Journal: PLoS ONE

Article Title: Characterization of neutralizing antibodies reacting with the 213-224 amino-acid segment of human galectin-9

doi: 10.1371/journal.pone.0202512

Figure Lengend Snippet: Western blot (WB) analysis of cold immuno-precipitation (IP) products obtained with various anti-gal-9 antibodies. The source of native gal-9 was a protein extract from a murine thymus. Concomitant IP were performed with non-specific mouse IgG1 (negative control), Gal-Nab1 and Gal-Nab2, 9M1 (a commercial antibody directed to human gal-9) and RG9-35 (a commercial antibody directed to murine gal-9). 15 ng of recombinant murine gal-9 M (lane 1) and 25 μg of crude murine thymus extract (lane 2) were used as WB controls. The primary antibody used for WB detection of murine gal-9 was a polyclonal goat serum. The secondary antibody reacted with this primary but also with the immunoglobulin heavy chains eluted from the beads giving strong bands at a molecular weight of about 55 Kd. These bands were of similar intensities for all five IP conditions, suggesting that bead capture of the immune complexes was in the same range of efficiency. The bands resulting from gal-9 staining (36 Kd) were assessed by densitometry (graphs for long and short exposure are presented at the bottom of the figure).

Article Snippet: A goat polyclonal antibody was used for detection of murine gal-9 by Western blotting (R&D System/Bio-Techne).

Techniques: Western Blot, Immunoprecipitation, Negative Control, Recombinant, Molecular Weight, Staining

Journal: Nature Communications

Article Title: Galectin-9 binds IgM-BCR to regulate B cell signaling

doi: 10.1038/s41467-018-05771-8

Figure Lengend Snippet: Galectin-9 regulates BCR microcluster formation and signaling. a Representative images of primary naive WT (top) and Gal9-KO B cells (bottom) fixed on bilayers containing anti-kappa as surrogate antigen (Ag) after 90 s of spreading and imaged by confocal microscopy. Brightfield (left) and confocal (right) visualizing antigen mapped to an 8-bit fire color scale (ImageJ). Scale bar 2 μm. Quantification of b area of spreading, c total antigen fluorescence intensity at the cell-bilayer contact, and d mean intensity of antigen for WT (black circles) and Gal9-KO (blue diamonds) cells (each dot represents 1 cell, 200 cells measured per condition), with the mean ± SEM indicated by the red bar, **** p < 0.0001, Mann-Whitney test. Data representative of at least three independent experiments. e – h Primary naive B cells from WT and Gal9-KO mice were settled onto anti-IgM-coated plates for the indicated time. Cells were lysed and subjected to SDS-PAGE followed by immunoblotting with e anti-phosphotyrosine and anti-ERK1/2, f anti-phospho-CD19, g anti-phospho-Akt, and h anti-phospho ERK1/2 (pERK) and anti-β tubulin. Data representative of at least three independent experiments. f Quantification of the fold increase in pCD19, pAkt, and pERK, with the mean ± SEM indicated by bar. Data were analyzed by two-way ANOVA, followed by Sidak’s multiple comparisons test; * p < 0.05, ** p < 0.01

Article Snippet: Primary murine B cells from WT and Gal9-KO mice were labeled with 4 ng/mL Attotec ® 633-labeled goat anti-mouse IgM Fab fragment (Jackson ImmunoResearch, Cat. No. 115-007-020) in 0.5% FBS in PBS for 15 min at 4 °C.

Techniques: Confocal Microscopy, Fluorescence, MANN-WHITNEY, SDS Page, Western Blot

