Distinct mechanisms trigger apoptosis in human immunodeficiency virus type 1-infected and in...

JOURNAL OF VIROLOGY,0022-538X/98/$04.0010

Jan. 1998, p. 660–670 Vol. 72, No. 1

Copyright © 1998, American Society for Microbiology

Distinct Mechanisms Trigger Apoptosis in HumanImmunodeficiency Virus Type 1-Infected and

in Uninfected Bystander T LymphocytesGEORGES HERBEIN, CARINE VAN LINT, JENNIE L. LOVETT, AND ERIC VERDIN*

The Picower Institute for Medical Research, Manhasset, New York 11030

Received 2 June 1997/Accepted 16 October 1997

Apoptosis is a main feature of AIDS pathogenesis and is thought to play a role in the progressive decreaseof CD41 T lymphocytes in infected individuals. To determine whether apoptosis occurs in infected and/or inuninfected peripheral blood T lymphocytes, we have used a recombinant human immunodeficiency virus type1 (HIV-1) infectious clone expressing the green fluorescent protein (GFP). Using flow cytometry, we havedetermined the incidence of apoptosis by either terminal transferase dUTP nick end labeling or annexin-Vassays in different cell subpopulations, i.e., in CD41 or CD81 T cells that were GFP positive or negative. AfterHIV-1 infection of purified peripheral blood lymphocytes, we observed that apoptosis occurred mostly ininfected CD41 peripheral blood lymphocytes. Remarkably, the presence of monocyte-derived macrophages inthe culture increased dramatically the apoptosis of uninfected bystander T lymphocytes, while apoptosis inHIV-infected T lymphocytes was not changed. We therefore demonstrate that HIV-induced apoptosis resultsfrom at least two distinct mechanisms: (i) direct apoptosis in HIV-infected CD41 T lymphocytes and (ii)indirect apoptosis in uninfected T cells mediated by antigen-presenting cells.

Human immunodeficiency virus type 1 (HIV-1) infection ischaracterized by the progressive depletion of CD41 T lympho-cytes (15). The drop in the number of CD41 T lymphocytes ispreceded by early T-cell functional defects characterized invivo by a loss of cell-mediated delayed-type hypersensitivityreactions and in vitro by a failure of T cells to proliferate inresponse to T-cell receptor stimulation by recall antigens ormitogens (15, 29, 39, 51). Several hypotheses have been ad-vanced to account for the loss of CD41 T lymphocytes. Theyinclude (i) direct lysis of the cells by the virus infection (53, 55),(ii) syncytium formation (34, 52, 63), (iii) autoimmunity (17),(iv) cellular and humoral virus-specific immune responses (65),(v) superantigen-mediated deletion of specific T-cell subpopu-lations (22), and (vi) apoptosis (4). The potential role of apo-ptosis in CD4 depletion has been examined in several studies(31, 35, 58), and increased apoptosis in freshly isolated CD41

and CD81 T lymphocytes in cultures grown with blood isolatedfrom HIV-positive individuals has been reported (18, 19, 32,38, 44, 48). Increased apoptosis in both CD41 mature T lym-phocytes and thymocytes after HIV infection in the hu-SCIDmouse model has also been described (3, 11, 41, 42, 56).

Using protease inhibitors to block virus replication, recentstudies have indicated that rapid turnover of circulating CD41

T lymphocytes occurs in HIV-1-infected individuals (21, 60).These studies have highlighted a dynamic inverse correlationbetween plasma virus levels and CD41 T-cell levels in patients(21, 60). While these observations suggested destruction ofHIV-infected cells in vivo, no direct evidence was provided forthis assumption, and the possibility remains that the HIV-mediated cell killing is indirect, i.e., that mostly uninfectedcells are killed. In fact, apoptosis occurs predominantly inbystander uninfected lymphocytes present in the vicinity ofHIV-infected cells in the lymph nodes of HIV-infected humans

and of SIV-infected monkeys (16). In lymph nodes, indirectapoptosis of uninfected T cells could result from CD4 cross-linking, secretion of apoptotic cytokines or viral proteins, orinvolvement of antigen-presenting cells (7–9, 33, 42, 45, 61,64).

To determine whether HIV-induced apoptosis occurs via adirect or an indirect mechanism, we generated a recombinantHIV-1 genome encoding the green fluorescent protein (GFP).Since GFP is expressed as an early viral product by this recom-binant virus, we have used flow cytometry to discriminate be-tween GFP-positive (infected) and GFP-negative (uninfected)peripheral blood lymphocytes (PBLs) to determine the inci-dence of apoptosis, as measured by terminal transferase dUTPnick end labeling (TUNEL) and annexin-V assays, in both cellsubpopulations. We observed that after infection of purifiedPBLs by HIV-1 in vitro, cells undergoing apoptosis are almostexclusively GFP-positive infected CD41 T lymphocytes. Incontrast, after HIV infection of a mixed population containingboth PBLs and monocyte-derived macrophages, cells undergo-ing apoptosis are essentially GFP-negative uninfected by-stander T lymphocytes.

MATERIALS AND METHODS

Cell lines. CEMx174 is a CD41 T-cell/B-cell hybrid line generated from thepolyethylene glycol-mediated fusion of 721.174 and CEM.3 cells (47). Jurkat is aCD41 T-cell line. Both CEMx174 and Jurkat cells were maintained in RPMImedium supplemented with 10% fetal calf serum (FCS).

