A Fusion Inhibitor Prevents Spread of Immunodeficiency Viruses, but Not Activation of Virus-Specific T Cells, by Dendritic Cells

Abstract

Dendritic cells (DCs) play a key role in innate immune responses, and their interactions with T cells are critical for the induction of adaptive immunity. However, immunodeficiency viruses are efficiently captured by DCs and can be transmitted to and amplified in CD4⁺ T cells, with potentially deleterious effects on the induction of immune responses. In DC-T-cell cocultures, contact with CD4⁺, not CD8⁺, T cells preferentially facilitated virus movement to and release at immature and mature DC-T-cell contact sites. This occurred within 5 min of DC-T-cell contact. While the fusion inhibitor T-1249 did not prevent virus capture by DCs or the release of viruses at the DC-T-cell contact points, it readily blocked virus transfer to and amplification in CD4⁺ T cells. Higher doses of T-1249 were needed to block the more robust replication driven by mature DCs. Virus accumulated in DCs within T-1249-treated cocultures but these DCs were actually less infectious than DCs isolated from untreated cocultures. Importantly, T-1249 did not interfere with the stimulation of virus-specific CD4⁺ and CD8⁺ T-cell responses when present during virus-loading of DCs or for the time of the DC-T-cell coculture. These results provide clues to identifying strategies to prevent DC-driven virus amplification in CD4⁺ T cells while maintaining virus-specific immunity, an objective critical in the development of microbicides and therapeutic vaccines.

Dendritic cells (DCs) provide innate immune defenses and play a critical role in the induction of protective immunity(54). Immature DCs entrap incoming pathogens in tissues and traffic them to the draining lymphoid tissues where as matured DCs they elicit immunity (1, 19, 33). Human and simian immunodeficiency viruses (HIV and SIV, respectively) exploit features of DC biology to facilitate their dissemination and propagation (41, 45, 60). Utilizing CD4 and chemokine receptors (primarily CCR5), virus fuses with immature DCs, allowing virus replication and low-level production of progeny virus (63, 64, 70). Although HIV can fuse with mature DCs, they do not support replication, most likely due to a block at the levels of reverse transcription and postintegration (5, 9, 12, 18). Alternatively, virus entry can be facilitated by C-type lectin receptors (CLRs) (CD206, macrophage mannose receptor; CD207, langerin; and CD209, DC-SIGN), unidentified trypsin-resistant CLRs (61, 62), and possibly other receptors (11, 21, 34, 70), which allow attachment and internalization of virus by both immature and mature DCs.

Exploiting normal DC-T-cell communication, immunodeficiency viruses are transmitted to CD4⁺ T cells, the primary cell substrates for virus replication. Two distinct phases of virus transfer exist (63, 70). The first involves the transfer of internalized virus by immature and/or mature DCs to T cells in the absence of DC infection (14, 18, 23, 36, 63, 67, 70). This phase likely contributes to the first round of DC-driven spread locally at the mucosa before DC-captured virus is transported to lymphoid tissue. The second phase involves de novo replication of CCR5-tropic viruses (R5 viruses) in infected immature DCs and probably serves as a longer lasting source of infectious virus (7, 42, 63). HIV and its entry receptors CD4, CCR5, and CXCR4 are recruited to the synapse of DC-T-cell conjugates, enhancing the likelihood of productive infection of T cells (36). Aggregation of these molecules is antigen independent, supporting earlier observations that DCs can facilitate transmission to quiescent/resting CD4⁺ T cells (3, 36, 46), probably as a result of the low-level signaling provided by the DCs within such conjugates (48).

Compounds targeting CD4, chemokine receptors, or virus envelope block different stages of the virus binding/fusion/entry process and inhibit DC-driven virus amplification in T cells (25, 26). This suggests that even if DCs can capture virus independent of these molecules, the transfer of DC-entrapped virus to CD4⁺ T cells remains vulnerable to inhibition by compounds capable of acting extracellularly. Similar observations have been made using mucosal explants, where CCR5 inhibitors readily blocked the infection of cells (potentially immature DCs, macrophages, and T cells) within the mucosal tissues (22) or where a fusion inhibitor reduced the ability of HIV-exposed Langerhans cells to transfer virus to cocultured T cells (56). The ability of DCs that migrated from mucosal tissues to transfer virus to T cells was most effectively blocked by agents that interfered with CLR-mediated capture (22), highlighting the involvement of the two DC-driven pathways in situ. The importance of CCR5-dependent infection during the early stages of infection is further emphasized in in vivo studies showing that topically applied CCR5-specific inhibitors protected macaques against vaginal simian/human immunodeficiency virus infection (29, 52, 66).

Here, we performed ultrastructural analyses of the first-phase transfer of DC-captured virions to CD4⁺ T cells across the synaptic clefts of DC-T-cell conjugates, studying the impact of the fusion inhibitor T-1249 on these events as well as on the ability of the DCs to stimulate SIV-specific T cells. These data provide important insight into how strategies that block DC-driven virus spread can be effective in limiting replication while not interfering with the stimulation of virus-specific immunity.

MATERIALS AND METHODS

Animals.

Adult male and female rhesus macaques (Macaca mulatta) were housed at the Tulane Regional Primate Research Center (Covington, LA) and tested negative by PCR for simian type D retroviruses and simian T cell leukemia virus. Macaques previously immunized with the attenuated SIV strain SIVmac239Δnef and successfully protected from challenge with wild-type SIVmac239 and SHIV162P3-infected macaques, both showing strong SIV-specific immune responses (16, 37, 57), were used as a source of virus-primed T cells to monitor virus-specific T-cell activation and CD14⁺ monocytes for the generation of DCs. All animals were healthy at the time of study with undetectable plasma viral loads and have not shown evidence of SIV-associated disease over the past years of follow-up. Animal care procedures were in compliance with the regulations detailed under the animal welfare act (2) and in the Guide for the Care and Use of Laboratory Animals (65).

Isolation and culture of primary cells.

