IFN-{alpha} mediates the up-regulation of HLA class I on melanoma cells without switching proteasome to immunoproteasome

Giuliana Cangemi1, Barbara Morandi2, Antonella D‘Agostino1, Cristiano Peri1, Romana Conte2, Gianluca Damonte3, Guido Ferlazzo1, Roberto Biassoni4 and Giovanni Melioli1

1 Laboratorio di Analisi, Istituto Giannina Gaslini, 16148 Genoa, Italy 2 Laboratorio di Immunologia, Istituto Nazionale per la Ricerca sul Cancro, Genoa, 16132 Italy 3 Sezione di Biochimica, Dipartimento di Medicina Sperimentale, Università di Genova, Genoa, 16132 Italy 4 Laboratorio di Medicina Molecolare, Istituto Giannina Gaslini, 16148 Genoa, Italy

Correspondence to: G. Melioli; E-mail: giovannimelioli{at}ospedale-gaslini.ge.it
Transmitting editor: L. Moretta


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Treatment of melanoma cell lines with IFN-{gamma} induces the switch from proteasome (PS) to immunoproteasome (iPS). This finding has profound implications for the immunobiology of melanoma cells since certain peptides (such as Melan-Amart127–35) are cleaved differently by iPS, thus implying a different ability to be presented by HLA class I molecules. IFN-{alpha} is a cytokine not only produced during infectious diseases, but also used in the treatment of certain cancers. Nevertheless, the effects of IFN-{alpha} on the switch of PS to iPS are largely unknown. A comparison of the effect of both IFN-{alpha} and IFN-{gamma} was thus carried out on melanoma cell lines. RT-PCR showed that mRNA for iPS subunits (i.e. LMP-2, LMP-7 and MECL-1) was detectable both in untreated and IFN-treated melanoma cells. Immunoblotting analysis revealed that while IFN-{gamma} was able to consistently induce the switch from PS to iPS, IFN-{alpha} treatment did not, possibly due to post-transcriptional event(s) blocking the expression of iPS-specific subunits. Finally, Melan-Amart127–35 peptide was found only in the HPLC-MS spectra from both untreated and IFN-{alpha}-treated cells, but not upon IFN-{gamma} treatment. Altogether, these data demonstrate that IFN-{alpha} does not induce the switch from PS to iPS.

