 proteins, which can potentially serve as target antigens for CTL (Chee et al., 1990). Recent studies showed that memory CTL isolated from HCMV-seropositive individuals display a broad spectrum of target specificity (Elkington et al., 2003; Khan et al., 2005; Sylwester et al., 2005). However, the virus also encodes proteins that interfere with the assembly and transport of MHC class I–peptide complexes and reduce class I surface expression (reviewed by Plachter, 1999; Reddehase 2000, 2002; Mocarski, 2004). The glycoprotein US3 (gpUS3) is expressed immediately after infection and prevents peptide loading of MHC class I complexes in the ER (Ahn et al., 1996; Jones et al., 1996; Park et al., 2004, 2006). The viral early glycoproteins US2 (gpUS2) and US11 (gpUS11) mediate retrograde translocation of MHC class I heavy chains from the ER to the cytoplasm, leading to their subsequent proteasomal degradation (Wiertz et al., 1996a, b). Finally, the early-late glycoprotein US6 (gpUS6) leads to a block in peptide transport from the cytosol to the ER through the TAP, thereby preventing the formation of MHC class I–peptide complexes in the ER (Ahn et al., 1997; Hengel et al., 1997; Lehner et al., 1997; Halenius et al., 2006). The question therefore arises as to how HCMV-specific CTL can recognize infected cells expressing the gpUS2–11 immunoevasion proteins.
Adoptive transfer of HCMV-specific CTL has been used successfully to prevent viral reactivation and disease in allogeneic bone marrow and haematopoietic stem-cell transplant recipients (Walter et al., 1995; Rauser et al., 2004; Cobbold et al., 2005). These and additional studies have provided circumstantial evidence that pp65-specific CTL are protective (Riddell et al., 1992; Walter et al., 1995). Therefore, pp65-specific CTL appear to recognize infected cells in the face of HCMV-mediated immunoevasion. In murine cytomegalovirus (MCMV), CTL against the immediate-early protein 1 (IE1) were shown to be protective after adoptive transfer (Reddehase et al., 1987; Pahl-Seibert et al., 2005) and the importance of IE1-specific CTL has recently been confirmed for HCMV (Kern et al., 1999; Bunde et al., 2005).
Considering the medical importance of the immunological mechanisms that lead to protection in HCMV infection, we decided to analyse processing and presentation of pp65 with CTL clones generated in HLA-A2 (human leukocyte antigen A2) transgenic mice. Using these CTL clones, it could be shown that presentation of pp65 by HLA-A2 was only partially impaired by gpUS2–11-mediated immunoevasion. Surprisingly, labelling of infected cells with pp65-derived peptides at later times after infection was only in part caused by stable MHC–peptide complexes on the cell surface. Rather, the presentation of pp65-derived peptides appeared to be the result of continuous translocation of peptide-loaded MHC complexes to the cell surface despite expression of immunoevasion proteins within the cell. pp65 is therefore the first example of a viral protein that is incompletely subjected to HCMV-mediated immunoevasion of MHC class I antigen presentation.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-mercaptoethanol.
CTL lines specific for the HLA-A0201 (A2)-restricted HCMV-derived peptides pp65495–503 (pp65NLV-CTL) (Wills et al., 1996; Diamond et al., 1997) and IE1297–305 (IE1TMY-CTL) (Gallez-Hawkins et al., 2003) were generated by immunizing HLA-A2/CD8 double-transgenic (tg) mice (a kind gift of L. Sherman, The Scripps Institute, La Jolla, CA, USA) with 100 µg peptide, suspended in incomplete Freund’s adjuvant. Ten days later, spleen cells of primed mice were cultured with irradiated A2-tg lipopolysaccharide-activated spleen cell stimulators that had been pulsed with the respective peptides at 5 µg ml–1 in supplement-free RPMI 1640 medium. CTL clones were established by limiting dilution and were cultured as previously described (Theobald et al., 1995).
Viruses and plasmids.
