The E41K Btk mutant displays increased, PI3K-independent membrane localization 26 and therefore we expected that even low-level expression of E41K-Btk would affect B-cell development. First, expression of constitutive activated Btk resulted in a copy-number dependent deletion of peripheral B cells Panobinostat order beyond the transitional B-cell stage, although absolute numbers of B-1 cells in the spleen were increased. Second, residual B cells were hyperresponsive, as evidenced by increased forward Scatter and expression of CD25 and CD69 and

sustained Ca2+ mobilization upon BCR stimulation. Third, residual B cells were efficiently driven into plasma cell differentiation, resulting in increased numbers of plasma cells in spleen and BM and increased serum IgM. Finally, we found anti-nucleosome autoantibodies and glomerular IgM deposition and enlarged glomeruli in aging mice. When comparing the phenotypes of E-Btk-2 and EY-Btk-5 Tg mice it is clear that expression of E-Btk-2 more profoundly affected B-cell differentiation than EY-Btk-5 did. The observed differences may originate from differential

effects of the two mutants or from expression level differences between the two Tg. The latter is most likely, because when EY-Btk-5 Tg mice were bred to homozygosity, we observed a more severe phenotype Selleck Kinase Inhibitor Library that was quite similar to that of E-Btk-2 mice, e.g. in terms of surface CD21/CD23 profiles of B cells (Fig. 2C) and micro-architecture of the spleen (data not shown). Moreover, we have previously found that Y223 phosphorylation is not essential for Btk function in vivo: Y223F-Btk can fully correct the 3-oxoacyl-(acyl-carrier-protein) reductase features of the Btk-deficient phenotype, including pre-B-cell B-1 cell development, serum IgM levels and TI-II responses 27. The complex phenotype of mice with constitutively activated Btk largely resembles that of other Tg or knock-out mice with

increased BCR signaling 12–19. These mice also contain fewer follicular B cells, increased numbers of B-1 B cells, together with B-cell hyperresponsiveness and autoimmunity. However, from the observed phenotypes it is not clear whether altered BCR signaling directly affects B-cell fate or affects selection, survival or differentiation of cells that are committed to a specific B-cell subset. Our crosses with 3-83μδ and VH81X BCR Tg mice clearly showed that constitutive active Btk expression did not change the follicular, MZ or B-1 B-cell fate, but resulted in selective expansion or survival. In this regard, the effects of constitutively activated Btk may be different from other genetic changes that enhance BCR signaling, because it was consistently associated with a profound reduction of total numbers of mature B cells and only a modest increase in the proportion of B-1 cells.