Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . Left , Representative immunostaining of macrophages in the splenic marginal zone expressing CD169 (red) and Tim4 (green); DAPI nuclear staining (blue). Right , FACS contour plots for CD169 and Tim4 expression in splenic CD45 + CD11b low F4/80 low cells and quantitation of Tim4 expression in CD169 + MMMs; n=4. B . Top Left , FACS pseudocolor plots of circulating Ly6C monocytes with histograms identifying CD169 + and CD169 − populations within Ly6C low monocytes, and corresponding cell quantitation; NTM, normalized to mode. Top Right , principal component analysis of top 500 differentially expressed genes (DEGs) from bulk RNAseq of sorted Ly6C low CD169 − and Ly6C low CD169 + cells, and heat maps with dendrograms for 334 DEGs (q<0.05) between the same sub-populations. Bottom , FACS pseudocolor plots of circulating Ly6C low CD169 + Tim4 + macrophages in naïve and spx mice blood with flow histograms identifying CD64 and MHCII surface expression (in black), together with quantitation of frequency or absolute number of the populations shown; n=5-7, statistics: unpaired t test. C. Top, Representative blood FACS dot plots and quantitation of tdTomato (Tdt) expression in blood Ly6C + monocytes from CX3CR1 CreERT ; Rosa26 tdTomato mice after a tamoxifen (TAM) pulse to induce Cre recombination. Shown are data from several time points after TAM and 14 d after splenectomy (Spx) performed at 26 d post-TAM; *p<0.05, n=3, statistics: unpaired t-test. Bottom , representative FACS plots showing CD169 expression in Tdt+ cells 26 d post-TAM pulse and overlay of the CD169 + Tdt + cells (green) on blood Ly6C + monocytes. D . Top , UMAP plots derived from single cell RNA sequencing of blood leukocytes from 3 naïve mice (∼400K total cells, 11 identified cell clusters), heat map demonstrating CD169 ( Siglec1 ) expression primarily in the monocyte cluster, and quantitative expression of select macrophage genes in cells with and without CD169 expression (adjusted FDR p values are shown). E . Left, FACS plots identifying CD169 + Tim4 + cardiac macrophages in intact and Spx mice, overlay of these macrophages on contour plots of CCR2 and LYVE1 expression (in intact mice), and quantitation of overall LYVE1 and CCR2 expression (n=6). Right , quantitation of frequency and number of cardiac CD169 + Tim4 + LYVE1 low macrophages naïve and Spx C57BL/6 mice; n=5-7/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Immunostaining, Expressing, Staining, Quantitation Assay, Derivative Assay, RNA Sequencing

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . FACS plots and group quantitation for circulating Ly6C low CD169 + Tim4 + macrophages in sham-operated and MI wild type (WT) C57BL/6 mice 1 d post-MI; n=4/group, statistics: unpaired t test. B. Left , Flow histograms demonstrating surface expression of CCR3 and CCR4 in Ly6C low CD169 + Tim4 + macrophages from the same groups; statistics: unpaired t test, NTM, normalized to mode, y-axis represents cell counts. Right , chemokine gene expression by RT-PCR (normalized to 18s) in the myocardial border zone (BZ) 1 d after MI or sham operation; n=4-5/group, statistics: unpaired t test. *p<0.05, **p<0.01, ***p<0.001 versus sham. C . Left , Circulating Ly6C low CD169 + Tim4 + cell frequency prior to and 1 d after MI in Spx mice; n=9, statistics: paired t test. NS, not significant. D. FACS density plots, histograms, and quantitation of cardiac Ly6C low CD169 + Tim4 + macrophages in WT and Spx mice 1 d post-MI or sham operation; n=4-7/group, statistics: unpaired t test. E . Immunostains and FACS dot plots of splenic CD169 + MMMs (red) 24 h after MI or sham operation, and quantitation of MMM frequency by FACS and spleen weight (Wt); n=5-7/group, statistics: unpaired t test. TL, tibia length. F . Left , FACS dot plots and histograms, and corresponding quantitation, of blood and heart Ly6C low CD169 + Tim4 + Bioparticle + cells from sham and MI mice given 10 mg/kg Texas Red-conjugated bioparticles i.v. 3 h before sacrifice; n=3-4/group, statistics: unpaired t test for blood and non-parametric Mann-Whitney U test for heart (non-normal distribution). Right, quantitation of splenic BioParticle + MMMs in the same experimental mouse groups; statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Quantitation Assay, Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A Left , Parabiosis schema joining CD45 isotype-mismatched host (spleen-intact or after splenectomy [Spx]) and donor parabiont mice, with MI induced in the host. Middle and Right , FACS plots and quantitation of donor chimerism in host mouse blood (total CD45 + leukocytes) and heart (Ly6C low CD169 + macrophages) in spleen-intact and Spx host mice 48 h after MI. n=4-7/group, statistics: unpaired t-test. B Top Left , Parabiosis schema joining CD169 DTR host and donor parabiont MaFIA mice, with host mice given either vehicle or diphtheria toxin (DT) at the time of MI. Right , Representative FACS dot plots of donor GFP + CD169 + macrophages in 48 h post MI hearts from host mice and flow histograms of CD169 expression in GFP + CD64 + MHCII + Tim4 + cells delineated as NTM or cell counts. Bottom Left , quantitation of GFP + frequency in host cardiac CD169 + Tim4 + macrophages and total CD169 + Tim4 + cells as a percentage of all autofluorescent(Auto) + macrophages in vehicle and DT treated host MI mice; n=3-4/group, statistics: unpaired t test. C. Left , Parabiosis schema joining CD169 DTR host mice and either spleen-intact or Spx MaFIA donor parabionts, with host mice given DT at the time of MI to deplete CD169 + macrophages. Right , Example FACS dot plots and quantitation of donor CD45 + Auto + CD64 + MHCII + GFP + CD169 + Tim4 + macrophages in the host MI heart 48 h post MI; n=3-4/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Quantitation Assay, Expressing