CD14⁺ monocytes were sorted (MACS [magnetism-activated cell sorting]; Miltenyi Biotec Inc., Auburn, CA) from human peripheral blood mononuclear cells (PBMCs) isolated from leukopaks obtained from the New York City Blood Bank or heparinized macaque blood by Ficoll-Hypaque density gradient (Amersham Pharmacia Biotech AB, Uppsala, Sweden) as previously described (15, 16). Cells were cultured in complete RPMI-1640 medium ([cRPMI] Cellgro; Fisher Scientific, Springfield, NJ) supplemented with 2 mM l-glutamine (GIBCO-BRL Life Technologies, Grand Island, NY), 50 μM 2-mercaptoethanol (Sigma Chemical Company, St. Louis, MO), 10 mM HEPES (GIBCO-BRL Life Technologies), penicillin (100 U/ml)-streptomycin (100 μg/ml) (GIBCO-BRL Life Technologies), and 1% heparinized human plasma (Innovative Research, Inc., Southfield, MI) containing 100 U/ml interleukin-4 (IL-4) and 1,000 U/ml granulocyte-macrophage colony-stimulating factor. On days 5 to 6, cells were activated for 2 days with the cocktail of prostaglandin E₂, IL-1β, IL-6, and tumor necrosis factor alpha (15). Immature and mature DCs were used after 7 to 8 days in culture. As needed, CD14⁻ cells were kept in culture (1 × 10⁷ cells/ml in cRPMI medium) for up to 8 days (adding additional fresh medium every 2 days). On the day of culture CD14⁻ cells were depleted of either CD8⁺ HLA-DR⁺ CD11b⁺ CD16⁺ or CD4⁺ HLA-DR⁺ CD11b⁺ CD16⁺ cells to obtain CD4⁺ or CD8⁺ T cells using directly conjugated beads or the phycoerythrin (PE)-conjugated anti-CD8 antibody ([Ab] clone BW135/80; Miltenyi Biotech Inc.) and anti-PE beads for macaque cells. For the depletion of human CD4⁺ T cells from DC-T-cell cocultures, CD3 beads were used, and HLA-DR beads were used to enrich for HLA-DR positive cells.

Flow cytometric analysis.

Phenotypic characterization of immature and mature DCs, including purity controls of generated DCs and T-cell subpopulations (purity of ≥97% for DCs and T-cell populations), was routinely monitored by two-color flow cytometry (16). A PE-conjugated anti-CD3 monoclonal Ab ([MAb] clone SK7 for human and clone SP34 for macaque cells; BD Biosciences, San Jose, CA) was used to detect contaminating T cells in DC preparations. To determine the frequency of DC-T-cell conjugates, DC-T-cell mixtures were stained with a fluorescein isothiocyanate-conjugated anti-HLA-DR (clone L243; BD Biosciences) and a PE-conjugated anti-CD3 MAb. Isotype controls were included in each experiment. Cell samples were acquired using a FACSCalibur flow cytometer (BD Biosciences), and data analysis was performed applying FlowJo software (Tree Star, Ashland, OR).

Virus loading of DCs and DC-T-cell coculture.

Sucrose gradient-purified infectious viruses and viruses whose infectivity had been inactivated by treatment with 2,2′-dithiodipyridine (aldrithiol-2 [AT-2]) were kindly provided by the AIDS Vaccine Program (SAIC-Frederick, National Cancer Institute at Frederick, Frederick, MD). To allow maximal virus capture by DCs (for visualization purposes) SIV CP-MAC, characterized by an approximately 10-fold increased level envelope glycoprotein expressed on the virion surface, was chosen as a model virus (16). SIV CP-MAC (AT-2-treated, lot no. P3866; infectious, lot no. P3759) produced from SUPT1 cells (CLN 131) and HIV_BaL (infectious, lot no. P3953) was propagated in SUPT1 cells expressing CCR5 CL.30 (SUPT1/CCR5 CL.30; CLN 204) (10) (generously provided by J. Hoxie, University of Pennsylvania) and prepared as described previously (4, 16). Virus content of purified concentrated preparations was determined using a p27 or p24 Gag enzyme-linked immunosorbent assay and/or by high-performance liquid chromatography (10). The infectious titer of SIV CP-MAC was calculated based on a viral titration in the cell lines in which the virus was produced (44). Virus stocks were diluted in 1% bovine serum albumin in phosphate-buffered saline ([PBS] Intergen, New York, NY) to 3 μg of p27/ml (AT-2 SIV) or 1 × 10⁶ the 50% tissue culture infective dose (TCID₅₀)/ml (infectious virus). Single-use aliquots were stored at −80°C.

DCs were pulsed with virus using slight modifications of an established protocol (15). DCs were exposed to AT-2 SIV CP-MAC (300 ng of p27/10⁶ cells) or infectious SIV CP-MAC or HIV_BaL (2,000 TCID₅₀/10⁶ cells) versus PBS with 1% bovine serum albumin for 1 h at 37°C before being washed, and the viable cells were recounted by trypan blue exclusion. DCs were aliquoted in serum-free RPMI medium alone or with autologous CD4⁺ or CD8⁺ T cells (3.75 × 10⁵ DCs with 1.5 × 10⁶ T cells in 300 μl) in the presence or absence of 25 μg/ml T-1249 (provided by Progenics Pharmaceutical Inc., Tarrytown, NY). The cells were then centrifuged (for 30 s at 2.8 × g or 500 rpm) (Centrifuge 5417 C/R; Eppendorf, Hamburg, Germany) and incubated on ice for 30 min to allow conjugate formation before being transferred to 37°C in 5% CO₂ for up to 2 h. After incubation, cells were gently resuspended, and cell fractions were analyzed as described above. Where indicated, 25 μg/ml of T-1249 was also included during the virus pulsing of the DCs. Aliquots of virus-loaded DCs were also used immediately in the infectious assays.

Microscopic analysis for virus at the DC-T-cell synapse.

SIV protein-positive T cells or DCs were detected by fluorescent microscopy as previously described (15). Briefly, cells were adhered to pretreated glass slides, fixed, and permeabilized before being incubated with the anti-SIV p27 MAb KK64 (1:1,000; hybridoma culture supernatant; NIH AIDS Research and Reference Reagent Program) versus the isotype control (mouse immunoglobulin G1; 2 μg/ml). Labeled cells were identified using the goat anti-mouse Alexa Fluor 555 (2.5 μg/ml; Invitrogen-Molecular Probes, Eugene, OR), and nuclei were stained with DAPI (4′,6′-diamidino-2-phenylindole) as cells were mounted (ProLong gold antifade reagent with DAPI; Molecular Probes, Eugene, OR) (38). Images were captured with a Plan-Apo 60× oil lens (numerical aperture, 1.4) using Image-Pro Plus, version 5.1, software (MediaCybernetics, San Diego, CA) or in z-series with a digital camera (SPOT RT Slider; Diagnostic Instruments, Sterling Heights, MI). Out-of-focus light was digitally removed using the AutoDeblur AutoVisualize deconvolution software. When applicable, the Image-Pro Plus (version 5.1) software was used for brightness and/or contrast intensity adjustments (equally applied to samples and controls) and to count T cells, which were discriminated from DCs on the basis of morphology and size (large, irregularly shaped DCs with kidney-shaped nuclei and considerable cytoplasm were readily distinguished from the small round T cells with round nuclei and little cytoplasm). For transmission electron microscopic (TEM) analyses, virus-loaded DCs or DC-T-cell mixtures were processed as described previously (15).

