BCR signaling during B cell development and maintenance

保持赠款的整体名称，工作的重点将转移到B细胞开发的后期阶段， 达到从幼稚、成熟B细胞到生发中心（GC）的反应和记忆B细胞的产生、维持 和回应。此外，虽然我们期望带来的BCR特异性的作用的分析，在形成和 为了在第一个资助期内完成B1和B2子集的维护，我们期望实验 为延长期提出的建议将通过确定关键的 细胞内信号级联。 我们在第一个资助期的工作已经证明，成熟B细胞的维持除了需要一个 通过BAFF-R的NFicB介导的信号（Sasaki等人，2006年），通过BCR的维护信号，涉及 Igo/p胞质尾（Kraus等，2004年）。然而，这种信号的分子性质仍然不确定。要求1 我们的延长期建议试图澄清这个问题，使用ITAM的靶向诱变 组合Igct/p异二聚体的胞质尾中的基序，磷酸酶的条件失活 SHP-1，以及一种结合遗传条件获得和功能丧失的方法，其中BCR是有条件的 在成熟B细胞中消除，同时激活候选细胞内信号传导途径，如PI 3和 MAP激酶信号传导。我们已经生成了编码这些途径的组成性活性成分的载体 根据我们开发的策略， 用于诱导组成型NFicB信号传导（Sasaki等，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，其中， some signaling信号pathways途径plays扮演a critical关键role作用. 目的2：阐明BCR信号在T细胞非依赖性和依赖性活化中的作用，特别是GC 反应我们在当前资助期之前和期间的工作（Kraus等人，2001;Patterson等人，2006年）成立 伊加胞质尾区中保守的ITAM和非ITAM酪氨酸在T依赖性和 - 独立的B细胞活化。虽然我们对非ITAM酪氨酸的分析已经得出结论，但ITAM酪氨酸仍然存在。 酪氨酸似乎起着一种至今仍是谜一样的双重作用，即与IG β ITAM结合，它们似乎对 BCR信号传导完全（Gazumyan等人，2006; Kraus等人，2001），而它们自身的突变导致B BCR交联后的细胞高反应性。我们假设伊加ITAM酪氨酸是BCR所必需的 信号起始和信号终止，前者的功能与Igp的ITAM酪氨酸是冗余的。 因此，我们的实验旨在识别针对伊加ITAM的负反馈回路 酪氨酸在BCR信号终止，使用生物化学方法与随后的遗传验证。我们将 考虑到B的T依赖性应答降低，我还评估了伊加ITAM在抗原呈递中的可能作用 携带突变的伊加ITAM的细胞（Kraus等，2001年）。使用GC B细胞特异性基因靶向（Casola等人， 2006）和我们已经产生的各种条件等位基因，我们将评估伊加/p* 异源二聚体的作用及其对免疫应答的影响。 ITAM和BAFF-R在体内GC反应的控制中的作用。 目的3探讨BCR信号通路在记忆B蛋白的维持和抗原攻击应答中的作用 细胞，这是一个过去基本上未被探索的问题。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追踪遗传标记的记忆B细胞， 连同BCR组分的条件等位基因和诱导型基因靶向系统，我们已经 建立在过去。在此基础上，我们将评估BCR的表达和信号传导以及BAFF-R在细胞凋亡中的作用。 记忆B细胞维持。特别关注的是伊加/p异二聚体对细胞凋亡的相对贡献。 另一方面是y亚类的IgH链的细胞质尾，因为大多数的BCR 与幼稚B细胞表达的BCR相反，记忆B细胞携带具有延长的胞质尾的IgH链 细胞我们已经构建了表达具有γ 2b胞质尾的Igyl链或Ign链的小鼠突变体 而不是Igm链，并在产生条件等位基因的过程中，允许Cre重组酶介导 用遗传标记的无尾突变体替换lyg 1链。最后，如果记忆B细胞的维护将 结果是BCR依赖的，我们将使用下面描述的组合增益和功能损失方法 目的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.