Keywords: IFN, HLA, peptide, proteasome


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Proteasomes (PS) are highly conserved structures present in both the cytoplasm and nucleus of the cell, and their role is to catalyze non-lysosomal protein degradation. This hydrolysis of polypeptides accounts for the removal of potentially dangerous substances (1). From an immunological point of view, PS have a central role in the generation of peptides that are supplied to HLA class I molecules in the endoplasmic reticulum (2,3). In the trimeric structure of HLA class I, 8- to 10-residue peptides acquire the capability of being presented to the immune system and evoking an immune response if recognized as non-self. The structure of PS is complex. They exist not only in a monomeric form (20S), but also in a more complex structure (26S) represented by two regions flanking the central 20S PS structure. The proteolytic component is the 20S core particle, a cylindrical structure made up of 28 subunits arranged in four stacked rings. The two central rings are composed of ß subunits, three of them, i.e. ß5(X), ß1(Y) and ß2(Z), containing the proteolytic active sites (4). While PS are detected in the vast majority of cells, in a few immunocompetent cells, such as mature dendritic cells (5,6), they have a different form and are called immunoproteasomes (iPS). Their structure differs from the original in that the three catalytic ß subunits are replaced by three different polypeptides, called low-molecular-weight protein (LMP)-7, LMP-2 and multicatalytic endopeptidase complex-like (MECL)-1 respectively, which are generated in an immature form. Following maturation and removal of the N-terminal propeptide, the LMP-2 subunit goes from its original 23.3 to 21.2 kDa, LMP-7 decreases from 30.3 to 22.6 kDa and MECL-1 changes from 28.9 to 24.6 kDa (7). Functionally, LMP-7 is required for the maturation of MECL-1 and LMP-2, and their insertion into the newly formed iPS (7). The switch from PS to iPS is primarily mediated by IFN-{gamma} in different types of cells. Recently, tumor necrosis factor-{alpha} was also shown to induce iPS assembly in melanoma cells (8). Of note, iPS is assembled only when all three of the above-mentioned different subunits are present. Furthermore, the switch from PS to iPS is a discrete phenomenon, not completely synchronized and requiring several hours (5). The functional difference between the two PS forms is the different cleavage sites for polypeptides. iPS cleaves polypeptides better than PS after hydrophobic and basic residues (such as leucine), particularly when they are followed by a small residue, such as alanine. On the other hand, the presence of acidic amino acids (such as glutamate and aspartate) apparently reduces the iPS chymotrypsin-like activity (9). Recently, a novel role of PS in the processing of endogenous peptides and that of iPS for exogenous peptides has been proposed (9). While the effects induced by IFN-{gamma} on PS are well known, the effects of IFN-{alpha} on the PS system are at present largely unknown. Nevertheless, IFN-{alpha} is endogenously produced during viral infections and it is used in humans not only for treatment of viral infectious diseases (such as hepatitis C), but also in the treatment of cancers, such as melanoma and kidney carcinoma (10). Both IFN-{gamma} and IFN-{alpha} significantly up-regulate HLA class I expression on the cell surface (11,12). On the other hand, it is still unclear whether treatment with IFN-{alpha} induces the switch of PS to iPS, thereby modifying the peptide repertoire presented to TCR. Along this line, we recently described that peptides eluted from HLA class I molecules are apparently only quantitatively and not qualitatively modified as a result of IFN-{alpha} treatment (13). More importantly, in some experiments of ours, no significant modification was observed when peptides such as MAGE and Melan-Amart132–40 were analyzed (13). However, in another experimental model (5), some differences were found analyzing Melan-Amart127–35 peptide in IFN-{gamma}-treated cells. For these reasons we analyzed the modification induced by IFN-{gamma} and IFN-{alpha} in PS subunits (in terms of mRNA and polypeptides) in three different melanoma cell lines. In one of them, we also analyzed the modification of the panel of peptides eluted from melanoma cell surface after treatment with both IFN-{alpha} and IFN-{gamma}. In this paper, we demonstrate that while IFN-{gamma} is capable of inducing the complete switch of PS to iPS in melanoma cells, a post-transcriptional event apparently inhibits the modification from PS to iPS in the presence of IFN-{alpha}. As a further control, it is shown that Melan-Amart127–35 eluted from IFN-{alpha}-treated cells completely disappeared following IFN-{gamma} treatment. This finding could have several implications for the planning and implementation of immunotherapy trials where immunogenic peptides as well as autologous cancer cells are used as immunogens.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell lines
Three melanoma cell lines (one, named 13443, kindly provided by G. Parmiani, Milan, Italy, HLA-A1/29, B37, B44; and two others, FR, HLA-A2/2, B8/35 and GI, HLA A9/24, B18/35 Bw6 from our cell line collection) were used in the study. Normal human fibroblasts (HuF) were derived from a punch biopsy of the skin of a healthy donor as described (14). The T2 cell line, a human T cell leukemia/B cell line hybrid defective in TAP1/TAP2 and iPS subunits (15), was employed as a control. The lymphoblastoid cell line (LCL)-2493, obtained by in vitro transformation of B cells from a healthy donor by the B95.8 EBV strain, was employed as a control in RT-PCR experiments. Repeated tests with Hoechst 33258 stain showed that the cell lines were mycoplasma free. All cell lines were maintained in RPMI 1640 media (Euroclone, Milan, Italy), supplemented with 2 mM L-glutamine (Euroclone), penicillin and streptomycin (Euroclone), and 10% heat-inactivated FCS (Boehringer Mannheim, Mannheim, Germany) at 37°C and 5% CO2 in a humidified incubator. Cells, grown on six-well plates were cultured with complete medium alone (hereafter referred to as ‘untreated’) or in combination with IFN-{gamma} (100 IU/ml) (Euroclone) or IFN-{alpha} (100 IU/ml) (Roferon; Roche, Milan, Italy). IFN was added daily for 8 consecutive days. These doses were identified as the plateau concentrations of IFN in up-regulating HLA class I on cell targets. IFN-{alpha} was also given at a higher dose (500 IU/ml) for 48 h in a series of different experiments.

