Laboratory of Vaccine Research1, Laboratory of Organic Analytical Chemistry2, Laboratory of Pathology and Immunobiology3, National Institute of Public Health and the Environment, PO Box 1, 3720 BA Bilthoven, The Netherlands
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands4
Author for correspondence: Carla Herberts. Fax +31 30 274 4429. e-mail carla.herberts{at}rivm.nl
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Abstract |
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Introduction |
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We then asked which processing events were underlying such successful epitope generation, especially since for cell membrane-targeted proteins, such as MV-F, no general MHC class I processing pathway has been described. The classic MHC class I processing pathway used by proteins present in the cytosol is relatively well understood. Key events are cytosolic degradation by proteasomes, transport of the proteolytic fragments from the cytosol into the ER by the transporter associated with antigen presentation (TAP) and peptide binding to nascent MHC class I molecules in the ER followed by exocytosis of the peptideMHC class I complexes to the cell membrane. CTL epitopes derived from transmembrane proteins have been described to exploit this classic processing pathway, requiring functional proteasomes and/or TAP for presentation (Ferris et al., 1996 ; Hammond et al., 1995
; Hombach et al., 1995
; Mosse et al., 1998
). These transmembrane proteins might gain access to the cytosol as a result of faulty synthesis on free ribosomes (Hammond et al., 1995
) or following an ER-associated degradation route involving retrograde transport of unproperly synthesized or folded proteins back into the cytosol (Ward et al., 1995
; Wiertz et al., 1996
). Alternatively, proteasomal- and TAP-independent MHC class I presentation pathways for transmembrane proteins are described. Alternative compartments for processing of the parental protein have been suggested, for example the ER itself (Hammond et al., 1995
) or endolysosomes, through which cell surface expressed MHC class I molecules can recycle and where peptide reloading can take place (Grommé et al., 1999
; Neumeister et al., 2001
).
We here find that processing of the abundant MV-F CTL epitope predominantly follows the classic MHC class I pathway, requiring functional proteasomes and TAP for presentation. In addition, we further describe which favourable events in this pathway lead to the successful presentation of this human viral CTL epitope.
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Methods |
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Plaque-purified MV (Edmonston B strain), cultured in Vero cells and containing 107 TCID50/ml, was used to infect GR, WH or Mel-JuSo cells at an m.o.i. of 1 in RPMI 1640 medium supplemented with antibiotics and 2% FCS.
Peptide synthesis.
Synthetic peptides were prepared by standard solid-phase Fmoc chemistry using an ABIMED AMS 422 simultaneous multiple peptide synthesizer.
Isolation and HPLC fractionation of MHC-bound peptides.
MHC class I molecules were immunoprecipitated from 48 h MV-infected and uninfected EBV-transformed B cells as described previously (van Els et al., 2000 ) using the HLA-B- and -C-specific monoclonal antibody B1.23.2 (Drouet et al., 1995
). Peptides were eluted and fractionated on a 2·1 mmx10 cm reversed-phase HPLC (rpHPLC) C2/C18 column (Pharmacia, SMART system) using an acetonitrile gradient (060%) and 0·1% trifluoroacetic acid in water (flow 100 µl/min). A part of each fraction was tested for its ability to sensitize EBV-transformed B cells to cytotoxic killing by clone WH-F40. Positive fractions were further analysed by mass spectrometry.
Standard CTL assay.
Cell-mediated cytotoxicity was measured in a standard chromium release assay using peptide-pulsed GR cells as target cells as described previously (van Binnendijk et al., 1992 ).
Mass spectrometry.
Peptide fractions were analysed using microcapillary-rpHPLC-electrospray ionization-mass spectrometry (µLC-ESI-MS) as previously described (van der Heeft et al., 1998 ). The presence or absence of specific peptides was determined by the presence or absence of their masses and confirmed by comparison with synthetic analogues. The number of epitopes per infected cell was calculated by first determining the molar amount of peptide in a sample (using several dilutions of the synthetic peptide and the sample), multiplication by Avogadros number and division by the number of cells.
Proteasomal dependency assay.
