HIV-1 enters target cells mainly via binding to CD4 and its coreceptors. The presence of HIV-1 in CD4 cells suggests, however, that there exist other mechanisms for viral entry. Here it is reported that HIV-1 DNA may be transferred from one cell to another by uptake of apoptotic bodies in a CD4-independent way. This was investigated by coculturing CD4, chemokine receptor CCR5 and CXCR4 human fetal fibroblasts with apoptotic HIV-1-infected HuT78 cells or apoptotic PBMC isolated from HIV-1-infected patients. After 2 wk of coculture, fibroblasts contained HIV-1 DNA and expressed HIV-1 proteins p24 and gp120. Transfer of HIV-1 DNA was verified by coculturing fibroblasts with apoptotic bodies derived from cells infected with a defective HIV-1 virus. These cells contain one integrated copy of a reverse transcriptase (RT)-negative HIV-1 strain (8E5/LAV RT cells) and consequently cannot produce free virus. Intracellular HIV-1 gag DNA was detected in both fibroblasts and dendritic cells after coculture with apoptotic 8E5/LAV RT cells. Transfer of viral DNA after uptake of apoptotic bodies may explain HIV-1 infection of CD4 cells in vivo and furthermore may be relevant for Ag presentation.

CD4, the primary HIV-1 receptor, is mainly expressed on populations of T cells, macrophages, and dendritic cells that also constitute the major target cells during primary HIV-1 infection (1, 2, 3, 4, 5). HIV-1 and SIV have, however, been detected in cell types lacking CD4 molecules on their surface, such as CD8+ T cells, fibroblasts, epithelial cells, and brain capillary endothelial cells, suggesting a CD4-independent infection pathway (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21). The possibility that some of these cells (e.g., CD8+ T cells) express low levels of CD4 is supported by the finding that neonatal CD8+ T cells, which are susceptible to productive HIV-1 infection in vitro, coexpress CD4 after activation (22).

Apart from CD4, coreceptors are also required for permissive cellular susceptibility to HIV-1 infection (1, 2, 3, 4, 5). Chemokine receptor CCR53 and CXCR4 are the major coreceptors for macrophage-tropic and T cell-tropic viruses, respectively. An explanation for viral entry in CD4 cells comes from studies with brain capillary endothelial cells, which were shown to be infected by a SIV by a CD4-independent, but CCR5-dependent, mechanism (20). Similarly, laboratory-adapted HIV and feline immunodeficiency virus strains were shown to productively infect cells in a CD4-independent, but CXCR4-dependent, manner (20, 23). Abs against galactosyl ceramide were, moreover, shown to inhibit CD4-independent entry of HIV-1 in neural cell lines (24). The list of HIV-1 coreceptors is constantly growing; it remains to be elucidated however which can be used in a CD4-independent way. Ab-mediated entry of HIV-1 into CMV-infected fibroblasts (25) and the formation of mixed phenotypes (pseudotypes) between HIV-1 and other viruses have also been proposed as potential mechanisms responsible for infection of CD4 cells (26).

Paradoxical viral infectibility without appropriate virus receptor expression has been reported also for EBV. EBV receptor (CD21)-negative cells such as epithelial cells in nasopharynx, fibroblasts from rheumatoid arthritis patients, and a variety of carcinomas and sarcomas were infected with EBV (27, 28, 29, 30, 31). We have recently shown that EBV DNA can be transferred from one cell to another by uptake of apoptotic bodies (32). This finding provides an explanation for how EBV DNA may enter cells by an EBV receptor-independent mechanism.

The present study was undertaken to examine whether HIV DNA can be transferred between cells by uptake of apoptotic bodies. Cells with phagocytosing capacity, such as human fibroblasts, endothelial cells and dendritic cells, were cocultured with apoptotic bodies derived from HIV-1-infected cells. The presence of HIV-1 gag DNA and protein expression of HIV-1-encoded genes was demonstrated in recipient cells after uptake of apoptotic bodies.

Human fetal lung fibroblasts and a human endothelial cell line (EaHy 926) (33) were cultured in DMEM (HyClone Europe, Perstorp, Sweden) supplemented with 2 mM l-glutamine (Life Technologies, Taby, Sweden), penicillin and streptomycin (Life Technologies), HEPES (Life Technologies), and 10% FCS (HyClone). These cells were treated with trypsin-EDTA (Life Technologies), washed twice in PBS supplemented with 10% FCS, and transferred to Lab-Tek chamber slides (Nunc, Naperville, IL; 5 × 104 cells/well) 1 day before addition of 1 × 105 apoptotic cells or cell-free primary T cell tropic virus isolates (100 tissue culture 50% infectious dose). Dendritic cells were generated from PBMC by culture in human rIL-4 (450 U/ml; Genzyme, Cambridge, MA) and GM-CSF (250 ng/ml; Leucomax, Shering-Plough, Brinny, Ireland) as previously described (34, 35, 36). Generated immature dendritic cells were used for coculture experiments on day 6 or 7. Apoptotic cells (5 × 105 cells/ml) were added to 3 × 105 dendritic cells/ml in a 24-well plate. The HIV-1 Ba-L isolate (500 tissue culture 50% infectious dose) (37), HuT78SF2 (38), and 8E5/LAV RT cells (39) were obtained through the AIDS Research and Reference Reagent Program, National Institutes of Health (McKesson BioServices, Rockville, MD), and HuT78 cells were obtained from American Type Culture Collection (Manassas, VA).