Various virus strains were used for infection experiments at an m.o.i. of 5, unless otherwise noted. For infections with viruses expressing both the US2–11 and the UL83 (pp65) gene regions, either the HCMV laboratory strain Ad169 or a bacterial artificial chromosome (BAC) derivative of that strain (AD169-BAC) were used (Hobom et al., 2000). For control, the US2–11 deletion mutant RV
US2–11 was employed (Falk et al., 2002). In addition, a pp65 deletion mutant, based on the AD169-BAC, was constructed using Red recombination in Escherichia coli strain EL250 as described by Lee et al. (2001). A kanamycin resistance gene flanked by FRT (FLP recognition target) sites was amplified from a derivative vector of pCP15 (Cherepanov & Wackernagel, 1995) using primers with 50 bp identity to the nucleotide sequence directly adjacent to the deletion (pp65-frt_fwd: 5'-GCTGCCGCACGAGACGCGACTCCTGCAGACGGGTATCCACGTACGCGTGAGGTACCCGGGGATCTTG-3', pp65-frt_rev: 5'-GGACGTGGGTTTTTATAGAGTCGTCCTAAGCGCGTGCGGCGGGTGGCTCATCGAGAAACGCAGCTTC-3'). In the resulting BAC clone pHd65, the complete coding sequence of pp65 was deleted except for 150 bp at the 5' end of UL83 and the stop codon. The reconstitution of BAC-vector-free mutant virus RVHd65 was carried out as described by Hobom et al. (2000) for AD169-BAC.
Dense body purification.
Dense bodies (DB) were purified from late-stage infected HFF by glycerol–tartrate gradient ultracentrifugation as originally published (Irmiere & Gibson, 1983) and described previously (Pepperl et al., 2000).
Gamma interferon (IFN-
)-based ELISPOT assays.
ELISPOT assays were performed according to published procedures (Miyahira et al., 1995; Frankenberg et al., 2002). The pp65NLV-CTL and IE1TMY-CTL were used as responder cells. HFF or T2 cells were used as stimulator cells at a number of 105 per assay. For inhibition of proteasome function, 10 µM lactacystin or 1 µM epoxomicin (both from Calbiochem) were used. Counting of spots was performed using an SZX-12 microscope (Olympus).
Cytotoxicity assays.
Functional consequences on antigen presentation by HLA-A2 were monitored using standard 5 h 51Cr release assays (CRA) with 5x103 target cells; pp65NLV-CTL were used as effector cells. Cells were employed at different effector to target ratios as indicated. Data represent percentages of specific lysis and are given as the mean value of duplicate cultures.
Peptide stripping by acid wash.
To remove MHC class I-bound peptides from the surface of infected HFF, cells were treated with citrate phosphate buffer, pH 3 (0.263 M citric acid and 0.123 M disodium phosphate), modified according to Sugawara et al. (1987). Fibroblasts grown in 10 mm dishes were washed twice with PBS and were then treated for 1 min with 2 ml citrate phosphate buffer, pH 3, at room temperature. Afterwards cells were washed twice with supplement-free MEM medium and once with MEM medium containing 10 % FCS, 2 mM L-glutamine, 50 mg gentamicin l–1 and 0.5 ng bFGF ml–1. Subsequently, cells were used for fluorescence-activated cell-sorting (FACS) or IFN--ELISPOT analysis or were further incubated to be analysed at a later time point.
Fixation of stimulator cells.
After washing with PBS, stimulator cells were resuspended in 200 µl 0.5 % paraformaldehyde and incubated for 30 min at room temperature. Cells were subsequently washed with PBS and RPMI 1640 medium, supplemented with 10 % FCS, 2 mM L-glutamine, 50 mg gentamicin l–1 and 5 µM -mercaptoethanol, and were then resuspended in an appropriate volume of RPMI 1640 medium, supplemented as mentioned before.
Cell viability assay.