Isolation and culture of PBLs and blood monocyte-derived macrophages.Human peripheral blood mononuclear cells were isolated from healthy donors asdescribed previously (12). In short, Ficoll-Hypaque (Pharmacia, Uppsala, Swe-den)-isolated peripheral blood mononuclear cells were incubated for 1 h on2% gelatin-coated plates. Adherent tissue culture-differentiated macrophages(TCDM), .94% CD141 by flow cytometry analysis, were cultured in RPMIsupplemented with 10% (vol/vol) pooled AB human serum (Sigma, St Louis,Mo.) for 48 h before transfer to six-well plates at a density of 5 3 106 cells perwell in a 3-ml total volume. Nonadherent cells, .98% which were PBLs asassessed by CD451 (Simultest Leucogate; Becton Dickinson, San Jose, Calif.)detection by flow cytometry analysis, were harvested after Ficoll-Hypaque isola-tion and adherence. PBLs were cultivated in RPMI with 10% FCS supplementedfor the first 48 h with phytohemagglutinin A (PHA; 5 mg/ml; Sigma) before theaddition of human recombinant interleukin-2 (hrIL-2; 20 IU/ml; Gibco-BRL,Gaithersburg, Md.). For coculture, PHA- and IL-2-activated PBLs were mixed

* Corresponding author. Mailing address: The Gladstone Institutefor Virology and Immunology/UCSF, 1310A Potrero Ave., San Fran-cisco, CA 94110. Phone: (415) 695-3815. Fax: (415) 826-8449. E-mail:[email protected].

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with 10% TCDM and cultivated in RPMI with 10% (vol/vol) FCS supplementedwith hrIL-2 as described above. Culture medium was replaced every 2 days.

Generation of 89.6 HIV-1 clones expressing both wild-type and bright mutatedGFP. To create the viruses designated HIV-GFPwt, HIV-GFPRS, and HIV-GFPS65T, we inserted the GFPwt, GFPRS, and GFPS65T genes amplified by PCRinto the nef open reading frame of HIV-89.6 (13, 54). The XhoI (nucleotide [nt]3353)-XhoI (nt 3866) fragment of 89.6-39Eco was modified as follows. The first110 nt of the nef gene were deleted, the ATG start codon of the nef gene wasmutated (into ATA), and two restriction sites corresponding to EagI and XmaIwere introduced into the nef gene by PCR with the following primers: 59- ACCGAG CTC GAG CGG CCG ACC ATG CGA CCC GGG CAC TTG CCA CCTATC TTA TAG CAAA-39 and 59-GAT GCA CTC GAG GGG ACC CGA CAGGCC CGA AGG-39. This modified XhoI-XhoI fragment was cloned into the89.6-39Eco plasmid digested by XhoI. We refer to this construct as HIV-89.6-EagI/XmaI. A fragment containing the cDNA for GFP was obtained afterdigestion of pGFP (Clontech) with SmaI and EagI and was cloned into theEagI-XmaI sites of plasmid HIV-89.6-EagI/XmaI. We refer to this construct asHIV-GFPwt. Fragments containing cDNAs for brighter GFPs were obtainedfrom plasmids pRSGFP-C1 and pS65T-C1 (Clontech) after digestion with AgeIand XmaI and cloned into plasmid HIV-89.6-EagI/XmaI digested by XmaI. Werefer to these constructs as HIV-GFPRS and HIV-GFPS65T, respectively.

Generation of viral stocks. The circular permutated infectious molecular cloneof HIV-1, pILIC19 (gift from A. Rabson), was digested by EcoRI and religatedon itself to eliminate the EcoRI (nt 5743; pILIC)-EcoRI (nt 400; pUC19) frag-ment. We refer to this construct as pILIC19DE. To generate vector pEV114, theApaI (nt 2010)-EcoRI (nt 5743) fragment from pNL4-3 (1) was cloned intovector pILIC19DE digested by ApaI and EcoRI. Wild-type and mutant HIV-1infectious DNAs were generated after ligation of a left hemigenome (EcoRI-digested pEV114) with the single-long terminal repeat (LTR)-containingright hemigenome constructs described above (EcoRI-digested HIV-89.6, HIV-GFPwt, HIV-GFPRS, or HIV-GFPS65T). Ten micrograms of concatemerizedproviral DNA was transfected into 107 Jurkat cells by the DEAE-dextran pro-cedure (59). Twenty-four hours posttransfection, Jurkat cells were cocultivatedwith 107 CEMx174 cells to allow rapid and efficient recovery of progeny virus.Virus stocks were prepared from supernatants after filtration through a 0.45-mm-pore-size filter, quantified by measuring reverse transcriptase (RT) activity,and stored at 280°C.

Infections. CEMx174 and Jurkat cells were cultivated in RPMI supplementedwith 10% FCS at a density of 106 cells/ml. TCDM, PHA-activated PBLs, orTCDM-PBL cocultures were cultivated in six-well plates at a density of 5 3 106

cells/well. The 3-ml final volume included RPMI supplemented with 10% (vol/vol) pooled AB human serum for the TCDM cultures or 10% FCS supplementedwith hrIL-2 (20 IU/ml) for the PHA-activated PBLs. After 2 h of exposure tovirus at 37°C, cells were washed three times with phosphate-buffered saline(PBS) to remove the unadsorbed inoculum and reincubated in fresh culturemedium at 37°C. Culture supernatants were collected every 2 days and assayedfor RT activity. Cell pellets were harvested and washed twice with PBS beforefixation with 3% paraformaldehyde in PBS for 30 min. Fixed cells were used toperform flow cytometry analysis.

