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: Cell Communication and Signaling : CCS

Article Title: The transcription factor MITF is a critical regulator of GPNMB expression in dendritic cells

doi: 10.1186/s12964-015-0099-5

Figure Lengend Snippet: PI3K/Akt-inhibition upregulates GPNMB gene expression in human moDC. Immature moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional TKI (3 μM imatinib or 3 μM nilotinib) or inhibitors of signal transduction (300 nM Akt inhibitor MK2206 (Akt-inh.), 300 nM Erk inhibitor FR180204 (Erk-inh.), 100 nM PI3K inhibitor LY294002 (PI3K-inh.), 20 nM c-Raf inhibitor 553003 (c-Raf-inh.)) and analyzed for GPNMB expression. Exemplary results from at least three independent experiments using different donors are presented. (A) qRT-PCR analysis: relative level of GPNMB mRNA. The mean (±SD) of duplicate measurements is shown. (B, C) GPNMB protein level of CD209 + moDC (of three different donors) was analyzed by flow cytometry. Where indicated, maturation of moDC was induced by LPS. Data were analyzed using FlowJo software and Difference in Median Fluorescence Intensity (DMFI) of CD209 + cells is shown in the upper right quadrants. (D) Phenotypic changes of immature moDC in the absence (4/GM) or presence of nilotinib or Akt inhibitor were analyzed by flow cytometry. Double stainings were performed with monoclonal antibodies recognizing CD209, CD1a or CD14. DMFI of CD209 + cells is shown in the upper right quadrants.

Article Snippet: After 7 days of culture, if necessary, DC-SIGN + (CD209 + ) moDC were enriched to > 90% purity prior to qRT-PCR and western blot analyses (CD209 MicroBead Kit, Miltenyi).

Techniques: Inhibition, Expressing, Generated, In Vitro, Transduction, Quantitative RT-PCR, Flow Cytometry, Software, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: The transcription factor MITF is a critical regulator of GPNMB expression in dendritic cells

doi: 10.1186/s12964-015-0099-5

Figure Lengend Snippet: The BCR-ABL TKI imatinib and nilotinib or IL-10 inhibit phosphorylation of Akt in human moDC. Western blot analysis of total Akt levels and its phosphorylated form in purified immature CD209 + moDC (of four different donors). moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional (A) imatinib (3 μM), (B) nilotinib (3 μM), (C) Akt inhibitor MK2206 (Akt-inh., 300 nM) or (D) IL-10 (10 ng/mL). Indicated time refers to further treatment of cells prior to cell lysis (see ). (E-G) GPNMB protein levels in moDC were analyzed by western blotting. GAPDH served as loading control. Exemplary results from at least three independent experiments using different donors are presented.

Article Snippet: After 7 days of culture, if necessary, DC-SIGN + (CD209 + ) moDC were enriched to > 90% purity prior to qRT-PCR and western blot analyses (CD209 MicroBead Kit, Miltenyi).

Techniques: Western Blot, Purification, Generated, In Vitro, Lysis

Journal: Cell Communication and Signaling : CCS

Article Title: The transcription factor MITF is a critical regulator of GPNMB expression in dendritic cells

doi: 10.1186/s12964-015-0099-5

Figure Lengend Snippet: Imatinib, nilotinib, IL-10 or Akt inhibitor prevent phosphorylation of GSK3ß in human moDC. Western blot analysis of total GSK3ß and GSK3α, as well as their phosphorylated forms in purified immature CD209 + moDC (of four different donors). moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional (A) imatinib (3 μM), (B) nilotinib (3 μM), (C) Akt inhibitor MK2206 (Akt-inh., 300 nM) or (D) IL-10 (10 ng/mL). Indicated time refers to further treatment of cells prior to cell lysis (see ). GAPDH served as loading control. Exemplary results from at least three independent experiments using different donors are presented.

Article Snippet: After 7 days of culture, if necessary, DC-SIGN + (CD209 + ) moDC were enriched to > 90% purity prior to qRT-PCR and western blot analyses (CD209 MicroBead Kit, Miltenyi).

Techniques: Western Blot, Purification, Generated, In Vitro, Lysis

Journal: Cell Communication and Signaling : CCS

Article Title: The transcription factor MITF is a critical regulator of GPNMB expression in dendritic cells

doi: 10.1186/s12964-015-0099-5

Figure Lengend Snippet: Upon treatment of moDC with imatinib, nilotinib, IL-10 or MK2206, MITF translocates into the nucleus. Western blot analysis of MITF level and phosphorylation status in the cytoplasmic or nuclear fraction of purified immature CD209 + moDC. moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with (A) imatinib (3 μM), (B) nilotinib (3 μM), (C) Akt inhibitor MK2206 (Akt-inh.; 300 nM) or (D) IL-10 (10 ng/mL). (E) Cells were treated with nilotinib (3 μM). Indicated time refers to further treatment of cells prior to cell lysis (see ). GAPDH served as loading control. Exemplary results from at least three independent experiments using different donors are presented.

