The Efficiency of Human Cytomegalovirus pp65 495−503 CD8+ T Cell Epitope Generation Is Determined by the Balanced Activities of Cytosolic and Endoplasmic This information is current as Reticulum-Resident Peptidases of September 26, 2021. Sabrina Urban, Kathrin Textoris-Taube, Barbara Reimann, Katharina Janek, Tanja Dannenberg, Frédéric Ebstein, Christin Seifert, Fang Zhao, Jan H. Kessler, Anne Halenius,

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

+ The Efficiency of Human Cytomegalovirus pp65495–503 CD8 T Cell Epitope Generation Is Determined by the Balanced Activities of Cytosolic and -Resident Peptidases

Sabrina Urban,* Kathrin Textoris-Taube,* Barbara Reimann,* Katharina Janek,* Tanja Dannenberg,† Fre´de´ric Ebstein,* Christin Seifert,† Fang Zhao,† Jan H. Kessler,‡ Anne Halenius,x Petra Henklein,* Julia Paschke,* Sandrine Cadel,{ Helga Bernhard,‖ Ferry Ossendorp,‡ Thierry Foulon,{ Dirk Schadendorf,† Annette Paschen,†,1 and ,#,1

Ulrike Seifert* Downloaded from

Control of human CMV (HCMV) infection depends on the cytotoxic activity of CD8+ CTLs. The HCMV phosphoprotein (pp)65 is a major CTL target Ag and pp65495–503 is an immunodominant CTL epitope in infected HLA-A*0201 individuals. As immuno- dominance is strongly determined by the surface abundance of the specific epitope, we asked for the components of the cellular Ag processing machinery determining the efficacy of pp65495–503 generation, in particular, for the proteasome, cytosolic peptidases,

and endoplasmic reticulum (ER)-resident peptidases. In vitro Ag processing experiments revealed that standard proteasomes and http://www.jimmunol.org/ immunoproteasomes generate the minimal 9-mer peptide epitope as well as N-terminal elongated epitope precursors of different lengths. These peptides are largely degraded by the cytosolic peptidases leucine and II, as evidenced by increased pp65495–503 epitope presentation after leucine aminopeptidase and tripeptidyl peptidase II knockdown. Additionally, with prolyl oligopeptidase and aminopeptidase B we identified two new Ag processing machinery components, which by destroying the pp65495–503 epitope limit the availability of the specific peptide pool. In contrast to cytosolic peptidases, silencing of ER 1 and 2 strongly impaired pp65495–503-specific T cell activation, indicating the importance of ER amino- peptidases in pp65495–503 generation. Thus, cytosolic peptidases primarily interfere with the generation of the pp65495–503 epitope, whereas ER-resident aminopeptidases enhance such generation. As a consequence, our experiments reveal that the combination of cytosolic and ER-resident peptidase activities strongly shape the pool of specific antigenic peptides and thus modulate MHC class I by guest on September 26, 2021 epitope presentation efficiency. The Journal of Immunology, 2012, 189: 529–538.

urface MHC class I molecules present peptide ligands of 8– The hierarchy of specific T cell responses is determined by 11 aa residues in length that are generated by intracellular different parameters, with one major factor being the abundance of S degradation. This allows CD8+ CTLs to specifi- the epitope presented on infected cells, which is determined by the cally recognize and eliminate infected cells based on their aberrant expression level of the viral Ag as well as by the activity of different MHC ligand repertoire derived from viral Ags. Human CMV components of the cellular Ag processing machinery (APM) that (HCMV)-specific CTLs recognize epitopes derived only from a act on the target Ag. Generation of MHC class I epitopes is initiated few viral Ags, including phosphoprotein (pp)65. A specific CTL by the proteolysis of protein Ags by proteasome complexes that response directed against the immunodominant HCMV pp65495–503 can produce peptides of the correct size for direct binding to MHC epitope, a dominant target in infected HLA-A*0201-positive indi- class I molecules (4) as well as N-terminally elongated epitope viduals (1), has been shown to be of importance for the control of precursors with a length of up to 20 aa residues (5). Proteasome acute and persistent infection caused by HCMV (2, 3). products are further substrates of cytosolic and/or endoplasmic