RNase protection analysis. HIV-1-specific transcripts were detected by RNaseprotection analysis after lysis of cells in guanidine thiocyanate (20) (LysateRibonuclease Protection kit; U.S. Biochemicals). An HIV-1-specific 32P-labeledantisense riboprobe was synthesized in vitro by transcription of pGEM23 (30)with SP6 polymerase according to standard protocols (6). A glyceraldehyde-3-phosphate dehydrogenase-specific antisense probe (provided with the LysateRibonuclease Protection kit; U.S. Biochemicals) was synthesized by the sameprotocol and used in the same reaction with the HIV-1 probe. The HIV-1antisense RNA probe protects two RNA fragments of 83 and 200 nt whichcorrespond to the 59 and 39 LTRs, respectively (30).

Western blotting. CEMx174 cells, uninfected or infected with either HIV-89.6or each of the three HIV-GFP clones, were harvested at the peak of RT activity.Cells were washed with PBS and lysed in 150 mM NaCl–10 mM EDTA–10 mMTris (pH8)–10 mM NaN3–1 mM phenylmethylsulfonyl fluoride–5 mM iodoacet-amide–1% (vol/vol) Nonidet P-40. Protein concentrations in cell lysates weredetermined (Protein Assay; Bio-Rad, Hercules, Calif.). Sample proteins wereseparated on sodium dodecyl sulfate–10% polyacrylamide gel and electroblottedonto nitrocellulose (Hybond-ECL; Amersham). Membranes were blocked inPBS with 3% (wt/vol) dried milk and 0.1% (vol/vol) Tween 20. The binding ofpolyclonal rabbit anti-GFP, used at 1:2,000 (Clontech), was determined by in-cubation with a horseradish peroxidase-conjugated goat anti-rabbit immunoglob-ulin G (IgG) used at 1:1,000 (Santa Cruz Biotechnology, Santa Cruz, Calif.) andby chemiluminescence (ECL kit; Amersham). For detection of virion-associatedproteins, HIV-1 lysates were prepared by ultracentrifugation of each virus stockand resuspension of pellets in Laemmli buffer at a concentration of 30,000 cpmof RT/ml. Lysates were heated at 95°C for 5 min and subjected to electrophoresison an 8% polyacrylamide gel, transferred to nitrocellulose, and probed with a1:2,000 dilution of purified human anti-HIV-1 IgG (National Institutes of HealthAIDS Research and Reagent Program). A second antibody, horseradish perox-idase-conjugated goat anti-human IgG (Pierce, Rockford, Ill.) was then used at1:10,000 for enhanced chemiluminescence detection (Amersham).

Flow cytometry analysis. Cells fixed in 3% paraformaldehyde in PBS werelabeled for either the TUNEL or annexin-V assay as described below and

analyzed by flow cytometry with a FACScan flow cytometer (Becton Dickinson,Mountain View, Calif.). CD4 and CD8 detections were performed with PerCP-labeled anti-human CD4 and CD8 mouse IgG1 monoclonal antibodies (BectonDickinson). Specific fluorescence was assessed by comparison with cells stainedwith isotype controls (Becton Dickinson). The purity of the PBL population wasconfirmed by the detection of CD451 antigen (Simultest Leucogate; BectonDickinson). The specific fluorescence of GFP was excited at 488 nm. Data from5 3 104 cells were collected, stored, and analyzed with Cellquest software(Becton Dickinson).

RT activity assay. HIV-1 production was measured by determining RT activityin culture supernatants by using a microassay (2).

TUNEL assay. After fixation with 3% paraformaldehyde in PBS for 30 min,5 3 106 cells per sample were washed three times with 0.3% (vol/vol) TritonX-100 in PBS. After being washed with terminal transferase buffer (BoehringerGmbH, Mannheim, Germany) in a 50-ml final volume, cells were incubated for3 h at 37°C in a 50-ml final volume containing 20 U of terminal transferase, 2.5mM CoCl2, 5 mM 16-dUTP biotin, 5 mM dUTP (all from Boehringer GmbH),and 0.3% (vol/vol) Triton X-100. After being washed (0.3% [vol/vol] TritonX-100 in PBS) for 1 h, cells were incubated for 1 h at room temperature inphycoerythrin-labeled streptavidin (Boehringer GmbH) diluted 1:200 in PBS.After one wash in 0.3% (vol/vol) Triton X-100 in PBS, cells were resuspended inPBS and then subjected to flow cytometry analysis. Apoptosis was measured inboth uninfected and infected PBLs isolated from the same donor, and values ofthe specific apoptosis in PBLs and in CD41 and CD81 subpopulations wereexpressed as follows: (percentage of apoptosis in infected culture) 2 (percentageof apoptosis in uninfected culture).

Annexin-V assay. Purified annexin-V (Biodesign International, Kennebunk,Maine) was biotinylated in vitro and purified according to the manufacturer’sprotocol (Sulfo-NHS-LC-Biotinylation Kit; Pierce). The modified protein wasexamined after sodium dodecyl sulfate-polyacrylamide gel electrophoresis byWestern blotting using horseradish peroxidase-labeled streptavidin diluted 1:2,000 in PBS and chemiluminescence (ECL kit; Amersham). This analysisshowed the presence of a single 35-kDa band on the gel corresponding to theknown molecular mass of annexin-V (data not shown). The annexin-V assay wasperformed as reported previously (27). After fixation in 3% paraformaldehyde inPBS for 30 min, 5 3 106 cells were washed three times in PBS. After incubationin HEPES buffer (10 mM HEPES–NaOH [pH 7.4], 140 mM NaCl, 5 mM CaCl2)supplemented with 2.5 mg of biotinylated annexin-V per ml for 15 min at roomtemperature, cells were washed twice with PBS before incubation for 1 h inphycoerythrin-labeled streptavidin (Boehringer GmbH) diluted 1:200 in PBS atroom temperature. To determine the integrity of the plasma membrane, 2 ml ofa 50-mg/ml solution of propidium iodide was added to each sample in a 100-mlfinal volume. After one wash in PBS, cells were resuspended in 500 ml of PBS forflow cytometry analysis. Apoptosis was measured in both uninfected and infectedPBLs isolated from the same donor, and specific apoptosis in PBLs and in CD41

and CD81 subpopulations was expressed as follows: (percentage of apoptosis ininfected culture) 2 (percentage of apoptosis in uninfected culture).

