Figure 6. Absence of CXCR5 on donor splenocytes alters hepatic IgM⁺ and IgG⁺ B cell number and B cell clustering.

(A) Frequency of B cells (plasma cells and plasmablasts) determined by FACS analysis on liver lymphocytes isolated 3 weeks after transfer of HBVEnvRag1^–/– or Rag1^–/– mice with WT or Cxcr5^–/– splenocytes. Percentage of IgG1, IgG2b, and IgG3 B cell (B220^loTCR-β^–CD44^hi) and IgM B cell (B220^loTCR-β^–) populations were identified in liver-derived lymphocytes by FACS (n = 3). (B) Representative images (original magnification, ×10) from liver tissue of HBVEnvRag1^–/– mice 21 days after adoptive transfer of WT or Cxcr5^–/– splenocytes, stained for IgM (green), IgG (red; IgG1 and IgG2b pooled) and DAPI (blue). (C and D) 15 random frames per section of n ≥ 3 mice per group were blinded and analyzed for (C) number of IgM⁺ and IgG⁺ cells and (D) number of IgG lymphocyte clusters (≥2 cells within 15 μm) and IgG⁺ cells associated with ≥1 IgM⁺ cell per frame. (E) Representative image (original magnification, ×40) from HBVEnvRag1^–/– mouse liver stained for F4/80 (green), IgG (red; IgG1 and IgG2b pooled), and DAPI (blue) 21 days after adoptive transfer of WT syngeneic splenocytes. Scale bars: 30 μm (B); 10 μm (E). *P < 0.05, **P < 0.01, ***P < 0.001, Tukey’s ANOVA multiple-comparison test (A) or unpaired 2-tailed Student’s t test (C and D).