Human Immunodeficiency Virus Type 1 Nef Induces Programmed Death 1 Expression through a p38...

JOURNAL OF VIROLOGY, Dec. 2008, p. 11536–11544 Vol. 82, No. 230022-538X/08/$08.00!0 doi:10.1128/JVI.00485-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Human Immunodeficiency Virus Type 1 Nef Induces ProgrammedDeath 1 Expression through a p38 Mitogen-Activated Protein

Kinase-Dependent Mechanism!

Karuppiah Muthumani,1*† Andrew Y. Choo,2† Devon J. Shedlock,1 Dominick J. Laddy,1Senthil G. Sundaram,1 Lauren Hirao,1 Ling Wu,1 Khanh P. Thieu,3 Christopher W. Chung,1

Karthikbabu M. Lankaraman,1 Pablo Tebas,4 Guido Silvestri,1 and David B. Weiner1

Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 191041;Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 021152; Department of Dermatology, Brigham and

Women’s Hospital, Harvard Medical School, Boston, Massachusetts 021153; and Division of Infectious Diseases,University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 191044

Received 5 March 2008/Accepted 3 September 2008

Chronic viral infection is characterized by the functional impairment of virus-specific T-cell responses.Recent evidence has suggested that the inhibitory receptor programmed death 1 (PD-1) is specifically upregu-lated on antigen-specific T cells during various chronic viral infections. Indeed, it has been reported thathuman immunodeficiency virus (HIV)-specific T cells express elevated levels of PD-1 and that this expressioncorrelates with the viral load and inversely with CD4! T-cell counts. More importantly, antibody blockade ofthe PD-1/PD-L1 pathway was sufficient to both increase and stimulate virus-specific T-cell proliferation andcytokine production. However, the mechanisms that mediate HIV-induced PD-1 upregulation are not known.Here, we provide evidence that the HIV type 1 (HIV-1) accessory protein Nef can transcriptionally induce theexpression of PD-1 during infection in vitro. Nef-induced PD-1 upregulation requires its proline-rich motif andthe activation of the downstream kinase p38. Further, inhibition of Nef activity by p38 MAPK inhibitoreffectively blocked PD-1 upregulation, suggesting that p38 MAPK activation is an important initiating event inNef-mediated PD-1 expression in HIV-1-infected cells. These data demonstrate an important signaling eventof Nef in HIV-1 pathogenesis.

Functional impairment of antigen-specific T cells is a hall-mark feature of chronic viral infections (1, 9). Accordingly,recent evidence has suggested that PD-1 upregulation isoften associated with various chronic viral infections, includ-ing lymphocytic choriomeningitis virus, hepatitis C virus,hepatitis B virus, cytomegalovirus (CMV), human immuno-deficiency virus (HIV), and tumors (1, 2, 4, 9, 23, 24). Theimmunoreceptor programmed death 1 (PD-1), a 55-kDatransmembrane protein containing an immunoreceptor ty-rosine-based inhibitory motif, was originally isolated from aT-cell line exhibiting high sensitivity to apoptosis (16). PD-1is one of the three identified inhibitory B7-recognizing im-munoreceptors of the CD28 family involved in signalingT-cell death and, similar to cytotoxic-T-lymphocyte antigen4 (CTLA4), negatively regulates T-cell function (8, 15, 16,19). Two PD-1 ligands, PD-L1 and PD-L2, have been iden-tified and show distinct roles in regulating the immune re-sponses. Engagement of PD-1 with its ligands, PD-L1 andPD-L2, inhibits T-cell proliferation and cytokine production(1, 8, 19, 22, 23). Also, in vivo administration of antibodiesthat blocked the interaction of PD-L1/PD-1 enhanced T-cell

responses in mice chronically infected with lymphocytic cho-riomeningitis virus (1).

In HIV-infected subjects, PD-1 expression was signifi-cantly increased on HIV-specific CD8! T cells comparedwith total CD8! T cells and was correlated with the viralload (1, 4, 9, 23, 31). Importantly, in HIV, treatment ofimpaired T cells with a blocking anti-PD-L1 antibody wassufficient to augment HIV-specific T-cell function (31). Therelationship between PD-1 expression on HIV-specificCD4! T cells and HIV disease is important to understandbecause functional impairment of HIV-specific CD4! Tcells during chronic HIV infection has been closely linked toHIV replication and disease progression (6, 4, 15, 23, 31).The association between PD-1 expression on HIV-specific Tcells, cellular exhaustion, and disease progression may rep-resent an important advance in our understanding of HIVpathogenesis. Targeting PD-1 may play a role in HIV dis-ease progression and development of new therapeutic ap-proaches. However, the mechanisms employed by HIV toregulate PD-1 expression remain unknown. Data clearlysupport the notion that PD-1 upregulation can be a functionof chronic immune activation (13). However, the observa-tion that PD-1 levels in HIV infection are higher than thoseobserved in other chronic infections suggests that additionalviral factors may play a more direct role in PD-1 expression.In order to provide an understanding, we assessed PD-1expression by directly looking at HIV-infected cells. Weobserved that HIV infection of T cells can drive increased

* Corresponding author. Mailing address: Department of Pathologyand Laboratory Medicine, University of Pennsylvania School of Med-icine, Philadelphia, PA 19104. Phone: (215) 662-2352. Fax: (215) 573-9436. E-mail: [email protected].

† K.M. and A.Y.C. contributed equally to this work.! Published ahead of print on 17 September 2008.

11536

at UNIVERSITY OF PENNSYLVANIA LIBRARY on Novem

ber 12, 2008 jvi.asm

.orgDownloaded from

PD-1 expression. This expression is a function primarily ofthe Nef gene product of HIV type 1 (HIV-1).

