Journal: iScience

Article Title: Single-cell RNA-seq analysis reveals dual sensing of HIV-1 in blood Axl + dendritic cells

doi: 10.1016/j.isci.2023.106019

Figure Lengend Snippet:

Article Snippet: CD169 (Siglec-1) Antibody, anti-human, PE-Vio770 Clone 7-239 , Miltenyi Biotec , Cat# 130-098-640, RRID: AB_2655549.

Techniques: Virus, Infection, Recombinant, Transfection, Electron Microscopy, Staining, Cell Isolation, Purification, Reverse Transcription, SYBR Green Assay, Sequencing, Software, FCAP Assay

Journal: Scientific Reports

Article Title: IgE-mediated enhancement of CD4 + T cell responses requires antigen presentation by CD8α − conventional dendritic cells

doi: 10.1038/srep28290

Figure Lengend Snippet: ( a ) BALB/c mice were immunized with 50 μg IgE anti-OVA pre-mixed with 20 μg OVA (n = 7), or 20 μg OVA alone (n = 7). Sera from d 7, 21, and 35 after immunization were analysed for IgG anti-OVA by ELISA. ( b ) BALB/c mice were adoptively transferred with splenocytes from DO11.10 mice one day before administration of 50 μg IgE anti-OVA pre-mixed with 20 μg OVA (n = 3) or 20 μg OVA alone (n = 3). Spleens were harvested 3 days after immunization and half of each spleen was analysed for proliferation of OVA-specific CD4 + T cells by flow cytometry. The gating strategy is shown in . Percentages of KJ1-26 + CD4 + T cells among total CD4 + T cells of each group were then quantified. ( c ) The other half of each spleen as in ( b ) was frozen and spleen sections were stained and analysed by confocal microscopy. B220, blue; CD169, grey; DO11.10 TCR, red. Images show T cell areas (640 μm × 640 μm) representative of 6 T cell zones from 2 non-consecutive sections per sample in each group. Scale bar represents 100 μm. ( a,b ) Data are representative of three independent experiments and are shown as mean + SEM. Significance was determined between the group immunized with IgE-OVA complexes and the group immunized with OVA alone by Student’s t -test. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Sections were fixed in 4% paraformaldehyde (Merck, Darmstadt, Germany) for 15 min before blocking with 5% horse serum (Sigma-Aldrich) for 30 min. Pacific Blue-labeled anti-CD45R/B220 (clone RA3-6B2; BD Biosciences) for staining B cells and FITC- or Alexa Fluor 647-labeled anti-CD169 (clone MOMA-1; AbD Serotec, Oxford, UK) for staining metallophilic macrophages in the marginal zone were used.

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Confocal Microscopy

Journal: Scientific Reports

Article Title: IgE-mediated enhancement of CD4 + T cell responses requires antigen presentation by CD8α − conventional dendritic cells

doi: 10.1038/srep28290

Figure Lengend Snippet: BALB/c mice were immunized with 50 μg IgE anti-OVA pre-mixed with 150 μg OVA-Alexa 647 (n = 2 per time point) ( a–h ), 150 μg OVA-Alexa 647 alone (n = 2 per time point) ( i–p ), or left unimmunized ( q ). Spleens were harvested 0.5, 2, 4, or 24 h after immunization. Non-consecutive sections of spleens were stained and analysed by confocal microscopy. B220 + B cells, blue; CD169 + metallophilic macrophages, green; OVA-Alexa 647, red. ( a–d , i–l,q ) All colors are shown. ( e–h,m–p ) All colors expect blue are shown. ( a–q ) Images show follicular areas (640 μm × 640 μm) representative of 3–4 follicular areas from 2 non-consecutive sections per sample in each group. Scale bar represents 100 μm. Data represent one experiment at 0.5 h and 2 h and two experiments at 4 h and 24 h. ( r ) Quantification of the Ag + area within the B220 + follicular area. ( s ) Percentages of Ag + cells among follicular B cells was analysed by flow cytometry on splenocytes from the other half of each spleen in ( a–p ). Follicular B cells are gated as B220 + CD21 + CD23 high cells . ( r,s ) Data are shown as mean + SEM. Significance was determined between the group immunized with IgE-OVA complexes and the group immunized with OVA alone by Student’s t -test. *p < 0.05; ***p < 0.001.

Article Snippet: Sections were fixed in 4% paraformaldehyde (Merck, Darmstadt, Germany) for 15 min before blocking with 5% horse serum (Sigma-Aldrich) for 30 min. Pacific Blue-labeled anti-CD45R/B220 (clone RA3-6B2; BD Biosciences) for staining B cells and FITC- or Alexa Fluor 647-labeled anti-CD169 (clone MOMA-1; AbD Serotec, Oxford, UK) for staining metallophilic macrophages in the marginal zone were used.