The biological effect of IFN-{alpha} and IFN-{gamma} treatment was monitored by calculating the up-regulation of the number of HLA class I molecules expressed on the cell surface on days 2, 4 and 8, as well as 4 and 6 days after IFN withdrawal, as described (14). For immunoblot analysis, cells expanded in different culture conditions were harvested on an ice bath with lysis buffer (1 M Tris–HCl, pH 6.8, 4 M NaCl, 10% glycerol, 1% Nonidet NP-40, 100 mM EDTA, 2 mM PMSF and protease inhibitor cocktail; Sigma, Milan, Italy) for 30 min. Lysates were collected and unsolubilized material was removed by centrifugation. Pellets (consisting of debris) were discharged and supernatants containing either PS or iPS were stored at –80°C until used.

RNA extraction and RT-PCR
Total RNA was extracted from 13443 melanoma cells either not treated or IFN-{alpha} and IFN-{gamma} treated for 48 h, HuF and LCL-RS using pepGOLD RNAPure System (Peqlab, Germany). mRNA was reverse transcribed using 2 µM oligo(dT) (TibMolbiol, Genova, Italy), 1.5 mM MgCl2, 200 µM dNTPs, 5 U RNase inhibitor (Genecraft, Munster, Germany), and 50 U MMLV reverse transcriptase (Genescript RT; Genecraft) at 42°C for 45 min and 50°C for 45 min. Then, 1 µl of each cDNA was added to 24 µl of PCR mix [1 x TAQ buffer, 200 µM dNTPs, 1.5 mM MgCl2, 1 U BioTherm DNA polymerase (GeneCraft) and 0.5 µM of each primer]. Sense and antisense primers used for PCR amplifications were: hß-actin: 5'-ACTCCATCATGAAGTGTGACG-3' and 5'-CATACTCCTGCTTGCTGATCC-3'(249 bp); hLMP-2: 5'-CGTTGTGATGGGTTCTGATTCC-3' and 5'-GTTCATTGCCCAAGATGACTCG-3' (547 bp); hLMP-7: 5'-AATGCAGGCTGTACTATCTGCG-3' and 5'-TGCAGCAGGTCACTGACATCTG-3' (406 bp) and h-MECL-1: 5'-CCTTCGAGAACTGCCAAAGAAATGC-3' and 5'-CAAGCTCTAAGCCTCAGCTTACTCC-3' (802 bp). PCR was performed on a GeneAmp PCR system 2700 thermal cycler (Applied Biosystems, Foster City, CA) under the following conditions: 94°C for 30 s (denaturation); 55 (hß-actin), 60 (hLMP-2 and hLMP-7) or 62°C (hMECL-1) for 30 s (annealing) and 72°C for 30 s (elongation) followed by 7 min at 72°C for 30 cycles. The PCR products were separated on a 1.5% agarose gel containing 0.5 µg/ml ethidium bromide and photographed under UV light.

Immunoblot analysis of PS subunits
Proteasome subunit-specific antibodies were purchased from Affiniti Research Products (Mamhead, UK). Mouse mAb, PW 8145 and PW8140, directed against the PS constitutive subunits ß2(Z) and ß1(Y), and PW8845, directed against the iPS subunit LMP-7, were used. In similar experiments, three other rabbit polyclonal antisera, PW8350, PW8345 and PW8355, directed against the iPS subunits MECL-1, LMP-2 and LMP-7 were used. These antisera were raised against fragments of the primary structure of the subunits in a region that is unaffected by the removal of the propeptide during subunit maturation. For the PS analysis, the protein concentration of cell lysates was determined by a modification of the Lowry method (Protein Assay; Bio-Rad, Milan, Italy). BSA was employed as a standard. 50 µg of each total cell lysate was mixed with electrophoresis sample buffer and separated by SDS–PAGE followed by transfer onto nitrocellulose membrane (Hybond-C; Amersham Pharmacia Biotech, Little Chalfont, UK). Membranes were soaked overnight in PBS containing 5% milk and 0.1% Tween 20 at 4°C, and were then probed for 2 h by gentle rocking at 4°C with the specific antibody in PBS containing 1% milk and 0.1% Tween 20 at the dilution indicated by the manufacturer. After extensive washing with PBS/1% Tween 20, membranes were incubated for 1 h in the dark at room temperature with horseradish peroxidase- conjugated monoclonal anti-mouse (1:2500) or anti-rabbit (1:10, 000) antibody (Amersham Pharmacia Biotech). Following extensive washing with PBS/1% Tween 20, the enzyme substrate (ECL; Amersham Pharmacia Biotech) was added and allowed to react in the dark for 1 min. Chemiluminescence was detected with autoradiographic film. The mol. wt of each subunit was calculated using reference proteins and was indicated using the mol. wt calculated on the basis of the subunit structure.