*Institut fu¨r Biochemie, Charite´–Universita¨tsmedizin, 10117 Berlin, Germany; Address correspondence and reprint requests to Dr. Ulrike Seifert, Institute of Mo- †Department of Dermatology, University Hospital Essen, 45147 Essen, Germany; lecular and Clinical Immunology, Otto von Guericke University at Magdeburg, Leip- ‡Department of Immunohematology and Blood Transfusion, Leiden University Med- ziger Strasse 44, 39120 Magdeburg, Germany. E-mail address: ulrike.seifert@med. x ical Center, 2333 ZA Leiden, The Netherlands; Institut fu¨r Virologie, Heinrich- ovgu.de { Heine-Universita¨tDu¨sseldorf, 40225 Du¨sseldorf, Germany; De´partement de Bio- The online version of this article contains supplemental material. chimie, Universite´ Pierre et Marie Curie–Universite´ Paris 6, Laboratoire Biogene`se des Signaux Peptidiques (ER3), 75005 Paris, France; ‖III. Medizinische Klinik, Kli- Abbreviations used in this article: AP-B, aminopeptidase B; APM, Ag processing nikum Rechts der Isar, Technische Universita¨tMu¨nchen, 81675 Mu¨nchen, Germany; machinery; BH, bleomycin ; ER, endoplamic reticulum; ERAP, endoplas- and #Institute of Molecular and Clinical Immunology, Otto von Guericke University mic reticulum aminopeptidase; FL, fluorescein; HA, hemagglutinin; HCMV, human at Magdeburg, 39120 Magdeburg, Germany CMV; HeLaA2+, HeLa cells expressing HLA-A*0201; HeLaA2+/iP, HeLaA2+ cells ex- pressing immunoproteasome; LAP, leucine aminopeptidase; LCL, lymphoblastoid 1A.P. and U.S. contributed equally to this work. cell line; POP, prolyl oligopeptidase; pp, phosphoprotein; PSA, puromycin- Received for publication June 27, 2011. Accepted for publication May 4, 2012. sensitive aminopeptidase; qRT-PCR, quantitative real-time PCR; siRNA, small inter- fering RNA; TOP, thimet oligopeptidase; TPP, tripeptidyl peptidase. This work was supported in part by Deutsche Forschungsgemeinschaft Grants SFB 421, SFB-TR36 (to U.S.), and SFB 456 (to H.B.) and Deutsche Krebshilfe Grant Ó 106861 (to A.P., D.S., and U.S.), as well as by Helmholtz-Gemeinschaft Deutscher Copyright 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 Forschungszentren Alliance Cancer Immunotherapy (to A.P. and D.S.). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101886 530 HCMV pp65 PROCESSING IN THE CYTOSOL AND ER COMPARTMENT reticulum (ER)-located aminopeptidases that either contribute to After coincubation of target cells and CTLs, IFN-g production was de- epitope generation by trimming N-terminally extended precursors termined by sandwich ELISA. Abs were purchased from Mabtech. MHC to the correct epitope length or counteract epitope generation by class I cell surface expression was assessed using an anti–HLA-ABC-PE Ab from Becton Dickinson. peptide degradation (6). Among the diverse cytosolic peptidases, the puromycin-sensitive aminopeptidase (PSA) (7), bleomycin hy- Immunoblotting drolase (BH) (7), thimet oligopeptidase (TOP) (8), tripeptidyl Immunoblotting was performed as described previously (18). Briefly, 25 mg peptidase (TPP)II (9), and leucine aminopeptidase (LAP) (10) have protein of cell lysate and 1 mg purified 20S proteasome per lane were been shown to contribute to Ag processing. Antigenic peptides are separated on 10% SDS-PAGE, transferred to nitrocellulose membranes, then translocated by TAPs into the ER, where they become potential and immunoblotted for proteasome subunits, POP and AP-B (all laboratory substrates of the ER-resident aminopeptidase (ERAP)1 (11) and stock), ubiquitin (FK2; Biomol), GAPDH (Santa Cruz Biotechnology), HA (Covance), and pp65 (B. Plachter). Membranes were developed with ECL ERAP2, also referred to as leukocyte-derived aminopep- (GE Healthcare). tidase (12, 13). During infection different components of the APM, such as ERAP1, ERAP2, and the proteasome immunosubunits, are RT-PCR and quantitative real-time PCR induced by IFNs to promote the production of peptide ligands for Total RNA from HeLa cells was isolated using the RNeasy Plus Mini kit MHC class I molecules (14, 15). Thus, a detailed knowledge of (Qiagen) in combination with a RNase-free DNase set (Qiagen). Reverse the function of APM components involved in the processing of transcription was performed with the a high-capacity cDNA reverse tran- scription kit (Applied Biosystems). Expression of aminopeptidases was viral Ags is a prerequisite for understanding anti-HCMV immune analyzed by RT-PCR and quantitative real-time PCR (qRT-PCR). Primers responses. as listed in Supplemental Table IA were employed for RT-PCR. For qRT- In this study, we analyzed the contribution of the proteasome, PCR, TaqMan Universal PCR Master Mix (23) and TaqMan Ex- Downloaded from cytosolic, and ER-resident proteolytic activities to the generation of pression Assays (Applied Biosystems) listed in Supplemental Table IB the HCMV pp65 immunodominant CTL epitope. Besides were used and the reactions were performed on the StepOnePlus real-time 495–503 PCR system (Applied Biosystems). PCR conditions were as follows: 95˚C known APM components, we identified two “new players” in- for 10 min, 40 cycles at 95˚C for 15 s, and 60˚C for 1 min. Target gene volved in Ag processing, prolyl oligopeptidase (POP) and ami- expression was normalized to endogenous GAPDH level. The relative gene 2 nopeptidase B (AP-B). We present evidence that besides the expression level was calculated by the 2 DDCt method. proteasome, both cytosolic and ER-proteolytic peptidases act on Proteasome purification and in vitro peptide digestion http://www.jimmunol.org/ the pp65 Ag and that their combined activity precisely dedicates the level of generated epitopes. Standard 20S proteasomes were isolated from T2 cells and immunoprotea- somes were from T2.27 cells as described previously (16). Eight micrograms peptide was digested with 2.5 mg purified proteasome in 60 ml digestion Materials and Methods buffer (20 mM HEPES/KOH [pH 7.8], 2 mM MgAc2, 2 mM DTT) at 37˚C. Peptides and plasmids The reaction was stopped at the indicated time points by addition of 0.5% trifluoroacetic acid. The pp65481-513 33-mer polypeptide VFTWPPWQAGILARNLVPMVAT- VQGQNLKYGEF, the 9-mer epitope pp65495–503 NLVPMVATV, and the Inhibition of peptidase expression by small interfering RNA 10-mer RNLVPMVATV, 11-mer ARNLVPMVATV, 12-mer LARNLVP- knockdown MVATV, 13-mer ILARNLVPMVATV, 14-mer GILARNLVPMVATV, and by guest on September 26, 2021 15-mer AGILARNLVPMVATV precursor peptides, as well as the enkeph- Specific small interfering RNA (siRNA) oligonucleotides obtained from alin neuropeptide substrates RYGGFL and YGGFL, were synthesized using Eurogentec were used as listed in Supplemental Table IC. As control, standard Fmoc methodology on an Applied Biosystems 433A automated nontargeting siRNA from Dharmacon was employed. Cells were transfected synthesizer at .90% purity. ICP47-hemagglutinin (HA) was cloned into with X-tremeGENE siRNA transfection reagent (Roche) and 100–200 nM the BamHI and EcoRI restriction sites of pcDNA3.1 using the primers 59- siRNA according to the manufacturer’s instructions and were analyzed at CGGGATCCTAATGCGGGTTGGGCCCAGG-39 and 59-CGGAATTCT- day 3 after transfection. Transfected cells were fixed by incubation with TCACGCATAATCCGGCACATCATACGGATACATCGGGTTACCGGA- 0.008% glutaraldehyde for 3 min on ice and thereafter coincubated with TTACG-39. The plasmid pcDNA6-pp65.35 encoding full-length pp65 of pp65495–503-specific CTLs in round-bottom 96-well plates overnight. HCMV was a gift from B. Plachter (Institute for Virology, Johannes siRNA knockdown was confirmed by RT-PCR and qRT-PCR. Activities of Gutenberg University, Mainz, Germany). POP and AP-B in 5 mg total cell lysates were determined with specific fluorogenic substrates as follows: AP-B activity was assayed in 50 mM Cell lines Tris-HCl (pH 7.4), 150 mM NaCl containing 50 mM H-Arg-AMC sub- strate (Bachem) in a reaction volume of 100 ml at 37˚C. POP activity was The TAP-defective cell lines T2 and T2.27 expressing LMP2/LMP7 (16) were assayed at 37˚C in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl with 1 mM grown in RPMI 1640 (Biochrom) containing 10% FCS, 2 mM L-glutamine, DTT using 50 mM Z-Gly-Pro-AMC substrate (Bachem). Fluorescence and 100 U/ml penicillin/streptomycin (additives from PAA Laboratories). A2+ intensity was measured at 360 nm excitation and 460 nm emission HeLa transfectants expressing HLA-A*0201 (HeLa ) were grown in medium supplemented with 2 mg/ml puromycin. HeLaA2+/pp65 cells and wavelengths using a spectrofluorimeter (Synergy HT; BioTek). A2+ A2+/iP HeLa cells expressing immunoproteasomes (HeLa ) were gener- Identification of cytosolic peptidases ated by transfection of HeLaA2+ cells with pcDNA6-pp65.35 and with the proteasomal immunosubunits (b1i/LMP2, b2i/MECL-1, and b5i/LMP7) Cytosolic fractions from T2 cells were separated. Briefly, 109 cells were (Ref. 17 and Supplemental Fig. 1A), respectively. lysed in 20 mM Tris-HCl (pH 7.5), 250 mM saccharose, 3 mM KCl, 5 mM MgCl2, and 1 mM DTT and homogenized with a Dounce homogenizer. T cell stimulation and FACS analysis Lysate was centrifuged at 4,000 3 g for 10 min and 100,000 3 g for 60 + min. Supernatants were applied to a Superose 6B column, and peptidase Phosphoprotein 65 -specific CD8 T cell clones were generated by two 495–503 and proteasome activity was determined in cytosolic fractions using 100 different protocols as described before (18, 19). Sensitivity of T lymphocytes mM of the following substrates: H-Pro-AMC, H-Arg-AMC, H-Leu-AMC, was assessed by peptide titration, and thus different amounts of pp65 495–503 and Suc-Leu-Leu-Val-Tyr-AMC (Bachem). Active fractions were sepa- synthetic peptide were loaded on T2 cells and were incubated for 2 h on ice. After two washing steps, 2 3 104 T2 cells were cocultivated with rated on SDS-PAGE, and protein bands were Coomassie stained, tryptic in- 2 3 104 CTLs for 6 h (Supplemental Fig. 1B). In CTL assays the following gel digested, and measured by mass spectrometry using a MALDI-TOF/ target cells were used: a HeLaA2+/pp65 cell clone transiently expressing TOF instrument (4700 Proteomics Analyzer; Applied Biosystems) (20). A2+ A2+/iP Data were analyzed using Swiss-Prot 57.9 and NCBInr 20090513 data- ICP47, or HeLa and HeLa cells transiently expressing HCMV pp65, bases. or T2 cells loaded with 0.5 mg/ml pp65495–503 peptide. Plasmid transfection using Lipofectamine 2000 (Invitrogen) was carried out according to the Purification of AP-B and POP and in vitro peptide digestion manufacturer’s instructions. Briefly, 3 mg pcDNA6-pp65.35 or ICP47-HA was transfected for 6 and 24 h, respectively. Cellular proteasome function DNA encoding human POP was subcloned into pRSET A (Invitrogen), was inhibited by adding 1 mM epoxomicin (Merck Biosciences) for 2 h. expressed in Escherichia coli BL21, and purified with Ni-NTA agarose The Journal of Immunology 531

