HIV Protease Inhibitor–Induced Cathepsin Modulation Alters Antigen Processing and Cross-Presentation

HIV Protease Inhibitor−Induced Cathepsin Modulation Alters Antigen Processing and Cross-Presentation

This information is current as Georgio Kourjian, Marijana Rucevic, Matthew J. Berberich, of September 29, 2021. Jens Dinter, Daniel Wambua, Julie Boucau and Sylvie Le Gall J Immunol 2016; 196:3595-3607; Prepublished online 23 March 2016;

doi: 10.4049/jimmunol.1600055 Downloaded from http://www.jimmunol.org/content/196/9/3595

Supplementary http://www.jimmunol.org/content/suppl/2016/03/22/jimmunol.160005 Material 5.DCSupplemental http://www.jimmunol.org/ References This article cites 61 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/196/9/3595.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision by guest on September 29, 2021

• 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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

HIV Protease Inhibitor–Induced Cathepsin Modulation Alters Antigen Processing and Cross-Presentation

Georgio Kourjian, Marijana Rucevic, Matthew J. Berberich, Jens Dinter, Daniel Wambua, Julie Boucau, and Sylvie Le Gall

Immune recognition by T cells relies on the presentation of pathogen-derived peptides by infected cells, but the persistence of chronic infections calls for new approaches to modulate immune recognition. Ag cross-presentation, the process by which pathogen Ags are internalized, degraded, and presented by MHC class I, is crucial to prime CD8 T cell responses. The original degradation of Ags is performed by pH-dependent endolysosomal cathepsins. In this article, we show that HIV protease inhibitors (PIs) prescribed to HIV-infected persons variably modulate cathepsin activities in human APCs, dendritic cells and macrophages, and CD4 T cells, three cell subsets infected by HIV. Two HIV PIs acted in two complementary ways on cathepsin hydrolytic activities: directly on cathepsins and indirectly on their regulators by inhibiting Akt kinase activities, reducing NADPH oxidase 2 Downloaded from activation, and lowering phagolysosomal reactive oxygen species production and pH, which led to enhanced cathepsin activities. HIV PIs modified endolysosomal degradation and epitope production of proteins from HIV and other pathogens in a sequence- dependent manner. They altered cross-presentation of Ags by dendritic cells to epitope-specific T cells and T cell–mediated killing. HIV PI-induced modulation of Ag processing partly changed the MHC self-peptidome displayed by primary human cells. This first identification, to our knowledge, of prescription drugs modifying the regulation of cathepsin activities and the

MHC-peptidome may provide an alternate therapeutic approach to modulate immune recognition in immune disease beyond http://www.jimmunol.org/ HIV. The Journal of Immunology, 2016, 196: 3595–3607.

uman immunodeficiency virus infection has been con- reticulum, where they can be further trimmed by endoplasmic sidered a chronic illness since the availability of highly reticulum–resident aminopeptidases 1 or 2 and, if they contain H active antiretroviral therapies (ART), a combination of appropriate anchor residues, loaded onto MHC-I and displayed nucleoside reverse-transcriptase inhibitors, nonnucleoside reverse- at the cell surface. HIV PIs ritonavir (RTV), saquinavir (SQV), transcriptase inhibitors, protease inhibitors (PIs), and integrase and nelfinavir (NFV) inhibit the proteolytic activities of the pro- inhibitors given to HIV-infected patients to suppress HIV repli- teasome, a threonine protease (5, 6), causing intracellular accu- by guest on September 29, 2021 cation (1). HIV PIs block the HIV aspartyl protease, preventing the mulation of polyubiquitinated proteins (7). In mice infected with cleavage of HIV Gag-Pol polyproteins and the conversion of HIV lymphocytic choriomeningitis virus and treated with RTV, CTL particles into mature infectious virions (2). The ability of pro- responses against lymphocytic choriomeningitis virus epitopes teasomes to cleave similar bonds as HIV-1 protease (3) raised were changed (6). We previously showed that HIV PIs altered questions about interactions between HIV PIs and proteasomal proteasome and aminopeptidase activities in primary human cells, catalytic sites. modified HIV protein degradation patterns, epitope production, Proteasomes play a central role in the generation of MHC class I and presentation, and altered CTL responses (8). (MHC-I) peptides (4). They degrade full-length proteins and de- Professional APCs, like dendritic cells (DCs) and macrophages fective ribosomal products into peptides that can be further short- (MFs), can cross-present exogenous Ags (9, 10). Ags transiting ened by cytosolic aminopeptidases and endopeptidases. Some through endosomes or phagosomes are partially degraded by ca- peptides are translocated by the TAP complex into the endoplasmic thepsins. Degradation fragments can escape into the cytosol for processing and presentation in the direct Ag-processing pathway (11) or be processed completely in the phagosomes by cathepsins Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139 before loading onto MHC-I (12, 13). The mechanisms of cross- ORCID: 0000-0003-2196-3757 (M.J.B.). presentation and connection with the direct presentation pathway Received for publication January 12, 2016. Accepted for publication March 1, 2016. remain incompletely understood (12–17). For instance, hen egg- This work was supported by R01 Grants AI084753, AI084106, and AI112493 from white lysozyme (HEL), targeted to early endosomes, is completely the National Institute of Allergy and Infectious Diseases (to S.L.G.). processed and loaded onto MHC-I in the endosomal compartments, Address correspondence and reprint requests to Dr. Sylvie Le Gall, Ragon Institute of MGH, MIT and Harvard, 400 Technology Square, Room 958, Cambridge, MA whereas HEL targeted to late endosomes is not cross-presented (15). 02139. E-mail address: [email protected] Soluble OVA escapes into the cytosol for proteasomal processing The online version of this article contains supplemental material. before cross-presentation, whereas HEL is processed and loaded Abbreviations used in this article: ART, antiretroviral therapies; DC, dendritic cell; within the endosome (15). The inhibition of cathepsin activities, DHR, dihydrorhodamine 123; DPI, diphenylene iodonium; DRV, darunavir; HCV, especially cathepsin S, altered epitope cross-presentation, showing hepatitis C virus; HEL, hen egg white lysozyme; LC, liquid chromatography; LPV, lopinavir; MF, macrophage; MFI, mean fluorescence intensity; MHC-I, MHC class the importance of cathepsins in this pathway (13, 15, 16). Cathepsin I; MHC-II, MHC class II; MS, mass spectrometry; MS/MS, tandem MS; NFV, activities are controlled through pH regulation. Upon phagocytosis, nelfinavir; NOX2, NADPH oxidase 2; PDK1, phosphoinositide-dependent kinase-1; phagosomes fuse with early and late endosomes and with lysosomes, PI, protease inhibitor; ROS, reactive oxygen species; RTV, ritonavir; SQV, saquinavir. thus acquiring progressively the acidification machinery (mainly the Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 V-ATPase complex) and lysosomal proteases (18). Acidification www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600055 3596 DRUG-INDUCED ALTERED Ag CROSS-PRESENTATION results in the activation of cathepsins with optimal proteolytic ac- Z-FL-COCHO for cathepsin S, and pepstatin A for cathepsin D and E. tivities between pH 4 and 6 (18, 19). The activation and recruitment The rate of fluorescence emission, which is proportional to the proteolytic of NADPH oxidase 2 (NOX2) at the phagosomal membrane gen- activity, was measured every 5 min at 37˚C in a VICTOR3 Plate Reader (Perkin Elmer). erates reactive oxygen species (ROS), increasing pH and limiting cathepsin activities and Ag degradation in DCs (20, 21). Protein expression, phosphorylation level, and kinase activity We showed previously that HIV PIs alter the hydrolytic activities measurement of nonaspartyl proteases, such as proteasomes and aminopeptidases Protein extracts (30 mg/lane) from treated (5 mM PIs and/or 0.4 mg/ml (8). However, the impact of HIV PIs on cathepsins, cysteine and PMA) or untreated DCs were subjected to SDS-PAGE on 4–12% gradient aspartyl proteases involved in cross-presentation, has never been gel. Protein bands were visualized using a dual infrared fluorophore Od- assessed. The aim of this study was to investigate the effect of five yssey Infrared Imaging System (LI-COR Biosciences), and densitometric quantification was performed as described (22). The Kinase Selectivity HIV PIs (RTV, SQV, NFV, the ubiquitously used lopinavir (LPV)/ Profiling System: AGC-1 was used in addition to the Kinase-Glo Lumi- RTV (Kaletra), and the most recently prescribed darunavir [DRV]) nescent kit (both from Promega) to measure kinase activities. on cathepsin activities in primary APCs and CD4 T cells, the main targets of HIV infection and subsets involved in clearing patho- Cross-presentation assay gens or priming immune responses. Our results showed that RTV Immature DCs were exposed to rHIV-1 p24 protein (Abcam) for 1 h at 37˚C. reduced cathepsin activities, whereas SQV and NFV enhanced DCs were washed thoroughly and cultured for 4 h before adding the them. We identified two complementary mechanisms: a direct effect epitope-specific CTL clones at a 4:1 E:T ratio in 96-well plates. DCs of PIs on cathepsins and an indirect effect on the regulatory pulsed with the indicated peptide concentrations were used as controls for Ag presentation and CTL specificity. Target cell lysis was measured with a pathway of lysosomal pH through reduction of kinase activities, fluorescent-based assay developed in the laboratory (26). Downloaded from NOX2 activation, phagolysosomal ROS production, and pH, leading to enhanced cathepsin activities. Altered cathepsin activities Isolation and identification of MHC-bound peptides modified Ag-degradation patterns and epitope production in a MHC-bound peptides (including MHC-I and MHC class II [MHC-II]) from sequence- and cell type–dependent manner and were linked to control and drug-treated live PBMCs were isolated directly from live cells variable cross-presentation of HIV epitopes, altered CTL recog- and identified by high-resolution tandem MS (MS/MS) (M. Rucevic, G. Kourjian, and S. Le Gall, unpublished observations). In brief, 5 3 107 nition, and partial modifications of the MHC self-peptidome. cells were exposed briefly to mild acid treatment (10% acetic acid, 5 min), http://www.jimmunol.org/ and the peptide pool obtained was immediately subjected to ultrafiltration Materials and Methods (3-kDa cut-off) for MHC peptides enrichment. The pools of eluted peptides were subjected to liquid chromatography (LC)-electrospray ioniza- Study approval tion–MS/MS analysis. They were loaded onto a ChromXP-C18 5-mm m 3 PBMCs were isolated from buffy coats collected from anonymous blood cHiPLC capillary column (75 m 15 cm; Eksigent) interfaced with an donors approved by the Partners Human Research Committee under pro- LTQ Orbitrap XL Mass Spectrometer (Thermo Fisher) and resolved with a tocol 2005P001218 (Boston, MA). PBMCs from HLA-typed blood donors gradient consisting of aqueous mobile phase A (0.1% formic acid in water) were obtained after written informed consent and approval under protocol and organic mobile phase B (0.1% formic acid in 100% acetonitrile). The 2010P002121 by the Partners Human Research Committee. 10 most abundant multiply charged ions were selected for high-resolution