RESULTS

Construction of HIV-1 molecular clones expressing eitherwild-type or bright mutated GFP. To distinguish between in-fected and uninfected cells, we introduced the GFP reportergene into the 89.6 infectious molecular clone of HIV-1 (13).This virus presents dual tropism for both primary humanmonocytes/macrophages and PBLs and is representative ofnatural isolates encountered in patients. We cloned genes en-coding distinct forms of GFP into a modified HIV-89.6 infec-tious molecular clone in which the first 110 nt of the nef openreading frame (nt 3757 to 3866), including ATG, had beendeleted and replaced with two unique cloning sites XmaI andEagI (Fig. 1). Three different forms of GFP were used: thewild-type form and two mutated forms characterized by in-creased fluorescence and called GFPRS and GFPS65T (Clon-tech). The three resultant recombinant HIV clones are calledHIV-GFPwt, HIV-GFPRS, and HIV-GFPS65T. The recom-binant HIV genomes were ligated and transfected intoCEMx174 cells, and virus stocks were harvested and quantifiedby RT activity measurement. Since the integrated fragmentcorresponding to GFP (780 nt) might interfere with the pack-aging of the HIV genome into particles, we used an RNaseprotection assay to determine whether all three virus genomes(HIV-GFPwt, HIV-GFPRS, and HIV-GFPS65T) were packagedwith the same efficiency as the parental clone (HIV-89.6).Similar amounts of virus particles, based on RT activity con-

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tent, were pelleted by centrifugation. Lysed pellets were usedboth in an RNase protection analysis using an antisense RNAprobe corresponding to the U3-R region of HIV (Fig. 2A) andin a Western blot analysis using a antiserum specific for HIVproteins (Fig. 2B). This experiment showed similar packagingof the HIV genome by all four viruses (Fig. 2A). Western blotanalysis of the same virus lysates confirmed that similaramounts of virus particles were used (Fig. 2B).

Replication of HIV-GFP clones in the CEMx174 cell line,in primary human PBLs, and in macrophages. Next, we as-sessed the ability of HIV-89.6 and HIV-GFP viruses toinfect CEMx174 cells, Jurkat T cells, human PBLs, andTCDM (Fig. 3). All four viruses replicated with the samekinetics in CEMx174 cells, demonstrating that the presence ofthe GFP gene within the viral genome did not impair viralgrowth in this cell line (Fig. 3). In contrast, the efficiencies ofreplication of the three HIV-GFP clones were diminished by afactor of 2 to 5 in primary PBLs in comparison to that ofHIV-89.6 (Fig. 3). This decrease in replication efficiency ismost likely secondary to the disruption of the nef gene and theresultant disruption of Nef, which, as previously reported, hasa role in HIV-1 replication in primary PBLs (24). In TCDM, allfour viruses replicated with similar kinetics (Fig. 3). As previ-ously reported, these viruses did not replicate efficiently inJurkat T cells (data not shown) (54).

GFP expression during infection with HIV-GFP. To exam-ine the expression of GFP during infection with HIV-GFPviruses, we harvested CEMx174 cells infected with either HIV-89.6 or the three GFP-recombinant HIVs at the peak of RTactivity. Cells were analyzed by Western blotting using an anti-GFP polyclonal antibody (Fig. 4A). No GFP protein could bedetected following infection with HIV-89.6. Instead, infectionwith HIV-GFP viruses resulted in the appearance of a newband of 27 kDa reacting with the anti-GFP antibody. The sizeof the GFP expressed in cells infected with HIV-GFPwt was

similar to that of the recombinant purified GFP (Fig. 4A). Incontrast, two distinct immunoreactive bands of 27 and 30 kDawere observed in cells infected with HIV-GFPRS or HIV-GFPS65T, respectively (Fig. 4A). Flow cytometry analysis ofCEMx174 cells, uninfected (data not shown) or infected withHIV-89.6, showed nonspecific autofluorescence (Fig. 4B).Cells infected with HIV-GFPwt showed a small shift in thefluorescence peak (Fig. 4B); however, discrimination betweenthe two cell subpopulations, GFP negative and GFP positive,was not possible. In contrast, in cells infected with HIV ex-pressing the brighter GFP variants, GFPRS and GFPS65T, twodistinct subpopulations were easily detected on the basis oftheir fluorescence (Fig. 4B). The percentage of GFP-positivecells present in the PBL culture following infection with HIV-GFPS65T was linearly correlated to the level of RT activity inthe culture supernatant (r2 5 0.98) (Fig. 4C). Therefore, cellfluorescence as a reflection of GFP expression allows the iden-tification of infected cells in a heterogeneous population com-prising both uninfected and infected cells.