PBMC from HIV-1-infected patients or HIV-1-seronegative blood donors were isolated from EDTA-blood by density centrifugation on Ficoll-Hypaque gradients (Pharmacia, Uppsala, Sweden). Plasma HIV-1 viremia was measured by a branch DNA assay (Chiron, Emeryville, CA). CD4 T cell counts were performed by routine clinical laboratory testing. Apoptosis was induced either by gamma irradiation (150 Gy) 1–3 h before addition to the cultures or by treatment with etoposide 16 μg/ml for 48 h.

Stainings were performed as previously described (40). In brief, cells were washed with PBS before fixation in 3.7% paraformaldehyde in PBS for 10 min. To reduce nonspecific Ab binding, cells were first incubated with 2% FCS in Earle’s balanced salt solution (Life Technologies) supplemented with 0.01 M HEPES buffer (Life Technologies). Cells were then permeabilized with 0.1% saponin dissolved in balanced salt solution to allow intracellular entrance of HIV-1-specific Abs. To prevent unspecific binding of secondary Abs, 1% goat serum (Dako, Glostrup, Denmark) was added during incubation with primary Abs. Primary Abs diluted in balanced salt solution-saponin were added and left to incubate for 45 min at 37°C. After several washes with balanced salt solution-saponin the secondary Ab and Hoechst 33258 (Sigma, Stockholm, Sweden) were added and left to incubate for 30 min at room temperature. Cells were examined in a Leica RXM microscope (Leica, Wetzlar, Germany). The following mouse mAbs were used: anti-p24 (KAL-1, IgG1, Dako), anti-gp120 (8835, IgG1, Chemicon, Temecula, CA), anti-CXCR4 (12G5, IgG2a, PharMingen, San Diego, CA), anti-CCR5 (2D7, IgG2a, PharMingen), anti-CD4 (IgG1, Becton Dickinson, San Diego, CA), and anti-vimentin (Dako). Secondary goat anti-mouse Abs were Oregon Green-conjugated anti-Ig (Molecular Probes, Eugene, OR).

Adherent cells (5–20 × 104 cells/sample) were trypsinized and washed in PBS twice before fixation in Permeafix (Ortho Diagnostics, Raritan, NJ). Dendritic cells (1 × 105 cells/sample) were washed in PBS and thereafter stored in Permeafix. HIV-1 gag DNA was detected by a fluorescent in situ 5′-nuclease assay (FISNA) (41, 42). The PCR was performed in cell suspension (1× PCR buffer II; 0.35 mM MgCl2; 200 μM each of dATP, dCTP, dGTP, and dTTP; 200 μM each of gag primers SK38/SK39, sequences 5′-ATAATCCACCTATCCCAGTAGGAGAAAT-3′ and 5′-TTTGGTCCTTGTCTTATGTCCAGAATGC-3′, and 100 nM of gag probe FTSK19 (FAM served as the reporter dye and TAMRA served as the quenching dye), sequence 5′- ATCCTGGATTAAATAAAATAGTAAGAATGTATAGCCCTAC-3′ (10 U AmpliTaq DNA Polymerase IS) using the Gene Amp PCR System 2400 (PE Applied Biosystems, Foster City, CA). Reaction tubes were heated to 95°C for 5 min followed by 30 cycles consisting of 94°C for 45 s and 56°C for 2 min, followed by a 15°C soak. The cells were thereafter washed in PBS and put on slides by cytospin. Autofluorescence was quenched by incubation with trypan blue. The cells were evaluated for the presence of HIV-1 gag DNA at the single-cell level by an ACAS 570 laser confocal microscope (Insight Biomedical, Manchester, NH). The frequencies were manually counted. The cut-off values were determined based on fluorescence emitted from noninfected cells.

