Enhances the Susceptibility to Viral Infection the Proteasome Inhibitor

The Proteasome Inhibitor Bortezomib Enhances the Susceptibility to Viral Infection Michael Basler, Christoph Lauer, Ulrike Beck and Marcus Groettrup This information is current as of September 27, 2021. J Immunol 2009; 183:6145-6150; Prepublished online 19 October 2009; doi: 10.4049/jimmunol.0901596 http://www.jimmunol.org/content/183/10/6145 Downloaded from References This article cites 50 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/183/10/6145.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 27, 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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The Proteasome Inhibitor Bortezomib Enhances the Susceptibility to Viral Infection1 Michael Basler,2*† Christoph Lauer,† Ulrike Beck,† and Marcus Groettrup*† The proteasome, a multicatalytic protease, is responsible for the generation of most MHC class I ligands. Bortezomib, a proteasome inhibitor, is clinically approved for treatment of multiple myeloma and mantle cell myeloma. In the present study, we investigated the effect of bortezomib on viral infection. Infection of bortezomib-treated mice with the lymphocytic choriomeningitis virus (LCMV) led to a decreased cytotoxic T cell response to several LCMV-derived CD8؉ T cell epitopes. Bortezomib treatment -caused a reduced expansion of CD8؉ T lymphocytes and increased viral titers in LCMV-infected mice. Administration of bort ezomib during expansion of CD8؉ T cells had no influence on the cytotoxic T cell response, suggesting that bortezomib interferes with priming of naive T cells. Indeed, determination of Ag load in spleen 4 days post infection, revealed a reduced presentation of LCMV-derived cytotoxic T cell epitopes on MHC class I molecules. In summary, we show that proteasome inhibition with Downloaded from bortezomib led to an increased susceptibility to viral infection, and demonstrate for the first time, that proteasome inhibitors can alter Ag processing in vivo. The Journal of Immunology, 2009, 183: 6145–6150. he proteasome is a large intracellular multicatalytic pro- Bortezomib (also named PS-341, or Velcade; Millennium Phar- tease found both in the cytoplasm and the nucleus (1). It maceuticals) is the first clinically approved proteasome inhibitor is the major cytosolic endoprotease in eukaryotes consist- for relapsed and/or refractory myeloma and mantle cell lymphoma. T http://www.jimmunol.org/ ing of ␣ and ␤ subunits that build a barrel-shaped complex of four The boronic acid dipeptide is a reversible proteasome inhibitor that rings with seven subunits each (called 20S proteasome) (2). The selectively and specifically inhibits the chymotrypsin-like activity outer two rings consist of ␣ subunits, and the inner two rings of ␤ of the proteasome 20S subunit (13, 14). subunits, which bear the proteolytically active centers. Three dif- Proteasome inhibitors were extensively used to study class-I Ag ferent ␤ subunits (␤1, ␤2, and ␤5) are responsible for at least three processing and presentation in vitro (15–19). Nevertheless, the in- peptidase activities: caspase-like, trypsin-like, and chymotrypsin- fluence of proteasome inhibition on Ag processing and the effect of like activities (3, 4). Multiple cellular processes like cell cycle proteasome suppression on a cytotoxic T cell response have not regulation (5), apoptosis (6), cell adhesion (7), transcription (8), been investigated in vivo. To address this issue, we used the in- and angiogenesis (9) are controlled by the proteasome. In addition, fection with lymphocytic choriomeningitis virus (LCMV) (3), a by guest on September 27, 2021 the proteasome plays a critical role in the generation of peptides noncytopathic arenavirus, to study the characteristics of Ag pro- from intracellular Ags that are presented to CTL by the MHC class cessing and presentation in PS-341 treated mice. The cytotoxic I (MHC-I)3 (10). immune response to LCMV is essential for the elimination of the Proteasome inhibitors represent novel drugs in anti-cancer ther- virus from infected mice (20). In C57BL/6 mice, this response is apy. They have been shown to induce apoptosis preferentially in shaped by CTLs specific for the dominant glycoprotein (GP)-de- transformed cells (11, 12). One mechanism of function attributed rived epitopes GP33–41/Db, GP34–41/Kb, GP276–286/Db, and to proteasome inhibitors