Journal: Nature Communications

Article Title: Galectin-9 binds IgM-BCR to regulate B cell signaling

doi: 10.1038/s41467-018-05771-8

Figure Lengend Snippet: Treatment with exogenous galectin-9 suppresses BCR signaling. a Representative flow cytometric histograms of WT (left) and Gal9-KO (middle) B cells either untreated or treated with various concentrations of recombinant galectin-9 (rGal9; 0.1, 0.2, 0.5, and 1 μM) followed by surface staining for galectin-9 and analyzed using flow cytometry. Overlay of endogenous galectin-9 surface expression in WT cells, and Gal9-KO cells treated with 0.1 μM rGal9 (right). b Naive B cells from WT and Gal9-KO mice treated with 0.1 μM rGal9 were settled onto anti-IgM-coated plates for the indicated time. Cells were lysed and subjected to SDS-PAGE followed by immunoblotting with anti-phospho ERK1/2 and anti-β tubulin (left panel). Quantification of the fold change in pERK over time, averaged over two independent experiments with the mean ± SEM indicated by the bar (right panel). c – f Naive B cells from WT mice were treated with 1 μM rGal9 and settled onto anti-IgM-coated plates for the indicated time. Cells were lysed and subjected to SDS-PAGE followed by immunoblotting with c anti-phosphotyrosine and ERK1/2, d anti-phospho-CD19, e anti-phospho-Akt, and f anti-phospho ERK1/2 and anti-β tubulin. Quantification of the fold change in pCD19, pAkt, and pERK over time, averaged over three independent experiments, with the mean ± SEM indicated by the bar is shown in the right panel. Statistical significance measure by two-way ANOVA followed by Sidak’s multiple comparisons test; **** p < 0.0001, ** p < 0.01, * p < 0.05

Article Snippet: Primary murine B cells from WT and Gal9-KO mice were labeled with 4 ng/mL Attotec ® 633-labeled goat anti-mouse IgM Fab fragment (Jackson ImmunoResearch, Cat. No. 115-007-020) in 0.5% FBS in PBS for 15 min at 4 °C.

Techniques: Recombinant, Staining, Flow Cytometry, Expressing, SDS Page, Western Blot

Journal: Nature Communications

Article Title: Galectin-9 binds IgM-BCR to regulate B cell signaling

doi: 10.1038/s41467-018-05771-8

Figure Lengend Snippet: Galectin-9 alters IgM-BCR nanoclusters. a TIRFM image of surface IgM and fluorescently labeled rGal9 before bleaching for image acquisition (two left panels respectively). dSTORM images reconstructed from single-molecule localization processed by Thunderstorm software mapped to a fire color scale as indicated; the magnified region (3 × 3 µm) from ROI (white box) is shown as 2D image (middle) and 3D surface plot (right) in the order of WT (top), Gal9-KO (middle), and Gal9-KO + 1 µM rGal9 (bottom). Scale bar represents 2 µm. b Quantification of the distribution of IgM by H function and c Hopkins index of localizations inside ROIs. d – g Reconstructed images were analyzed by a model-based Bayesian approach to identify nanoclusters and their physical properties. d Number of clusters (one point per ROI). e Cluster radii (one point per cluster). f Number of molecules (one point per cluster). g Percentage of localization in clusters (one point per ROI). Each category contains at least 15 ROIs from three independent experiments (at least four cells per experiment). Statistical analysis was performed using Kruskal-Wallis test with Dunn’s multiple comparison test ( d , e , f ) and one-way ANOVA with Tukey’s multiple comparison test ( b , g ). Red bars indicate mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Primary murine B cells from WT and Gal9-KO mice were labeled with 4 ng/mL Attotec ® 633-labeled goat anti-mouse IgM Fab fragment (Jackson ImmunoResearch, Cat. No. 115-007-020) in 0.5% FBS in PBS for 15 min at 4 °C.

Techniques: Labeling, Software

Journal: Nature Communications

Article Title: Galectin-9 binds IgM-BCR to regulate B cell signaling

doi: 10.1038/s41467-018-05771-8

Figure Lengend Snippet: Galectin-9 immobilizes IgM-BCR and attenuates BCR microclustering. a Diffusion coefficients and b frequency distribution histogram of single-particle tracking of IgM in WT (black circle) or Gal9-KO (blue diamond) primary B cells with the median indicated in red. Five hundred representative diffusion coefficients from a total of at least 1500 tracks from three independent experiments. c Representative TIRF image of fluorescently labeled rGal9 on primary B cell (left) and mask (right) created to differentiate tracks inside Gal9 regions (lower left, red lines) and tracks outside Gal9 regions (lower right, yellow lines). d Diffusion coefficients and e frequency distribution inside Gal9 regions (black circle) and outside Gal9 regions (black triangle) with the median indicated in red. In all, 250 representative diffusion coefficients from a total of at least 900 tracks from three independent experiments. f Representative TIRF microscopy images of WT cell (top) and WT cells treated with 1 μM rGal9 (bottom) on artificial planar lipid bilayers containing anti-kappa mapped to an 8-bit fire color scale (ImageJ). g Quantification of the total antigen intensity at the cell-bilayer interface in WT (black circles) and WT treated with rGal9 (open circles), with the mean ± SEM indicated by the red bar. Scale bar represents 2 μm. Statistical significance assessed by Mann-Whitney; **** p < 0.0001, *** p < 0.001, ** p < 0.01

Article Snippet: Primary murine B cells from WT and Gal9-KO mice were labeled with 4 ng/mL Attotec ® 633-labeled goat anti-mouse IgM Fab fragment (Jackson ImmunoResearch, Cat. No. 115-007-020) in 0.5% FBS in PBS for 15 min at 4 °C.