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . Left, FACS pseudocolor plots of cardiac CD169 + Tim4 + macrophages and separation based on LYVE1 surface expression in naïve and 1 d post-MI mice. Right , quantitation of LYVE1 hi and LYVE1 low CD169 + Tim4 + macrophages in hearts from naïve and 1 d post-MI mice. n=4-6/group; statistics: unpaired t test. B . Heat map of 462 significant (p adjusted<0.05) DEGs by RNAseq analysis in sorted LYVE1 hi and LYVE1 low CD169 + Tim4 + cardiac macrophages 1 d post-MI. C. PCA plots using the top 500 DEGs after rlog transformation of RNAseq data from these macrophages sorted from the indicated sites in naïve and 1 d post-MI mice. D. Left , Flow histograms depicting LYVE1 surface expression on cardiac CD169 + Tim4 + macrophages (green) and total Autofluorescence + CD64 + MHCII + macrophages (brown) in WT and Spx mice, 1 d post-MI. Right , FACS quantitation of LYVE1 hi and LYVE1 low CD169 + Tim4 + macrophages in the hearts of WT and Spx mice, 1 d post-MI; n=5-6/group. Statistics: unpaired t test. E. Representative confocal micrograph of border zone (BZ) myocardium immunostained for CD169 (red) and Tim4 (green) 1 d post-MI in WT and Spx mice; nuclear staining with DAPI (blue). Scale bar, 200 μm. Inset shows magnified images of CD169 and Tim4 staining. Yellow arrows indicate double positive cells; scale bar 10 μm.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Expressing, Quantitation Assay, Transformation Assay, Staining

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: FACS plots and quantitation of Ly6C low CD169 + Tim4 + macrophages and total Ly6C low cells in blood ( A ) and in heart ( B ) 1 d post-MI in wild-type (WT) and splenectomized (Spx) WT mice, and CD169 DTR mice given diphtheria toxin (CD169 DTR /DT) at the time of MI; n=5-7/group, statistics: one-way ANOVA, Bonferroni post-test. Isotype antibody is shown in gray. C . FACS plots and group data for blood CD45 + CD11b + Ly6G + neutrophils and ICAM-1/CD54 + neutrophils 1 d post MI in WT, Spx, and CD169 DTR /DT mice; n=6-8/group; statistics: one-way ANOVA, Bonferroni post-test. D . Left, Representative confocal images of immunofluorescent Ly6G staining in WT, Spx, and CD169 DTR /DT hearts 1 d post-MI demonstrating Ly6G + neutrophil (red) infiltration (arrows); nuclear staining with DAPI (blue). Scale bar 20 μm. Right , FACS plots and corresponding quantitation of cardiac CD45 + CD11b + Ly6G + neutrophils (red) and annexin V + apoptotic neutrophils (blue) in the same groups 1 d post-MI; n=3-4/group, statistics: one-way ANOVA, Dunnett’sT3 post-test. E . FACS density plots for Lin − c-kit + CD34 + CD16/32 + granulocyte monocyte precursors (GMPs) in bone marrow from WT, Spx, and CD169 DTR /DT mice 1 d post MI, together with quantitation. Flow gates were based on isotype antibody control. n=5-7/group; statistics: one-way ANOVA, Tukey’s post-test. F . Left , Representative FACS dot plots identifying cardiac neutrophils as CD11b + Ly6G + cells in WT mice and as Ly6G + tdTomato + cells in Catchup mice at baseline and 1 d post-MI. Right Top , Representative histograms of Ly6G and tdTomato fluorescence intensity in heart mononuclear cells from the same groups. Right Bottom , FACS dot plots gated on cardiac CD169 + Tim4 + macrophages illustrating tdTomato expression in Catchup mice 1 d post-MI. Auto, autofluorescence. G . Representative FACS histograms of intracellular IL4 and IL10 staining in cardiac Ly6C low cells 1 d post MI in WT, Spx and CD169 DTR /DT mice, together with cell quantitation of the Ly6C low subsets. N=6-7/group; statistics: one-way ANOVA, Bonferroni post-test. H . Representative FACS pseudocolor plots for intracellular TGFβ and IL10 staining in cardiac CD169 + macrophages 1 d post MI in WT and Spx mice, with accompanying quantitation; n=4-5/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Quantitation Assay, Staining, Control, Fluorescence, Expressing