MATERIALS AND METHODS

Plasmid construction. The HIV-1 proviral infectious constructs pNL4-3WtHSA (14) and pNL4-3HSA/"Env (12) were obtained through the AIDS Re-search and Reference Reagent Program, Division of AIDS, National Institute ofAllergy and Infectious Diseases, NIH. HIV-1 proviral DNA genes were individ-ually mutated specifically by inactivating the start codon without affecting thereading frames of other viral proteins that use the same transcript. Mutationswere made by in vitro site-directed mutagenesis using a QuickChange mutagen-esis kit (Stratagene, La Jolla, CA) (12, 14). All mutations were confirmed bysequencing, and all the mutated constructs were tested by Western blot analysisfor loss of gene expression. Constructs containing accessory gene-deficient vari-ants and p38Wt and dominant-negative plasmids were generated as describedbefore (3, 7, 20).

Patients. HIV-infected individuals’ cells and sera or plasma were obtainedfrom the University of Pennsylvania Center for AIDS Research immunologyclinical core for our study. Heparinized blood was obtained in accordance withprotocols approved by the Institutional Review Board of the Hospital of theUniversity of Pennsylvania. The median viral load for these samples was 15,401HIV-1 RNA copies per ml plasma (range, 121 to 204,575), and the medianabsolute CD4 T-cell count was 612 (range, 211 to 1,432). Peripheral bloodmononuclear cells (PBMCs) were separated and cryopreserved in liquid nitrogenuntil assay time. The HIV-1 RNA level was determined from plasma using theRoche Amplicor 1.5 kit (Roche Diagnostic Systems, New Jersey) according tothe manufacturer’s recommendations. HIV-infected subjects were serologicallyidentified as having the HLA-A2! genotype and were determined by PCR-sanitation standard operating procedure using sequence-specific primers (29).

Cell culture, virus production, and viral infection. Leukopacks from individualdonors were obtained from the immunology clinical core facility at the Universityof Pennsylvania School of Medicine, and PBMCs were isolated by Ficoll-Hypaque (Pharmacia, Piscataway, NJ) density centrifugation. The cells weremaintained in RPMI 1640 medium supplemented with 10% fetal bovine serum,2 mM L-glutamine, 20 mM HEPES, 100 U/ml penicillin, and 100 #g/ml strep-tomycin. All cells were cultured in lipopolysaccharide-free medium in the pres-ence of interleukin 2 in the medium. HIV-1 stocks were produced in 293T cellsand pseudotyped by using vesicular stomatitis virus G to replace Env (14, 20).HIV-1 expresses mouse heat-stable antigen (HSA) in place of Vpr (14) or Nef(28) to produce this clone, allowing infected cells to be identified by flow cytom-etry. HSA expression indicates completion of steps in the viral life cycle up to andincluding de novo viral-gene expression (30). HIV-1 pseudeoviral particles wereproduced by transfecting 293T cells (obtained from the ATCC) with FuGene 6transfection reagent (Roche Applied Science, Nutley, NJ) by using vectors en-coding vesicular stomatitis virus G envelope (5 #g). Virus-containing superna-tants were harvested 60 to 72 h after transfection, viral titers were determined byinfection of the human T-cell line Jurkat, and p24Gag antigen was measured bycapture enzyme-linked immunosorbent assay (ELISA) using a p24 ELISA kit(Coulter, Miami, FL). For infection studies, human PBMCs were isolated fromhealthy HIV-1-negative donors as described above. PBMCs (2 $ 105 cells/well)were mock infected (with media from the cell cultures used to grow the cells) orinfected with cell-free HIV-1 at a concentration of 100 50% tissue cultureinfective doses/106 cells/ml (14, 20, 30). After 4 to 6 h of incubation at 37°C, thecells were gently washed, resuspended with complete medium, and maintainedfor the indicated time periods. At the end of the incubation period, culturesupernatants and cells were harvested for p24Gag antigen determinations, as wellas other fluorescence-activated cell sorter (FACS) analysis (20, 30).

Tetramer and antibody staining. The following directly conjugated antibodieswere used: CD3-phycoerythrin (PE)/fluorescein isothiocyanate (FITC)/allophy-cocyanin (APC)/Pacific Blue (PB), CD4-PE/FITC/PB, CD8-PE/FITC/PB, andstreptavidin-FITC or PE-Cy5 with their respective isotype control antibodies(BD Biosciences, San Jose, CA); CD4-APC, CD8-APC, and PD-1–FITC/PE/APC with their respective isotype control antibodies (eBiosciences, San Diego,CA); and biotinylated anti-human PD-1 and PD-L1 (R&D Systems). Phospho-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182)-Alexa Fluor 647or -Alexa Fluor 488 was obtained from Cell Signaling Technology, Danvers, MA.The p38 MAPK inhibitor RWJ67657 has been previously described (20, 33).

Tetramers for HIV in this study were HLA-DRB1* 0101-type alleles (29)complexed to the peptides p24.17 (amino acids 294 to 313; FRDYVDRFYKTLRAEQASQD) (iTag major histocompatibility complex class II HIV-specifictetramers, PE conjugated) and were purchased from Beckman Coulter, Fuller-ton, CA. Cryopreserved PBMCs were stained for 2 h at room temperature with

PE-conjugated major histocompatibility complex class II tetramer. For staining,cells were incubated with 1 #g PE-labeled tetramer in 100 #l FACS stainingbuffer (1$ phosphate-buffered saline (PBS), 0.02% NaN3, and 0.2% fetal calfserum) for 1 h at 37°C and subsequently with combinations of fluorochrome-labeled antibodies for 30 min on ice (29). For intracellular staining, cells werepermeabilized using BD FixPerm (BD) following staining. The percentages ofcells expressing intracytoplasmic HIV-1 Gag-related products were evaluatedusing KC57-RD1/PE- or KC57/FITC-conjugated anti-HIV-1 Gag monoclonalantibody (Beckman Coulter, Miami, FL). Electronic compensation was con-ducted with antibody capture beads (BD Biosciences, San Jose, CA) stainedseparately with individual monoclonal antibodies used in the test samples. For-ward scatter area versus forward scatter height was used to gate out the cellaggregates. In addition, ViViD dye staining was used to exclude the dead anddying cells (13). Cells were analyzed with a modified LSRII flow cytometry (BDImmunocytometry Systems, San Jose, CA) or Coulter Epics Flow Cytometer(Beckman Coulter, Miami, FL) using FlowJo software (TreeStar, Ashland, OR)(20, 29).

