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0022-538X/07/$08.00⫹0 doi:10.1128/JVI.02572-06

Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Maturation of Blood-Derived Dendritic Cells Enhances Human

Immunodeficiency Virus Type 1 Capture and Transmission

Nuria Izquierdo-Useros,

1

Julia

` Blanco,

1

Itziar Erkizia,

1

Maria Teresa Ferna

´ndez-Figueras,

2

Francesc E. Borra

`s,

3

Mar Naranjo-Go

´mez,

3

Margarita Bofill,

1,4

Lidia Ruiz,

1

Bonaventura Clotet,

1

and Javier Martinez-Picado

1,4

*

IrsiCaixa Foundation,1Department of Pathology,2and Laboratory of Immunobiology for Research and Application to Diagnosis,3 Blood and Tissue Bank, Institut d’Investigacio´ en Cie`ncies de la Salut Germans Trias i Pujol, Universitat Auto`noma de

Barcelona, Badalona, Spain, and Institucio´ Catalana de Recerca i Estudis Avanc¸ats, Barcelona, Spain4

Received 21 November 2006/Accepted 20 April 2007

Dendritic cells (DCs) are specialized antigen-presenting cells. However, DCs exposed to human immu-nodeficiency virus type 1 (HIV-1) are also able to transmit a vigorous cytopathic infection to CD4T cells, a process that has been frequently related to the ability of DC-SIGN to bind HIV-1 envelope glycoproteins. The maturation of DCs can increase the efficiency of HIV-1 transmission through trans infection. We aimed to comparatively study the effect of maturation in monocyte-derived DCs (MDDCs) and blood-derived myeloid DCs during the HIV-1 capture process. In vitro capture and transmission of envelope-pseudotyped HIV-1 and its homologous replication-competent virus to susceptible target cells were assessed by p24gag

detection, luciferase activity, and both confocal and electron microscopy. Maturation of MDDCs or myeloid DCs enhanced the active capture of HIV-1 in a DC-SIGN- and viral envelope glycoprotein-independent manner, increasing the life span of trapped virus. Moreover, higher viral transmission of mature DCs to CD4T cells was highly dependent on active viral capture, a process mediated through cholesterol-enriched domains. Mature DCs concentrated captured virus in a single large vesicle staining for CD81 and CD63 tetraspanins, while immature DCs lacked these structures, suggesting different intracellular trafficking processes. These observations help to explain the greater ability of mature DCs to transfer HIV-1 to T lymphocytes, a process that can potentially contribute to the viral dissemination at lymph nodes in vivo, where viral replication takes place and there is a continuous interaction between susceptible T cells and mature DCs.

Dendritic cells (DCs) are specialized antigen-presenting cells derived from CD34⫹progenitors in bone marrow. Imma-ture DCs are scattered throughout the peripheral tissues, ready to recognize a wide range of pathogens. When infection takes place, immature DCs capture the pathogen that ends up in the intracellular lytic pathway, allowing the cells to process it into antigens. This way, immature DCs are activated and migrate from the periphery to lymph nodes, where cell activation cul-minates with the maturation of DCs that are then able to present processed antigens to T cells, promoting specific im-mune responses (6).

It has been known for years that DCs exposed to human immunodeficiency virus type 1 (HIV-1) transmit a vigorous cytopathic infection to CD4⫹ T cells (4). However, HIV-1 replication in DCs is generally less productive than that in CD4⫹T cells or macrophages, and the frequency of HIV-1-infected DCs found in vivo is also lower (10, 22). Although limited, the small proportion of HIV-infected DCs could spread newly synthesized virus to T cells in a highly efficient way (14). On the other hand, maturation of DCs is known to

reduce the ability of these cells to support HIV-1 replication (10). Interestingly, DCs do not need to be productively in-fected to transmit the virus and spread it in an infectious form, contrasting in this way with other HIV-1 target cells, like CD4⫹ T lymphocytes or macrophages (9). This particular viral trans-mission mechanism is known astransinfection and involves the binding and capture of HIV-1, the traffic of internalized virus, and its final release, allowing the transfer to CD4⫹T cells (9). Thetransinfection process has been related to the ability of DC-SIGN (DC-specific ICAM-3-grabbing nonintegrin; for-mally CD209), a C-type lectin expressed on the surface of monocyte-derived DCs (MDDCs), to tightly bind to the HIV-1 surface envelope (Env) glycoprotein 120 (gp120) (5). However, recent studies also suggest that HIV-1 binding, uptake, and transfer from DCs to CD4⫹T lymphocytes may involve alter-native DC-SIGN pathways such as mannose binding C-type lectin receptors (24) or other molecules associated with lipid rafts (12). Moreover, the functional relevance of the high-affinity interaction between HIV-1 and DC-SIGN has been hampered in several studies by the use of monomeric gp120 instead of HIV-1 virions. Therefore, different factors have con-tributed to make the functional role of DC-SIGN in HIV-1 pathogenesis controversial.

It is well documented that the efficiency of HIV-1 transmis-sion throughtransinfection can be increased upon DC matu-ration (10, 15, 21). This observation has led to the suggestion that mature MDDC (mMDDC)-enhanced transmission corre-lates with viral distribution during infectious synapse or with an

* Corresponding author. Mailing address: IrsiCaixa Foundation, Hospital Germans Trias i Pujol, Ctra. de Canyet s/n, 08916 Badalona, Spain. Phone: 34 93 4656374. Fax: 34 93 4653968. E-mail: jmpicado @irsicaixa.es.

† Supplemental material for this article may be found at http://jvi .asm.org/.

Published ahead of print on 2 May 2007.

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increase in cell surface expression of the intercellular adhesion molecule ICAM-1, the ligand of T-cell-expressed leukocyte function antigen LFA1, which in turn facilitates viral transfer from mMDDCs to T cells (15, 21). However, the mechanisms that underlie this increase in viral transmission have not been well defined yet, despite the possible implications in augment-ing viral dissemination at lymphoid tissue in vivo.

We aimed to comparatively study the effect of maturation in MDDCs and blood-derived myeloid DCs during the HIV-1 capture process. We show how the maturation of MDDCs or myeloid DCs enhances active capture of HIV-1 in a DC-SIGN-independent manner, increasing the life span of trapped virus. We also found that viral envelope glycoprotein is not required to efficiently internalize virus into a CD81- and CD63-positive compartment in mature myeloid DCs. These observations con-tribute to explain the greater ability of mature DCs to transfer HIV-1 to T lymphocytes, a process that can potentially con-tribute to the viral dissemination at lymph nodes in vivo, where viral replication takes place and there is a continuous interac-tion between susceptible T cells and mature DCs.