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . Kaplan-Meier survival curves over 10 d following MI or sham operation in WT, Spx, and CD169 DTR /DT mice, and after MI in CD45.2 Spx mice with adoptive transfer of naïve splenic CD169 + Tim4 + cells from syngeneic CD45.1 WT mice 24 h post-MI (Spx-MI+AT). Statistical comparisons: log-rank test. B . Gross images of post-MI cardiac rupture with hemothorax or hemopericardium, and Kaplan-Meier curves for freedom from rupture over 10 d post-MI in WT, Spx, CD169 DTR /DT, and Spx-MI+AT mice. Statistical comparisons: log-rank test. C . Left , Representative post-mortem whole hearts and end-diastolic long-axis 2-dimensional echocardiograms from WT-MI, Spx-MI, CD169 DTR /DT-MI and Spx-MI+AT mice (10 d post-MI). Right , Quantitation of LV ejection fraction (EF) and end-diastolic and end-systolic volume (EDV and ESV) at 10 d post-MI; n=4-6/group, statistics: one-way ANOVA, Bonferroni post-test. D . Left , Representative confocal images of immunofluorescent staining for CD206 (green) and iNOS (red) in the heart infarct border zone (BZ) from WT-MI, Spx-MI, CD169 DTR /DT-MI, and Spx-MI+AT mice 10 d post-MI. DAPI (blue) nuclear staining. iNOS + CD206 + cells appear yellow. Higher magnification is shown in the middle panel. Right , quantitation of iNOS + CD206 + and iNOS − CD206 + cells/mm 2 in the hearts. N=3/group, statistics: one-way ANOVA, Bonferroni post-test. NS, not significant. E . Top Left , Confocal images of MMP-9 immunostaining (red) in infarct BZ of hearts from WT-MI, Spx-MI, CD169 DTR /DT-MI, and Spx-MI+AT mice 10 d post-MI. Nuclear staining with DAPI (blue). Top Right , Quantitative group data for total MMP-9 mean fluorescence intensity per region of interest (ROI). AU, arbitrary units. N=4/group, statistics: one-way ANOVA, Bonferroni post-test. Bottom , Representative Masson’s trichrome stains of infarcted hearts (10 d post-MI) from the same groups demonstrating MI border zone (BZ) fibrosis, together with BZ fibrosis quantitation. n=4/group; statistics: one-way ANOVA, Bonferroni post-test. F . FACS quantitation of Ly6C hi blood monocytes and serum IL-10 levels in WT-MI, Spx-MI, CD169 DTR /DT-MI, and Spx-MI+AT mice at 10 d post-MI. N=4-7/group, statistics: one-way ANOVA, Bonferroni post-test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Adoptive Transfer Assay, Quantitation Assay, Staining, Immunostaining, Fluorescence

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . Protocol for LXRα agonist T0901317 treatment (40 mg/kg i.p.) from 1 d prior to 5 d post-MI, with 10 d post-MI follow-up, in WT and Spx mice. B . FACS contour plots and quantitation of cardiac CD169 + Tim4 + macrophages 1 d post-MI and blood CD169 + Tim4 + macrophages 10 d post-MI in untreated and T0901317-treated WT and Spx mice. N=5-6/group, statistics: one-way ANOVA, Bonferroni post-test. C . Kaplan-Meier survival curves post-MI in untreated and T0901317-treated WT and Spx mice. Statistical comparisons by log-rank test, group sizes as indicated. D . Representative end-diastolic long-axis 2-dimensional echocardiograms and group data for LV ejection fraction (EF) and end-diastolic and end-systolic volume (EDV and ESV) in the same mouse groups at 10 d post-MI; n=5-10/group, statistics: one-way ANOVA, Bonferroni post-test. E . Top , Kaplan-Meier survival curves over 8 w post-MI in WT mice treated with either vehicle or T0901317 from 1 d before MI to 5 d post-MI (statistical comparison by log-rank test, n=12-17/group as indicated) and group data for LVEF, LVEDV, LVESV, and normalized heart and lung weight at 8 w post-MI; n=8-9/group for echocardiography, n=4-7/group for gravimetry. Statistical comparisons: unpaired t test. HF, heart failure; TL, tibia length; NS, not significant. Bottom Left , Representative Masson’s trichrome staining of LV short-axis sections (2x magnification) and infarct border zone (BZ, scale bar 500 μm), along with quantitation of cardiac fibrosis (BZ and remote zone [RZ], blue staining) in vehicle- and T0901317-treated WT HF mice. Also shown are confocal images of immunofluorescent staining for CD206 (green) and iNOS (red) in the RZ of hearts from vehicle-and T0901317-treated HF mice (8 w post-MI) and quantitation of iNOS + CD206 + and iNOS − CD206 + macrophages (Mφ) per mm 2 . Double positive (CD206 + iNOS + ) cells appear yellow (arrows). DAPI (blue) was used for nuclear staining. N=4-8/group, statistics: unpaired t test. Bottom Right , Representative FACS contour plots to identify Ly6C hi monocytes in vehicle- and T0901317-treated WT HF mice (8 w post-MI), and corresponding quantitation. N=5-10/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Quantitation Assay, Comparison, Staining

