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 30, 2021. Sabrina Urban, Kathrin Textoris-Taube, Barbara Reimann, Katharina Janek, Tanja Dannenberg, Frédéric Ebstein, Christin Seifert, Fang Zhao, Jan H. Kessler, Anne Halenius, Petra Henklein, Julia Paschke, Sandrine Cadel, Helga Downloaded from Bernhard, Ferry Ossendorp, Thierry Foulon, Dirk Schadendorf, Annette Paschen and Ulrike Seifert J Immunol published online 15 June 2012 http://www.jimmunol.org/content/early/2012/06/15/jimmun ol.1101886 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2012/06/15/jimmunol.110188 Material 6.DC1 Why The JI? Submit online. by guest on September 30, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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. Published June 15, 2012, doi:10.4049/jimmunol.1101886 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 Endoplasmic Reticulum-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 aminopeptidase and tripeptidyl peptidase 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 aminopeptidases 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 30, 2021 epitope presentation efficiency. The Journal of Immunology, 2012, 189: 000–000. 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 protein 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 hydrolase; 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 2 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 arginine 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