Apoptosis occurs in both uninfected and HIV-infected cul-tures. To detect apoptosis in human PBLs, we used two assayswhich examine two different markers of apoptosis. TheTUNEL assay detects the presence of nicks in DNA, a lateevent in the apoptotic process, whereas the annexin-V assaydetects the appearance of phosphatidyl serine at the cell sur-faces of apoptotic cells and represents an earlier marker (36).We performed a time course analysis following infection ofpurified human PBLs with HIV-GFPS65T. Three different vari-ables were monitored as a function of time: (i) RT activity inculture supernatants; (ii) apoptosis level, by the TUNEL orannexin-V assay; and (iii) HIV gene expression by GFP mea-surement. In three independent experiments, no apoptosis wasdetected at the time of PBL isolation (Fig. 5). Starting at day4 following stimulation with PHA plus IL-2, we observed sig-nificant apoptosis even in uninfected PBLs, as previously re-

FIG. 1. Construction of HIV-1 molecular clones expressing either wild-type or mutated GFP. The parental virus, HIV-89.6, is shown at the top, and the insertionof GFPwt, GFPRS, or GFPS65T into the nef open reading frame is illustrated below the sequence. HIV-GFPwt, HIV-GFPRS, and HIV-GFPS65T contain an insertedGFPwt, GFPRS, or GFPS65T gene, respectively, at the 39 end of env. Open boxes indicate open reading frames of genes expressed in the virus construct.

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ported (Fig. 5) (44). Interestingly, levels of absolute apoptosiswere higher in infected PBLs than in uninfected PBLs usuallyat days 9 (P 5 0.04; t test) and 11 (P 5 0.001; t test) postin-fection, demonstrating specific HIV-dependent apoptosis (Fig.5). No difference in apoptosis between infected and uninfectedpopulations was noted earlier (day 4) or later (days 13 and 15).Similar results were obtained with several other donors, whosecells displayed significant specific HIV-dependent apoptosisfrom days 9 to 17 postinfection (data not shown).

Apoptosis occurs predominantly in infected cells after HIVinfection of purified human PBLs. To determine whetherHIV-specific apoptosis occurred in GFP-positive (productivelyinfected) or GFP-negative (uninfected or unproductively in-fected) cells, we combined the measurement of GFP fluores-cence and TUNEL or annexin-V assays of PBLs at each timepoint following infection (Fig. 6). Both GFP-negative andGFP-positive PBLs underwent specific apoptosis, but specificapoptosis occurred at a significantly higher rate in GFP-posi-tive than in GFP-negative PBLs (Fig. 6). To measure the rateof apoptosis specifically dependent on HIV infection, we com-pared the rates of apoptosis in infected and uninfected cultures

of cells from the same donor. For example, the fractions ofcells located in the upper right quadrant (UR) or upper leftquadrant (UL) were compared for infected (URi or ULi) anduninfected (URu or ULu) cultures. HIV-specific apoptosis isdefined as the difference between these two values. For exam-ple, ULi2ULu represents the percentage of cells undergoingHIV-specific apoptosis in GFP-negative cells, whereas URi2URu represents HIV-specific apoptosis in GFP-positive cells.The peak of specific apoptosis in both GFP-positive and GFP-negative PBLs was concomitant with the peak of viral replica-tion as determined by measurement of RT activity in culturesupernatants (Figure 6B). Interestingly, a subpopulation ofGFP-positive PBLs was resistant to apoptosis and usually wasdetected at the same time as or following the appearance of

FIG. 2. Packaging of recombinant HIV-GFP genomes. (A) RNase protec-tion analysis of HIV genomic RNA purified after ultracentrifugation of culturesupernatants. Two protected fragments corresponding to the 39 and 59 LTRs areshown. (B) Western blot analysis of ultracentrifuged virion-associated proteinsperformed with an IgG fraction purified from HIV-infected individuals. Thecontrol lane shows results for the resuspended pellet obtained following ultra-centrifugation of supernatant harvested from uninfected cells.

FIG. 3. Replication of HIV-GFPwt, HIV-GFPRS, and HIV-GFPS65T clonesin CEMx174 cells, primary human PBLs, and TCDM. CEMx174 cells, PHA- andIL-2-activated PBLs, and TCDM were either left uninfected (}) or infected withHIV-89.6 (■), HIV-GFPwt (Œ), HIV-GFPRS (3), or HIV-GFPS65T (E) clones(5 3 105 cpm of RT/5 3 106 cells). RT activity was measured in culture super-natants. Results are representative of three independent experiments. p.i., post-infection.



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GFP-positive apoptotic PBLs (Fig. 6B). All cell populationswere also tested for trypan blue exclusion and propidium io-dide staining under nondetergent condition (36) and were uni-formly negative by both tests (data not shown). The finding ofa positive annexin-V assay result combined with a negativepropidium iodide staining in nondetergent conditions is there-fore consistent with apoptosis.

Apoptosis occurs almost exclusively in HIV-infected CD41

T lymphocytes and not in CD81 T lymphocytes after infectionof purified PBLs. To determine the PBL subpopulation whichundergoes apoptosis during HIV infection, we infected puri-fied PBLs with HIV-GFPS65T and monitored viral replicationby measurement of RT activity in culture supernatants. Afterlabeling cells with anti-human CD4 and anti-human CD8monoclonal antibodies, we measured GFP expression and ap-optosis by both TUNEL and annexin-V assays in both CD41

and CD81 T-lymphocyte populations (Fig. 7). Using three-

color flow cytometry, we observed that specific apoptosis oc-curred almost exclusively in CD41 T lymphocytes and not inCD81 T lymphocytes (Fig. 7). In CD41 T lymphocytes, anincrease in apoptosis was observed during HIV infection ofGFP-negative cells (5.38 versus 1.46%); the extent of the in-crease was much larger for GFP-positive cells (55.78 versus32.26%). The peak of specific apoptosis in CD41 T lympho-cytes was concomitant with the peak of viral replication orfollowed it by 24 to 48 h (Fig. 7B and data not shown). Nosignificant apoptosis of PBLs was observed prior to the viralreplication peak (Fig. 7 and data not shown). NonapoptoticGFP-positive CD41 T lymphocytes could be detected by bothTUNEL and annexin-V assays at the time of or following themain peak of apoptosis (Fig. 7). CD81 T lymphocytes showedless than 1% specific apoptosis even at the peak of viral rep-lication (Fig. 7). The percentage of CD41 T lymphocytes un-dergoing specific apoptosis ranged from 10 to 30% dependingon the donors (Fig. 7 and data not shown). Using either theTUNEL or the annexin-V assay to measure apoptosis, wefound as a rule that 90% of apoptotic CD41 T lymphocyteswere GFP positive, while only 10% were GFP negative afterHIV-1 infection of purified PBLs (data not shown).