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . FACS plots and characterization of human CD45 + CD14 + HLA- DR + CD64 + CD169 + Tim4 + circulating macrophages from a subject with acute STEMI. The accompanying overlaid contour plot illustrates CD14 + HLA-DR + (red) and CD64 + CD169 + Tim4 + (green) subsets superimposed on all CD45 + leukocytes in an SSC-A versus FSC-A gate. B . Top , FACS gating strategy for sorting CD45 + CD14 + HLA-DR + CD169 + cells from human peripheral blood for further characterization using ImageStream analysis. Bottom , ImageStream visualization of FACS-sorted CD45 + CD169 + blood cells from STEMI patients and control subjects undergoing elective percutaneous coronary intervention (PCI), and group data for size distribution of CD169 + cells; scale bar 10 μm. C . FACS contour plots and quantitation of circulating CD45 + CD14 + HLA-DR + CD64 + CD169 + Tim4 + macrophages in STEMI and control PCI subjects. n=11-14/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Control, Quantitation Assay

Journal: International Journal of Molecular Sciences

Article Title: Multiple Immunostainings with Different Epitope Retrievals—The FOLGAS Protocol

doi: 10.3390/ijms23010223

Figure Lengend Snippet: Secondary antibodies, chromogens, fluorophores and metals. Except DAPI, the fluorophores not directly coupled to antibodies are linked to streptavidin for binding of biotinylated antibodies. Abbreviations: RTU: ready to use, AP: alkaline phosphatase, HRP: horseradish peroxidase. Tb: Terbium, Er: Erbium.

Article Snippet: , 170Er (conjugated to CD163 clone EDHu-1) , , - , Fluidigm (SKU 201300).

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: Dendritic Cells From the Cervical Mucosa Capture and Transfer HIV-1 via Siglec-1

doi: 10.3389/fimmu.2019.00825

Figure Lengend Snippet: Myeloid cells from human cervical mucosa express Siglec-1. FACS analysis and representative gating strategy of the cervicovaginal myeloid cell subsets. Colored gates and arrows indicate populations analyzed, and corresponding matching colors are used to identify bar graphs showing frequencies of those populations. (A) Hematopoietic cells were identified by their CD45 expression, and single-live cells were selected by doublet discrimination and live/dead staining. (B) Representative dot plot and frequency of HLA-DR + and CD3 − on hematopoietic cells. (C) Representative dot plot showing that HLA-DR − cells do not express Siglec-1. (D) Representative dot plot and frequency of Siglec-1 expression analyzed in HLA-DR + myeloid cells compared to matched isotype control. (E) Representative dot plot and frequency of CD11c + CD14 + cells among myeloid HLA-DR + cells expressing Siglec-1. (F) Representative dot plot of CD11b expression analyzed in CD11c + CD14 + Siglec-1 + myeloid cells. (G) Representative dot plot of CD14 and CD11c among the myeloid HLA-DR + cells that do not express Siglec-1. All bar graphs show mean values and SEM from 14 donors. Statistical differences were assessed with a Mann-Whitney test.

Article Snippet: The mean number of Siglec-1 Ab binding sites per monocyte-derived DC from men and women was obtained with a Quantibrite kit (Becton-Dickinson) as previously described ( ).

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Dendritic Cells From the Cervical Mucosa Capture and Transfer HIV-1 via Siglec-1

doi: 10.3389/fimmu.2019.00825

Figure Lengend Snippet: Siglec-1 + cells accumulate in the submucosa of the ectocervix and endocervix. (A) Representative immunofluorescent stainings on four ectocervix and endocervix of CD14 (AF488) and Siglec-1 (AF647), or CD11c (AF488) and Siglec-1 (AF647). Scale bars 20 μm. (B) Representative images of Siglec-1 immunostaining (40x) on 10 ectocervix and endocervix. Scale bars 100 μm. Insets show Siglec-1 + cells magnification (100x). Bar graph shows the mean values and SEM of Siglec-1 + cells per field counted in 5 consecutive fields. (C) Representative images of Siglec-1 immunostaining (40x) on 10 endocervix displaying a low and high grade of inflammation. Scale bars 100 μm. Bar graph shows the mean values and SEM of Siglec-1 + cells per field counted in 5 consecutive fields of 10 endocervix with different inflammation grades. Statistical differences were assessed with a Mann-Whitney test.

Article Snippet: The mean number of Siglec-1 Ab binding sites per monocyte-derived DC from men and women was obtained with a Quantibrite kit (Becton-Dickinson) as previously described ( ).