Transfer and amplification of infectious virus.

DCs (1.5 × 10⁵) loaded with infectious virus in the presence (see Fig. 6 and 7, Pre) or absence of 25 μg/ml of T-1249 were cultured with T cells (4.2 × 10⁴ SUPT1/CCR5 CL.30 cells in cRPMI medium with 10% fetal calf serum [Mediatech, Inc., Herndon, VA] or 6 × 10⁵ autologous CD4⁺ T cells in cRPMI medium in 96-well round-bottomed plates). Various concentrations of T-1249 were also added to the cocultures as indicated (see Fig. 6 and 7, Post). Samples were set up in duplicate. The cocultures were typically kept for 3 to 4 (SUPT1/CCR5 CL.30 cells) or 7 (CD4⁺ T cells) days before the cells were collected, lysed, and assayed for full-length SIV or HIV-1 gag DNA by quantitative PCR (qPCR). Additional kinetic studies were performed on the DC-SUPT1/CCR5 CL.30 cocultures, and samples were collected for PCR on days 2, 4, 6, and 8 of culture. To monitor the infectivity of the DCs after coculture with CD4⁺ T cells, 3.75 × 10⁵ virus-loaded DCs were mixed with 1.5 × 10⁶ autologous CD4⁺ T cells (in 300 μl; 1.5-ml conical Eppendorf tube) and incubated for 2 h in the presence or absence of 25 μg/ml of T-1249. T cells were then removed using anti-CD3 MACS depletion technology, and the cells were washed thoroughly to remove free drug. After T-cell removal (verified by microscopy and flow cytometry), 1.5 × 10⁵ DCs were cultured with 4.2 × 10⁴ SUPT1/CCR5 CL.30 cells, and infection was measured.

FIG. 6.

DC-induced SIV-specific CD4⁺ and CD8⁺ T-cell responses are sustained in the presence of T-1249. Immature (Imm) and mature (Mat) DCs isolated from healthy SIV-infected animals were exposed to AT-2 SIV in the absence (PBS) or presence (Pre) of T-1249 (25 μg/ml) (as described in the legend of Fig. 1), washed, seeded into the ELISPOT plate, and cultured overnight with autologous CD4⁺ or CD8⁺ T cells (1 DC to 10 T cells). T-1249 (25 μg/ml) was also directly added to DC-T-cell mixtures containing DCs loaded with virus in the absence of T-1249 (Post). Results are expressed as mean differences in SIV-specific responses (± standard error of the mean) relative to the responses stimulated by the virus-loaded DCs treated with PBS (set as 1) from 14 (Mat DC+CD4 T cell), 6 (Mat DC+CD8 T cell), and 7 (Imm DC+CD4 T cell) independent experiments. Virus-loaded mature DCs cocultured with CD4⁺ T cells in the presence of T-1249 (Post) yielded a statistically significant decrease in the activation of CD4⁺ T cells (P < 0.05). No other changes in the levels of SIV-specific responses were significantly different.

FIG. 7.

T-1249 blocks DC-driven transfer and amplification of virus in DC-T cell mixtures. Human (a) or macaque (b) DCs were exposed to infectious SIV (2,000 TCID₅₀/10⁶ cells) in the absence (PBS) or presence of T-1249 (25 μg/ml; Pre), washed, and mixed with SUPT1/CCR5 CL.30 cells. T-1249 (0.04 to 25 μg/ml) was also added to cocultures containing DCs loaded with virus in the absence of T-1249 (Post), and 25 μg/ml of T-1249 was added to a replicate of the T-1249-pretreated DC mixture (Pre/Post). (c) Virus-pulsed immature or mature DCs were cocultured with SUPT1/CCR5 CL.30 cells in the presence of the indicated doses of T-1249 (μg/ml). (d) Virus-loaded DCs were incubated with autologous CD4⁺ T cells for 2 h in the presence (+) or absence (−) of 25 μg/ml T-1249. CD3⁺ T cells were removed from the 2-h-cultured DC-T-cell mixtures, and the isolated DCs (1.5 × 10⁵) or isolated T cells (4.5 × 10⁵) were washed and recultured with SUPT1/CCR5 CL.30 cells (4.2 × 10⁴). Virus-pulsed DCs were incubated for 2 h with or without 25 μg/ml T-1249 alongside the DC-T-cell mixtures and (after washing) cultured with SUPT1/CCR5 CL.30 cells as an additional control (DC). The cocultures of DCs or T cells with SUPT1/CCR5 CL.30 cells were kept for 4 (a, b, and d) or 2 to 8 (c) days before the cells were collected, lysed, and assayed for full-length SIV gag DNA by qPCR. Results are shown as SIV gag DNA copies per cell from three to four different donors (mean ± standard error of the mean) in panels a and b, the mean (± standard error of the mean) of duplicate samples from a representative experiment (of three) in panel c, and the mean (± standard error of the mean) of three to five experiments in panel d.

qPCR.