Monocyte-derived macrophages increase apoptosis in by-stander uninfected T lymphocytes but not in HIV-infected Tlymphocytes. To determine whether antigen-presenting cellscould modify the phenotype of apoptosis observed after HIV

FIG. 4. Cells infected with HIV-GFP express GFP. (A) Detection by West-ern blot analysis of GFPs in CEMx174 cells infected with HIV-GFPwt, HIV-GFPRS, and HIV-GFPS65T clones but not with HIV-89.6. Recombinant GFP(500 ng; Clontech) was loaded as a positive control. Uninfected cells were loadedas a negative control (Control). (B) Flow cytometry analysis of CEMx174 cellsuninfected (data not shown) or infected with HIV-89.6, HIV-GFPwt, HIV-GF-PRS, and HIV-GFPS65T viruses. Cells were harvested at day 15 postinfection,fixed with 3% paraformaldehyde, and analyzed by flow cytometry. Mean fluo-rescence values (in the defined gate) for HIV-89.6, HIV-GFPwt, HIV-GFPRS,and HIV-GFPS65T were 10.1, 15.9, 46.2, and 48.9, respectively. (C) Percentage ofGFP-positive PBLs correlates linearly with the RT levels in the culture super-natants (r2 5 0.98).



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infection of purified PBLs, we combined measurements ofGFP fluorescence, apoptosis by the TUNEL assay, and RTactivity at several time points following infection of eitherpurified PBLs or of a PBL–monocyte-derived macrophage co-culture (Fig. 8). In contrast to purified PBLs, in which apopto-sis occurred mostly in GFP-positive PBLs and to a lesser extentin GFP-negative PBLs, the presence of monocyte-derived mac-rophages dramatically enhanced the rate of apoptosis in GFP-negative PBLs but did not modify the rate of apoptosis inGFP-positive PBLs (Fig. 8). Therefore, in the presence ofmonocyte-derived macrophages and PBLs, HIV-specific apo-ptosis occurred at a significantly higher rate in GFP-negativethan in GFP-positive PBLs. Specific apoptosis in both GFP-negative and GFP-positive PBLs was concommitant with thepeak of viral replication, as determined by measurement of RTactivity in culture supernatants (Fig. 8). A subpopulation ofGFP-positive cells resistant to apoptosis was detected both inthe presence and in the absence of monocyte-derived macro-phages (Fig. 8).

DISCUSSION

We have used an HIV reporter virus expressing GFP todistinguish between infected and uninfected cells in a hetero-geneous population. We observed that apoptosis occurs almostexclusively in CD41 and not in CD81 T lymphocytes duringinfection of purified PBLs. Ninety percent of CD41 T lympho-cytes undergoing apoptosis expressed GFP and therefore wereinfected, while 10% of cells did not and therefore representeither uninfected bystander cells or cells early in the infectiouscycle. Monocyte-derived macrophages triggered apoptosis inGFP-negative uninfected bystander T lymphocytes and did notaffect apoptosis in GFP-positive HIV-infected T lymphocytes.These observations demonstrate that HIV alone is capable oftriggering apoptosis in infected CD41 peripheral blood T lym-phocytes and that, in purified PBLs, apoptosis is limited almostexclusively to infected cells. In contrast, antigen-presentingcells such as monocyte-derived macrophages are a key regula-tor of apoptosis in uninfected bystander T lymphocytes.

Previous studies have reported increased apoptosis during

HIV infection: independently of the phase of HIV infection(acute, asymptomatic, or AIDS), lymphocytes isolated frominfected individuals undergo accelerated apoptosis in compar-ison to lymphocytes from uninfected individuals (14, 18, 38).Interestingly, this increased apoptosis is much less pronouncedwhen lymphocytes from HIV-2-infected individuals are exam-ined. Since HIV-2 infection usually runs a milder clinicalcourse, these observations suggest that apoptosis has a directbearing on HIV pathogenesis and the development of immu-nodeficiency (23). Since productively infected cells are rare inthe peripheral blood of infected individuals (46, 50), most ofthe lymphocytes undergoing apoptosis are probably uninfectedand a direct cytopathic effect of HIV is probably not the solemechanism. This possibility is supported by recent in vivo stud-ies showing that apoptosis occurs predominantly in uninfectedbystander T cells in lymph nodes of HIV-infected children andin SIV-infected monkeys (16).

Recent studies using inhibitors of HIV-1 protease showedthat the turnover of HIV-infected CD41 T lymphocytes inperipheral blood was higher than previously estimated andstrictly dependent on virus production (21, 60), demonstratinga direct link between virus production and CD41 T-lympho-cyte depletion. However, these data do not allow a distinctionbetween direct and indirect cytopathic effects of HIV infection,and this question has therefore remained unanswered. Whileboth direct and indirect mechanisms of HIV-induced apoptosisshould respond therapeutically to a blockage of HIV replica-tion, one can envision that an indirect mechanism could bemodulated therapeutically without affecting virus replication.Such treatment would therefore circumvent the problem ofHIV genome variability and the appearance of virus-resistantstrains.

