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* Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland;
Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne Branch, University Hospital, Lausanne, Switzerland;
Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche ScientifiqueS, Toulouse, France;
Department of Biochemistry, University of Lausanne, Epalinges, Switzerland; and
¶ National Center of Competence in Research-Molecular Oncology, Epalinges, Switzerland
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In recent years much attention has been paid to the melanoma-associated Ag Melan-A/MART-1 (hereafter termed Melan-A), an intracellular transmembrane protein expressed in melanocytes and melanomas, because of the identification of an immunodominant epitope recognized frequently by CD8+ tumor-infiltrating T lymphocytes (TIL)3 from HLA-A*0201+ melanoma patients (6). The antigenic peptide is located within the transmembrane domain of Melan-A and requires the activities of the standard proteasomes for its presentation (7, 8). Although the nonamer peptide Melan-A27–35 AAGIGILTV has been isolated from HLA-A*0201+ melanoma cells (9), many tumor-reactive CTL recognize the decamer Melan-A26–35 (EAAGIGILTV) with equal or increased efficiency (10, 11). Therefore, the cognate antigenic peptides will be referred to as peptides Melan-A26/27–35 throughout the text. Earlier work had also shown that the substitution of Ala27 for Leu within the decamer sequence (position 2 of the decamer) increased the binding affinity of the peptide to HLA-A*0201 and led to a more efficient recognition by specific CTL and a stronger induction of CTL responses (10, 12). Most importantly, these CTL were tumor-reactive.
In this study we analyzed the degradation of wild-type and mutated Melan-A26/27–35 precursors by standard proteasomes in vitro. We observed that the proteasomes produced the antigenic peptides (nona- and decamers) in their final sizes along with N-terminally extended intermediates. We then asked which pool of the fully or incompletely processed intermediates was the source of the antigenic peptides eventually presented by HLA-A*0201 molecules at the surface of melanoma cells. Our results indicate that antigenic peptides directly produced in their final form by the proteasomes are preferentially selected for presentation by HLA-A*0201 molecules. Inefficient postproteasomal trimming of the N-terminally extended intermediates by cytosolic peptidases contributes to this preference.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wild-type and A27L substituted precursor peptides (3.5 µg), Melan-A15–40 and Melan-A15–40A27L, respectively, were digested by 4 µg of proteasomes isolated from human erythrocytes in 13 µl of 25 mM Tris, pH 7.6. Proteasome affinity purification and quantification were performed as previously described (8, 13, 14). The purity of the proteasome preparations was assessed by SDS-PAGE and staining. The reaction was stopped after 1 h with 1% trifluoroacetic acid (TFA). After lyophilization, the digests were resuspended in 12 µl of H2O/CH3CN (50/50, v/v) and diluted to 48 µl in CH3CN/H2O/HCOOH (5/95/0.05, v/v/v). Twelve microliters of a 20 µM solution of an internal standard synthetic peptide in CH3CN/H2O/HCOOH (5/95/0.05, v/v/v) was then added to the digest. Fifteen microliters was analyzed by online HPLC-UV-electro-spray ionization-MS/MS. Peptides were separated on a Pepmap LC Packings (Dionex, Amsterdam, The Netherlands) C18 column (1 mm x 15 cm) at a flow rate of 40 µl/min. The separation was achieved with a gradient elution of 10–50% for 40 min (A is 5/95/0.05 CH3CN/H2O/HCOOH, B is 70/30/0.05 CH3CN/H2O/HCOOH; v/v/v). UV absorption was measured online at 215 nm with electro-spray ionization-MS/MS detection performed on an LCQ ion trap mass spectrometer (ThermoFinningan, San Jose, CA). Data acquisition was performed in a data-dependent mode consisting of a full-scan MS over the mass range m/z 220-2000, followed by a zoom-scan MS and a full-scan MS/MS in a dynamic exclusion mode of the most intense ion detected in the full-scan MS. MS/MS data were acquired using a 3 m/z ion isolation window, a relative collision energy of 35%, and a dynamic exclusion duration of 0.5 min. After identification by mass spectrometry, quantification of the peptides was performed by measuring the surface area (millivolts per second) of the relevant peaks on the UV chromatograms and by measuring the ion current (arbitrary units) corresponding to a specific m/z value in the full-scan MS. For nonoverlapping UV peaks, the variations in relative abundances of different peptide species determined by UV and MS analyses were similar. In case of peptide coelution during the HPLC separation, quantification was based on MS data only.