according to the manufacturer’s instructions (Qiagen). Human amino- 1C). The pp65495–503 minimal epitope was obtained by proteaso- peptidase B (AP-B) was purified from HeLa cells. Cytosol was separated mal cleavage between R494 and N495 defining the correct N- by glycerol gradient fractionation, followed by purification on a HiTrap terminus. Additionally, both isoforms liberated N-terminal– DEAE column (GE Healthcare) (buffer composition, 50 mM Tris-HCl [pH extended precursor peptides, the 14-mer pp65 , the 11-mer 7.5], 10 mM MgCl2, 10% glycerol, for elution 1 M NaCl). Fractions with 490-503 peak specific fluorogenic activities were purified on a MonoQ 5/50 GL pp65493-503, and the 10-mer pp65494-503 from the 33-mer poly- column (GE Healthcare). Purity of was verified by Coomassie peptide. Kinetic analysis of substrate turnover demonstrated that staining and were identified by immunoblot. AP-B depletion from immunoproteasomes degraded the 33-mer polypeptide more effi- purified fraction was performed using AP-B–specific Abs coupled to a mixture of protein A- and protein G-Sepharose beads (GE Healthcare) in ciently than did standard proteasomes (Fig. 1D), releasing higher buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 10% amounts of the epitope and its precursor peptides in vitro (Fig. glycerol. After overnight incubation at 4˚C, beads were pelleted and the 1E). Analyzing the relative abundance of the pp65495–503 epitope supernatant was analyzed by immunoblot. For in vitro digestion experi- and its N-terminal–extended precursors revealed that the 9-mer ments, 5 mg peptide was incubated with 2.4 mg purified POP in 50 ml epitope was preferentially liberated by both proteasome types. The digestion buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM DTT) at 37˚C. One microgram peptide was digested with 300 ng purified AP-B or extended precursor peptides were produced at lower amounts, the corresponding volume of fraction depleted from AP-B in 20 ml buffer with the 14-mer peptide most efficiently generated by immuno- (50 mM Tris-HCl [pH 7.5], 150 mM NaCl) at 37˚C. The reaction was proteasomes (Fig. 1E). stopped at indicated time points by addition of 0.5% trifluoroacetic acid. Samples were separated by reversed-phase chromatography on a mRPC Destruction of the HCMV pp65495–503 epitope by cytosolic C2/C18 2.1/10 column (Pharmacia Biotech) and analyzed online with peptidases electrospray ionization-mass spectrometry (LCQ and DECA; Thermo Fisher Scientific). Peptides were identified by tandem mass spectrometry The 9-mer pp65495–503 as well as its N-terminally elongated pre- Downloaded from experiments and the relative amount of generated fragments was calculated cursors liberated by the proteasome may be substrates of cytosolic using synthetic control peptides. peptidases. To study this, we downregulated the expression of the Peptide translocation assay peptidases PSA, BH, TPPII, TOP, and LAP already known to be involved in epitope processing. After exposure of HeLaA2+/pp65 A modified TAP translocation protocol using microsomes prepared from a B lymphoblastoid cell line (LCL) was performed according to Neisig et al. transfectants to aminopeptidase-specific siRNAs, pp65495–503