To differentiate between infected and uninfected cells, wehave constructed a recombinant virus expressing GFP. Sincethe GFP cDNA replaces the nef open reading frame, we an-ticipate that GFP is expressed with kinetics identical to thoseof nef with respect to the virus life cycle, i.e., within the first 6 hpostinfection (26). In addition, since GFP is not packagedwithin virus particles, GFP detection is strictly limited to in-fected cells, thereby alleviating a problem encountered whenother viral proteins were used as markers for the specific de-tection of infected cells (10, 20a). Introduction of the GFPcDNA within our infectious HIV clone results in the deletionof the nef open reading frame. While nef is nonessential forvirus replication in vitro, nef is a critical factor for HIV and SIVpathogenesis in SCID-hu mice and macaques, respectively (24,25), and its deletion could therefore modify the ability of HIVto induce apoptosis. Since nef expression is restricted to theinfected cell, any nef-mediated effect on apoptosis is likely tobe limited to the infected cell. Therefore, it is unlikely that theindirect apoptosis mediated by monocytes/macrophages thatwe have observed is dependent on nef. In support of thiscontention, we have detected the same macrophage-dependentincrease in apoptosis with several other isolates of HIV ex-pressing a functional nef protein (20a). However, nef is likely tocontribute to the direct apoptosis associated with virus repli-cation since nef is an important factor in the replication of HIVin primary lymphocytes (40). To test these predictions, we willreconstruct the nef open reading frame in the context of ourGFP-expressing recombinant virus and directly test the role ofnef expression in HIV-induced apoptosis.

Our data demonstrate for the first time that HIV itself cankill CD41 T lymphocytes directly via induction of apoptosis.The peak of apoptosis was concomitant with the peak of viralreplication or followed it by 24 to 48 h, suggesting a direct linkbetween viral replication and apoptosis. This form of apoptosis

FIG. 5. Apoptosis occurs in both uninfected and HIV-infected human PBLs.Purified PBLs isolated and activated with PHA and IL-2 were either left unin-fected (h) or infected by HIV-GFPS65T (■). A total of 5 3 106 cells wereharvested at each time point, fixed with 3% paraformaldehyde, and labeled byTUNEL assay to measure apoptosis. By single-color flow cytometry the levels ofapoptotic cells were determined in parallel in both uninfected and infected PBLs.The averages 6 the standard deviations from three independent experiments areshown. p.i., postinfection.



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is mediated in a direct fashion by one of the viral products inthe infected cell and does not involve the secretion of a toxicproduct in culture supernatant since uninfected cells are un-affected. We also observed that this form of apoptosis is inde-pendent of the presence of monocytes/macrophages in the

culture system. As discussed above, given the enhancing role ofnef expression in HIV replication in PBLs (20a, 40), we antic-ipate that direct apoptosis will be enhanced when examined inthe context of a nef-expressing virus. Our results thereforedemonstrate that the CD41 T-lymphocyte depletion observed

FIG. 6. Apoptosis occurs predominantly in HIV-infected GFP-positive cellsafter infection of purified PBLs. (A) Two-color flow cytometry analysis of apo-ptosis in PBLs infected with HIV-GFPS65T as measured by the TUNEL assay.Purified PBLs were left uninfected or were infected with HIV-GFPS65T. At day11 postinfection, cells were harvested, fixed with 3% paraformaldehyde, andlabeled by TUNEL assay (TdT labeling) as reported in Materials and Methods.By two-color flow cytometry, the cell population distribution was determined inboth uninfected (ULu, URu, LRu) and infected (ULi, URi, LRi) PBLs. (B)Measurement of specific apoptosis in human PBLs infected with HIV-GFPS65Tas determined by TUNEL and annexin-V assays. Purified PBLs were infectedand analyzed as described for panel A. The histograms represent the PBLpopulation distribution: apoptotic GFP-positive cells (solid bars; URi 2 URu),apoptotic GFP-negative cells (shaded bars; ULi 2 ULu), and nonapoptoticGFP-positive cells (open bars; LRi 2 LRu). RT activity (graphs) was measuredin culture supernatants harvested from uninfected (E) and infected (■) PBLs.Panels of TUNEL and annexin-V assays represent data obtained from twodifferent donors, and each panel is representative of the results observed in fiveindependent experiments using different donors. p.i., postinfection.



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FIG. 7. Apoptosis occurs almost exclusively in HIV-infected GFP-positiveCD41 T lymphocytes and not in CD81 T lymphocytes after infection of purifiedPBLs. (A) Three-color flow cytometry analysis of apoptosis in CD41 and CD81

T lymphocytes infected with the HIV-GFPS65T clone as measured by theTUNEL assay. Purified PBLs were left uninfected or were infected with theHIV-GFPS65T clone. At day 15 postinfection, cells were harvested, fixed with 3%paraformaldehyde and analyzed by the TUNEL assay. CD41 and CD81 Tlymphocytes were labeled (TdT labeling) with anti-human CD4 and anti-humanCD8 PerCP-labeled monoclonal antibodies. The cell population distribution(UL, UR, LR) for both uninfected and infected CD41 and CD81 T lymphocyteswas determined by three-color flow cytometry. (B) Measurement of specificapoptosis in human peripheral blood CD41 and CD81 T lymphocytes infectedwith the HIV-GFPS65T clone by the TUNEL and annexin-V assays. PurifiedPBLs were infected with the HIV-GFPS65T clone. RT activity was measured(graphs) in culture supernatants harvested from uninfected (E) and infected (■)PBLs. A total of 5 3 106 cells were harvested at each time point, fixed with 3%paraformaldehyde, and labeled concomitantly by either TUNEL or annexin-Vassays with anti-human CD4 or anti-human CD8 monoclonal antibodies. The cellpopulation distribution for both uninfected and infected CD41 and CD81 PBLswas determined by three-color flow cytometry. The histograms represent theCD41 and CD81 T-lymphocyte population distribution: apoptotic GFP-positivecells (solid bars; URi 2 URu), apoptotic GFP-negative cells (shaded bars; ULi 2ULu), and nonapoptotic GFP-positive cells (open bars; LRi 2 LRu). Panelsshowing the results of TUNEL and annexin-V assays represent data obtainedfrom two different donors, and each panel is representative of results observed infive independent experiments with different donors. p.i., postinfection.