Construction of plasmids and recombinant vaccinia
The ubiquitin/protein/reference (UPR)-based pEGFP vector used in this study is identical with the vector described previously (12), except that the GFP coding sequence was replaced by the enhanced GFP (EGFP). All constructs were obtained by annealing complementary synthetic oligonucleotides containing at their 5' and 3' ends SacII and BamHI restriction sites, respectively. The oligonucleotides contained two codons for Gly at the 5' end (except for construct shown in Fig. 2B; see below) and a stop codon at the 3' end. Insertion of these fragments into the SacII-BamHI sites of pEGFP/ubiquitin (Ub) led to the generation of plasmids directing the expression of the reference protein EGFP-Ub and the Melan-A fragments spanning aa 26–35 (M26–35A27L), aa 26–40 (M26–40A27L), aa 20–35 (M20–35A27L), and aa 22–35 (M22–35A27L; Fig. 2A). In the construct Ub-M26–35A27L (Fig. 2B), the two C-terminal Gly of ubiquitin were removed, inhibiting the cleavage of EGFP-Ub by ubiquitin peptidase (15). In all constructs, the amino acid Ala27 of Melan-A was substituted by a leucine (L). The sequence of all inserts was confirmed by DNA sequencing.
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Cell lines
The human melanoma cell line NA8-MEL was maintained in RPMI 1640 medium (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% FCS, 10 mM HEPES (Invitrogen Life Technologies), and 1% penicillin-streptomycin (Invitrogen Life Technologies). The CTL used in the IFN-
release assay is a murine CTL line obtained by weekly restimulations of cells after immunization of HLA-A*0201/Kb transgenic mice (16) with vaccinia virus coding for Melan-A26–35A27L. The CTL line was maintained in DMEM (Invitrogen Life Technologies) supplemented with 10% FCS, 10 mM HEPES (Life Technologies), 1% penicillin-streptomycin (Life Technologies), 2% 2-ME, and 30 U/ml IL-2 (produced at our institute).
The human CTL clone A42 was provided by Dr. L. Rivoltini (Unit of Immunotherapy of Human Tumors, Istituto Nazionale Tumori, Milan, Italy) and has been described previously (17). The line EAA and the clone A42 were maintained by culture in DMEM supplemented with 8% human A+ pooled sera and 103 U/ml human rIL-2.
CTL assay, IFN- release assay, and ELISA
NA8-MEL cells were seeded at a density of 2 x 104 cells/well in a 96-well plate the day before transfection. Plasmids (0.1 µg/well) were transfected in duplicate into cells with FuGene according to the manufacturer’s protocol (FuGene transfection reagent; Amersham Biosciences, Arlington Heights, IL). Half the cells were pulsed with exogenous Melan-A26–35A27L peptide (1 µM final concentration) for 30 min at 37°C before the addition of CTL at an E:T cell ratio of 3:1 in DMEM containing 10% FCS, penicillin-streptomycin/HEPES, 5% IL-2, and 2% 2-ME. Twenty-four hours later, supernatants were harvested, and the concentration of IFN- in the supernatant was measured by ELISA. Microtiter plates were coated with 2 µg/ml anti-mouse IFN- mAbs (R46A2, produced at the institute) and blocked with 1% BSA in PBS. Serial 2-fold dilutions of the cell supernatant were added to the wells, and the concentration of IFN- was revealed using 1 µg/ml anti-murine IFN--biotinylated rat Ig (ANI-803, produced at the institute) and HRP-conjugated streptavidin (Invitrogen Life Technologies, Gaithersburg, MD). The OD was measured at 490 nm. Duplicate values were generally within 15% of the mean.
Western blot analysis
An aliquot of each infected target cell (2 x 106 cells) used in the CTL assay presented in Fig. 5C was lysed in 100 µl of lysis buffer containing 20 mM Tris (pH 7.6), 150 mM NaCl, 5 mM EDTA, and 1% Nonidet P-40. The lysate was cleared by centrifugation at 11,000 x g for 10 min at 4°C. Ten microliters of the cleared lysate was separated by SDS-12% PAGE and subjected to immunoblotting. Due to the fact that the reference protein GFPha-Ub contained an influenza hemagglutinin (ha) epitope tag between the sequence of GFP and Ub, its expression was detected with the anti-ha mAb (1/2,000; Roche, Indianapolis, IN) and HRP-conjugated anti-mouse Ab (1/2,500; Amersham Biosciences) and revealed by ECL (Amersham Biosciences). The x-ray film was scanned and the bands corresponding to GFPha-Ub were quantitated densitometrically using AIDA Image Analyzer software (Raytest, Straubenhardt, Germany).