To determine the number of viable cells after treatment with proteasome inhibitors, a CellTiter 96 AQueous One Solution cell proliferation assay kit (Promega) was used. The absorbance was measured at 492 nm in a 96-well plate reader.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Diamond et al., 1997; Gillespie et al., 2000; Gallez-Hawkins et al., 2003). To provide a reproducible experimental setting, murine CTL clones rather than human CTL lines were generated in HLA-A2 transgenic mice. The resulting CTL clones pp65NLV-CTL and IE1TMY-CTL were tested for their specificity in IFN--ELISPOT assays (Fig. 1). Firstly, T2 cells were labelled with both peptides. Both CTL clones were stimulated by their cognate MHC–peptide complex and failed to recognize cells labelled with the mismatched peptide (Fig. 1a). Secondly, pp65NLV-CTL were tested for their recognition of HLA-A2-positive HFF that were infected either with the laboratory strain Ad169 (AD169-BAC) or with a pp65-deletion mutant (RVHd65; Fig. 1b). Only those cells infected with the pp65-expressing strain Ad169 stimulated IFN- secretion. To test for MHC restriction of the CTL clones, HLA-A2-positive and HLA-A2-negative cells were infected and used as stimulators. Infection was performed with a US2–11-negative HCMV strain (RVUS2–11) to avoid suppression of antigen presentation. Only HLA-A2-expressing cells stimulated IFN- secretion by both pp65NLV-CTL and IE1TMY-CTL (Fig. 1c). No detectable stimulation was triggered by infected HLA-A2-negative or by mock-infected cells. This demonstrated that the transgenic CTL-clones were both antigen- and HLA-A2-specific.
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). MRC-5 human fibroblasts were infected with the HCMV laboratory strain Ad169 and were subjected to CRA (Fig. 2a). MRC-5 cells that had been infected for 16 h proved to be good targets for pp65NLV-CTL. Since only low-level de novo expression of pp65 can be expected to occur at this time p.i. (Depto & Stenberg, 1989), the pp65NLV peptide must be primarily derived from virion-associated pp65. In contrast, 96 h p.i., cells were no longer lysed by pp65NLV-CTL. These results matched the downregulation of MHC class I molecules on infected cells at this time p.i. (Fig. 2b). To test whether there might be a stimulation of the CTL by cells 96 h p.i. which did not result in killing, IFN--ELISPOT analyses were carried out. As expected, MRC-5 cells 16 h p.i. stimulated pp65NLV-CTL significantly (Fig. 2c). In contrast to CRA, some spots also appeared when 96 h p.i. cells were used for stimulation. This indicates that, in the face of immunoevasion, there are still sufficient HLA-A2-pp65NLV complexes on the surface of fibroblasts 96 h p.i. to stimulate IFN- production in CTL.
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). It remained unclear whether the tegument protein was processed by the proteasome or in a proteasome-independent fashion (Gil-Torregrosa et al., 2000; Del-Val & Lopez, 2002). We found that processing of the pp65NLV after de novo synthesis in cells transfected with a pp65 expression plasmid was sensitive to proteasomal inhibition (data not shown). To investigate whether particle-associated pp65 is processed by the same or an alternative processing pathway, HFF were incubated with DB (30 µg per 5x105 HFF) from strain RVUS2–11 for 12 h while treated with proteasomal inhibitors. Cells were then fixed with paraformaldehyde to prevent further processing and subjected to IFN--ELISPOT analyses using pp65NLV-CTL as responder cells (Fig. 3a). Treatment of DB-loaded cells with lactacystin or epoxomicin resulted in marked reduction of spot numbers, demonstrating that processing of particle-associated pp65 was indeed proteasome dependent. To verify that MHC class I presentation of de novo-synthesized pp65 resulting from residual infectious virus in the DB preparation was excluded, parallel cultures were tested with IE1TMY-CTL as responders. The expression of IE1 precedes that of pp65 during infection. No significant numbers of spots were observed (Fig. 3b). Cell viability subsequent to proteasome inhibitor treatment and DB exposure was found to be unaffected (Fig. 3c). Furthermore, for control of the impact of fixation on antigen presentation, cells were also labelled with peptide, fixed with paraformaldehyde and used as stimulators in IFN--ELISPOT analysis. Both pp65NLV-CTL and IE1TMY-CTL were stimulated by peptide-labelled cells that were fixed with paraformaldehyde (data not shown).