HPLC-MS analysis of HLA class I eluted peptides
HLA class I antigen-associated peptides were obtained and analyzed from the HLA-A2+ FR cell line using a previously described technique (13,16). Briefly, the peptides were obtained from subsequent elutions of 1 x 109 cells (untreated, IFN-{gamma}-treated or IFN-{alpha}-treated) with citrate phosphate buffer at pH 3.3. Eluates were purified and then analyzed by HPLC-MS using an atmospheric pressure ionization electrospray source (59897A; Hewlett Packard) and a single quadrupole HP engine 5989-A. Using this approach, the presence of the peptide MelanAMart127–35 (AAGIGILTV, mol. wt = 813.5) was checked in full scan spectra, from untreated, IFN-{alpha}-treated or IFN-{gamma}-treated cells.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IFN-{alpha} and IFN-{gamma} effects on HLA class I expression on cultured melanoma cells
The three melanoma cell lines were cultured in vitro in the presence of IFN-{alpha} and IFN-{gamma} for 2, 4 and 8 days. After IFN withdrawal, cells were cultured for 4 and 6 additional days. Biological effects of IFN on HLA class I expression were evaluated at the specified culture times. IFN-{gamma} was more efficient than IFN-{alpha} in the up-regulation of HLA class I molecules as demonstrated by flow cytometric analysis. After IFN withdrawal, the effect of IFN-{gamma} on the up-regulation of HLA class I continued for at least 4 days in two of the cell lines analyzed, while the IFN-{alpha} effect decreased (Fig. 1).



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Fig. 1. One representative cytofluorimetric analysis on melanoma cell line 13443. HLA class I expression was evaluated using w6/32 mAb on days 2, 4 and 8 of culture in untreated (NT), IFN-{alpha}-treated and IFN-{gamma}-treated cells, and after IFN withdrawal on days 12 and 14.

 
RT-PCR analysis
The mRNA expression of the three subunits was evaluated after 48 h by RT-PCR analysis. As shown in Fig. 2, no differences between untreated and IFN-{alpha}- or IFN-{gamma}-treated 13443 melanoma cells were evident for LMP-2, LMP-7 and MECL-1. Identical results were also obtained using the GI and FR cells (not shown). LCL-2493 (positive control) amplified products of the same size as those displayed by melanoma cells. On the other hand, untreated HuF, used as negative control for iPS-specific subunits, were negative for LMP-2 and LMP-7, while the presence of MECL-1 mRNA was clearly detected. Following treatment with IFN-{gamma}, the mRNA of the three subunits was also evident in HuF (not shown).



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Fig. 2. Analysis of iPS subunit transcription by RT-PCR in untreated (NT), IFN-{alpha}-treated and IFN-{gamma}-treated 13443 melanoma cell line, LCL-2493 and HuF. Negative RT-PCR control is shown. The mol. wt markers (M) are {phi}x174/HaeIII digested on the left and pBR322/AluI digested on the right. Amplicon sizes are 547, 406 and 802 bp for LMP-2, LMP-7 and MECL-1 respectively.

