Structural/Function B Cell Differentiation Antigens

描述（由申请人提供）：本研究的主要目的是研究静止B细胞的调控机制，这些细胞可以处于细胞周期的GO阶段（GO B细胞），也可以离开GO进入细胞周期。我们已经发现半胱天冬酶--一个参与B细胞程序性死亡的半胱氨酸蛋白酶家族--也调节B细胞进入细胞周期。半胱天冬酶-6抑制剂VEID阻断GO B细胞的增殖。然而，半胱天冬酶抑制剂不阻断细胞凋亡抑制剂（IAP）的诱导。我们的数据表明半胱天冬酶是诱导B细胞进入细胞周期的所有模式所共有的途径所必需的。我们的目的是：目的1：确定B细胞中半胱天冬酶活化的要求，即，B细胞中caspase激活的上游信号通路。我们将测试不同的受体是否激活GO B细胞中相同或不同的半胱天冬酶组。我们将检验T细胞依赖性（TD）与T细胞非依赖性（TI）信号传导受体激活GO B细胞中相似或不同的半胱天冬酶途径的假设。我们将定义激活B细胞中caspase-6和-8所需的上游途径。使用显性阴性（DN）形式的FADD或半胱天冬酶-6和来自半胱天冬酶-6敲除小鼠的B细胞，我们将测试半胱天冬酶-6的激活是否是半胱天冬酶-8的激活所必需的，反之亦然。我们还将测试当经由⑶ 40激活时，cIAP是否有助于半胱天冬酶-6和半胱天冬酶-8的选择性激活以及细胞周期进入。目标二。我们将检验这样的假设，即当GO B细胞进入细胞周期并激活存活程序时，胱天蛋白酶-6切割GO B细胞中的一组功能性底物。首先，我们将表征在抑制半胱天冬酶-6后观察到的细胞周期阻滞。其次，我们将定义caspase-6的底物，并评估它们在B细胞中的作用。我们将确定caspase-6在B细胞中被激活后的位置，以及caspase-6底物SATB 1在B细胞中如何受caspase-6调节。目标3：我们将描述半胱天冬酶-6-/-小鼠中B细胞缺陷的特征，并比较野生型与CD 40-/-或半胱天冬酶-6-/-小鼠中B细胞亚群的存活率。我们预测caspase-6和CD 40是最佳B细胞存活所必需的。我们还将测试半胱天冬酶是否选择性地调节B细胞亚群的稳态和扩增。这些研究将为阐明正常B细胞、恶性B细胞以及自身免疫性疾病中B细胞的生存调节机制提供新的思路

DESCRIPTION (provided by applicant): In this grant the major goal is to investigate mechanisms regulating quiescent B cells either poised in the GO stage of the cell cycle (GO B cells) or leaving GO to enter the cell cycle. We have found that caspases- a family of cysteine proteases involved in programmed death of B cells- also regulates B cell entry into the cell cycle. The caspase-6 inhibitor VEID blocks proliferation of GO B cells. However, caspase inhibitors do not block induction of inhibitors of apoptosis (lAPs). Our data suggest caspases are required for a pathway common to all modes of inducing B cells into the cell cycle. Our Aims are: Aim 1: To define the requirements for caspase activation in B cells, i.e., the signaling pathways upstream of caspase activation in B cells. We will test whether or not different receptors activate the same or distinct sets of caspases in GO B cells. We will test the hypothesis that T cell dependent (TD) vs. T cell independent (TI) signaling receptors activate similar or distinct caspase pathways in GO B cells. We will define the upstream pathways required for activating caspase-6 and -8 in B cells. Using a dominant negative (DN) form of FADD or caspase-6 and B cells from caspase-6 knockout mice, we will test if activation of caspase-6 is required for activation of caspase-8 or vice versa. We will also test if clAPs, when activated via CD40, contribute to the selective activation of caspases -6 and -8 and ceil cycle entry. Aim 2. We will test the hypothesis that caspase-6 cleaves a functional set of substrates in GO B cells as they enter the cell cycle and activate a survival program. First, we will characterize the cell cycle block observed upon inhibition of caspase-6. Second, we will define substrates for caspase-6 and assess their roles in B cells. We will define where caspase-6 goes in B cells after they are activated and how the caspase-6 substrate, SATB1, is regulated by caspase-6 in B cells. Aim 3. We will characterize the B cell defects in caspase-6 -/- mice and compare the survival of B cell subsets in wildtype vs. CD40 -/- or caspase-6 -/- mice. We predict that caspase-6 and CD40 are required for optimal B cell survival. We will also test if caspases selectively regulate homeostasis and expansion of B cell subsets. These studies will provide new insights into the mechanisms, which regulate the survival of normal B cells, malignant B cells and B cells contributing to autoimmune diseases