The efficient deletion of immature B cells in mice expressing high-levels of E41K-mutated Btk (Fig. 1C and Ref. 28) indicates that E-Btk Tg immature B cells are subject to efficient clonal deletion. We did not detect any defects in receptor editing when 3-83μδ, E-Btk-2 double Tg B cells were crossed on a central deleting C57BL/6 background (R.K., unpublished results). Because a significant fraction of circulating mature B cells is thought to be auto- or poly-reactive 36, such B cells may become activated because constitutive active Btk suppresses inhibitory effects of FcγRIIb

or SHIP (as was previously shown for a membrane-associated Btk chimera, which led to sustained elevation click here of intracellular calcium 37). In this context, it is conceivable that the E-Btk-2 Tg can also counteract inhibitory signals generated by FcγRIIb crosslinking that were recently found to induce apoptosis and thereby govern differentiation and maintenance of plasma cells 38. Persistence of plasma cells in E-Btk-2 Tg mice would be supported by our finding of increased numbers of these cells in the BM. Importantly, the complex phenotype of mice with constitutive Btk activation shows that Btk signals are essential for appropriate

regulation of B-cell activation. Since successful treatment of patients with autoimmune disorders such as lupus and rheumatoid arthritis have demonstrated the importance of B cells in disease pathology 39, it should be worthwhile developing treatment strategies for autoimmune diseases PI3K Inhibitor Library in vivo based

on Btk-specific small molecule inhibitors. Btk-deficient, Slp65-deficient, VH81X or 3-83μδ Tg mice have been described 8, 24, 29, 30. We previously reported CD19-driven E41K-Btk and E41K-Y223F-Btk mice 28, 40. Additional low-copy number acetylcholine Tg mice on the FVB background were generated using the same constructs and crossed onto the Btk-deficient background 28, 40. Tg mice were analyzed together with non-Tg littermates at the age of 8–16 wk. Mice were bred and maintained under specific pathogen-free conditions. Experimental protocols were reviewed and approved by the Erasmus MC Committee of animal experiments. Preparations of single-cell suspensions, flow cytometry and Ca2+ measurements upon anti-IgM F(ab)2 stimulation have been described previously 25, 41. For comparison of Ca2+ fluxes between samples, background values of indo-1 acetoxymethylester (AM) loaded cells were set at equal height and plots were analyzed using the same fluorescence ratios (FL-5/FL-4) scales. Correct comparisons were confirmed by using the signals of 2 μg/mL ionomycin as a control. The 35-1 and 54-1 anti-idiotypic antibodies were kindly provided by J. F. Kearney (Birmingham, USA) and D. Nemazee (La Jolla, USA), respectively; 1-5×105 Events were scored using a FACSCalibur flow cytometer and analyzed using CellQuest (BD Biosciences, Mountain View, USA) or FlowJo (Tree Star, Ashland, OR) software.