RNA was purified from fibroblasts cocultured with apoptotic HuT78SF2, 8E5/LAV RT or noninfected HuT78 cells for 1 or 2 wk as well as adherent PBMC (macrophages) cultured in RPMI-10% FCS for 1 wk, by Trizol reagent (Life Technologies) according to the manufacturer’s protocol. RNA pellets were resuspended in 1× transcription buffer (Promega, Madison, WI) with 2 U RQ1 RNase-free DNase (Promega) and incubated for 30 min at 37°C to remove contaminating DNA. The mixture was extracted once with phenol/chloroform/isoamyl alcohol and once with chloroform/isoamyl alcohol. The aqueous layer was removed, and the RNA was precipitated in 3 vol of ethanol and 1/40 vol of 3 mol/L sodium acetate overnight at −20°C. Quantitative kinetic RT-PCR (43) was performed by adding 45 μl of reaction mix (1× RT Taqman EZ buffer (PE Applied Biosystems, Foster City, CA), 4.0 mmol/L Mn(O)Ac2, 300 μmol/L dATP, 300 μmol/L dCTP, 300 μmol/L dGTP, 300 μmol/L dTTP, 200 nmol/L upstream primer, 200 nmol/L downstream primer, 200 nmol/L internally conserved fluorogenic probes, and 10 U of TTH polymerase) directly to 100 ng of total RNA in 5 μl of Rnase- and DNasefree water (Ambion, Austin, TX). Input RNA was normalized using glyceraldehyde-3-phosphate dehydrogenase mRNA quantification (PE Applied Biosystems). RT and thermal amplification were performed using the following linked profile: RT, 30 min at 60°C; cDNA denaturation, 5 min at 95°C, 40 cycles of denaturation (95°C for 15 s); and annealing/extension, 60°C for 1 min in a 7700 sequence detection system (PE Applied Biosystems). Duplicate standard curves with copy number controls ranging from 10 to 105 copies were run with each optical 96-well plate (PE Applied Biosystems). In addition, no template controls were included with each plate. The primers and their respective probes were previously described (43).

Culture supernatants from dense cultures with 8E5/LAV RT, HuT78SF2, and noninfected HuT78 cells were tested for the presence of RT using a sensitive Lenti-RT activity assay (Cavidi Tech, Uppsala, Sweden) according to the manufacturer’s protocol.

Irradiated or etoposide-treated PBMC, HuT78, or 8E5/LAV RT cells were stained with annexin V-FITC (Boehringer Mannheim, Mannheim, Germany) and propidium iodide (PI) according to the manufacturer’s protocol. Early apoptosis was defined by annexin V+PI staining as determined by FACScan or FACSCalibur (Becton Dickinson). The kinetics of cell death after irradiation (2, 4, 10, 18, 24, 48 h) or etoposide treatment (12, 24, 48 h) wer studied in noninfected cells, since HIV-1-infected cells were always fixed in paraformaldehyde before analyses by flow cytometry. Fluorescence intensity was measured using a log10 scale, and 10,000 events were analyzed per sample.

Human fetal fibroblasts that lacked detectable mRNA and protein expression of CD4, CCR5, and CXCR4, as shown by immunofluorescent stainings and quantitative kinetic RT-PCR (Table I and data not shown), were used to study HIV-1 receptor-independent transfer of HIV-1. Freshly isolated PBMC or HuT78 cells were used as positive controls for immunofluorescent stainings of CD4, CCR5, and CXCR4, while macrophages (adherent PBMC cultured for 1 wk) were used as a positive control for the expression of CCR5 and CXCR4 mRNA (Table I). To investigate whether HIV-1 DNA could be transferred by the uptake of apoptotic bodies in coculture experiments, HIV-1-infected and noninfected T cell lymphomas as well as PBMC were induced to undergo apoptosis before addition to fibroblast cultures. Apoptosis, as detected by annexin V binding was induced by either gamma irradiation (150 Gy) or treatment with etoposide (Fig. 1). Freshly isolated PBMC contained some debris and dead cells that were annexin V+PI+ and a few cells bound annexin V but did not take up PI (Fig. 1,A). HIV-1-infected T cell lymphomas contained about 10–20% annexin-V+ debris and cells before induction of apoptosis (Fig. 1,B). Almost all HIV-1-infected T lymphomas (Fig. 1, D and F) and around 50% of PBMC (Fig. 1, C and E) bound annexin V after 48 h of etoposide treatment or 18–24 h after irradiation. Approximately 15–20% of PBMC also took up PI, a sign of secondary necrosis. Fibroblasts were cocultured with apoptotic HIVSF2-infected or noninfected HuT78 cells, and the first analyses were performed after 2 wk of culture. The presence of HIV-1 DNA after uptake of apoptotic bodies was detected using FISNA (41). Evaluation showed that fibroblasts cocultured with apoptotic HuT78SF2 cells contained intracellular localized HIV-1 gag DNA, which remained throughout the culture period of 8 wk. However, fibroblasts cocultured with a cell-free primary T cell-tropic virus isolate or apoptotic noninfected HuT78 cells showed no presence of HIV-1 gag DNA (Fig. 2,a and Table II). An immortalized human endothelial cell line was analyzed following coculture with apoptotic HuT78SF2 cells in parallel with fibroblasts, but did not show any gag DNA-positive cells. The endothelial cells thus served as a negative control for fibroblast cocultures analyzed by FISNA (Table II).

Table I.