Cells were washed in RPMI and resuspended at 1 × 10⁷ cells/ml in DNA lysis buffer (10 mM Tris [pH 8.3] 2.5 mM MgCl₂, 50 mM KCl, 0.45% Tween 20, 0.45% Igepal-630; all from Sigma Chemicals, St. Louis, MO) with 1 mg/ml proteinase K (Roche Biochemicals, Indianapolis, IN). Cell lysis was carried out for 90 min at 56°C, and proteinase K was inactivated by a final 10-min cycle at 95°C. qPCR for HIV gag DNA and an estimation of cell numbers using albumin DNA copy numbers was performed using primers and molecular probes (13). For SIV, quantification was also done using primers and probes for late-stage SIV gag DNA. Primers and probes were designed on the published SIVmac239 sequence with the aid of Primer Express software (Applied Biosystems, Branchburg, NJ). In the following primers and probe, the locations of sequences on the published genome of SIVmac239 (accession number M33262) are given in parentheses: SIV Gag Forward, 5′-GGTTGCACCCCCTATGACAT-3′ (1846 to 1865); SIV Gag Reverse,5′-TGCATAGCCGCTTGATGGT-3′ (1892 to 1910); SIV probe, 5′-FAM-AATCAGATGTTAAATTGTGTGGGA-TAMRA-3′ (1867 to 1890; FAM is 6-carboxyfluorescein, and TAMRA is 6-carboxytetramethylrhodamine). HIV/SIV DNA was quantified by qPCR with an ABI 7000 PCR machine (PerkinElmer Life and Analytical Sciences, Boston, MA) using 5 μl of cell lysate for 40 cycles and the ABI master mix (TaqMan Universal PCR Master Mix; Applied Biosystems). Quantification of viral copy numbers was carried out as follows. Titrated doses of the plasmid pSIVmac1A11 or the 8e5 cell line that contains 1 copy of HIV per cell (NIH AIDS Research and Reference Reagent Program) were serially diluted into a constant genomic background of SUPT1/CCR5 CL.30 cells or PBMCs. The region amplified in pSIVmac1A11 matches that of SIVmac239 with 100% identity. To quantify cell numbers, titrated numbers of uninfected SUPT1/CCR5 CL.30 cells or PBMCs were lysed as above, and after the lysis reaction, they were serially diluted in lysis buffer.

IFN-γ ELISPOT assay.

SIV-specific T-cell responses were measured by gamma interferon (IFN-γ) secretion using an IFN-γ enzyme-linked immunospot (ELISPOT) assay (16, 30). Virus-loaded macaque DCs (±25 μg/ml T-1249) (see Fig. 6, Pre) were mixed with autologous CD4⁺ or CD8⁺ T cells (1 × 10⁴ DCs with 1 × 10⁵ T cells) in an anti-IFN-γ precoated ELISPOT plate (duplicates or triplicates per condition). T-1249 was also added directly to the cocultures as noted (see Fig. 6, Post). To control for the responsiveness of the T cells, 1 μg/ml phytohemagglutinin-P (Sigma Chemical) was added to DC-T-cell mixtures with or without the inhibitor. After an 18-h incubation at 37°C, the assay was developed (57), and IFN-γ spot-forming cells were enumerated by an AID ELISPOT reader using optimized settings.

Statistical analysis.

Results of experiments have been graphed to exhibit the arithmetic mean and standard error. When two groups were compared, the probabilities of differences were evaluated by using the nonparametric Mann-Whitney test or, in the case of paired samples, the Wilcoxon matched pairs test. When the desired comparisons arose from more than one set of experiments and more than one experimental factor varied, analysis of variance was employed. We used the conventional measure of a P value of <0.05 to judge whether experimental differences were statistically significant.

RESULTS

Rapid movement to and release of virus at DC-T-cell synapses is CD4⁺ T cell dependent.

Supporting a recent report (67), structurally intact virions were found to accumulate at or near synapses forming between SIV-loaded immature or mature DCs and CD4⁺ T cells (Fig. 1). Well-delineated contact zones formed between immature DCs and T cells, creating only a narrow intercellular space for virus to accumulate. Looser synapses were often apparent with mature DCs where long veils and/or dendrites extended to T cells. Overall, however, the movement of virus from immature and mature DCs to T cells was similar. DC-T-cell conjugates containing virus in the intercellular spaces were present already after 5 min of coculture at 37°C (www.popcouncil.org/pdfs/2008FrankJVirolFigs.pdf). Immunofluorescent microscopy confirmed that approximately twofold more conjugates had formed in mature DC-T-cell mixtures at this time point (12.2% versus 6.5% for immature DC-T-cell mixtures; on average, a total of 695 T cells per sample were counted). Although virus rapidly accumulated at or near the synapses, unequivocal fusion of virions at the T-cell surface was observed only once, and uptake of virus by T cells via receptor-mediated endocytosis, i.e., via clathrin-coated pits or via sequestration in vacuoles or endosomes, was also very rarely detected (www.popcouncil.org/pdfs/2008FrankJVirolFigs.pdf). We frequently observed that the curvature of the cell membrane followed the curvature of the virus where the contact was made, as recently described in association with putative virus en try complexes (53) (www.popcouncil.org/pdfs/2008FrankJVirolFigs.pdf). However, the analyses performed lack the resolution to visualize these putative virus entry structures (“claws”) (53). Due to the very low frequency of such events, quantitative judgments as to which of the mechanisms for virus uptake by the T cells predominate could not be made.

FIG. 1.

Accumulation of whole virions at DC-T-cell synapses. Human DCs exposed to AT-2 SIV (300 ng of p27/10⁶ cells; 60 min at 37°C) were thoroughly washed and cocultured with resting CD4⁺ T cells (1 DC to 4 T cells) for 120 min at 37°C in serum-free medium. Immediately after culture, samples were processed for TEM. Typical ultrastructural views of DC-T-cell synapses are shown, highlighting the concentration of whole structurally intact virus at the contact sites and intercellular spaces filled with virus particles that can be best appreciated in the enlarged areas in the right panels. Note compartments that open toward the T cell and release their viral contents (bottom right and left panels). Representative results of at least 10 different donors, comprising a total of more than 600 inspected DC-T-cell contacts, are shown. Images in the top row show immature DCs, and the middle and bottom rows show mature DCs. Magnifications range from ×7,000 to ×45,000. Scale bars correspond to 200 nm in the top and middle right panels and to 1 μm in all the other panels. Boxed areas in the left panels delineate parts of the synapses; they are enlarged on the images opposite at right. Some examples of intact virions are marked with asterisks. T, T cell; DC, dendritic cell.

