DC-SIGN mediates cell-free infection and transmission of human T-cell lymphotropic virus type 1 by dendritic cells

Abstract

Despite the susceptibility of dendritic cells (DCs) to human T-cell lymphotropic virus type 1 (HTLV-1) infection and the defined role of these cells in disease pathogenesis, the mechanisms of viral binding to DCs have not been fully delineated. Recently, a glucose transporter, GLUT-1, heparan sulfate proteoglycans (HSPGs), and neuropilin-1 (NRP-1) were demonstrated to facilitate HTLV-1 entry into T cells. DCs express their own array of antigen receptors, the most important being the DC-specific intercellular adhesion molecule-3 (ICAM-3)-grabbing nonintegrin (DC-SIGN) with respect to retrovirus binding. Consequently, the role of DC-SIGN and other HTLV-1 attachment factors was analyzed in viral binding, transmission, and productive infection using monocyte-derived DCs (MDDCs), blood myeloid DCs, and B-cell lines expressing DC-SIGN. The relative expression of DC-SIGN, GLUT-1, HSPGs, and NRP-1 first was examined on both DCs and B-cell lines. Although the inhibition of these molecules reduced viral binding, HTLV-1 transmission from DCs to T cells was mediated primarily by DC-SIGN. DC-SIGN also was shown to play a role in the infection of MDDCs as well as model B-cell lines. The HTLV-1 infection of MDDCs also was achieved in blood myeloid DCs following the enhancement of virus-induced interleukin-4 production and subsequent DC-SIGN expression in this cell population. This study represents the first comprehensive analysis of potential HTLV-1 receptors on DCs and strongly suggests that DC-SIGN plays a critical role in HTLV-1 binding, transmission, and infection, thereby providing an attractive target for the development of antiretroviral therapeutics and microbicides.

FIG. 1.

Analysis of HTLV-1 binding to B-THP-1 cells. (A) Parental B-THP-1 cells or cells transduced to stably express DC-SIGN (B-THP-1/DC-SIGN) were incubated with an FITC-labeled anti-CD19 Ab in combination with APC-labeled anti-DC-SIGN, Alexa Fluor 647-labeled anti-GLUT-1, or anti-HSPGs, and PE-labeled anti-NRP-1 Abs and were analyzed by flow cytometry. A total of 50,000 events collected for each sample were gated to include the CD19⁺ population. Isotype controls are represented by dotted-line histograms, whereas solid-line histograms represent Ab reactivity with respect to the indicated receptor molecules. (B) A QDot-based binding assay was employed to determine the effect of blocking DC-SIGN and other receptor molecules on HTLV-1 binding to B-THP-1 cells. Target cells (1 × 10⁶) either were left untreated or were treated (30 min, room temperature) with a blocking Ab against DC-SIGN (clone 507; 20 μg/ml), the GLUT-1 inhibitor Cyto B (20 μM), the HSPG inhibitor Hep (20 mU), or NRP-1 ligand (VEGF; 50 ng/ml). Cells subsequently were incubated with Biot-HTLV-1 (125 ng/10⁶ cells) for 45 min on ice, and the binding was quantified as described in Materials and Methods. Cells incubated with QDots or biotin alone were used as negative controls. HTLV-1 binding was estimated as the fluorescence measured at 400 or 605 nm. The results shown represent the mean fluorescence ± standard deviations (T bars) from three independent experiments each performed in duplicate. An asterisk denotes a statistically significant decrease in fluorescence compared to maximum binding without any inhibitor (P ≤ 0.05).

FIG. 2.

HTLV-1 colocalizes with DC-SIGN on B-THP-1 cells. (A) A fraction of cells from the QDot binding assay were analyzed by confocal microscopy. Biot-HTLV-1/Strep-QDot-bound DCs were plated on poly(D)-lysine-coated slides, fixed, and incubated with an anti-DC-SIGN MAb (1:80; clone 507) and then by an Alexa Fluor 488 (1:200)-conjugated secondary Ab. Confocal microscopy at 100× magnification demonstrated that HTLV-1 (red) colocalized specifically with DC-SIGN (green) in a uniformly but occasionally patchy configuration. Hoechst 33342 (blue) was used to demarcate the nucleus. (B) FACS analyses were performed to reconfirm the DC-SIGN-mediated binding of HTLV-1 to B-THP-1 cells. Parental and DC-SIGN-transduced B-THP-1 cells were incubated with HTLV-1 in the absence or presence of a blocking Ab against DC-SIGN (clone 507; 20 μg/ml). Unbound virus particles were removed by washing, and cells were incubated with an Alexa Fluor 647-labeled MAb against HTLV-1 gp46. A total of 50,000 collected events were gated to include the CD19⁺ population. The dotted-line histogram represents the isotype Ab; a solid-line histogram indicates the results obtained with untreated cells, and the filled histogram represents B-THP-1 cells positive for HTLV-1 gp46. The results shown are representative of one of the two independent experiments.

