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Journal of Virology, December 2008, p. 11536-11544, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.00485-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Human Immunodeficiency Virus Type 1 Nef Induces Programmed Death 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,¹ Senthil 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 19104,¹ Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115,² Department of Dermatology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115,³ Division of Infectious Diseases, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104⁴

Received 5 March 2008/ Accepted 3 September 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 upregulated on antigen-specific T cells during various chronic viral infections. Indeed, it has been reported that human immunodeficiency virus (HIV)-specific T cells express elevated levels of PD-1 and that this expression correlates with the viral load and inversely with CD4⁺ T-cell counts. More importantly, antibody blockade of the PD-1/PD-L1 pathway was sufficient to both increase and stimulate virus-specific T-cell proliferation and cytokine 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 the expression of PD-1 during infection in vitro. Nef-induced PD-1 upregulation requires its proline-rich motif and the activation of the downstream kinase p38. Further, inhibition of Nef activity by p38 MAPK inhibitor effectively blocked PD-1 upregulation, suggesting that p38 MAPK activation is an important initiating event in Nef-mediated PD-1 expression in HIV-1-infected cells. These data demonstrate an important signaling event of Nef in HIV-1 pathogenesis.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Functional impairment of antigen-specific T cells is a hallmark feature of chronic viral infections (1, 9). Accordingly, recent evidence has suggested that PD-1 upregulation is often associated with various chronic viral infections, including lymphocytic choriomeningitis virus, hepatitis C virus, hepatitis B virus, cytomegalovirus (CMV), human immunodeficiency virus (HIV), and tumors (1, 2, 4, 9, 23, 24). The immunoreceptor programmed death 1 (PD-1), a 55-kDa transmembrane protein containing an immunoreceptor tyrosine-based inhibitory motif, was originally isolated from a T-cell line exhibiting high sensitivity to apoptosis (16). PD-1 is one of the three identified inhibitory B7-recognizing immunoreceptors of the CD28 family involved in signaling T-cell death and, similar to cytotoxic-T-lymphocyte antigen 4 (CTLA4), negatively regulates T-cell function (8, 15, 16, 19). Two PD-1 ligands, PD-L1 and PD-L2, have been identified and show distinct roles in regulating the immune responses. Engagement of PD-1 with its ligands, PD-L1 and PD-L2, inhibits T-cell proliferation and cytokine production (1, 8, 19, 22, 23). Also, in vivo administration of antibodies that blocked the interaction of PD-L1/PD-1 enhanced T-cell responses in mice chronically infected with lymphocytic choriomeningitis virus (1).

In HIV-infected subjects, PD-1 expression was significantly increased on HIV-specific CD8⁺ T cells compared with total CD8⁺ T cells and was correlated with the viral load (1, 4, 9, 23, 31). Importantly, in HIV, treatment of impaired T cells with a blocking anti-PD-L1 antibody was sufficient to augment HIV-specific T-cell function (31). The relationship between PD-1 expression on HIV-specific CD4⁺ T cells and HIV disease is important to understand because functional impairment of HIV-specific CD4⁺ T cells during chronic HIV infection has been closely linked to HIV replication and disease progression (6, 4, 15, 23, 31). The association between PD-1 expression on HIV-specific T cells, cellular exhaustion, and disease progression may represent an important advance in our understanding of HIV pathogenesis. Targeting PD-1 may play a role in HIV disease progression and development of new therapeutic approaches. However, the mechanisms employed by HIV to regulate PD-1 expression remain unknown. Data clearly support the notion that PD-1 upregulation can be a function of chronic immune activation (13). However, the observation that PD-1 levels in HIV infection are higher than those observed in other chronic infections suggests that additional viral factors may play a more direct role in PD-1 expression. In order to provide an understanding, we assessed PD-1 expression by directly looking at HIV-infected cells. We observed that HIV infection of T cells can drive increased PD-1 expression. This expression is a function primarily of the Nef gene product of HIV type 1 (HIV-1).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Plasmid construction. The HIV-1 proviral infectious constructs pNL4-3Wt_HSA (14) and pNL4-3_HSA/Env (12) were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH. HIV-1 proviral DNA genes were individually mutated specifically by inactivating the start codon without affecting the reading frames of other viral proteins that use the same transcript. Mutations were made by in vitro site-directed mutagenesis using a QuickChange mutagenesis kit (Stratagene, La Jolla, CA) (12, 14). All mutations were confirmed by sequencing, and all the mutated constructs were tested by Western blot analysis for loss of gene expression. Constructs containing accessory gene-deficient variants and p38Wt and dominant-negative plasmids were generated as described before (3, 7, 20).