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Flow cytometry was performed on FACScan devices using CellQuest software (BD Biosciences, San Jose, CA). Transfected cells were washed twice, resuspended in PBS/3% FCS, and directly analyzed. Identification of live cells was based on the forward and side scatter of mock-transfected cells. Sorting of transfected cells was performed using a FACSAria cell sorter (BD Biosciences). Cells (107) were washed twice, resuspended in PBS/3% FCS, and directly sorted according to their EGFP expression level into EGFPhigh and EGFPlow subpopulations. Sorted cells were allowed to recover for 60 min in complete medium before the addition of specific CTL.
Peptide digestion by incomplete cytosol, HPLC separation, and MS
Purified synthetic peptides were quantified by amino acid analysis. Five nanomoles of peptide Melan-A26–35, Melan-A26–35A27L, Melan-A22–35, Melan-A22–35A27L, and RU129–42 were digested in 40 µl of incomplete cytosol (cytosol without proteasome) corresponding to 3 x 106 cell equivalents. Preparation of proteasome-depleted cytosol was described previously (15) and contained <5% of the remaining proteasomal activities, based on the proteolysis of fluorogenic substrates (data not shown). For each peptide, an aliquot was removed at time zero and served as the reference for quantitative analysis. Digestions were allowed to proceed for 2.5, 5, 10, and 20 min at 37°C. At each time point, the reaction was stopped with 1% TFA. The digests were diluted with acetic acid/water (50/50, v/v) to a final volume of 120 µl, and 100 µl was injected on the HPLC. HPLC separations were performed on an Agilent system (1100 series) using a C18 small pore Vydac (250 x 4 mm; 5 µm; 201SP54; Hesperia, CA) column. The column was eluted at a flow rate of 1 ml/min by a linear gradient of acetic acid/TFA/acetonitrile/H2O (0.1/0.005/80/20, v/v/v/v) on acetic acid/TFA/H2O (0.1/0.005/100, v/v/v/), rising within 60 min from 0 to 100%. The OD of the eluate was monitored at 214 nm. Fractions were collected using an Agilent (G1364A) fraction collector on 96-well plates and subjected to MALDI-TOF mass spectrometric analysis as previously described (15).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first analyzed and compared degradation of the wild-type and mutated Melan-A26/27–35 precursors by purified proteasomes in vitro. The 26-aa-long precursors, encompassing aa 15–40, were digested with standard proteasomes and subjected to on-line HPLC-UV-MS/MS analyses. As shown in Fig. 1, the major single proteasomal cleavage site was detected at the exact C terminus of the wild-type and mutated antigenic peptides, producing the fragments Melan-A15–35 and Melan-A27–35A27L (and its complementary fragment ILGVL). Five fragments were generated with that C terminus: the final antigenic peptides Melan-A26–35 and Melan-A27–35, two N-terminally extended fragments corresponding to the peptide Melan-A22–35 and Melan-A20–35, and the 8-mer Melan-A28–35, a fragment too short to bind to HLA-A*0201 (Fig. 1, A and B). The peptides Melan-A28–35, Melan-A27–35, Melan-A26–35, and Melan-A22–35 were produced in similar amounts, whereas less of the fragment Melan-A20–35 was generated. The same cleavages were also observed with the mutated precursor Melan-A15–40A27L, although the relative ratio of Melan-A26–35A27L vs Melan-A27–35A27L was strongly biased in favor of the 10-mer (Fig. 1, C and D). The N-terminally extended precursors were also produced, as was the internal 8-mer Melan-A28–35. We concluded from this experiment that both wild-type and mutated minimal antigenic Melan-A peptides were produced by the proteasomes along with two N-terminally extended intermediates.
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Recognition of cells expressing Melan-A26–35A27L and extended precursors by specific CTL
Based on the peptide fragments described in Fig. 1D, we constructed a series of plasmids to express the corresponding fragments in mammalian cells (Fig. 2). We selected four sequences, two corresponding to the fragments carrying additional four and six N-terminal amino acids, respectively, and one corresponding to the final antigenic decamer. Because the proteasomes have been shown to limit the supply of antigenic peptides, we also compared the presentation efficiency of a peptide that only required proteasomal processing with those that only required N-terminal trimming. Therefore, we included a plasmid coding for a fragment that displayed the final N terminus, but carried an additional five amino acids at the C terminus of the antigenic peptide. All constructs were expressed from a UPR-based plasmid, which produces equimolar amounts of the reference protein EGFP-Ub and the fragment of interest (14, 20). This strategy circumvents the need for N-terminal Met and allows measurement of the transfection efficiency and expression levels of the EGFP-Ub moieties by flow cytometry. We focused on the processing of Melan-A peptides carrying the A27L mutation because we had previously shown that the recognition of transiently transfected cells expressing the wild-type antigenic peptide Melan-A26–35 did not efficiently stimulate the production of cytokines by specific CTL (7, 12).