 
Analysis of the specificity of polyclonal and mAb directed against PS and iPS
In order to study the three iPS specific subunits at the post-translational level, immunoblotting analyses were carried out. The specificity of the polyclonal anti-MECL-1 and LMP-2 antisera and monoclonal anti-LMP-7 antibody was evaluated on untreated or IFN-{gamma}-treated 13443 melanoma cells. The T2 cell line and either untreated or IFN-{gamma}-treated HuF were used as controls. As shown in Fig. 3, anti MECL-1 polyclonal antiserum clearly shows reactivity in both the untreated and IFN-{gamma}-treated 13443 melanoma cell line (Fig. 3A) and HuF (Fig. 3B). Interestingly, untreated cells display only the mature MECL-1 (24.6 kDa) polypeptide, while IFN-{gamma}-treated cells also display the MECL-1 precursor (28.9 kDa) and an increased level of the processed form. On the other hand, no reactivity was observed in T2 cells (genetically defective for iPS), thus assuring the specificity of recognition (Fig. 3A). LMP-2 polyclonal antiserum displays the processed form on untreated or IFN-{gamma}-treated 13443 melanoma cells. IFN-{gamma}-treated 13443 melanoma cells also display the LMP-2 unprocessed subunit (23.3 kDa, Fig. 3A). The T2 cell line expressed only the immature LMP-2 unprocessed subunit (Fig. 3A), while HuF, both untreated and IFN-{gamma} treated, did not display any reactivity (Fig. 3B). Finally, reactivity of LMP-7 mAb was analyzed. A band of 22.6 kDa relative to the mature LMP-7 polypeptide was found on both the IFN-{gamma}-treated 13443 melanoma cells (Fig. 3A) and HuF (Fig. 3B) while untreated cells were negative. The T2 cell line did not show any reactivity (Fig. 3A). Interestingly, the LMP-7 polyclonal antiserum showed a faint band, related to the immature form of LMP-7 (30.3 kDa) on IFN-{gamma}-treated HuF (Fig. 3C).



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Fig. 3. Immunoblotting analysis of the specificity of both mAb and polyclonal antisera directed against the iPS subunits. Analysis was performed on T2 cells, the melanoma cell line 13443 (both untreated and IFN-{gamma}-treated) (A) and HuF (both untreated and IFN-{gamma}-treated) (B). (C) Reactivity of polyclonal anti LMP-7 antiserum is shown on untreated and IFN-{gamma}-treated HuF.

 
Detection of PS and iPS subunits in untreated melanoma cells
Immunoblotting analysis in unstimulated cells revealed that the constitutive Y and Z subunits were expressed in all melanoma cell lines analyzed: 13443 (Fig. 4), FR (Fig. 5) and GI (data not shown). Of note, the mol. wt of the analyzed subunits (Y and Z) corresponded to those of the unprocessed form (25.3 and 29.9 kDa respectively). In the same cells, the presence of iPS-specific subunits was heterogeneous. The MECL-1 and LMP-2 subunits were detected in all three cell lines in their processed form, while LMP-7 was undetected. These data suggest that in the absence of the LMP-7 subunit and the immature form of LMP-2 and MECL-1, iPS could not be assembled in untreated melanoma cells.



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Fig. 4. Immunoblotting analysis of PS (Y and Z) and iPS (LMP-7, LMP-2 and MECL-1) subunits on untreated (NT), IFN-{alpha}-treated and IFN-treated 13443 melanoma cells. D0, D2, D4 and D8 indicate the days of treatment. At D2, two different IFN-{alpha} doses are shown ({alpha}500 = 500 UI/ml and {alpha}100 = 100 U/ml).

 


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Fig. 5. Immunoblotting analysis of PS (Y and Z) and iPS (LMP-7, LMP-2 and MECL-1) subunits on untreated (NT), IFN-{alpha}-treated and IFN-{gamma}-treated FR melanoma cells. D0, D2, D4 and D8 indicate the days of treatment. At D2, two different IFN-{alpha} doses are shown ({alpha}500 = 500 IU/ml and {alpha}100 = 100 IU/ml).

 
Detection of iPS subunits in melanoma cell lines treated with IFN-{gamma}
LMP-7 was clearly detected in melanoma cells, upon IFN-{gamma} treatment. Of note, mAb against LMP-7 revealed only the mature form of the subunit, while the polyclonal antiserum raised against the same antigen revealed not only the mature form, but also a faint band related to the immature subunit (not shown). In addition, LMP-2 as well as MECL-1 unprocessed precursors appeared. This is consistent with data obtained by analyzing specific mRNA. The time course revealed that in the GI melanoma cell line the expression of LMP-7 was induced only after 4 days of stimulation (not shown), while in the other two the expression was evident after 2 days (Figs 4 and 5). After IFN-{gamma} removal, the immune subunit expression returned to basal levels in one cell line only (GI), while in the other two it remained at detectable levels until day 6 after IFN removal (not shown). The contemporary presence of all three subunits suggested that the iPS could be correctly assembled in IFN-{gamma}-treated melanoma cells.

