Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DC-SIGN is a receptor for human herpesvirus 8 on dendritic cells and macrophages.

doi: 10.4049/jimmunol.176.3.1741

Figure Lengend Snippet: FIGURE 3. Infection of DCs with HHV-8 is blocked by anti DC-SIGN mAb. A, Immunofluorescence results on DCs that were left untreated or treated with either anti-DC-SIGN mAb (clone 120507) or anti-CD11a mAb, infected with HHV-8, incubated, and stained with anti-K8.1A/B mAb (red) at 24 h. Uninfected DCs were used as controls. B, Immunoflu- orescence results on DCs that were either treated with anti-DC-SIGN mAb (clone 120507) or left untreated, infected, and stained at 24 h with anti- DC-SIGN mAb (green) and anti-ORF 59 mAb (red). The overlay of com- bined colors for anti-DC-SIGN and ORF 59 is shown. Cells were coun- terstained with DAPI (blue) (600). Data are from one experiment representative of eight independent experiments.

Article Snippet: For blocking studies, cells were pretreated with 20 g/ml anti-human-DC-SIGN mAb (clone 120507; R&D Systems, or clone DCN46; BD Biosciences), anti-CD11a mAb (BD Biosciences), mouse IgG (Sigma-Aldrich), or 100 g/ml mannan (Sigma-Aldrich), for 1 h at 4°C before exposure to HHV-8.

Techniques: Infection, Incubation, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DC-SIGN is a receptor for human herpesvirus 8 on dendritic cells and macrophages.

doi: 10.4049/jimmunol.176.3.1741

Figure Lengend Snippet: FIGURE 4. DC-SIGN expression renders resistant cells susceptible to HHV-8 infection. A, Immunofluorescence results on K562 and K562-DC- SIGN cells that were infected with HHV-8 and stained with anti-K8.1A/B mAb at 24 h (red). B, Immunofluorescence results on B-LCL and B-LCL DC-SIGN that were infected with HHV-8 and stained after 24 h with anti- K8.1A/B mAb (red). Cells were counterstained with DAPI (600). Data are from one experiment representative of five independent experiments.

Article Snippet: For blocking studies, cells were pretreated with 20 g/ml anti-human-DC-SIGN mAb (clone 120507; R&D Systems, or clone DCN46; BD Biosciences), anti-CD11a mAb (BD Biosciences), mouse IgG (Sigma-Aldrich), or 100 g/ml mannan (Sigma-Aldrich), for 1 h at 4°C before exposure to HHV-8.

Techniques: Expressing, Infection, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DC-SIGN is a receptor for human herpesvirus 8 on dendritic cells and macrophages.

doi: 10.4049/jimmunol.176.3.1741

Figure Lengend Snippet: FIGURE 5. HHV-8 binds to DC-SIGN. A, Inhibition of binding of radio- actively labeled HHV-8 by treatment of target cells with anti-DC-SIGN mAb. DCs or B-LCL-DC-SIGN were pretreated with anti-DC-SIGN mAb (clone 120507) or mannan, or left untreated. Each bar represents the mean percent of binding inhibition ( SE) (compared with untreated cells) from two duplicate determinations. B, Inhibition of binding of radioactively labeled HHV-8 by treatment of virus with soluble DC-SIGN. Results are the mean ( SE) per- centage of inhibition of binding of soluble DC-SIGN-treated HHV-8 com- pared with binding of radiolabeled untreated virus to each cell type from two determinations. C, Dose response of inhibition of virus binding to DCs by treatment with anti-DC-SIGN mAb. Each bar represents the mean of duplicate reactions ( SE) from duplicate determinations. Data are from one experiment representative of three independent experiments.

Article Snippet: For blocking studies, cells were pretreated with 20 g/ml anti-human-DC-SIGN mAb (clone 120507; R&D Systems, or clone DCN46; BD Biosciences), anti-CD11a mAb (BD Biosciences), mouse IgG (Sigma-Aldrich), or 100 g/ml mannan (Sigma-Aldrich), for 1 h at 4°C before exposure to HHV-8.

Techniques: Inhibition, Binding Assay, Labeling, Virus

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DC-SIGN is a receptor for human herpesvirus 8 on dendritic cells and macrophages.

doi: 10.4049/jimmunol.176.3.1741

Figure Lengend Snippet: FIGURE 6. HHV-8 infection of IL-13-activated macrophages is related to DC-SIGN expression. A, Flow cytometric analysis showing expression of DC-SIGN on HHV-8-infected (empty histogram, broken line) or un- infected (empty histogram, solid line) IL-13-activated macrophages. Full histogram, isotype controls. B, Im- munofluorescence results on IL-13-activated macro- phages that were infected with HHV-8 for 24 h and stained for ORF 59 (red) and DC-SIGN (green). The overlay of combined colors for anti-DC-SIGN and ORF59 is shown. C, Immunofluorescence results on IL- 13-activated macrophages that were pretreated with anti- DC-SIGN mAb (clone 120507) or mouse IgG, infected with HHV-8 for 24 h and stained for anti-K8.1 mAb (red). Cells were counterstained with DAPI (600). Data are from one experiment representative of four in- dependent experiments.