MATERIALS AND METHODS

Primary cell cultures and cell lines.PBMCs were obtained from HIV-1-seronegative donors by Ficoll-Hypaque density gradient centrifugation of heparin-treated venous blood. Highly purified monocyte populations (⬎97% CD14⫹) were isolated with CD14⫹selection magnetic beads (Miltenyi Bio-tec). The addition of 1,000 U/ml of granulocyte-macrophage colony-stimu-lating factor (GM-CSF) and interleukin-4 (R&D) to 8⫻105

cells/ml cultured in RPMI containing 10% fetal bovine serum (FBS) (Invitrogen) differenti-ated monocytes into immature MDDCs (iMDDCs) after 7 days. Culture medium containing interleukin-4 and GM-CSF was replaced every 2 to 3 days. mMDDCs were obtained by culturing iMDDCs at day 5 for two more days in the presence of 100 ng/ml of lipopolysaccharides (LPS) (Sigma). After the depletion of CD3⫹cells with a RosseteSep CD3 Depletion kit (Stem-Cell), myeloid DCs were isolated from the PBMCs fraction of HIV-1-sero-negative donors by employing the CD1c⫹(BDCA-1) isolation kit (Miltenyi Biotec). Enriched populations of myeloid DCs were cultured at a final con-centration of 8 ⫻105

cells/ml for 2 days in the presence of 20 U/ml of GM-CSF. After isolation, we obtained an average of 92% CD11c⫹cells with traces of CD3 (⬍1%), where the main contaminants were monocytes and B lymphocytes. However, plastic adherence after 2 days of culture in the pres-ence of GM-CSF reduced the prespres-ence of monocytes in suspension to 3% of CD14⫹cells. Furthermore, when MDDCs and myeloid blood DC were ob-tained from the same donor, CD14⫹cells were removed before BDCA-1 purification, resulting in reduced levels of contaminating CD14⫹cells. We obtained mature myeloid DCs by adding 100 ng/ml of LPS during those 2 days of culture. Myeloid DCs were immunophenotyped at day 2, and MDDCs were stained at day 7 using the following monoclonal antibodies (mAbs): CD3-peridinin chlorophyll protein (PerCP), CD4-PerCP, and HLA-DR–PerCP; CD19-phycoerythrin (PE) (BD); CD11c-fluorescein isothiocyanate (FITC) (Serotec); CD14-FITC, CD83-PE, and CD86-FITC (Pharmingen); and DC-SIGN-PE (R&D). Adequate differentiation from monocytes to iMDDCs was based on the loss of CD14 and the acquisition of DC-SIGN, while maturation upregulated the expression of CD83, CD86, and HLA-DR in MDDCs and myeloid DCs. The review board on biomedical research of the Hospital Germans Trias i Pujol approved this study.

The Raji DC-SIGN B-cell line (kindly provided by Y. van Kooyk) was grown in RPMI with 10% FBS plus 1 mg/ml of geneticin (Invitrogen). The human T-cell line Hut CCR5⫹(kindly provided by V. N. KewalRamani) was grown in RPMI with 10% FBS plus geneticin (300␮g/ml) and puromycin (1␮g/ml) (all from Invitrogen). The Ghost CCR5 green fluorescent protein (GFP) indicator cell line (obtained through the NIH AIDS Research and Reference Reagent Program from V. N. KewalRamani and D. R. Littman) was maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, geneticin (500␮g/ml), hygromycin (100␮g/ml; Invitrogen), and puromycin (1␮g/ml). Hut 78 CCR5-negative and Raji cell lines were grown in RPMI with 10% FBS. HEK-293T cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with

10% FBS. All media contained 100 U/ml of penicillin and 10␮g/ml of strepto-mycin (Invitrogen).

Viral stocks and plasmids.Infectious single-round pseudotyped HIV-1 stocks (HIVJRFL/NL43-Luc) were generated by cotransfecting the envelope-deficient pro-viral vector pNL4-3.Luc.R⫺E⫺containing the firefly luciferase reporter gene (obtained through the NIH AIDS Research and Reference Reagent Program from N. Landau) with plasmid pJRFL expressing the envelope glycoprotein of the CCR5-tropic strain HIV-1JRFL(kindly provided by V. N. KewalRamani). HIV⌬env-NL43, lacking the envelope glycoprotein, was generated by transfecting vector pNL4-3.Luc.R⫺E⫺ alone. Replication-competent full-length HIV-1 stocks (HIVNFN-SX) were generated by transfecting the proviral construct NFN-SX (19), an HIV-1NL43provirus that expresses the HIV-1JRFLenvelope glycoprotein (kindly provided by W. O’Brien). HIVNL43/eGFPvirus was obtained by cotransfecting vector pNL43 with plasmid vpr-eGFP as previously described (29). Briefly, 293T cells were transfected with calcium phosphate (CalPhos; BD), adding up to 20␮g of plasmid DNA. Supernatants containing virus were col-lected 2 days later, filtered (Millex HV, 0.45␮m; Millipore), and frozen at⫺80°C until use. Titers of HIVJRFL/NL43-Luc, used fortransinfection assays, were de-termined by using the Ghost CCR5 indicator cell line containing an HIV-2 long terminal repeat linked to a GFP gene (18), resulting in a viral stock of 6.1⫻105 50% tissue culture infective doses/ml. Viral stocks contained 1 to 2␮g of p24gag per ml as measured by enzyme-linked immunosorbent assay (ELISA) (Perkin-Elmer).

Virus binding and capture assays.A total of 3⫻105myeloid DCs, MDDCs, Raji DC-SIGN cells, and Raji cells were incubated at 37°C for 2 h with 80 ng of HIVNFN-SXp24gagper 3⫻105cells at a final concentration of 1⫻106cells/ml, washed with phosphate-buffered saline (PBS), and lysed with 0.5% Triton X-100 (at a constant concentration of 5⫻105cells per ml). Lysates were cleared of cell debris by centrifugation (10,000⫻gfor 5 min) to measure p24gag

antigen content by ELISA. Viral capture and subsequent transfer to target cells were compared by pulsing MDDCs, Raji DC-SIGN cells, and Raji cells at 37°C or 4°C for 2 h with 300 ng of HIVJRFL/NL43-Lucp24gagper 3⫻105cells at a final concentration of 1⫻106cells/ml. Of note, after 48 h of viral pulsing, neither iMDDCs nor mMDDCs displayed phenotypic changes, as determined by CD83, CD86, and HLA-DR expression levels, excluding the presence of a potential maturation stimulus in the viral stocks, like carryover plasmid DNA.

To potentially inhibit virus capture, cells were preincubated for 30 min at 4°C with mannan (500␮g/ml; Sigma) or the anti-DC-SIGN mAb MR-1 (kindly provided by A. Corbı´) at saturating concentrations determined using Raji DC-SIGN cells and then processed as described above. The envelope requirement for viral capture was assessed by pulsing cells with equal amounts of HIVNFN-SX and HIV⌬env-NL43as described above.

Kinetic analysis was assessed by pulsing 2⫻106to 4106MDDCs with 20 ng of p24gag

per 5⫻105

cells at a final concentration of 2⫻106

cells/ml. To asses the time course of viral capture, part of the cells were maintained in the presence of the virus at 37°C up to 10 h before they were lysed right after an extensive wash. The rest of the cells were used to monitor viral fate after capture: 2 h after the pulse, cells were washed and kept in culture at 37°C for up to 48 h before lysing them and re-collecting the cell supernatants.