Techniques: Immunostaining, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Dendritic Cells From the Cervical Mucosa Capture and Transfer HIV-1 via Siglec-1

doi: 10.3389/fimmu.2019.00825

Figure Lengend Snippet: DCs from cervical mucosa mediate viral uptake via Siglec-1 and are detected in vivo . (A) Cervical mononuclear cells isolated from the ectocervix and endocervix of benign hysterectomies were pulsed with VLPs for 18 h at 37°C, extensively washed, labeled with the indicated mAbs and assessed by FACS. Colored gates and arrows indicate populations analyzed, and corresponding matching colors are used to identify bar graphs showing frequencies of those populations. Representative dot plot and frequency of HLA-DR + and Siglec-1 + cells on hematopoietic cervical cells. (B) Representative dot plot and frequency of cells capturing HIV-1 Gag−eGFP VLPs among the myeloid HLA-DR + fraction. Smaller dot plot in between depicts the control without VLPs. (C) Representative dot plot showing reduced expression of Siglec-1 in the myeloid HLA-DR + cells not capturing HIV-1 Gag−eGFP VLPs. (D) Representative dot plot of Siglec-1 + cells among the cells capturing HIV-1 Gag−eGFP VLPs. Bar graphs show mean values and SEM from the ectocervix and endocervix of 4 to 5 donors. (E) Images of Siglec-1 + cervical cells pulsed and labeled as in (A) . Cells were acquired by Amnis-imaging FACS, and showed green fluorescent HIV-1 Gag−eGFP VLPs accumulation within a sac-like virus-containing compartment enriched in Siglec-1 (labeled in red). (F) Paraffin-embedded cervical tissue from one viremic HIV-infected woman stained for HIV-1 p24 antigen (labeled in red), Siglec-1 (in green), and nucleus (in blue). Scale bar 50 μm. (Inset panels) zoom in of squared region with distinct fluorescences (scale bar 20 μm). (G) 3D volumetric x-y-z data fields reconstruction of Siglec-1 + cells from four distinct areas of the cervical tissue of the viremic HIV-infected woman. Opacity representation of DAPI stained nuclei and fluorescence of the sac-like virus-containing compartment (VCC; white arrows). Right bottom image displays a characteristic cell pattern with p24 + dots reflecting viral production (Infection).

Article Snippet: The mean number of Siglec-1 Ab binding sites per monocyte-derived DC from men and women was obtained with a Quantibrite kit (Becton-Dickinson) as previously described ( ).

Techniques: In Vivo, Isolation, Labeling, Expressing, Imaging, Infection, Staining, Fluorescence

Journal: Frontiers in Immunology

Article Title: Dendritic Cells From the Cervical Mucosa Capture and Transfer HIV-1 via Siglec-1

doi: 10.3389/fimmu.2019.00825

Figure Lengend Snippet: pDCs exposed to HIV-1 induce Siglec-1 expression on DCs via IFNα secretion. (A) IFNα release measured by ELISA on supernatants from blood derived pDCs co-cultured 24 h alone, with an uninfected MOLT CD4 + T cell line or an HIV-1 infected MOLT CD4 + T cell line that chronically produces R5-tropic BaL viruses in the presence or absence of 10 μg/ml of an anti-CD4 or an isotype mAb. Bar graph shows mean values and SEM from at least 6 donors and 3 independent experiments. Statistical differences were assessed with a Wilcoxon matched-pairs signed rank test. (B) Induction of Siglec-1 on monocyte-derived DCs incubated with supernatants isolated from pDCs co-cultured as in (A) and assessed by FACS. (C) Representative histograms of Siglec-1 expression on monocyte-derived DCs exposed to medium, recombinant IFNα, supernatants from uninfected pDCs, supernatants from HIV-1-exposed pDCs on mock treated DCs or on DCs previously incubated with the type-I interferon blocking receptor B18R. (D) IFNα release measured by ELISA on supernatants of pDCs isolated from women or men and co-cultured 24 h with an HIV-1 infected MOLT CD4 + T cell line. Bar graph shows mean values and SEM from 19 donors and 4 independent experiments. Statistical differences were assessed with a Mann-Whitney test. (E) Quantification of Siglec-1 expression levels on monocyte-derived DCs from men and women assessed by FACS. Bar graph shows mean values and SEM from 12 donors and 3 independent experiments. Prentice Rank Sum Test was used to assess statistical differences, which did not reach statistical significance ( P = 0.073).

Article Snippet: The mean number of Siglec-1 Ab binding sites per monocyte-derived DC from men and women was obtained with a Quantibrite kit (Becton-Dickinson) as previously described ( ).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay, Cell Culture, Infection, Incubation, Isolation, Recombinant, Blocking Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Dendritic Cells From the Cervical Mucosa Capture and Transfer HIV-1 via Siglec-1

doi: 10.3389/fimmu.2019.00825

Figure Lengend Snippet: IFNα enhances viral capture and trans -infection on Siglec-1 + cervical DCs. (A) Percentage of Siglec-1 + DCs within the myeloid HLA-DR + CD14 + CD11c + fraction from small pieces of ectocervix or endocervix cultured in the presence or absence of increasing concentrations of IFNα (1,000; 10,000; and 100,000 IU/ml) and assessed by FACS as in . Bar graph shows mean values and SEM from 5 donors and 4 independent experiments. Colors depict each particular donor. Statistical differences were assessed with a one-way repeated measures ANOVA test. (B) Percentage of Siglec-1 + DCs within the myeloid HLA-DR + CD14 + CD11c + fraction from cellular suspensions obtained from tissue digestion and cultured in the presence or absence of IFNα. Statistical differences were assessed with a paired t -test. (C) Percentage of cells capturing HIV-1 Gag−eGFP VLPs among the myeloid HLA-DR + CD14 + CD11c + fraction from cellular suspensions obtained from ectocervix or endocervix digestion and cultured in the presence or absence of IFNα. Statistical differences were assessed with a paired t -test. (D) Percentage of cells capturing HIV-1 Gag−eGFP VLPs as in (C) on cells that had been previously pre-incubated with 20 μg/ml of 7D2 anti-Siglec-1 mAb or isotype control. Bar graph shows mean values and SEM from 2-3 donors. Dot plots showing representative inhibition are also depicted. (E) Relative R5 tropic HIV-1 NFN−SX transmission to CD4 + target cells from cervical CD45 + CD3 − CD19 − HLA-DR + sorted cells pre-incubated with 20 μg/ml of isotype or anti-Siglec-1 mAbs before viral exposure. Values are normalized to isotype-treated cells (set at 100%). Statistical differences were assessed with a one sample t -test. Mean values and SEM from two experiments include cells from 3 or 4 donors.