Human fetal lung fibroblasts lack expression of CCR5 and CXCR4 mRNA

CCR5 mRNAaCXCR4 mRNAa
MeanSDMeanSD
One week of coculture     
Fibroblast+ apoptotic 8E5/LAV RT 
Fibroblast+ apoptotic HuT78SF2 0.1 0.2 71 24 
Fibroblast+ apoptotic noninfected HuT78 0.2 0.2 247 51 
Two weeks of coculture     
Fibroblast+ apoptotic 8E5/LAV RT 
Fibroblast+ apoptotic HuT78SF2 
Fibroblast+ apoptotic noninfected HuT78 
Macrophagesb 66 13 1966 537 
CCR5 mRNAaCXCR4 mRNAa
MeanSDMeanSD
One week of coculture     
Fibroblast+ apoptotic 8E5/LAV RT 
Fibroblast+ apoptotic HuT78SF2 0.1 0.2 71 24 
Fibroblast+ apoptotic noninfected HuT78 0.2 0.2 247 51 
Two weeks of coculture     
Fibroblast+ apoptotic 8E5/LAV RT 
Fibroblast+ apoptotic HuT78SF2 
Fibroblast+ apoptotic noninfected HuT78 
Macrophagesb 66 13 1966 537 
a

Human fetal lung fibroblasts were transferred to Lab-Tek chamber slides (5 × 104 cells/well) before addition of apoptotic (1 × 105 cells/well) 8E5/LAV RT T cells infected with a defective RT HIV-1 isolate, or apoptotic HIV-1-infected HuT78SF2 T cells or apoptotic noninfected HuT78 T cells. After 1 or 2 wk of coculture, chemokine receptor mRNA was quantified by RT-PCR. Values (mean and SD from duplicates) equal number of chemokine receptor mRNA copies per 10,000 G3PDH mRNA copies.

b

PBMC were cultured for 1 wk. The recovered adherent cells were analyzed for CCR5 and CXCR4 expression and used as positive control.

FIGURE 1.

Induction of apoptosis in PBMC and HIV-1-infected T lymphomas. PBMC isolated from healthy HIV-1-seronegative blood donors by Ficoll separation (A, C, and E) were stained with annexin V-FITC and PI and analyzed by flow cytometry. No gates were used. HIV-1-infected 8E5/LAV RT cells (B, D, and F) were stained with annexin V-FITC and then fixed in 4% paraformaldehyde before analysis by flow cytometry. PBMC or 8E5/LAV RT cells were untreated in A and B, etoposide treated (16 μg/ml) for 48 h in C and D, or irradiated (150 Gy) 18–24 h before staining in E and F. Results are expressed as log10 of fluorescence, and 10,000 events were collected per sample. One representative experiment of three is shown.

FIGURE 1.

Induction of apoptosis in PBMC and HIV-1-infected T lymphomas. PBMC isolated from healthy HIV-1-seronegative blood donors by Ficoll separation (A, C, and E) were stained with annexin V-FITC and PI and analyzed by flow cytometry. No gates were used. HIV-1-infected 8E5/LAV RT cells (B, D, and F) were stained with annexin V-FITC and then fixed in 4% paraformaldehyde before analysis by flow cytometry. PBMC or 8E5/LAV RT cells were untreated in A and B, etoposide treated (16 μg/ml) for 48 h in C and D, or irradiated (150 Gy) 18–24 h before staining in E and F. Results are expressed as log10 of fluorescence, and 10,000 events were collected per sample. One representative experiment of three is shown.

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FIGURE 2.

Fibroblasts cocultured with apoptotic HIV-1-infected cells contain HIV-1 DNA and express p24 and gp120. a, Fibroblasts were cocultured with apoptotic HIV-1-infected HuT78SF2 (A), apoptotic noninfected HuT78 (B), a cell-free primary T cell tropic virus isolate (C), or apoptotic HIV-1-infected cells infected with a defective RT isolate (8E5/LAV RT cells; D) (39 ) at a ratio of 1:2. After 2 wk of coculture, fibroblasts were treated with trypsin and washed in PBS before fixation in Permeafix. HIV-1 gag DNA was detected by FISNA (41 ). Cells containing HIV-1 DNA appear yellow-red-white, with a peak fluorescence intensity of 4095. Uninfected cells appear purple-blue-green, with a peak fluorescence intensity of 2303. Pictures are representative of three experiments. b, Fibroblasts were cocultured for 2 wk on Lab-Tek chamber slides with apoptotic HIV-1-infected HuT78SF2 (A and B), apoptotic 8E5/LAV RT (39 ) (C and D), or apoptotic noninfected HuT78 cells (E and F) at a ratio of 1:2. Fibroblasts were washed in PBS before they were fixed in paraformaldehyde, permeabilized with saponin, and stained with mAb directed against either p24 (KAL-1, IgG1; A, C, and E) or gp120 (8835, IgG1; B, D, and F) followed by goat anti-mouse Oregon Green-conjugated anti-Ig and Hoechst. One representative experiment of five is shown. The frequency of p24-positive fibroblasts after coculture with apoptotic HuT78SF2 or apoptotic 8E5/LAV RT cells ranged between 0.3–1.7%. One thousand cells per sample were counted. Size bar = 20 μm.

FIGURE 2.