HIVNL43/eGFPwas used to monitor viral capture with flow cytometry, puls-ing 0.5⫻106

MDDCs with 150 ng of p24gag

up to 12 h before fixing them. Analysis was done in the living cell gate determined by forward- and side-scatter light.

To analyze viral degradation in iMDDCs and mMDDCs, cells were prein-cubated with 250 nM of bafilomycin A1 and 10␮M ofclasto-lactacystin ␤-lactone (both from Sigma) during 30 min at 37°C and then exposed to HIVNFN-SXas previously described. After extensive washing, part of the cells were lysed and analyzed for p24gag. The rest of the cells were left 4 h more at 37°C in the presence of these blocking agents and in the absence of virus before lysing them. Degradation was measured by subtracting cell-associated p24gagvalues obtained 2 h immediately after the viral pulse to cell-associated p24gagvalues obtained 4 h later. The percentage of degradation was calcu-lated relative to the viral decay of p24gagvalues of untreated cells during 4 h, normalized to 100%.

Viral transmission assay.To characterize viral transmission efficiencies of different DCs, immature and mature cells from the same seronegative donors were pulsed with HIVJRFL/NL43-Lucas described previously (26), with some modifications. Briefly, 3⫻105

to 5⫻105

MDDCs, Raji DC-SIGN cells, and Raji B cells were counted with Perfectcount cytometer beads (Cytognos), pulsed with 180 to 300 ng of HIVJRFL/NL43-Lucp24

gag

for 3 h at 37°C or 4°C, washed five times with PBS, and counted again with beads on the flow cytometer to ensure viability. In experiments done with myeloid DCs, 2⫻105 cells were pulsed with 120 ng of HIVJRFL/NL43-Lucp24gagfor 3 h at 37°C. All

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pulsed cells were cocultured in duplicate with the target Hut CCR5⫹cell line at a ratio of 1:1 (up to 1⫻105

cells per well in a 96-well plate) in the presence of 10␮g/ml of polybrene (Sigma). Cells were then assayed for luciferase activity 48 h later (BrightGLo luciferase system; Promega) in a Fluoroskan Ascent FL luminometer. To detect possible productive infection of pulsed cells, CCR5⫹target cells in the cocultures were substituted with CCR5⫺ target cells in all the experiments.

DC-SIGN quantification.The mean number of DC-SIGN antibody binding sites (ABS) per cell was obtained with a Quantibrite kit (BD) according to the manufacturer’s instructions. The mean number of PE-labeled DC-SIGN ABS per cell (antibody-to-PE ratio of 1:1; R&D) was quantified by a standard linear regression curve built with four different precalibrated beads conjugated with fixed amounts of PE molecules per bead. Geometrical mean fluorescence-ob-tained labeling with goat anti-mouse immunoglobulin G2b (IgG2b) PE was used as an isotype control (BD), and ABS-per-cell values were subtracted from values of corresponding samples. All cells were previously blocked with 1 mg/ml of human IgG (Baxter, Hyland Immuno) to prevent binding through the Fc portion of the antibody. Rainbow calibration particles (BD) were used before quantita-tion to ensure the consistency of fluorescence intensity measurements through-out all the experiments. All cells were stained at 4°C for 20 min, washed, and resuspended in PBS containing 2% formaldehyde. Samples were analyzed with FACSCalibur (BD) using CellQuest software to evaluate collected data. For-ward-angle and side-scatter light gating were used to exclude dead cells and debris from the analysis.

Viral capture and transmission followed by microscopy.MDDCs and myeloid DCs were pulsed with 50 ng of p24gag

of HIVNL43/eGFPduring 3 h, washed with PBS, stained with DAPI (4⬘,6⬘-diamidino-2-phenylindole) (Sigma), fixed with 2% formaldehyde, and cytospun onto glass slides. Cells were mounted in Dako fluorescent medium and sealed with nail polish to analyze them using a confocal microscope (Laser Optic Leica TCS SP2 AOBS). Colocalization experiments were done by pulsing mature myeloid DCs with equal amounts of HIVNFN-SX and HIV⌬env-NL43during 4 h as described above. Cells were then fixed, perme-abilized (Caltag), and stained with p24gag-RD1 mAb (Beckman Coulter), DAPI, and CD81, CD63, or LAMP-1 (all conjugated with FITC) (BD). Viral kinetics of mMDDCs (n⫽3) were analyzed with fluorescence microscopy by comparing HIVNL43/eGFP-pulsed cells with the same cells left in the absence of virus for 4 h more.

Monocytes were negatively selected from PBMCs (Miltenyi Biotec) to avoid magnetic bead interference during electron microcopy observation. Subsequent differentiation to iMDDCs and mMDDCs was performed as stated above. MDDCs were pulsed for 30 min or 24 h with 200 ng of p24gagof HIV

JRFL/NL43-Luc per 5⫻105

cells, extensively washed in PBS, and fixed in 2.5% glutaraldehyde for 1 h. Cells were then processed as described elsewhere previously (3) for analysis of ultrathin sections using a Jeol JEM 1010 electron microscope. In order to monitor viral transmission, extensively washed MDDCs pulsed for 24 h were cocultured for an extra hour with Hut CCR5⫹cells at a 1:1 ratio before they were fixed.

Macropinocytic characterization and-methyl-cyclodextrin treatment of MDDCs.iMDDCs and mMDDCs were incubated with Alexa 488-labeled dex-tran (Molecular Probes) for 2 h at 4°C or 37°C and then fixed for fluorescence-activated cell sorting analysis. mMDDCs were treated with␤ -methyl-cyclodex-trin (Sigma) at the concentrations indicated for 30 min at 37°C before they were pulsed with HIVNFN-SXand assayed for p24gagas described above. mMDDC viability in the presence of␤-methyl-cyclodextrin was assessed with a flow cy-tometer by labeling cells with propidium iodide and DIOC-6 (Sigma and Mo-lecular Probes, respectively) as previously described (2). Cells were incubated with Alexa 555-conjugated transferrin (25␮g/ml) from Molecular Probes for 1 h at 4°C, washed, and then shifted to 37°C for 30 min. Cells were then stained with DAPI, fixed with 2% formaldehyde, and cytospun onto glass slides for analysis by confocal microscopy.

RESULTS

Mature DCs capture higher amounts of HIVNFN-SXthan

im-mature DCs. To compare viral capture abilities of mMDDCs and iMDDCs of the same seronegative donors, we pulsed both cell types with equal amounts of HIVNFN-SXat 37°C for 2 h.

