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

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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 © 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 hybrid- presence of various PS-341 concentrations. Twenty-four hours later, dead omas. MC57 were incubated with indicated peptide concentrations (x-axis)

cells were stained with propidium iodide (BD Biosciences) and analyzed and used as stimulator cells for GP276–286-specific T cell hybridomas in Downloaded from by flow cytometry. lacZ assays. The y-axis shows the absorbance of enzymatically converted Intracellular cytokine staining (ICS) for IFN-␥ chromogen at 570 nm in lacZ-assays. Analysis of T cell responses was performed as previously detailed (25). In brief, splenocytes were incubated in round-bottom 96-well plates with 10Ϫ7 Mor10Ϫ6 M of the specific peptide in 100 ␮l IMDM 10% plus using epoxomicin and lactacystin to investigate Ag presentation brefeldin A (10 ␮g/ml) for5hat37°C. The staining, fixation, and perme-

(18). Hence, bortezomib is capable of inhibiting Ag presenta- http://www.jimmunol.org/ abilization of the cells were performed exactly as detailed previously (26). tion in vitro. LacZ assay Reduced cytotoxic T cell response in bortezomib-treated mice MC57 (C57BL/6-derived methylcholantrene-induced fibrosarcoma cell line) cells were infected with LCMV-WE (M.O.I. of 0.05). An acid To analyze the impact of PS-341 on the cytotoxic T cell response, wash procedure was performed to remove pre-existing peptide/MHC we used the well-characterized LCMV as model system for viral complexes from the surface. Cells were resuspended in acid wash buffer infection. C57BL/6 mice were infected with LCMV-WE (day 0) (131 mM citric acid and 66 mM disodium phosphate (pH 3.1)) for 3 and treated three times with bortezomib (days Ϫ1, 1, and 3). Start- min. Cells were washed three times with ice-cold medium. Removal of MHC was confirmed by staining for H-2Db and analysis by flow cy- ing dose of bortezomib to treat multiple myeloma patients is cur-

2 by guest on September 27, 2021 tometry. Cells were incubated for 6 h with indicated concentrations of rently 1.3 mg/m , which leads to an ϳ60–80% inhibition of the PS-341. Cells were washed twice and incubated with GP276-specific T proteasome’s activity in the blood 1 h post treatment (27, 28). To 5 cell-hybridomas (18). Five ϫ 10 T cell-hybridomas were cocultured achieve a comparable inhibition in mice, we used a PS-341 dose ϫ 5 overnight with 5 10 stimulator cells in 96-well plates overnight. The of 0.75 mg/kg, which inhibits the proteasome to ϳ80% (29). lacZ-based color reaction was performed and measured as detailed else- where (18). Eight days post LCMV-WE infection, the cytotoxic T cell response in LCMV-infected PS-341-treated and untreated mice Peptide-specific CTL-lines was assayed for their responses to four LCMV-derived epitopes LCMV-specific CTL-lines were generated exactly as previously described by ICS for IFN-␥ (Fig. 2A). CTL-responses to all epitopes were (19). An additional density centrifugation step was conducted 1–2 days significantly reduced to ϳ50%. To control whether the LCMV- before using CTL in Ag presentation experiments. CTL were used in ICS WE-specific Th cell response was similarly affected by bort- at an E:S ratio of 0.2 in the first dilution, and serial 3-fold dilution of ϩ stimulators were performed. ezomib, GP61–80-specific CD4 T cells were analyzed by ICS for IFN-␥ (Fig. 2B). In contrast to the LCMV-specific CTL- Statistical analysis response, no significant difference could be observed for the

Statistical significance of differences was determined using Student’s t test. CD4 T cell epitope GP61–80. LCMV induces a massive cyto- Differences were considered significant when p Ͻ 0.05. toxic T cell proliferation leading to huge numbers of LCMV- specific CTLs (30, 31). To investigate whether bortezomib Results treatment affects the LCMV-induced T cell expansion, CD4ϩ Bortezomib reduces Ag presentation in vitro and CD8ϩ lymphocytes were analyzed in spleen by flow cy- The proteasome plays a critical role in the generation of peptides tometry (Fig. 3). PS-341 led to a 30% reduction (from 45 to ϩ from intracellular Ags that are presented to CTL by the class-I 30%) of the percentage of CD8 of the total lymphocyte pop- ϩ MHC (10). To investigate whether the proteasome inhibitor bort- ulation, whereas CD4 lymphocytes were even slightly in- ezomib is able to alter Ag presentation in vitro, the fibroblast line creased. Taken together, bortezomib administration impairs the MC57 was infected with LCMV-WE for 24 h. Subsequently, the LCMV-WE specific cytotoxic T cell response. pre-existing epitopes from class-I molecules were removed by acid wash procedure and incubated for 8 h with bortezomib concentra- Impaired viral clearance in bortezomib-treated mice tions ranging from 12.5 to 2000 nM. The amount of LCMV-de- Viral load in LCMV-WE infected mice, peaking at day 4 post rived GP276–286 presented on MHC-I was detected by the help of infection, is controlled by the CTLs response, leading to barely