Western blot analysis. Cell lysates (50 #g protein) were resolved on 10%sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, transferred tonitrocellulose membranes, and processed according to the standard protocols.The antibodies used were polyclonal anti-human PD-1 (R&D Systems, Minne-apolis, MN) and %-actin (Cell Signaling Technology, Danvers, MA). The primaryantibodies were used at dilutions of 1:1,000. The secondary antibodies wereanti-rabbit or anti-mouse immunoglobulin G conjugated to horseradish peroxi-dase (dilution, 1:5,000). Signals were detected using enhanced chemilumines-cence (Amersham Life Sciences Inc., Piscataway, NJ) (21).

PD-1 promoter construction and luciferase reporter assay. For assessment ofPD-1 transcription by viral genes, luciferase reporter plasmids expressing PD-1were assembled from synthetic oligonucleotides (Geneart, Germany) and clonedby inserting 510-bp promoter sequences derived from PD-1 genes into the KpnI/SacI cloning site of the pTA-Luc vector (Clontech, Mountain View, CA). Thepromoter sequence used started from a putative transcription start site andextended to the 5& upstream regions. All new constructs and mutations wereconfirmed by DNA sequencing. In brief, Jurkat cells were seeded onto a six-wellculture plate at a density of 0.5 million cells per milliliter of medium andtransiently transfected as described previously (21) with a constant amount of theluciferase reporter PD-1 promoter and various viral-gene expression plasmids.The total amount of DNA was kept constant by adding empty vector. Cells wereharvested 48 h after transfection and lysed in cell lysis buffer, and luciferaseactivities were assayed with the luciferase assay kit (Promega, Madison, WI)using Lumat-LB9501 (Berthold, Bad Wildbad, Germany). The transfection ef-ficiency was normalized by cotransfection with pEF-lacZ and assay for %-galac-tosidase expression (21).

Determination of soluble Nef antigen by ELISA. A sandwich ELISA proce-dure was used to detect the soluble Nef antigen in serum (10). Briefly, 100 #l wasmeasured with sandwich-type capture ELISA plates; 96-well ELISA plates werecoated with 100 #l of 1.0-#g/ml rabbit HIV-1 Nef antiserum (AIDS Researchand Reference Reagent Program, Division of AIDS, National Institute of Al-lergy and Infectious Diseases, NIH) overnight at 4°C. After incubation withblocking buffer (0.25% bovine serum albumin/0.05% Tween 20 in PBS) at 37°Cfor 1 h, experimental sera and purified entire Nef recombinant protein (Immu-noDiagnostics, Inc., Woburn, MA) as a standard at twofold dilutions (100 #l)were added to wells in duplicate. The plates were incubated overnight at 4°C andthen washed three times. After the washing, 100 #l of 0.5-#g/ml mouse mono-clonal antibody to HIV-1 Nef (1:5,000; Abcam, Cambridge, MA) was added, andthe plates were incubated at 37°C for 1 h. After six washes with PBS plus 0.05%Tween 20 (PBST), 100 #l of horseradish peroxidase-conjugated anti-mousesecondary antibodies (1:5,000) was added, and the plates were incubated for 1 hat 37°C. After being washed eight times with PBST, the substrate (o-phenylene-diamine [Sigma, St. Louis, MO], 0.4 mg/ml in 0.1 mol/liter citrate/phosphatebuffer, pH 5.5, 0.04% H2O2) was added, and the reaction was stopped 20 minlater by adding 50 #l of 12.5% (vol/vol) H2SO4. RPMI medium and controlhuman immunoglobulin G supernatants were used as negative controls, andPBST was used as a zero standard. Absorbance was measured with an ELISAreader at 405 nm, and the concentrations of soluble Nef protein in the sampleswere calculated by interpolation from the standard curve (10).

RNA extraction and Northern blot analysis. Total RNA was extracted using anRNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer’s pro-tocol. Twenty-five micrograms of total RNA was subjected to electrophoresis ona 1.2% denaturing agarose gel and transferred to nitrocellulose. The PD-1expression construct was used as the probe and was random-prime labeled using['-P32]dCTP and an oligonucleotide labeling kit (Amersham Pharmacia Biotech,Piscataway, NJ). The blots were probed as described previously (21) and washed

VOL. 82, 2008 HIV-1 Nef INDUCES PD-1 EXPRESSION THROUGH p38 MAPK 11537



.orgDownloaded from

five times at 42°C with 2$ SSC (1$ SSC is 0.15 M NaCl plus 0.015 M sodiumcitrate)-0.1% SDS, once with 0.5$ SSC-0.1% SDS, and once at 55°C with 0.1$SSC-0.1% SDS in a minihybridization oven. The membrane was exposed on adeveloping screen for 16 to 24 h and scanned using a PhosphorImager (Molec-ular Dynamics, Piscataway, NJ). The transcripts were quantified with Image-QuaNT (version 4.0) software.

Statistical analysis. All data were analyzed using Prism software (GraphPadSoftware, Inc., San Diego, CA). Statistical comparisons between groups wereanalyzed using the Wilcoxon matched pairs t test. Correlations between variableswere evaluated using the Spearman rank correlation test. For all tests, a two-sided P value of (0.05 was considered significant.

RESULTS

Nef is necessary and sufficient to drive PD-1 upregulationduring HIV-1 infection. The mechanisms employed by HIVresulting in PD-1 upregulation on a per cell basis are not wellcharacterized. To address this question, we infected humanPBMCs with HIV-1 (NL4-3) and analyzed the upregulation ofPD-1 on HIV-infected (CD24HSA-positive) cells (12, 14, 20,30). As shown in Fig. 1, HIV infection was sufficient to up-regulate PD-1 expression on T cells. We next individually de-leted the viral Env gene and the accessory genes Vif, Nef, Vpr,and Vpu and utilized these mutant viruses in a pseudoviralsystem as tools to probe PD-1 expression. Loss of Nef, but notEnv and the other accessory genes, led to an attenuation ofHIV-mediated PD-1 upregulation. This effect did not appearto be a consequence of the efficiency of infection, as the mutantviruses all exhibited similar levels of p24Gag production (datanot shown). Therefore, in the context of HIV infection of

target cells, Nef drives the surface expression of PD-1 on CD4!

