Fig 2: 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

Fig 3: 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

Fig 4: Model depicting how N-glycosylation regulates CD22 organization and function. (A) Top and side view of IgM and CD22 organization on the surface of resting WT B cells (left) and 5Q-mutant Daudi B cells (right). In resting WT B cells, galectin-9 mediates basal interaction between IgM and CD22 to allow for regulated BCR signaling. Mutation of five N-linked glycosylation sites from asparagine to glutamine on CD22 leads to increased CD22 clustering, decreased IgM clustering, decreased CD22 phosphorylation, and increased BCR signaling. (B) Top and side view of IgM and CD22 organization on the surface of recombinant galectin-9 (rGal9) treated WT (left) and 5Q-mutant (right) B cells. In resting WT B cells, treatment with rGal9 increases IgM and CD22 nanoclusters and increases IgM-CD22 colocalization leading to suppression of BCR signaling upon antigen stimulation. Treatment of 5Q-mutant with rGal9 increases IgM cluster size and decreases IgM-CD22 colocalization. rGal9 treatment does not reduce BCR signaling in 5Q-mutant B cells as observed in WT B cells, demonstrating that galectin-9 mediated regulation of BCR signaling is dependent on CD22 N-glycans.

Fig 5: 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 (Gal9high) and low galectin-9 (Gal9low). d Mean fluorescence intensity of CD45 (left) and IgM (right) in Gal9high and Gal9low 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 Gal9high and Gal9low 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 Gal9low and Gal9high 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