Fig 2: B1 B cells but not macrophages specifically phagocytose PtC beads. (A) PerC cells were incubated with Rhodamine green (RG)-PtC beads and TRITC (red)-control beads. Cells were then stained for IgM (blue) and CD5 (magenta). IgM+CD5+ B1 B cells were compared with IgM-CD5- large macrophages for internalized red and green beads. (B) Representative flow cytometry gating of PerC cells after incubation with the beads. Red arrows indicate that most macrophages were double positive with red control beads and green PtC beads, whereas phagocytic B1 B cells were exclusively single positive with green PtC beads. (C) Percentages of B1 B cells and macrophages that have PtC-specific and non-specific (both beads) phagocytosis are compared. Results are mean ± SEM of 12 mice from 3 independent experiments.

Fig 3: Loss of Gal9 in NZB/W mice leads to accelerated autoimmunity.(A) Titers of dsDNA-specific IgG in sera of NZB/W mice (as indicated) monitored over time. (B) Protein secretion in the urine of NZB/W mice monitored over time. (C) Representative plots and (D) summary proportion of splenic GC B cells in NZB/W mice (as indicated). (E) Representative plots and (F) summary proportion of antibody secreting cells (ASC) in the spleen of NZB/W mice. (G) Proportion of long-lived ASC lacking B220 expression. (H) Circulating IgG-containing immune complex (ICs) titers. (I) Proportion of CD5+ B-1a cells in the peritoneal cavity of NZB/W mice. (J) Proportion of CD19+ CD43+ B-1 cells in the spleen of NZB/W mice. (K) Proportion of splenic CD5+ B-1a cells. (L) Circulating phosphoryl-choline specific IgM titers in NZB/W mice. Data in (A) and (B) are representative of at least 14 biological replicates, data in (C–L) are representative of eight biological replicates. Data represent mean and SEM. Statistical significance was assessed by Mann–Whitney, *p=0.05, **p=0.01, ***p=0.001.

Fig 4: Loss of Gal9 leads to enhanced activation and accumulation of B-1a cells at steady-state.(A) Representative plots of IgM expressing cells in the PerC of aged WT and Gal9KO mice, as indicated. (B) Summary proportion of CD5 expressing B-1a cells in the PerC shown in A. (C) Representative histograms of CD86 expression at steady state on PerC cells shown in (A). (D) Summary gMFI of CD86 expression shown in C. (E) Representative plots of ASCs in the PerC of aged mice. (F) Summary proportion of ASCs shown in (E). (G) Anti-phosphorylcholine (PC) specific IgM in sera of aged mice. (H) Representative plots of B-1 cells of IgM expressing cells in the spleen of aged mice. (I) Proportion of B-1 cells shown in H. (J) Representative plots of B-1a and B-1b cells of H. (K) Summary proportion of CD5 expressing B-1a cells in the spleen of aged mice shown in (I). Data are representative of ten biological replicates over three independent experiments. Statistical significance was assessed by Mann–Whitney ****p<0.0001.

Fig 5: Gal9 regulates BCR signal transduction in B-1a cells through interactions with IgM and the negative co-receptor CD5.(A) Representative histograms of CD86 expression on B-1a cells from WT (black) and Gal9KO (red) mice stimulated with titrated concentrations of anti-IgM F(ab’)2. (B) Summary proportion of CD86 expressing cells as a function of F(ab’)2 concentration as in (A). (C) EC50 of F(ab’)2 titration shown in (A). (D) Representative histogram of p-Tyr level in B-1a cells from WT (black, open) or Gal9KO (red, filled), and WT B-1a cells treated with lactose (blue, open) and Gal9KO B-1a cells treated with rGal9 (orange, filled) stimulated with anti-IgM (Fab’)2 for 5 min (left), summary gMFI of p-Tyr levels (right). (E) Representative histograms of Gal9 expression in B-1a cells (green), B-1b cells (white), and splenic B-2 cells (purple) from WT mice (left), summary gMFI of Gal9 expression (right). (F) Representative histograms of CD5 detection on rGal9-coated beads incubated with lysates from different B cell subsets, as indicated (left), summary gMFI of CD5 enrichment on beads incubated with lysates (right). (G) Reconstructed dual-dSTORM images of IgM (yellow) and CD5 (cyan) on the surface of B-1a cells from WT and Gal9KO mice, ROI (3 µm x 3 µm) used for analysis is expanded in zoom. Scale bars represent 1 µm. (H) Quantification of IgM mean cluster area from dSTORM images. (I) Quantification of IgM clustering tendency using Hopkins index. (J) Quantification of CD5 mean cluster area from dSTORM images. (K) Quantification of CD5 clustering tendency using Hopkins index. (L) Frequency distribution of coordinate-based colocalization (CBC) between IgM and CD5. (M) Mean CBC value per ROI. (N) Mean distance between nearest neighbors. Data in (A–F) are representative of nine biological replicates over three independent experiments. Data in (G–N) are representative of 30 ROIs acquired over at least three independent experiments. Data represent mean ± SEM. Statistical significance for C, H, I, J, K, M, and N was assessed by Mann–Whitney, statistical significance for D, E, and F was assessed by Kruskal–Wallis. *p≤0.05, **p≤0.01, ***p≤0.001, ****p<0.0001.