BCR hyperresponsiveness of CD19-hBtk B cells is kinase-dependent. (A) Ca²⁺ influx on F(ab′)₂ α-IgM stimulation of splenic naive B cells from the indicated mouse groups, 3 hours after oral administration of PCI-32765 or placebo. Signals were normalized for maximum influx on stimulation with ionomycin. Plots are representative for 2 to 4 mice of each genotype. (B) Flow cytometric analysis of expression of activation markers of MACS-purified B cells from spleens of the indicated mouse groups after culture for 24 hours with or without F(ab′)₂ α-IgM. (C) Flow cytometric analysis of CD86 on splenic CD19⁺ B cells of the indicated mice, after 8 days of treatment with PCI-32765 or placebo. B cells were evaluated 1 or 23 days after termination of treatment (days 9 and 31, respectively). (D) Flow cytometric analysis of splenic CD95⁺PNA⁺ GC B cells, gated from CD19⁺ B cells. Data are shown as dot plots and the percentages GC B cells are given. (E) Numbers of IgM⁺ and IgG⁺ splenic plasma cells 9 and 31 days after start of treatment of the indicated mouse groups. Collective data from 3 independent experiments are shown. (F) Autoimmunity in Btk overexpressing mice is dependent on Btk kinase activity, but not on Btk Y223 autophosphorylation. Flow cytometric quantification of splenic GC B cells (CD95⁺PNA⁺), IgM⁺ and IgG⁺ plasma in 10- to 14-week-old mice that overexpress autophosphorylation-defective Btk (CD19-hBtk^Y223F) or kinase-dead Btk (CD19-hBtk^K430R). (G) Quantification of serum IgG anti-nucleosome auto-antibodies, determined by ELISA, whereby sera of diseased MRL/lpr mice were used as a reference. Each symbol represents an individual mouse.