GP276–286-speciﬁc T cell-hybridomas in lacZ assays (Fig. 1). detectable virus titers on day 8 post infection (32). To investigate Increasing inhibitor concentrations led to a reduced GP276–286 whether the impaired T cell response in inhibitor treated mice presentation in a concentration-dependent manner, conﬁrming manifests in increased viral load, C57BL/6 mice were infected the proteasome dependency of GP276–286 of an earlier study with LCMV-WE (day 0) and treated three times with bortezomib The Journal of Immunology 6147

FIGURE 2. Impaired LCMV-specific T cell responses in PS-341 treated mice. C57BL/6 mice were infected with LCMV-WE on day 0. On days Ϫ1, 1, and 3, mice were treated with PS-341 (0.75 mg/kg, i.p.) (ϩ) or were left untreated (Ϫ). Eight days post infection, spleen cells were harvested, stimulated in vitro with the indicated peptides for 5 h, and analyzed by flow cytometry after staining for CD8 and intracellular IFN-␥. Shown are the percentages of IFN-␥-positive cells of CD8ϩ cells (y-axis) as determined by flow cytometry. Unstimulated cells (no peptide) were used as a negative control. The experiments have been repeated three times, yielding similar results. Downloaded from

(days Ϫ1, 1, and 3). On day 6 post infection, virus titers in spleen No difference in early T cell activation in PS-341-treated mice of LCMV-infected PS-341-treated and untreated mice were as- PS-341 treatment of mice in the beginning of an LCMV-WE ϳ sayed (Fig. 4). Virus load in spleen was increased by 10 times in infection led to a tremendous reduction in the LCMV-speciﬁc PS-341-treated mice. cytotoxic T cell response (Fig. 2 and 3). To address whether the

Bortezomib treatment during expansion phase is not affecting activation of T cells is altered in the presence of bortezomib, http://www.jimmunol.org/ ␣ ␣ LCMV-specific CTL-response CD3/ CD28 stimulated T cells were treated with indicated concentrations of PS-341 in vitro. Twenty-four hours later, cell Priming of LCMV-specific T cells takes place during the first death was analyzed by propidium iodide staining (Fig. 6A). In days of infection leading to detectable LCMV-specific CTLs on contrast to unstimulated T cells, CD3/CD28-activated T cells dem- day 5 post infection, followed by a massive expansion, which onstrated increasing cell death in a dose-dependent manner at con- leads to an apex in the number of LCMV-specific T cells at day centrations between 2.5 and 20 nM. These results are in agreement 8 post infection (31, 32). To investigate whether massively pro- with an earlier publication, showing enhanced cell death in PHA- liferating T cells are sensitive to bortezomib in vivo, mice were treated T lymphocytes (33). To investigate whether bortezomib infected with LCMV-WE (day 0) and treated with PS-341 on alters activation of lymphocytes in vivo, the early activation by guest on September 27, 2021 days 4 and 6. The cytotoxic T cell response was assayed on day marker CD69 was analyzed. Bortezomib treated or untreated mice 8 post infection for responses to four defined LCMV-derived were infected with LCMV-WE and CD69 expression on CD4 and ␥ epitopes by intracellular cytokine staining for IFN- (Fig. 5). In CD8 cells was analyzed by flow cytometry 1 day post infection contrast to the administration of PS-341 during the priming (Fig. 6B). No difference in percentage of either CD4ϩCD69ϩ or phase (Fig. 2), the cytotoxic T cell response was not reduced in CD8ϩCD69ϩ could be observed between PS-341-treated and un- this experimental setup. Unexpectedly, the T cell response was treated mice. Therefore, we conclude that in vivo proteasome in- even slightly increased to all epitopes in PS-341-treated mice. hibition does not alter early T cell activation. Hence, administration of bortezomib does not hamper the expansion of CTLs during LCMV-WE infection if treatment com- Reduced Ag presentation in PS-341-treated mice mences after completion of the priming phase. Apart from impeded T cell activation of PS-341 treated T lymphocytes, proteasome inhibition in bortezomib-treated mice might negatively affect Ag presentation in vivo. The number of peptide- MHC-I-complexes required to optimally activate naive CD8ϩ T

