Btk overexpression in vivo enhances GC and plasma cell formation. (A) Quantification of intracellular flow cytometric detection of Btk protein in WT splenic B cells after 3 days of in vitro culture in the presence of the indicated stimuli (supplemental Figure 1). Levels were calculated relative to average Btk expression levels (MFI) in unstimulated B cells (set to 1) and background signals in unstimulated Btk^−/− B cells (set to 0). Data are mean values ± SD (n = 3) from 1 of 3 representative experiments, asterisks indicate significant increases in Btk expression (P < .05). (B) Intracellular flow cytometric Btk detection in splenic CD19⁺ B cells, shown as histogram overlays from Btk^−/− (shaded histogram), WT (black line) and CD19-hBtk (red line) mice. (C) Immunohistochemical analysis of spleen tissue sections of CD19-hBtk and WT mice. Sections were stained for CD21 (red), and metallophilic macrophage marker MOMA-1 (blue), or for GL7 (red) and CD138 (blue); arrows indicate clusters of GL7⁺ GC B cells (objective 10×). (D) Flow cytometric analysis of GC cells in the spleens of the indicated mice. CD19⁺ B cells were gated and analyzed for IgM/IgD profiles. IgD^lowIgM^low cells were subsequently gated and analyzed for PNA and CD95. Data are displayed as dot plots and the percentages of cells in the indicated quadrants are given. (E-F) Flow cytometric quantification of GC B cells (PNA⁺CD95⁺IgD^lowIgMl^owCD19⁺) and IgM⁺CD138^high or IgG⁺CD138^high plasma cells in spleen (E) and BM (F). Horizontal lines indicate median per group; each symbol represents and individual mouse. Mice were 10 to 14 weeks old.