MS/MS sequencing. Data were analyzed with Proteome Discoverer 1.4 by guest on September 29, 2021 Antiretroviral drugs software (Thermo Fisher) and searched against the Swiss-Prot human database using MASCOT search engine. To ensure high accuracy of HIV PIs were obtained from Selleckchem (RTV, LPV, and DRV), Sigma identified sequences, the mass tolerance on precursor was set to 5 ppm and (SQV), and Santa Cruz Biotechnology (NFV). All drugs were dissolved in 0.02 Da for fragment ions, with all assignments made at 1% false-discovery 100% DMSO. Kaletra is a combination of LPV and RTV at a 5:1 ratio. rate. The candidate MHC peptides identified were selected based on their Kaletra concentrations mentioned in this article correspond to LPVamount. presence in triplicate samples. All identified MHC peptides were blasted 2 Aliquots of stock solutions of 10 mM were kept at 20˚C. against the nonredundant Swiss-Prot database restricted to human entries Measurement of phagosomal pH and ROS production to identify their corresponding source proteins. Monocytes and CD4 T cells were isolated from PBMCs by immuno- Statistics magnetic enrichment (STEMCELL Technologies). Monocytes were dif- Empirical data were analyzed using GraphPad Prism version 6. We com- ferentiated into DCs and MFs, as described (22). Phagosomal pH and ROS pared two variables with t tests. All p values are two-sided, and p values , production were measured, as described (23). Briefly, beads were cova- 0.05 were considered significant. Multiple comparisons were done using lently coupled with FITC and FluoProbes 647 Dihydrorhodamine 123 one-way ANOVA and the Dunnett posttest. (DHR; Life Technologies). Cells treated for 30 min with different HIV PIs (5 mM) or inhibitors (10 mM diphenylene iodonium [DPI] or 1 mM bafilomycin) were pulsed with beads for 20 min and washed extensively Results in cold PBS. Then cells were incubated at 37˚C and analyzed by flow HIV PIs alter cathepsin activities cytometry at different times. We aimed to assess the effect of five HIV PIs (RTV, SQV, NFV, In vitro peptide-degradation assay and mass spectrometry Kaletra, and DRV) on omnicathepsin activities (which measure the analysis combined activities of cathepsin S, L, and B) and cathepsin S, D, and A total of 2 nmol pure peptides was digested with 15 mg whole-cell extracts E, which are expressed in APCs important for Ag processing in the at 37˚C in pH 4 degradation buffer (24). The degradation was stopped with phagosomes (8, 13, 19, 21, 27, 28). The PI concentrations used in m 2.5 l 100% formic acid, and peptide fragments were purified by 5% tri- this study correspond to plasma levels of ART-treated persons and chloroacetic acid precipitation. Degradation peptides were identified by in- house mass spectrometry (MS), as previously described (8, 19). are not toxic for primary cells (8, 29). The cleavage of a peptidase- specific fluorogenic peptide substrate was measured over time. The Proteolytic activities in live cells and cell extracts specificity of substrate cleavage was checked by preincubation of Proteolytic activity measurement was performed as we described previously cells with cognate inhibitors of omnicathepsin (E64) or cathepsin (8, 19, 25). Briefly, 5 3 104 DCs, MFs, or CD4 T cells were pretreated with S (Z-FL-COCHO) (1A). The hydrolysis kinetics was calculated as the indicated PIs, and omnicathepsin, cathepsin S, cathepsin D, or the maximum slope of fluorescence emission after background cathepsin E activities were measured using Z-FR-AMC–specific, Z-VVR- AMC–specific, Ac-RGFFP-AMC–specific, or MOCAc-GSPAFLAK-Dnp- subtraction (Fig. 1A). 100% represents the maximum slope of R–specific fluorogenic substrates, respectively. Specific inhibitors were fluorescence emission by cells preincubated with DMSO (maxi- used to confirm the specificity of the reactions: E64 for omnicathepsin, mum slope of 67 for control DMSO, 430 for SQV, 36 for RTV, 5 The Journal of Immunology 3597 for E64) (Fig. 1B). The effects of each HIV PI on omnicathepsin was reduced by RTV and increased by SQV and NFV (Fig. 1H). and cathepsin S activities were assessed in freshly isolated Cathepsin D and E activities measured in live DCs showed PBMCs, CD4 T cells, monocyte-derived DCs, and MFs from at similar alterations upon PI treatment as did purified enzymes least six HIV negative donors (Fig. 1C–F). Baseline cathepsin (data not shown). We then tested whether HIV PIs affect the activities were lower in CD4 T cells and PBMCs than in DCs and maturation of inactive procathepsins into cathepsins. Activation is highest in MFs. In PBMCs, RTV reduced both omnicathepsin triggered by a decrease in pH in endolysosomes, which induces and cathepsin S activities by 3.3-fold. In contrast, SQV and NFV the proteolytic removal of a prodomain blocking the catalytic site increased omnicathepsin activity by .3-fold, but only SQV (30). Procathepsin K was treated with 5 mM of different PIs at increased cathepsin S activity. DRV did not change cathepsin different pHs before measuring cathepsin K activity (Fig. 1I). At activities (Fig. 1C). Similar alterations were seen in CD4 T cells, pH 7, only SQV and NFV activated procathepsin K, resulting in a DCs, and MFs, albeit with different fold changes (Fig. 1D–F). 7.5-fold increase in activity. At pH 5.5, SQV and NFV activated We tested whether HIV PIs can directly affect cathepsins. The procathepsin K by 1.8-fold. Thus, HIV PIs have a direct effect on pH of PBMC extracts was reduced to 4–5.5 to specifically ac- cathepsin maturation and activities. They may bind directly to the tivate cathepsins (24). Similar PI-induced alterations in cathep- catalytic site of cathepsins and inhibit the activity or interact with sin activities were observed in PBMC extracts, albeit with a noncatalytic effector sites, allosterically modulate the conforma- lower fold change (G. Kourjian and S. Le Gall, unpublished tion of the enzyme (31), and decrease or increase hydrolytic ac- observations). The effect of PIs was also tested on recombinant tivities. However, differences in fold changes between live cells cathepsin S and D. RTV, NFV, and Kaletra reduced cathepsin S and recombinant enzymes suggest that other mechanisms might activity, whereas SQV increased it (Fig. 1G). Cathepsin D activity alter cathepsin activities. Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. HIV PIs alter cathepsin activities in human CD4 T cells, DCs, and MFs. (A) Omnicathepsin activity was monitored with fluorogenic substrate every 5 min in live PBMCs pretreated with DMSO, 5 mMRTV,5mM SQV, or 10 mM E64. (B) The maximum slope of fluorescence emission over 1 h in the presence of DMSO was represented as 100%, and the effect of each PI was calculated as a percentage of control. PBMCs (C), CD4 T cells (D), DCs (E), or MFs(F) were pretreated for 30 min with DMSO (control) or with 5 mM of the indicated PIs before adding specific cathepsin substrate. Data are average 6 SD of cells from six healthy donors. Recombinant cathepsin S (G) or recombinant cathepsin D (H) was pretreated with DMSO or 5 mM of the indicated PIs or inhibitor (10 mM ZFL-COCHOO for cathepsin S, 100 mM Pepstatin A for cathepsin D) before adding specific substrate for each activity. In each panel, 100% represents the maximum slope of DMSO-treated enzyme. Data are average 6 SD of three independent experiments. (I) Procathepsin K was incubated with 5 mM of DMSO or the indicated PIs for 30 min at different pHs before adding specific substrate. pH 3.5 was used to trigger the maximal procathepsin maturation. The maximum slope of fluorescence emission is represented. *p , 0.05, **p , 0.01, ***p , 0.001. 3598 DRUG-INDUCED ALTERED Ag CROSS-PRESENTATION