Fibroblasts cocultured with apoptotic HIV-1-infected cells contain HIV-1 DNA and express p24 and gp120. a, Fibroblasts were cocultured with apoptotic HIV-1-infected HuT78SF2 (A), apoptotic noninfected HuT78 (B), a cell-free primary T cell tropic virus isolate (C), or apoptotic HIV-1-infected cells infected with a defective RT isolate (8E5/LAV RT cells; D) (39 ) at a ratio of 1:2. After 2 wk of coculture, fibroblasts were treated with trypsin and washed in PBS before fixation in Permeafix. HIV-1 gag DNA was detected by FISNA (41 ). Cells containing HIV-1 DNA appear yellow-red-white, with a peak fluorescence intensity of 4095. Uninfected cells appear purple-blue-green, with a peak fluorescence intensity of 2303. Pictures are representative of three experiments. b, Fibroblasts were cocultured for 2 wk on Lab-Tek chamber slides with apoptotic HIV-1-infected HuT78SF2 (A and B), apoptotic 8E5/LAV RT (39 ) (C and D), or apoptotic noninfected HuT78 cells (E and F) at a ratio of 1:2. Fibroblasts were washed in PBS before they were fixed in paraformaldehyde, permeabilized with saponin, and stained with mAb directed against either p24 (KAL-1, IgG1; A, C, and E) or gp120 (8835, IgG1; B, D, and F) followed by goat anti-mouse Oregon Green-conjugated anti-Ig and Hoechst. One representative experiment of five is shown. The frequency of p24-positive fibroblasts after coculture with apoptotic HuT78SF2 or apoptotic 8E5/LAV RT cells ranged between 0.3–1.7%. One thousand cells per sample were counted. Size bar = 20 μm.

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Table II.

Detection of HIV-1 DNA in cocultures by FISNAa

Coculturesb% HIV-1 DNA Containing Cells
2-wk coculture8 wk coculture
Fibroblast+ apoptotic 8E5/LAV RT 19 5.2 
Fibroblast+ apoptotic HuT78SF2 46 68 
Fibroblast+ apoptotic noninfected HuT78 0.3 0.1 
Fibroblast+ apoptotic PBMC HIV-1 infected donor 51 7.1 
Fibroblast+ apoptotic PBMC seronegative donor 0.1 0.1 
Fibroblast+ T cell tropic primary virus isolate 0.1 0.6 
   
Dendritic cells+ apoptotic 8E5/LAV RT 18 ND 
Dendritic cells+ apoptotic noninfected HuT78 0.1 ND 
Dendritic cells + Ba-L isolate 8.9 ND 
Dendritic cells 0.1 ND 
   
Endothelial+ apoptotic 8E5/LAV RT 1.3 0.3 
Endothelial+ apoptotic noninfected HuT78 0.1 ND 
Endothelial+ apoptotic PBMC HIV-1 infected donor 0.9 0.9 
Endothelial+ apoptotic PBMC seronegative donor 0.1 0.1 
Endothelial+ T cell tropic primary virus isolate 0.3 0.1 
Coculturesb% HIV-1 DNA Containing Cells
2-wk coculture8 wk coculture
Fibroblast+ apoptotic 8E5/LAV RT 19 5.2 
Fibroblast+ apoptotic HuT78SF2 46 68 
Fibroblast+ apoptotic noninfected HuT78 0.3 0.1 
Fibroblast+ apoptotic PBMC HIV-1 infected donor 51 7.1 
Fibroblast+ apoptotic PBMC seronegative donor 0.1 0.1 
Fibroblast+ T cell tropic primary virus isolate 0.1 0.6 
   
Dendritic cells+ apoptotic 8E5/LAV RT 18 ND 
Dendritic cells+ apoptotic noninfected HuT78 0.1 ND 
Dendritic cells + Ba-L isolate 8.9 ND 
Dendritic cells 0.1 ND 
   
Endothelial+ apoptotic 8E5/LAV RT 1.3 0.3 
Endothelial+ apoptotic noninfected HuT78 0.1 ND 
Endothelial+ apoptotic PBMC HIV-1 infected donor 0.9 0.9 
Endothelial+ apoptotic PBMC seronegative donor 0.1 0.1 
Endothelial+ T cell tropic primary virus isolate 0.3 0.1 
a

HIV-1 DNA was detected by FISNA (41). The cells were evaluated for presence of HIV-1 DNA at the single cell level by an ACAS 570 laser confocal microscope. Values above 1.5% are considered positive. One representative experiment of at least two is shown.

b

Human fetal lung fibroblasts and human endothelial cells were transferred to Lab-Tek chamber slides (5 × 104/well) 1 day before addition of indicated apoptotic cells (1 × 105 cells/well) or cell-free virus isolates 100 TCID50. Dendritic cells were generated from PBMC from blood donors by culture in medium containing human rIL-4 and GM-CSF. Generated immature dendritic cells were used for coculture experiments at day 6 or 7. Apoptotic cells (5 × 105 cells/ml) were added to 3 × 105 dendritic cells/ml. HIV-infected HuT78SF2 (38), noninfected HuT78, 8E5/LAV RT cells (39), and PBMC isolated from HIV-1 infected patients or healthy blood donors, were irradiated 150 Gy, before addition to the cultures.