Patients. HIV-infected individuals' cells and sera or plasma were obtained from the University of Pennsylvania Center for AIDS Research immunology clinical core for our study. Heparinized blood was obtained in accordance with protocols approved by the Institutional Review Board of the Hospital of the University of Pennsylvania. The median viral load for these samples was 15,401 HIV-1 RNA copies per ml plasma (range, 121 to 204,575), and the median absolute CD4 T-cell count was 612 (range, 211 to 1,432). Peripheral blood mononuclear cells (PBMCs) were separated and cryopreserved in liquid nitrogen until assay time. The HIV-1 RNA level was determined from plasma using the Roche Amplicor 1.5 kit (Roche Diagnostic Systems, New Jersey) according to the manufacturer's recommendations. HIV-infected subjects were serologically identified 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 individual donors were obtained from the immunology clinical core facility at the University of Pennsylvania School of Medicine, and PBMCs were isolated by Ficoll-Hypaque (Pharmacia, Piscataway, NJ) density centrifugation. The cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 20 mM HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin. All cells were cultured in lipopolysaccharide-free medium in the presence of interleukin 2 in the medium. HIV-1 stocks were produced in 293T cells and 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 cytometry. HSA expression indicates completion of steps in the viral life cycle up to and including de novo viral-gene expression (30). HIV-1 pseudeoviral particles were produced by transfecting 293T cells (obtained from the ATCC) with FuGene 6 transfection reagent (Roche Applied Science, Nutley, NJ) by using vectors encoding vesicular stomatitis virus G envelope (5 µg). Virus-containing supernatants were harvested 60 to 72 h after transfection, viral titers were determined by infection of the human T-cell line Jurkat, and p24^Gag antigen was measured by capture enzyme-linked immunosorbent assay (ELISA) using a p24 ELISA kit (Coulter, Miami, FL). For infection studies, human PBMCs were isolated from healthy HIV-1-negative donors as described above. PBMCs (2 x 10⁵ cells/well) were mock infected (with media from the cell cultures used to grow the cells) or infected with cell-free HIV-1 at a concentration of 100 50% tissue culture infective doses/10⁶ cells/ml (14, 20, 30). After 4 to 6 h of incubation at 37°C, the cells were gently washed, resuspended with complete medium, and maintained for the indicated time periods. At the end of the incubation period, culture supernatants and cells were harvested for p24^Gag antigen determinations, as well as other fluorescence-activated cell sorter (FACS) analysis (20, 30).