Techniques: Staining, Confocal Microscopy, Flow Cytometry

Journal: Scientific Reports

Article Title: IgE-mediated enhancement of CD4 + T cell responses requires antigen presentation by CD8α − conventional dendritic cells

doi: 10.1038/srep28290

Figure Lengend Snippet: Spleens from BALB/c mice (n = 2 per time point) immunized with 250 μg IgE anti-OVA pre-mixed with 100 μg OVA or with 100 μg OVA alone were harvested after 0.5, 4, 8, 24, or 48 h. One unimmunized mouse (Nil) was used as control. ( a–k ) Half of each spleen was snap-frozen and non-consecutive spleen sections were stained and analyzed by confocal microscopy. Localization of DCIR2 + cDCs in spleens harvested at indicated time points after immunization was followed. B220 + B cells, blue; CD169 + metallophilic macrophages, grey; DCIR2 + cDCs, red. Marginal zone bridging channels are indicated with arrows in ( a,g ). Images show representative areas (640 μm × 640 μm) of 3–4 T cell zones from 2 non-consecutive sections of each sample in every group. Scale bar represents 100 μm. Data represent one experiment where mice were immunized with IgE-OVA or OVA alone and one where they were immunized with IgE-OVA. ( l ) The other halves of the spleens from mice immunized with IgE-OVA complexes in ( a–e ) were prepared into single cell suspensions and 6 × 10 5 cells were used as APCs in co-cultures with 10 5 CFSE-labeled CD4 + T cells isolated from DO11.10 splenocytes. Percentages of divided cells among OVA-specific CD4 + T cells after incubation for 3 days with APCs taken from an unimmunized mouse (Nil) or from mice immunized with IgE-OVA complexes are quantified by flow cytometry as shown in . CD4 + T cells cultured alone were used as negative control. Each circle represents one mouse and the lines represent the mean values.

Article Snippet: Sections were fixed in 4% paraformaldehyde (Merck, Darmstadt, Germany) for 15 min before blocking with 5% horse serum (Sigma-Aldrich) for 30 min. Pacific Blue-labeled anti-CD45R/B220 (clone RA3-6B2; BD Biosciences) for staining B cells and FITC- or Alexa Fluor 647-labeled anti-CD169 (clone MOMA-1; AbD Serotec, Oxford, UK) for staining metallophilic macrophages in the marginal zone were used.

Techniques: Staining, Confocal Microscopy, Labeling, Isolation, Incubation, Flow Cytometry, Cell Culture, Negative Control

Journal: Multiple sclerosis (Houndmills, Basingstoke, England)

Article Title: CD169 is a marker for highly pathogenic phagocytes in multiple sclerosis.

doi: 10.1177/1352458517698759

Figure Lengend Snippet: Figure 1. CD169-expressing phagocytes are increased in the circulation and CNS of EAE animals. (a and b) Spinal cord tissue of EAE animals (20 dpi) stained for (a) CD169 and Iba1 or (b) CD169 and CD11b/c (both 20× magnification). (c) Spinal cord tissue of EAE animals (30 dpi) stained for CD169 and Iba1 (40× magnification). (d) Quantification of the amount of CD169+ cells per image and (e) the percentage of Iba1+ cells expressing CD169 in spinal cord tissue of control and EAE mice (acute (20 dpi) and chronic phase (30 dpi)). For quantification, 20 images per animal with 9 animals per time point were used (20× magnification). (f and g) PBMC isolated from control (n = 7) and EAE mice (acute phase (20 dpi), n = 6; chronic phase (30 dpi), n = 9) were stained for CD14 and CD169. Flow cytometry was used to assess the (f) % of CD14+ cells expressing CD169 and (g) the abundance of CD169 on these cells. Data are presented as mean ± SEM.

Article Snippet: For flow-cytometric analysis of PBMCs, the following antibodies were used: rat-anti-mouse CD169 FITC (Abd Serotec), rat-anti mouse CD14 PE (eBioscience, San Diego, CA), mouse anti-human CD14 PerCP (BD Biosciences, Erembodegem, Belgium), mouse anti-human CD56 PE-Cy7 (BD biosciences), mouse anti-human CD169 FITC (Abd Serotec), and mouse anti-human CD7 PE (BD Biosciences).