Certain HIV PIs increase phagolysosomal acidification Dextran-pHrodo was used to measure endosomal pH in CD4 The activity of endolysosomal cathepsins is controlled, in part, T cells (Fig. 2A). Standard curves were obtained by permeabilizing through pH regulation (18, 19). We assessed whether HIV PI- and incubating cells in predetermined pH media. FITC intensity induced alterations in cathepsin activities are due to changes in decreased and pHrodo intensity increaseduponacidification phagolysosomal pH in DCs, MFs, and CD4 T cells. We used a (Fig. 2B). The pH values in phagosomes were determined by com- flow cytometry–based measurement of phagosomal pH using latex paring the mean fluorescence intensity (MFI) in the cell pop- beads coated with pH-sensitive (FITC) and pH-insensitive (FluoProbe ulation to the pH standard curves. As shown previously (20), the 647) fluorescent dyes (23). The fluorescence intensity was quan- pH in DC phagosomes was stable at 6.5 during the 90-min tified by flow cytometry at different times. The pH-insensitive dye monitoring (Fig. 2C, left panel). In contrast, the phagosomal allowed gating on cells that phagocytosed only one bead, whereas pH in MFs decreased more quickly and reached pH 5 (Fig. 2C, the fluorescence intensity of the pH-sensitive dye reflected the middle panel). Bafilomycin, a specific inhibitor of vacuolar H+ phagosomal pH (Fig. 2A). HIV PI treatment did not affect bead ATPase, increased the pH by 1.3 pH points in all cell types uptake (G. Kourjian and S. Le Gall, unpublished observations). tested. SQV and NFV, but not RTV and Kaletra, reduced DC Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. HIV PIs alter phagosomal and endosomal pH. (A) Experimental method of phagosomal and endosomal pH measurement. (B) pH titration curves obtained using fluorochrome-coated beads in DCs (left panel) and MFs(middle panel) or using dextran pHrodo in CD4 T cells (right panel). (C) Phagosomal pH measurement over time after a 30-min pretreatment of cells with 5 mM of different PIs or 1 mM Bafilomycin A1. Fluorescence measured by flow cytometry was compared to the pH titration curves in (B) to determine pH values. (D) Difference in pH between phagosomes of cells treated with 5 mM of different PIs or 1 mM Bafilomycin A1 compared with phagosomes of cells treated with DMSO 60 min after phagocytosis. Data are average 6 SD of cells from five healthy donors. ***p , 0.001. The Journal of Immunology 3599 phagosomal pH by ∼1 pH point (Fig. 2C, 2D, left panel). HIV PIs we measured ROS generation by quantifying the fluorescent in- did not significantly affect the already lower phagosomal pH in tensity of DHR on cells that phagocytosed only one bead (Fig. 3D). MFs (Fig. 2C, 2D, middle panel). CD4 T cell endolysosomal pH As expected, DC phagosomes produced $2-fold more ROS than was reduced ∼1 pH point in 60 min by SQV and NFV treatment. did MFs, and ROS generation was strongly inhibited by DPI RTV and Kaletra did not change the pH significantly (Fig. 2C, 2D, treatment in both cell types (20) (Fig. 3E, 3F). RTV and Kaletra did right panel). Thus SQV and NFV, but not RTV and Kaletra, re- not significantly alter ROS production in DCs and MFs. In contrast, duced phagosomal pH, which may contribute to the PI-induced SQV and NFV significantly reduced ROS generation in DCs (263 increase in cathepsin activities in live cells. and 272 RFU, respectively) and, to a lesser extent, in MFs(233 and 242 RFU, respectively) (Fig. 3E–H), showing that SQV and Certain HIV PIs reduce ROS generation in phagolysosomes NFV inhibition of ROS generation in DCs and MFs led to phago- The phagosomal pH is regulated, in part, by the consumption of somal acidification and increased cathepsin activities. protons through the generation of ROS by NOX2 (20, 32). To The activation of NOX2 is triggered by the phosphorylation of confirm this finding in our experimental model, we incubated DCs one of its subunits, phox-p47, at multiple locations and is regulated with DPI, a specific inhibitor of flavin-containing enzymes, such as by different kinases, including Akt and PKC (33). To examine NOX2. DPI reduced phagosomal pH by ∼1 pH point (Fig. 3A). DPI whether an SQV- or NFV-induced reduction in NOX2 activity is treatment increased omnicathepsin and cathepsin S activities in DCs linked to changes in phox-p47, we used Western blot to analyze by .2-fold, confirming that phagosomal ROS affect cathepsin ac- the phosphorylation of phox-p47 at serine 345 and 359 in DCs tivities (Fig. 3B). We hypothesized that HIV PIs might modulate after PI treatment (Fig. 4A). A reduction in the phosphorylation

ROS generation through NOX2, subsequently affecting phagosomal through a point mutation at either serine reduced NOX2 activity Downloaded from pH and cathepsin activities (Fig. 3C). Latex beads were coated with by at least half (34). SQV and NFV reduced p47 Ser359 phos- DHR, a dye that only emits fluorescence under oxidative conditions phorylation by 1.7-fold but did not affect p47 Ser345 phosphory- (23), and an oxidation-insensitive (FluoProbe 647) fluorescent dye lation (Fig. 4A, 4B). To test the significance of this inhibition, we was added to MFs or DCs. At different times after phagocytosis, assessed whether SQV and NFV could counteract the strong http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. HIV PIs alter ROS production in DC phagosomes. (A) Measurement of DC phagosome pH over time after a 30-min treatment with DMSO (black) or 10 mM DPI (gray). Average 6 SD of three independent experiments. (B) Omnicathepsin and cathepsin S activities in DCs after a 30-min treatment with 10 mM DPI. 100% represents the maximum slope of DMSO-treated DCs. Data are average 6 SD of three independent experiments. (C) Representation of the hypothesis: PIs inhibit ROS production, leading to phagosome acidification and cathepsin activation. (D) Method used to measure phagosomal ROS. ROS measurement in DCs (E) and MFs(F) after a 30-min pretreatment with 5 mMPIor10mM DPI. Differences in ROS production in phagosomes of DCs (G) and macrophages (H) after treatment with different PIs or DPI compared with DMSO 60 min after phagocytosis. Data represent the average of cells from five healthy donors. *p , 0.05, **p , 0.01, ***p , 0.001. 3600 DRUG-INDUCED ALTERED Ag CROSS-PRESENTATION stimulatory effect of PMA on p47 phosphorylation (Fig. 4C). As were used to degrade a synthetic 35-mer peptide in HIV-1 Gag previously reported (34, 35), PMA increased p47 Ser359 phos- p24 (MVHQAISPRTLNAWVKVVEEKAFSPEVIPMFAALS, aa phorylation by 1.5-fold. SQV and NFV pretreatment blocked the 10–44 in Gag p24) containing the HLA-B57–restricted epitopes stimulatory effect of PMA, showing that PMA and PIs exert op- ISW9 and KF11 (36). Supplemental Fig. 1 shows all degradation posite effects on pathways leading to p47 phosphorylation peptides from the p24 35-mer identified by LC-MS/MS after a 60- (Fig. 4D). To assess whether this reduction in phosphorylation was min degradation in DC, MF, or CD4 T cell extracts in the absence caused by direct inhibition of kinases of the PI3K/Akt kinase of PIs; each bar corresponds to a peptide with a defined peak pathway involved in the upstream regulation of phox-p47, we intensity. We quantified the relative amount of each fragment by measured the activities of Akt, PKA, PKC, and phosphoinositide- measuring its contribution to the total intensity of all degradation dependent kinase-1 (PDK1) in the presence or absence of different fragments, as done previously (19). We displayed the relative PIs. SQV and NFV, but not RTV and Kaletra, moderately reduced amount of peptides starting at any N-terminal residue or ending at all four kinase activities by 1.5-fold (Fig. 4E), suggesting that any C-terminal residue (top and bottom bars, respectively), thus SQV- and NFV-induced inhibition of PDK1/Akt reduced phox-p47 showing the relative frequency of cleavage sites in the fragment phosphorylation, leading to lower NOX2 activity and reduced (Fig. 5A). For instance, a frequent N-terminal cleavage site is ROS production in phagosomes. This mechanism is complemen- observed at a valine in between boxed epitopes ISW9 and KF11. tary to the direct enhancement of cathepsin activities induced by We observed differences in the frequency of cutting sites among these two PIs. DC, MF, and CD4 T cell extracts, confirming the differential Ag- processing activities by different cell types previously reported by