Tetramer and antibody staining. The following directly conjugated antibodies were used: CD3-phycoerythrin (PE)/fluorescein isothiocyanate (FITC)/allophycocyanin (APC)/Pacific Blue (PB), CD4-PE/FITC/PB, CD8-PE/FITC/PB, and streptavidin-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 647 or -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-specific tetramers, PE conjugated) and were purchased from Beckman Coulter, Fullerton, 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 µg PE-labeled tetramer in 100 µl FACS staining buffer (1x phosphate-buffered saline (PBS), 0.02% NaN₃, and 0.2% fetal calf serum) for 1 h at 37°C and subsequently with combinations of fluorochrome-labeled antibodies for 30 min on ice (29). For intracellular staining, cells were permeabilized using BD FixPerm (BD) following staining. The percentages of cells expressing intracytoplasmic HIV-1 Gag-related products were evaluated using KC57-RD1/PE- or KC57/FITC-conjugated anti-HIV-1 Gag monoclonal antibody (Beckman Coulter, Miami, FL). Electronic compensation was conducted with antibody capture beads (BD Biosciences, San Jose, CA) stained separately with individual monoclonal antibodies used in the test samples. Forward scatter area versus forward scatter height was used to gate out the cell aggregates. In addition, ViViD dye staining was used to exclude the dead and dying cells (13). Cells were analyzed with a modified LSRII flow cytometry (BD Immunocytometry 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 µg protein) were resolved on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and processed according to the standard protocols. The antibodies used were polyclonal anti-human PD-1 (R&D Systems, Minneapolis, MN) and β-actin (Cell Signaling Technology, Danvers, MA). The primary antibodies were used at dilutions of 1:1,000. The secondary antibodies were anti-rabbit or anti-mouse immunoglobulin G conjugated to horseradish peroxidase (dilution, 1:5,000). Signals were detected using enhanced chemiluminescence (Amersham Life Sciences Inc., Piscataway, NJ) (21).

PD-1 promoter construction and luciferase reporter assay. For assessment of PD-1 transcription by viral genes, luciferase reporter plasmids expressing PD-1 were assembled from synthetic oligonucleotides (Geneart, Germany) and cloned by 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). The promoter sequence used started from a putative transcription start site and extended to the 5' upstream regions. All new constructs and mutations were confirmed by DNA sequencing. In brief, Jurkat cells were seeded onto a six-well culture plate at a density of 0.5 million cells per milliliter of medium and transiently transfected as described previously (21) with a constant amount of the luciferase reporter PD-1 promoter and various viral-gene expression plasmids. The total amount of DNA was kept constant by adding empty vector. Cells were harvested 48 h after transfection and lysed in cell lysis buffer, and luciferase activities were assayed with the luciferase assay kit (Promega, Madison, WI) using Lumat-LB9501 (Berthold, Bad Wildbad, Germany). The transfection efficiency was normalized by cotransfection with pEF-lacZ and assay for β-galactosidase expression (21).

Determination of soluble Nef antigen by ELISA. A sandwich ELISA procedure was used to detect the soluble Nef antigen in serum (10). Briefly, 100 µl was measured with sandwich-type capture ELISA plates; 96-well ELISA plates were coated with 100 µl of 1.0-µg/ml rabbit HIV-1 Nef antiserum (AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH) overnight at 4°C. After incubation with blocking buffer (0.25% bovine serum albumin/0.05% Tween 20 in PBS) at 37°C for 1 h, experimental sera and purified entire Nef recombinant protein (ImmunoDiagnostics, Inc., Woburn, MA) as a standard at twofold dilutions (100 µl) were added to wells in duplicate. The plates were incubated overnight at 4°C and then washed three times. After the washing, 100 µl of 0.5-µg/ml mouse monoclonal antibody to HIV-1 Nef (1:5,000; Abcam, Cambridge, MA) was added, and the plates were incubated at 37°C for 1 h. After six washes with PBS plus 0.05% Tween 20 (PBST), 100 µl of horseradish peroxidase-conjugated anti-mouse secondary antibodies (1:5,000) was added, and the plates were incubated for 1 h at 37°C. After being washed eight times with PBST, the substrate (o-phenylenediamine [Sigma, St. Louis, MO], 0.4 mg/ml in 0.1 mol/liter citrate/phosphate buffer, pH 5.5, 0.04% H₂O₂) was added, and the reaction was stopped 20 min later by adding 50 µl of 12.5% (vol/vol) H₂SO₄. RPMI medium and control human immunoglobulin G supernatants were used as negative controls, and PBST was used as a zero standard. Absorbance was measured with an ELISA reader at 405 nm, and the concentrations of soluble Nef protein in the samples were calculated by interpolation from the standard curve (10).