Detection of iPS subunits in melanoma cell lines treated with IFN-{alpha}
In the 13443 (Fig. 4), FR (Fig. 5) and GI (not shown) melanoma cell lines, IFN-{alpha} treatment failed to induce the LMP-7 subunit even after prolonged treatment (8 days) with 100 IU/ml. Increasing the concentration of IFN-{alpha} (500 IU/ml) still failed to induce the LMP-7 subunit. LMP-2 was clearly present in the mature form for all 8 days of treatment and its expression remained stable after IFN-{alpha} removal (not shown). MECL-1 was detected in the mature form in all three cell lines following IFN-{alpha} treatment. All these data are consistent with the evidence obtained by RT-PCR analysis. Surprisingly, despite the presence of clearly detectable LMP-7 mRNA in RT-PCR experiments and the availability of highly specific mAb, LMP-7 was consistently undetected in all three cell lines. Thus, the absence of the expression of the immature forms of LMP-2 and MECL-1 as well as the absence of the LMP-7 subunit suggested that the iPS could not be assembled in IFN-{alpha}-treated melanoma cells.

Detection of Y and Z PS subunits in melanoma cell lines treated with IFN-{alpha} and IFN-{gamma}
As shown in Figs 4 and 5, the expression of the constitutive subunits Y and Z was detected during stimulation with both cytokines. Following IFN-{gamma} treatment the expression of Y and Z was reduced. In particular, the expression of the Y subunit was markedly reduced from day 4 of treatment (more evident in FR cell line, Fig. 5). Treatment with IFN-{alpha} did not significantly modify Y and Z expression in treated cells.

Analysis of melanoma-associated peptides eluted from HLA class I molecules
The conflicting data regarding the presence of specific mRNA for LMP-7 and the absence of the relevant subunit in protein analysis using the immunoblot technique led to the planning of a different set of experiments to elucidate whether iPS could be present in IFN-{alpha}-treated cells. For this reason, the HLA-A2-bound MelanAMart127–35 peptide was eluted from the HLA-A2+ melanoma cell line FR and analyzed using HPLC-MS. This nonapeptide is particularly interesting because treatment with IFN-{gamma} of MelanAMart127–35+ cells resulted in a clear resistance of target cells to specific cytotoxic T lymphocytes (5). The presence of iPS, induced by IFN-{gamma} treatment, could account for the inability to generate such a peptide, thus suggesting an explanation for the resistance to lysis (5). As shown in Fig. 6, HPLC-MS clearly detected the presence of the whole MelanAMart127–35 peptide on untreated and IFN-{alpha}-treated cells. On the contrary, no ion with an m/z value corresponding to that of MelanAMart127–35 could be extracted from the total ion current chromatogram derived from IFN-{gamma}-treated cells. These findings were consistent with the presence of the PS in untreated and IFN-{alpha}-treated cells, and the presence of iPS in IFN-{gamma}-treated melanoma cells.