Article Snippet: The mean number of Siglec-1 Ab binding sites per monocyte-derived DC from men and women was obtained with a Quantibrite kit (Becton-Dickinson) as previously described ( ).

Techniques: Infection, Cell Culture, Incubation, Inhibition, Transmission Assay

Journal: OncoImmunology

Article Title: CD169 identifies an activated CD8+T cell subset in regional lymph nodes that predicts favorable prognosis in colorectal cancer patients

doi: 10.1080/2162402x.2016.1177690

Figure Lengend Snippet: Figure 1. CD169+CD8+ T cells are selectively present in the regional LNs and decreased

Article Snippet: Frozen LN tissue sections were stained with sheep anti-human CD169 (R&D Systems, Minneapolis, MN, USA), mouse anti-human CD3 or mouse anti-human CD68 (Thermo Fisher Scientific, Waltham, MA, USA) and rabbit anti-human CD8 (Thermo Fisher Scientific), followed by specimen-paired immunofluorescence secondary antibodies (Life Technologies, Carlsbad, CA, USA).

Techniques:

Journal: PLoS Biology

Article Title: Siglec-1 Is a Novel Dendritic Cell Receptor That Mediates HIV-1 Trans -Infection Through Recognition of Viral Membrane Gangliosides

doi: 10.1371/journal.pbio.1001448

Figure Lengend Snippet: (A) (Left) Comparative HIV-1 capture of LPS and ITIP mDCs: cells were cultured with HIV-1, washed, and lysed to measure viral p24 Gag antigen by ELISA. (Right) Comparative transmission of captured HIV-1 from LPS and ITIP mDCs to a reporter CD4 + cell line. Graphs show mean values and standard error of the means (SEMs) from two independent experiments including cells from six donors. (B) Plot of SIGLEC genes (in open circles), CD86 and DC-SIGN (in grey circles) computing the fold change in LPS mDCs compared to ITIP mDCs, and the average gene expression across all samples. Circle size is inversely proportional to adjusted p values. Highlighted in red are statistically differentially expressed genes. Analysis was performed with DCs from four donors matured in parallel with the different stimuli. (C) Relative quantification of SIGLEC1 mRNA expression levels in distinct DCs analyzed by qRT-PCR. Measurements were normalized using the endogenous control housekeeping gene Beta Glucuronidase . Data show means and SEMs of samples from six donors. (D) Cell surface expression of Siglec-1 in distinct DCs analyzed by FACS with mAb 7–239-PE. (Left graph) Geometric mean fluorescence intensity (MFI) of Siglec-1. (Right graph) Percentage of Siglec-1 positive cells. Data show mean values and SEM from two experiments, including cells from six donors. (Histograms) Representative profiles of Siglec-1 staining in distinct DCs derived from one donor.

Article Snippet: DCs were blocked with 1 mg/ml of human IgG (Baxter, Hyland Immuno) and stained with α-Siglec-1-PE 7–239 mAb (AbD Serotec) following the manufacturer's instructions at 4°C for 20 min.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Transmission Assay, Expressing, Quantitative RT-PCR, Fluorescence, Staining, Derivative Assay

Journal: PLoS Biology

Article Title: Siglec-1 Is a Novel Dendritic Cell Receptor That Mediates HIV-1 Trans -Infection Through Recognition of Viral Membrane Gangliosides