HIV PIs alter degradation patterns of antigenic peptides our group (19, 22, 25) (Fig. 5A, left panel). A similar analysis was Downloaded from We used our in vitro Ag-degradation assay to analyze how PI- performed for degradations done in the presence of different HIV induced changes in cathepsin activities alter Ag processing in PIs. In DCs, SQV introduced new cutting sites within the ISW9 compartments involved in cross-presentation (19, 24). We showed epitope (e.g., N-terminal of proline position 8 and C-terminal of previously that the reduction in cellular extracts’ pH to 4 activates arginine position 9). RTV and SQV also changed the frequency of cathepsins, inhibits proteasome and aminopeptidase activities, and certain cutting sites (e.g., N-terminal of valine position 17 and

mimics degradation conditions in puriﬁed endolysosomes (19, 24). C-terminal of proline position 29) (Fig. 5A, right panel). We gener- http://www.jimmunol.org/ DC, MF, and CD4 T cell whole-cell extracts placed at acidic pH ated heat maps showing the percentage changes in the frequency by guest on September 29, 2021

FIGURE 4. HIV PIs inhibit kinase activity, reduce p47 phosphorylation, and counteract PMA stimulatory effect on p47 phosphorylation. (A) Western Blot using extracts of DCs pretreated with DMSO, SQV, or NFV and Abs against tubulin, p47, p47-pSer345, and p47-pSer359. (B) Quantiﬁcation of tubulin-normalized bands. 100% represents the normalized band value of DMSO control. Data are average 6 SD of four independent experiments using DCs from four healthy donors. (C) Western blot analysis of tubulin, p47, and p47-pSer359 in extracts of DCs pretreated with DMSO, PMA, SQV+PMA, or NFV+PMA. (D) Quantiﬁcation of tubulin-normalized bands. 100% represents the normalized band value of DMSO control. Data are the average 6 SD of four independent experiments using DCs from four healthy donors. (E) Kinase activity measurement after a 30-min pretreatment with different PIs (5 mM). 100% represents the baseline activity of the control. Data are average 6 SD of three independent experiments. The Journal of Immunology 3601 Downloaded from http://www.jimmunol.org/

FIGURE 5. HIV PIs change protein-degradation patterns and epitope production in lysosomal compartments. (A) Cleavage patterns of p24-35mer in- by guest on September 29, 2021 cubated with DC, MF, or CD4 cell extracts for 60 min at pH 4 (left panel). Relative amount of fragments starting or ending at each amino acid (top,N- terminal; bottom, C-terminal) after a 60-min degradation in DC extracts preincubated with 5 mM DMSO (gray), RTV (blue), or SQV (red) at pH 4 (right panel). (B) Cleavage patterns of p24-35mer incubated with DCs (top panel), MFs(middle panel), or CD4 T cells (bottom panel) shown as heat maps of cleavage site intensity compared with DMSO control. Red represents increased cleavage and blue represents reduced cleavage compared with control. (C) Degradation products from (B), identified by LC-MS/MS, were grouped into fragments containing B57-ISW9 and B57-KF11 epitopes (black), only B57- KF11 epitope (blue), only B57-ISW9 epitope (red), or neither epitope (gray). The contribution of each category of peptides to the total intensity of all degradation products is shown for each condition. (D) All peptides from (B) were grouped according to their lengths of fragments. The contribution of each category of peptides to the total intensity of all degradation products is shown for each condition. All data are representative of three independent experiments using DCs from three different donors. of cleavages at each amino acid induced by each PI over DMSO. amount of ISW9- and KF11-containing fragments, HIV PI treat- Fig. 5B shows that HIV PIs increased cleavages at some locations ments changed the size distribution of the degradation products. and decreased them at others. For instance, SQV increased the The amount of 8–12-aa-long peptides was reduced by all PIs by an cleavage within the ISW9 epitope in DCs (Fig. 5B, top panel), average of 2-fold in DCs and MFs. In CD4 T cells, the amount of potentially leading to the destruction of ISW9. Similar changes 13–18-aa-long peptides was reduced 2.7-fold by RTV, 1.7-fold by were observed when the full HIV p24 protein was used for deg- SQV, 1.4-fold by NFV, and 3-fold by Kaletra (Fig. 5D). Because radation (Supplemental Fig. 2). To further analyze the effect of cathepsins are involved in MHC-II epitope processing (37), we PIs on epitope production, we measured the amount of epitope- assessed the effect of PIs on MHC-II epitope production. DC containing fragments. In DCs, SQV reduced the production of whole-cell extracts placed at acidic pH were used to degrade a B57-ISW9–containing fragments by 1.7-fold, which was expected synthetic 24-mer peptide in HIV-1 Gag p24 (FRDYVDR- because of the increased cleavage within the epitope (Fig. 5B, top FYKTLRAEQASQEVKNW, aa 161–185 in Gag p24) rich in MHC- panel). RTV reduced the production of B57-KF11–containing II epitopes (36), in the presence or absence of PIs. Supplemental fragments by 1.8-fold and B57-ISW9–containing fragments by Fig. 3 shows that SQV and NFV increased cleavages at some lo- 6-fold. In MFs, RTV and SQV reduced the amount of B57-ISW9– cations and decreased them at others. These changes in the cutting containing fragment by 2-fold. The amount of B57-KF11–con- sites patterns altered the amount of MHC-II epitope production. taining fragments was reduced 2.6-fold by RTV and increased For instance, the amount of MHC-II DQA1*01/DQB1*05-restricted 2-fold by SQV, 2-fold by NFV, and 1.4-fold by Kaletra. In CD4 FT11 (FRDYVDRFYKT) was increased upon SQV treatment, T cells, the amount of B57-ISW9–containing fragments was re- and the amount of MHC-II DRB1*01/DRB1*04-restricted FE13 duced 6-fold by SQV. The amount of B57-KF11 containing (FYKTLRAEQASQE) was decreased by NFV (Supplemental fragments was increased 2-fold by RTV and 3-fold by SQV, NFV, Fig. 3). Together, these results suggest that, by altering cathep- and Kaletra (Fig. 5C). In addition to the PI-induced changes in the sin activities, PIs changed the patterns and frequency of cutting 3602 DRUG-INDUCED ALTERED Ag CROSS-PRESENTATION sites, affecting the amount of MHC-I and MHC-II epitopes the exception of NFV reduced the cleavage within the RA15 produced. (RAQDDFSGWDINTPA 47–61 aa) epitope, which, as expected, led to a 2–5-fold increase in RA15 epitope production (Fig. 6B). HIV PIs alter degradation patterns of Ags from other In contrast, all of the PIs tested increased the cleavage within the pathogens coinfecting HIV-positive individuals LM19 (LQANRHVKPTGSAVVGLSM 113–131 aa) epitope, leading One third of HIV-infected individuals on ART are coinfected with to a 2–4-fold decrease in epitope production. In addition to alter- other pathogens, such as Mycobacterium tuberculosis or hepatitis ations in epitope production, by introducing a new cleavage site C virus (HCV) (38, 39). We hypothesized that, by modifying within the AT15 (AMSGLLDPSQAMGPT 152–166 aa) epitope, cathepsin activities, PIs might alter the processing of epitopes RTV and SQV completely abolished its production (Fig. 6B). PI from coinfecting pathogens in APCs. We assessed the effect of the treatment also altered the degradation of HCV NS3 protein, PIs on the processing of two Ags: M. tuberculosis Ag85 and HCV causing increased or decreased cutting frequencies at various lo- NS3, which both contain multiple MHC-I and MHC-II epitopes cations (data not shown). Together, these results suggest that, by (40–45). DC whole-cell extracts placed at acidic pH were used to altering cathepsin activities, PIs changed the patterns and fre- degrade M. tuberculosis Ag85 and HCV NS3 in the presence of quency of cutting sites in M. tuberculosis and HCVAgs, leading to different PIs. The frequency of cleavage sites (Fig. 6A) and the changes in epitope production. How these PI-induced variations in percentage changes in the frequency of each cutting site upon PI epitope production will modify the hierarchy or specificity im- treatment (Fig. 6B) were analyzed as previously described. HIV mune responses against coinfecting pathogens in HIV-positive PIs increased the cleavage at some locations, decreased it at individuals remains to be determined. others, and created new cleavages sites. These alterations in the Downloaded from degradation patterns changed the amount of epitopes produced HIV PIs alter the cross-presentation of Gag p24 epitopes during M. tuberculosis Ag85 and HCV NS3 degradation in the To understand whether these PI-induced alterations in epitope presence of different PIs. Of the seven epitopes exactly generated production measured in vitro also affect endogenous processing during M. tuberculosis Ag85 degradation, PIs changed the and presentation by DCs, we analyzed the cross-presentation of amounts produced of each one. For instance, all of the PIs with the three optimally defined HLA-B57–restricted HIV epitopes http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. HIV PIs change M. tuberculosis Ag85 degradation patterns and epitope production in lysosomal compartments. M. tuberculosis Ag85 was degraded for 60 min at pH 4 in DC extracts preincubated or not with different PIs (5 mM). Degradation products were analyzed by LC-MS/MS, and the contribution of the cleavage of each amino acid position (N-ter and C-ter) to the total intensity of all degradation products was quantified. (A) Cleavage pattern of M. tuberculosis Ag85 by DCs without PI treatment. (B) Heat map representing the increase (red) or decrease (blue) in cutting intensity at each amino acid of M. tuberculosis Ag85 by DCs induced by each PI compared with control. The dashed areas represent MHC-I and MHC-II epitopes produced following the degradation. AT15 (red) represents the epitope whose production was abolished by RTV and SQV. Data are representative of two independent experiments with DCs from two different donors each analyzed in duplicate by LC-MS/MS. The Journal of Immunology 3603 originating from HIV-1 p24 protein: HLA-B57–restricted ISW9 DCs to CTLs (Fig. 7D). These results demonstrate the link between (ISPRTLNAW, aa 15–23 in Gag p24), HLA-B57–restricted KF11 drug-induced alteration of epitope production and subsequent (KAFSPEVIPMF, aa 30–40 in Gag p24), and HLA-B57–restricted changes in epitope-specific CTL responses to cross-presented TW10 (TSTLQEQIGW, aa 108–117 in Gag p24) (36). Monocyte- epitopes. derived DCs from HLA-B57+ patients loaded with HIV-1 p24 protein in the presence of different HIV PIs were challenged with HIV PI SQV alters PBMC MHC self-peptidome epitope-specific CTLs (19). Target cell killing was monitored This and our previous study (8) showed that HIV PIs affect the using a fluorescent-based assay developed in the laboratory (26). direct endogenous Ag-processing and cross-presentation path- As previously seen in B cell lines (8), HIV PIs had no effect on the ways. In addition, these alterations were not limited to HIV pro- level of MHC-I surface expression in DCs (Fig. 7A). The three teins; they also affected the processing of M. tuberculosis and epitopes tested were restricted by HLA-B57 and shown to have HCV Ags. Therefore, we wondered whether HIV PIs also affect similar affinities (36, 46). DCs pulsed with increasing amount of self-derived MHC-bound peptidome (MHC-I and MHC-II) of peptides were increasingly lysed after the addition of cognate uninfected cells. We analyzed the self-derived total MHC-bound CTLs, showing the similar sensitivity of these CTL clones for peptidome (MHC-I and MHC-II) of healthy PBMCs that were their cognate peptides (Fig. 7B). RTV reduced B57-KF11 cross- pretreated with DMSO or SQV for 2 d. MHC-bound peptides were presentation by DCs by 1.4-fold, whereas the other PIs had no eluted by acid from live PBMCs, and 686 and 680 human peptides effect (Fig. 7C). B57-ISW9 cross-presentation was reduced by were identified in triplicate runs with high mass accuracy by in- RTV and SQV (3.2- and 1.6-fold, respectively). In contrast, NFV house LC-MS/MS on Ctrl PBMCs and SQV-treated PBMCs, re- increased killing by 1.35-fold (Fig. 7D). RTV reduced B57-TW10 spectively (M. Rucevic, G. Kourjian, and S. Le Gall, unpublished Downloaded from cross-presentation by 2.4-fold, whereas SQV increased it by 1.4- observations). We performed an in-depth comparative analysis of fold (Fig. 7E). During endogenous processing and presentation of the peptides commonly identified with the highest confidence p24, the effect of PIs, the asynchronous timing of epitope pro- scores among repeated MS runs (148 Ctrl PBMCs and 155 SQV- duction (19, 47), the relative amount of each epitope, and the treated PBMC peptides listed in Supplemental Table I). The com- number of B57 molecules available for loading also define the parative mapping revealed that 68% of peptides were commonly