Article Snippet: After 7 days of culture, if necessary, DC-SIGN + (CD209 + ) moDC were enriched to > 90% purity prior to qRT-PCR and western blot analyses (CD209 MicroBead Kit, Miltenyi).

Techniques: Western Blot, Purification, Generated, In Vitro, Lysis

Journal: Traffic (Copenhagen, Denmark)

Article Title: Differential Use of the C-Type Lectins L-SIGN and DC-SIGN for Phlebovirus Endocytosis.

doi: 10.1111/tra.12393

Figure Lengend Snippet: Figure 6: Efficiency of DC-SIGN and L-SIGN in mediating UUKV infection. Expression of either A) DC-SIGN or B) L-SIGN at the surface of Raji and HeLa cell lines (Raji DC-SIGN+, HeLa DC-SIGN+, Raji L-SIGN+ and HeLa L-SIGN+) was detected using flow cytometry after immunostaining with the Fab 1621P, which recognizes the CRD of both L-SIGN and DC-SIGN. C) DC-SIGN- and L-SIGN-expressing Raji cells were infected with UUKV at different MOIs and analyzed 20 h post-infection using flow cytometry after immunostaining against the UUKV nucleoprotein N. Infection is expressed as the percentage of N-positive cells (% of total). D) Infection of HeLa cell lines expressing either DC-SIGN or L-SIGN with UUKV was performed, monitored and analyzed following the same procedure as that in (C).

Article Snippet: The anti-DC-SIGN/L-SIGN mouse mAb 1621 and the anti-DC-SIGN mouse mAb 162 as well as the phycoerythrin (PE)- and fluorescein isothiocyanate (FITC)-conjugated Fab 1621P (anti-DC-SIGN/L-SIGN) and Fab 162F (anti-L-SIGN) were all purchased from R&D Systems.

Techniques: Infection, Expressing, Cytometry, Immunostaining

Journal: Traffic (Copenhagen, Denmark)

Article Title: Differential Use of the C-Type Lectins L-SIGN and DC-SIGN for Phlebovirus Endocytosis.

doi: 10.1111/tra.12393

Figure Lengend Snippet: Figure 7: UUKV internalization and endosomal escape into DC-SIGN- and L-SIGN-expressing cells. A) Expression of DC-SIGN and its endocytosis-defective mutant was detected on HeLa cells (HeLa DC-SIGN+ or HeLa DC-SIGNLL+ respectively) using flow cytometry after immunostaining with the Fab 1621P. B) HeLa cells that express L-SIGN, DC-SIGN, and the corresponding endocytosis-defective mutants (L-SIGNLL or DC-SIGNLL, respectively) were assessed for UUKV replication 7 h post-infection (MOI ∼1). Cells positive for the UUKV protein N were quantified using flow cytometry. Infection was normalized to the infected HeLa cells expressing the WT lectin (% of control). C) HeLa DC-SIGN+ and HeLa-L-SIGN+ cells were exposed to UUKV (MOI ∼3) on ice, washed and rapidly warmed. NH4Cl (50 mM) was added at different times to block further penetration. Infection was detected using flow cytometry 16 h later and normalized to infection in the absence of NH4Cl.

Article Snippet: The anti-DC-SIGN/L-SIGN mouse mAb 1621 and the anti-DC-SIGN mouse mAb 162 as well as the phycoerythrin (PE)- and fluorescein isothiocyanate (FITC)-conjugated Fab 1621P (anti-DC-SIGN/L-SIGN) and Fab 162F (anti-L-SIGN) were all purchased from R&D Systems.

Techniques: Expressing, Mutagenesis, Cytometry, Immunostaining, Infection, Control, Blocking Assay

Journal: Cells

Article Title: SARS-CoV-2 Spike Protein and Its Receptor Binding Domain Promote a Proinflammatory Activation Profile on Human Dendritic Cells

doi: 10.3390/cells10123279

Figure Lengend Snippet: Dendritic cell activation in response to Spike protein and the RBD is DC-SIGN independent. iDCs were preincubated with an anti-DC-SIGN antibody, stimulated as indicated for 24 h, and then stained and analyzed by flow cytometry. ( A ) CD83 and ( B ) CD86 expression in DCs with the indicated treatment are shown. Graphs show percentage of positivity (left) and GMFI (right) of each marker. Graphs show mean ± SEM; n = 5 different donors.

Article Snippet: Blocking assay was performed incubating iDCs (1 × 10 6 /mL) in complete medium with anti-DC-SIGN antibody (40 μg/mL) (Cat# MAB16211, RD Systems) at 37 °C, for 30 min. After two washes, cells were stimulated as indicated during 24 h and then analyzed.