Article Snippet: For blocking studies, cells were pretreated with 20 g/ml anti-human-DC-SIGN mAb (clone 120507; R&D Systems, or clone DCN46; BD Biosciences), anti-CD11a mAb (BD Biosciences), mouse IgG (Sigma-Aldrich), or 100 g/ml mannan (Sigma-Aldrich), for 1 h at 4°C before exposure to HHV-8.

Techniques: Infection, Expressing, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DC-SIGN is a receptor for human herpesvirus 8 on dendritic cells and macrophages.

doi: 10.4049/jimmunol.176.3.1741

Figure Lengend Snippet: FIGURE 7. Effect of HHV-8 in- fection of DCs on expression of DC- SIGN and costimulatory molecules. A, DC-SIGN expression on unin- fected or HHV-8 infected DCs. Data are mean MFI (SE) from seven in- dependent experiments. B, Expres- sion of HLA-ABC, HLA-DR, CD83, and DC-SIGN on infected DCs. Blue histogram, HHV-8-infected DCs; yellow histogram, uninfected DCs; empty histogram, fine line, and bro- ken line isotype controls for the infected and uninfected DCs, respec- tively. Data are from one experiment representative of 10 independent ex- periments. C, Confocal microscopy of HHV-8-infected DCs stained with anti-DC-SIGN (green) and anti-ORF 59 (red) mAbs at 24 h (left panel) and 48 h (center panel) after infection. Uninfected DCs served as controls (right panel). Data are from one ex- periment representative of two inde- pendent experiments.

Article Snippet: For blocking studies, cells were pretreated with 20 g/ml anti-human-DC-SIGN mAb (clone 120507; R&D Systems, or clone DCN46; BD Biosciences), anti-CD11a mAb (BD Biosciences), mouse IgG (Sigma-Aldrich), or 100 g/ml mannan (Sigma-Aldrich), for 1 h at 4°C before exposure to HHV-8.

Techniques: Expressing, Infection, Confocal Microscopy, Staining

Journal: Cellular and Molecular Life Sciences

Article Title: Glucosylceramide in bunyavirus particles is essential for virus binding to host cells

doi: 10.1007/s00018-023-05103-0

Figure Lengend Snippet: Glucosylceramide (GlcCer) in viral particles promotes Uukuniemi virus (UUKV) binding. A UUKV particles derived from BHK-21 cells in the presence of DL- threo -phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, 2.5 µM) were bound to freshly seeded naïve BHK-21 cells for 2 h on ice before fixation and western blot analysis with an antibody recognizing the UUKV N protein. B N was semi-quantified from the cells described in A , and the value is presented as a percentage of the N level measured in the sample corresponding to virus binding in the absence of PPMP ( n = 8). C Alternatively, UUKV particles produced in the presence or the absence of PPMP were allowed to bind to BHK-21 cells for 2 h on ice, and binding was assessed by measuring the BHK-21 cell-associated S viral segment by RT-qPCR ( n = 6). D Fluorescently labeled UUKV particles (UUKV-ATTO488) were bound to BHK-21 cells [multiplicity of infection (MOI) ~ 4] on ice for 1 h, and viral binding was evaluated by flow cytometry analysis. E and F BHK-21 cells, A549 human lung epithelial cells, and BHK-21 cells expressing the UUKV receptor DC-SIGN (BHK-21 DC-SIGN +) were preincubated with varying amounts of soluble C6-GlcCer E or C6-Cer F for 2 h and then exposed to UUKV-ATTO488 (MOI ~ 4) on ice for 1 h. Virus binding was measured by flow cytometry, and the data were normalized to those in control samples processed in the absence of soluble C6-GlcCer or C6-Cer. An one-way ANOVA with Dunnett’s multiple comparison test was applied ( n ≥ 3). *, p < 0.05; **, p < 0.01; ns, not significant; RFI, relative fluorescence intensity. G BHK-21 cells were transduced with a retroviral vector system to express DC-SIGN (BHK-21 DC-SIGN +). DC-SIGN expression was measured by flow cytometry analysis using phycoerythrin-conjugated anti-DC-SIGN mAb. H Soluble C6-GlcCer was allowed to bind BHK-21 cells on ice for 2 h before exposure to UUKV for 1 h (MOI ~ 0.5). After virus binding on ice, unbound UUKV particles were washed away, and the cells were incubated at 37 ℃ for 8 h. Infection was quantified by flow cytometry after immunostaining for UUKV N protein. Values are presented as the percentage of the control sample without prebinding of soluble C6-GlcCer ( n = 4)

Article Snippet: The location of DC-SIGN was assessed at the surface of BHK-21 cells (not permeabilized) by flow cytometry using an anti-DC-SIGN phycoerythrin-conjugated antibody (FAB1621P R&D Systems) according to a standard procedure [ ].