At 12 h post-MV-infection the cell surface of WH cells was stripped by incubation with 4 mg/ml Pronase in the presence of 1 µg/ml DNase for 30 min at 37 °C. Membrane stripping was stopped by addition of culture medium (containing 10% FCS), followed by two washing steps with PBS. Protease inhibitors lactacystin, CbzL3 or leupeptin were added at the indicated concentrations in RPMI with 2% FCS. After 6 h, cells were washed thoroughly with PBS and fixed with 1% paraformaldehyde and washed with 0·2 M glycine in PBS. These cells were then used as stimulator cells for clone WH-F40 in a TNF release assay (Traversari et al., 1992
).
Peptide translocation by TAP.
Peptide translocation was performed as previously described by Neisig et al. (1995) . Briefly, WH cells were harvested and permeabilized by streptolysin O treatment. For each translocation assay, radioiodinated reference peptide (TVNTERAY) containing a glycosylation sequence, competing peptide (final concentration as indicated) and ATP were added to the permeabilized cells. Peptide translocation was performed at 37 °C for 5 min, stopped by lysing the cells with lysis mix containing Triton X-100, and glycosylated radiolabelled reference peptide was recovered with ConASepharose. Associated peptides were quantified by gamma counting, after extensive washing with lysis mix.
Confocal microscopy.
For microscopical studies enzymatically dispersed Mel-JuSo cells were infected with MV and allowed to grow on 8-well Lab-Tek glass chamber slides for 24 h. Then cells were washed in serum-free PFHM medium and subsequently fixed in 2% paraformaldehyde (pH 7·6). Lactacystin (10 µM) was added 1 h before fixation. Immunostaining was done with polyclonal rabbit anti-MV-F or mouse monoclonal anti-MV-haemagglutinin (anti-MV-H) antibodies. These primary antibodies were stained with FITC-labelled F(ab)2 DonkeyRabbit-IgG and Cy5-labelled Goat
Mouse-IgG (Jackson ImmunoRes Lab), respectively. After washing, slides were mounted in Vectashield and directly observed under a NIKON Optiphot 2 microscope. Fluorescence was imaged with a Bio-Rad 1024 confocal laser-scanning microscope equipped with an argon/krypton laser. Wavelengths of 488 nm and 645 nm were used to excite fluorescein and Cy5, respectively. Confocal images were recorded with a x60 Plan Apo objective lense with identical settings between images. Images were processed with Lasersharp software (Bio-Rad). Fluorescence of fluorescein and Cy5 was pseudocoloured as green or red, respectively, and high intensity fluorescence (channels 175255) was pseudocoloured as yellow.
Proteasome purification and in vitro proteasomal digestions.
Partially purified proteasomes were derived from U937 cells. Aliquots of approximately 1x108 cells were washed twice with PBS and lysed in distilled water. After homogenization with a Potter-Elvejem homogenizer, compensation buffer was added to a final concentration of 20 mM Tris, 2 mM ATP, 5 mM MgCl2, 1 mM DTT, pH 7·5. Post-nuclear supernatants (1 ml) were loaded on a sucrose gradient (1040%, w/v, in compensation buffer) and centrifuged for 21 h at 200000 g. Fractions of 0·65 ml were recovered and 10 µl of each fraction was assayed for proteolytic activity by measuring hydrolysis of Suc-LLVY-MCA (100 µM; 40 min; 37 °C; assay buffer, 50 mM Tris, 5 mM MgCl2, 5 mM DTT, pH 7·5) in a fluorometric assay. Active fractions were pooled and dialysed overnight against compensation buffer followed by concentration to approximately 50 µl with a 100 kDa centrifugal concentrator, after which the activity was assayed again. Approximately 1000 U of proteasome activity (1 U=hydrolysis of 1·5 nmol Suc-LLVY-MCA per hour) was used per digestion of 10 µg of RRYPDAVYL containing polypeptide as indicated in assay buffer (50 mM Tris, 5 mM MgCl2, 5 mM DTT, 2 mM ATP, pH 7·5). Digestions were performed at 37 °C for 1 and 3 h. Digest samples were subjected to rpHPLC; peptide-containing fractions were pooled, dried under vacuum and analysed by mass spectrometry. Individual peptides were identified by their molecular masses and quantified by the intensity of their peaks in the mass spectra. A proteasomal cleavage site was considered to be major if it generated peptide fragments with a relative amount above 9% of the total amount of RRYPDAVYL-containing polypeptide digested by the proteasome.