Human NA8-MEL melanoma cells (HLA-A*0201 positive, Melan-A negative) were transiently transfected with those constructs. Transfection efficiency and expression level were assessed by flow cytometry (Fig. 3A). Approximately 20% of the cells expressed EGFP-Ub, and the level of expression was similar for each construct, as judged by the mean fluorescence of the fluorescent cell pool. Nevertheless, the expression level in individual cells varied significantly within a transfected population, as evidenced by the width of the fluorescence histogram.
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by the CTL was measured by ELISA. Cells expressing the antigenic decamer (M26–35A27L) were efficiently recognized, as indicated by the level of IFN- release. This level was similar to that obtained by incubating CTL with cells loaded with saturating amounts of exogenous Melan-A26–35A27L peptide. The recognition of cells expressing the C-terminally extended precursor M26–40A27L, which requires a single proteasomal cleavage for the production of the antigenic peptide, was lower, confirming that the proteasomal cleavage is limiting the availability of the antigenic peptide. Surprisingly, cells expressing the two N-terminally extended precursors, M20–35A27L and M22–35A27L, were recognized less efficiently than cells expressing M26–35A27L. These results suggest that conversion of the N-terminally extended intermediates into the final antigenic Melan-A peptide is inefficient in these cells. Alternatively, rapid proteolytic cleavage within the antigenic peptide could limit presentation of the N-terminally extended products.
Sorting of the subpopulations of transfected cells expressing M26–35A27L or the full-length protein Melan-AA27L and exhibiting bright or dim fluorescence intensities by FACS (Fig. 3C) showed that both populations could be recognized by anti-Melan-A CTL, but that the amount of IFN- secreted by CTL was 3 times higher when incubated with bright cells than with dim cells (Fig. 3D). In both subpopulations, cells expressing M26–35A27L were recognized more efficiently than those expressing the full-length protein.
To control that the efficient presentation of cells expressing M26–35A27L was caused by release of the EGFP-Ub moiety from the final antigenic peptide and not by rapid degradation of the uncleaved fusion protein, we introduced a mutation in the plasmid M26–35A27L, which resulted in the production of an uncleavable fusion protein, EGFP-Ub-M26–35A27L (Fig. 2B). This mutation, which removes the two C-terminal Gly residues of ubiquitin, prevents cleavage of the EGFP-Ub moiety by ubiquitin proteases. The effects of this mutation have been described previously (15). NA8-MEL cells were transfected with this plasmid and exposed to anti-Melan-A CTL (Fig. 3B). Recognition of the transfected cells by CTL was poor, confirming that the efficient recognition of cells expressing M26–35A27L was due to the efficient release of the antigenic peptide in its final form. This also confirmed that the differences observed between the fully processed peptide and the N-terminally extended ones were due to the peptide-flanking sequences liberated in the cytosol.