Raji B cells stably transfected with DC-SIGN were routinely employed to measure DC-SIGN viral capture efficiency, and the parental Raji cell line was used to determine the viral capture background. Interestingly, viral capture determined by

p24gagELISA was much more efficient in mMDDCs than in

the rest of the cells tested, suggesting a differential active capture process of HIVNFN-SXin this cell type (P ⬍0.0001,

pairedttest) (Fig. 1A). This enhanced mMDDC viral capture correlated with an increased viral transmission efficiency, as observed when setting 48-h cocultures of MDDCs pulsed with pseudotyped HIVJRFL/NL43-Lucand target Hut CCR5⫹T cells

(P⫽0.048, pairedttest) (Fig. 1B). Raji DC-SIGN cells were employed to confirm viral transfer efficiency, and parental Raji cells were used as a negative control, showing the same relative light unit (RLU) values as those of nonpulsed cells (Fig. 1B, dotted line). Notably, cocultures with CCR5-negative target cells did not show any infection 48 h postpulse, indicating that neither Raji DC-SIGN cells nor MDDCs were productively infected during this experimental period (Fig. 1B, clear bars). Results were analogous by using HIVNFN-SXto pulse the cells

and then measuring viral transmission by intracellular staining of cocultures with an anti-p24 mAb (data not shown). In this case, viral transfer, expressed as a percentage of p24⫹cells, was also more efficient when cocultures were done with mMDDCs than when they were done with iMDDCs (P ⬍ 0.0001, pairedttest).

We next investigated whether myeloid DCs expressing CD1c⫹(BDCA-1) antigen would behave similarly to MDDCs in terms of viral capture andtransinfection ability. We isolated the myeloid dendritic CD1c⫹subset from PBMCs of seroneg-ative donors. A fraction of them were subsequently LPS ma-tured. We performed the same viral capture assay employed with MDDCs, and, as reported above for mMDDCs, viral capture was more efficient in mature myeloid DCs (P⫽0.039, pairedttest) (Fig. 1C). Regarding viral transmission, we tested the transfer capacity of HIVJRFL/NL43-Luc-pulsed mature and

immature myeloid DCs of the same seronegative donors cocul-tured with Hut CCR5⫹ cells. As observed for MDDCs, the mean viral transmission ability of mature myeloid DCs was 50 times more efficient than that of immature myeloid DCs (P⫽ 0.013, pairedttest) (Fig. 1D). Overall, these data show that mMDDCs and mature CD1c⫹myeloid DCs capture and trans-mit HIV-1 to a higher extent than do iMDDCs and immature myeloid DCs.

Enhanced viral transmission of mMDDCs depends on the amount of virus actively captured.To test whether the higher viral capture of mMDDCs accounted for the increased HIV transmission observed, we pulsed both immature and mature MDDCs with high concentrations of HIVJRFL/NL43-Lucat 4°C

and 37°C (Fig. 2). After extensive washing, we lysed part of the cells and determined the cell-associated virus fraction by p24gagELISA. The rest of the washed cells were cocultured at

37°C with Hut CCR5⫹target cells and assayed for luciferase activity. Of note, viral binding at 4°C in mMDDCs was similar to viral capture at 37°C observed in iMDDCs (Fig. 2A). Inter-estingly, the subsequent viral transmission of these two cell subsets was analogous (Fig. 2B). These results indicate that initial viral capture correlates with final viral transmission, regardless of the maturation status of DCs. It is noteworthy that Raji DC-SIGN viral capture and transmission were also temperature sensitive, showing the same behavior at 4°C as the Raji parental cell line. Again, HIVJRFL/NL43-Luccapture and

transmission observed in mMDDCs pulsed at 37°C were the highest of all cell types tested.

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In order to exclude the possibility that mMDDCs could better stimulate target cells in the cocultures and thereby lead to a greater readout in RLUs, we also estimated the prolifer-ation capacity of target cells in cultures with mMDDCs or

[image:4.585.113.471.68.323.2]

iMDDCs. We found, however, no differences in Hut cell pro-liferation when cultured with any type of MDDCs (data not shown). These results suggest that higher viral transmission of mMDDCs is highly dependent on active viral capture.

FIG. 1. Maturation of MDDCs and myeloid DCs enhances HIVNFN-SXcapture and HIVJRFL/NL43-Luctransmission. (A) Comparative capture of

HIVNFN-SXby distinct cell types. A total of 3⫻10

5MDDCs, Raji DC-SIGN cells, and Raji cells were pulsed for 2 h at 37°C with 80 ng p24gagin 0.3

ml, washed with PBS, and lysed with 0.5% Triton (at a final concentration of 5105cells per ml) to measure p24gagby ELISA. Results are expressed

in pg of p24gagbound per ml of cell lysate. Data show mean values and standard errors of the means (SEM) from six independent experiments including

cells from 11 donors. mMDDCs significantly capture higher amounts of virus than iMDDCs (P0.0001, pairedttest). (B) HIVJRFL/NL43-Luctransmission

from MDDCs, Raji DC-SIGN cells, and Raji cells to Hut CCR5⫹cells (dark bars). Cocultures were assayed for luciferase activity 48 h postpulse. No productive infection of MDDCs or Raji DC-SIGN cells was detected, as shown when using Hut CCR5-negative target cells (clear bars). The dotted line shows background levels of RLUs observed when nonpulsed cells were lysed. mMDDCs transmit higher amounts of HIVJRFL/NL43-Lucthan iMDDCs

derived from the same seronegative donors (P0.048, pairedttest). (C) Comparison of HIVNFN-SXcapture by myeloid DCs as described above (A),

including cells from four different donors. Mature myeloid DCs (m Myeloid) significantly capture higher amounts of virus than immature myeloid DCs (i Myeloid) (P0.039, pairedttest). (D) HIVJRFL/NL43-Luctransmission from myeloid DCs, Raji DC-SIGN cells, and Raji cells to Hut CCR5⫹cells (dark

bars) and to Hut CCR5⫺cells (clear bars) as described above (B). Mature myeloid DCs transmit higher amounts of HIVJRFL/NL43-Lucthan immature

myeloid DCs derived from the same seronegative donors (P0.013, pairedttest).

FIG. 2. Enhanced viral transmission of mMDDCs depends on the amount of HIVJRFL/NL43-Lucactively captured. (A) Comparative capture at 37°C

and binding at 4°C of HIVJRFL/NL43-Lucby distinct cell types. A total of 3⫻105MDDCs, Raji DC-SIGN cells, and Raji cells were incubated at 37°C (dark

bars) or 4°C (clear bars) with 300 ng of HIVJRFL/NL43-Lucp24

gagin a final volume of 0.3 ml, washed five times with PBS, and lysed with 0.5% Triton (at

a final concentration of 5⫻105cells per ml) to measure p24gagby ELISA. (B) HIV

JRFL/NL43-Luctransmission to Hut CCR5⫹target cells of pseudotyped

virus captured at 37°C (dark bars) or bound at 4°C (clear bars). Different cell types pulsed as described above (A) were cocultured with target cells at 37°C and assayed for luciferase activity 48 h postpulse. A and B show a representative experiment including mean values and SEM for three different donors.

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mMDDC viral capture increases over time, and captured virus has a longer life span than in iMDDCs. To further characterize the viral capture process, we monitored mMDDCs and iMDDCs exposed to HIVNL43/eGFP fluorescent virus or

HIVNFN-SX during 10 to 12 h at 37°C (Fig. 3A to D). After

extensive washing, the percentage of GFP-positive cells was analyzed by flow cytometry, and the cell-associated virus frac-tion was determined by p24gag ELISA at each of the time

points indicated. Interestingly, viral capture in mMDDCs in-creased over time (Fig. 3A and C) while remaining constant in iMDDCs (Fig. 3B and D). These data show that mMDDCs actively capture HIV-1 and that viral capture differences with respect to iMDDCs increase over time.