T cells. We next transiently transfected Jurkat T cells with theindividual HIV genes Vif, Nef, Vpr, Vpu, and Env and evaluatedPD-1 upregulation. As shown in Fig. 2A, only Nef was sufficientto upregulate PD-1, and using its mutants, we observed thatNef required its proline-rich (PXXP) motif (3, 26) for thisactivity (Fig. 2B).

Nef expression induces PD-1 production. To examine therole of Nef in the regulation of PD-1, we first developed PD-1/Luc constructs and analyzed the transcriptional activity ofPD-1 using cotransfection of each of the viral-gene constructswith the PD-1 luciferase reporter plasmid into Jurkat cells. Thetranscription activity of PD-1 was significantly increased incells transfected with pNef compared with cells transfectedwith the other viral genes or a mock control (Fig. 3A). ThisPD-1 effect of Nef was through transcriptional activation of thePD-1 promoter, which led to both PD-1 mRNA and proteinupregulation (Fig. 3B and C). These data also suggest thatincreased surface translocation of cytoplasmic PD-1 may notbe a major contributor to the observed phenotype, despiteNef’s previous role in modulating the surface expression ofvarious proteins, including CD4 (25, 26, 30). Taken together,our data suggest that Nef mediates PD-1 upregulation in HIV-infected cells.

PD-1 expression is upregulated on HIV-specific CD4! Tcells. We next examined infection of primary PBMCs to studythe effects of infection on primary CD4 T cells. To answer this

FIG. 1. Nef is necessary for PD-1 upregulation during HIV-1 infection. (A) Flow cytometric analysis of cell surface expression of PD-1 onhuman PBMCs mock infected or infected with HIV-1 (NL4-3) with different viral genes deleted as indicated. Seventy-two hours postinfection, thecells were stained for CD4/FITC, CD24HSA/APC (infection marker), and PD-1/PE. The histograms depict the PD-1 expression staining gated onCD4!/CD24HSA! T cells. The shaded histograms represent staining with isotype control, the thin-line histograms represent the uninfectedcontrol, and the thick-line histograms represent staining with PD-1 antibody.

11538 MUTHUMANI ET AL. J. VIROL.



.orgDownloaded from

question, we took HIV-1-positive patient PBMCs and stainedthese samples to identify virus-specific CD4! T cells with HIVclass II tetramers. HIV-specific CD4! T cells were both posi-tive and negative for infection (p24Gag positive). In addition,PD-1 was upregulated on HIV-specific and positive CD4 Tcells (Fig. 4A), suggesting that its upregulation in CD4! T cellswas not controlled autonomously by viral infection of the hostcell. Next, we infected HIV-negative PBMCs with the HIV-1NL4-3-Wt virus or virus with Nef deleted and measured PD-1upregulation in CD3!/CD4! T cells. When CD4! T cells wereanalyzed, PD-1 was upregulated by 2 days postinfection (Fig.4B). Kinetically, PD-1 upregulation was minimal in early in-fection but gradually increased by day 2 and was fully upregu-lated by day 10 (Fig. 4C). This upregulation of PD-1 on CD4!

T cells by HIV required Nef, suggesting that Nef was directlyinvolved in the ability of HIV-1 to upregulate PD-1 on CD4!

T cells.p38 MAPK activation by Nef is required for the transcrip-

tional upregulation of PD-1. We had previously reported that

Nef was both necessary and sufficient for HIV-1 to activate thep38' kinase in T cells (Fig. 5A) (20). Similar to PD-1 upregula-tion, Nef also required its proline-rich motif (PXXP) to stimulatethe activation of p38. Therefore, we examined whether the acti-vation of p38 was also involved in the ability of Nef to upregulatePD-1. As shown in Fig. 5B, pharmacologic inhibition of the p38'kinase specifically downregulated HIV-induced PD-1 upregula-tion in a concentration-dependent manner. Similar results wereachieved with small interfering RNA (siRNA)-mediated knock-down of p38' kinase and with an overexpression of a dominant-negative p38' (Fig. 5C). The siRNA clone 352, which fails toknock down p38', failed to prevent Nef-mediated upregulation ofPD-1. However, siRNA clone 61, which efficiently knocks downp38' (20), prevented Nef-mediated PD-1 upregulation. Consis-tently, pharmacologic inhibition of p38 also inhibited Nef-inducedtranscriptional activation of PD-1 (Fig. 5D). Therefore, it appearsthat upon Nef’s entry into T cells, the p38 pathway is activatedand requires its proline-rich motif for PD-1 transcriptional up-regulation.

FIG. 2. Nef is sufficient for PD-1 upregulation. (A and B) Analysis of PD-1 expression in transfected cells. Flow cytometric analysis of PD-1was performed on Jurkat cells transfected with HIV-1 viral genes as indicated and pGFP. The cells were collected 3 days later, and PD-1 expressionwas measured. Gates were set to include green fluorescent protein (GFP)-positive cells only. The data are representative of three or more separatestudies. The shaded histograms represent staining with isotype control, and the open histograms represent staining with PD-1 antibody. FSC-A,forward scatter area.