FIG. 3.

DC-SIGN interacts with HTLV-1 envelope glycoprotein. (A) To examine the role of the HTLV-1 envelope glycoprotein gp46 in the DC-SIGN-mediated binding of HTLV-1, target cells (1 × 10⁶) were left untreated or were treated (30 min, room temperature) with carbohydrate mannan (20 μg/ml; Sigma), a MAb specific for DC-SIGN (clone 507; 20 μg/ml), or a MAb directed against L-SIGN (clone 604; 20 μg/ml) and then incubated with HTSU-IgG (200 ng/ml) or SUA-IgG (negative control) for 30 min on ice. In a parallel experiment, HTSU was preincubated either with a pool of three anti-HTSU neutralizing MAbs (PRH-4, PRH-7A, and PRH-11; 10 μg/ml) or with the nonneutralizing MAb PRH-1. A total of 50,000 events collected for each sample were gated to include live cells. (A) The numbers shown indicate the percentages of CD19⁺/HTSU-IgG⁺ cells. (B) The results shown represent the MFI values ± standard deviations (T bars) from the triplicate samples.

FIG. 4.

Expression of DC-SIGN on B-THP-1 cells enhances HTLV-1 infection. Target cells were incubated with purified HTLV-1 (3 μg/10⁶ cells) at the indicated time periods. (A) At increasing times after treatment, total RNA was isolated, converted to cDNA, and subjected to real-time PCR using pX-specific primers. The threshold cycle values obtained for duplicate samples were averaged and normalized to levels of β-actin, and the change (n-fold) in mRNA expression with respect to the control was calculated as described in Materials and Methods. (B) Productive infection was analyzed in supernatants of infected cells collected on days 2, 4, and 6 after infection using an HTLV-1 p19 (gag)-specific ELISA. A standard curve generated for p19 antigen was used to determine p19 levels (in picograms/milliliter) and is represented as the means ± standard deviations from triplicate samples. An asterisk denotes a statistically significant increase in the amount (in picograms/milliliter) of p19 in the presence of DC-SIGN (P ≤ 0.05).

FIG. 5.

Analyses of HTLV-1 binding to MDDCs. Immature DCs were differentiated from highly purified monocytes, and their phenotype was determined using a FITC-labeled lineage cocktail (Lin-1) to determine the absence of other leukocytes. (A) To detect the presence of various HTLV-1-binding receptors on DCs, immature DCs were incubated with a FITC-labeled Lin-1 Ab in combination with APC-labeled anti-DC-SIGN, Alexa Fluor 647-labeled anti-GLUT-1, or anti-HSPGs and PE-labeled anti-NRP-1 Abs and was analyzed by flow cytometry. A total of 50,000 events collected for each sample were gated to include the Lin-1⁻ population. Isotype controls are represented by dotted-line histograms, whereas filled histograms represent the corresponding Ab binding on untreated MDDCs. Solid-line histograms represent receptor expression postincubation (30 min) with the respective inhibitor. The results are representative of MDDCs differentiated from six different donors. (B) Immature DCs (1 × 10⁶) either were left untreated or were treated (30 min, room temperature) with a DC-SIGN-blocking Ab (clone 507; 20 μg/ml), Cyto B (20 μM), Hep (20 mU), or VEGF (50 ng/ml). Cells subsequently were incubated with cell-free HTLV-1 (125 ng/10⁶ cells) for 45 min on ice. Unbound virus particles were removed by excessive washing, and viral binding was examined by flow cytometry using an Alexa Fluor 647-labeled MAb with the HLTV-1 envelope glycoprotein gp46. Following appropriate color compensation, live cells collected for each sample were gated to include the Lin-1⁻/HTLV-1 gp46⁺ population. The control sample represents non-virus-pulsed DCs to exclude the nonspecific effects of the anti-gp46 Ab. The results are representative of one of four independent experiments.

FIG. 6.