Techniques: Expressing, Staining, Control, Isolation, Flow Cytometry

Journal: Multiple sclerosis (Houndmills, Basingstoke, England)

Article Title: CD169 is a marker for highly pathogenic phagocytes in multiple sclerosis.

doi: 10.1177/1352458517698759

Figure Lengend Snippet: Figure 2. Transient depletion of CD169-expressing cells reduces EAE severity. (a) CD169-DTR mice and WT mice were sacrificed 2 or 8 days after intraperitoneal DTR administration. Splenic sections were stained with CD169 and DAPI (20× magnification). One representative image is shown. (b) Spinal cord tissue of PBS- and DT-treated CD169- DTR EAE animals (30 dpi) stained for CD169 and CD11b/c (20× magnification). EAE animals were treated with PBS or DT every other 5 days, starting 5 dpi. (c) Images show CD169 immunoreactivity on phagocytes 5 days post the last DT treatment. MOG-immunized CD169-DTR animals were treated every other 5 days (arrows) starting 5 dpi with PBS (n = 4; black) or diphtheria toxin (DT, n = 8). Neurological score and weight was assessed daily. Data represent the mean ± SEM. (d) The cumulative disease index and (e) disease onset of immunized CD169-DTR mice treated with PBS or DT. Data represent the mean ± SEM.

Article Snippet: For flow-cytometric analysis of PBMCs, the following antibodies were used: rat-anti-mouse CD169 FITC (Abd Serotec), rat-anti mouse CD14 PE (eBioscience, San Diego, CA), mouse anti-human CD14 PerCP (BD Biosciences, Erembodegem, Belgium), mouse anti-human CD56 PE-Cy7 (BD biosciences), mouse anti-human CD169 FITC (Abd Serotec), and mouse anti-human CD7 PE (BD Biosciences).

Techniques: Expressing, Staining

Journal: Multiple sclerosis (Houndmills, Basingstoke, England)

Article Title: CD169 is a marker for highly pathogenic phagocytes in multiple sclerosis.

doi: 10.1177/1352458517698759

Figure Lengend Snippet: Figure 3. The number of circulating CD169+ phagocytes is increased in MS patients. (a and b) PBMCs isolated from MS patients (n = 57) and healthy controls (n = 19) were stained for CD14 and CD169. Flow cytometry was used to assess (a) the percentage of CD14+ cells expressing CD169 and (b) the abundance of CD169 on these cells. MS patients were subdivided based on their treatment regime: no treatment (n = 19), natalizumab (n = 10), beta-interferon (n = 22), and alemtuzumab (n = 6). Data are presented as mean ± SEM.

Article Snippet: For flow-cytometric analysis of PBMCs, the following antibodies were used: rat-anti-mouse CD169 FITC (Abd Serotec), rat-anti mouse CD14 PE (eBioscience, San Diego, CA), mouse anti-human CD14 PerCP (BD Biosciences, Erembodegem, Belgium), mouse anti-human CD56 PE-Cy7 (BD biosciences), mouse anti-human CD169 FITC (Abd Serotec), and mouse anti-human CD7 PE (BD Biosciences).

Techniques: Isolation, Staining, Flow Cytometry, Expressing

Journal: Multiple sclerosis (Houndmills, Basingstoke, England)

Article Title: CD169 is a marker for highly pathogenic phagocytes in multiple sclerosis.

doi: 10.1177/1352458517698759

Figure Lengend Snippet: Figure 4. CD169 is highly expressed in MS brain tissue. (a–g) CNS tissue of non-neurological controls (white matter) and (chronic) active MS lesions were stained for CD169. Overview images of (a) an active and (b) chronic active MS lesion (2.5× magnification). (c) NAWM of non-demented control stained for CD169 (40× magnification). Representative images of (d) perilesional area of active MS lesion, (e) lesion center of active MS lesion, (f) perivascular cuff of active MS lesion, (g) active rim of chronic active MS lesion, and (h) inactive center of chronic active MS lesion (all 40× magnification).

Article Snippet: For flow-cytometric analysis of PBMCs, the following antibodies were used: rat-anti-mouse CD169 FITC (Abd Serotec), rat-anti mouse CD14 PE (eBioscience, San Diego, CA), mouse anti-human CD14 PerCP (BD Biosciences, Erembodegem, Belgium), mouse anti-human CD56 PE-Cy7 (BD biosciences), mouse anti-human CD169 FITC (Abd Serotec), and mouse anti-human CD7 PE (BD Biosciences).

Techniques: Staining, Control

Journal: Multiple sclerosis (Houndmills, Basingstoke, England)

Article Title: CD169 is a marker for highly pathogenic phagocytes in multiple sclerosis.

doi: 10.1177/1352458517698759

Figure Lengend Snippet: Figure 5. CD169 is highly expressed on myelin-containing phagocytes in MS lesions. (a) Fluorescent staining of active MS lesion (green, Iba1; red; CD169; 20× magnification). (b) Fluorescent staining of active MS lesion (green, PLP; red; CD169; 20× magnification). (c) Quantification of the percentage of Iba1+ cells expressing CD169 in active MS lesions. For quantification, 10 images per tissue or MS lesions were used (20× magnification). Data are presented as mean ± SEM.