.orgDownloaded from

Activation of p38 by Nef correlates with PD-1 expression.Although it appears that Nef is important for HIV-1 to up-regulate PD-1 in infection in vitro, we investigated whether thiseffect was involved in the upregulation of PD-1 in HIV-1-positive patients in vivo. In determining whether an associationexists between PD-1 expression on CD4! T cells and HIVdisease progression, we found a strong correlation between theHIV plasma viral load and PD-1 expression on HIV-specificCD4! T cells (r ) 0.6249; P ) 0.0032) (Fig. 6A). Further, wefound a strong positive correlation on PD-1 expression onHIV-positive and tetramer-specific CD4! T cells (P ) 0.0001)and a weaker but not statistically significant correlation ontetramer-negative CD4! T cells (P ) 0.0582) compared toHIV-negative cells (Fig. 6B). As previously reported, there is adirect inverse correlation between CD4! T-cell counts andPD-1 expression in the tetramer-positive CD4 T-cell popula-tion among HIV-1-positive patients (reference 6 and data notshown). Consistent with the hypothesis that Nef directly regu-lates PD-1 expression, there was a direct correlation betweenPD-1 expression on HIV-specific CD4! T cells and levels ofNef in patient serum (r ) 0.2960; P ) 0.0131) (Fig. 6C;). Thissuggests that Nef produced and released from cells in vivocould correlate with PD-1 expression in patients. In support ofin vitro data suggesting Nef stimulates p38 to activate PD-1transcription, we observed p38 MAPK activation on HIV-spe-cific cells inversely correlated with CD4 T-cell counts (r )*0.3077; P ) 0318), suggesting that p38 MAPK activationantagonizes the maintenance of patient CD4! T-cell levels(Fig. 6D). Indeed, the activation of p38 also directly correlatedwith the levels of PD-1 on CD4!/tetramer! T cells (r ) 0.3604;P ) 0.0390) (Fig. 6E and F), suggesting that p38 MAPK maybe involved in PD-1 upregulation in vivo, which may contributeto the CD4! T-cell depletion and immune dysfunction.

DISCUSSION

This study provides the first mechanistic insight into howHIV-1 regulates PD-1 expression on HIV-1-infected cells. Weobserved that infection of CD4! T cells by HIV-1 upregulatesPD-1 expression and that the accessory protein Nef is bothnecessary and sufficient for this phenotype in vitro. We alsoobserved that Nef associates with viral antigens to specificallyincrease PD-1 expression through a p38 MAPK-dependentmechanism. The results of this study are in agreement withobservations in both humans and primates that suggest Nef isimportant for disease progression and AIDS-like disease (5,11, 18). In the context of simian immunodeficiency virus (SIV)infection, Nef was shown to be the critical factor determiningthe persistence of infection and pathogenesis, thus distinguish-ing acute and chronic infection (27).

Recent evidence from several groups has suggested that viralpersistence and chronic T-cell receptor (TCR) stimulation maycontribute to virus-specific increase of PD-1 on T cells (6, 32).Accordingly, a transgenic mouse model from Hanna et al. (11)has shown that T cells transgenically expressing Nef led to ahyperactivated phenotype that was associated with CD3 hyper-responsiveness and constitutive activation of growth factor-regulated kinases. More interestingly, splenocytes from theseNef-expressing mice exhibited a decrease in TCR-activatedproliferation, suggesting an “exhausted” T-cell phenotype.Therefore, we hypothesized that serum levels of Nef (10) may“potentiate” the effects of viral antigens to hyperstimulate vi-rus-specific T cells to increase PD-1 upregulation. Indeed,when we treated HIV-positive PBMCs with a Gag peptide andincreasing concentrations of recombinant Nef, a dramatic in-crease in the mean fluorescence intensity (MFI) of PD-1 onHIV-specific T cells could be seen (data not shown). There-

FIG. 3. Nef expression induces PD-1 production. (A) Jurkat T cells were transiently transfected with a PD-1/Luc reporter construct (10 #g) andequal amounts of either empty vector or vectors containing accessory genes as indicated. Forty-eight hours posttransfection, PD-1 transcriptionalactivity was examined by luciferase assay as described in Materials and Methods. Values and bars represent means (n ) 3) and standard deviations.(B and C) Characterization of PD-1 expression. Total RNA and proteins were extracted from the samples in panel A and analyzed for PD-1expression. (B) Northern blot of 20 #g of total RNA isolated from transfected cells (top).Shown is hybridization with '-32P-labeled human PD-1cDNA probe. The same blot was subsequently hybridized with %-actin cDNA probe (bottom) as a loading control. (C) Western blot analysis ofPD-1 expression from the transfected cells using specific PD-1 antibody (top) or %-actin antibody (bottom). HIV-1-infected samples were used asa positive control.




.orgDownloaded from

fore, Nef appears to synergize with viral antigens to dramati-cally increase PD-1 expression levels on virus-specific T cells.This effect may help us to better understand the mechanism bywhich Nef exerts SIV-driven pathogenesis in vivo (17). Forinstance, deletion of Nef in SIV has minimal effects on virus

replication in cultured cells but is required for maintaininghigh viral loads and pathological potential in vivo (17). Thesestudies suggest that Nef dictates the ability of SIV infection tobecome either an acute or a chronic infection.

These data suggest that p38 inhibition and/or anti-Nef ther-

FIG. 4. HIV-1 Nef stimulates upregulation of PD-1 in infected cells. (A) Phenotypic analysis of PD-1 on HIV-specific and positive CD4! T cells using classII tetramer and p24Gag staining in viremic patients. PBMCs from the HIV-1-positive and -negative patients were stained directly ex vivo and were assessed byfive-color flow cytometry on gated CD3!/CD4! lymphocytes. Representative dot plots (panel-I) show positive/negative class II tetramer staining in HIV-infectedindividuals gated on CD3! T cells. The inset boxes indicate the tetramer-positive cells. The percentage of tetramer-positive cells is indicated in each plot. Furtherrepresentative dot plots (panel-II and -III) show the staining of HIV-1 p24Gag-positive and -negative cells from the tetramer-positive and -negative cells. Theoverlay histograms (panel-IV) represent the MFI of PD-1 expression. The shaded histograms represent the tetramer-negative/HIV-1-positive cells, and the openhistograms represent tetramer-positive/HIV-1-positive cells. (B) Cell surface expression of PD-1 on human PBMCs infected with HIV-1Wt or HIV-1"Nef virus.Infected cells (after 2 days and 6 days of infection) were analyzed for PD-1 expression in the CD3!/CD4!/CD24HSA! populations. (C) Longitudinal analysisof PD-1 on human PBMCs infected with wild-type virus at different time periods as indicated. PD-1 expression on CD3!/CD4!/CD24HSA! cells was measuredin HIV-1-infected and -uninfected control cells. Representative data show the MFI of PD-1 expression (n ) 3). The bars show mean values. Error bars showstandard deviations.