To verify whether the presence of gag DNA in fibroblasts was due to transfer of HIV-1 DNA and not to uptake of free viruses released from apoptotic HuT78SF2 cells, cocultures with apoptotic bodies derived from a T lymphoma infected with a defective virus were analyzed. 8E5/LAV RT cells contain one integrated copy of an HIV-1 strain that lacks RT and consequently cannot produce complete viral particles, but can produce some HIV-1-encoded proteins (39). Culture supernatants from 8E5/LAV RT cells were analyzed by a sensitive RT assay at several time points to assure that the cells lacked RT (data not shown). Fibroblasts cocultured up to 8 wk with apoptotic bodies from 8E5/LAV RT cells contained gag DNA, showing that the intracellular presence of HIV-1 DNA was not due to infection with viral particles derived from the apoptotic T lymphomas (Fig. 2 a).

To exclude that HIV-1 detected in fibroblasts were due to remaining apoptotic bodies, the expressions of CXCR4 and CCR5 mRNA, originating from HuT78 cells, were followed by quantitative kinetic RT-PCR. Fibroblasts that were cocultured with apoptotic 8E5/LAV RT, HuT78SF2, or noninfected HuT78 cells for 1 wk had detectable CCR5 and CXCR4 mRNA expression as assessed by quantitative kinetic RT-PCR. Apoptotic bodies were also detected by Hoechst staining in the cytoplasm of fibroblasts after 1 wk of cocultivation with apoptotic HuT78 or 8E5/LAV RT cells (data not shown) (32). After 2 wk of coculture, however, no remaining apoptotic bodies were found in the fibroblasts as detected by either Hoechst staining or quantitative kinetic RT-PCR (Table I).

To investigate whether the transferred HIV-1 DNA was transcribed, fibroblasts were analyzed for protein expression of HIV-1 p24 and gp120 Ags. Immunofluorescent labelings showed expression of the HIV-1-encoded gene products p24 and gp120 in fibroblasts after 2 wk of coculture with apoptotic 8E5/LAV RT and apoptotic HuT78SF2 cells (Fig. 2,b). The staining pattern was characterized by the accumulation of protein in the cytosol. Fibroblasts cocultured with noninfected HuT78 cells did not express p24 or gp120 as expected (Fig. 2 b). The frequency of p24 Ag-positive fibroblasts after coculture with apoptotic 8E5/LAV RT and apoptotic HuT78SF2 cells ranged between 0.3–1.7% in five independent experiments.

Dendritic cells can present Ag derived from apoptotic cells, stimulating MHC class I-restricted Ag-specific CD8+ cytotoxic T cells (44). We therefore investigated whether HIV-1 DNA could be transferred to dendritic cells by uptake of apoptotic bodies. Dendritic cells express HIV-1 receptors (45), an expression pattern that seems to be tightly regulated during dendritic cell maturation (46). Apoptotic 8E5/LAV RT cells infected with the defective, RT-negative virus were therefore used in cocultures with dendritic cells. Dendritic cells were prepared from peripheral blood precursors of healthy donors by in vitro culture in the presence of rIL-4 and GM-CSF (34, 35, 36). Apoptotic 8E5/LAV RT cells were added to the in vitro differentiated dendritic cells after 6–7 days of culture. At this time dendritic cells were CD14, HLA-DR+, CD83, and CD86 (36), characteristic of an immature phenotype with phagocytosing capacity (47). HIV-1 gag DNA could be detected by FISNA in approximately 18% of dendritic cells after 2 wk of coculture with apoptotic 8E5/LAV RT cells and in 9% after infection with a cell-free macrophage-tropic Ba-L isolate (Fig. 3,a and Table II). Dendritic cells cocultured with noninfected cells or without any virus isolate did not emit positive signals for gag DNA. These results show that HIV-1 DNA can be transferred to dendritic cells by uptake of apoptotic bodies.

FIGURE 3.