Next, we asked whether captured virus would follow dif-ferent intracellular pathways in distinct MDDCs. Kinetic analyses of both the cell-associated and the cell-free virus fractions were longitudinally determined by p24gag in

cultures of immature and mature MDDCs exposed to HIVNFN-SXand extensively washed to remove unbound

vi-rus (Fig. 3E and F). Immediately following viral exposition and washing, cell-associated virus was approximately 25 times more abundant in mMDDCs than in iMDDCs. HIVNFN-SXp24gagantigen associated with iMDDCs became

quickly undetectable in the first 4h (Fig. 3F). Although these results suggest that iMDDCs may lead the virus to an intra-cellular lytic pathway, showing 50% ⫾ 10% viral degrada-tion, the steady increase in soluble p24gag also indicated

some degree of virus release to the culture supernatant (22%⫾ 3% of captured virus). Conversely, mMDDCs de-graded 30% ⫾ 10% of captured virus during the first 4 h

(Fig. 3E) without a substantial release of trapped virions (7% ⫾ 3%). p24gag associated with mMDDCs decreased

50%⫾12% during the first day of culture without a further increase in soluble antigen, indicating the persistence of detectable cell-associated p24gag.

Finally, to exclude that the greater viral degradation of iMDDCs than of mMDDCs (P⫽0.024, pairedttest) could account for the viral capture differences observed, we treated cells with bafilomycin A1 (an inhibitor of lysosomic degradation) and clasto-lactacystin ␤-lactone (an inhibitor of proteasome activity). We found no significant increase in viral capture of drug-treated cells compared to mock-treated cells during 2 h, as previously reported (16). After 4 h in the absence of virus but in the presence of these drugs, mMDDCs degraded a mean of 51%⫾19% of the lost virus throughout lysosomes and 55%⫾15% through the protea-some machinery. Retained viral particles in iMDDCs were processed in a similar way. These data suggest that greater viral degradation in iMDDCs does not account for the viral capture differences observed between both cell types. Over-all, these observations support a more efficient capture pro-cess and a longer life span of captured HIV-1 in mMDDCs than in iMDDCs.

[image:5.585.45.535.67.325.2]

Higher viral capture in mature DCs than in immature DCs can occur independently of DC-SIGN and does not require viral envelope glycoprotein.Since the ability of DCs to bind and transmit HIV-1 has been previously related to DC-SIGN expression levels in the 293 transfected cell line (20), we used a fluorescence quantitation method to obtain absolute num-bers of DC-SIGN ABS in MDDCs and Raji DC-SIGN cells

FIG. 3. mMDDC viral capture increases over time, and captured virus has a longer life span than in iMDDCs. (A to D) Time course of HIVNL43/eGFPor HIVNFN-SXcapture by mMDDCs and iMDDCs showed that mMDDC viral capture increases over time while remaining constant

in iMDDCs. (E and F) Fate of HIVNFN-SXcaptured for 2 h by mMDDCs and iMDDCs, respectively, monitored during 2 days. Cell-associated

p24gagmeasured by ELISA is shown as closed symbols, while p24gagreleased to the cell culture medium is shown as open symbols. Captured virus

showed a prolonged life span in mMDDCs. Data (means and SEM from two independent experiments) include cells from six different donors.

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(Fig. 4A). We focused on MDDCs because the immunophe-notype of myeloid DCs showed almost no expression of DC-SIGN (Fig. 4B). Monocytes from 30 HIV-1-seronegative do-nors were consecutively differentiated into immature and mature MDDCs and assayed in parallel for DC-SIGN expres-sion. The mean number of DC-SIGN ABS per DC-SIGN-positive cell in mMDDCs (4⫻104) was half that of iMDDCs

(8⫻104) (P0.0001, pairedttest) (Fig. 4A). Raji DC-SIGN

cells displayed a mean number of DC-SIGN ABS comparable to that of mMDDCs. Thus, we found no correlation between DC-SIGN expression levels and viral capture efficiency of mMDDCs and iMDDCs.

We decided to further address the impact of DC-SIGN on the viral capture process mediated by MDDCs and blood my-eloid DCs by testing mAb MR-1 against DC-SIGN or mannan (a C-type lectin competitive inhibitor) to see if they could impair HIVNFN-SXcapture at 37°C. Figure 4C shows the

per-centage of HIVNFN-SXcaptured in the presence of different

[image:6.585.43.540.66.387.2]

inhibitors relative to untreated cells normalized to 100% of viral capture. Pretreatment with these compounds efficiently inhibited Raji DC-SIGN viral capture to levels similar to those displayed by Raji cells (greater than 85% for any of the inhib-itors;P⬍0.0001, pairedttest). We also observed an inhibitory effect in iMDDCs, the DC subset displaying higher amounts of

FIG. 4. Higher viral capture in mature DCs than in immature DCs can occur independently of DC-SIGN and does not require viral envelope glycoprotein. (A) MDDCs of 30 seronegative donors were stained with anti-DC-SIGN mAb labeled with PE (1:1 ratio of mAb to PE) and analyzed for DC-SIGN surface expression using Quantibrite PE standard beads to calculate the number of DC-SIGN ABS displayed per PE-positive cell. Mean values and SEM are represented. iMDDCs had twice the mean number of DC-SIGN ABS per cell compared to those of mMDDCs and Raji DC-SIGN cells (P ⬍ 0.0001, paired t test). (B) Expression of DC-SIGN in MDDCs, myeloid DCs, Raji DC-SIGN cells, and Raji cells. Isotype-matched mouse IgG controls are also indicated (empty peaks). (C) Percentage of p24gagHIV

NFN-SXcaptured at 37°C in the presence of

different DC-SIGN inhibitors relative to untreated cells normalized to 100% of viral capture. Raji cells were compared to untreated Raji DC-SIGN cells. Viral capture by cells with no inhibitor (dark bars) or preincubated with mannan (light gray bars) or MR-1 (white bars) is depicted. Significant inhibition in the Raji DC-SIGN cell line reached 85% (P ⬍0.0001, paired t test). Mean p24gag(pg/ml) values of untreated cells used for

normalization were 3,960 for mMDDCs, 1,154 for iMDDCs, 1,497 for mature myeloid cells (mMyeloid), 765 for immature myeloid cells (iMyeloid), 566 for Raji DC-SIGN cells, and 105 for Raji cells. Data show mean values and SEM from two independent experiments, including cells from six different donors. (D) Percentage of p24gagHIV

⌬env-NL43virus lacking the envelope glycoprotein captured by MDDCs, myeloid DCs, Raji cells, and

Raji DC-SIGN cells relative to HIVNFN-SXcapture normalized to 100%. Cells were pulsed with equal amounts of both viruses, and the Raji cell

line was compared to Raji DC-SIGN cells. The envelope requirement for mMDDC and myeloid DC viral capture was not significant, while it reached significance in iMDDCs (P ⫽ 0.0007, pairedt test). Raji DC-SIGN cells captured mainly HIVNFN-SX enveloped virus and bound

HIVenv-NL43virus only to levels comparable to the background seen by employing the Raji cell line (P⫽0.0001, pairedttest). Mean p24

gag(pg/ml)

values in cells exposed to HIVNFN-SXand used for normalization were 4,880 for mMDDCs, 1,562 for iMDDCs, 4,737 for mature myeloid cells,

1,280 for immature myeloid cells, 895 for Raji DC-SIGN cells, and 243 for Raji cells. Data show mean values and SEM from two independent experiments, including cells from five different donors.

