Regulation of B Cell Gene Expression Patterns by Btk

描述（由申请人提供）：布鲁顿成分B酪氨酸激酶（Btk）是调节B细胞发育和功能的信号通路的关键。Btk的突变导致人类细胞免疫缺陷的x连锁γ球蛋白血症（XLA）和小鼠细胞免疫缺陷的x连锁γ球蛋白血症（xid）。Btk对于自身抗体的产生和肥大细胞的有效脱颗粒也是必需的。对Btk下游信号通路的深入了解可能会揭示B细胞免疫缺陷、自身免疫和过敏的新治疗靶点。本研究将验证在Btk缺失的情况下，BCR交联导致基因表达调控受损导致B细胞发育和功能缺陷的假设。将野生型和Btklo小鼠的B细胞进行比较，这两种小鼠表达的Btk是有限剂量的。与Btk-/-或xid小鼠不同，Btklo小鼠具有正常数量的成熟B细胞。由于Btk水平不足，这些细胞仍然功能受损，允许在成熟B细胞中识别Btk依赖的信号事件。我们已经开始使用cDNA微阵列分析表征bcr诱导的基因表达中btk依赖性的变化。我们的第一个目标是使用实时RT-PCR和Western blots来确认这些结果，并确定Btk信号通路的哪些分支参与了这些基因的调控。btk调控基因在介导btk启动信号的各种功能结果中的作用将被评估如下。Btk调控基因将在Btk-/-型、Btk-型和野生型小鼠中以转基因形式表达，并观察到它们恢复常规B细胞发育和功能中Btk依赖性缺陷的能力。为了评估Btk调控基因在B-1细胞分化中的作用，我们将表达Btk调控基因的转基因与携带VH12抗磷脂酰胆碱Ig转基因的Btk-/-、Btklo和野生型小鼠杂交。这种转基因驱动了磷脂酰胆碱特异性B细胞的克隆扩增，并以btk依赖的方式向B-1区区分化。这些研究将确定btk介导的BCR信号传递调节常规B细胞和B-1细胞发育和功能的机制，并可能确定涉及B细胞失调的疾病的新治疗方法。

DESCRIPTION( provided by the applicant): Bruton's component B tyrosine kinase (Btk) is a critical of signaling pathways that regulate B cell development and function. Mutations in Btk cause the cell immunodeficiency's X-linked a gammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. Btk is also required for the production of autoantibodies and the efficient degranulation of mast cells. A thorough understanding of the signaling pathways downstream of Btk is likely to reveal new therapeutic targets for B cell immunodeficiencies,autoimmunity, and allergy. This study will test the hypothesis that impaired regulation of gene expression in response to BCR crosslinking results in the B cell developmental and functional defects observed in the absence of Btk. B cells from wild type and Btklo mice, which express a limiting dosage of Btk, will be compared. Unlike Btk-/- or xid mice, Btklo mice have normal numbers of mature B cells. These cells remain functionally impaired due to insufficient levels of Btk, allowing Btk-dependent signaling events to be identified in mature B cells. We have begun to characterize Btk-dependent changes in BCR-induced gene expression using cDNA microarray analysis. Our first aim is to confirm these results using real-time RT-PCR and Western blots and to define which branches of Btk signaling pathways are involved in the regulation of these genes. The role of Btk-regulated genes in mediating various functional outcomes of Btk-initiated signals will be assessed as follows. Btk-regulated genes will be expressed as transgenes in Btk-/-, Btk lo, and wild type mice and their ability to restore Btk-dependent defects in conventional B cell development and function observed. To assess the role of Btk-regulated genes in B-1 cell differentiation, transgenes expressing Btk regulated genes will be crossed to Btk-/-, Btklo, and wild type mice carrying a VH12 anti-phosphatidylcholine Ig transgene. This transgene drives the clonal expansion of phosphatidylcholine-specific B cells and their differentiation into the B-1 compartment in a Btk-dependent manner. These studies will define the mechanism by which Btk-mediated transmission of BCR signals regulates the development and function of both conventional B and B-1 cells and potentially identify novel therapeutic approaches for diseases involving B cell disregulation.