Generation of BCL10 Tg mice and analysis of B-cell subsets. (A) BCL10 transgenic (Tg) construct and expression in spleen by Western blotting. The full-length, wild-type (WT) human BCL10 cDNA was placed under control of the EμSRα enhancer/promoter upstream of a polyA signal element¹⁶  with an N-terminal FLAG tag fused to the BCL10 protein. Expression of Tg protein in spleen was examined using cell lysates from total splenocytes or purified splenic T and B cells from WT and BCL10 Tg mice analyzed by immunoblotting with an anti-BCL10 Ab that recognizes both human and mouse proteins (a rabbit polyclonal generated by the Morris Laboratory). The human Tg BCL10 protein migrates slightly slower than mouse BCL10 because of the presence of the N-terminal FLAG tag. (B) Distribution of Hardy fraction (Fr) A to F B cells in BM of BCL10 Tg and WT littermates. Fluorescence-activated cell sorting (FACS) analyses of BM B-cell subsets. Cells were gated on lymphocytes (top), IgM⁻ lymphocytes (middle), or B220⁺CD43⁺ lymphocytes (bottom). The numbers are percentages of cells falling within each gate. (C) Distribution of transitional (Tr), follicular (FO), and marginal zone (MZ) B cells in the spleen. The cells were gated on lymphocytes. The numbers are percentages of cells falling within each gate and are representative of analyses of 6 Tg and WT mice. (D) Expression of CD9 and CD1d on FO (filled line) and MZ (open line) B cells. The gates used for defining FO and MZ B cells are shown in panel C. All histograms are gated on CD19⁺ cells. (E) FACS comparisons of mean fluorescence intensities (MFIs) of CD23 expression on FO and MZ B cells from Tg and WT mice. Each triangle represents data from analysis of cells from an individual mouse of the indicated genotype. Note the marked decrease in CD23 expression on the Tg FO B cells (P < .001). (F) Analysis of B2, B1a, and B1B cells in the peritoneal cavity. The numbers are percentages of cells falling within each gate. All cells are gated on the CD19⁺ population.