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DC-SIGN, but it only reached 50% (P⫽0.049, pairedttest) (Fig. 4C). However, neither compound had any significant effect on viral capture mediated by mMDDCs (expressing SIGN) or immature and mature myeloid DCs (lacking DC-SIGN expression).

Since DC-SIGN inhibitors had no blocking effect on mature DC viral capture, and this C-type lectin is known to bind with high affinity to the gp120 viral envelope glycoprotein (5), we next analyzed the envelope requirement during the viral cap-ture process. We pulsed MDDCs and myeloid DCs with equal amounts of a viral construct lacking the envelope glycoprotein (HIVenv-NL43) and its counterpart expressing the envelope

protein (HIVNFN-SX). Figure 4D shows the percentage of

HIVenv-NL43captured by each cell type relative to cells pulsed

with HIVNFN-SX, normalized to 100% of viral capture. As

expected, Raji DC-SIGN cell viral capture was totally depen-dent on the envelope glycoprotein, as seen by the percentage of delta envelope virus captured compared to that of the wild-type virus (P⫽0.0008, pairedttest) (Fig. 4D). However, the envelope requirement for viral capture decreased in iMDDCs and was totally absent in myeloid DCs and mMDDCs. Overall, these results suggest that myeloid DC and mMDDC viral cap-ture is independent of viral envelope glycoprotein, data that

correlate with the lack of a blocking effect displayed by DC-SIGN inhibitors in these cell types.

Confirmation of viral capture, turnover, and transmission results by microscopy. To further address viral capture, HIVNL43/eGFP-pulsed MDDCs and myeloid DCs were

moni-tored by confocal microscopy. In agreement with our capture observations, a considerably greater amount of virions were found in mature DCs than in immature DCs after 4 h of viral exposition (Fig. 5A and B). Many mMDDCs and mature my-eloid DCs showed a large single GFP-positive vesicle-like structure, observed by reconstructing a series ofx-y sections collected through the nucleus of the cells to project thez-xand z-yplanes (Fig. 5C and D). Of note, iMDDCs and immature myeloid DCs did not present any of these large vesicles (data not shown), suggesting differential intracellular viral trafficking in mature DCs.

To better understand viral kinetics, HIVNL43/eGFP-pulsed

mMDDCs were analyzed by fluorescence microscopy. After 4 h of viral capture, 97%⫾3% of the pulsed mMDDCs were GFP positive, presenting captured virus in one single vesicle (55% ⫾ 8%), polarized to one side of the cell membrane (25% ⫾ 7%), and randomly distributed throughout the cell surface (14%⫾9%). The same pulsed mMDDCs, washed and

FIG. 5. Confocal and electron microscopy of HIV1 capture and transfer mediated by mMDDCs. (A) Confocal images of a section of mMDDCs exposed to HIVNL43/eGFPfor 4 h and stained with DAPI. Merged images of the section showing the cells and the green and blue fluorescence are

shown. (B) Composite of a series ofx-ysections collected through the entire thickness of the cell nucleus of mature myeloid DCs (m Myeloid) exposed to HIVNL43/eGFPand projected onto a two-dimensional plane. A three-dimensional reconstruction of a series ofx-ysections collected

through part of the cell nucleus can be found as movie S1 in the supplemental material. (C) Composition of a series ofx-ysections of mMDDCs collected through part of the cell nucleus and projected onto a two-dimensional plane to show thex-zplane (bottom) and they-zplane (right) at the points marked with the dotted white axes. (D) Confocal image composition of mature myeloid DCs constructed as described above (C). (E to G) mMDDCs presented large amounts of viral particles associated with the cell membrane outside dendrites (E and F) or initiating endocytosis (G). (H and I) Large vesicles containing numerous viral particles could be found proximal to the plasma membrane surface or deep inside the cellular cytoplasm. (J) Infectious synapse could also be observed in mMDDC and Hut CCR5⫹cell cocultures. The marked box on the picture shows the attachment of mMDDCs (with light cytoplasm at the bottom) to a Hut CCR5⫹cell (with granulated cytoplasm and a large nucleus at the top). (K) Magnification of the marked box in J is shown on the right, where viral particles are polarized to the cell-to-cell contact area, to form what has been described as a virological synapse.

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analyzed 4 h later, presented a similar viral distribution pat-tern, although the mean percentage of captured virus in one single vesicle increased up to 75%⫾4%, and randomly dis-tributed virus decreased down to 4%⫾6%. These data indi-cate that viral vesicles represent a more stable compartment, as they contain most of the captured virus at the time when viral degradation reaches the plateau (Fig. 3E). However, the dif-ferences observed were not statistically significant, suggesting

that viral turnover was comparable in each of the cell popula-tions found.

To gain further insights into the mechanism of viral capture and transmission, HIVJRFL/NL43-Luc-pulsed MDDCs were

[image:8.585.80.505.67.552.2]

monitored by electron microscopy. After 24 h of exposition, viral particles in mMDDCs attached to the extracellular cell membrane proximal to the soma between dendrites (Fig. 5E and F). Initial endocytic events were also observed (Fig. 5G),

FIG. 6. Mature myeloid DCs retain HIVNFN-SXand HIV⌬env-NL43in a CD81- and CD63-positive compartment. (Top) Confocal images of a

section of mature myeloid DCs exposed to HIVNFN-SXfor 4 h, fixed, and permeabilized to stain them with DAPI and p24

gag(RD1). Cells were

labeled in parallel for CD81, CD63, or LAMP-1 (all conjugated with FITC). Images show, from left to right, individual green and red fluorescence and the combination of both either alone, merged with DAPI, or with bright-field cellular shape. (Bottom) Confocal images of a section of mature myeloid DCs exposed to HIVenv-NL43for 4 h, fixed, and permeabilized to stain them with DAPI and p24gag(RD1). Cells were labeled in parallel

for CD81, CD63, or LAMP-1 (all conjugated with FITC). Images show, from left to right, individual green and red fluorescence and the combination of both either alone, merged with DAPI, or with bright-field cellular shape.