Article Snippet: For flow-cytometric analysis of PBMCs, the following antibodies were used: rat-anti-mouse CD169 FITC (Abd Serotec), rat-anti mouse CD14 PE (eBioscience, San Diego, CA), mouse anti-human CD14 PerCP (BD Biosciences, Erembodegem, Belgium), mouse anti-human CD56 PE-Cy7 (BD biosciences), mouse anti-human CD169 FITC (Abd Serotec), and mouse anti-human CD7 PE (BD Biosciences).

Techniques: Staining, Expressing

Journal: Vaccines

Article Title: Targeting Toll-Like Receptor 2: Polarization of Porcine Macrophages by a Mycoplasma-Derived Pam2cys Lipopeptide

doi: 10.3390/vaccines9070692

Figure Lengend Snippet: Effect of Mag-Pam2Cys on moMΦ surface marker expressions. Then, 24 h post-stimulation with scalar doses of Mag-Pam2Cys (10 or 100 ng/mL), surface expression of CD14, MHC II DR, CD25, CD163, MHC I, and CD169 were assessed by flow cytometry. In Panel ( a ), mean fluorescence intensity (MFI) data are presented as fold-change relative to the untreated control (moMΦ). In Panel ( b ), percentages of positive cells are shown. For both Panels, mean data and SD from four independent experiment using different blood donor pigs are displayed. Values of Mag-Pam2Cys-treated samples were compared to the untreated control (moMΦ) using a one-way ANOVA followed by a Dunnett’s multiple comparison test. *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Cells were then resuspended in 0.1 mL of PBS, and staining with monoclonal antibodies was performed using mouse anti human CD14-PerCP (Tuk4; Miltenyi Biotec, Bergisch Gladbach, Germany), mouse anti pig MHC class II DR-FITC (2E9/13, Bio-Rad Antibodies, Kidlington, UK), MHC class I (JM1E3, Bio-Rad Antibodies), CD163-PE (2A10/11, Bio-Rad Antibodies), CD169-FITC (MCA2316F, Bio-Rad Antibodies), and CD25 (K231.3B2, Bio-Rad Antibodies).

Techniques: Marker, Expressing, Flow Cytometry, Fluorescence, Control, Comparison

Journal: Vaccines

Article Title: Targeting Toll-Like Receptor 2: Polarization of Porcine Macrophages by a Mycoplasma-Derived Pam2cys Lipopeptide

doi: 10.3390/vaccines9070692

Figure Lengend Snippet: Modulation of moMΦ surface marker expressions by Mag-Pam2Cys and classical M1 polarizing factors. Porcine moMΦ were left untreated (MΦ) or stimulated with Mag-Pam2Cys (100 ng/mL) or IFN-γ + LPS (M1). At 24 h post-stimulation, surface expression of CD14, MHC II DR, CD25, CD163, MHC I, and CD169 were assessed by flow cytometry. In Panel ( a ), mean fluorescence intensity (MFI) data are presented as fold-change relative to the untreated control (moMΦ). In Panel ( b ), percentages of positive cells are shown. For both Panels, mean data and SD from three independent experiments using different blood donor pigs are displayed. Values of Mag-Pam2Cys-treated samples were compared to bona fide moM1 (M1), using a Mann–Whitney test. *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Cells were then resuspended in 0.1 mL of PBS, and staining with monoclonal antibodies was performed using mouse anti human CD14-PerCP (Tuk4; Miltenyi Biotec, Bergisch Gladbach, Germany), mouse anti pig MHC class II DR-FITC (2E9/13, Bio-Rad Antibodies, Kidlington, UK), MHC class I (JM1E3, Bio-Rad Antibodies), CD163-PE (2A10/11, Bio-Rad Antibodies), CD169-FITC (MCA2316F, Bio-Rad Antibodies), and CD25 (K231.3B2, Bio-Rad Antibodies).

Techniques: Marker, Expressing, Flow Cytometry, Fluorescence, Control, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: CD169-Mediated Trafficking of HIV to Plasma Membrane Invaginations in Dendritic Cells Attenuates Efficacy of Anti-gp120 Broadly Neutralizing Antibodies

doi: 10.1371/journal.ppat.1004751

Figure Lengend Snippet: (A) Cells incubated with HIV-1 particles were lysed, and cell lysates used for measuring cell-associated p24 gag . The data shown are the mean percent of captured p24 gag (virus) ± SEM of independent experiments performed in triplicates (n = 3 for DC, n = 5 for THP-1, n = 3 for Raji and HeLa). (B) CD169 expressing cell lines and mature DCs were incubated with Gag-mCherry VLPs and stained for plasma membrane bound CD169 (Surface, top panel) or total CD169 (+ Tx100, bottom panel). CD169 (green), Gag-mCherry VLP (red) and nucleus (blue). Representative deconvolved images of single slices of cells are shown. Scale bars represent 5 μm. (C) Cells incubated with HIV-1 particles were co-cultured with CD4 + T cells to monitor HIV-1 trans-infection. Cells were lysed two days post initiation of co-culture and lysates used for measurement of luciferase activity. Values were normalized to luciferase activity observed in control cells (immature DCs or CD169 low/null control cell lines). The data shown are the means ± SEM of independent experiments performed in triplicates with CD4 + T cells from different donors (n = 3 for DC, n = 4 for THP-1, n = 3 for Raji and n = 4 for HeLa).