Transmission of HTLV-1 from DCs to T cells. The flow-cytometric observations shown in Fig. 4B were further confirmed by a QDot-based binding assay as described in Materials and Methods. (A) MDDCs were pretreated with the specific inhibitors of the receptor and subsequently were incubated with biotin-HTLV-1 (125 ng/10⁶ cells) for 45 min on ice, washed three times, and incubated with Strep-QDot for 30 min. After being washed, cells were fixed and analyzed for HTLV-1 binding as determined by fluorescence measured at 400 or 605 nm. Cells exposed to QDot or biotin alone were used as negative controls. The results shown represent mean fluorescence ± standard deviations from three independent experiments, each performed in duplicate. The asterisk denotes a statistically significant decrease in fluorescence compared to that of maximum binding without any inhibitor (P ≤ 0.05). (B) To examine the role of the cellular receptor molecules in the cell-free transmission of HTLV-1 from DCs to T cells, immature DCs were pretreated with inhibitors as described for panel A and incubated with HTLV-1 (3 μg/10⁶ cells) for 2 h at 37°C, washed, and mixed with equal numbers of autologous T cells. Following 6 days of coculture, cells were incubated with PE-Cy5-labeled anti-CD3 Ab, fixed, permeabilized, and incubated with the Alexa Fluor 647-labeled anti-p19 Ab. A total of 50,000 events collected for each sample were gated to include live CD3⁺/HLTV-1 p19⁺ T cells. The numbers shown indicate the percentage of cells positive for both CD3 and HLTV-1 p19⁺. The results are representative of one of the three independent experiments.

FIG. 7.

Silencing of DC-SIGN expression inhibits HTLV-1 infection of DCs. DC-SIGN was silenced by transfecting DCs with siRNA targeted against DC-SIGN. Nontargeting siRNA was used as a negative control, whereas DCs only with transfection reagent were considered the mock control. (A) To confirm gene silencing, DC-SIGN mRNA levels were determined in GeneSilencer (mock)-transfected cells and cells transfected with nontargeting siRNA or siDC-SIGN. Real-time PCR analysis using DC-SIGN-specific primers to amplify a 370-bp product spanning the silenced region confirmed DC-SIGN silencing in transfected cells. β-Actin was used as an internal control. (B) The effect of silencing also was monitored by the cell surface expression of DC-SIGN. A total of 50,000 events collected for each sample were gated to include the Lin-1⁻/DC-SIGN⁺ population. The dotted-line histogram represents the isotype control, while filled and solid-line histograms represent DC-SIGN expression on the mock-transfected and siDC-SIGN-transfected DCs. (C) DCs silenced for DC-SIGN were infected with cell-free HTLV-1 for 3 days, washed, and cultured in fresh medium for an additional 3 days. The level of HTLV-1 gag protein (p19) in the supernatant of infected DCs was determined by ELISA. Supernatant from uninfected DCs was used as a negative control. A standard curve generated for HTLV-1 p19 antigen was used to determine productive infection in each sample. p19 levels (in picograms/milliliter) are represented as the means ± standard deviations from triplicate samples. The experiment was repeated twice. An asterisk denotes a statistically significant decrease in HTLV-1 p19 levels in siDC-SIGN-transfected DCs compared to that of mock-transfected DCs (P ≤ 0.05).

FIG. 8.

Analysis of DC-SIGN expression following HTLV-1 infection and the role of DC-SIGN in HTLV-1 infection of PBMCs and mDCs. (A) PBMCs obtained from healthy donors either were left untreated, incubated in the absence or presence of cell-free HTLV-1 (3 μg/10⁶ cells) for 6 days at 37°C, or pulsed with recombinant IL-4 (10 ng/ml) for 24 h. Following incubation with the indicated stimuli, cells were examined to determine the effect of HTLV-1 infection and IL-4 pulsing on the expression of DC-SIGN on PMBCs and mDCs (gated on the Lin-1⁻/CD11c⁺ population). A total of 300,000 events were analyzed to determine the geometric MFI and the percentage of cells positive for DC-SIGN. The values of MFI and percent positive cells are shown as means ± standard deviations from six different donors. An asterisk denotes a statistically significant increase in DC-SIGN expression in the presence of HTLV-1 or IL-4 compared to that of untreated control mDCs (P ≤ 0.05). (B) PBMCs also were analyzed for the expression of IL-4 following HTLV-1 infection. Following the fixation and permeabilization of infected cells, the intracellular expression of IL-4 was detected using an APC-labeled MAb. A total of 300,000 events collected for each sample were gated to include live PBMCs shown to be positive for IL-4. The numbers shown indicate the percentage of cells shown to be positive for IL-4 in uninfected and HTLV-1-infected PBMCs, respectively. (C) The role of DC-SIGN in HTLV-1 entry into PBMCs and mDCs was examined by infecting cells with HTLV-1 (3 μg/10⁶ cells) in the absence or presence of the DC-SIGN-blocking Ab (20 μg/ml) for 6 days. The level of intracellular HTLV-1 p19 was determined with Alexa Fluor 647-labeled anti-p19. A total of 300,000 events collected for each sample were gated to include live cells for PBMCs and the Lin-1⁻/CD11c⁺ population for mDCs. The numbers shown indicate the percentage of HLTV-1 p19⁺ cells. The results are representative of three independent experiments.