HIV-1 receptor-independent uptake of HIV-1 DNA by immature dendritic cells. a, Immature dendritic cells (3 × 105/ml) were cocultured with apoptotic 8E5/LAV RT cells (5 × 105/ml; A), cell-free Ba-L isolate (B), or apoptotic noninfected HuT78 cells (5 × 105/ml; C) for 2 wk. The presence of HIV-1 gag DNA was detected by FISNA (41 ) using the gag primers SK38/SK39 and the gag probe FTSK19. Cells were examined for the presence of HIV-1 DNA at the single-cell level by a laser confocal microscope. Cells containing HIV-1 DNA appear green-yellow-red, with a peak fluorescence intensity of 3327. Uninfected cells appear purple-blue with a peak fluorescence intensity of 1536. Pictures are representative of two experiments. b, Functional transfer of HIV-1 DNA by apoptotic PBMC isolated from HIV-1-infected patients. Fibroblasts expressed the HIV-1 Ag p24 after 2 wk of coculture with irradiated (150 Gy), apoptotic PBMC isolated from HIV-1-infected patients (donor 1: HIV RNA, 3.7 log10 copies/ml; CD4 count, 50 cells/mm3; A; donor 2: HIV RNA, <2.7 log10 copies/ml; CD4 count, 220 cells/mm3; B), while coculture with irradiated apoptotic PBMC from healthy blood donors did not give any staining signal (C). Immunofluorescent stainings were performed with anti-p24 (KAL1 IgG1) followed by Oregon Green-conjugated goat anti-mouse Ig and Hoechst. The frequency of p24 Ag-positive fibroblasts after coculture with apoptotic PBMC isolated from HIV-1-infected patients ranged between 0.6–2.7%. One thousand cells were counted per sample. Size bar = 20 μm. c, Fibroblasts contain HIV-1 DNA after coculture with apoptotic PBMC from HIV-1-infected patients. The presence of gag DNA (yellow-red-white) was analyzed by FISNA (41 ) in fibroblasts cocultured with nonirradiated (A), irradiated (150 Gy) apoptotic PBMC isolated from an HIV-1-infected patient (donor 3; HIV RNA. 6.5 log10 copies/ml; CD4 count, 25 cells/mm3;B), irradiated (150 Gy) apoptotic PBMC isolated from another HIV-1-infected patient (higher magnification of donor 4; HIV RNA, 4.2 log10 copies/ml; CD4 count, 190 cells/mm3; C), and irradiated apoptotic PBMC from a healthy HIV-1-seronegative blood donor (D) at a ratio of 1:2. After 2 wk in culture, the presence of HIV-1 gag DNA (yellow-red-white) was detected by FISNA using the gag primers SK38/SK39 and the gag probe FTSK19. Cells containing HIV-1 DNA appear yellow-red-white, with a peak fluorescence intensity of 4095. Uninfected cells appear purple-blue-green, with a peak fluorescence intensity of 2303.

FIGURE 3.

HIV-1 receptor-independent uptake of HIV-1 DNA by immature dendritic cells. a, Immature dendritic cells (3 × 105/ml) were cocultured with apoptotic 8E5/LAV RT cells (5 × 105/ml; A), cell-free Ba-L isolate (B), or apoptotic noninfected HuT78 cells (5 × 105/ml; C) for 2 wk. The presence of HIV-1 gag DNA was detected by FISNA (41 ) using the gag primers SK38/SK39 and the gag probe FTSK19. Cells were examined for the presence of HIV-1 DNA at the single-cell level by a laser confocal microscope. Cells containing HIV-1 DNA appear green-yellow-red, with a peak fluorescence intensity of 3327. Uninfected cells appear purple-blue with a peak fluorescence intensity of 1536. Pictures are representative of two experiments. b, Functional transfer of HIV-1 DNA by apoptotic PBMC isolated from HIV-1-infected patients. Fibroblasts expressed the HIV-1 Ag p24 after 2 wk of coculture with irradiated (150 Gy), apoptotic PBMC isolated from HIV-1-infected patients (donor 1: HIV RNA, 3.7 log10 copies/ml; CD4 count, 50 cells/mm3; A; donor 2: HIV RNA, <2.7 log10 copies/ml; CD4 count, 220 cells/mm3; B), while coculture with irradiated apoptotic PBMC from healthy blood donors did not give any staining signal (C). Immunofluorescent stainings were performed with anti-p24 (KAL1 IgG1) followed by Oregon Green-conjugated goat anti-mouse Ig and Hoechst. The frequency of p24 Ag-positive fibroblasts after coculture with apoptotic PBMC isolated from HIV-1-infected patients ranged between 0.6–2.7%. One thousand cells were counted per sample. Size bar = 20 μm. c, Fibroblasts contain HIV-1 DNA after coculture with apoptotic PBMC from HIV-1-infected patients. The presence of gag DNA (yellow-red-white) was analyzed by FISNA (41 ) in fibroblasts cocultured with nonirradiated (A), irradiated (150 Gy) apoptotic PBMC isolated from an HIV-1-infected patient (donor 3; HIV RNA. 6.5 log10 copies/ml; CD4 count, 25 cells/mm3;B), irradiated (150 Gy) apoptotic PBMC isolated from another HIV-1-infected patient (higher magnification of donor 4; HIV RNA, 4.2 log10 copies/ml; CD4 count, 190 cells/mm3; C), and irradiated apoptotic PBMC from a healthy HIV-1-seronegative blood donor (D) at a ratio of 1:2. After 2 wk in culture, the presence of HIV-1 gag DNA (yellow-red-white) was detected by FISNA using the gag primers SK38/SK39 and the gag probe FTSK19. Cells containing HIV-1 DNA appear yellow-red-white, with a peak fluorescence intensity of 4095. Uninfected cells appear purple-blue-green, with a peak fluorescence intensity of 2303.

