BCR signaling during B cell development and maintenance

保持赠款的总体头衔，工作重点将转移到B细胞开发的后期阶段， 从幼稚、成熟的B细胞到生发中心(GC)的反应和记忆B细胞的生成、维持 和回应。此外，虽然我们希望对BCR特异性在形成和发展中的作用进行分析 在第一个资助期内完成对B1和B2子集的维护，我们预计实验 拟议的延长期将进一步有助于了解这一问题，因为 细胞内信号级联。 我们在第一个资助期的工作表明，维持成熟的B细胞除了需要 NFicB通过BAFF-R介导的信号(Sasaki等人，2006)，通过BCR的维护信号，涉及 Igo/p胞质尾巴(Kraus等人，2004年)。然而，这一信号的分子性质仍未确定。目标1 我们关于延长期限的提议的一部分试图通过对ITAM进行定向突变来澄清这一问题 Igct/p异源二聚体结合后胞质尾部的基序，条件失活的磷酸酶 SHP-1，以及遗传条件获得和功能丧失相结合的方法，其中BCR是有条件的 在成熟的B细胞中被消融，并激活候选的细胞内信号通路，如PI3和 MAP激酶信号转导。我们已经生成了编码这些通路的构成活性成分的载体 根据我们开发的一种策略，正在将它们定位到rosa26基因座上 用于诱导构成NFicB信号(Sasaki等人，2006年)。项目的这一部分将我们的工作与 B细胞亚群分化的区域以及B细胞淋巴瘤的发病机制 某些信号通路起着关键作用。 目的2探讨BCR信号在T非依赖性和非依赖性B细胞活化中的作用，特别是GC 反应。我们在本供资期间之前和之后的工作(Kraus等人，2001年；Patterson等人，2006年)已经确定 IGA胞浆尾部保守的ITAM和非ITAM酪氨酸在T依赖性和 -独立的B细胞激活。虽然我们对非ITAM酪氨酸的分析得出了结论，但ITAM 酪氨酸似乎扮演着一个神秘的双重角色，与Ig(3ITAM)结合在一起，它们似乎对 BCR信号转导(Gazumyan等人，2006；Kraus等人，2001)，而它们本身的突变会导致B BCR交联时细胞的高反应性。我们假设IGA、ITAM、酪氨酸对BCR是必需的 信号的启动和终止，前者的功能与免疫球蛋白的ITAM酪氨酸是多余的。 因此，我们的实验旨在识别针对IGA ITAM的负反馈环路 Bcr信号终止中的酪氨酸，使用生化方法和随后的遗传验证。我们会 也评估免疫球蛋白ITAM在抗原提呈中的可能作用，考虑到B细胞的T依赖反应减少 含有突变的IGA ITAM的细胞(Kraus等人，2001年)。使用GC B细胞特异性基因打靶(Casola等人， 2006)和我们产生的各种条件等位基因，我们将评估IGA/p*异源二聚体及其 ITAM和BAFF-R在体内控制GC反应中的作用。 目的3探讨BCR信号在记忆B的维持和对抗原挑战的反应性中的作用 细胞，这个问题在过去很大程度上仍然没有被探索。我们将使用细胞转移系统来解决这个问题， 这是我们最近建立的，它允许在体内选择性地追踪标记了基因的记忆B细胞， 以及BCR组件的条件等位基因和可诱导基因打靶系统，我们有 在过去建立起来的。在此基础上，我们将评估BCR的表达和信号转导以及BAFF-R在 内存B单元维护。一个特别的焦点将是免疫球蛋白/p异源二聚体在 一方面和y亚类IgH链的细胞质尾巴，给出了大多数 记忆B细胞携带带有延长的细胞质尾巴的IgH链，与幼稚的B细胞表达的BCR相反 细胞。我们已经构建了表达具有y2b细胞质尾巴的Igyl链或ign链的小鼠突变体 而不是IGM链，并且正在产生允许Cre重组酶介导的条件等位基因 用基因标记的无尾突变体取代赖氨酸链。最后，如果存储器B单元维护将 证明是依赖于BCR的，我们将使用下面描述的组合增益和损失函数方法 目标1和2确定BCR下游有助于细胞维持的信号通路。

Maintaining the overall title of the grant, the focus of the work will shift to later phases of B cell development, reaching from the naive, mature B cell to the germinal center (GC) reaction and memory B cell generation, maintenance and response. In addition, while we expect to bring the analysis of the role of BCR specificity in the formation and maintenance of the Bl and B2 subsets to completion within the first funding period, we expect that the experiments proposed for the extension period will further contribute to the understanding of this problem by identifying critical intracellular signaling cascades. Our work in the first funding period has demonstrated that the maintenance of mature B cells requires, apart from an NFicB-mediated signal through the BAFF-R (Sasaki et al., 2006), a maintenance signal through the BCR, involving the Igo/p cytoplasmic tail (Kraus et al., 2004). However, the molecular nature of this signal has remained undefined. Aim 1 of our proposal for the extension period attempts to clarify this issue, using targeted mutagenesis of the ITAM motifs in the cytoplasmic tails of the Igct/p heterodimer in combination, conditional inactivation of the phosphatase SHP-1, and a combined genetic conditional gain- and loss-of-function approach, in which the BCR isconditionally ablated in mature B cells, together with the activation of candidate intracellular signaling pathways like PI3 and MAP kinase signaling. We have generated vectors encoding constitutively active components of these pathways and are in the process of targeting them into the ROSA26 locus, according to a strategy, which we have developed for the induction of