Article Snippet: To determine if VLP containing compartments remained connected to the cell surface, HeLa/CD169 cells (seeded on coverslips in a 24-well tissue culture plate on the day before), THP-1/CD169 cells, Raji/CD169 cells or mature DCs (2x10 5 cells) were incubated with 10 ng p24 gag of VLP Gag-mCherry for 2 hours at 37°C, washed extensively to remove unbound VLPs, chilled to 4°C and stained with Alexa488-conjugated mouse anti-CD169 mAb (AbD Serotec) on ice for 1 hour, prior to fixation with 4% PFA.

Techniques: Incubation, Expressing, Staining, Cell Culture, Infection, Co-Culture Assay, Luciferase, Activity Assay

Journal: PLoS Pathogens

Article Title: CD169-Mediated Trafficking of HIV to Plasma Membrane Invaginations in Dendritic Cells Attenuates Efficacy of Anti-gp120 Broadly Neutralizing Antibodies

doi: 10.1371/journal.ppat.1004751

Figure Lengend Snippet: (A) Sequences of wild type and mutant CD169 CTs. The asterisks represent stop codons introduced into the ORFs of the two CT mutants. (B) Western blot analysis of THP-1 cell lysates expressing either wild type or mutant CD169. (C) Cell surface expression of CD169 on THP-1 cells was measured by flow cytometry. (D) Relative cell surface expression of CD169 CT mutants was quantified and normalized to that observed with THP-1/CD169 cells. (E) Cells were challenged with HIV-1, washed and cell-associated p24 gag was measured. The data shown is the virus capture by THP-1/CD169 CT mutants (ΔCT or ΔCT4R) normalized to that observed with THP-1/CD169 cells. (F) THP-1/CD169- or THP-1/CD169 CT mutant-mediated trans-infection was determined by measuring luciferase activity in THP—CD4 + T cell co-cultures 2 days post initiation of co-culture. The data shown is the relative virus transmission by THP-1/CD169 CT mutants (ΔCT or ΔCT4R) to that observed with THP-1/CD169 cells. (G) Efficacy of trans-infection was calculated as trans-infection (luciferase activity) per amount of virus captured (cell-associated p24 gag ) and normalized to that observed with THP-1/CD169 cells (set as 100). The data shown are the means ± SEM of three (D to F) or four (G) independent experiments. (H) THP-1/CD169 or THP-1/CD169ΔCT4R cells were incubated with Gag-mCherry VLPs (red), washed, fixed and stained for CD169 (green) and nucleus (blue). Representative deconvolved images of single slices of cells are shown. Scale bar represents 5 μm. WT: THP-1/CD169, ΔCT: THP-1/CD169ΔCT, ΔCT4R: THP-1/CD169ΔCT4R and Vec: empty vector transduced THP-1.

Article Snippet: To determine if VLP containing compartments remained connected to the cell surface, HeLa/CD169 cells (seeded on coverslips in a 24-well tissue culture plate on the day before), THP-1/CD169 cells, Raji/CD169 cells or mature DCs (2x10 5 cells) were incubated with 10 ng p24 gag of VLP Gag-mCherry for 2 hours at 37°C, washed extensively to remove unbound VLPs, chilled to 4°C and stained with Alexa488-conjugated mouse anti-CD169 mAb (AbD Serotec) on ice for 1 hour, prior to fixation with 4% PFA.

Techniques: Mutagenesis, Western Blot, Expressing, Flow Cytometry, Infection, Luciferase, Activity Assay, Co-Culture Assay, Transmission Assay, Incubation, Staining, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: CD169-Mediated Trafficking of HIV to Plasma Membrane Invaginations in Dendritic Cells Attenuates Efficacy of Anti-gp120 Broadly Neutralizing Antibodies