(21). Translocation efficiency of test peptides was measured by competi- presentation was determined by IFN-g release of specific CTLs. http://www.jimmunol.org/ tion of translocation of a fluorescein (FL)-labeled reference peptide that As shown in Fig. 2A, knockdown of BH, PSA, and TOP revealed contains a consensus site for N-linked glycosylation in the ER. Samples no significant effect on T cell stimulation, whereas silencing of were prepared containing titrated amounts of the competing test peptide and 0.5 mM FL-labeled reference peptide C(-FL)VNKTERAY in the LAP and TPPII resulted in considerably enhanced T cell activation, presence of 10 mM ATP. Per sample, 40 ml microsomes (obtained from suggesting that both peptidases are involved in postproteasomal 3 6 1.3 10 cells) was added and samples were incubated for 10 min at 37˚C. destruction of the pp65495–503 epitope and/or its precursor pep- After stop of peptide translocation, glycosylated FL reference peptide was tides. Efficient downregulation of peptidases upon siRNA trans- recovered by incubation for 2 h at 4˚C with 100 ml packed Con A-Sepharose fection was confirmed at the RNA level (Fig. 2B, 2C). 4B (GE Healthcare). Fluorescence was measured using excitation at 485 nm and emission at 535 nm. AP-B and POP activity negatively affect pp65495–503 epitope by guest on September 26, 2021 presentation Results Because a large percentage of proteasome-generated peptides is 20S proteasomes generate the HCMV pp65495–503 epitope and rapidly destroyed in the cytosol, we searched for additional ami- N-terminal extended precursor peptides nopeptidases that may modulate pp65 epitope presentation efficacy. To analyze the involvement of proteasomes in pp65495–503 Therefore, we analyzed fractionated cytosolic extracts from T2 cells epitope (NLVPMVATV) generation, HeLaA2+ cells expressing for their activity against the three fluorogenic peptidase substrates either predominantly standard proteasomes or immunoprotea- H-Pro-AMC, H-Arg-AMC, and H-Leu-AMC. The proteins of frac- somes (HeLaA2+/iP) in an IFN-independent manner were used as tions revealing strong peptidase activity were analyzed by mass Ag epitope-generating cells and APCs (Supplemental Fig. 1A). At spectrometry to identify the relevant peptidases. In addition to BH, first, proteasome inhibitor-treated and nontreated pp65-transfected LAP, PSA, TOP, and TPPII we identified POP and AP-B as potential HeLaA2+ cells were compared for their stimulatory capacity against new candidates relevant for Ag processing (Fig. 3). pp65495–503-specific CTLs. Cloned CTLs were obtained from the To test the involvement of AP-B and POP in the processing of + + peripheral blood of normal HCMV , HLA-A*0201 individuals the pp65495–503 epitope, we first performed siRNA knockdown and were characterized by a high affinity toward the HLA- experiments (Fig. 4). POP is known to hydrolyze oligopeptide + A*0201 /pp65495–503 peptide complex (Supplemental Fig. 1B). substrates at the carboxyl side of internal residues. Ac- As shown in Fig. 1, epoxomicin-treated target cells stimulated cordingly, POP activity should result in pp65495–503 epitope pp65495–503-specific T cells less efficiently than did control target destruction by cleavage after P498. Indeed, siRNA-mediated cells, as measured by IFN-g release, indicating that the proteasome downregulation of POP in HeLaA2+/pp65 cells improved epitope is essentially involved in pp65495–503 epitope generation (Fig. 1A, presentation to pp65495–503-specific T cells, indicating that POP A2+/iP 1B). Importantly, HeLa cells expressing the immunoprotea- may destroy the pp65495–503 epitope or its precursors (Fig. 4A). some independent of IFN exhibited a higher pp65495–503-specific AP-B catalyzes the cleavage of basic residues such as arginine and T cell stimulatory capacity than did cells expressing the standard from the N terminus of peptides (22). Therefore, AP-B proteasome (Fig. 1A, Supplemental Fig. 1C). should generate the correct N terminus of the pp65495–503 epi- To determine proteasomal cleavage preferences within the tope by cleaving after R494. However, knockdown of AP-B in A2+/pp65 pp65495–503 epitope-containing Ag region, synthetic pp65-derived HeLa cells improved pp65495–503 epitope presentation, 33-mer polypeptides (pp65481–513) were applied to in vitro di- suggesting that AP-B as well as POP destroyed the pp65 epitope gestion experiments with either 20S standard proteasomes or (Fig. 4A). As control for peptidase knockdown, extracts obtained immunoproteasomes. Mass spectrometric analysis of the digestion from cells silenced for POP or AP-B expression showed a strong products revealed strong proteasomal cleavage after V503 for both decrease in the degradation of Pro-AMC and Arg-AMC sub- proteasome isoforms, generating the epitope’s C terminus (Fig. strates, respectively (Supplemental Fig. 2C). 532 HCMV pp65 PROCESSING IN THE CYTOSOL AND ER COMPARTMENT Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

A2+ A2+/iP FIGURE 1. HCMV pp65495–503 epitope and precursor peptides are generated by proteasomes in vitro. (A) HeLa and HeLa cells were transiently transfected with pcDNA-pp65 for 6 h, treated with epoxomicin (1 mM) for 2 h or left untreated (c), and were cocultivated with pp65495–503-specific CTLs. Control cells were transfected with pcDNA3.1 vector (+vector), loaded with pp65495–503 peptide (peptide), or were transfected with pcDNA-pp65, treated with epoxomicin, and loaded with pp65495–503 peptide (+pp65+epoxomicin+peptide). Values are the means 6 SD of duplicate determinations. (B) Pro- teasome inhibition was verified by immunoblotting with FK2 Ab against ubiquitin. Expression level of pp65 in HeLaA2+ and HeLaA2+/iP cells was de- termined by immunoblot. GAPDH served as internal control. (C–E) Precursor polypeptide pp65481–513 encompassing the CTL epitope pp65495–503 was digested for 2, 4, 8, and 24 h with purified 20S proteasomes and cleavage preferences were determined by mass spectometry. (C) Cleavage sites and fragments are indicated by the vertical lines and the horizontal bars, respectively. Arrow thickness is proportional to the relative cleavage intensity. (D)

Analysis of pp65481-513 polypeptide turnover by 20S standard proteasomes and immunoproteasomes. (E) Relative abundance of cleavage products generated by 20S standard proteasomes and immunoproteasomes obtained by pp65481-513 digestion. Evaluation of two independent series 6 SD is shown. c, Control.