Trimming of Melan-A26–35 and Melan-A26–35A27L precursors by cytosolic proteases
A possible cause of the apparent inefficient presentation of the antigenic Melan-A peptide in cells expressing the N-terminally extended precursors could be their different sensitivities to cytosolic peptidases. To address this question, we incubated synthetic peptides corresponding to the 14-mers Melan-A22–35 and Melan-A22–35A27L as well as the 10-mers Melan-A26–35 and Melan-A26–35A27L with NA8-MEL cytosol depleted of proteasomes. As control, we incubated an N-terminally extended 14-mer precursor of the HLA-B51-restricted peptide RU134–42. This precursor was shown previously to be trimmed to the antigenic nonamer by the sequential activity of two different cytosolic peptidases, tripeptidyl peptidase II and puromycin-sensitive aminopeptidase (13, 15). The two 14-mer Melan-A wild-type and mutated peptides were degraded at a significantly lower rate than the RU1 peptide and the 10-mer Melan-A peptides (Fig. 4, A and B, respectively). The half-life of the 14-mer Melan-A peptides was 
10 min, whereas the half-life of the 14-mer RU1 peptide was 5 min, similar to the half-life of the antigenic 10-mers. At each time point, the peptide digest was separated by HPLC, and the content of each detected peak was identified by mass spectrometry. As shown in Fig. 4B, the species corresponding to the final antigenic peptide sequence 26–35 was produced from the precursor Melan-A22–35 and was detectable after 20 min degradation. For Melan-A22–35A27L, the final antigenic peptide could be identified after 5 min. In sharp contrast, the fragment corresponding to the final antigenic peptide RU134–42 was already clearly quantifiable after 2.5 min. The exact composition of the fractions containing the final antigenic peptides for each of the precursors is detailed in Fig. 4C. For Melan-A22–35 (upper row), deletion fragments were already detected at time zero. However, several other fragments appeared over time, including the final antigenic peptide at 20 min. For Melan-A22–35A27L (middle row), the final antigenic peptide was already detectable at 5 min, among other fragments. Finally, the final antigenic peptide produced from the RU1 precursor (lower row) was detected at 0, 2.5, and 5 min. Despite its detection by mass spectrometry at time zero, the final antigenic peptide is nevertheless produced in the course of the digestion, and its actual amount can be derived from the HPLC profile shown in Fig. 4B, because no other fragment coeluted. The RU129–42 peptide was completely digested at 20 min. The antigenic 9-mer Melan-A27–35 (m/z 815) and Melan-A27–35A27L (m/z 857) could not be detected by MALDI-TOF MS, because this region contains several intense peaks corresponding to matrix ions. Because MALDI-TOF mass spectrometry does not allow the identification of fragments below a mass of 850 Da, we cannot completely rule out that a rapid internal cleavage could take place in the cytosol. Nevertheless, the degradation kinetics of the Melan-A precursors correlated well with the efficiency by which the final antigenic peptides were generated.
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To confirm that the expression of an N-terminally extended Melan-A precursor leads to poor recognition of target cells by specific CTL, we took advantage of the fine specificity of a previously described CTL clone, clone A42 (17). This clone was shown to recognize Melan-A27–35 more efficiently than Melan-A26–35, which can be considered, under these circumstances, as an N-terminally extended intermediate. We first established that this tumor-reactive clone indeed recognizes the nonamer Melan-A27–35 more efficiently than the decamer Melan-A26–35 (Fig. 5A). We then tested whether melanoma cells expressing the nonamer Melan-A27–35 or the decamer Melan-A26–35 would be recognized with similar efficiency. To this end, we infected NA8-MEL cells with recombinant vaccinia virus coding for the wild-type versions of the decamer Melan-A26–35, the nonamer Melan-A27–35, and the full-length Melan-A protein and incubated them with CTL clone A42 (Fig. 5B). As expected, the cells expressing the antigenic nonamer were efficiently recognized by the CTL clone A42, but not cells expressing the decamer Melan-A26–35. NA8-MEL cells infected with recombinant vaccinia virus coding for the 10-mer Melan-A26–35A27L were not recognized either (Fig. 5C). However, cells expressing the full-length proteins Melan-A and Melan-AA27L were recognized by the CTL clone A42. To ensure that the cells used for the experiment depicted in Fig. 5C were expressing similar amounts of Melan-A proteins or peptides, we took advantage of our UPR-based constructs, in which Melan-A is expressed as C-terminal fusions of the ha-tagged reporter GFP-Ub (12, 20). We therefore performed a Western blot analysis on an aliquot of cells used in Fig. 5C. As shown in Fig. 5D, infected cells were expressing similar amounts of the reporter proteins. Because the reporter and the Melan-A products are expressed from the same mRNA, the amount of detected reporter is equimolar to the amount of Melan-A in the cells. This observation led us to conclude that although the decamers Melan-A26–35 and Melan-A26–35A27L are expressed, they are not efficiently converted into the antigenic nonamers in infected cells, supporting our previous findings that cells expressing the N-terminally elongated precursors of Melan-A were inefficiently recognized by CTL. However, we cannot completely rule out that the decamer Melan-A26–35 escapes N-terminal trimming by rapid binding to HLA-A*0201 molecules. Cells expressing the full-length protein were also recognized by the CTL clone A42, suggesting that along with other fragments, these cells produce the antigenic nonamer from the full-length protein. In parallel, cells infected with the same constructs were incubated with the CTL line EAA that indiscriminately recognizes the nonamer and decamer peptides. Contrary to what was observed with clone A42, cells expressing the different constructs were recognized with similar efficiency, indicating that infected NA-8 MEL can present Melan-A26–35 as well as Melan-A27–35 (Fig. 5B).