.orgDownloaded from

apy could potentially prime antiviral immune responses. Anobvious advantage of anti-PD-L1 therapy is that inhibiting Nefcould minimize potential autoimmune side effects manifestingfrom PD-1 inhibition, which has been observed in anti-CTLAtherapies (15, 16, 22). However, several clarifications regardingthe time and nature of Nef-mediated PD-1 upregulation stillremain to be investigated. For instance, although serum con-centrations of Nef correlate with PD-1 upregulation, it is un-known whether this serum Nef can sufficiently enter randomlyactivated or HIV-specific T cells to stimulate PD-1 and facili-tate an “exhaustive” phenotype, as has been described by oth-ers (4, 31). Furthermore, in HIV-positive patients, the upregu-lation of PD-1 appears to be exclusive to HIV-specific T cells

and not CMV-specific T cells (31). Thus, having sufficientquantities of Nef at the time of T-cell programming and inspatial proximity to antigen presentation may be important forhaving selectivity toward different viruses. At least in the caseof CMV, HIV infection may also have minimal effects because,in most instances, CMV infection and its memory T-cell de-velopment may have occurred prior to HIV infection. There-fore, it remains to be determined if HIV, and specifically Nef,can modulate the expression of PD-1 on other virus-specificT-cells after HIV infection. Such modulation would be ex-pected to contribute to immune dysregulation and T-cell dys-function (4, 6, 15, 32). Although antigen persistence andchronic TCR stimulation have also been implicated as causes

FIG. 5. p38 MAPK activation by Nef is required for the transcriptional upregulation of PD-1. (A) p38 MAPK activation by Nef. Shown isWestern blot analysis of protein extracted from Jurkat cells transfected with vector control (5 #g) or pNef (5 #g) and immunoblotted with totalp38 MAPK- and phospho (P)-p38 MAPK-specific antibodies. The histograms represent FACS analysis of phospho-p38 MAPK expression.(B) Blockade of PD-1 expression by p38 MAPK inhibition. Human PBMCs (1 $ 106) were infected with NL4-3Wt virions and treated with orwithout increasing doses of p38 MAPK inhibitor (RWJ67657) as indicated. Four days postinfection and posttreatment, equal number of cells wereassayed for surface PD-1 expression in a CD3!/CD4! population by flow cytometry. The data are representative of two independent experiments.Observations of similar suppression of PD-1 were obtained. Wt, wild type; Inhi, inhibitor. (C) Jurkat T cells negative for p38 MAPK activity bysiRNA or a dominant-negative phenotype with pNef. At 48 h after transfection, the surface levels of PD-1 expression were determined by flowcytometry using a PD-1-specific antibody. The shaded histograms show the isotype-matched control antibodies, and the open histograms representPD-1 expression. Nef-induced PD-1 induction in clone p38 siRNA (clone 61) (top) and p38 MAPK-DN cells was inhibited (bottom). Similar resultswere obtained in two independent experiments. The transfection efficiency was monitored by cotransfection of a pCMV plasmid encoding greenfluorescent protein, which also served as a marker for gating on transfected cells. (D) Jurkat T cells were transiently transfected with the reporterconstruct PD-1/Luc and the empty vector or pNef and cultured for 2 days in the presence or absence of p38 inhibitor, and luciferease activity wasmeasured as described in Materials and Methods. Values and bars represent means (n ) 3) and standard deviations. AU, arbitrary units.




.orgDownloaded from

of PD-1 upregulation, inhibition of PD-1 was sufficient to re-store the antiviral immune response, resulting in virus clear-ance (1). Therefore, PD-1 upregulation may not only be theeffect of failed virus clearance, but may also contribute to viralpersistence.

Considering that both acute and chronic virus infectionsprovide antigens for the host, ascertaining the impetus drivinga chronic infection is of great interest. Our findings suggestthat Nef, a factor implicated in driving chronic infections inSIV, stimulates the expression of PD-1 during HIV infection.

ACKNOWLEDGMENTS

We thank Jean D. Boyer, David A. Hokey, Michele A. Kutzler, andDaniel Schullery for technical assistance, helpful comments on themanuscript, and providing reagents. We thank Farida Shaheen, Centerfor AIDS Research, University of Pennsylvania, Philadelphia, for as-sistance with virus generation. We thank Jeffrey S. Faust, The WistarInstitute flow cytometry facility, for FACS analysis and Michael J.Merva and Denise Dixon for administrative assistance. We thank ScottWadsworth and John Siekierka from Johnson & Johnson Pharmaceu-tical Research and Development, New Jersey, for their useful com-ments and reagents.

This work was supported by a Johnson & Johnson PharmaceuticalResearch and Development grant to D.B.W. and K.M. Support fromthe National Institutes of Health AIDS Research and Reference Re-agents Program is also acknowledged.

REFERENCES

1. Barber, D. L., E. J. Wherry, D. Masopust, B. Zhu, J. P. Allison, A. H. Sharpe,G. J. Freeman, and R. Ahmed. 2006. Restoring function in exhausted CD8 Tcells during chronic viral infection. Nature 439:682–687.

2. Boni, C., P. Fisicaro, C. Valdatta, B. Amadei, P. Di Vincenzo, T. Giuberti, D.Laccabue, A. Zerbini, A. Cavalli, G. Missale, A. Bertoletti, and C. Ferrari.

2007. Characterization of hepatitis B virus (HBV)-specific T-cell dysfunctionin chronic HBV infection. J. Virol. 81:4215–4225.

3. Collins, K. L., B. K. Chen, S. A. Kalams, B. D. Walker, and D. Baltimore.1998. HIV-1 Nef protein protects infected primary cells against killing bycytotoxic T lymphocytes. Nature 391:397–401.