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Fig. 6. Ions extracted from the total ion current chromatogram obtained from HPLC-MS analysis of the peptides eluted from FR melanoma cell line. Vertical axis: m/z abundance. Horizontal axis: retention time (in min). MelanAMart127–35 peptide (m/z value = 813.5) was found in untreated (NT) and IFN-{alpha}-treated cells, but not in IFN-{gamma}-treated cells.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this paper, we obtained direct evidence that the treatment of melanoma cells with IFN-{alpha} did not result in the switch of PS to iPS. The immature forms of the three subunits were not detected during IFN-{alpha} treatment, despite the presence of mRNAs specific for LMP-2, LMP-7 and MECL-1. Propeptides of iPS subunits are required for the correct assembly of the complete structure (17), and, along this line, the immature forms of LMP-2, MECL-1 and (in certain experimental conditions) LMP-7 were evident in IFN-{gamma}-treated cells, where iPS is generated. To further support this finding, the Y and Z subunit concentration was reduced during treatment correlating inversely with the presence of the relevant iPS subunits (LMP-2 and MECL-1 respectively). At present, it is not clear why the mRNA coding for iPS subunits is not translated in IFN-{alpha}-treated cells. However, the capacity of IFN-{alpha} to inhibit the translation of peptides through the IFN-{alpha}-induced protein kinase R has been described both during infection (18) and in cancer cells (19). The presence of both mRNA and the mature forms of LMP-2 and MECL-1 in melanoma cells is intriguing. It could be related to the deep perturbation of the cellular regulatory circuits in neoplastic cells. Nevertheless, a very rapid turnover of the immature forms or the lack of clearing of the mature forms cannot be ruled out. Whatever the reason, the constitutive presence of mature LMP-2 and MECL-1 cannot per se suggest the presence of iPS. To further support the finding that iPS cannot be induced by IFN-{alpha}, the analysis of peptides eluted from HLA class I molecules on the cell surface indicated that the MelanAMart127–35 peptide, susceptible to the proteolytic activity of iPS, could be detected in the peptide mixtures eluted from untreated cells and was strongly enhanced in IFN-{alpha}-treated cells, but completely disappeared in IFN-{gamma}-treated cells. These two different pieces of evidence, obtained on the same cells using different approaches, strongly suggest that treatment with IFN-{alpha} does not induce the switch from PS, constitutively present in melanoma cells, to iPS, detectable in melanoma cells only upon IFN-{gamma} treatment. The reason why these two different possibilities occur is unclear. An active control of anti-self-immunity is obtained through the continuous exposure of self-peptides to effector cells in the absence of co-stimulatory signals (20). Thus, the switch from PS to iPS by producing novel peptides should allow more specific recognition of non-self or modified-self in cells circumvented by a pro-inflammatory environment where IFN-{gamma}, secreted by activated T cells, is present. In this report we suggest that, at least under the experimental conditions used, IFN-{alpha} does not allow the assembly of iPS subunits. Under these conditions, cells involved in an anti-viral response (where IFN-{alpha} is extensively produced by both epithelial and white blood cells) continue to express the PS-specific panel of peptides. In addition, IFN-{alpha} enhances the expression of HLA class I on the cell surface, thus increasing the potency of the antigenic signal on treated cells. Two different conditions result in the high concentration of IFN-{alpha} near target cells: viral infections and therapeutic IFN-{alpha} administration. In the former situation, provided that the above-described melanoma model is representative of other non-neoplastic cells, it is probable that the presence of IFN-{alpha}, secreted during the first phases of viral infection, mediates both the inhibition of virus proliferation and the up-regulation of HLA class I expression. HLA class I, even in the absence of the PS to iPS switch, presents a large proportion of viral-derived peptides, easily recognized as non-self signals by effector cells. Nevertheless, during the primary immune response, the frequency of specific T cells is low and this mechanism of disease control is only starting. When the clonal expansion of specific effectors is complete, it is also highly probable that the viral load inside the cell has been significantly reduced by the effect of IFN-{alpha}. Under these conditions, specific cytotoxic T lymphocytes would create a pro-inflammatory environment and the presence of iPS-specific peptides would be relevant in distinguishing between self and non-self. Thus, it may be that during viral infections, IFN-{alpha} is present during the early phases of the disease in the absence of iPS, while IFN-{gamma} appears later, switching PS to iPS to allow more accurate recognition of non-self.

A different situation can be foreseen for cancer cells, such as melanomas, treated with IFN-{alpha}. In this case, IFN-{alpha} up-regulated the expression of HLA class I, thus allowing more accurate recognition and lysis of autologous melanoma cells. The absence of any PS to iPS switch accounts for the ‘stability’ of the peptide signal, thus allowing the use of integrated strategies (such as vaccination with autologous melanoma cells, melanoma proteins or PS-specific peptides) in the experimental therapy of melanoma in humans.


    Acknowledgements
 
This work was supported in part by grants from Associazione Italiana per la Ricerca sul Cancro, Consiglio Nazionale delle Ricerche, Ministero della Sanità and Istituto Superiore di Sanità.


    Abbreviations
 
iPS—immunoproteasome

HuF—human fibroblast

LCL—lymphoblastoid cell line

LMP—low-molecular-weight protein

MECL—multicatalytic endopeptidase complex-like

PS—proteasome


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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