doi: 10.1371/journal.pbio.1001448

Figure Lengend Snippet: (A) Relative capture of VLP HIV-Gag-eGFP by LPS mDCs that had been pre-incubated with 10 µg/ml of the indicated mAbs or 500 µg/ml of mannan before VLP exposure for 30 min at 37°C. Values are normalized to the level of VLP capture by mock-treated LPS mDCs (set at 100%). Data show mean values and SEMs from three experiments including cells from nine donors. (B) Relative capture of GM1 containing LUV HIV-tRed by LPS mDCs as described in (A). Data show mean values and SEMs from two experiments including cells from six donors. (C) Relative capture of Exosomes DiI by LPS mDCs that had been pre-incubated with 10 µg/ml of the indicated mAbs before exosome exposure for 4 h at 37°C. Values are normalized to the level of exosome capture by isotype-treated LPS mDCs (set at 100%). Data show mean values and SEMs from two experiments including cells from five donors. (D) Capture of VLP HIV-Gag-eGFP by LPS mDCs that had been pre-incubated with decreasing concentrations of α-Siglec-1 mAb 7D2 before VLP exposure for 30 min at 37°C. Titration of α-Siglec-1 mAb 7–239 is shown in . Data show mean values and SEMs from three experiments including cells from six donors. (E) Capture of VLP HIV-Gag-eGFP by LPS mDCs that had been pre-incubated with or without 2 µg/ml of α-Siglec-1 mAb 7D2 previously treated or not with at least a 100-fold molar excess of the indicated human recombinant proteins. Of note, Siglec-14 shares 100% of amino acid homology with Siglec-5 in the V-set domain. Data show mean values and SEMs from three experiments including cells from nine donors. (F) Kinetics of VLP HIV-Gag-eGFP capture by iDCs (left graph) and LPS mDCs (right graph) compared to the expression of Siglec-1 over time, assessed after LPS addition to mDCs. Cells were pulsed for 1 h at 37°C with VLP HIV-Gag-eGFP and labeled for Siglec-1 and HLA-DR in parallel at the indicated time points. For comparative purposes, the maximum geometric MFI values obtained by FACS for each donor were set at 100%. Data show mean values and SEMs including cells from three donors. (G) Positive correlation (ρ = 0.9695) between the geometric MFI of captured VLPs and the mean number of Siglec-1 Ab Binding Sites per cell in different DC subtypes (see also to compare VLP capture capacity among LPS mDCs derived from the same donor). Data show values from three experiments including cells from nine donors.

Article Snippet: DCs were blocked with 1 mg/ml of human IgG (Baxter, Hyland Immuno) and stained with α-Siglec-1-PE 7–239 mAb (AbD Serotec) following the manufacturer's instructions at 4°C for 20 min.

Techniques: Incubation, Titration, Recombinant, Expressing, Labeling, Binding Assay, Derivative Assay

Journal: PLoS Biology

Article Title: Siglec-1 Is a Novel Dendritic Cell Receptor That Mediates HIV-1 Trans -Infection Through Recognition of Viral Membrane Gangliosides

doi: 10.1371/journal.pbio.1001448

Figure Lengend Snippet: (A) Comparative capture of HIV-1 by distinct DCs that had been pre-incubated with 10 µg/ml of the indicated mAbs or 500 µg/ml of mannan for 30 min before viral exposure. Cells were cultured with HIV-1 in the presence of the indicated reagents, washed, and lysed to measure p24 Gag by ELISA. Viral binding at 4°C in LPS mDCs is shown in . Data show mean values and SEMs from two experiments including cells from six donors. (B) Comparative capture of HIV-1 by distinct DCs first exposed to the virus and then treated with the indicated reagents for 30 min before washing. Cells were lysed and assessed by p24 Gag ELISA. Data show mean values and SEMs from two experiments including cells from six donors. (C) Comparative capture of HIV-1 by distinct blood myeloid DCs that had been pre-incubated with 10 µg/ml of the indicated mAbs for 30 min before viral exposure as in panel A. depicts Siglec-1 surface expression levels of blood myeloid cells. Data show mean values and SEMs from two experiments including cells from six donors. (D) Confocal microscopy analysis of LPS mDCs pulsed for 4 h with GM1-containing LUV HIV-tRed , VLP HIV-Gag-Cherry or HIV-1 Cherry , fixed, permeabilized, and then stained for Siglec-1 with mAb 7–239-Alexa 488. (Inset) Merge of the bright field and maximun fluorescence intensity (scale bar: 5 µm). (3D images) Isosurface representation of DAPI stained nucleus and maximum fluorescence intensity of the sac-like compartment where particles and Siglec-1 accumulate are shown in a 3D volumetric x-y-z data field. (Bar graphs) Quantification of the percentage of GM1-containing LUV HIV-tRed , VLP HIV-Gag-Cherry or HIV-1 Cherry co-localizing with Siglec-1-Alexa 488 7–239 and vice versa, obtained analyzing at least 50 compartments from LPS mDCs of two donors. The mean and standard deviation of the thresholded correlation coefficient of Pearson (obtained considering all the images) were 0.77±0.07, indicating co-localization. See also , , or to observe the compartment in relation to the plasma membrane or the cytoplasm of the cells. (E) Confocal microscopy analysis showing the sac-like compartment pattern of Siglec-1 in LPS mDCs after internalization of the α-Siglec-1 mAb 7D2. Cells were labeled with the mAb for 30 min at 16°C, revealed with an Alexa 488 secondary Ab, shifted to 37°C for 4 h, and analyzed. (3D image) 3D reconstruction (representative of 69% of the analyzed DCs) was done as in (D). (Inset) Merge of the bright field and maximun fluorescence intensity (scale bar: 5 µm).

Article Snippet: DCs were blocked with 1 mg/ml of human IgG (Baxter, Hyland Immuno) and stained with α-Siglec-1-PE 7–239 mAb (AbD Serotec) following the manufacturer's instructions at 4°C for 20 min.

Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Binding Assay, Expressing, Confocal Microscopy, Staining, Fluorescence, Standard Deviation, Labeling

Journal: PLoS Biology

Article Title: Siglec-1 Is a Novel Dendritic Cell Receptor That Mediates HIV-1 Trans -Infection Through Recognition of Viral Membrane Gangliosides

doi: 10.1371/journal.pbio.1001448

Figure Lengend Snippet: (A) HIV-1 transmission from distinct DCs to a reporter CD4 + cell line. DCs were pre-incubated as in , washed, and co-cultured with reporter cells for 48 h. HIV-1 infection of reporter cells was determined by induced luciferase activity in relative light units (RLUs). Data show mean values and SEMs from two experiments including cells from six donors. (B) HIV-1 transmission from distinct DCs first exposed to the virus and then treated with the indicated reagents for 30 min before washing and co-culture with reporter cells. Data show mean values and SEMs from two experiments including cells from six donors. (C) Confocal microscopy analysis of LPS mDCs pulsed with HIV-1 Cherry and then co-cultured with CD4 + T cells to reveal Siglec-1 localization. Co-cultures were stained with α-CD4-Alexa 647 mAb to identify the membrane of CD4 + T cells, fixed, permeabilized, and labeled with α-Siglec-1-Alexa 488 7–239 mAb. (Left images) Merge of the bright field and the fluorescence of an x-y plane (scale bar: 5 µm). (Right images) Isosurface representation of DAPI-stained nucleus and maximum fluorescence intensity of the compartment where HIV-1 Cherry and Siglec-1 accumulate in the contact zone with a CD4 + T cell, shown in a 3D volumetric x-y-z data field. (D) HIV-1 transmission to reporter cells from distinct blood myeloid DCs that had been pre-incubated with 10 µg/ml of the indicated mAbs for 30 min before viral exposure as in (A). Data show mean values and SEMs from three experiments including cells from twelve donors.

Article Snippet: DCs were blocked with 1 mg/ml of human IgG (Baxter, Hyland Immuno) and stained with α-Siglec-1-PE 7–239 mAb (AbD Serotec) following the manufacturer's instructions at 4°C for 20 min.

Techniques: Transmission Assay, Incubation, Cell Culture, Infection, Luciferase, Activity Assay, Co-Culture Assay, Confocal Microscopy, Staining, Labeling, Fluorescence

Journal: PLoS Biology

Article Title: Siglec-1 Is a Novel Dendritic Cell Receptor That Mediates HIV-1 Trans -Infection Through Recognition of Viral Membrane Gangliosides

doi: 10.1371/journal.pbio.1001448

Figure Lengend Snippet: (A) Interference of SIGLEC1 . Percentage of LPS mDCs positive for CD14, HLA-DR, Siglec-1, or VLP capture following mock transduction or transduction with nontarget or two different SIGLEC1 -specific shRNAs. Data show mean values and SEMs from four experiments including cells from at least four donors. (B) Representative cell surface expression levels of CD14, HLA-DR, or Siglec-1 and VLP HIV-Gag-eGFP capture profile of LPS mDCs transduced with nontarget shRNA (blue), SIGLEC1 target shRNA (red), or mock transduced (grey). (C) HIV-1 transmission to CD4 + reporter cells of LPS mDCs that had been mock-transduced or transduced with nontarget or SIGLEC1 -specific shRNA. DCs were pulsed with HIV-1, washed, and co-cultured with reporter cells for 48 h. HIV-1 infection of CD4 + reporter cells was determined by induced luciferase activity in RLUs. Data show mean values and SEMs from two experiments including cells from four donors. (D) Transfection of Siglecs in Raji B cells (see also for transfection efficiencies). Capture of VLP HIV-Gag-eGFP by Raji cells transfected with the indicated expression plasmids for Siglecs or mock transfected. Transfected Raji cells were pre-incubated with 10 µg/ml of the indicated mAbs and exposed to VLPs. See for blocking effect of sialyllactose. Data show mean values and SEMs from two experiments including cells from four transfections. shows results for Siglec transfections in HEK-293T. (E) Representative dot plots from Siglec-1, Siglec-5, and Siglec-7 transfected Raji cells pre-incubated with the indicated mAbs and subjected to VLP capture. (F) HIV-1 transmission from Raji cells transfected with the indicated expression plasmids for Siglecs to reporter CD4 + cells. Transfected cells were pre-incubated with the indicated mAbs as in (C) and then exposed to HIV-1. Data show mean values and SEMs from two experiments including cells from four transfections. depicts viral capture and transmission of viruses with or without the envelope glycoproteins.

Article Snippet: DCs were blocked with 1 mg/ml of human IgG (Baxter, Hyland Immuno) and stained with α-Siglec-1-PE 7–239 mAb (AbD Serotec) following the manufacturer's instructions at 4°C for 20 min.

Techniques: Transduction, Expressing, shRNA, Transmission Assay, Cell Culture, Infection, Luciferase, Activity Assay, Transfection, Incubation, Blocking Assay

Journal: iScience

Article Title: Immunometabolic adaptation in monocytes underpins functional changes during pregnancy

doi: 10.1016/j.isci.2024.109779

Figure Lengend Snippet:

Article Snippet: CD169 human antibody (clone 7-239) , Miltenyi , Cat#130-098-654; RRID: AB_2655545.

Techniques: Recombinant, Adhesive, Sequencing, Modification, DC Protein Assay, Staining, Imaging, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Gene Expression, Software, Saline