Figure 3. Extrafollicular J558 Id+ CD138+ early proliferation is balanced by apoptosis later in the response to E. cloacae.

(A) Spleen sections from V_HJ558 TG mice immunized with E. cloacae strain MK7 were harvested at 0, 3, 7, 14, and 28 days after immunization and stained with antibodies specific for DEX-specific B cells bearing the J558 idiotype (red), CD4+ T cells (green), and metallophilic macrophages (blue) revealing the presence of J558 idiotype high plasma cells within extrafollicular foci in the red pulp (white arrows). (B) Many more extrafollicular J558 Id+ CD138+ B cells express Ki67 at 3 days compared to 21 days after immunization with E. cloacae. Sections of spleen from V_HJ558 TG mice immunized with E. cloacae strain MK7 and stained with Ki67-FITC (green) and anti-J558 Id (red). (C) Ki67+ J558 Id+ plasmablasts in extrafollicular foci at 3, 14, and 21 days after E. cloacae strain MK7 immunization. Numbers were calculated by counting Ki67 staining cells in 5 individual extrafollicular foci per 3 mice. (D) DEX-specific plasmablast homeostasis is balanced by proliferation and apoptosis. Analysis of division and apoptosis of J558 Id+ CD138 + B cells using intracellular PI cell to determine cycle status (black line; left y axis) and surface annexin V+/7AAD+ staining to determine late apoptotic cells (grey line; right y axis). Data shown is from 3 independent experiments (n= 3 mice per experiment). Asterisks show statistical significance in comparison to the day 0 time point.