RNA extraction and Northern blot analysis. Total RNA was extracted using an RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Twenty-five micrograms of total RNA was subjected to electrophoresis on a 1.2% denaturing agarose gel and transferred to nitrocellulose. The PD-1 expression 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 five times at 42°C with 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS, once with 0.5x SSC-0.1% SDS, and once at 55°C with 0.1x SSC-0.1% SDS in a minihybridization oven. The membrane was exposed on a developing screen for 16 to 24 h and scanned using a PhosphorImager (Molecular Dynamics, Piscataway, NJ). The transcripts were quantified with ImageQuaNT (version 4.0) software.

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

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Nef is necessary and sufficient to drive PD-1 upregulation during HIV-1 infection. The mechanisms employed by HIV resulting in PD-1 upregulation on a per cell basis are not well characterized. To address this question, we infected human PBMCs with HIV-1 (NL4-3) and analyzed the upregulation of PD-1 on HIV-infected (CD24HSA-positive) cells (12, 14, 20, 30). As shown in Fig. 1, HIV infection was sufficient to upregulate PD-1 expression on T cells. We next individually deleted the viral Env gene and the accessory genes Vif, Nef, Vpr, and Vpu and utilized these mutant viruses in a pseudoviral system as tools to probe PD-1 expression. Loss of Nef, but not Env and the other accessory genes, led to an attenuation of HIV-mediated PD-1 upregulation. This effect did not appear to be a consequence of the efficiency of infection, as the mutant viruses all exhibited similar levels of p24^Gag production (data not 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 the individual HIV genes Vif, Nef, Vpr, Vpu, and Env and evaluated PD-1 upregulation. As shown in Fig. 2A, only Nef was sufficient to upregulate PD-1, and using its mutants, we observed that Nef required its proline-rich (PXXP) motif (3, 26) for this activity (Fig. 2B).

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

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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-1 was performed on Jurkat cells transfected with HIV-1 viral genes as indicated and pGFP. The cells were collected 3 days later, and PD-1 expression was measured. Gates were set to include green fluorescent protein (GFP)-positive cells only. The data are representative of three or more separate studies. The shaded histograms represent staining with isotype control, and the open histograms represent staining with PD-1 antibody. FSC-A, forward scatter area.

Nef expression induces PD-1 production. To examine the role of Nef in the regulation of PD-1, we first developed PD-1/Luc constructs and analyzed the transcriptional activity of PD-1 using cotransfection of each of the viral-gene constructs with the PD-1 luciferase reporter plasmid into Jurkat cells. The transcription activity of PD-1 was significantly increased in cells transfected with pNef compared with cells transfected with the other viral genes or a mock control (Fig. 3A). This PD-1 effect of Nef was through transcriptional activation of the PD-1 promoter, which led to both PD-1 mRNA and protein upregulation (Fig. 3B and C). These data also suggest that increased surface translocation of cytoplasmic PD-1 may not be a major contributor to the observed phenotype, despite Nef's previous role in modulating the surface expression of various proteins, including CD4 (25, 26, 30). Taken together, our data suggest that Nef mediates PD-1 upregulation in HIV-infected cells.

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FIG. 3. Nef expression induces PD-1 production. (A) Jurkat T cells were transiently transfected with a PD-1/Luc reporter construct (10 µg) and equal amounts of either empty vector or vectors containing accessory genes as indicated. Forty-eight hours posttransfection, PD-1 transcriptional activity 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-1 expression. (B) Northern blot of 20 µg of total RNA isolated from transfected cells (top).Shown is hybridization with -32P-labeled human PD-1 cDNA probe. The same blot was subsequently hybridized with β-actin cDNA probe (bottom) as a loading control. (C) Western blot analysis of PD-1 expression from the transfected cells using specific PD-1 antibody (top) or β-actin antibody (bottom). HIV-1-infected samples were used as a positive control.