4. Day, C. L., D. E. Kaufmann, P. Kiepiela, J. A. Brown, E. S. Moodley, S.Reddy, E. W. Mackey, J. D. Miller, A. J. Leslie, C. DePierres, Z. Mncube, J.Duraiswamy, B. Zhu, Q. Eichbaum, M. Altfeld, E. J. Wherry, H. M. Coova-dia, P. J. Goulder, P. Klenerman, R. Ahmed, G. J. Freeman, and B. D.Walker. 2006. PD-1 expression on HIV-specific T cells is associated withT-cell exhaustion and disease progression. Nature 443:350–354.

5. Deacon, N. J., A. Tsykin, A. Solomon, K. Smith, M. Ludford-Menting, D. J.Hooker, D. A. McPhee, A. L. Greenway, A. Ellett, C. Chatfield, V. A. Lawson,S. Crowe, A. Maerz, S. Sonza, J. Learmont, J. S. Sullivan, A. Cunningham,D. Dwyer, D. Dowton, and J. Mills. 1995. Genomic structure of an attenuatedquasi species of HIV-1 from a blood transfusion donor and recipients.Science 270:988–991.

6. D’Souza, M., A. P. Fontenot, D. G. Mack, C. Lozupone, S. Dillon, A. Meditz,C. C. Wilson, E. Connick, and B. E. Palmer. 2007. Programmed death 1expression on HIV-specific CD4! T cells is driven by viral replication andassociated with T cell dysfunction. J. Immunol. 179:1979–1987.

7. Eyers, P. A., P. van den IJssel, R. A. Quinlan, M. Goedert, and P. Cohen.1999. Use of a drug-resistant mutant of stress-activated protein kinase 2a/p38to validate the in vivo specificity of SB 203580. FEBS Lett. 451:191–196.

8. Freeman, G. J., A. J. Long, Y. Iwai, K. Bourque, T. Chernova, H. Nishimura,L. J. Fitz, N. Malenkovich, T. Okazaki, M. C. Byrne, H. F. Horton, L. Fouser,L. Carter, V. Ling, M. R. Bowman, B. M. Carreno, M. Collins, C. R. Wood,and T. Honjo. 2000. Engagement of the PD-1 immunoinhibitory receptor bya novel B7 family member leads to negative regulation of lymphocyte acti-vation. J. Exp. Med. 192:1027–1034.

9. Freeman, G. J., E. J. Wherry, R. Ahmed, and A. H. Sharpe. 2006. Reinvig-orating exhausted HIV-specific T cells via PD-1-PD-1 ligand blockade. J.Exp. Med. 203:2223–2227.

10. Fujii, Y., K. Otake, M. Tashiro, and A. Adachi. 1996. Soluble Nef antigen ofHIV-1 is cytotoxic for human CD4! T cells. FEBS Lett. 393:93–96.

11. Hanna, Z., D. G. Kay, N. Rebai, A. Guimond, S. Jothy, and P. Jolicoeur.1998. Nef harbors a major determinant of pathogenicity for an AIDS-likedisease induced by HIV-1 in transgenic mice. Cell 95:163–175.

12. He, J., S. Choe, R. Walker, P. Di Marzio, D. O. Morgan, and N. R. Landau.1995. Human immunodeficiency virus type 1 viral protein R (Vpr) arrests

FIG. 6. Activation of p38 by Nef correlates with PD-1 expression and inversely with CD4 counts in HIV! patients. (A) MFI of PD-1 expressionon tetramer-positive CD4! cells and viral-RNA counts. The FACS plots are gated on CD3!/CD4!/p24.17-DR1 tetramer-positive T cells (n ) 20).(B) MFI of PD-1 expression on total CD4! (Tet*/HIV!) and HIV-1-specific CD4! (Tet!/HIV!) T cells (n ) 12) from the infected patients. Thelines show mean values. (C) Correlation between MFI of PD-1 expression and the serum Nef level. There is a correlation between the serum Nefconcentration and PD-1 expression (n ) 20). (D and F) Intracellular staining for phospho-p38 MAPK in HIV-1 patients was determined by FACSanalysis; the plots are gated on CD3!/CD4! T cells or CD3!/CD4!/tetramer! T cells. (D) There is an inverse correlation between phospho-p38MAPK expression and CD4 T-cell counts (n ) 15). (E) There is no correlation between p38 MAPK activation and PD-1 expression on total CD4T cells (n ) 15). However, a positive correlation exists with PD-1 expression (MFI) on HIV-specific CD4! T cells (F). These relationships wereevaluated using the Spearman correlation test using the Prism 4 GraphPad software.




.orgDownloaded from

cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J. Virol.69:6705–6711.

13. Hokey, D. A., F. B. Johnson, J. Smith, J. L. Weber, J. Yan, L. Hirao, J. D.Boyer, M. G. Lewis, G. Makedonas, M. R. Betts, and D. B. Weiner. 2008.Activation drives PD-1 expression during vaccine-specific proliferation andfollowing lentiviral infection in macaques. Eur. J. Immunol. 38:1435–1445.

14. Jamieson, B. D., and J. A. Zack. 1998. In vivo pathogenesis of a humanimmunodeficiency virus type 1 reporter virus. J. Virol. 72:6520–6526.

15. Kaufmann, D. E., D. G. Kavanagh, F. Pereyra, J. J. Zaunders, E. W. Mackey,T. Miura, S. Palmer, M. Brockman, A. Rathod, A. Piechocka-Trocha, B.Baker, B. Zhu, S. Le Gall, M. T. Waring, R. Ahern, K. Moss, A. D. Kelleher,J. M. Coffin, G. J. Freeman, E. S. Rosenberg, and B. D. Walker. 2007.Upregulation of CTLA-4 by HIV-specific CD4! T cells correlates withdisease progression and defines a reversible immune dysfunction. Nat. Im-munol. 8:1246–1254.

16. Keir, M. E., M. J. Butte, G. J. Freeman, and A. H. Sharpe. 2008. PD-1 andits ligands in tolerance and immunity. Annu. Rev. Immunol. 26:677–704.

17. Kestler, H. W. III, D. J. Ringler, K. Mori, D. L. Panicali, P. K. Sehgal, M. D.Daniel, and R. C. Desrosiers. 1991. Importance of the nef gene for mainte-nance of high virus loads and for development of AIDS. Cell 65:651–662.