Techniques: Diffusion-based Assay, Single-particle Tracking, Labeling, Microscopy, MANN-WHITNEY

Journal: Nature Communications

Article Title: Galectin-9 binds IgM-BCR to regulate B cell signaling

doi: 10.1038/s41467-018-05771-8

Figure Lengend Snippet: The galectin-9 lattice increases the molecular density of IgM-BCR and co-receptors. a Representative confocal images of primary WT B cells treated with 1 µM rGal9 and immuostained for CD45 (cyan), IgM (magenta), and galectin-9 (Gal9; yellow). b Fluorescence intensity profile of CD45, IgM, and Gal9 along the cell membrane. c Representative example of masking output of algorithm to detect regions of high galectin-9 (Gal9 high ) and low galectin-9 (Gal9 low ). d Mean fluorescence intensity of CD45 (left) and IgM (right) in Gal9 high and Gal9 low regions. e Representative confocal images of WT B cells treated with 1 µM rGal9 and immunostained for CD22 (cyan), IgM (magenta), and Gal9 (yellow). f Fluorescence intensity profile of CD22, IgM, and Gal9 along the cell membrane. g Mean fluorescence intensity of CD22 (left) and IgM (right) in Gal9 high and Gal9 low regions. h Representative confocal images of WT B cells treated with 1 μM rGal9 and immunostained for CD19, IgM, and Gal9. i Fluorescence intensity profile of CD19, IgM, and Gal9 along the cell membrane. j Mean fluorescence intensity of CD19 (left) and IgM (right) in Gal9 low and Gal9 high regions. Data representative of at least three independent experiments. Each dot represents 1 cell, at least 30 cells measured per condition per experiment. Mean ± SEM indicated by the red bar. Statistical significance assessed by Mann-Whitney; **** p < 0.0001 *** p < 0.001, * p < 0.05. Scale bar 2 μm

Article Snippet: Primary murine B cells from WT and Gal9-KO mice were labeled with 4 ng/mL Attotec ® 633-labeled goat anti-mouse IgM Fab fragment (Jackson ImmunoResearch, Cat. No. 115-007-020) in 0.5% FBS in PBS for 15 min at 4 °C.

Techniques: Fluorescence, MANN-WHITNEY

Journal: Nature Communications

Article Title: Galectin-9 binds IgM-BCR to regulate B cell signaling

doi: 10.1038/s41467-018-05771-8

Figure Lengend Snippet: Galectin-9 increases colocalization between CD22 and IgM in primary B cells. a Representative merged TIRF (top), dSTORM (middle), and dSTORM zoom (bottom) images showing surface CD45 (magenta) and IgM-BCR (green) on primary wild-type (WT) (left) and galectin-9 knockout (Gal9-KO) (right) B cells. dSTORM ROI (3 × 3 μm) is outlined in yellow (middle) and magnified in dSTORM zoom (bottom). b – e Quantification of at least 20 ROIs from WT and Gal9-KO B cells pooled from three independent experiments. b Hopkin’s index showing randomness of CD45 organization (one point per ROI). c H function derived from Riplely’s K showing degree of CD45 clustering. d Mean diameter of CD45 clusters (one point per ROI). e Mean area of CD45 clusters (one point per ROI). f , g Quantification of at least 15 ROIs from WT and Gal9-KO B cells pooled from three independent experiments. f Coordinate-based colocalization (CBC) histograms of the single-molecule distributions of colocalizations between CD45 and IgM. g Nearest-neighbor distance (NND) analysis of the data shown in f . Symbol represents the median NND of all paired single-molecule localizations from one ROI. h Representative merged TIRF and dSTORM images showing surface CD22 (magenta) and IgM-BCR (green) on primary WT (left) and Gal9-KO (right) B cells. i – l Quantification of at least 30 ROIs from WT and Gal9-KO B cells pooled from three independent experiments. i Mean Hopkin’s index showing randomness of CD22 organization (one point per ROI). j H function showing degree of CD22 clustering. k Mean diameter of CD22 clusters (one point per ROI). l Mean area of CD22 clusters (one point per ROI). m , n Quantification of at least 20 ROIs from WT and Gal9-KO B cells pooled from three independent experiments. m CBC histograms of the single-molecule distributions of colocalizations between CD22 and IgM. n NND analysis of the data shown in m . Colocalization between channels shown in white. Scale bars represent 2 and 1 μm (zoom). Mean ± SEM indicated by the red bar. Statistical significance assessed by Mann-Whitney, * p < 0.05