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The pp65-derived peptide is presented by MHC class I despite gpUS2–11 expression
Recently, HCMV mutants lacking the US2–11 genes have been used to investigate the impact of immunoevasion proteins on MHC class I presentation during infection (Manley et al., 2004; Khan et al., 2005). We used the US2–11 deletion mutant RVUS2–11 in comparison with an evasion-competent strain (AD169-BAC) in IFN--ELISPOT analysis to test for the kinetics of MHC class I presentation of pp65NLV. Infection of HFF with the US2–11-competent strain resulted in a gradual decrease in spot numbers over time p.i., using pp65NLV-CTL as responder cells. In contrast, infection with RVUS2–11 resulted in stimulation of IFN- secretion by pp65NLV-CTL at a comparable level at all time points p.i. tested (Fig. 4a). Thus, the deletion of the US2–11 genes completely restored pp65NLV recognition by CTL, indicating that the impairment of pp65-derived peptide presentation was mediated through immunoevasion proteins. To analyse whether this time frame of MHC class I–peptide presentation was specific to pp65 or was intrinsic to our test system, we repeated the experiment using IE1TMY-CTL. In accordance with what had been reported before by others using human IE1-specific CTL lines (Manley et al., 2004; Khan et al., 2005), IE1TMY-CTL were not stimulated by Ad169-infected fibroblasts at any time p.i. tested (Fig. 4b). In contrast, RVUS2–11-infected fibroblasts were excellent stimulators for IFN- production by IE1TMY-CTL. Both pp65NLV-CTL and IE1TMY-CTL induced comparable numbers of spots when incubated with the evasion-negative strain, indicating comparable sensitivity of the CTL clones in this assay. These experiments showed that expression of US2–11 immunoevasion proteins incompletely blocks MHC class I presentation of pp65-derived peptides.
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). The capacity of these cells to restore pp65NLV presentation was analysed by IFN--ELISPOT. In a first control experiment, it could be shown that removal of MHC class I was complete immediately after acid treatment, and restoration of MHC class I to the level of untreated cells was entirely accomplished 24 h later, as shown by FACS analysis (Fig. 5a). To ensure the complete removal of functional MHC class I–peptide complexes from the cell surface by acid treatment, HFF were infected for 48 h with the US2–11-negative strain RVUS2–11. Cells were fixed using paraformaldehyde, or acid-stripped and subsequently fixed to prevent further processing and MHC translocation. Fixed cells were good CTL targets (Fig. 5b). Acid treatment and paraformaldehyde fixation, however, completely abrogated CTL recognition, demonstrating the efficiency of MHC class I stripping. To test whether the restoration of MHC class I surface expression correlated with the capacity of the cells to present pp65NLV, HFF were infected for 24, 48 and 72 h, respectively, with RVUS2–11 (Fig. 5c left panel). Subsequently, cells were either left untreated (control, dark grey bars) or were stripped with acid (light grey bars). In both cases, cells were incubated for another 24 h prior to ELISPOT analysis. The pp65-peptide presentation could be fully restored to the level of untreated cells in each 24 h period after acid wash. This demonstrated that peptide loading and surface translocation of MHC class I–pp65NLV complexes was efficient subsequent to acid wash and was not a limiting step in the chosen experimental setting.
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, right panel). However, in repeated experiments, acid washes at 24 h resulted in a more than half-maximal restoration of antigen presentation during the following 24 h. Even when acid washes were performed at 48 h p.i., there was restoration of presentation, yet failing to reach the level of untreated cells. pp65NLV presentation was completely abrogated when the acid washes were carried out at 72 h p.i. For negative control, a parallel experiment was conducted with a pp65-negative strain (RVHd65). No recognition of cells infected with this strain was detectable at any of the time points tested (Fig. 5c).
These experiments showed that pp65-derived peptide presentation could be restored in early- and late-infected fibroblasts despite the expression of gpUS2–11 and that this process was, at least in part, due to continuing translocation of MHC class I–pp65NLV complexes to the cell surface. It should be emphasized at this point that IE1TMY presentation was not detectable on HFF infected with an US2–11-competent virus, showing that immunoevasion was fully operative (see Fig. 4). Taken together, these results demonstrated that immunoevasion is only partly effective in preventing pp65NLV presentation.