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and vacuoles containing numerous viral particles consistent with our previous confocal microscopy observation were found proximal to the plasma membrane or deep inside the cytoplasm (Fig. 5H and I). Quantitative differences between iMDDCs and mMDDCs confirmed our previous results: among 113 mMDDCs analyzed, 33% of them showed at least one virus in the cell surface, and 28% had endocytosed virus. Conversely, among 90 iMDDCs analyzed, only 9% of them showed at least one virus in the cell surface, and 6% had endocytosed virus. Virions were associated with dendrites in only two cases (one for mMDDCs and one for iMDDCs).

Viral transmission was also monitored by using MDDCs cocultured with Hut CCR5⫹ cells. mMDDCs filled with HIVJRFL/NL43-Luc established associations with Hut CCR5⫹

cells, and virus could be found to be restricted to the site of the cell contact area (Fig. 5J and K). Polarization of viral particles to the cell-to-cell interface, where intimate contact between mMDDCs and Hut CCR5⫹ cells takes place, suggested the formation of an infectious synapse.

Mature myeloid DCs retain HIVNFN-SXand HIVenv-NL43in

a compartment similar to that of mMDDCs.Previous work has demonstrated that in mMDDCs, HIV1 accumulates in intra-cellular vacuoles containing CD81 and CD63 tetraspanins (8). We extended these observations to mature myeloid DCs and found that HIVNFN-SXcolocalizes with CD81 and CD63 (Fig.

6, top) but not with the LAMP-1 lysosomic marker. Further-more, by employing HIVenv-NL43, we observed accumulation

in the same intracellular compartments (Fig. 6, bottom). Hence, in mature myeloid DCs, the process of viral accumu-lation in vesicles is not directed by the envelope glycoprotein, in agreement with our previous data (Fig. 4D). Furthermore, both types of captured viruses are retained in a single CD81-and CD63-positive compartment.

mMDDC viral capture is not mediated through pinocytosis and requires cholesterol.We next asked whether pinocytosis events could determine the enhanced viral capture efficiency of mature DCs. We confirmed a greater macropinocytic capacity of iMDDCs than of mMDDCs by analyzing the capture of dextran particles labeled with Alexa 488 (Fig. 7A). Fluores-cence-activated cell sorting analysis revealed that both cell types showed similar capacities to bind to dextran at 4°C. However, active dextran pinocytosis was higher at 37°C in iMDDCs. These preliminary data suggested that pinocytosis does not account for the enhanced viral capture observed in mature DCs.

Previous works have shown that ␤-methyl-cyclodextrin, a cholesterol-sequestering reagent, efficiently blocks iMDDC vi-ral binding and capture (12). Therefore, we addressed whether cholesterol could also play a role during mMDDC viral cap-ture. We first checked mMDDC viability in the presence of

␤-methyl-cyclodextrin with a flow cytometer, labeling cells with propidium iodide and DIOC-6 to analyze the drug induction of necrosis and apoptosis. We confirmed that mMDDCs were not affected by␤-methyl-cyclodextrin until we reached a concen-tration of 10 mM (data not shown). We then measured the effect of ␤-methyl-cyclodextrin on mMDDC viral capture at 37°C by employing nontoxic increasing concentrations of the drug (Fig. 7B). We found a dose-dependent inhibition of mMDDC active viral capture, reaching levels similar to those of viral binding observed at 4°C (Fig. 7B). Thus, mMDDC viral

capture is a temperature-sensitive process that can be blocked by␤-methyl-cyclodextrin.

To assess the specificity of ␤-methyl-cyclodextrin and ex-clude the possibility that it could be affecting other endocytic cellular pathways aside from lipid rafts, we analyzed the effect that this drug had on mMDDC pinocytosis by using dextran labeled with Alexa 488 (Fig. 7C). We found no significant effect of␤-methyl-cyclodextrin on mMDDC macropinocytosis when we compared fluorescence intensities of pinocytosed dextran in mMDDCs exposed to increasing concentrations of the drug to those of mock-treated cells. This finding further supported our preliminary data indicating that mMDDC viral capture does not take place through pinocytosis. Finally, to analyze␤-methyl-cyclodextrin activity regarding clathrin-medi-ated endocytosis, we employed transferrin labeled with Alexa 555, a control that is known to enter the cell through a clathrin-mediated pathway. We pulsed mMDDCs previously exposed to␤-methyl-cyclodextrin or mock treated with transferrin at 4°C to allow binding to the cellular surface. We then washed away unbound transferrin, leaving part of the cells at 37°C to incorporate labeled ligands for 30 min and keeping the rest of the cells at 4°C to obtain binding controls. Cells were then fixed and analyzed by confocal microscopy (Fig. 7D). mMDDCs exposed to␤-methyl-cyclodextrin at 2.5 mM had almost no inhibition of transferrin uptake, being absent in some of the donors tested and never exceeding 20% of inhibition (Fig. 7D). However, cells exposed to ␤-methyl-cyclodextrin at 5 mM showed an inhibition of transferrin uptake, indicating that this drug has collateral effects on mMDDC clathrin-mediated en-docytosis. Thus, we can conclude that␤-methyl-cyclodextrin at 2.5 mM is blocking mMDDC viral capture by affecting choles-terol pathways to a higher extent than clathrin-mediated endo-cytosis.

DISCUSSION

Soon after HIV-1 exposure, both mature and immature DCs are able to transfer the virus preferentially to antigen-specific CD4⫹T cells in the absence of productive infection (14, 17). This viral transmission process, known as trans infection, is enhanced when DCs are matured in the presence of CD40 ligand, gamma interferon, poly(I:C), and lipopolysaccharide (21).

The present study compares the effect of LPS maturation on MDDCs and myeloid DCs during HIV-1 viral capture, under-scoring new insights into a mechanism that might have relevant implications during pathogenesis, given the interaction of ma-ture DCs with CD4⫹T cells at lymph nodes. Previous works have shown that iMDDCs capture virus to a higher or similar extent compared to mMDDCs (7, 21). When we performed binding experiments at 4°C, no major differences between these cell types were observed. Strikingly, when cells were exposed to virus at 37°C, higher amounts of virus were found in mMDDCs, and this difference increased over time. We ex-tended our observations to blood-derived DCs of myeloid line-age and found that mature myeloid DCs also capture higher amounts of virus than immature myeloid DCs. Thus, con-versely to previous studies, our cell culture system and the viral isolates that we employed underscore a viral capture mecha-nism that is dramatically enhanced upon maturation that takes

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place independently of fusion events and does not require the envelope glycoprotein. Hence, although cell-associated p24gag

values provided in Fig. 1 to 3 do not distinguish between viral capture and fusion events, the uptake of virus lacking the envelope (Fig. 4D and 6, bottom) helps to discriminate this nonfusogenic viral entry pathway. This mechanism could ex-plain why mature DCs, with limited antigen capture activity, sequester significantly larger numbers of intact whole viral particles than immature DCs, as previously reported (7).