These data demonstrate 1) that Melan-A26–35, which can be considered an N-terminally extended precursor of Melan-A27–35, is not converted to the antigenic 9-mer in melanoma cells; and 2) that both 10- and 9-mer Melan-A peptides can be presented by HLA-A*0201 at the surface of melanoma cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Over the last few years an increasing number of peptidases have been implicated in the postproteasomal processing of antigenic peptides, including tripeptidyl peptidase II, puromycin-sensitive aminopeptidase, bleomycin hydrolase in the cytosol (1, 15, 21, 22, 23), and ERAPI/ERAAP in the endoplasmic reticulum (24, 25). However, none of these studies determined the roles of these peptidases when antigenic peptides were produced by the proteasomes both in their final form and as N-terminally extended intermediates. It has been shown that the degradation of OVA precursors by 20S proteasomes in vitro produces both fully processed and N-terminally extended antigenic peptides (26). Based on the analysis of the OVA fragments produced by 26S proteasomes in vitro and in cells, it was later suggested that most antigenic peptides are produced as N-terminally extended intermediates that require postproteasomal trimming (4, 5). Although this conclusion was consistent with the idea that such extensions would protect the antigenic core sequences from premature destruction by cytoplasmic proteases, it was not determined experimentally which pool of the N-terminally elongated or minimal peptides of OVA served as the source of CTL epitopes. Similar to OVA, Melan-A is degraded by the proteasomes into minimal antigenic peptides and extended intermediates. In the case of Melan-A, our results suggest that peptides directly produced by the proteasomes are preferentially selected for presentation by HLA-A*0201 molecules in melanoma cells. This preference could be imposed by the limiting amount of specific peptidases present within melanoma cells or by the presence of unfavorable amino acids within the N-terminal extension of Melan-A.
Evidence of possible negative effects of N-terminal extensions was reported previously (27). In this study the authors analyzed the presentation of a subdominant CTL epitope derived from OVA and showed that the subdominance was directly linked to the flanking N-terminal amino acid sequence. These results led the authors to suggest that N-terminal trimming played a significant role in this process. Other studies also showed that amino acids preceding the antigenic sequences could affect the presentation of epitopes (28, 29). More recently, it was shown that cytosolic peptidases can limit the availability of antigenic peptides by overdigesting the precursors (30). As shown in this study, it appears that the limitation in the availability of Melan-A26/27–35 antigenic peptides is caused by an increased resistance of the N-terminally elongated intermediates to proteolytic degradation, although a higher sensitivity to postproteasomal peptidases could also contribute to the poor presentation of N-terminally extended intermediates.
Altogether, our results indicate that the postproteasomal processing of Melan-A peptides does not contribute significantly to the pool of antigenic peptides loaded and presented by HLA-A*0201 molecules at the surface of melanoma cells. Moreover, our findings suggest that the presentation of the Melan-A epitope in melanoma cells is affected not only by the efficiency by which proteasomes generate the appropriate C terminus, but also by the ratio between N-terminally elongated fragments and final antigenic peptides produced by the proteasomes. It is therefore reasonable to postulate that aside from proteasomes, postproteasomal processing peptidases impose a second limiting step in the availability of antigenic peptides for presentation.
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Acknowledgments |
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Footnotes |
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1 This work was supported in part by a grant from the Swiss National Fund (to L.C. and F.L.), the National Center of Competence in Research-Molecular Oncology, a research instrument of the Swiss National Science Foundation (to C.S., P.R., and F.L.), grants from the Centre National de la Recherche Scientifique and the Région Midi Pyrénées (to B.M.), and an Investigator Award from the Cancer Research Institute (to F.L.). 
2 Address correspondence and reprint requests to Dr. Frédéric Lévy, Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland. E-mail address: frederic.levy{at}isrec.unil.ch
3 Abbreviations used in this paper: TIL, tumor-infiltrating T lymphocyte; EGFP, enhanced GFP; ha, hemagglutinin; MS, mass spectrometry; TFA, trifluoroacetic acid; Ub, ubiquitin; UPR, ubiquitin/protein/reference.
Received for publication January 21, 2004. Accepted for publication September 3, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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–induced aminopeptidase in the ER, ERAP1, trims precursors to MHC class I–presented peptides. Nat. Immunol. 3:1169.[Medline]
-galactosidase by the proteasome to yield antigenic peptides. J. Immunol. 152:3884.[Abstract]
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