De novo generation and MHC class I loading of pp65-derived peptides in the face of immunoevasion
One possible explanation for the presentation of pp65NLV in the face of immunoevasion was that pp65 degradation, MHC class I peptide loading of pp65NLV and the translocation of the complex to the cell surface were continuing despite gpUS2–11 expression. Alternatively, recycling of preformed complexes to the cell surface could occur despite immunoevasion. To be able to distinguish between these alternatives, we tested whether restoration of pp65NLV presentation after acid wash was dependent on the functionality of the proteasome. Proteasome functionality would be required for de novo processing of pp65NLV from pp65, but not for recycling of preformed peptides. Cells were infected for 24 h at an m.o.i. of 10 with the US2–11-competent strain AD169-BAC. Subsequently, they were acid-washed and analysed in a 20 h IFN--ELISPOT assay (Fig. 6). During the assay period, cells were either mock treated (with DMSO) or treated with the proteasomal inhibitors lactacystin or epoxomicin. Inhibition of proteasomal function almost completely abrogated pp65NLV presentation. To control for the impact of proteasomal inhibitors on CTL function, HFF were labelled with pp65NLV, fixed with paraformaldehyde and tested with pp65NLV-CTL in IFN--ELISPOT analysis. The CTL were only partially influenced by proteasomal inhibitors. This could not explain the significant reduction of spots seen after treatment of infected cells with these inhibitors. Furthermore, target cell viability was controlled using a commercial proliferation assay (data not shown). Taken together, these experiments showed that pp65NLV can be proteasomally processed, loaded on MHC class I and translocated to the cell surface despite the expression of the US2–11 genes of HCMV.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). pp65 appears to be exceptional, being an immunodominant antigen that mediates protective immunity against HCMV (reviewed by Reddehase, 2000; Wills et al., 2006). However, this contrasts with in vitro findings, demonstrating the efficacy of HCMV immunoevasion proteins (Barnes & Grundy, 1992; Jones et al., 1995; Ahn et al., 1996; Reddehase, 2002). From these investigations, the key question that arose was how infected cells could present pp65-derived peptides despite immunoevasion, thereby providing the basis for immune recognition and protection mediated by pp65-specific CTL.
Early studies provided evidence that pp65-specific CTL could lyse infected cells from 2 to 66 h p.i. (McLaughlin-Taylor et al., 1994). Here we extended previous findings by showing that (i) pp65-derived peptides are presented by MHC class I in the course of infection, yet to a decreasing extent; (ii) this decrease is completely abrogated by preventing gpUS2–11 expression, indicating that the effect was mediated by these immunoevasion proteins; and (iii) presentation of pp65-derived peptides at early and late times after infection is, at least in part, the result of continuing proteasomal processing and MHC class I loading despite immunoevasion.
MHC class I presentation of pp65-derived peptides can be observed as early as 2 h p.i. (McLaughlin-Taylor et al., 1994) and continues up to 96 h p.i. (this study). However, the capability of cells to present these peptides declined over time. This decrease was shown to be dependent on expression of the US2–11 gene region. It remains to be determined which of the immunoevasion proteins (gpUS2, gpUS3, gpUS6 or gpUS11), or which combination of them, is responsible for the enhanced suppression of pp65-derived peptide presentation by MHC class I over time. In contrast to pp65, we found that presentation of the IE1 peptide was completely suppressed by US2–11 expression, thereby confirming results from previous studies by others (Manley et al., 2004; Khan et al., 2005).
One possible explanation for the presentation of pp65-derived peptide persisting during productive infection could be attributed to the expression of this gene preceding immunoevasion gene expression. However, transcriptional analyses indicate that UL83 mRNA synthesis is restricted to the early and late phases of virus replication (Depto & Stenberg, 1989). Consequently, pp65 is synthesized de novo within infected cells only when immunoevasion proteins are present.
As infectious virions contain a significant amount of pp65 (Varnum et al., 2004), particle-associated pp65 may be introduced into the MHC class I presentation pathway before immunoevasion become operative (McLaughlin-Taylor et al., 1994; Pepperl et al., 2000; Pepperl-Klindworth et al., 2003). Virion-associated pp65 could potentially sensitize cells for a prolonged period of time. The half-lives (t1/2) of peptide-loaded MHC class I complexes vary considerably, ranging up to over 30 h. Affinity to MHC class I has been found to be one crucial parameter for immunogenicity of a given peptide (Yu et al., 2004; Kambayashi et al., 2004). pp65NLV binds with high affinity to HLA-A2 (Frankenberg et al., 2002). Thus it was theoretically possible that particle-derived pp65NLV was presented throughout infection, while further processing and presentation were inhibited by immunoevasion. However, acid stripping of surface peptide could only transiently prevent pp65NLV presentation, indicating that peptide-loaded MHC class I complexes almost completely replenished the pool that had been removed from the surface. This argues against a significant role for the persistence of stable MHC class I–pp65NLV complexes in the recognition of early and late infected cells by CTL.