HLA-B*2705 peptide-binding assay.
TAP-deficient T2 cells transfected with HLA-B*2705 were incubated overnight in 200 µl with serial dilutions of peptides in PFHM supplemented with antibiotics in V-bottom 96-wells at 37 °C. Cells were washed with cold (4 °C) FACS buffer (1% BSA in PBS) and assembled surface HLA-B*2705 molecules were detected by flow cytometry using the monoclonal antibody B1.23.2 and FITC-labelled rat anti-mouse IgG (Dako) using FACScan (Becton Dickinson). Only peptides binding to the otherwise unstable HLA-B*2705 molecules on these cells will increase their cell surface expression. In this assay, binding of a peptide is quantified as its fluorescence index (FI), FI=[(mean fluorescence in the presence of the peptide)-(mean fluorescence without the peptide)]/(mean fluorescence without the peptide). At a peptide concentration of 50 µM an FI of 0·5 or higher is considered as positive and an FI above 1·0 is indicative of high affinity binding of HLA-B*2705 to the peptide.
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Results |
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To determine the affinity of binding of the MV-F438446 epitope to its restriction element, we used an assay based on the capacity of a peptide to stabilize the cell surface expression of empty unstable HLA-B molecules on HLA-B*2705-transfected T2 cells. The measured affinity was high, and comparable to that of other known HLA-B*2705-restricted CTL epitopes (Fig. 7). Based on these findings we conclude that formation of the HLA-B*2705MV-F438446 complex is efficient and is probably highly favoured in the ER during MV infection.
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Discussion |
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Further studies were aimed at the processing events leading to the abundant presentation of this MV-F438446, since no general MHC class I processing pathway for glycoproteins is known. Previously, van Binnendijk et al. (1992) suggested that the presentation of the MV-F438446 epitope requires TAP, based on the absence of WH-F40 CTL recognition of MV-infected, HLA-B*2705-transfected and TAP-deficient T2 cells. In contrast, Grommé et al. (1999)
, recently reported a residual WH-F40 recognition of the same target cells, suggesting that expression of this epitope in HLA-B*2705 is not fully dependent on TAP. In addition they reported that recognition was inhibitable by treatment with NH4Cl, a lysosomotropic agent. Therefore these authors propose that alternative processing of MV-F438446 may occur in endolysosomal compartments followed by presentation by MHC class I molecules, which recycle from the cell surface through these endolysosomes. In contrast, we find only residual recognition of another TAP-deficient cell line (BM36.1; Urban et al., 1994
) which was transfected with HLA-B*2705 and infected with MV by clone WH-F40 (K. Stittelaar & C. Herberts, unpublished observations). Moreover, we found that presentation of the HLA-B*2705-associated MV-F epitope is profoundly hampered by proteasomal inhibitors and not an inhibitor of lysosomal proteases. From these combined data we conclude that, even though a small portion of the MV-F438446 epitope may be processed via an alternative MHC class I loading pathway, the majority is formed through the classic proteasome- and TAP-dependent pathway.
While examining the processing pathway of the MV-F438446 epitope, we found some typical processing features which might explain its abundance and its outnumbering of the MV-H-derived MHC class I CTL epitope (Table 1). Immunostaining experiments indicated that MV-F products were not predominantly localized at the cell surface, but were mainly present in the cytosol. In contrast, MV-H proteins were highly associated with the cellular membrane. Furthermore, we found a strong cytosolic accumulation of MV-F, but not of MV-H, within 1 h of lactacystin treatment, indicating extensive proteasomal breakdown of MV-F. So the cytosol appears to be more readily accessible for MV-F than MV-H, implying that the availability for proteasomal degradation also differs markedly for these two transmembrane proteins. Recently, it was postulated that a considerable part of all newly synthesized proteins (up to 30%) is targeted for proteasomal degradation, because of errors in translation or in post-translational processes necessary for proper protein folding (Schubert et al., 2000
). Some proteins may be more predisposed than others towards becoming these so-called defective ribosomal products (DRiPs), on the basis of size or inherent difficulties in folding or assembly. We like to propose that the abundant cytosolic MV-F forms are in fact DRiPs, failing to attain their correct structure, and that the biogenesis and assembly of MV-F are more sensitive to errors than those of MV-H. While both proteins require folding and glycosylation for full maturation, MV-F also needs to be cleaved postranslationally into two disulphide-linked subunits, F1 and F2, to generate the functional form of this transmembrane protein.
