Analysis of Direct and Cross-Presentation of Antigens in TPPII Knockout Mice 1 Elke Firat, Jisen Huai, Loredana Saveanu, Simone Gaedicke, Peter Aichele, Klaus Eichmann, Peter van Endert and This information is current as Gabriele Niedermann of September 29, 2021. J Immunol 2007; 179:8137-8145; ; doi: 10.4049/jimmunol.179.12.8137 http://www.jimmunol.org/content/179/12/8137 Downloaded from References This article cites 37 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/179/12/8137.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • 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 by guest on September 29, 2021 *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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Analysis of Direct and Cross-Presentation of Antigens in TPPII Knockout Mice1 Elke Firat,2* Jisen Huai,2* Loredana Saveanu,† Simone Gaedicke,* Peter Aichele,‡ Klaus Eichmann,§ Peter van Endert,† and Gabriele Niedermann3* Tripeptidyl peptidase II (TPPII) is an oligopeptidase forming giant complexes in the cytosol that have high exo-, but also, endoproteolytic activity. Immunohistochemically, the complexes appear as distinct foci in the cytosol. In part controversial bio- chemical and functional studies have suggested that TPPII contributes, on the one hand, positively to Ag processing by generating epitope carboxyl termini or by trimming epitope precursors, and, on the other, negatively by destroying potentially antigenic peptides. To clarify which of these roles is predominant, we generated and analyzed TPPII-deficient mice. Cell surface levels of MHC class I peptide complexes tended to be increased on most cell types of these mice. Although presentation of three individual ؊ ؊ epitopes derived from lymphocytic choriomeningitis virus was not elevated on TPPII / cells, that of the immunodominant OVA Downloaded from epitope SIINFEKL was significantly enhanced. Consistent with this, degradation of a synthetic peptide corresponding to the OVA epitope and of another corresponding to a precursor thereof, both being proteasomally generated OVA fragments, was delayed in TPPII-deficient cytosolic extracts. In addition, dendritic cell cross-presentation of phagocytosed OVA and of OVA internalized as an immune complex was increased to about the same level as direct presentation of the Ag. The data suggest a moderate, predominantly destructive role of TPPII in class I Ag processing, in line with our finding that TPPII is not induced by IFN-␥, which up-regulates numerous, predominantly constructive components of the Ag processing and presentation machinery. The http://www.jimmunol.org/ Journal of Immunology, 2007, 179: 8137–8145. ells present samples of their protein content to CTLs via (Ct) (4) aa. Their cleavage preferences thus perfectly match the the proteolytic generation of peptides in the cytosol, preferences of human TAP and of the vast majority of MHC class C which after transport to the endoplasmic reticulum (ER)4 I alleles for the Ct peptide position. Thus, in many cases, protea- are loaded onto MHC class I molecules, which then migrate to the somes produce the epitope as well as epitope precursors, as has cell surface. The displayed peptides are 8–10 aa long. The TAP repeatedly been shown in in vitro digestion experiments (1, 3, 5). transporter in the ER membrane prefers peptides of 8–15 aa, im- Of the downstream peptidases, the ER-aminopeptidase associ- by guest on September 29, 2021 plying that both peptides corresponding to epitopes and epitope ated with Ag processing or endoplasmic reticulum-associated ami- precursors are translocated into the ER, where final trimming of nopeptidase (ERAP)1, has been investigated most intensively. epitope precursors can occur (1–3). ERAP1 appears to be the only ER-associated trimming peptidase Cytosolic endo- and aminopeptidases and ER-resident amin- in mice. Although the knockout (KO) of ERAP1 significantly re- opeptidases, but not carboxypeptidases, operate in the classical duces cell surface MHC class I expression, only a subset of epitope class I Ag processing pathway (4). Proteasomes are in many cases precursor peptides is trimmed down to final epitope size by involved in the initial processing steps. They preferentially gener- ERAP1, whereas other epitope or epitope precursor peptides are ate peptides of 3–20 aa with a hydrophobic or charged C-terminal destroyed or