PD-1 expression is upregulated on HIV-specific CD4⁺ T cells. We next examined infection of primary PBMCs to study the effects of infection on primary CD4 T cells. To answer this question, we took HIV-1-positive patient PBMCs and stained these samples to identify virus-specific CD4⁺ T cells with HIV class II tetramers. HIV-specific CD4⁺ T cells were both positive and negative for infection (p24^Gag positive). In addition, PD-1 was upregulated on HIV-specific and positive CD4 T cells (Fig. 4A), suggesting that its upregulation in CD4⁺ T cells was not controlled autonomously by viral infection of the host cell. Next, we infected HIV-negative PBMCs with the HIV-1 NL4-3-Wt virus or virus with Nef deleted and measured PD-1 upregulation in CD3⁺/CD4⁺ T cells. When CD4⁺ T cells were analyzed, PD-1 was upregulated by 2 days postinfection (Fig. 4B). Kinetically, PD-1 upregulation was minimal in early infection but gradually increased by day 2 and was fully upregulated by day 10 (Fig. 4C). This upregulation of PD-1 on CD4⁺ T cells by HIV required Nef, suggesting that Nef was directly involved in the ability of HIV-1 to upregulate PD-1 on CD4⁺ T cells.

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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 class II tetramer and p24Gag staining in viremic patients. PBMCs from the HIV-1-positive and -negative patients were stained directly ex vivo and were assessed by five-color flow cytometry on gated CD3+/CD4+ lymphocytes. Representative dot plots (panel-I) show positive/negative class II tetramer staining in HIV-infected individuals gated on CD3+ T cells. The inset boxes indicate the tetramer-positive cells. The percentage of tetramer-positive cells is indicated in each plot. Further representative dot plots (panel-II and -III) show the staining of HIV-1 p24Gag-positive and -negative cells from the tetramer-positive and -negative cells. The overlay histograms (panel-IV) represent the MFI of PD-1 expression. The shaded histograms represent the tetramer-negative/HIV-1-positive cells, and the open histograms represent tetramer-positive/HIV-1-positive cells. (B) Cell surface expression of PD-1 on human PBMCs infected with HIV-1Wt or HIV-1Nef 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 analysis of 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 measured in 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 show standard deviations.

p38 MAPK activation by Nef is required for the transcriptional upregulation of PD-1. We had previously reported that Nef was both necessary and sufficient for HIV-1 to activate the p38 kinase in T cells (Fig. 5A) (20). Similar to PD-1 upregulation, Nef also required its proline-rich motif (PXXP) to stimulate the activation of p38. Therefore, we examined whether the activation of p38 was also involved in the ability of Nef to upregulate PD-1. As shown in Fig. 5B, pharmacologic inhibition of the p38 kinase specifically downregulated HIV-induced PD-1 upregulation in a concentration-dependent manner. Similar results were achieved with small interfering RNA (siRNA)-mediated knockdown of p38 kinase and with an overexpression of a dominant-negative p38 (Fig. 5C). The siRNA clone 352, which fails to knock down p38, failed to prevent Nef-mediated upregulation of PD-1. However, siRNA clone 61, which efficiently knocks down p38 (20), prevented Nef-mediated PD-1 upregulation. Consistently, pharmacologic inhibition of p38 also inhibited Nef-induced transcriptional activation of PD-1 (Fig. 5D). Therefore, it appears that upon Nef's entry into T cells, the p38 pathway is activated and requires its proline-rich motif for PD-1 transcriptional upregulation.

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FIG. 5. p38 MAPK activation by Nef is required for the transcriptional upregulation of PD-1. (A) p38 MAPK activation by Nef. Shown is Western blot analysis of protein extracted from Jurkat cells transfected with vector control (5 µg) or pNef (5 µg) and immunoblotted with total p38 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 x 106) were infected with NL4-3Wt virions and treated with or without increasing doses of p38 MAPK inhibitor (RWJ67657) as indicated. Four days postinfection and posttreatment, equal number of cells were assayed 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 by siRNA or a dominant-negative phenotype with pNef. At 48 h after transfection, the surface levels of PD-1 expression were determined by flow cytometry using a PD-1-specific antibody. The shaded histograms show the isotype-matched control antibodies, and the open histograms represent PD-1 expression. Nef-induced PD-1 induction in clone p38 siRNA (clone 61) (top) and p38 MAPK-DN cells was inhibited (bottom). Similar results were obtained in two independent experiments. The transfection efficiency was monitored by cotransfection of a pCMV plasmid encoding green fluorescent protein, which also served as a marker for gating on transfected cells. (D) Jurkat T cells were transiently transfected with the reporter construct 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 was measured as described in Materials and Methods. Values and bars represent means (n = 3) and standard deviations. AU, arbitrary units.