Early works proposed that iMDDC viral capture protects virions against degradation (9, 13), but we and others have shown that iMDDCs show rapid degradation of captured viral particles (16, 25). It is noteworthy that we found still greater amounts of virus 48 h postpulse in mMDDCs than in iMDDCs immediately after pulsing. Thus, possible explanations for

en-hanced viral transmission in mMDDCs versus iMDDCs in-clude an increased ability for capturing the virus and a longer life span of trapped virions. Furthermore, our results correlate net viral capture with final viral transmission, regardless of the MDDC maturation status (Fig. 2). Therefore, in our experi-mental settings, mMDDC differences in ICAM-1 expression or viral distribution during the infectious synapse cannot exclu-sively account for the enhanced HIV-1 transmission displayed by this cell type (15, 21). In vivo, however, viral capture would need to be considered along with DCs for primary CD4 T-cell contact and any subsequent stimulation arriving from this.

[image:10.585.50.539.66.421.2]

Although DC-SIGN has been proposed to be the most im-portant HIV-1 attachment factor that concentrates virus par-ticles on the surface of DCs, recent studies also suggest that HIV-1 binding, uptake, and transfer from DCs to CD4⫹ T

FIG. 7. mMDDC viral capture is not mediated through pinocytosis and depends on cholesterol-enriched domains. (A) Increased macropino-cytosis of dextran labeled with Alexa 488 in iMDDCs versus mMDDCs. Thin histograms represent dextran binding at 4°C, and thick histograms represent dextran capture at 37°C. Numbers above the peaks of the histograms indicate geometric mean fluorescence intensities. The negative control was done in the absence of dextran (dotted histograms). (B) mMDDCs were preincubated with increasing concentrations of␤ -methyl-cyclodextrin, a cholesterol-sequestering reagent, below observed toxic concentrations. Viral capture (performed as described in the legend of Fig. 1A) was inhibited in a dose-dependent manner, reaching statistical significance at 2.5 mM (P⫽ 0.001, paired t test). (C) mMDDCs were preincubated with increasing concentrations of␤-methyl-cyclodextrin for 30 min at 37°C, and dextran was then added to measure macropinocytosis at 37°C. Geometric mean (Geo mean) fluorescence intensity for each condition is graphed, subtracting negative controls done in the absence of dextran but in the presence of the␤-methyl-cyclodextrin concentrations indicated. (D) Confocal images of a single plane of mMDDCs exposed to transferrin Alexa 555 at 4°C for 1 h and then shifted to 37°C (where transferrin bound to its receptor is able to enter through clathrin-mediated endocytosis) or left at 4°C (as a control for transferrin external binding). Previous treatment of mMDDCs with 2.5 mM␤-methyl-cyclodextrin did not substantially affect transferrin clathrin-mediated endocytosis.

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lymphocytes may involve alternative pathways (11) such as mannose binding C-type lectin receptors (23, 24) or other molecules associated with lipid rafts (12). Overall, our results further confirm these observations. Despite the fact that DC-SIGN expression is almost absent in myeloid DCs and that mMDDCs display half the number of DC-SIGN ABS com-pared to iMDDCs, HIV-1 capture and transfer to target T cells are augmented upon maturation in both cell types. Even though the efficiency of DC-SIGN-transfected cell lines trans-mitting HIV-1 has been previously related to DC-SIGN ex-pression levels (1, 20), we confirm that the cellular context in which DC-SIGN is expressed determines capture and trans-mission efficiency (27, 28). Furthermore, DC-SIGN-blocking agents had minimal effects on mature DC viral capture while completely abrogating Raji DC-SIGN cell viral capture. Re-garding HIV-1 envelope requirements during the capture pro-cess, we found that this glycoprotein is not necessary for my-eloid DC and mMDDC viral uptake. Thus, analyzing the role of adhesion receptors dragged from the membrane of infected cells during viral budding might aid in identifying the molec-ular determinants that lead to viral capture in mature DCs. These factors might be ubiquitous, because we observed an increased viral uptake pattern in mature DCs compared to immature DCs exposed to viral stocks produced in 293T cells, MOLT-4 cells, and stimulated PBMCs (N. Izquierdo-Useros, unpublished results) (P⫽0.006, pairedttest).

We have also shown how MDDC maturation induces a rapid decrease in macropinocytic activity of fluid-phase markers such as dextran particles. Therefore, mMDDC-enhanced viral cap-ture through a pinocytic pathway is highly unlikely, since this transportation system is less active in mMDDCs than in iMDDCs. Recent studies showed brief colocalization of HIV with transferrin (13), the archetypical cargo of clathrin-medi-ated endocytosis. However, virus-containing compartments in mMDDCs colabel with lipid raft-associated tetraspanins CD81, CD82, and CD9 (8). We have extended these observa-tions and found that blood myeloid DCs also accumulate HIV1 in a CD81- and CD63-positive compartment and that this process is not mediated by the viral envelope glycoprotein. In addition, it has been shown previously that depleting choles-terol from membranes with agents such as␤ -methyl-cyclodex-trin inhibits iMDDCs viral capture (12). That is why we have focused on cholesterol-mediated pathways of entry, further confirming their role during viral capture. Although we found

␤-methyl-cyclodextrin concentrations that blocked mMDDC viral capture without affecting transferrin clathrin-mediated endocytosis, higher concentrations did affect this process. We have seen, however, that␤-methyl-cyclodextrin does not affect dextran pinocytosis. It is then more likely that cholesterol-enriched lipid rafts account for mMDDC viral capture, as has been previously suggested for iMDDCs, although further work should address this issue.

In conclusion, our results suggest that the unique capability of mature DCs to increase HIV-1 infection could play a key role in augmenting viral dissemination at the lymphoid tissue, a major site of viral replication and continuous interaction between susceptible T cells and mature DCs. Thus, elucidating the mechanisms underlying mature DC-enhanced HIV-1 transmission is required to design effective strategies for ther-apeutic intervention aimed at blocking viral spread.

Further-more, this knowledge would indeed increase the success ex-pectations of therapeutic vaccines based on mature DC immune response-priming capacities.

ACKNOWLEDGMENTS

We thank M. Ferna´ndez for technical assistance with flow cytometry quantification, L. Tam for manuscript editing, M. Rolda´n from the UAB Microscopy Service, and P. Parrales from the Pathology Depart-ment of our hospital.

J.M.-P. and J.B. are supported by the Institute for Research on Health Sciences Germans Trias i Pujol in collaboration with the Cata-lan Health Department and research grants SAF2004-06991, FIS 05/ 504, and FIPSE 36536/05, the Spanish AIDS Network grant RD06/006, and the HIVACAT program. N.I.-U. is supported by DURSI from the Generalitat de Catalunya.

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Figure

FIG. 2. Enhanced viral transmission of mMDDCs depends on the amount of HIVand binding at 4°C of HIVbars) or 4°C (clear bars) with 300 ng of HIVa final concentration of 5virus captured at 37°C (dark bars) or bound at 4°C (clear bars)
FIG. 3. mMDDC viral capture increases over time, and captured virus has a longer life span than in iMDDCs
FIG. 4. Higher viral capture in mature DCs than in immature DCs can occur independently of DC-SIGN and does not require viral envelopeglycoprotein
FIG. 6. Mature myeloid DCs retain HIVenv-NL43NFN-SX�labeled in parallel for CD81, CD63, or LAMP-1 (all conjugated with FITC)
+2

References

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