FIGURE 3. Reduced expansions of CTLs in PS-341 treated mice. C57BL/6 mice were infected with LCMV-WE on day 0. On days Ϫ1, 1, and 3, mice were treated with PS-341 (0.75 mg/kg, i.p.) (ϩ) or were left FIGURE 4. Increased viral titers in PS-341-treated mice. C57BL/6 mice untreated (Ϫ). Eight days post infection, spleen cells were harvested, were infected with LCMV-WE on day 0. On days Ϫ1, 1, and 3, mice were stained for CD4 or CD8, and analyzed by ﬂow cytometry. Shown are the treated with PS-341 (0.75 mg/kg, i.p.) (ϩ) or were left untreated (Ϫ). Six percentages of CD4- or CD8-positive cells of lymphocytes (y-axis) as de- days post infection, LCMV titers were determined in the spleen. Titers are termined by ﬂow cytometry. The experiments have been repeated three given in PFU LCMV-WE per spleen (y-axis). The experiments have been times, yielding similar results. repeated twice, yielding similar results. 6148 BORTEZOMIB INCREASES VIRAL SUSCEPTIBILITY

FIGURE 5. LCMV-specific T cell responses in mice treated with PS-341 during the T cell expansion phase. C57BL/6 mice were infected with LCMV-WE on day 0. On d4 and d6 mice were treated with PS-341 (0.75 mg/ kg, i.p.) (ϩ) or were left untreated (Ϫ). Eight days post infection, spleen cells were harvested, stimulated in vitro with the indicated peptides for 5 h and analyzed by flow cytometry after staining for CD8 and intracellular IFN-␥. Shown are the percentages of IFN-␥-positive cells of CD8ϩ cells (y-axis) as determined by flow cytometry. Unstimulated cells (no peptide) were used as a negative control. The experiments have been repeated twice, yielding similar results. Downloaded from cells can greatly limit the immunogenicity of such complexes (34, as stimulators for IFN-␥ production (ICS) by in vitro generated

35). Hence, administration of bortezomib during infection might mono-specific CTL-lines specific for GP33–41 or NP396–404 (Fig. ϩ inhibit the proteasome in APCs and thereby reduce the peptide 7). To exclude that activated TCD8 in infected spleens falsify the supply to class-I complexes, which limits the activation of naive T result, an intracellular IFN-␥ staining (ICS) with infected spleno- cells. Numerous LCMV-derived class-I ligands are processed in a cytes but without mono-specific CTLs was performed. No IFN-␥- http://www.jimmunol.org/ proteasome-dependent manner (17–19). As shown in Fig. 1, in producing CTLs were detected on day 4 after LCMV-WE infec- vitro experiments demonstrated that PS-341 could reduce presen- tion in these spleens, thus indicating that they did not contribute to tation of an LCMV-derived peptide. To determine the amount of the number of IFN-␥ϩ cells (data not shown). As shown in Fig. 7,

Ag presented directly in vivo in bortezomib treated mice, C57BL/6 GP33–41 and NP396–404 are presented differently in spleens on day mice were infected with LCMV-WE (day 0) and treated with bort- 4 post-LCMV-WE infection in treated compared with untreated ezomib on day 1 and 3. The status of GP33–41 and NP396–404 mice, whereas splenocytes derived from uninfected mice were not presentation was analyzed in the spleen on day 4 after infection. able to activate mono-speciﬁc CTLs. APCs derived from PS-341

Splenocytes from bortezomib treated or untreated mice were used treated mice are hampered to produce and present GP33–41 and by guest on September 27, 2021 NP396–404 in similar amounts as splenocytes derived from untreated mice. Hence, the reduced T cell response in bortezomib treated mice seen in Fig. 2, is probably due to a reduced Ag presentation in proteasome inhibited APCs, which leads to an impaired activation of naive CTL.

Discussion Bortezomib is a proteasome inhibitor with signiﬁcant clinical activity in multiple myeloma and mantle cell myeloma. In this study,

FIGURE 6. Effect of bortezomib on T cell activation. A, Thy1.2ϩ splenocytes derived from C57BL/6 mice were treated with the indicated

