2A and B). Analysis of the CD21/CD23 profile of

E-Btk-2 Tg splenic B cells revealed an apparently learn more normal population of CD21−CD23− immature B cells, but the follicular B cells were significantly reduced in number and manifested low surface expression of both CD21 and CD23 (Fig. 2A and B). CD21highCD23low MZ B cells were completely lacking in E-Btk-2 mice. As Btk-deficient B cells appear to have slightly increased CD21 expression levels (Fig. 2A), it was conceivable that in E-Btk-2 mice MZ B cells were still present but lacked CD21 expression. However, almost complete absence of MZ B cells in the spleen of E-Btk-2 mice was confirmed both by CD1d FACS staining (Supporting Information. Fig. S1) and by immunohistochemical analysis that demonstrated the absence of IgM+ B cells outside the rim of MOMA-1+ metallophilic macrophages (Fig. 5B, left panels). In contrast, EY-Btk-5 Tg mice had significantly reduced numbers of follicular B cells and apparently normal numbers of immature B cells. Due to CDK inhibitor the reduction in follicular B cells, relative proportions of MZ cells were increased (Fig. 2A), but their absolute numbers were in the normal range (Fig. 2B). The milder phenotype in EY-Btk-5 Tg mice,

as compared with E-Btk-2 transgenic mice might originate from differential effects of the E41K single and the E41K-Y223F double mutation or alternatively from the ∼2 times higher expression levels of the E-Btk-2 mutant, as compared with EY-Btk-5. To investigate this, we generated mice homozygous for the EY-Btk-5 Tg and analyzed the B-cell compartment by flow cytometry. Strikingly, homozygous EY-Btk-5 mice manifested a phenotype reminiscent of that found in E-Btk-2 mice, with severely reduced numbers of B cells, a complete lack of CD21highCD23low MZ B cells and a significant reduction in the numbers of follicular B cells, whereby residual B cells were CD21lowCD23low (Fig. 2C). Taken together, these findings show

that expression of constitutive active Btk significantly affected B-cell differentiation beyond the transitional B-cell stage, resulting in reduced numbers of follicular B cells and the absence of MZ B cells in E-Btk-2 Tg mice and in homozygous EY-Btk-5 Tg mice. Because mutant mice with enhanced BCR signaling often show increased numbers of B-1 B cells 12–19, we evaluated Cyclin-dependent kinase 3 the expression of the B-1-associated surface markers CD5 and CD43 in spleen, MLN and peritoneal cavity. We identified significant proportions of B220lowCD5+CD43+ B-1 B cells in the spleens of E-Btk-2 and EY-Btk-5 mice, in contrast to spleens of WT and Btk-deficient mice, which contained only minor fractions of B-1 cells or completely lacked B-1 cells, respectively (Fig. 3A and B). In MLN of both E-Btk-2 and EY-Btk-5 mice, the proportions of B cells were significantly reduced, whereby B220lowCD5+CD43+ B-1 B cells, which are normally not present in MLN (Supporting Information Fig. S2A), were prominent.