relative amount of the three epitopes presented by HLA-B57. presented by Ctrl- and SQV-treated PBMCs. Thirty-two percent of http://www.jimmunol.org/ Although we cannot quantify the relative amount of each of the peptides were uniquely presented by Ctrl- or SQV-treated cells three B57 epitopes endogenously processed and presented by in- (Fig. 8A). As expected, the majority of common MHC-bound fected cells, the changes in CTL recognition induced by HIV PIs peptides were 8–12 aa long (compatible with MHC-I loading), are in accordance with changes in epitope production measured and 40% were 13–18 aa long (compatible with MHC-II loading) with our degradation assays. For instance, SQV enhanced cleav- (Fig. 8B). Intriguingly, .80% of peptides uniquely presented on ages within the B57-ISW9 epitope (Fig. 5B), leading to reduced SQV-treated PBMCs were longer than 13 aa compared with only production of B57-ISW9–containing fragments (Fig. 5C), which 60% of peptides presented uniquely on Ctrl PBMCs (Fig. 8B). resulted in lower endogenous cross-presentation of B57-ISW9 by Because SQV treatment did not change the surface expression of by guest on September 29, 2021

FIGURE 7. HIV PIs variably alter the processing and cross-presentation of HIV p24. (A) Flow cytometry MFI representing surface MHC-I expression levels of DCs pretreated with 5 mM the indicated PIs. (B) Lysis percentage of DCs pulsed with different concentrations of peptides B57-TW10, B57-KF11, or B57-ISW9 in a fluorescence-based killing assay with epitope-specific CTLs. Data are the average 6 SD of three experiments. (C–E) Monocyte-derived DCs from HLA-B57+ donors were pretreated with 5 mM the indicated PIs and loaded with HIV p24 for 1 h. After extensive washing and a 4-h incubation, B57-KF11 (C), B57-ISW9 (D), or B57-TW10 (E) CTLs were added, and cell lysis was measured using a fluorescence-based assay. Data are the average 6 SD of five independent experiments with five different donors. *p , 0.05, **p , 0.01, ***p , 0.001. 3604 DRUG-INDUCED ALTERED Ag CROSS-PRESENTATION