Typically, when in contact with a CD4⁺ T cell, DCs discharged their internalized viruses and became empty, while virus-pulsed DCs cultured for 2 h with CD8⁺ T cells or in the absence of T cells mostly retained the internalized viruses. The average percentages of virus-positive DCs among at least 50 ultrastructurally evaluated DC profiles were 25% (donor 1) and 32% (donor 2) in cocultures with CD4⁺ T cells versus 38% (donor 1) and 40% (donor 2) in cocultures with CD8⁺ T cells. Significantly higher levels of virus-positive T cells were also detected by immunofluorescent microscopy of cocultures of virus-loaded DCs with CD4⁺ T cells than with CD8⁺ T cells (Fig. 2a) (P < 0.05 for immature and mature DCs). This was not due to the lack of conjugate formation between DCs and CD8⁺ T cells (Fig. 2b and c). As expected (6), there were (approximately twofold) more mature than immature DC-T-cell conjugates. An increase in immature DC-T-cell conjugates in the presence of SIV was seen with CD4⁺ and CD8⁺ T cells by flow cytometry but did not reach statistical significance (Fig. 2b). Ultrastructurally, conjugate and synapse formation between DCs and CD8⁺ T cells was comparable to conjugates with CD4⁺ T cells (Fig. 2c).

FIG. 2.

Virus release from DCs is triggered by CD4⁺ but not CD8⁺ T cells in spite of similar conjugate formation with CD8⁺ T cells. Human DCs were exposed to AT-2 SIV and cocultured with autologous CD4⁺ (a and b) or CD8⁺ (a to c) T cells for 120 min (as described in the legend of Fig. 1). Immediately after culturing, cells were washed, and fractions of cells were processed for immunofluorescent microscopy (a), flow cytometry (b), or TEM (c). (a) Mature DC-T-cell mixtures were fluorescently labeled for SIV Gag p27 protein (red), and the nuclei were stained with DAPI (blue). Representative images from 1 out of 8 (CD8) and 13 (CD4) different donors are shown to highlight how virus-positive T cells were scored (right panels). Original magnification, ×60. The mean percentages (± standard error of the mean) of Gag p27-positive small CD4⁺ or CD8⁺ T cells (among all small cells) were determined by counting an average of 550 T cells per sample. The percentages of p27-positive CD4⁺ T cells were significantly greater in cocultures with immature (P < 0.05) and mature (P < 0.05) DCs than cultures with CD8⁺ T cells. (b) Cocultures with virus-treated (SIV) or untreated (No SIV) immature and mature DCs were analyzed by fluorescence-activated cell sorting to monitor the frequency of conjugate formation. The mean percentages (± standard error of the mean) of CD3⁺HLA-DR⁺ conjugates in the DC-T-cell cocultures from six (No SIV) to eight (SIV) (CD4⁺ T cells) and two (No SIV) to four (SIV) (CD8⁺ T cells) different donors are shown. (c) TEM of a mature DC-CD8⁺ T-cell conjugate. Magnification, ×8,000. Scale bar, 2 μm. Imm, immature; Mat, mature.

The fusion inhibitor T-1249 blocks virus transfer to CD4⁺ T cells.

While TEM verified the release of considerable amounts of virus at DC-CD4⁺ T-cell contact points and fluorescent microscopy documented the numerous virus protein-positive CD4⁺ T cells in virus-loaded DC-T-cell mixtures, entry of virus into T cells (via fusion or internalization) was rarely seen. Presumably, this was because the fusion phase of virus entry, once initiated, was too rapid to be readily observed (20, 43), and the fused virions were no longer visible by TEM while the viral proteins of the fused particles were readily detected by immunofluorescent microscopy. In an attempt to limit this rapid fusion, we added the fusion inhibitor T-1249 to the DC-T-cell cocultures so that the virus released by the DCs would not be able to fuse with the CD4⁺ T cells and might therefore accumulate and be more readily observed at T-cell surfaces. T-1249 is a 39-amino-acid gp41-based peptide inhibitor of fusion that is composed of sequences derived from HIV-1, HIV-2, and SIV and blocks fusion mediated by the envelope glycoproteins of these viruses (27). We verified the cross-reactivity of T-1249 with SIV by demonstrating that 25 μg/ml of T-1249 completely prevented the induction of syncytia in CEMx174 cells by the highly fusogenic AT-2 SIV E11S_mne (fusion from without [38]) (data not shown). Addition of T-1249 to the virus-loaded immature or mature DC-T-cell mixtures significantly reduced the numbers of virus-positive CD4⁺ T cells approximately 3.5- to 5-fold (from 34% to 7% [P < 0.05 for immature DC-T-cell mixtures] and from 29% to 8% [P < 0.05 for mature DC-T-cell mixtures]) (Fig. 3a). The reduction of virus-positive T cells was not due to T-1249 impairing DC-T-cell conjugate formation, since the frequency of DC-T-cell conjugates was unchanged in the presence of T-1249 (Fig. 3b).

FIG. 3.

T-1249 blocks virus transfer to CD4⁺ T cells. Virus-loaded human DCs were cocultured with autologous CD4⁺ cells in the absence (PBS) or presence of T-1249 (25 μg/ml) (as described in the legend of Fig. 1). After incubation a fraction of cells was used for immunofluorescent microscopy (a), and the remaining cells were analyzed by fluorescence-activated cell sorting (b). (a) The mean percentages (± standard error of the mean) of Gag p27-positive T cells from 7 (immature DCs) and 12 (mature DCs) donors are shown (an average of 450 T cells were counted per sample). T-1249 significantly reduced the percentage of p27-positive CD4⁺ T cells in cocultures with immature (P < 0.05) and mature (P < 0.05) DCs. (b) Conjugate formation in cocultures of untreated (No SIV) or virus-pulsed (SIV) DCs with CD4⁺ T cells (with or without T-1249) was monitored. The mean percentages (± standard error of the mean) of CD3⁺HLA-DR⁺ conjugates from six (immature DCs) and eight (mature DCs) different donors are shown. The frequency of conjugates with untreated (No SIV) mature DCs was significantly increased compared to untreated (No SIV) immature DCs (P < 0.05). Imm, immature; Mat, mature.

Virus accumulation in DCs of T-1249-treated DC-T-cell cultures.

Knowing that T-1249 prevented virus transfer to CD4⁺ T cells, we wanted to determine whether T-1249 impacted the fate of viruses within the DCs, as well as the movement of viruses toward the DC-T-cell synapses. TEM analysis revealed that, although large virion-containing organelles were present in the cocultures not treated with T-1249, there were significantly more DCs carrying virus in large organelles in cocultures exposed to T-1249 (1.3- to 7-fold) (Fig. 4). This was the case for free nonconjugated DCs (48% ± 6% versus 21% ± 3%; P < 0.05; 570 and 620 cell profiles analyzed in the presence versus absence of T-1249, respectively) (Fig. 4a and b) and DCs bound to T cells (36% ± 7% versus 8% ± 3%; P < 0.05) (Fig. 4c). Sequestration of virus within large intracellular compartments in mature DCs made it much easier to visualize and enumerate by TEM (15). Therefore, extensive analysis to demonstrate statistically significant differences was conducted only with mature DCs although qualitatively similar observations were also made for immature DCs (Fig. 4b).