PS-341 concentrations (x-axis) and stimulated with plate bound CD3/CD28 FIGURE 7. Reduced GP33–41 and NP396–404 presentation in PS-341 or left untreated (unstimulated). Twenty-four hours later, the cells were treated mice. C57BL/6 mice were infected with LCMV-WE on day 0. On stained with propidium iodide (PI) and analyzed by flow cytometry. Shown days 1 and 3, mice were treated with PS-341 (0.75 mg/kg, i.p.) (ϩPS-341) are the percentages of PI-positive cells of lymphocytes (y-axis). The ex- or were left untreated (ϪPS-341). Four days post infection, splenocytes periments have been repeated twice, yielding similar results. B, C57BL/6 were used as APC for GP33–41-orNP396–404-specific CTL lines. Activa- mice were infected with LCMV-WE on day 0. On day Ϫ1, mice were tion of CTL-lines was analyzed by staining for CD8 and intracellular treated with PS-341 (0.75 mg/kg, i.p.) (ϩPS-341) or were left untreated IFN-␥. Shown are the percentages of IFN-␥-positive cells of CD8ϩ cells as (ϪPS-341). One day post infection, spleen cells were harvested, stained for determined by flow cytometry. The percentage of intracellular IFN-␥ (y- CD4 and CD69 or CD8 and CD69, and analyzed by flow cytometry. axis) produced by CTL-lines is plotted vs E:S ratio (effector (CTL lines) to Shown are the percentages of CD69ϩ or CD4 or CD8 as determined by stimulators (splenocytes)). Uninfected splenocytes were used as a negative flow cytometry. Uninfected mice were used as negative control (naive). control. The experiments have been repeated twice, yielding similar results. The Journal of Immunology 6149 we investigated the impact of bortezomib on a virus-induced cy- were adoptively transferred into mice to trigger a CTL response, totoxic T cell response. CTL-responses to four different LCMV- the intensity of the generated CTL response increased directly with derived class-I epitopes were markedly reduced in bortezomib- the quantity of pulsed peptide (44). Apart from an impaired CTL treated mice (Fig. 2A), whereas the CD4ϩ response to the class II induction, an altered Ag presentation can modulate neutral killer epitope GP61–80 was not altered. Associated with the impaired cell (NK) activity. It was reported that bortezomib down-regulates CTL response was the reduced number of CD8ϩ T cells in spleen class I in a time- and dose dependent fashion on multiple myeloma (Fig. 3). It has been reported that bortezomib, apart from direct cell lines and on multiple myeloma patients-derived cells (45). anti-tumor activity, is also immunomodulatory with effects on the Thereby, bortezomib significantly enhanced the sensitivity of these survival and function of lymphocytes and dendritic cells (DC) (36, cells to allogeneic and autologous NK cell-mediated lysis, whereas 37). Exposure to the proteasome inhibitor bortezomib was found to clinically relevant PS-341 concentrations did not affect NK cell promote apoptosis of human monocyte-derived DCs and to reduce function. the yield of viable DCs when given to monocytes early during The present study suggests a higher susceptibility of bort- differentiation to DCs (36, 38). Proteasome inhibition in mice dras- ezomib-treated patients to viral infections. Indeed, several pub- tically but reversibly impairs murine T lymphocyte development in lications report on an increased incidence of varicella herpes the thymus (39). Additionally, bortezomib induces selective apo- zoster (46–49). Analysis of herpes zoster events among bort- ptosis and decreases Th1 responses among alloreactive T lympho- ezomib-treated patients in the phase III APEX study revealed a cytes while barely affecting unstimulated T cells (33). The reduced significantly higher incidence of herpes zoster compared with T cell response (Fig. 2 and 3) manifested in increased viral load at dexamethasone treatment (49). During primary infection with day 6 post infection in bortezomib-treated mice (Fig. 4). It has the neurotropic human varicella zoster virus (VZV), viral rep- Downloaded from been published that PS-341 can directly influence viral replication lication in the skin or the respiratory mucosa is followed by a in vitro (40, 41). Whereas poliovirus replication was less sensitive lifelong latent infection of neurons within sensory ganglia. to proteasome inhibition, bortezomib abrogated vesicular stomati- VZV-specific T cells are important in controlling infection. tis virus replication (VSV). Proteasome targeting suppressed VSV CD8ϩ T cells that recognize the VZV encoded IE62, which is protein synthesis and protected infected cells from toxic effect critical for VZV replication, are present in immune individuals ϩ of VSV replication (40). Similar findings were observed for or- (50). In case such CD8 T cells are involved in controlling http://www.jimmunol.org/ thopoxviruses (41), where bortezomib treatment of cells had a VZV reactivation from latency, proteasome inhibition might re- marked effect on viral titers, severely blocking viral replication and duce class-I presentation of VZV-derived peptides and thereby propagation. In contrast, no diminished replication could be ob- promote viral escape from the immune system. served in vivo in our study with LCMV, where increased viral In summary, the present study is the first investigating the im- titers were observed most likely due to a restrained cytotoxic T cell pact of proteasome inhibition on Ag presentation in vivo. Infection response (Fig. 4). of bortezomib-treated mice with LCMV revealed increased viral Activation of LCMV-specific CTLs is followed by a massive load due to an impaired activation of CTLs, a fact that has impor- expansion of primed T cells (31, 32). When bortezomib was ap- tant implications for the clinical use of bortezomib. Our results plied to mice during this expansion phase, no reduction in the suggest considering antiviral prophylaxis of bortezomib treated by guest on September 27, 2021 CTL-activity could be observed (Fig. 5), suggesting that bort- patients. ezomib is not affecting proliferating T cells. Additionally, proliferation of adoptively transferred CFSE-labeled THY1.2ϩ P14 Disclosures

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