constitutive NFicB signaling (Sasaki et al., 2006). This part of the project connects our work to the areas of B cell subset differentiation as well as the pathogenesis of B cell lymphomas, where the activation of certain signalingpathways plays a critical role. Aim 2 addressesthe role of BCR signaling in T-independent and -dependentB cell activation and in particular the GC reaction. Our work before and in the present funding period (Kraus et al., 2001;Patterson et al., 2006) has established that conserved ITAM and non-ITAM tyrosines in the Iga cytoplasmic tail play distinct roles in T-dependent and -independent B cell activation. While our analysis of the non-ITAM tyrosines has come to a conclusion, the ITAM tyrosines appear to play an as yet enigmatic dual role, in that in combination with the Ig(3 ITAM they appear be crucial for BCR signaling altogether (Gazumyan et al., 2006; Kraus et al., 2001), whereas their mutation by themselves results in B cell hyper-reactivity upon BCR cross-linking. We hypothesize that the Iga ITAM tyrosines are essential for BCR signal-initiation as well as -termination, and that the former function is redundant with the ITAM tyrosines ofIgp\ Accordingly, our experiments aim at the identification of a negative feedback loop targeting the Iga ITAM tyrosines in BCR signal termination, using a biochemical approach with subsequent genetic verification. We will also assess a possible role of the Iga ITAM in antigen presentation, given the reduced T-dependent response of B cells harboring a mutated Iga ITAM (Kraus et al., 2001). Using GC B cell-specific gene targeting (Casola et al., 2006) and the various conditional alleles we have generated, we will assess the role of the Iga/p* heterodimer and its ITAMs and of BAFF-R in the control of the GC reaction in vivo. Aim 3 addressesthe role of BCR signaling in the maintenance and responsiveness to antigenic challenge of memory B cells, an issue that has remained largely unexplored in the past. We will address this issue using a cell transfer system, which we have recently established and which allows the selective tracing of genetically labeled memory B cells in vivo, together with conditional alleles of components of the BCR and systems of inducible gene targeting, which we have established in the past. On this basis, we will assess the role BCR expression and signaling as well as of BAFF-R in memory B cell maintenance. A particular focus will be on the relative contributions of the Iga/p heterodimer on the one hand and the cytoplasmic tails of IgH chains of y subclasses on the other, given that the BCRs of most memory B cells carry IgH chains with extended cytoplasmic tails, in contrast to the BCRs expressed by naive B cells. We have already constructed mouse mutants expressing Igyl chains or Ign chains with a y2b cytoplasmic tail instead of Igm chains, and are in the process of generating a conditionalallele allowing Cre recombinase-mediated replacement of an lygl chain by a genetically labeled tailless mutant. Finally, if memory B cell maintenance will turn out to be BCR dependent, we will use the combined gain- and loss-of-function approach described under Aims 1 and 2 to identify signalingpathways downstream of the BCR that contribute to cellular maintenance.