doi: 10.1371/journal.ppat.1004751

Figure Lengend Snippet: (A) Amino acid sequences of the CTs of wild type (WT) CD169 and mutant CD169YF are shown. Alanine to tyrosine mutation at position 1683 (in red) creates a di-aromatic motif, YF (underlined). (B) Western blot analysis for CD169 expression in THP-1/CD169 and THP-1/CD169YF cell lysates. (C) Representative FACS analysis of cell surface expression of CD169 on wild type and YF mutant expressing THP-1 cells. (D) The mean fluorescence intensity of cell surface expression of CD169 on YF mutant expressing THP-1 cells was quantified and normalized to that observed with THP-1/CD169 (wt) cells (set at 100). (E) Cells were incubated with Gag-mCherry VLPs and stained for CD81, CD63 or Lamp1 and nucleus. CD81, CD63 or Lamp1 (green), Gag-mCherry VLP (red) and nucleus (blue). Representative deconvolved images of single slices of cells are shown. Scale bar represents 5 μm. (F) Co-localization between green (CD81, CD63 or Lamp1) and red (VLPs) signals is reported as mean Pearson’s coefficient ± SEM. Each dot represents a single cell. Two-tailed P values were calculated using unpaired t-test in GraphPad Prism 5. ***: P < 0.0001. (G) Cells were challenged with HIV-1, washed and cell-associated p24 gag was measured. Virus capture observed with THP-1/CD169YF cells was normalized to that observed with THP-1/CD169 cells (WT; set as 100). (H) Cells challenged with HIV-1/Bal-luc, were washed, co-cultured with CD4 + T cells and lysed at two days post initiation of co-culture for measurement of luciferase activity. The level of virus transmission observed in THP-1/CD169 (wt)—CD4 + T cell co-cultures was set as 100. (I) Efficacy of trans-infection was calculated as trans-infection (luciferase activity) per virus capture (cell-associated p24 gag ) and is shown relative to that observed with THP-1/CD169 cells (set as 100). The data shown are the means ± SEM of four (D) or six (G to I) independent experiments.

Article Snippet: To determine if VLP containing compartments remained connected to the cell surface, HeLa/CD169 cells (seeded on coverslips in a 24-well tissue culture plate on the day before), THP-1/CD169 cells, Raji/CD169 cells or mature DCs (2x10 5 cells) were incubated with 10 ng p24 gag of VLP Gag-mCherry for 2 hours at 37°C, washed extensively to remove unbound VLPs, chilled to 4°C and stained with Alexa488-conjugated mouse anti-CD169 mAb (AbD Serotec) on ice for 1 hour, prior to fixation with 4% PFA.

Techniques: Mutagenesis, Western Blot, Expressing, Fluorescence, Incubation, Staining, Two Tailed Test, Cell Culture, Co-Culture Assay, Luciferase, Activity Assay, Transmission Assay, Infection

Journal: PLoS Pathogens

Article Title: CD169-Mediated Trafficking of HIV to Plasma Membrane Invaginations in Dendritic Cells Attenuates Efficacy of Anti-gp120 Broadly Neutralizing Antibodies

doi: 10.1371/journal.ppat.1004751

Figure Lengend Snippet: (A) Representative FACS analysis for CD169 expression on LPS or IFN-α-matured DCs. (B) HIV-1 capture by immature (NT), IFN-α or LPS-matured DCs was determined by measuring cell-associated p24 gag in cell lysates. (C) HIV-1 transfer to CD4 + T cells, by immature (NT), IFN-α or LPS-matured DCs was determined by measuring luciferase activity in DC—CD4 + T cell co-cultures. HIV-1 capture and transfer experiments were performed in triplicates with DCs isolated from eight independent donors. The individual dot represents a single donor and the means ± SEM are depicted. (D) LPS or IFN-α-matured DCs were incubated with fluorescent HIV-1 particles (green) and stained for CD169 (red) and nucleus (blue). Representative deconvolved images of single slices of cells are shown. Scale bar represents 5 μm. (E) Representative electron micrographs of LPS or IFN-α-matured DCs incubated with HIV-1. The bottom panels are higher magnification pictures of the area depicted within the highlighted squares in the top panels. Arrows indicate virus particles. Scale bar represents 1 μm for top panels and 500 nm for bottom panels. (F) Cells were incubated with HIV-1 and stained for HIV-1 p24 gag (green) and CD169 (red). Cells were imaged by FPALM super resolution microscopy. The top panels represent a single LPS or IFN-α matured DC while the middle panels show cross sections along the a — b line indicated in the top panels. The bottom panels are pictures enlarged from the area depicted within the highlighted (dotted) squares in the middle panels. Scale bars represent 1 μm in the top and middle panels and 500 nm in the bottom panels. LPS: LPS-treated DCs, IFN-α: IFN-α-treated DCs, Immature: immature DCs.

Article Snippet: To determine if VLP containing compartments remained connected to the cell surface, HeLa/CD169 cells (seeded on coverslips in a 24-well tissue culture plate on the day before), THP-1/CD169 cells, Raji/CD169 cells or mature DCs (2x10 5 cells) were incubated with 10 ng p24 gag of VLP Gag-mCherry for 2 hours at 37°C, washed extensively to remove unbound VLPs, chilled to 4°C and stained with Alexa488-conjugated mouse anti-CD169 mAb (AbD Serotec) on ice for 1 hour, prior to fixation with 4% PFA.