FIG. 4.

Blocking virus spread to CD4⁺ T cells by T-1249 results in accumulation of virus in a higher proportion of the DCs. Virus-loaded immature (b) or mature (a to c) DCs were cocultured with CD4⁺ T cells in the presence or absence of T-1249 (as described in the legend of Fig. 1) before being processed for TEM. (a) Typical examples for singly dispersed mature DCs from cocultures treated with PBS (left) versus T-1249 (right) are shown. Note the presence of virus particles (some marked with asterisks) in the DCs from the T-1249-treated DC-T-cell cultures. Such large virus-containing organelles also occurred in the absence of T-1249, albeit at a significantly lower frequency. The boxed area is further magnified in the inset. Magnifications: ×6,900 (left), ×7,400 (right), and ×58,000 (inset). Scale bars represent 1 μm in the micrographs at magnifications of ×6,900 and ×7,400 and 100 nm in the inset. (b) The mean percentages (± standard error of the mean) of virus-carrying single DC profiles (not conjugated to T cells) in the differently treated (PBS versus T-1249) DC-T-cell cultures were determined by counting an average of 80 DCs per TEM sample from two to seven different donors. Each symbol indicates a different donor, with the mean indicated by the black bars. The frequency of virus-positive mature DC profiles (left) is significantly increased in the presence of T-1249 compared to control cells (P < 0.05). A similar tendency was observed for immature DCs (right panel). (c) The percentages of virus-carrying mature DCs conjugated to T cells (virus-positive versus negative [Pos DCs versus Neg DCs]) and the percentages of DC-T-cell contact points that had virus accumulated at the synapse were enumerated. Per experiment an average of 10 conjugates were counted. Results are expressed as mean (± standard error of the mean) from four different donors. T-1249 significantly increased (P < 0.05) the frequency of virus-positive DC-T-cell conjugates while its absence significantly increased (P < 0.05) the frequency of virus-negative DC-T-cell conjugates. Imm, immature; Mat, mature.

The larger numbers of virus-carrying DCs in T-1249-treated DC-T-cell mixtures suggested that T-1249 interfered with virus release at the synapses and/or that viruses released at the synapses were taken back up by the DCs since fusion-dependent entry into the T cells was prevented. TEM examination revealed that T-1249 did not prevent the release of virus at the DC-T-cell contact points (Fig. 4c, Synapse) although we cannot rule out subtle differences that may not be appreciated via this method. The sustained release of viruses in the presence of T-1249 suggests that the larger numbers of virus-carrying DCs likely reflects the reuptake of released virions by the DCs in the presence of T-1249. The capture and internalization of virus by DCs in the presence of T-1249 was confirmed by microscopy (Fig. 5). In addition, the intracellular distribution of virus captured by either immature or mature DCs was unaffected by T-1249 (Fig. 5a and c).

FIG. 5.

T-1249 does not interfere with virus capture by DCs. Immature (Imm; a) or mature (Mat; a to c) DCs were pulsed with AT-2 SIV in the absence (PBS) or presence of T-1249 (as described in the legend of Fig. 1) and immunofluorescently stained for the SIV Gag p27 protein (as described in the legend of Fig. 2) (a). Remaining cells were processed for TEM (b and c). (a) Virus-positive immature and mature DCs are shown in red, and nuclei are in blue. The distinct localization of virus close to the periphery of immature DCs and deeper within larger vacuoles of mature DCs is apparent. Images were collected in z-series and deconvoluted. Original magnification, ×60. Representative results from one out of five different donors are shown. (b) The percentages of virus-carrying DCs were determined by counting an average of 60 profiles per TEM sample. Mean percentages (± standard error of the mean) from three different donors are shown. (c) A representative TEM image of a mature DC that captured virus in the presence of T-1249 is shown. T-1249 does not impair the uptake of large numbers of virions into spacious intracellular compartments (delimited with > < symbols). High-magnification inset shows a view from another cell. Some virions are indicated by asterisks. Magnification, ×14,000 (large image) and ×26,000 (inset). Scale bar, 2 μm (large image) and 200 nm (inset).

DC-induced SIV-specific CD4⁺ and CD8⁺ T-cell responses are sustained in the presence of T-1249.

Since virus-bearing DCs are able to process and present viral (HIV/SIV) antigens and stimulate virus-specific CD4⁺ and CD8⁺ T-cell responses (16, 49), we wanted to determine whether T-1249 had any impact on these responses. To monitor this, we utilized our macaque DC-T-cell system with which we showed previously that AT-2 SIV-loaded mature DCs stimulate both CD4⁺ and CD8⁺ T-cell responses while immature DCs preferentially trigger CD4⁺ T-cell responses (16). There is no obvious difference between AT-2 and infectious SIV or HIV in terms of antigen uptake, processing, and presentation (8, 15, 28, 49) or in the transfer from DCs to T cells (63). However, using AT-2 virus in this assay allowed us to measure DC-mediated T-cell responses in the absence of viral infection.

We measured the ability of immature or mature DCs loaded with AT-2 SIV to stimulate SIV-specific IFN-γ release in the presence or absence of T-1249. Mimicking the design of DC-to-T-cell transfer experiments (above), T-1249 was added to the DC-T-cell mixtures (Fig. 6, Post). We also compared the ability of DCs loaded with AT-2 SIV in the presence of T-1249 prior to being mixed with the T cells (Fig. 6, Pre) in order to simulate the presentation of antigens from viruses recaptured by DCs in the presence of the inhibitor. SIV-specific CD4⁺ and CD8⁺ T-cell responses were maintained in the presence of T-1249, independent of the timing or duration of treatment (Fig. 6). While slight variations in the magnitudes of CD4⁺ and CD8⁺ T-cell responses were observed, only the CD4⁺ T-cell responses induced by virus-loaded mature DCs when T-1249 was added during the time of coculture were significantly reduced from untreated controls (P < 0.05). The slight increases seen with the immature DCs when T-1249 was present during the culture period or the mature DCs when exposed to T-1249 during virus treatment were not statistically significant.