MHC-I or MHC-II (G. Kourjian and S. Le Gall, unpublished treated PBMCs was divided into subgroups according to the observations), these results suggest that SQV may increase the diversity of peptide location. Peptides presented commonly or diversity of MHC-II peptidome and/or lead to the presentation of uniquely were derived from the same location in 64% of proteins longer peptides by MHC-I. The total peptidome of Ctrl- and SQV- represented by treated and untreated PBMCs (Fig. 8D, Similar). In treated PBMCs was sampled from 56 host proteins. Many of the 22% of the proteins from this group, SQV treatment narrowed the commonly identified MHC peptides were derived from the same locations from which the peptides were derived (Fig. 8D, Narrow- proteins and corresponded to nested peptides, with an average of ing), whereas in the remaining 14% of the proteins, SQV broadened three peptides/protein (Fig. 8C, 8D). However, peptides uniquely the locations of the peptides (Fig. 8D, Broadening). We concluded identified from SQV- or Ctrl-treated PBMCs were sampled from that SQV-induced changes in peptidase activities and degradation diverse proteins, with one peptide/protein (Fig. 8C). patterns contributed, in part, to change the MHC self-peptidome. Sixty-four percent of all proteins identified were represented in untreated and SQV-treated peptidome, whereas 18% were uniquely Discussion represented on Ctrl PBMCs, and another 18% were represented only Any perturbation of protease activities could modify protein- on SQV-treated PBMCs; this suggested that SQV partially changed degradation patterns and, consequently, epitope presentation and the origin of proteins represented on the surface (Fig. 8D). This immune recognition. In this study, we showed that several HIV PIs might be explained, in part, by an SQV-induced alteration of tran- modified cathepsin activities through two complementary mech- scription and expression. However, we also identified changes in the anisms and altered the MHC-peptidome and recognition of infected number and location of peptides originating from a common pro- cells by epitope-specific CTLs. tein, in line with PI-induced changes in protein-degradation patterns Four of the five PIs tested variably affected cathepsin activities. Downloaded from (Fig. 8D). The group of proteins represented by treated and un- RTV mostly reduced all cathepsin activities tested, whereas NFV http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 8. HIV PIs alter the MHC self-peptidome. PBMCs were pretreated for 2 d with DMSO or SQV. Total surface peptides (MHC-I and MHC-II peptides) were isolated by acid elution and identified by LC-MS/MS. (A) Venn diagram showing the number and percentage of peptides commonly or uniquely present on PBMCs treated with DMSO or SQV (peptides are listed in Supplemental Table I). (B) Uniquely or commonly presented peptides were grouped according to their lengths. (C) Number of total, common, or uniquely presented peptides on PBMCs treated with DMSO or SQV and the number of proteins from which they originate. (D) The proteins of origin of the surface peptides were grouped into proteins represented in DMSO and SQV treatment, proteins represented uniquely in DMSO treatment, and proteins represented uniquely in SQV treatment. The group of proteins represented in each treatment was divided into three subgroups: similar (unique and common peptides coming from similar location on the protein), narrowing (SQV narrowing the number of peptides coming from this group of proteins), and broadening (SQV broadening the number of peptides coming from this group of proteins). This experiment is representative of two independent peptide elutions performed on PBMCs from two donors. The Journal of Immunology 3605 reduced cathepsin S activity and enhanced cathepsin D activity, production. It altered MHC-I and MHC-II epitope production and suggesting different interactions between each drug and cathepsins. the self-derived MHC peptidome. The increase in 13–18-aa-long It is intriguing that HIV PIs designed to inhibit the HIV aspartyl peptides presented by PBMCs upon SQV treatment suggests that protease alter the activities of aspartyl proteases like cathepsin D SQV might broaden the MHC-II surface peptidome. It is unknown and E, as well as cysteine proteases, such as cathepsin S. HIV PIs whether this HIV PI-induced modulation of the self-peptidome is might bind directly to the catalytic site of cathepsins, interact with sufficient to induce autoimmune symptoms. No increase in auto- noncatalytic effector or allosteric sites that are known to regulate immune disease in HIV patients receiving ART, including Kaletra, the hydrolytic activities (31), or induce the maturation of proca- has been reported in the literature. Thymic selection of T cells thepsin into cathepsin. These off-target effects of HIV PIs might involves negative and positive selection in medullary and cortical explain the inhibition or enhancement of cathepsin activities, al- thymic epithelial cells. The unique expression of thymoprotea- though molecular modeling and structural studies are required to some in cortical thymic epithelial cells, but not in medullary test this hypothesis. thymic epithelial cells (56), contributes to shaping the immuno- An increase in cathepsin activity could also be triggered by a competent repertoire of CD8 T cells (57) and determines the Ag drug-induced maturation of inactive procathepsins into cathepsins. responsiveness of CD8 T cells (58). One can speculate that the At neutral pH, glycoaminoglycans can loosen the binding of the variable and broad peptide displayed in the thymus during selec- propeptide on the catalytic site and accelerate its autocatalytic tion (59) might cover some variations in the self-peptidome. A removal, subsequently activating cathepsins (48, 49). We discov- direct comparison of the thymus peptidome and PI-treated or ered that SQV and NFV, but not RTV, facilitated the maturation of untreated PBMCs is needed to address this question. procathepsin K, even at neutral pH. SQV and NFV might be using The alterations in degradation patterns were drug and sequence Downloaded from the same mechanism as glycoaminoglycans to trigger cathepsin dependent and varied according to cell subsets, in line with our maturation at neutral pH. Cathepsin deregulation and the extra- previous findings on cell subset–specific variations in peptidase cellular presence of active cathepsins are linked to several dis- activities (22, 25). Given the sequence specificity of cathepsins eases, including damage to the mucosal barrier in inflammatory (30), each PI may variably increase the cleavage of certain motifs bowel disease (50), osteoporosis and osteopenia, cancer, rheu- and decrease others. Interestingly, HIV PIs altered the degradation

matoid arthritis, atherogenesis, and muscular dystrophy (30). of HIV, HCV, and M. tuberculosis proteins in accordance with http://www.jimmunol.org/ Additional research is needed to investigate whether PI-induced sequence-specific, rather than virus-specific, modifications in cathepsin alterations contribute to chronic disease development in degradation patterns. A computational analysis of the degradation patients receiving long-term ART. patterns, as done previously (8, 60), may allow us to predict the First-generation PIs, like RTV, SQV, and NFV, had stronger effect of each PI on epitope production. effects on cathepsin activity and its upstream regulators than did PI-induced changes in cathepsin, proteasome, and aminopep- newer PIs like DRV. First-generation PIs also induced more rapid tidase activities (this study) (8) modified protein degradation and and profound adverse effects on lipid metabolism (51). The half- the MHC-peptidome and altered CTL recognition of HIV cross- life of perilipin, a protein involved in regulating adipocyte lipol- presenting or infected cells. In ART-treated persons with ongoing ysis, was reduced by 6-fold in NFV-treated adipocytes, an effect replication of drug-resistant viruses or coinfected with M. tuberculosis by guest on September 29, 2021 reversed by NH4Cl but not by proteasome inhibition (52). The or HCV, PIs might alter the presentation of pathogen-derived NFV-induced increase in lysosomal cathepsin activity identified in epitopes by APCs or target cells. A drug-induced reduction or this study might provide the missing link between NFV and per- impairment of epitope presentation could render some pathogen- ilipin lysosomal proteolysis in lipid metabolism disorders. specific T cell responses useless or less efficient at recognizing SQV and NFV have pleiotropic anticancer activities and are target cells, or it might affect the T cell memory compartment if being repurposed for cancer treatment (53). The anticancer effects an epitope is never presented. Conversely, increased presenta- of some PIs observed in vitro were linked to the suppression of the tion of other peptides or presentation of new peptides may Akt signaling pathway, but the actual molecular targets remain broaden the T cell compartment. The effects would be determined, unknown (54, 55). The direct inhibition of multiple members of in part, by the amount of MHC peptides displayed and the bind- the protein kinase–like superfamily (PDK1, Akt, and PKA/C) by ing affinity of peptides for their MHC and corresponding TCRs. SQV and NFV lends support to a drug-induced inhibition of cel- Although the lack of appropriate clinical samples precludes us lular processes vital for carcinogenesis and metastasis and pro- from testing this hypothesis, the PI-induced modifications in vides a molecular basis to explain the broad-spectrum anticancer M. tuberculosis and HCV Ag-degradation patterns and epitope effect of SQV and NFV (53, 55). Akt and PKC are also involved production suggest that HIV PIs may modify the spectrum of in regulating activation of the NOX2 complex (33). NOX2 pro- the pathogen-specific T cell compartment. Mouse models of viral duces ROS in the phagosomes of neutrophils, eosinophils, mono- or bacterial infections will allow us to test this hypothesis. Shock- cytes, and MFs. It contributes to the destruction of engulfed and-kill approaches to clear HIV reservoirs propose to pharma- pathogens (32), as well as to the regulation of phagosomal pH in cologically reactivate provirus in the presence of ART to induce DCs (20). Whether PI-induced NOX2 inhibition affects the ca- immune clearance (61). If ART includes PIs, such as Kaletra, it pacity of MFs to kill phagocytosed pathogens requires further will be important to assess the impact of HIV PIs on epitope investigation. presentation by CD4 T cells and DCs when designing therapies to SQV and NFV have the unique ability to activate cathepsin eliminate reactivated HIV-infected cells (62). activities in APCs through two independent mechanisms: inhibiting By acting directly on cathepsins and altering the physiology of NOX2 activity and increasing phagosome acidification and directly phagocytic compartments, HIV PIs modify Ag processing, the enhancing cathepsin activities. The mechanism underlying PI- MHC peptidome, and immune recognition. If HIV PIs allow the induced enhanced cathepsin activity is less clear in CD4 T cells, diversification of epitope presentation, a temporary PI treatment but it involves direct activation of cathepsins and pH acidification. that would not induce the adverse effects observed in long-term Enhanced cathepsin activity modified the degradation patterns highly active ART may provide a complementary approach to of HIV, HCV, and M. tuberculosis proteins, altering the frequency modify or improve immune recognition in the context of various of cleavage sites, size distribution of the fragments, and epitope immune diseases. 3606 DRUG-INDUCED ALTERED Ag CROSS-PRESENTATION