unaffected (6–9). ERAP2, which is exclusively ex- pressed in humans, forms in part complexes with ERAP1, and both aminopeptidases are required to efficiently trim certain epitope pre- cursor sequences (10). *Clinic for Radiotherapy, University Hospital of Freiburg, Freiburg, Germany; †In- Of the many cytosolic peptidases, leucine aminopeptidase stitut National de la Sante´et de la Recherche Me´dicale, Universite´Paris Descartes, Faculte´deMe´decine Rene´Descartes, Paris, France; ‡Institute for Medical Microbi- (LAP), thimet oligopeptidase (TOP), puromycin sensitive amino- ology and Hygiene, Department of Immunology, University of Freiburg, Freiburg, peptidase (PSA), bleomycin hydrolase, and tripeptidyl peptidase II Germany; and §Max-Planck Institute of Immunobiology, Freiburg, Germany (TPPII) have been implicated in class I Ag processing (4, 11). The Received for publication August 7, 2007. Accepted for publication October 15, 2007. endo-oligopeptidase TOP prefers peptides of 9–16 aa and prefer- 1 This work was supported by the Deutsche Forschungsgemeinschaft (NI 368/4–2), entially destroys potential epitopes and epitope precursors (12). In the Clotten Foundation, and a grant from the Forschungskommission of the University of Freiburg Medical Faculty (NIE346/04). vitro digestion and protease inhibitor experiments suggested that the aminopeptidases LAP, bleomycin hydrolase, and PSA signif- 2 E.F. and J.H. contributed equally to this work. icantly contribute to cytosolic epitope precursor trimming, but 3 Address correspondence and reprint requests to Dr. Gabriele Niedermann, Clinic for Radiotherapy, University Hospital of D-79106 Freiburg, Freiburg, Germany. E-mail studies performed in KO mice could not confirm this (13, 14). address: [email protected] With a molecular mass of ϳ6 MDa, TPPII forms the largest 4 Abbreviations used in this paper: ER, endoplasmic reticulum; Ct, C-terminal; protease complex of eukaryotic cells. These complexes are com- ERAP, endoplasmic reticulum-associated aminopeptidase; LAP, leucine aminopepti- posed of 40 identical subunits with a molecular mass of 140 kDa, dase; TOP, thimet oligopeptidase; PSA, puromycin sensitive aminopeptidase; TPPII, tripeptidyl peptidase II; KO, knockout; AAF-CMK alanine-alanine-phenylalanine- arranged in two twisted strands (15). Degradation of short and long chloromethyl ketone; DCs, dendritic cells; BM, bone marrow; LCMV, lymphocytic (up to 40 aa) oligopeptides but not proteins has been demonstrated choriomeningitis virus; VV, vaccinia virus; ID, immunodominant; WT, wild type. with purified TPPII complexes in vitro. The complexes have a Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 highly active aminotripeptidase, and some endopeptidase activity. www.jimmunol.org 8138 Ag PROCESSING IN TPPII KNOCKOUT MICE TPPII’s endopeptidase preferentially cleaves after lysine and argi- incubation with LPS (10 ␮g/ml; Sigma-Adrich) or IFN-␥ (200 U/ml; eBio- nine in long substrates (16–18). Peptides with Ct lysine residues science). Cell lysates were separated by SDS-PAGE. The blots were are not efficiently produced by proteasomes, but are preferred by probed with rabbit peptide-specific anti-TPPII Abs and goat polyclonal anti-actin Abs and developed by ECL (Amersham Biosciences). some HLA class I alleles such as HLA-A3 and HLA-A11, whose ligands may therefore be produced by alternative proteases (19). Cloning of EGFP-TPPII Purified TPPII precisely excises an HLA-A3/A11-restricted HIV- The murine TPPII c-DNA was amplified from genomic DNA by long- Nef epitope with a Ct lysine from its flanking sequences and TPPII range PCR. After subcloning into the Bluescript vector (Stratagene), the knockdown significantly reduces recognition of the epitope by insert was sequenced. EGFP was fused to the N terminus of TPPII and the CTLs (17). TPPII is thus the first and so far only nonproteasomal EGFP-TPPII fusion protein was cloned into pcDNA3 (Invitrogen) by stan- protease to be shown to generate the C terminus of a CTL epitope. dard cloning procedures. COS-7 cells were transfected using DEAE dex- tran and analyzed after 36 h by fluorescence microscopy. TPPII seems also to be required for generation of the influenza Separation of cell extracts on gel filtration columns NP147–155 epitope as suggested by siRNA and protease inhibitor experiments using the broadly specific serine protease inhibitor High molecular