To directly assess the cleavage pattern of the two peptidases AP-B (pp65496-503) was not recognized by pp65495–503-specific CTLs as and POP we performed in vitro digestion experiments of the 14-mer demonstrated by a deficiency of IFN-g release (data not shown). (pp65490-503) proteasomal processing product using recombinant Additionally, cleavages after P498 and M499 and a cut after A501 human POP and human AP-B purified from HeLa cells (Supple- were detected, resulting in pp65495–503 epitope destruction (Fig. mental Fig. 2). The human AP-B activity has not been character- 4B, Supplemental Fig. 3B, 3C). In contrast to the 14-mer peptide, ized so far, but the peptidase displayed activities similar to those of in vitro digestion of the pp65495–503 9-mer synthetic peptide by AP- recombinant rat AP-B, resulting in a strong cleavage behind an B revealed only minor degradation (data not shown). arginine residue as observed for the RYGGFL enkephalin neuro- Analysis of POP cleavage activity revealed a predominant peptide substrate (Supplemental Fig. 3A) (22). Mass spectrometric destructive cut after P498 and a cleavage site behind A493 (Fig. analysis of digestion products obtained from 14-mer pp65490-503 4C). In summary, these results explain why silencing of AP-B digestion identified several cleavage sites in the N-terminal part of and POP lead to an increased pp65495–503 epitope presentation on the 14-mer peptide besides a cleavage after R494 that leads to the HeLaA2+/pp65 cells (Fig. 4A). Finally, we assessed the effect of AP-B generation of the pp65495–503 epitope. However, AP-B cleavage and POP knockdown on the overall MHC class I expression on activity did not stop at the N terminus of the pp65495–503 epitope, HeLa cells. As shown in Fig. 4D, in comparison with HeLa cells but proceeded and consequently destroyed the epitope by a treated with control siRNA, AP-B downregulation had no effect, cleavage behind N495. The N-terminal truncated 8-mer peptide whereas POP knockdown slightly decreased MHC class I levels. The Journal of Immunology 533

AB

FIGURE 2. Effect of cytosolic APs on A2+/pp65 pp65495–503 epitope generation. (A) HeLa cells were transfected with control siRNA (siC) or siRNA specific for the indicated peptidases.

After 72 h, cells were evaluated for pp65495–503 epitope presentation to specific CTLs. Activation of T cells was determined by IFN-g ELISA. T2 C cells without and with pp65495–503 peptide served as an additional control. Values are the means 6 SD of duplicate determinations. One experiment Downloaded from out of three is shown. (B) RT-PCR amplification and (C) qRT-PCR specific for the indicated transcripts confirmed siRNA knockdown of the specific peptidase. GAPDH and pp65 expres- sion levels were determined as controls. http://www.jimmunol.org/ by guest on September 26, 2021

TAP mediates transport of the pp65495–503 epitope and its derived peptides are transported by TAP into the ER and might be 10- and 11-mer precursors into the ER a substrate for ERAP1 and ERAP2. Therefore, TAP-dependent peptide translocation was analyzed using purified microsomes. The observation that pp65495–503 epitope and/or its precursors are destroyed in the cytosolic compartment by the activity of TPPII, The translocation efficiency of pp65 peptides was determined by LAP, POP, and AP-B raised the question as to which of the pp65- their capability to interfere with the translocation of a simulta- neously added FL-labeled reference peptide (21). Interestingly, short peptides, such as the minimal pp65495–503 epitope and also 10- and 11-mer precursor peptides, were efficiently transported by TAP (Fig. 5A), whereas translocation of longer precursor peptides, for example, 12–15 mers, was strongly diminished (Fig. 5B). To confirm TAP-dependent transport of pp65 peptides in a cellular environment, we specifically inhibited TAP activity by ex- pression of the HSV immediate early protein ICP47. Activation of pp65495–503-specific CTLs was significantly reduced, although not completely abrogated, after coincubation with ICP47-expressing target cells compared with control cells (Fig. 5C).

Activity of ER-resident aminopeptidases enhances pp65495–503 epitope presentation

Because 10- and 11-mer precursors of the pp65495–503 epitope are efficiently transported by TAP into the ER, we tested the role of ERAP1 and EARP2 in precursor trimming. Downregulation of ERAP1 or ERAP2 expression in HeLaA2+/pp65 cells by siRNAs FIGURE 3. Identification of cytosolic peptidase activities. Cytosol from reduced epitope presentation to pp65 -specific CTLs (Fig. T2 cells was purified and fractionated. Fractions were analyzed for post- 495–503 prolyl (Pro-AMC), postarginine (Arg-AMC), postleucine (Leu-AMC), and 6A) and silencing of both ERAP1 and ERAP2 did not enhance this proteasome (Suc-Leu-Leu-Val-Tyr-AMC) activity and fractions displaying effect. This suggests that 1) ERAPs predominantly act on 10- and peptidase activity were analyzed by mass spectrometry. The following 11-mer precursors to support the generation of the minimal epi- peptidases could be identified and attributed to the different fraction pools: tope, and 2) ERAPs do not degrade substantial amounts of the BH, LAP, PSA, TPPII, AP-B, POP, and TOP. 9-mer epitope, which is also efficiently transported into the ER. 534 HCMV pp65 PROCESSING IN THE CYTOSOL AND ER COMPARTMENT