Article Snippet: Primary murine B cells from WT and Gal9-KO mice were labeled with 4 ng/mL Attotec ® 633-labeled goat anti-mouse IgM Fab fragment (Jackson ImmunoResearch, Cat. No. 115-007-020) in 0.5% FBS in PBS for 15 min at 4 °C.

Techniques: Knock-Out, Derivative Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: Galectin-9 binds IgM-BCR to regulate B cell signaling

doi: 10.1038/s41467-018-05771-8

Figure Lengend Snippet: Schematic model of galectin-9 regulation of B-cell activation. a In resting primary naive WT B cells, galectin-9 facilitates interactions between BCRs and either the inhibitory proteins CD45 or CD22 through binding to N-linked glycans, providing a basal attenuation of B-cell signaling upon antigen stimulation. b BCR signaling is enhanced in Gal9-KO B cells due to loss of association of BCR with inhibitory co-receptors. c Treatment of WT B cells with rGal9 induces the association of IgM-BCR with CD45 and CD22 to suppress B-cell signaling

Article Snippet: Primary murine B cells from WT and Gal9-KO mice were labeled with 4 ng/mL Attotec ® 633-labeled goat anti-mouse IgM Fab fragment (Jackson ImmunoResearch, Cat. No. 115-007-020) in 0.5% FBS in PBS for 15 min at 4 °C.

Techniques: Activation Assay, Binding Assay

Journal: PLoS ONE

Article Title: Graves’ Disease Is Associated with a Defective Expression of the Immune Regulatory Molecule Galectin-9 in Antigen-Presenting Dendritic Cells

doi: 10.1371/journal.pone.0123938

Figure Lengend Snippet: Triple immunofluorescence microscopy analysis of thyroid tissue from a representative AITD patient (A,B) and goiter (C,D) for the expression of Gal-1 or Gal-9 (green), HLA-DR (purple) and CD11c (red); nuclei were counterstained with Hoechst (blue); arrowheads marks some of the triple-positive cells. E) Percent of conventional dendritic cells expressing Gal-1 or Gal-9, a minimum of 100 cells (CD11c+) per slide were analyzed. F, G. Flow cytometry analysis of Gal-1 and Gal-9 expression in cDCs. F) Dot plot strategy to select cDCs; lineage negative cells (CD3, CD14, CD16, CD19 and CD20), CD45+, HLA-DR+, CD11c+. G) Representative histogram of Gal-1 and Gal-9 expression in cDCs from the thyroid gland. The percentage of Gal-1 and Gal-9 positive cells in cDCs in a representative GD patient (blue line) compared to the negative control (gray line) is shown. The apparent discrepancy between IHC and flow cytometry studies is very likely due to the different strategy to select cDCs. The Gal-1 and Gal-9 positive intrathyroidal cDCs (blue line) are compared to peripheral blood expression in the same GD patient (red line).

Article Snippet: To analyze galectin surface expression on peripheral blood DCs, the following mouse anti-human monoclonal antibodies (mAb) were used: PerCP-HLA-DR, FITC-CD3, FITC-CD14, FITC-CD16, FITC-CD19, FITC-CD20, V450-CD11c (BD Biosciences) and PE-CD123 (Biolegend, San Diego, CA,USA), and goat polyclonal anti-Gal-1 or anti-Gal-9 (R&D systems, McKinley, MN,USA), followed by an AlexaFluor 647 donkey-anti-goat (DAG) (Applied Biosystems, Carlsbad, CA,USA).

Techniques: Immunofluorescence, Microscopy, Expressing, Flow Cytometry, Negative Control