Activation of p38 by Nef correlates with PD-1 expression. Although it appears that Nef is important for HIV-1 to upregulate PD-1 in infection in vitro, we investigated whether this effect was involved in the upregulation of PD-1 in HIV-1-positive patients in vivo. In determining whether an association exists between PD-1 expression on CD4⁺ T cells and HIV disease progression, we found a strong correlation between the HIV plasma viral load and PD-1 expression on HIV-specific CD4⁺ T cells (r = 0.6249; P = 0.0032) (Fig. 6A). Further, we found a strong positive correlation on PD-1 expression on HIV-positive and tetramer-specific CD4⁺ T cells (P = 0.0001) and a weaker but not statistically significant correlation on tetramer-negative CD4⁺ T cells (P = 0.0582) compared to HIV-negative cells (Fig. 6B). As previously reported, there is a direct inverse correlation between CD4⁺ T-cell counts and PD-1 expression in the tetramer-positive CD4 T-cell population among HIV-1-positive patients (reference 6 and data not shown). Consistent with the hypothesis that Nef directly regulates PD-1 expression, there was a direct correlation between PD-1 expression on HIV-specific CD4⁺ T cells and levels of Nef in patient serum (r = 0.2960; P = 0.0131) (Fig. 6C;). This suggests that Nef produced and released from cells in vivo could correlate with PD-1 expression in patients. In support of in vitro data suggesting Nef stimulates p38 to activate PD-1 transcription, we observed p38 MAPK activation on HIV-specific cells inversely correlated with CD4 T-cell counts (r = –0.3077; P = 0318), suggesting that p38 MAPK activation antagonizes the maintenance of patient CD4⁺ T-cell levels (Fig. 6D). Indeed, the activation of p38 also directly correlated with the levels of PD-1 on CD4⁺/tetramer⁺ T cells (r = 0.3604; P = 0.0390) (Fig. 6E and F), suggesting that p38 MAPK may be involved in PD-1 upregulation in vivo, which may contribute to the CD4⁺ T-cell depletion and immune dysfunction.

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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 expression on 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. The lines show mean values. (C) Correlation between MFI of PD-1 expression and the serum Nef level. There is a correlation between the serum Nef concentration and PD-1 expression (n = 20). (D and F) Intracellular staining for phospho-p38 MAPK in HIV-1 patients was determined by FACS analysis; the plots are gated on CD3+/CD4+ T cells or CD3+/CD4+/tetramer+ T cells. (D) There is an inverse correlation between phospho-p38 MAPK expression and CD4 T-cell counts (n = 15). (E) There is no correlation between p38 MAPK activation and PD-1 expression on total CD4 T cells (n = 15). However, a positive correlation exists with PD-1 expression (MFI) on HIV-specific CD4+ T cells (F). These relationships were evaluated using the Spearman correlation test using the Prism 4 GraphPad software.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This study provides the first mechanistic insight into how HIV-1 regulates PD-1 expression on HIV-1-infected cells. We observed that infection of CD4⁺ T cells by HIV-1 upregulates PD-1 expression and that the accessory protein Nef is both necessary and sufficient for this phenotype in vitro. We also observed that Nef associates with viral antigens to specifically increase PD-1 expression through a p38 MAPK-dependent mechanism. The results of this study are in agreement with observations in both humans and primates that suggest Nef is important 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 determining the persistence of infection and pathogenesis, thus distinguishing acute and chronic infection (27).