Close modal

PBMC from HIV-1-infected patients contain cells that are latently infected and in which the viral cDNA is integrated within host cell DNA (3). To assess whether PBMC isolated from HIV-1-infected patients could transfer HIV-1 by uptake of apoptotic bodies, fibroblasts were cocultured with apoptotic PBMC isolated from HIV-1-infected patients. PBMC were isolated from five patients with HIV RNA levels of <2.7–6.5 log10 copies/ml of plasma and CD4 cell counts between 25–220/mm3. Fibroblasts cocultured with apoptotic bodies derived from PBMC isolated from HIV-1-infected donors contained gag DNA after 2–8 wk of culture at a frequency of 6–51% (Fig. 3,c and Table II). Freshly isolated PBMC (from the same HIV-1-infected patients) that had not been induced to undergo apoptosis by irradiation as well as apoptotic PBMC from HIV-1 seronegative donors did not transfer HIV-1 DNA to cocultured fibroblasts (Fig. 3,c and Table II).

Fibroblasts cocultured with apoptotic PBMC isolated from HIV-1-infected patients also expressed the HIV-1 Ag p24 (Fig. 3 b). The frequency of intracellular p24 Ag-positive fibroblasts detected by immunofluorescence after 2 wk of coculture with apoptotic PBMC isolated from HIV-1-infected patients ranged between 0.6–2.7%, thus similar to the frequency detected in cocultures with apoptotic 8E5/LAV RT and apoptotic HuT78SF2 cells (0.3–1.7%).

In the present study it was shown that fibroblasts and dendritic cells cocultured with apoptotic bodies derived from HIV-1-infected cells, resulted in uptake of HIV-1 DNA and expression of HIV-1 proteins p24 and gp120. Two lines of evidence indicated that the transfer of HIV-1 was CD4-, CCR5-, and CXCR4-independent. First, the fibroblasts used in these experiments did not express detectable mRNA or protein levels of CD4, CCR5, or CXCR4. Second, transfer of HIV-1 DNA was possible using apoptotic bodies from HIV-1-infected T lymphoma with one integrated copy of a defective RT virus. This defective RT RNA virus cannot produce HIV-1 viral particles (39). The results presented here, hence, are consistent with transfer of HIV-1 DNA to the fibroblasts and dendritic cells. The transferred HIV-1 DNA was, furthermore, transcribed and resulted in expression of HIV-1-encoded proteins p24 and gp 120.

The in vitro infectibility of peripheral CD8+ T cells from adults was shown to be dependent on the presence of CD4+ T cells in the initial culture exposed to HIV-1 (12). The CD8+ T cells were also infected with HIV-1 after coculture with autologous HIV-1-infected CD4+ T cells (12). CD8+ cytotoxic T cells were, moreover, shown to become infected in vitro in the process of killing HIV-1-infected target cells, a process involving apoptosis of the infected target cell (48). It remains to be elucidated whether the infection of CD8+ T cells was caused by transfer of HIV DNA after uptake of apoptotic bodies derived from either CD4+ T cells or HIV-1-infected target cells.

CD4 cells infected with HIV-1 have been observed at several anatomic locations in HIV-1-infected individuals (6, 7, 8, 17, 18, 19). Findings presented here raise the question of whether apoptotic bodies derived from HIV-1-infected cells can transfer viral DNA to HIV-1 receptor-negative, phagocytosing cells in vivo. Such a mechanism could play a role in virus persistence in the infected individual and may explain the spread of HIV-1 to, for example, endothelial cells in the brain as well as epithelial cells. The pathogenesis of HIV-1 infection is characterized by increased frequency of apoptosis (49). The finding that PBMC isolated from HIV-1-infected patients could transfer HIV-1 DNA supports the hypothesis that viral transfer by apoptotic bodies could also play a role in vivo. It was, however, necessary to induce apoptosis in the infected PBMC, since freshly isolated PBMC could not transfer HIV-1 to HIV-1 receptor-negative cells. This suggests that apoptotic PBMC may transfer HIV-1 DNA to CD4 cells in vivo.

Dendritic cells have been shown to acquire Ag from apoptotic cells and induce MHC class I-restricted CTL (44) as well as present phagocytosed cellular fragments on MHC class II molecules (50). The source(s) of the peptides presented by MHC molecules remains unresolved. Are they derived from processed phagocytosed proteins that have been stored in dendritic cells after internalization of apoptotic bodies (50), and/or do they derive from endogenously produced peptides after transfer of viral DNA? Follicular dendritic cells have been shown to be able to present Ag for long periods of time after Ag exposure (51, 52). In the current study transferred HIV DNA could be detected in high frequencies for up to 8 wk after initiation of cocultures. We speculate that transfer of DNA, leading to expression of proteins and processing of peptides in the APC, could account for prolonged capacity of Ag presentation.

We thank Lena Radler for expert technical assistance, Margit Halvarsson and Ann-Sofie Löfstrand for blood samples, and Dr. Anders Sönnerborg (Department of Clinical Virology, Huddinge Hospital) for help and advice.

1

This work was supported by the Swedish Physicians Against AIDS Research Foundation, the Swedish Medical Council (Grant 10850), the National Cancer Institute (Grant 2766), the National Institutes of Health (Grant U01 AI41536-02), and the Swedish Cancer Society.

3

Abbreviations used in this paper: CCR, chemokine receptor; FISNA, fluorescent in situ 5′-nuclease assay; PI, propidium iodide.

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