Techniques: Virus, Binding Assay, Derivative Assay, Western Blot, Produced, Quantitative RT-PCR, Labeling, Infection, Flow Cytometry, Expressing, Control, Comparison, Fluorescence, Transduction, Retroviral, Plasmid Preparation, Incubation, Immunostaining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Internalizing antibodies to the C-type lectins, L-SIGN and DC-SIGN, inhibit viral glycoprotein binding and deliver antigen to human dendritic cells for the induction of T cell responses.

doi: 10.4049/jimmunol.176.1.426

Figure Lengend Snippet: FIGURE 1. Screening of phage-displayed Fabs for L-SIGN reactivity. A, Ninety-six Fab phage clones selected from three rounds of pan- ning on recombinant DC-SIGN (negative selec- tion) and L-SIGN (positive selection) proteins were screened for binding to K562 (negative control) and K562/L-SIGN-transfected cells by flow cytometry using goat anti-mouse IgG PE conjugate for detection. Only clones showing at least a 5-fold higher binding to K562/L-SIGN cells are shown. B, To select Fab phage clones uniquely reactive with L-SIGN, but not DC- SIGN, K562/L-SIGN cell-reactive clones were screened for reactivity with DC-SIGN-Fc and L- SIGN-Fc fusion proteins in a capture ELISA. mAb162 (L-SIGN specific), mAb1621, and mAb16211 (DC-SIGN/L-SIGN cross-reactive) were used as positive controls. Data are repre- sentative of two independent experiments.

Article Snippet: Before their use in assays, RBC were lysed and any remaining dead cells were further depleted using a dead cell removal kit (Miltenyi Biotec; catalog 130-090-101), per the manufacturer’s instructions. mAbs mAb162 (reactive only with L-SIGN), mAb1621, and mAb16211 (cross-reactive with DC-SIGN and L-SIGN) were purchased from R&D Systems.

Techniques: Clone Assay, Recombinant, Selection, Binding Assay, Negative Control, Transfection, Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Internalizing antibodies to the C-type lectins, L-SIGN and DC-SIGN, inhibit viral glycoprotein binding and deliver antigen to human dendritic cells for the induction of T cell responses.

doi: 10.4049/jimmunol.176.1.426

Figure Lengend Snippet: FIGURE 2. Receptor specificity analysis of soluble L-SIGN Fabs. A, 5 105 K562, K562/DC-SIGN, or K562/L-SIGN cells were incubated with pu- rified soluble Fabs (20 g/ml) representing six unique clones for 1 h, and the extent of their binding was assessed by flow cytometry using goat anti-mouse IgG PE conjugate. Values represent mean (bars, SD) of two independent ex- periments. B, Histograms showing similar expression of SIGN molecules on K562 cells after staining with SIGN-cross-reactive mAb16211 (20 g/ml) and detecting with goat anti-mouse IgG PE conjugate.

Article Snippet: Before their use in assays, RBC were lysed and any remaining dead cells were further depleted using a dead cell removal kit (Miltenyi Biotec; catalog 130-090-101), per the manufacturer’s instructions. mAbs mAb162 (reactive only with L-SIGN), mAb1621, and mAb16211 (cross-reactive with DC-SIGN and L-SIGN) were purchased from R&D Systems.

Techniques: Incubation, Clone Assay, Binding Assay, Cytometry, Expressing, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Internalizing antibodies to the C-type lectins, L-SIGN and DC-SIGN, inhibit viral glycoprotein binding and deliver antigen to human dendritic cells for the induction of T cell responses.

doi: 10.4049/jimmunol.176.1.426

Figure Lengend Snippet: FIGURE 5. Conversion of Fab to IgG improves receptor binding and blocking of viral protein adhesion. A, K562, K562/DC-SIGN, and K562/ L-SIGN cells were incubated with purified Abs, and the extent of their binding was assessed by flow cytometry using PE-conjugated goat anti- mouse (Fab detection) or goat anti-human (IgG detection) secondary Abs. mAb162 (L-SIGN specific) and mAb16211 (DC-SIGN/L-SIGN cross-re- active) were used as positive controls. B, K562/L-SIGN and K562/DC- SIGN cells were incubated with fluorescent beads coated with HIVgp120 in the presence of Abs and assessed by flow cytometry. C, Same as in B, except that Ebola envelope glycoprotein-coated beads were used instead of HIVgp120-coated beads. mAb162 (L-SIGN specific) and mAbAZND1 (DC-SIGN specific) were used as controls for comparison. Percentage of binding is determined as 100 times the number of cells bound to protein- coated beads with Ab divided by the number of cells bound to protein- coated beads without Ab. Values represent mean (bars, SD) of four inde- pendent experiments. , Highly significant values (p 0.00005) compared with samples not treated with Abs.

Article Snippet: Before their use in assays, RBC were lysed and any remaining dead cells were further depleted using a dead cell removal kit (Miltenyi Biotec; catalog 130-090-101), per the manufacturer’s instructions. mAbs mAb162 (reactive only with L-SIGN), mAb1621, and mAb16211 (cross-reactive with DC-SIGN and L-SIGN) were purchased from R&D Systems.

Techniques: Binding Assay, Blocking Assay, Incubation, Cytometry, Comparison