Recent evidence from several groups has suggested that viral persistence and chronic T-cell receptor (TCR) stimulation may contribute 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 a hyperactivated phenotype that was associated with CD3 hyperresponsiveness and constitutive activation of growth factor-regulated kinases. More interestingly, splenocytes from these Nef-expressing mice exhibited a decrease in TCR-activated proliferation, suggesting an "exhausted" T-cell phenotype. Therefore, we hypothesized that serum levels of Nef (10) may "potentiate" the effects of viral antigens to hyperstimulate virus-specific T cells to increase PD-1 upregulation. Indeed, when we treated HIV-positive PBMCs with a Gag peptide and increasing concentrations of recombinant Nef, a dramatic increase in the mean fluorescence intensity (MFI) of PD-1 on HIV-specific T cells could be seen (data not shown). Therefore, Nef appears to synergize with viral antigens to dramatically increase PD-1 expression levels on virus-specific T cells. This effect may help us to better understand the mechanism by which Nef exerts SIV-driven pathogenesis in vivo (17). For instance, deletion of Nef in SIV has minimal effects on virus replication in cultured cells but is required for maintaining high viral loads and pathological potential in vivo (17). These studies suggest that Nef dictates the ability of SIV infection to become either an acute or a chronic infection.

These data suggest that p38 inhibition and/or anti-Nef therapy could potentially prime antiviral immune responses. An obvious advantage of anti-PD-L1 therapy is that inhibiting Nef could minimize potential autoimmune side effects manifesting from PD-1 inhibition, which has been observed in anti-CTLA therapies (15, 16, 22). However, several clarifications regarding the time and nature of Nef-mediated PD-1 upregulation still remain to be investigated. For instance, although serum concentrations of Nef correlate with PD-1 upregulation, it is unknown whether this serum Nef can sufficiently enter randomly activated or HIV-specific T cells to stimulate PD-1 and facilitate an "exhaustive" phenotype, as has been described by others (4, 31). Furthermore, in HIV-positive patients, the upregulation of PD-1 appears to be exclusive to HIV-specific T cells and not CMV-specific T cells (31). Thus, having sufficient quantities of Nef at the time of T-cell programming and in spatial proximity to antigen presentation may be important for having selectivity toward different viruses. At least in the case of CMV, HIV infection may also have minimal effects because, in most instances, CMV infection and its memory T-cell development may have occurred prior to HIV infection. Therefore, it remains to be determined if HIV, and specifically Nef, can modulate the expression of PD-1 on other virus-specific T-cells after HIV infection. Such modulation would be expected to contribute to immune dysregulation and T-cell dysfunction (4, 6, 15, 32). Although antigen persistence and chronic TCR stimulation have also been implicated as causes of PD-1 upregulation, inhibition of PD-1 was sufficient to restore the antiviral immune response, resulting in virus clearance (1). Therefore, PD-1 upregulation may not only be the effect of failed virus clearance, but may also contribute to viral persistence.

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

 
We thank Jean D. Boyer, David A. Hokey, Michele A. Kutzler, and Daniel Schullery for technical assistance, helpful comments on the manuscript, and providing reagents. We thank Farida Shaheen, Center for AIDS Research, University of Pennsylvania, Philadelphia, for assistance with virus generation. We thank Jeffrey S. Faust, The Wistar Institute flow cytometry facility, for FACS analysis and Michael J. Merva and Denise Dixon for administrative assistance. We thank Scott Wadsworth and John Siekierka from Johnson & Johnson Pharmaceutical Research and Development, New Jersey, for their useful comments and reagents.

This work was supported by a Johnson & Johnson Pharmaceutical Research and Development grant to D.B.W. and K.M. Support from the National Institutes of Health AIDS Research and Reference Reagents Program is also acknowledged.

 
* Corresponding author. Mailing address: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104. Phone: (215) 662-2352. Fax: (215) 573-9436. E-mail: muthuman{at}mail.med.upenn.edu

Published ahead of print on 17 September 2008.

K.M. and A.Y.C. contributed equally to this work.

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Journal of Virology, December 2008, p. 11536-11544, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.00485-08
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