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in the absence of antigen-presenting cells occurs as a directconsequence of virus infection. Additionally, other mecha-nisms besides those studied here are at play in infected indi-viduals since all our studies were performed on peripheralblood cells isolated from uninfected individuals, who were notexperiencing an anti-HIV immune response. In vivo, cytotoxicT cells and antibodies directed against gp120 and other viralproducts can play additional and critical role in the inductionof apoptosis. In future studies, we will examine the role ofdistinct HIV-encoded proteins by introducing mutations in theopen reading frames of their genes.

Interestingly, we have reproducibly observed the emergenceof a population of nonapoptotic HIV-infected (GFP-positive)cells. Such cells typically emerged after most of the infected Tcells had undergone apoptosis and probably represent a sub-population of T cells resistant to apoptosis induction by HIV.Previous reports have documented the existence of a subpopu-lation of T cells expressing low levels of CD4 that can supportHIV-1 replication without demonstrating significant cytopathiceffects (5, 28). Another possibility is that these T cells resistantto HIV-induced apoptosis constitute a pool of memory T cellsrefractory to apoptosis (57). Since apoptotic cells are short-lived and hard to detect in vivo, the emergence of infected andapoptosis-resistant cells in infected individuals could accountfor the observation that most infected T cells are nonapoptoticin vivo (16).

We demonstrate that HIV infection is associated with anincrease in apoptosis in uninfected T cells. Surprisingly, wefound that this increase is strictly dependent on the presence ofmonocytes/macrophages. Preliminary experiments indicatethat this increase in apoptosis affects both uninfected CD41

and CD81 T-cell populations (20a). Previous studies have doc-

umented that, independently of HIV infection, monocytes/macrophages are required to prime peripheral blood T cells toundergo apoptosis (43, 62). Recent studies also indicate thatmacrophages play a critical role in inducing apoptosis in CD41

cells isolated from HIV-infected individuals but not in CD41

cells isolated from healthy individuals (7, 8). In this system,apoptosis was mediated by tumor necrosis factor and the Fasligand (7, 8). Several other mechanisms have been proposed toexplain the induction of apoptosis in uninfected bystander lym-phocytes: cross-linking of surface CD4 (9, 44); secretion ofcytokines such as tumor necrosis factor alpha, lymphotoxin,and transforming growth factor b (49, 64); and secretion ofsoluble Tat protein (33, 37, 61). Interestingly, CD4 cross-link-ing has been reported to induce apoptosis in normal CD41 Tlymphocytes when the cross-linking is performed in unfraction-ated peripheral blood mononuclear cells but not in purifiedPBL preparations (44). These data therefore support our ob-servations on the critical role of macrophages in apoptosisinduction.

In conclusion, we have established a new infection systemfor the study of HIV pathogenesis. This system is based on thein vitro infection of primary cells isolated from healthy indi-viduals with a GFP-expressing recombinant HIV genome. Wehave used this system to systematically dissect the mechanismof HIV-induced apoptosis. Tagging of infected cells via expres-sion of GFP has allowed the direct determination of apoptosisrates in both infected and uninfected populations. We demon-strate that distinct mechanisms underlie HIV-mediated apo-ptosis in infected and uninfected cells. In future studies, we willexamine in greater detail the role of accessory cells and specificHIV-encoded gene products in these processes. It is antici-pated that through such a systematic approach, the role of

FIG. 8. Monocyte-derived macrophages induce apoptosis in uninfected GFP-negative bystander T lymphocytes, but not in HIV-infected GFP-positive T lympho-cytes. Measurement of specific apoptosis in human PBLs after infection of either purified PBLs (PBL) or PBLs and TCDM (PBL1TCDM) with HIV-GFPS65T asdetermined by a TUNEL assay. Either purified PBLs or cocultivated PBLs and TCDM were infected and analyzed as described in the legend for Fig. 6. The histogramsrepresent the PBL population distribution: apoptotic GFP-positive cells (solid bars; URi 2 URu), apoptotic GFP-negative cells (shaded bars; ULi 2 ULu), andnonapoptotic GFP-positive cells (open bars; LRi 2 LRu). RT activity was measured (graphs) in culture supernatants harvested from uninfected (E) and infected (■)cells. Panels A and B represent data obtained from cells of different donors. p.i., postinfection.



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various factors, both viral and host dependent, will be closelydefined.

ACKNOWLEDGMENTS

We are grateful to C. A. Amella for help with cell culture and to H.Schmidmayerova and G. Zybarth for providing the primers (LTR andhuman tubulin) and recombinant GFP, respectively. We thank theAIDS Research and Reference Reagent Program (NIAID, NIH, PHS)for reagents used in this study. We thank M. Laspia and A. Robson forreagents used in this study.

This work was supported in part by a grant from the NIH of theUnited States Public Health Service (AI40847-01A1) and by Institu-tional Funds from the Picower Institute for Medical Research. C. VanLint is “Charge de Recherches” of the Fond National de la RechercheScientifique (FNRS, Belgium).

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