Btk overexpression in mature B cells is sufficient to induce autoimmunity. (A) Intracellular flow cytometric Btk detection in splenic CD19⁺ B cells, shown as histogram overlays from Btk^−/− (shaded histogram), WT (black line), and MHCII-hBtk (green line) mice. (B) Immunohistochemical analysis of CD21⁺ B cells (red), MOMA-1⁺ metallophilic macrophages (blue; left panel) and GL7⁺ GC B cells (red), and CD138⁺ plasma cells (blue; right panel) in the spleens of young (10-14 weeks) MHCII-hBtk mice. (C) Flow cytometric quantification of splenic IgM⁺ and IgG⁺ plasma cell numbers in young MHCII-hBtk mice and nontransgenic littermates (WT). (D) WT (black line) and MHCII-hBtk (green line) B cells were stimulated with α-IgM F(ab′)₂ fragments and Ca²⁺ mobilization was monitored and normalized for maximum influx on stimulation with ionomycin. Plots are representative for 4 mice of each genotype. (E) HEp2 reactivity of serum IgG antibodies in aging MHCII-hBtk mice. Percentages indicate proportion of HEp2 reactive serum samples per group (objective 40×). (F) Quantification of IgG antinucleosome and anti-dsDNA autoantibodies in the indicated mice, as determined by ELISA. (G) IgG immune complex deposition in glomeruli, detected by immunohistochemistry on kidney tissue sections from old (> 40 weeks) MHCII-hBtk mice. (H) Immunohistochemical analysis for dendritic cells (CD11c⁺, red) and B cells (B220⁺, blue; left panel), GC B cells (PNA⁺, red) and T cells (CD3⁺, blue; middle panel), or GC B cells (PNA⁺, red) and IgG1⁺ plasma cells (IgG1⁺, blue; right panel) in lungs from old MHCII-hBtk mice (objective 10×). Data are representative of 6 MHCII-hBtk mice.