Techniques: Activation Assay, Staining, Flow Cytometry, Expressing, Marker

Journal: bioRxiv

Article Title: The unique ORF8 protein from SARS-CoV-2 binds to human dendritic cells and induces a hyper-inflammatory cytokine storm

doi: 10.1101/2022.06.06.494969

Figure Lengend Snippet: a) Experimental set up: monocytes were differentiated into immature dendritic cells in presence or absence of ORF8; b) Flow cytometry analysis of the effect of ORF8 on the expression of MHCII, CD80, CD83, CD86, CD40 and CD11c during differentiation of monocytes into immature dendritic cells with IL-4 and GM-CSF compared to only IL-4+GM-CSF differentiated monocytes (blue line); control: monocytes without IL-4+GM-CSF treatment (black line); representative flow cytometry histograms (n=6 donors) c) Flow cytometry analysis of the effect of ORF8 on the expression of DC-SIGN during differentiation of monocytes into immature dendritic cells with IL-4 and GM-CSF (red line) compared to only IL-4+GM-CSF differentiated monocytes (blue line); control: monocytes without IL-4+GM-CSF treatment (black line); representative flow cytometry histograms (n=3 donors) d) Flow cytometry analysis of the effect of the blockade of ORF8 by an inhibitory anti-ORF8 antibody (α-ORF8-iAB) on the expression of DC-SIGN on differentiated dendritic cells compared to only IL-4+GM-CSF differentiated monocytes (blue line) and IL-4+GM-CSF+ORF8 differentiated monocytes (red line); control: monocytes without IL-4+GM-CSF treatment (black line; for isotype control see supplemental ); representative flow cytometry histograms (n=3 donors); e) Immunoprecipitations (IP) of recombinant DC-SIG with 1) no protein control, recombinant ORF8, ORF8 + an inhibitory anti-ORF8 antibody (αORF8), 2) single recombinant proteins (single protein) ORF8 and recombinant human DC-SIGN (positive control) were immunoblotted (IB) with anti-DC-SIGN antibody; * indicates reduced signal intensity compared to ORF8 alone

Article Snippet: For protein-protein interaction, pre-incubated beads were incubated a second time with 2 µg of a recombinant human DC-SIGN protein with a human Fc tag (Sino Biological, Köln, Germany) for 1 h at 4°C with agitation.

Techniques: Flow Cytometry, Expressing, Recombinant, Positive Control

Journal: Cell reports

Article Title: TRIM5α Restricts Flavivirus Replication by Targeting the Viral Protease for Proteasomal Degradation

doi: 10.1016/j.celrep.2019.05.040

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Expression plasmids for Langerin (HG13040-UT) and DC-SIGN (HG10200-UT) were purchased from Sino Biological.

Techniques: Transduction, Recombinant, Modification, Protease Inhibitor, Staining, Transfection, Clone Assay, Construct, shRNA, Sequencing, Luciferase, Plasmid Preparation, Expressing, Software, Imaging, Western Blot

Journal: mSphere

Article Title: Term Human Placental Trophoblasts Express SARS-CoV-2 Entry Factors ACE2, TMPRSS2, and Furin

doi: 10.1128/mSphere.00250-21

Figure Lengend Snippet: The ACE2-dependent entry of SARS-CoV-2 pseudovirus into cultured PHT cells. (A) PHT (five donors) and 293T-ACE2 cells inoculated with SARS-CoV-2 protein pseudotyped lentiviruses containing SMNE proteins (spike, membrane, nucleocapsid, and envelope) or MNE proteins, as detailed in Materials and Methods. Cells, harvested on day 2 postinoculation with the virus, were washed three times with PBS and trypsinized to remove adherent virus, and the cell pellet was lysed to release intracellular p24. Shown is p24 content (in picograms per milliliter) in cell lysates. *, P < 0.01 (paired t test). (Inset) 293T cells expressing ACE2, assessed at 2 days or 5 days ( n = 2 for each) and serving as a positive control. (B) PHT were preincubated or not with 20 μg/ml of anti-ACE-2 or anti-DC-SIGN antibody (Ab) and then inoculated with pvSARS-CoV-2 S+. The PHT cells were also inoculated with pseudovirus lacking S protein (pvS-) or with inactivated HIV-1 as negative controls. Cells were harvested on day 2 postinoculation, and p24 content (in picograms per milliliter) was measured in cell lysates, as detailed in Materials and Methods. *, P < 0.05 (ANOVA and Tukey test, n = 5).

Article Snippet: For antibody inhibition experiments, PHT cells were preexposed to 20 μg/ml anti-ACE2 (catalog no. AF933; R&D Systems, Minneapolis, MN ) or anti-DC-SIGN (R&D catalog no. MAB161) for 30 min at 37°C before inoculation with the viruses ( , ).

Techniques: Cell Culture, Expressing, Positive Control