is the induction of apoptosis via inhibition nucleoprotein (NP)-derived NP396–404/Db, as well as the sub- of activation of NF-␬B. Due to the role of NF-␬B in multiple dominant epitope NP205–212/Kb (21–23). Our results indicate that cellular processes like cell invasion, oncogenesis, proliferation, an- bortezomib reduces class-I presentation of virus-derived peptides, giogenesis, and suppression of apoptosis, inhibition of the NF-␬B and thereby suppresses the cytotoxic T cell response, leading to pathway is an attractive strategy to anti-cancer therapy. augmented viral titers. *Biotechnology Institute Thurgau (BITg) at Constance University, CH-8280 Kreu- Materials and Methods zlingen, Switzerland; and †Division of Immunology, Department of Biology, Uni- Mice, viruses, and medium versity of Constance, Konstanz, Germany C57BL/6 mice (H-2b) were originally purchased from Charles River Lab- Received for publication May 20, 2009. Accepted for publication September 10, 2009. oratories. Mice were kept in a specific pathogen-free facility and used at 6–10 wk of age. Animal experiments were approved by the review board The costs of publication of this article were defrayed in part by the payment of page of Regierungspra¨sidium Freiburg. LCMV-WE was originally obtained advertisement charges. This article must therefore be hereby marked in accordance from F. Lehmann-Grube (Heinrich-Pette-Institute, Hamburg, Germany) with 18 U.S.C. Section 1734 solely to indicate this fact. and propagated on the fibroblast line L929. LCMV was titrated on MC57 1 This work was funded by the German National Science Foundation (DFG) Grant Nr. cells as previously described (24). Mice were infected with 200 PFU GR1517/4-2. LCMV-WE i.v. All medium were purchased from Invitrogen Life Tech- 2 Address correspondence and reprint requests to: Dr. Michael Basler, Department of nologies and contained GlutaMAX, 10% FCS, and 100U/ml Biology, Division of Immunology, University of Constance, P1101 Universita¨- penicillin/streptomycin. tsstrasse 10, Konstanz, Germany. E-mail address: [email protected] 3 Abbreviations used in this paper: MHC-I, MHC class I; LCMV, lymphocytic cho- Synthetic peptides riomeningitis virus; GP, glycoprotein; NP, nucleoprotein; ICS, intracellular cytokine staining; DC, dendritic cell; VSV, vesicular stomatitis virus; NK, neutral killer; VZV, The synthetic peptides GP33–41 (KAVYNFATC), GP276–286 (SGVEN varicella zoster virus. PGGYCL), NP396–404 (FQPQNGQFI), NP205–212 (YTVKYPNL), and GP61–80 (GLNGPDIYKGVYQFKSVEFD) were obtained from P. Hen- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 klein (Charite´, Berlin, Germany). www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901596 6146 BORTEZOMIB INCREASES VIRAL SUSCEPTIBILITY Proteasome inhibitor PS-341 (bortezomib, or Velcade; Millennium Pharmaceuticals) was purchased from a local pharmacy. The proteasome inhibitor was dissolved in DMSO at 1 mg/ml and stored at Ϫ80°C. For further applications, the inhibitor was diluted in medium or PBS. For animal experiments, PS-341 was administered to mice i.p. at 0.75 mg/kg in PBS. Flow cytometry Splenocytes from LCMV-infected C57BL/6 mice were incubated for 30 min with anti-CD4 (BD Biosciences), anti-CD8 (BD Biosciences), or anti- FIGURE 1. Reduced presentation of GP in LCMV-infected PS- CD69 (BD Biosciences) Abs at 4°C. After two washes, cells were acquired 276–286 341-treated cells. A, Effect of PS-341 on GP presentation. Acid with the use of FACScan flow cytometer (BD Biosciences) and analyzed 276–286 wash-treated LCMV-infected cells were incubated with various concentra- with the FlowJo software (Tree Star). b tions of PS-341 (x-axis). The GP276–286 presentation on H-2D was ana- Propidium iodide staining lyzed with a GP276–286-specific T cell-hybridoma. Uninfected cells were used as negative control. The y-axis shows the absorbance of enzymatically T cells from splenocytes of Thy1.2-positive mice (C57BL/6) were isolated with anti-CD90.2 (Thy1.2)-coated microbeads (Miltenyi Biotec). Cells converted chromogen at 570 nm in lacZ-assays. The values are the means (4 ϫ 105/well) were stimulated with plate bound anti-CD3 (4 ␮g/ml, clone of three replicate cultures. The experiments have been repeated twice, 145–2C11) and anti-CD28 (4 ␮g/ml, clone 37.51; BD Biosciences) in the yielding similar results. B, Peptide titration for GP276-specific

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