A siC siAP-B siPOP AP-B >800 POP FIGURE 4. AP-B and POP destroy pp65 the pp65495–503 epitope. (A) Left, 400 GAPDH A2+/pp65 HeLa cells were transfected with (pg/ml) γ 300 AP-B POP control siRNA (siC) or siRNA specific 1.6 1.8 IFN- for the indicated peptidases. After 72 h, 200 1.4 1.6 1.2 1.4 cells were evaluated for pp65495–503 100 1.2 1 1 epitope presentation to specific CTLs. 0.8 0 0.8

Activation of T cells was determined by relative 0.6 0.6 0.4 IFN-g ELISA. T2 cells without and with siC AP-B POP T2 T2 +peptide 0.4 gene expression 0.2 0.2 pp65495–503 peptide served as an addi- 0 0 tional control. Values are the means 6 siC siAP-B siPOP siC siAP-B siPOP SD of duplicate determinations. One experiment out of three is shown. Right, AP-B POP B 100 C 100 RT-PCR amplification and qRT-PCR analysis of the indicated transcripts con- 80 80 Downloaded from firming siRNA-mediated knockdown. 60 60 GAPDH and pp65 cDNA amplifications were performed as controls. (B and C) 40 40

The 14-mer pp65490-503 peptide was 20 20 digested with AP-B (B) or POP (C) for substrate (%) pp65-14mer the indicated time points and substrate substrate (%) pp65-14mer 0 0 0 2 4 8 0 20 45 60 120

turnover was determined by mass spec- http://www.jimmunol.org/ time of digest (h) time of digest (min) trometry. Lower panels indicate cleav- age sites and fragments by arrows and horizontal bars, respectively. Dominant 490 495 503 490 495 503 cleavages are marked by thick arrows. G I L A R N L V P M V A T V G I L A R N L V P M V A T V Evaluation of two independent series 6 SD is shown. (D) Left, MHC class I ex- pression on HeLa cells transfected with siRNA specific for AP-B, POP, and TOP AP-B POP was measured by flow cytometry. Ex- D 1.6 1.2 1.4

1 by guest on September 26, 2021 pression on HeLa cells transfected with 1.2 0.8 control siRNA (siC) was set to 0; bars 1 0.8 0.6 3535 0.6 indicate deviation of MHC class I ex- 3030 relative 0.4 2525 0.4 pression on HeLa cells transfected with 2020 0.2 0.2 gene expression the indicated peptidase. Evaluation of 1515 0 0 1010 siC siAP-B siPOP siTOP siC siAP-B siPOP siTOP two independent series is shown. Right, 55 00 -5 TOP siRNA knockdown was verified by qRT- -5 1.2 -10-10 deviation of PCR analysis. -15-15 1 -20-20 -25-25 0.8 -30-30

HLA classHLA expression I (%) siAP-B siTOP siPOP 0.6 0.4 relative 0.2

gene expression 0 siC siAP-B siPOP siTOP

Additionally, in vitro peptide trimming experiments revealed that hanced liberation of the 9-mer pp65495–503 epitope by immuno- rERAP1 did not degrade the pp65495–503 epitope (S. Urban, proteasomes compensates in part the reduced ERAP1 trimming K. Textoris-Taube, B. Reimann, A. Halenius, F. Ebstein, activity. D. Schadendorf, A. Paschen, U. Seifert, and P.-M. Kloetzel, unpublished observations). Discussion As indicated above, our results demonstrate that immunopro- This study sought to elucidate the multistep processing pathway of teasomes release higher amounts of the pp65495–503 epitope and its the pp65495–503 epitope to define those components contributing to precursors from the pp65 Ag (see Fig. 1A, 1E). Thus, we inves- the generation of an immunodominant CTL target structure. We tigated the influence of ERAPs on pp65495–503 epitope generation followed the fate of this specific CTL epitope by analyzing all in HeLaA2+/iP cells, which constitutively express the proteasomal components that have been attributed to the Ag processing path- subunits b1i, b2i, and b5i and that were transiently transfected way so far. Our analysis revealed that in a first step standard and to express the pp65 protein. Similar to our results obtained for immunoproteasomes directly generate the 9-mer epitope as well as HeLaA2+/pp65 cells containing mainly standard proteasomes, knock- N-terminally elongated precursors. Although the cleavage pattern of down of either ERAP1 or ERAP2 in HeLaA2+/iP cells resulted in both proteasome types is similar, substrate turnover by the immu- impaired pp65495–503 presentation (Fig. 6B). However, the ef- noproteasome occurs much faster. Accordingly, cells expressing fect of ERAP1 knockdown on pp65 epitope generation in the immunoproteasome showed an increased capacity to stimulate A2+/iP+pp65 HeLa cells was less prominent, suggesting that the en- pp65495–503-specific CTLs. Previous studies on other epitopes The Journal of Immunology 535

AB

FIGURE 5. Efficient translocation of pp65495–503 and its 10- and 11-mer precursors by TAP. The pp65495–503 9-mer epitope and precursors of differ- ent length were tested for their ability to compete for translocation by TAP with an FL-labeled reference peptide using microsomes of B LCL cells. (A) Ef- ficient translocation of the 9-mer epitope and 10- and 11-mer precursors by TAP. (B) No translocation of 12-, 13-, 14-, and 15-mer peptides. One repre- C sentative experiment out of three is shown. (C) Left, HeLaA2+/pp65 cells were transiently transfected with ICP47-HA for 24 h or left untreated and were cocultivated with pp65495–503-specific CTLs. Con- trol cells were transfected with pcDNA3.1 vector (+vector), and ICP47-HA–expressing cells (white bar) as well as T2 cells were loaded with pp65495–503 peptide (+peptide). Values are the means 6 SD of duplicate determinations. One experiment out of two Downloaded from is shown. Right, protein expression was verified by immunoblotting using anti-pp65 and anti-HA Abs. GAPDH served as loading control. Lower panel, Inhibition of peptide translocation by ICP47 was verified by detection of reduced MHC class I cell surface expression on HeLaA2+/pp65 by flow cytom- http://www.jimmunol.org/ etry. Background fluorescence (c, nonlabeled); MHC expression on nontransfected cells (c), on control transfected cells (+vector), on ICP47-HA transfected cells (+ICP47-HA). c, Control.