Acknowledgments 23. Savina, A., P. Vargas, P. Guermonprez, A. M. Lennon, and S. Amigorena. 2010. Measuring pH, ROS production, maturation, and degradation in dendritic cell phago- We thank Drs. F. Pereyra, D. Kavanagh, B. Walker, and S. Pillai for stim- somes using cytofluorometry-based assays. Methods Mol. Biol. 595: 383–402. ulating discussions and input on the manuscript. 24. Vaithilingam, A., N. Y. Lai, E. Duong, J. Boucau, Y. Xu, M. Shimada, M. Gandhi, and S. Le Gall. 2013. A simple methodology to assess endolysosomal protease activity involved in antigen processing in human primary cells. Disclosures BMC Cell Biol. 14: 35. The authors have no financial conflicts of interest. 25. Lazaro, E., S. B. Godfrey, P. Stamegna, T. Ogbechie, C. Kerrigan, M. Zhang, B. D. Walker, and S. Le Gall. 2009. Differential HIV epitope processing in monocytes and CD4 T cells affects cytotoxic T lymphocyte recognition. J. In- fect. Dis. 200: 236–243. References 26. Gourdain, P., J. Boucau, G. Kourjian, N. Y. Lai, E. Duong, and S. Le Gall. 2013. 1. Autran, B., G. Carcelain, T. S. Li, C. Blanc, D. Mathez, R. Tubiana, C. Katlama, A real-time killing assay to follow viral epitope presentation to CD8 T cells. J. P. Debre´, and J. Leibowitch. 1997. Positive effects of combined antiretroviral Immunol. Methods 398–399: 60–67. therapy on CD4+ T cell homeostasis and function in advanced HIV disease. 27. Chain, B. M., P. Free, P. Medd, C. Swetman, A. B. Tabor, and N. Terrazzini. Science 277: 112–116. 2005. The expression and function of cathepsin E in dendritic cells. J. Immunol. 2. Flexner, C. 1998. HIV-protease inhibitors. N. Engl. J. Med. 338: 1281–1292. 174: 1791–1800. 3. Diez-Rivero, C. M., E. M. Lafuente, and P. A. Reche. 2010. Computational 28. Zaidi, N., T. Herrmann, D. Baechle, S. Schleicher, J. Gogel, C. Driessen, analysis and modeling of cleavage by the immunoproteasome and the consti- W. Voelter, and H. Kalbacher. 2007. A new approach for distinguishing cathepsin tutive proteasome. BMC Bioinformatics 11: 479. E and D activity in antigen-processing organelles. FEBS J. 274: 3138–3149. 4. Neefjes, J., M. L. Jongsma, P. Paul, and O. Bakke. 2011. Towards a systems 29. Acosta, E. P., T. N. Kakuda, R. C. Brundage, P. L. Anderson, and C. V. Fletcher. understanding of MHC class I and MHC class II antigen presentation. Nat. Rev. 2000. Pharmacodynamics of human immunodeficiency virus type 1 protease Immunol. 11: 823–836. inhibitors. Clin. Infect. Dis. 30(Suppl. 2): S151–S159. 5. Gaedicke, S., E. Firat-Geier, O. Constantiniu, M. Lucchiari-Hartz, 30. Turk, V., V. Stoka, O. Vasiljeva, M. Renko, T. Sun, B. Turk, and D. Turk. 2012. M. Freudenberg, C. Galanos, and G. Niedermann. 2002. Antitumor effect of the Cysteine cathepsins: from structure, function and regulation to new frontiers. human immunodeficiency virus protease inhibitor ritonavir: induction of tumor- Biochim. Biophys. Acta 1824: 68–88. Downloaded from cell apoptosis associated with perturbation of proteasomal proteolysis. Cancer 31. Novinec, M., M. Korenc, A. Caflisch, R. Ranganathan, B. Lenarcic, and A. Baici. Res. 62: 6901–6908. 2014. A novel allosteric mechanism in the cysteine peptidase cathepsin K dis- 6. Andre´, P., M. Groettrup, P. Klenerman, R. de Giuli, B. L. Booth, Jr., covered by computational methods. Nat. Commun. 5: 3287. V. Cerundolo, M. Bonneville, F. Jotereau, R. M. Zinkernagel, and V. Lotteau. 32. Segal, A. W. 2005. How neutrophils kill microbes. Annu. Rev. Immunol. 23: 197– 1998. An inhibitor of HIV-1 protease modulates proteasome activity, antigen 223. presentation, and T cell responses. Proc. Natl. Acad. Sci. USA 95: 13120–13124. 33. El-Benna,J.,P.M.Dang,M.A.Gougerot-Pocidalo, J. C. Marie, and F. Braut-Boucher. 7. Pajonk, F., J. Himmelsbach, K. Riess, A. Sommer, and W. H. McBride. 2002. 2009. p47phox, the phagocyte NADPH oxidase/NOX2 organizer: structure, phos-