18. Kirchhoff, F., T. C. Greenough, D. B. Brettler, J. L. Sullivan, and R. C.Desrosiers. 1995. Brief report: absence of intact nef sequences in a long-termsurvivor with nonprogressive HIV-1 infection. N. Engl. J. Med. 332:228–232.

19. Latchman, Y., C. R. Wood, T. Chernova, D. Chaudhary, M. Borde, I. Cher-nova, Y. Iwai, A. J. Long, J. A. Brown, R. Nunes, E. A. Greenfield, K.Bourque, V. A. Boussiotis, L. L. Carter, B. M. Carreno, N. Malenkovich, H.Nishimura, T. Okazaki, T. Honjo, A. H. Sharpe, and G. J. Freeman. 2001.PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat.Immunol. 2:261–268.

20. Muthumani, K., A. Y. Choo, D. S. Hwang, A. Premkumar, N. S. Dayes, C.Harris, D. R. Green, S. A. Wadsworth, J. J. Siekierka, and D. B. Weiner.2005. HIV-1 Nef-induced FasL induction and bystander killing requires p38MAPK activation. Blood 106:2059–2068.

21. Muthumani, K., A. Y. Choo, W. X. Zong, M. Madesh, D. S. Hwang, A.Premkumar, K. P. Thieu, J. Emmanuel, S. Kumar, C. B. Thompson, andD. B. Weiner. 2006. The HIV-1 Vpr and glucocorticoid receptor complex isa gain-of-function interaction that prevents the nuclear localization ofPARP-1. Nat. Cell Biol. 8:170–179.

22. Nishimura, H., M. Nose, H. Hiai, N. Minato, and T. Honjo. 1999. Develop-ment of lupus-like autoimmune diseases by disruption of the PD-1 geneencoding an ITIM motif-carrying immunoreceptor. Immunity 11:141–151.

23. Petrovas, C., J. P. Casazza, J. M. Brenchley, D. A. Price, E. Gostick, W. C.Adams, M. L. Precopio, T. Schacker, M. Roederer, D. C. Douek, and R. A.

Koup. 2006. PD-1 is a regulator of virus-specific CD8! T cell survival in HIVinfection. J. Exp. Med. 203:2281–2292.

24. Radziewicz, H., C. C. Ibegbu, M. L. Fernandez, K. A. Workowski, K.Obideen, M. Wehbi, H. L. Hanson, J. P. Steinberg, D. Masopust, E. J.Wherry, J. D. Altman, B. T. Rouse, G. J. Freeman, R. Ahmed, and A.Grakoui. 2007. Liver-infiltrating lymphocytes in chronic human hepatitis Cvirus infection display an exhausted phenotype with high levels of PD-1 andlow levels of CD127 expression. J. Virol. 81:2545–2553.

25. Roeth, J. F., and K. L. Collins. 2006. Human immunodeficiency virus type 1Nef: adapting to intracellular trafficking pathways. Microbiol. Mol. Biol. Rev.70:548–563.

26. Saksela, K., G. Cheng, and D. Baltimore. 1995. Proline-rich (PxxP) motifs inHIV-1 Nef bind to SH3 domains of a subset of Src kinases and are requiredfor the enhanced growth of Nef! viruses but not for down-regulation ofCD4. EMBO J. 14:484–491.

27. Sawai, E. T., M. S. Hamza, M. Ye, K. E. Shaw, and P. A. Luciw. 2000.Pathogenic conversion of live attenuated simian immunodeficiency virusvaccines is associated with expression of truncated Nef. J. Virol. 74:2038–2045.

28. Schaeffer, E., R. Geleziunas, and W. C. Greene. 2001. Human immunodefi-ciency virus type 1 Nef functions at the level of virus entry by enhancingcytoplasmic delivery of virions. J. Virol. 75:2993–3000.

29. Scriba, T. J., H. T. Zhang, H. L. Brown, A. Oxenius, N. Tamm, S. Fidler, J.Fox, J. N. Weber, P. Klenerman, C. L. Day, M. Lucas, and R. E. Phillips.2005. HIV-1-specific CD4! T lymphocyte turnover and activation increaseupon viral rebound. J. Clin. Investig. 115:443–450.

30. Swingler, S., B. Brichacek, J. M. Jacque, C. Ulich, J. Zhou, and M. Steven-son. 2003. HIV-1 Nef intersects the macrophage CD40L signalling pathwayto promote resting-cell infection. Nature 424:213–219.

31. Trautmann, L., L. Janbazian, N. Chomont, E. A. Said, S. Gimmig, B.Bessette, M. R. Boulassel, E. Delwart, H. Sepulveda, R. S. Balderas, J. P.Routy, E. K. Haddad, and R. P. Sekaly. 2006. Upregulation of PD-1 expres-sion on HIV-specific CD8! T cells leads to reversible immune dysfunction.Nat. Med. 12:1198–1202.

32. Velu, V., S. Kannanganat, C. Ibegbu, L. Chennareddi, F. Villinger, G. J.Freeman, R. Ahmed, and R. R. Amara. 2007. Elevated expression levels ofinhibitory receptor programmed death 1 on simian immunodeficiency virus-specific CD8 T cells during chronic infection but not after vaccination.J. Virol. 81:5819–5828.

33. Wadsworth, S. A., D. E. Cavender, S. A. Beers, P. Lalan, P. H. Schafer, E. A.Malloy, W. Wu, B. Fahmy, G. C. Olini, J. E. Davis, J. L. Pellegrino-Gensey,M. P. Wachter, and J. J. Siekierka. 1999. RWJ 67657, a potent, orally activeinhibitor of p38 mitogen-activated protein kinase. J. Pharmacol. Exp. Ther.291:680–687.




.orgDownloaded from

Human Immunodeficiency Virus Type 1 Nef Induces Programmed Death 1 Expression through a p38...

Documents

Transcript of Human Immunodeficiency Virus Type 1 Nef Induces Programmed Death 1 Expression through a p38...