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， 从幼稚、成熟B细胞到生发中心（GC）反应和记忆B细胞的产生、维持和应答。此外，虽然我们期望在第一个资助期内完成BCR特异性在B1和B2亚群的形成和维持中的作用的分析，但我们期望为延长期提出的实验将通过鉴定关键的细胞内信号级联来进一步有助于理解该问题。 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通过BAFF-R的NF κ B介导的信号外（Sasaki et al.，2006），通过BCR的维持信号，涉及Igo/p胞质尾（Kraus et al.，2004年）。然而，这种信号的分子性质仍然不确定。 我们延长期提案的目标1试图通过ITAM的靶向突变来澄清这一问题 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探讨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。我们在本资助期之前和期间的工作（Kraus等人，2001;Patterson等人，2006）已经确定伊加胞质尾区中保守的ITAM和非ITAM酪氨酸在T依赖性和非依赖性B细胞活化中起不同的作用。虽然我们对非ITAM酪氨酸的分析已经得出结论，但ITAM酪氨酸似乎起着迄今为止神秘的双重作用，因为与IG β ITAM组合，它们似乎对BCR信号传导完全至关重要（Gazumyan等人，2006; Kraus等人，2001），而它们自身的突变导致BCR交联后的B细胞高反应性。我们假设伊加ITAM酪氨酸对于BCR信号启动和终止至关重要，并且前者的功能与Igp的ITAM酪氨酸是多余的。因此，我们的实验旨在鉴定针对BCR信号终止中的伊加ITAM酪氨酸的负反馈环，使用生物化学方法并随后进行遗传验证。我们还将评估伊加ITAM在抗原呈递中的可能作用，考虑到携带突变的伊加ITAM的B细胞的T依赖性应答降低（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* 异二聚体及其ITAM和BAFF-R在体内GC反应控制中的作用。 目的3阐明BCR信号在记忆B细胞抗原攻击的维持和应答中的作用，这一问题在过去很大程度上未被探索。我们将解决这个问题，使用细胞转移系统，我们最近建立，并允许选择性跟踪的遗传标记的记忆B细胞在体内，连同条件等位基因的组件的BCR和系统的诱导基因靶向，我们已经建立了在过去。在此基础上，我们将评估BCR表达和信号传导以及BAFF-R在记忆B细胞维持中的作用。考虑到大多数记忆B细胞的BCR携带具有延长的胞质尾的IgH链，与幼稚B细胞表达的BCR相反，将特别关注一方面伊加/p异二聚体和另一方面y亚类的IgH链的胞质尾的相对贡献。我们已经构建了表达Igyl链或Ign链的小鼠突变体，其具有y2 b胞质尾而不是Igm链，并且正在产生条件等位基因，该条件等位基因允许Cre重组酶介导的由遗传标记的无尾突变体替换lygl链。最后，如果记忆B细胞的维持被证明是BCR依赖的，我们将使用目标1和2中描述的功能获得和功能丧失相结合的方法来识别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.