T-1249 blocks DC-driven transfer and amplification of virus in DC-T-cell mixtures.

The persistence of the smallest amounts of infectious virus in a milieu that promotes the activation of SIV-reactive CD4⁺ T cells (Fig. 6) might actually exacerbate virus spread. We therefore investigated the efficiency of T-1249 to block viral infection. DCs were loaded with infectious SIV, washed, and mixed with SUPT1/CCR5 CL.30 cells (to serve as a maximally sensitive permissive recipient for even small amounts of infectious virus) (64), and various doses of T-1249 were added (Fig. 7, Post). T-1249 inhibited SIV replication driven by human (Fig. 7a) and macaque DCs (Fig. 7b), as well as HIV_BaL replication driven by human DCs (data not shown) in a dose-dependent manner. Similar results were obtained for SIV-loaded DCs cocultured with autologous resting CD4⁺ T cells even though the virus amplification was markedly lower (data not shown). Since DCs capture virus when T-1249 is present during the loading of the DCs (Fig. 5), it was not surprising that these cells were still able to transfer and amplify virus in the cocultures (Fig. 7a and b, Pre) although the level of infection was always lower than that driven by untreated DCs. While different levels of infection inhibition may reflect the potency of T-1249 to inhibit SIV versus HIV infection (reductions of 1.1- to 2.9-fold for SIV and 8.3- to 29-fold for HIV), SIV captured in the presence of T-1249 was not resistant to inhibition by T-1249 per se because addition of the inhibitor to the pretreated DC cocultures readily inhibited infection (Fig. 7a and b, Pre/Post).

The levels of infection amplified by virus-bearing mature DCs were approximately 2.2-fold higher than those of infections driven by immature DCs (P < 0.05), and the lower doses of T-1249 were less efficient in blocking mature DC-driven infection. To examine this more closely, we measured infection levels over time in the presence of limiting T-1249 doses. As expected, at each time point mature DCs consistently promoted higher levels of infection (increased over time), and the dose-dependent inhibition by T-1249 was reduced accordingly, although 5 μg/ml of T-1249 still completely blocked infection (Fig. 7c).

Finally, we wanted to determine if the DCs within the DC-T-cell cocultures still retained infectious virus and whether the larger numbers of virus-carrying DCs in the T-1249-treated cocultures (Fig. 4) were more infectious. To do this, virus-loaded DCs were incubated with autologous CD4⁺ T cells for 2 h in the presence or absence of T-1249 before the T cells were depleted from DC-T-cell cocultures. The isolated DCs (versus isolated T cells; washed free of drug) were then recultured with SUPT1/CCR5 CL.30 cells to assess their infectivity. DCs isolated from T-1249-treated cocultures exhibited 3.1-fold less infectivity than DCs isolated from the untreated mixtures (Fig. 7d). Similar observations were made when the HLA-DR⁺ cells were enriched from the cocultures (data not shown). As expected, only the isolated T cells from the untreated cocultures were infectious (0.56 copies/cell versus 0.02 copies/cell for T cells from the untreated versus treated cocultures), but these were 3.8-fold less infectious than the isolated DCs (even though three times more T cells were added to the SUPT1/CCR5 CL.30 cocultures). Freshly pulsed DCs that were cultured for 2 h alongside were more infectious than the DCs isolated from the DC-T-cell mixtures (2.3-fold for untreated cells and 3.6-fold for T-1249-treated cells). Inclusion of T-1249 during the 2-h culture of the DCs (washed out before coculture with SUPT/CCR5 CL.30 cells) reduced the infectivity of the cells by twofold, indicating that some T-1249 is passively absorbed by the cells. Addition of T-1249 directly to the DC-SUPT1/CCR5 CL.30 mixtures was able to prevent virus amplification driven by all virus-bearing cells (data not shown). Therefore, even though there were significantly more virus-carrying DCs in T-1249-treated DC-T-cell mixtures, these DCs do not represent a more infectious source of virus.

DISCUSSION

DCs are central to the activation of potent innate and effector immunity (54) and are in prime locations to interact with HIV crossing the body surfaces. However, HIV takes advantage of the normal DC-T-cell interplay to establish and disseminate HIV infection (41, 45, 60). While immature DCs support low-level R5 HIV replication, newly produced viruses as well as those internalized by immature or mature DCs are efficiently transmitted to CD4⁺ T cells, providing a perfect niche for prolific virus expansion. A better understanding of this biology should enable identification of ways to restrict DC-driven spread of HIV while maintaining or even boosting DC-mediated activation of antiviral immunity. Herein, we used microscopy to analyze the movement of DC-captured viruses to T cells and explored how to limit this process and the amplification of infection while not interfering with the activation of virus-specific T-cell responses.

DCs promote virus growth in autologous DC-T-cell mixtures, even in the absence of antigenic stimulation (3, 36, 46, 68), and virus movement from DCs to CD4⁺ T cells in the absence or presence of specific antigen occurs at a virologic or infectious synapse (3, 17, 36, 63). CD4, CCR5, and CXCR4 are recruited to these synapses to facilitate virus spread to the T cells (3, 17, 36). Virus movement has been largely visualized by confocal microscopy (with fluorescently labeled viruses or by staining for viral proteins), and ultrastructural aspects of virus transfer have been less well studied. Our studies using TEM with immunofluorescent microscopy support the recent observations from Wang et al. definitively showing the release of intact virions across the DC-T-cell synapse by electron microscopy (67). We were also able to demonstrate that virus mostly remained within the DCs when the DCs encountered CD8⁺ T cells (or when cultured in the absence of T cells). Occasionally, we found a few virions in the synaptic cleft of mature DC-CD8⁺ T cells, or we observed that some parts of a mature DC opened toward the extracellular space when DCs were cultured in the absence of T cells. In both instances even minor contamination of CD4⁺ T cells (from the DC preparation) could account for these observations. Although both CD4⁺ and CD8⁺ T cells conjugate with DCs, primarily CD4⁺ T cells provided the signal(s) needed to trigger virus release from the DCs. This is consistent with the observation that in antigen-independent DC-T-cell synapses (with CD4⁺ or CD8⁺ T cells in absence of virus), DCs more readily triggered responses in CD4⁺ than in CD8⁺ T cells (48). Viral movement and discharge in response to CD4⁺ T-cell contact suggest that resting T cells can also deliver signals to DCs even in the absence of antigen (46).