from viral Ags demonstrated a stronger activation of Ag-specific compensate for the negative effects of TPPII (see also Fig. 1). by guest on September 26, 2021 CD8+ T cells by immunoproteasome-expressing target cells owing Overall, the role of TPPII in Ag processing is discussed contro- to enhanced epitope liberation (23, 24). However, the proteasomal versially, indicating limitation of the peptide pool on the one hand cleavage is only the initial step in the processing cascade of (31) and epitope enhancing effects on the other hand (30, 32–34). pp65495–503, as we observed that pp65495–503-containing peptides Importantly, we identified the two peptidases AP-B and POP are substrates of the cytosolic peptidases LAP, TPPII, AP-B, and as new cytosolic APM components. So far, their physiological POP. Activity of all these peptidases destroys the pp65495–503 function is unclear. Besides their cytosolic localization, AP-B and epitope. POP can be localized in intracellular vesicles as well as in nuclei In general, the relative contribution of cytosolic peptidases to and on the plasma membrane, which depends on the cell type epitope processing as well as the crosstalk between cytosolic and analyzed as well as on cellular activity and developmental status ER-resident peptidases to generate one specific epitope has not yet (35–37). AP-B is implicated to play a role in the processing of been determined. Several studies in mice being deficient in certain glucagon (38) and in the conversion of cholecystokinin 9 (39). cytosolic peptidases revealed only marginal effects of peptidase Moreover, AP-B has a close structural relationship with leuko- deficiency on overall Ag presentation (25–28). This suggested that triene A4 hydrolase, suggesting a putative function in the gener- APs have redundant functions in Ag processing and can com- ation of inflammatory lipid mediators (40). When analyzing the pensate each other in the processing of MHC class I-presented role of AP-B in pp65495–503 epitope processing, we detected a peptides. However, in contrast to the indifferent data obtained in negative effect of AP-B on epitope generation when using the AP-deficient mice, a detailed analysis of human cells provided 14-mer peptide as a substrate. However, the pp65495–503 9-mer evidence that the cytosolic aminopeptidases PSA and BH affect synthetic peptide was not efficiently degraded by AP-B, which the peptide loading and MHC class I surface expression in a dif- might be explained by the absence of the arginine residue R494 ferential allele-specific manner (29). This is consistent with our located at the epitope’s N terminus. results that show a balanced pattern of peptidase activity to gen- POP is expressed in various tissues with high concentration erate defined amounts of epitope. Our results indicate that similar detectable in brain and is involved in the cleavage of neuropeptides to LAP, TPPII also negatively affects pp65495–503 epitope gener- (41). POP activity has been shown to negatively affect the gen- ation as demonstrated by enhanced pp65 epitope presentation after eration of a subset of CD4+ T cell epitopes derived from gluten TPPII knockdown. In contrast, previous observations by Die- (42). Our data obtained from pp65490-503 in vitro digestion ex- kmann et al. (30) revealed no effect of TPPII on pp65 processing periments with rPOP show a dominant cleavage after P498 within in EBV-transformed B LCLs. An explanation for the observed the epitope. Interestingly, downregulation of POP slightly de- discrepancy might be the analysis of different cell lines since creased MHC class I expression on HeLa cells, in contrast to TOP, LCLs primarily contain immunoproteasomes, thereby providing which showed the opposite effect as described before (8) (see also enhanced amounts of proteasomal cleavage products that may Fig. 4D). Thus, we assume that POP might contribute to the 536 HCMV pp65 PROCESSING IN THE CYTOSOL AND ER COMPARTMENT

A >400

(pg/ml) 200 γ IFN- 100

0 siC E1 E2 E1+E2 T2 T2+peptide

FIGURE 6. ERAP activity is important for efficient HeLaA2+/pp65 pp65495–503 epitope presentation. (A) Upper panel, A2+/pp65 HeLa cells were transfected with control ERAP1 ERAP2 siRNA (siC) or siRNA specific for the indicated pep- 1.2 1.6 siERAP1 siERAP2 si C siE1+siE2 1.4 1 tidases. After 72 h, cells were evaluated for pp65495–503 ERAP1 1.2 epitope presentation to specific CTLs. Activation of 0.8 1 T cells was determined by IFN-g ELISA. T2 cells ERAP2 0.6 0.8 0.6 without and with pp65495–503 peptide served as an ad- pp65 0.4 A2+/iP 0.4 B 0.2 Downloaded from ditional control. ( ) Upper panel, HeLa cells GAPDH 0.2 were transiently transfected with a plasmid encoding 0 0 relative generelative expression pp65 after incubation with control siRNA (siC) or siC siE1 siE2 siE1+E2 siC siE1 siE2 siE1+E2 siRNA specific for the indicated peptidases. After >1000 72 h, cells were evaluated for pp65495–503 epitope B presentation to specific CTLs. T2 cells without and with pp65495–503 peptide served as control as well as

A2+/iP A2+/iP cells http://www.jimmunol.org/ nontransfected HeLa cells (2)andHeLa 600 transiently transfected with pcDNA3.1 vector (+V). (pg/ml) Values are the means 6 SD of duplicate determi- γ 400

nations. One representative out of two is depicted. (A IFN- and B) Lower panels, RT-PCR amplification and qRT- 200 PCR analysis of the indicated transcripts confirming siRNA knockdown of the specific aminopeptidase. 0 GAPDH- and pp65-specific RT-PCRs were performed siC E1 E2 E1+E2 +V (-) T2 T2+peptide as controls. HeLaA2+/iP +pp65 by guest on September 26, 2021

ERAP1 ERAP2 1.2 2.0 1.8 si C siERAP1 siERAP2 siE1+siE2 1 1.6 1.4 ERAP1 0.8 1.2 ERAP2 0.6 1 0.8 0.4 pp65 0.6 0.2 0.4 GAPDH 0.2 0 0 relative generelative expression siC siE1 siE2 siE1+E2 siC siE1 siE2 siE1+E2