However, the results cannot discriminate between whether MHC class I presentation of pp65NLV at later stages of infection was due to prolonged processing and presentation of particle-derived pp65 or de novo synthesis of the tegument protein. Particle-associated pp65 appears to be efficiently degraded as evidenced by the rapid loss of staining signal following the exposure of HFF to large amounts of subviral DB (data not shown). It remains questionable, at least, whether particle-associated pp65 could be a relevant source of antigen at early and late times when the UL83 gene is highly active and newly synthesized pp65 is accumulating. Using proteasome inhibition, processing of both particle-derived and de novo-synthesised pp65 was observed to be proteasome-dependent. Consequently, following infection, particle-associated pp65 would serve as a source for proteasomal processing and MHC class I loading, whilst, at later stages, the large amounts of de novo-synthesized pp65 provide the most abundant potential source of antigenic peptides.
Recycling of MHC class I molecules from the cell surface has been reported (Jondal et al., 1996; Gromme et al., 1999). Reappearance of pp65NLV presentation after acid stripping could have been explained by such a recycling mechanism of peptide-loaded complexes. However, inhibition of proteasomal function abrogated pp65NLV presentation following acid stripping. Therefore, it appears that pp65 degradation by the proteasome and subsequent de novo formation of MHC class I–peptide complexes is a prerequisite for pp65NLV presentation in the face of immunoevasion.
Several studies have recently emphasized the diversity of the CTL response against HCMV with respect to target antigens (Elkington et al., 2003; Manley et al., 2004; Khan et al., 2005; Sylwester et al., 2005). Cross-presentation by professional antigen-presenting cells (pAPC) rather than direct presentation of viral antigens has been suggested to explain how CTL could be primed against HCMV proteins which are not presented by infected cells (Sigal et al., 1999; Arrode et al., 2000). This may also explain the high frequencies of IE1-specific CTL in some seropositive donors (Kern et al., 1999; Khan et al., 2002). However, it could not be the reason for the predominance of IE1-specific CTL found in some of the donors as cross-priming of IE1 and pp65, synthesized in permissively infected cells, should be equally effective for both antigens. Recent data from the MCMV model, however, may provide a rationale to understand this phenomenon. Simon et al. (2006) showed that CD8+ T cells specific for an antigenic peptide from the MCMV IE1 protein rapidly sense and terminate transcriptional reactivation from viral latency in the lungs. Consequently, downstream early or late gene expression is prevented and antigenic peptides originating from these genes are not presented. Similar transcriptional reactivation events may occur in humans, allowing for frequent restimulation of pre-existing IE1-specific CTL. Depending on the HLA haplotype of the individual, IE1-derived peptides may be immunodominant and consequently may promote efficient CTL restimulation. Such CTL may then predominate in frequency over pp65-specific CTL, which may require complete reactivation to virus replication for restimulation.
Both pp65 and IE1 have been labelled as immunodominant CTL antigens, based on CD8+ T-cell frequencies in seropositive donors (Wills et al., 2006). However, in the MCMV model, CTL against an immunodominant peptide from the M45 protein failed to protect against infection after adoptive transfer (Holtappels et al., 2004). Consequently, the protective function of CTL against IE1 and pp65 of HCMV cannot be solely deduced from cell frequencies. Yet, both for vaccine development as well as for the design of immunointerventive strategies, it appears important to discriminate between ‘protective antigens’ and ‘non-protective antigens’. Adoptive transfer experiments have provided circumstantial evidence that pp65-specific CTL are protective in vivo (Walter et al., 1995; Cobbold et al., 2005). This work shows, for the first time, that loading and cell-surface transport of MHC class I–peptide complexes derived from HCMV antigens can occur in infected cells despite immunoevasion. This was even true in the absence of IFN-, known to compensate for the suppression of MHC surface expression by immunoevasion proteins (Benz & Hengel, 2000). Although at this stage it cannot be discriminated whether resistance to immunoevasion was intrinsic to the pp65-derived peptide or whether other structural or non-structural proteins could also evade evasion, the findings may serve as a rationale for future studies on the role of individual HCMV proteins as CTL antigens in the course of immune protection against disease.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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