Despite the distinct patterns of sequestration of entrapped virus within immature versus mature DCs (15, 23, 63) and subtle differences in the formation of DC-T-cell conjugates, comparable levels of virus transfer to CD4⁺ T cells were ultimately seen for both DC types. However, when we used infectious virus, it was apparent that virus transferred by mature DCs resulted in greater virus amplification in the target cells. This is probably due to a greater efficiency in DC-T-cell communication and conjugate formation (signaling the T cell) that is characteristic for mature DCs (47) and would augment infection in the recipient T cells (18, 23, 36, 50, 68, 70). Differences in the cellular trafficking of HIV in immature and mature DCs, discharge of a larger number of virions at synapses by mature DCs, and increased life span of internalized virus in mature DCs (23, 63) might also contribute to differences in the potential to transmit internalized virus. The extent to which the increased efficiency of virus transmission by mature DCs is due to enhanced capture of virus (23) remains to be clarified, as preceding works have shown that immature DCs take up virus to a similar or greater extent than mature DCs (15, 50).

The distinct nonconventional endocytic compartments in which immature and mature DCs sequester virus (15, 23, 63) probably represent multivesicular bodies, based on staining for several tetraspanins (CD81, CD82, CD9, CD53, and CD63) (17, 23, 69). SIV and HIV also colocalize with CD81 and the CLR DEC-205 (CD205) in mature DCs and, to a lesser extent, with CD81 in immature DCs (data not shown). In contrast to a recent report that endocytosed virions are released into the extracellular milieu associated with endocytic vesicles (69), we rarely observed release of virions from immature or mature DCs in the absence of CD4⁺ T-cell contact, suggesting that an alternative mechanism was involved in viral discharge. The movement and emptying of large virus-containing compartments at the cell membrane (the present study) mimics the intracellular translocation of multivesicular bodies. This suggests that the virus uses the exosome release pathway in immature and mature DCs upon CD4⁺ T-cell contact, facilitating the first phase of virus transmission (51, 55, 58, 59).

Virus transfer at the infectious synapse between DCs and T cells involves CD4 and chemokine receptors on the T cells (3, 17, 36). Once virus was released at the synapse from immature or mature DCs and engaged receptors on a cocultured CD4⁺ T cell, fusion with T-cell membrane was presumably rapid (20, 43) since we were not able to visualize virions fusing with the T-cell surface. T-1249 readily prevented the movement of virus from the DC to the T cells and limited DC-driven SIV/HIV replication. The lack of viruses visualized at the T-cell surface in the presence of T-1249 is similar to recent observations where a fusion inhibitor reduced the presence of viruses seen in close proximity of the T-cell membrane (53). Coincident with limiting DC-to-T-cell spread, T-1249 favored the accumulation of virus particles in the DCs. Despite the presence of larger numbers of virus-carrying DCs, the DCs from T-1249-treated cocultures were less infectious than those isolated from untreated cocultures. DCs from the cocultures were less infectious than freshly virus-pulsed DCs cultured alongside (independent of the presence of T-1249). In the case of the DC-T-cell cocultures not treated with T-1249, the reduced infectivity mediated by the isolated DCs compared to the freshly pulsed DCs most likely reflected the prior transfer of significant amounts of virus to the T cells. The lower infectivity mediated by DCs isolated from the T-1249-treated cocultures suggests that the drug present in the cocultures was able to interfere with the infectivity of these accumulated viruses. T-1249 prevents cell-virus fusion by binding to the heptad repeats within the fusion domain of gp41 after the initial conformational changes have occurred upon envelope binding to CD4/CCR5 (27). A possible scenario could be that virus released from DCs in the presence of T-1249 binds to CD4/CCR5 on the T cells, T-1249 immediately interacts with gp41 to prevent fusion, and the T-1249-bearing virus particles released from the T cells (since we saw no T-cell-associated virus or proteins) were taken back up by the DCs. These viruses presumably remain noninfectious due to the presence of bound T-1249, thereby not resulting in enhanced infectivity of the larger numbers of virus-bearing DCs. It is also possible that some drug was taken up with the reinternalized virus, thereby further limiting subsequent virus spread to the permissive T cells (as seen when the drug is present during the virus pulsing of the DCs and upon reculture of virus-loaded DCs in the presence of T-1249).

While preventing virus spread to limit the destruction of the immune system is critical, maintaining or boosting immune function is equally important in controlling HIV infection. DC-captured AT-2-inactivated or infectious HIV/SIV is rapidly processed and presented for T-cell activation (16, 24, 28, 31, 32, 37, 40). Mature DCs stimulate CD4⁺ and CD8⁺ T cells while immature DCs primarily stimulate CD4⁺ T cells (16, 28, 49). Productive infection is not required, but stimulation of major histocompatibility complex class I (MHC-I)-restricted responses by CD8⁺ T cells has been described as being largely dependent on functional envelope proteins on the virions (40). The fusion inhibitor T-1249 did not significantly affect the ability of the virus-bearing DCs to stimulate virus-specific CD4⁺ or CD8⁺ T-cell responses. Thus, internalization of virus via endocytosis appeared to be the prime route for MHC-II-restricted presentation of whole virus-derived antigens by immature and mature DCs (24, 40, 49). DCs are also especially effective at cross-presenting exogenous viral antigens via MHC-I (28, 35, 71), and this is partially (∼40 to 50%) dependent on the endocytic internalization of infectious or AT-2 HIV, suggesting that fusion may occur in the endosomal compartments of DCs through which viral proteins gain access to the cytosol (39, 49). The availability of these routes of antigen processing/presentation likely explain how T-1249 did not impede the activation of SIV-specific T cells.

Altogether, these data demonstrate that a fusion inhibitor can prevent first-phase transfer of virus from DCs to T cells while preserving the ability of the DCs to present viral antigens to virus-specific T cells. However, because DCs are so efficient in transmitting virus to resting and activated CD4⁺ T cells, virus amplification may still be fostered, especially if the inhibitor levels become limiting in the more permissive mature DC-T-cell milieu. Therefore, particularly stringent and broad-acting approaches are needed to inhibit DC-driven infection during the earliest moments after HIV enters the body as well as in the deeper tissues where virus is further amplified and disseminated. Continued research is required to identify combined strategies that will target multiple modes of virus-cell interactions to efficiently prevent virus amplification by DCs while preserving or preferably augmenting the activation and expansion of virus-specific T-cell immunity.