Techniques: Expressing, Luciferase, Activity Assay, Isolation, Incubation, Staining, Microscopy

Journal: PLoS Pathogens

Article Title: CD169-Mediated Trafficking of HIV to Plasma Membrane Invaginations in Dendritic Cells Attenuates Efficacy of Anti-gp120 Broadly Neutralizing Antibodies

doi: 10.1371/journal.ppat.1004751

Figure Lengend Snippet: (A) Experimental procedure utilized for testing the susceptibility of HIV-1 particles captured by CD169 to extracellular protease treatment is depicted. (B) Representative FACS analysis of cell surface expression of CD169 on LPS-DCs. (C) Relative cell surface expression of CD169 expression was measured by flow cytometry and normalized to that observed with untreated control (NT, set at 1). The data shown are the means ± SD of five (LPS-DCs, Trp), nine (LPS-DCs, Prn) and two (IFN-DCs, Typ and Prn) independent experiments with DCs from different donors. (D) LPS or IFN-α-matured DCs, incubated with HIV-1, were treated with pronase or trypsin. The amount of virus particles left associated with cells following protease treatments was determined by measuring cell-associated p24 gag and the values were normalized to that observed with untreated cells (NT). The data shown are the means ± SD of four (LPS-DCs, Trp), seven (LPS-DCs, Prn) and two (IFN-DCs, Typ and Prn) independent experiments with DCs from different donors. (C and D) Two-tailed P values were calculated using one sample t-test in GraphPad Prism 5. *: P < 0.05, ***: P < 0.0001. Trp: trypsin-treated sample, Prn: pronase-treated sample.

Article Snippet: To determine if VLP containing compartments remained connected to the cell surface, HeLa/CD169 cells (seeded on coverslips in a 24-well tissue culture plate on the day before), THP-1/CD169 cells, Raji/CD169 cells or mature DCs (2x10 5 cells) were incubated with 10 ng p24 gag of VLP Gag-mCherry for 2 hours at 37°C, washed extensively to remove unbound VLPs, chilled to 4°C and stained with Alexa488-conjugated mouse anti-CD169 mAb (AbD Serotec) on ice for 1 hour, prior to fixation with 4% PFA.

Techniques: Expressing, Flow Cytometry, Incubation, Two Tailed Test

Journal: PLoS Pathogens

Article Title: CD169-Mediated Trafficking of HIV to Plasma Membrane Invaginations in Dendritic Cells Attenuates Efficacy of Anti-gp120 Broadly Neutralizing Antibodies

doi: 10.1371/journal.ppat.1004751

Figure Lengend Snippet: (A and B) LPS or IFN-α-matured DCs incubated with fluorescent HIV-1 particles, were stained for either surface-exposed gp120 (A) or CD169 (B) on living cells (Surface, top panels) or total gp120 (A) or CD169 (B) on cells after fixation and TritonX-100 treatment (+ Tx100, bottom panels). HIV-1 particles (green), gp120 or CD169 (red) and nucleus (blue). The arrowheads indicate green HIV-1 particles in VCCs that were not stained by surface-applied antibodies. Representative deconvolved images of single slices of cells are shown. Scale bar represents 5 μm. (C and D) Co-localization between HIV-1 particles and gp120 (C) or CD169 (D) is reported as Manders’ coefficients. Each dot represents a single cell and the means ± SEM are shown. The data shown is a representative experiment using DCs isolated from two different donors. (E and F) HIV-1 exposed LPS or IFN-α-matured DCs or cell-free (CF) HIV-1 particles were incubated with increasing concentrations of VRC01 (E), NIH45–46 G54W (F) or sCD4–183 (G) prior to initiation of CD4 + T cell infections. The x-axis shows the concentration of input VRC01 (E), NIH45–46 G54W (F) and sCD4–183 (G) in log μg/ml, and the y-axis shows the percentage inhibition relative to infection without any antibody. The data shown are the means ± SEM of a representative experiment performed in triplicate. (H, I and J) IC50 values for VRC01 (H), NIH45–46 G54W (I) or sCD4–183 (J) are shown as mean ± SEM and each dot represents data obtained from cells derived from an independent donor. Two-tailed P values were calculated using unpaired (C and D) or paired (H, I and J) t-test in GraphPad Prism 5. * P<0.05, **: P < 0.01, ***: P < 0.0001, n.s.: not significant.

Article Snippet: To determine if VLP containing compartments remained connected to the cell surface, HeLa/CD169 cells (seeded on coverslips in a 24-well tissue culture plate on the day before), THP-1/CD169 cells, Raji/CD169 cells or mature DCs (2x10 5 cells) were incubated with 10 ng p24 gag of VLP Gag-mCherry for 2 hours at 37°C, washed extensively to remove unbound VLPs, chilled to 4°C and stained with Alexa488-conjugated mouse anti-CD169 mAb (AbD Serotec) on ice for 1 hour, prior to fixation with 4% PFA.