The human immunodeficiency virus (HIV)-1 protease inhibitor saquinavir in- phorylation and implication in diseases. Exp. Mol. Med. 41: 217–225. http://www.jimmunol.org/ hibits proteasome function and causes apoptosis and radiosensitization in non- 34. Faust, L. R., J. el Benna, B. M. Babior, and S. J. Chanock. 1995. The phos- HIV-associated human cancer cells. Cancer Res. 62: 5230–5235. phorylation targets of p47phox, a subunit of the respiratory burst oxidase. 8. Kourjian, G., Y. Xu, I. Mondesire-Crump, M. Shimada, P. Gourdain, and Functions of the individual target serines as evaluated by site-directed muta- S. Le Gall. 2014. Sequence-specific alterations of epitope production by HIV genesis. J. Clin. Invest. 96: 1499–1505. protease inhibitors. J. Immunol. 192: 3496–3506. 35. Johnson, J. L., J. W. Park, J. E. Benna, L. P. Faust, O. Inanami, and B. M. Babior. 9. Guermonprez, P., J. Valladeau, L. Zitvogel, C. The´ry, and S. Amigorena. 2002. 1998. Activation of p47(PHOX), a cytosolic subunit of the leukocyte NADPH Antigen presentation and T cell stimulation by dendritic cells. Annu. Rev. oxidase. Phosphorylation of ser-359 or ser-370 precedes phosphorylation at other Immunol. 20: 621–667. sites and is required for activity. J. Biol. Chem. 273: 35147–35152. 10. Bernhard, C. A., C. Ried, S. Kochanek, and T. Brocker. 2015. CD169+ macro- 36. Yusim, K., B. T. M. Korber, C. Brander, D. Barouch, R. de Boer, B. F. Haynes, phages are sufficient for priming of CTLs with specificities left out by cross- R. Koup, J. P. Moore, B. D. Walker, and D. I. Watkins. 2013. HIV Molecular priming dendritic cells. Proc. Natl. Acad. Sci. USA 112: 5461–5466. Immunology 2013. Los Alamos National Laboratory, Theoretical Biology and 11. Kovacsovics-Bankowski, M., and K. L. Rock. 1995. A phagosome-to-cytosol Biophysics, Los Alamos, NM. Available at: http://www.hiv.lanl.gov/content/ by guest on September 29, 2021 pathway for exogenous antigens presented on MHC class I molecules. Science immunology/contents2013.html. Accessed: March 16, 2016. 267: 243–246. 37. Blum, J. S., P. A. Wearsch, and P. Cresswell. 2013. Pathways of antigen pro- 12. Bertholet, S., R. Goldszmid, A. Morrot, A. Debrabant, F. Afrin, C. Collazo- cessing. Annu. Rev. Immunol. 31: 443–473. Custodio, M. Houde, M. Desjardins, A. Sher, and D. Sacks. 2006. Leishmania 38. Soriano, V., E. Vispo, P. Labarga, J. Medrano, and P. Barreiro. 2010. Viral antigens are presented to CD8+ T cells by a transporter associated with antigen hepatitis and HIV co-infection. Antiviral Res. 85: 303–315. processing-independent pathway in vitro and in vivo. J. Immunol. 177: 3525–3533. 39. Getahun, H., C. Gunneberg, R. Granich, and P. Nunn. 2010. HIV infection- 13. Shen, L., L. J. Sigal, M. Boes, and K. L. Rock. 2004. Important role of cathepsin associated tuberculosis: the epidemiology and the response. Clin. Infect. Dis. S in generating peptides for TAP-independent MHC class I cross-presentation 50(Suppl. 3): S201–S207. in vivo. Immunity 21: 155–165. 40. Huygen, K. 2014. The Immunodominant T-Cell Epitopes of the Mycolyl- 14. Giodini, A., C. Rahner, and P. Cresswell. 2009. Receptor-mediated phagocytosis Transferases of the Antigen 85 Complex of M. tuberculosis. Front. Immunol. elicits cross-presentation in nonprofessional antigen-presenting cells. Proc. Natl. 5: 321. Acad. Sci. USA 106: 3324–3329. 41. Smith, S. M., R. Brookes, M. R. Klein, A. S. Malin, P. T. Lukey, A. S. King, 15. Belizaire, R., and E. R. Unanue. 2009. Targeting proteins to distinct subcellular G. S. Ogg, A. V. Hill, and H. M. Dockrell. 2000. Human CD8+ CTL specific for compartments reveals unique requirements for MHC class I and II presentation. the mycobacterial major secreted antigen 85A. J. Immunol. 165: 7088–7095. Proc. Natl. Acad. Sci. USA 106: 17463–17468. 42. Tabatabai, N. M., T. H. Bian, C. M. Rice, J. Yoshizawa, J. Gill, and D. D. Eckels. 16. Rock, K. L., and L. Shen. 2005. Cross-presentation: underlying mechanisms and 1999. Functionally distinct T-cell epitopes within the hepatitis C virus non- role in immune surveillance. Immunol. Rev. 207: 166–183. structural 3 protein. Hum. Immunol. 60: 105–115. 17. Chatterjee, B., A. Smed-So¨rensen, L. Cohn, C. Chalouni, R. Vandlen, B. C. Lee, 43. Ciuffreda, D., D. Comte, M. Cavassini, E. Giostra, L. Buhler,€ M. Perruchoud, J. Widger, T. Keler, L. Delamarre, and I. Mellman. 2012. Internalization and M. H. Heim, M. Battegay, D. Genne´, B. Mulhaupt, et al. 2008. Polyfunctional endosomal degradation of receptor-bound antigens regulate the efficiency of HCV-specific T-cell responses are associated with effective control of HCV cross presentation by human dendritic cells. Blood 120: 2011–2020. replication. Eur. J. Immunol. 38: 2665–2677. 18. Claus, V., A. Jahraus, T. Tjelle, T. Berg, H. Kirschke, H. Faulstich, and 44. Wertheimer, A. M., C. Miner, D. M. Lewinsohn, A. W. Sasaki, E. Kaufman, and G. Griffiths. 1998. Lysosomal enzyme trafficking between phagosomes, endo- H. R. Rosen. 2003. Novel CD4+ and CD8+ T-cell determinants within the NS3 somes, and lysosomes in J774 macrophages. Enrichment of cathepsin H in early protein in subjects with spontaneously resolved HCV infection. Hepatology 37: endosomes. J. Biol. Chem. 273: 9842–9851. 577–589. 19. Dinter, J., E. Duong, N. Y. Lai, M. J. Berberich, G. Kourjian, E. Bracho-Sanchez, 45. Vita, R., J. A. Overton, J. A. Greenbaum, J. Ponomarenko, J. D. Clark, D. Chu, H. Su, S. C. Zhang, and S. Le Gall. 2015. Variable processing and cross- J.R.Cantrell,D.K.Wheeler,J.L.Gabbard,D.Hix,A.Sette,andB.Peters. presentation of HIV by dendritic cells and macrophages shapes CTL immuno- 2015. The immune epitope database (IEDB) 3.0. Nucleic Acids Res. 43: dominance and immune escape. PLoS Pathog. 11: e1004725. D405–D412. 20. Savina, A., C. Jancic, S. Hugues, P. Guermonprez, P. Vargas, I. C. Moura, 46. Kloverpris, H. N., A. Stryhn, M. Harndahl, M. van der Stok, R. P. Payne, A. M. Lennon-Dumeńil, M. C. Seabra, G. Raposo, and S. Amigorena. 2006. P. C. Matthews, F. Chen, L. Riddell, B. D. Walker, T. Ndung’u, et al. 2012. HLA- NOX2 controls phagosomal pH to regulate antigen processing during cross- B*57 Micropolymorphism shapes HLA allele-specific epitope immunogenicity, presentation by dendritic cells. Cell 126: 205–218. selection pressure, and HIV immune control. J. Virol. 86: 919–929. 21. Delamarre, L., M. Pack, H. Chang, I. Mellman, and E. S. Trombetta. 2005. 47. Le Gall, S., P. Stamegna, and B. D. Walker. 2007. Portable flanking sequences Differential lysosomal proteolysis in antigen-presenting cells determines antigen modulate CTL epitope processing. J. Clin. Invest. 117: 3563–3575. fate. Science 307: 1630–1634. 48. Caglic, D., J. R. Pungercar, G. Pejler, V. Turk, and B. Turk. 2007. Glycosami- 22. Dinter, J., P. Gourdain, N. Y. Lai, E. Duong, E. Bracho-Sanchez, M. Rucevic, noglycans facilitate procathepsin B activation through disruption of propeptide- P. H. Liebesny, Y. Xu, M. Shimada, M. Ghebremichael, et al. 2014. Different mature enzyme interactions. J. Biol. Chem. 282: 33076–33085. antigen-processing activities in dendritic cells, macrophages, and monocytes 49. Vasiljeva, O., M. Dolinar, J. R. Pungercar, V. Turk, and B. Turk. 2005. lead to uneven production of HIV epitopes and affect CTL recognition. J. Recombinant human procathepsin S is capable of autocatalytic processing at Immunol. 193: 4322–4334. neutral pH in the presence of glycosaminoglycans. FEBS Lett. 579: 1285–1290. The Journal of Immunology 3607

50. Menzel, K., M. Hausmann, F. Obermeier, K. Schreiter, N. Dunger, F. Bataille, 56. Tomaru, U., A. Ishizu, S. Murata, Y. Miyatake, S. Suzuki, S. Takahashi, W. Falk, J. Scholmerich, H. Herfarth, and G. Rogler. 2006. Cathepsins B, L and T. Kazamaki, J. Ohara, T. Baba, S. Iwasaki, et al. 2009. Exclusive expression of D in inflammatory bowel disease macrophages and potential therapeutic effects proteasome subunit beta5t in the human thymic cortex. Blood 113: 5186–5191. of cathepsin inhibition in vivo. Clin. Exp. Immunol. 146: 169–180. 57. Nitta, T., S. Murata, K. Sasaki, H. Fujii, A. M. Ripen, N. Ishimaru, S. Koyasu, 51. Carr, A., K. Samaras, S. Burton, M. Law, J. Freund, D. J. Chisholm, and K. Tanaka, and Y. Takahama. 2010. Thymoproteasome shapes immunocompe- D. A. Cooper. 1998. A syndrome of peripheral lipodystrophy, hyperlipidaemia tent repertoire of CD8+ T cells. Immunity 32: 29–40. and insulin resistance in patients receiving HIV protease inhibitors. AIDS 12: 58. Takada, K., F. Van Laethem, Y. Xing, K. Akane, H. Suzuki, S. Murata, F51–F58. K. Tanaka, S. C. Jameson, A. Singer, and Y. Takahama. 2015. TCR affinity for 52. Kovsan, J., R. Ben-Romano, S. C. Souza, A. S. Greenberg, and A. Rudich. 2007. thymoproteasome-dependent positively selecting peptides conditions antigen Regulation of adipocyte lipolysis by degradation of the perilipin protein: nelfi- responsiveness in CD8(+) T cells. Nat. Immunol. 16: 1069–1076. navir enhances lysosome-mediated perilipin proteolysis. J. Biol. Chem. 282: 59. Adamopoulou, E., S. Tenzer, N. Hillen, P. Klug, I. A. Rota, S. Tietz, M. Gebhardt, 21704–21711. S. Stevanovic, H. Schild, E. Tolosa, et al. 2013. Exploring the MHC-peptide matrix 53. Gantt, S., C. Casper, and R. F. Ambinder. 2013. Insights into the broad cellular of central tolerance in the human thymus. Nat. Commun. 4: 2039. effects of nelfinavir and the HIV protease inhibitors supporting their role in 60. Lazaro, E., C. Kadie, P. Stamegna, S. C. Zhang, P. Gourdain, N. Y. Lai, cancer treatment and prevention. Curr. Opin. Oncol. 25: 495–502. M. Zhang, S. A. Martinez, D. Heckerman, and S. Le Gall. 2011. Variable HIV 54. Plastaras, J. P., N. Vapiwala, M. S. Ahmed, D. Gudonis, G. J. Cerniglia, peptide stability in human cytosol is critical to epitope presentation and immune M. D. Feldman, I. Frank, and A. K. Gupta. 2008. Validation and toxicity of escape. J. Clin. Invest. 121: 2480–2492. PI3K/Akt pathway inhibition by HIV protease inhibitors in humans. Cancer 61. Siliciano, J. D., and R. F. Siliciano. 2013. HIV-1 eradication strategies: design Biol. Ther. 7: 628–635. and assessment. Curr. Opin. HIV AIDS 8: 318–325. 55. Gills, J. J., J. Lopiccolo, and P. A. Dennis. 2008. Nelfinavir, a new anti-cancer 62. Deng, K., M. Pertea, A. Rongvaux, L. Wang, C. M. Durand, G. Ghiaur, J. Lai, drug with pleiotropic effects and many paths to autophagy. Autophagy 4: H. L. McHugh, H. Hao, H. Zhang, et al. 2015. Broad CTL response is required to 107–109. clear latent HIV-1 due to dominance of escape mutations. Nature 517: 381–385. Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021