1A). Median fluorescence intensities from Tg, WT and Btk-deficient mice were used to calculate the relative Btk expression in immature and mature B cells in BM (Fig. 1B). Btk expression of the appropriate molecular weight was confirmed by Western blot of B-cell-enriched splenic or BM cell suspensions (data not shown). The mouse lines exhibited a wide range of Btk protein expression levels that correlated with the Tg copy numbers. Overall, Btk expression increased during B-cell development (Fig. 1B). To examine the effects of E-Btk and EY-Btk expression on B-cell development, BM and
spleen from 8-wk-old Tg mice were analyzed Navitoclax datasheet by flow cytometry and compared with WT and Btk-deficient littermates (Fig. 1C). As previously described 23, 24, Btk-deficient mice had a specific defect in B220high mature recirculating cells in the BM and exhibited relatively increased IgMhighIgDlow transitional B-cell fractions with impaired maturation into IgMlowIgDhigh mature follicular B cells in the spleen. We have previously reported that high expression of E41K-Btk (E-Btk-3) resulted in an almost complete arrest of B-cell development
at the B220lowIgMlow immature B-cell stage in the BM 28. In Tg lines expressing a lower dose of the E41K-Btk mutant (E-Btk-1 and E-Btk-2) the B220lowIgMlow immature B-cell fractions were less affected, but the fractions of recirculating B220high B cells were still severely reduced (Fig. 1C). Accordingly, in the spleen of E-Btk Tg mice a dose-dependent reduction in the PD-0332991 supplier proportions of B cells
was observed (Fig. 1D). For the EY-Btk double mutant Tg mice a similar dose-dependent phenotype was found. The severe block of B-cell development at the immature B-cell stage in the BM of E-Btk-3 Tg mice was suggestive of clonal deletion. This was confirmed by an in vivo kinetic study using the thymidine analogue BrdU, which showed that the absolute numbers Cyclin-dependent kinase 3 of Ig μ+immature B cells generated in the BM were limited and decreased after 24 h (data not shown), indicating a short life span of immature E-Btk-3 Tg B cells. Taken together, these findings show that low-level expression of the E41K-Btk single or the E41K-Y223F-Btk double mutant resulted in an arrest of B-cell development at the immature B-cell stage in the BM and subsequently a dose-dependent reduction of peripheral B cells. For the remainder of our study we focused on the mouse lines E-Btk-2 and EY-Btk-5, because these lines expressed detectable levels of Tg Btk, while deletion in the BM was limited (Fig. 1C), resulting in splenic B-cell numbers that were in the range of Btk-deficient mice (∼30×106 for EY-Btk-5 mice) or markedly lower (∼12×106 for E-Btk-2; compare WT mice: ∼70×106 and Btk-deficient mice: ∼24×106; Fig. 2B). Next, we determined the B-cell subset composition of spleen, peritoneal cavity and MLN in E-Btk-2 and EY-Btk-5 Tg mice.