Techniques: Incubation, Staining, Isolation, Concentration Assay, Inhibition, Infection, Derivative Assay, Two Tailed Test

Journal: PLoS Pathogens

Article Title: CD169-Mediated Trafficking of HIV to Plasma Membrane Invaginations in Dendritic Cells Attenuates Efficacy of Anti-gp120 Broadly Neutralizing Antibodies

doi: 10.1371/journal.ppat.1004751

Figure Lengend Snippet: Capture of HIV-1 particles by CD169 leads to the formation of CD169 + VCCs in LPS-matured DCs. Lateral membrane movement of CD169-bound HIV-1 can result in accumulation of HIV-1 particles in plasma membrane microdomains in LPS-DCs. Multivalent interactions between multiple CD169 and HIV-1 particles and co-factor(s) recruitment might induce localized stress and strain to which the plasma membrane responds by forming invaginations. Arrow indicates lateral movement of CD169-bound HIV-1 particles into the VCC.

Article Snippet: To determine if VLP containing compartments remained connected to the cell surface, HeLa/CD169 cells (seeded on coverslips in a 24-well tissue culture plate on the day before), THP-1/CD169 cells, Raji/CD169 cells or mature DCs (2x10 5 cells) were incubated with 10 ng p24 gag of VLP Gag-mCherry for 2 hours at 37°C, washed extensively to remove unbound VLPs, chilled to 4°C and stained with Alexa488-conjugated mouse anti-CD169 mAb (AbD Serotec) on ice for 1 hour, prior to fixation with 4% PFA.

Techniques:

Journal: Vaccines

Article Title: Targeting Toll-Like Receptor 2: Polarization of Porcine Macrophages by a Mycoplasma-Derived Pam2cys Lipopeptide

doi: 10.3390/vaccines9070692

Figure Lengend Snippet: Effect of Mag-Pam2Cys on moMΦ surface marker expressions. Then, 24 h post-stimulation with scalar doses of Mag-Pam2Cys (10 or 100 ng/mL), surface expression of CD14, MHC II DR, CD25, CD163, MHC I, and CD169 were assessed by flow cytometry. In Panel ( a ), mean fluorescence intensity (MFI) data are presented as fold-change relative to the untreated control (moMΦ). In Panel ( b ), percentages of positive cells are shown. For both Panels, mean data and SD from four independent experiment using different blood donor pigs are displayed. Values of Mag-Pam2Cys-treated samples were compared to the untreated control (moMΦ) using a one-way ANOVA followed by a Dunnett’s multiple comparison test. *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Cells were then resuspended in 0.1 mL of PBS, and staining with monoclonal antibodies was performed using mouse anti human CD14-PerCP (Tuk4; Miltenyi Biotec, Bergisch Gladbach, Germany), mouse anti pig MHC class II DR-FITC (2E9/13, Bio-Rad Antibodies, Kidlington, UK), MHC class I (JM1E3, Bio-Rad Antibodies), CD163-PE (2A10/11, Bio-Rad Antibodies), CD169-FITC (MCA2316F, Bio-Rad Antibodies), and CD25 (K231.3B2, Bio-Rad Antibodies).

Techniques: Marker, Expressing, Flow Cytometry, Fluorescence

Journal: Vaccines

Article Title: Targeting Toll-Like Receptor 2: Polarization of Porcine Macrophages by a Mycoplasma-Derived Pam2cys Lipopeptide

doi: 10.3390/vaccines9070692

Figure Lengend Snippet: Modulation of moMΦ surface marker expressions by Mag-Pam2Cys and classical M1 polarizing factors. Porcine moMΦ were left untreated (MΦ) or stimulated with Mag-Pam2Cys (100 ng/mL) or IFN-γ + LPS (M1). At 24 h post-stimulation, surface expression of CD14, MHC II DR, CD25, CD163, MHC I, and CD169 were assessed by flow cytometry. In Panel ( a ), mean fluorescence intensity (MFI) data are presented as fold-change relative to the untreated control (moMΦ). In Panel ( b ), percentages of positive cells are shown. For both Panels, mean data and SD from three independent experiments using different blood donor pigs are displayed. Values of Mag-Pam2Cys-treated samples were compared to bona fide moM1 (M1), using a Mann–Whitney test. *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Cells were then resuspended in 0.1 mL of PBS, and staining with monoclonal antibodies was performed using mouse anti human CD14-PerCP (Tuk4; Miltenyi Biotec, Bergisch Gladbach, Germany), mouse anti pig MHC class II DR-FITC (2E9/13, Bio-Rad Antibodies, Kidlington, UK), MHC class I (JM1E3, Bio-Rad Antibodies), CD163-PE (2A10/11, Bio-Rad Antibodies), CD169-FITC (MCA2316F, Bio-Rad Antibodies), and CD25 (K231.3B2, Bio-Rad Antibodies).

Techniques: Marker, Expressing, Flow Cytometry, Fluorescence, MANN-WHITNEY