generation of some epitopes owing to its capacity to remove the two amino acids at the position next to the final epitope’s N proline residues from precursor peptides, therefore enhancing their terminus takes place predominantly in the ER (44). Furthermore, affinity to TAP transporter molecules (21). our observations correlate with the model that ERAP1 works via Altogether, the destructive activity of the four cytosolic pepti- a “molecular ruler” mechanism that protects 9-mer epitopes from dases LAP, TPPII, AP-B, and POP on pp65495–503-containing further degradation and with the structural composition of ERAP1 sequences supports the view of the cytosol as a highly peptide that is supposed to act mainly on antigenic precursor peptides (45, degradative compartment. It has been demonstrated that .99% of 46). Importantly, double knockdown of ERAP1 and ERAP2 the cytosolic peptide pool is destroyed before peptides are trans- revealed no further impairment of pp65495–503 epitope presenta- ported by TAP (43). Our in vitro translocation assays suggest that tion, suggesting no additive effects of ERAP1 and ERAP2 on the 9-mer pp65495–503 epitope as well as 10- and 11-mer pre- pp65 epitope processing. However, it has been demonstrated that cursors might escape from cytosolic degradation most efficiently both aminopeptidases form complexes (13). With regard to our as they are preferentially transported via TAP in contrast to 12–15 results on pp65 epitope processing, this suggests that ERAP1 and mers. Within the ER the 10- and 11-mer epitope precursors are ERAP2 act more efficiently in complexes, and therefore removing substrates of ERAP1 and ERAP2, as knockdown of either of the one would be similarly efficient as removing both. two peptidases strongly impaired pp65495–503 presentation. Thus, For ERAP1, several studies in the human system in vitro as well both peptidases do not seem to act substantially on the pp65495–503 as in mice demonstrated that it can trim precursor peptides to their 9-mer epitope, because otherwise an increase in specific epitope correct length for efficient MHC class I binding (11, 47) and that its presentation after ERAP knockdown would have been expected. deficiency leads to reduced surface expression of MHC class I This is supported by previous data demonstrating that trimming of molecules (47–49). The role of ERAP1 in determining antiviral The Journal of Immunology 537 immunohierarchies has been analyzed in knockout mice, but this third member of the oxytocinase subfamily of aminopeptidases. J. Biol. Chem. 278: 32275–32283. is still under debate (48, 49). However, a recent study by Tenzer 13. Saveanu, L., O. Carroll, V. Lindo, M. Del Val, D. Lopez, Y. Lepelletier, F. Greer, et al. (50) demonstrated that ERAP1, besides the proteasome and L. Schomburg, D. Fruci, G. Niedermann, and P. M. van Endert. 2005. Concerted TAP, controls the abundance of immunodominant HIV epitopes in peptide trimming by human ERAP1 and ERAP2 aminopeptidase complexes in the endoplasmic reticulum. Nat. Immunol. 6: 689–697. humans. Thus, ERAPs might be of specific importance for the 14. Shin, E. C., U. Seifert, S. Urban, K. T. Truong, S. M. Feinstone, C. M. Rice, generation of immunodominant CTL epitopes. In contrast, our P. M. Kloetzel, and B. Rehermann. 2007. Proteasome activator and antigen- data suggest that enhanced epitope generating capacity, which is processing aminopeptidases are regulated by virus-induced type I interferon in the hepatitis C virus-infected liver. J. Interferon Cytokine Res. 27: 985–990. mediated by immunoproteasomes, can at least in part compensate 15. Strehl, B., T. Joeris, M. Rieger, A. Visekruna, K. Textoris-Taube, for ERAP1 deficiencies. S. H. Kaufmann, P. M. Kloetzel, U. Kuckelkorn, and U. Steinhoff. 2006. Immunoproteasomes are essential for clearance of Listeria monocytogenes In summary, following the fate of the pp65495–503 CTL epitope in nonlymphoid tissues but not for induction of bacteria-specific CD8+ we demonstrate that a series of APM components are decisive for T cells. J. Immunol. 177: 6238–6244. the generation of the epitope. Our data suggest that the surface 16. Kuckelkorn, U., S. Frentzel, R. Kraft, S. Kostka, M. Groettrup, and P. M. Kloetzel. 1995. Incorporation of major histocompatibility complex: abundance of the epitope is determined by the balanced combi- encoded subunits LMP2 and LMP7 changes the quality of the 20S protea- nation of cytosolic and ER-resident proteolytic activities. Ac- some polypeptide processing products independent of interferon-g. Eur. J. cordingly, disturbance of this balance can impair the processing of Immunol. 25: 2605–2611. 17. Seifert, U., L. P. Bialy, F. Ebstein, D. Bech-Otschir, A. Voigt, F. Schro¨ter, immunodominant epitopes, resulting in an inefficient elimination T. Prozorovski, N. Lange, J. Steffen, M. Rieger, et al. 2010. Immunoproteasomes of infected cells or can lead to an enhanced generation of sub- preserve protein homeostasis upon interferon-induced oxidative stress. Cell 142: 613–624. dominant epitopes from viral and host Ags, with the latter being of 18. Ebstein, F., N. Lange, S. Urban, U. Seifert, E. Kru¨ger, and P. M. Kloetzel. 2009. potential relevance for the development of autoimmune diseases. Maturation of human dendritic cells is accompanied by functional remodelling Downloaded from How the interplay of these components in general defines CTL of the ubiquitin-proteasome system. Int. J. Biochem. Cell Biol. 41: 1205–1215. 19. Bernhard, H., B. Schmidt, D. H. Busch, and C. Peschel. 2005. 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C. J. Melief, and J. J. Neefjes. 1995. Major differences in transporter associated with http://www.jimmunol.org/ Acknowledgments antigen presentation (TAP)-dependent translocation of MHC class I-presentable We thank Peter-M. Kloetzel for a critical reading of the manuscript and for peptides and the effect of flanking sequences. J. Immunol. 154: 1273–1279. 22. Cadel, S., A. R. Pierotti, T. Foulon, C. Cre´minon, N. Barre´, D. Segre´tain, and helpful discussions. We thank E. Bu¨rger and A. Niewienda for excellent P. Cohen. 1995. Aminopeptidase-B in the rat testes: isolation, functional prop- technical assistance. erties and cellular localization in the seminiferous tubules. Mol. Cell. Endo- crinol. 110: 149–160. 23. Sijts, A. J., T. Ruppert, B. Rehermann, M. Schmidt, U. Koszinowski, and Disclosures P. M. Kloetzel. 2000. Efficient generation of a hepatitis B virus cytotoxic The authors have no financial conflicts of interest. T lymphocyte epitope requires the structural features of immunoproteasomes. J. Exp. Med. 191: 503–514. 24. van Hall, T., A. Sijts, M. Camps, R. Offringa, C. Melief, P. M. Kloetzel, and

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