Figure 3.

COMMD8 deficiency impairs B cell migration and humoral immune responses. (A) Chemotactic responses of control and Commd8Δ B cells were assessed by transwell migration of Fo B cells toward CXCL12, CXCL13, CCL19, 7α,25-HC, and S1P. Data are shown as the mean ± SD of triplicates. (B) Immunofluorescence microscopy of spleen tissues from control and Commd8Δ mice to detect B cells (IgD; green) and T cells (CD4; red). (C) Distribution of control and Commd8Δ B cells in the spleen of mixed bone marrow chimeras (20% Igh^b control or Commd8Δ plus 80% Igh^a WT). Sections were stained to detect Igh^b B cells (IgD^b; green), Igh^a B cells (IgD^a; red), and T cells (CD4; blue). (D) Control (Ctrl) and Commd8Δ (Δ) B cells were labeled with CFSE and transferred into WT mice. At 24 h after cell transfer, density of CFSE⁺ cells (green) in the follicles relative to the red pulp was assessed in the spleen sections stained for CD4 (blue) and MAdCAM-1 (red). The boxed areas are magnified to show CFSE⁺ cells (circled) and the perimeter of the follicles (dashed line). Each symbol represents an individual analyzed field, and bars indicate means (n = 12). (E and F) A mixture of CFSE-labeled LN cells (50% CD45.2 control or Commd8Δ plus 50% CD45.1 WT) were transferred into WT mice. LN entry of CD45.2 relative to CD45.1 B cells (CD19⁺) was assessed at 90 min after transfer (E). B cell egress from LNs was assessed at 12 h after blockade of LN entry (F). Transferred CD4⁺ T cells were tracked as an additional control. (G) Distribution of NP-binding B cells (green) in the spleen of control and Commd8Δ B1-8^hi mice was analyzed at the indicated times after NP-PE injection. (H–J) Control (Ctrl) and Commd8Δ (Δ) mice were immunized by i.p. injection of NP-CGG on days 0 and 28. Serum antibody titers (H, shown as the mean ± SEM of 10 mice) and the generation of GC B cells and plasmablasts in the spleen (I, shown as the percentages among B cells and total lymphocytes, respectively) were measured at the indicated times. Spleen sections were analyzed on day 7 to detect GCs (GL7; red), follicles (IgD; green), and T cell zones (CD4; blue; J). Littermate Commd8^f/+Mb1^Cre/+ (C–E) or Commd8^f/fMb1^+/+ (A, B, and F–J) mice served as the control. Data are representative of three (A and G) or two (B, C, F, and J) independent experiments, or pooled from two (D and E) or three (H and I) independent experiments. Each symbol represents an individual mouse, and bars indicate means (E, F, and I). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. The P values were obtained by two-tailed unpaired (A, D–F, and I) or paired (H) t test. Bars, 200 µm.