In addition to the cytosolic availability of MV-F, we studied the efficiency of several processing steps further downstream in the classic MHC class I pathway. In vitro proteasomal liberation of the precise MV-F438446 epitope from MV-F precursor polypeptides did not occur, but an assumed important requirement for the generation of CTL epitopes, proteasomal cleavage at the exact C terminus of the epitope (Craiu et al., 1997 ; Snyder et al., 1998
), was met. In fact, the C terminus was liberated very efficiently by the proteasome. So, N-terminally extended versions of the MV-F438446 epitope are the presumed in vivo post-proteasomal processing intermediates. The fact that the proteasome did not generate the exact N terminus of the CTL epitope is not likely to limit its abundant presentation since redundant trimming systems acting downstream of the proteasome have been described both in the cytosol and in the ER (Craiu et al., 1997
; Roelse et al., 1994
; Snyder et al., 1994
; Stoltze et al., 2000
), and the epitope itself as well as the extended length variants are transported by TAP with comparable efficiencies.
In addition to the major C-terminal proteasomal cleavage site we also found a major cleavage site within the MV-F438446 epitope. In contrast to some CTL epitopes where the presence of an internal cleavage site was found to abolish presentation altogether (Luckey et al., 1998 ; Niedermann et al., 1995
; Ossendorp et al., 1996
), here this still allows abundant expression of the MV-F438446 epitope. One explanation could be that once the N-terminal length variants of the epitope are formed by cleavage at the major cleavage sites, C-terminal and upstream of the epitope, the internal cleavage site is ignored by the proteasome in vivo. Alternatively, the presence of an intra-epitopic cleavage site may be of little significance for the presentation of viral CTL epitopes generated during acute infection, due to the high level of viral protein synthesis and subsequent turnover during lytic infection. Overall, from the observed digestion pattern, we conclude that proteasomal degradation is an efficient step in the generation of the MV-F-derived CTL epitope. Similar assumptions, with respect to the relevance of proteasomal cleavage sites, were made by Kessler et al. (2000)
, who successfully identified four novel naturally presented CTL epitopes derived from the tumour antigen PRAME utilizing an improved reverse immunology strategy, including the analysis of proteasomal digestions. Candidate CTL epitopes were primarily selected on the basis of efficient liberation of their precise C terminus, while intactness of the candidate epitope was evaluated as a secondary factor and no significance was assigned to exact N-terminal excision of the epitope.
The next two processing events following proteasome digestion, i.e. transport by TAP and peptide binding to HLA-B*2705, were efficient and probably non-limiting in the generation of MHC class IMV-F438446 complexes. Moreover, given the high affinity binding of synthetic MV-F438446 to HLA-B*2705 and the unique score of this epitope when screening the whole MV proteome using a peptide-binding algorithm, we propose that, once in the ER, the MV-F438446 epitope does not encounter any strong competition from other potential MV-derived 9- or 10-mers for binding to HLA-B*2705.
Altogether, we found that this particular glycoprotein-derived CTL epitope is predominantly processed and presented via the classic MHC class I loading pathway, and that due to the unexpected extensive presence and turnover of its precursor protein in the cytosol, in combination with multiple other excellent intrinsic processing features, the MV-F438446 epitope reaches a relatively high level of cell surface expression when compared to other examples of human viral CTL epitopes (Table 1). In general, viral glycoproteins, including MV-F and MV-H, appear to be a major source of target proteins for antiviral CTL responses (Jaye et al., 1998
; Rammensee et al., 1997
). Therefore more insight into their individual processing requirements and their naturally occurring MHC class I epitopes can be instrumental in the development of new generations of viral vaccines.
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Acknowledgments |
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Footnotes |
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References |
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Received 27 February 2001;
accepted 30 April 2001.