BCR signaling during B cell development and maintenance

B 细胞发育和维持期间的 BCR 信号传导

• 批准号: 8212170
• 负责人: Michael Craig Carroll
• 金额: $ 43.27万
• 依托单位: IMMUNE DISEASE INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-15 至 2012-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8212170
• 关键词: 1-Phosphatidylinositol 3-Kinase; Address; Alleles; Antigen Presentation; Applications Grants; Area; B-Cell Activation; B-Cell Development; B-Cell Lymphomas; B-Lymphocyte Subsets; B-Lymphocytes; Biochemical; Cell Maintenance; Cells; Cytoplasmic Tail; Feedback; Funding; Gene Targeting; Generations; Genetic; Grant; ITAM; Label; Maintenance; Mature B-Lymphocyte; Mediating; Memory B-Lymphocyte; Mitogen-Activated Protein Kinase Kinases; Molecular; Mutagenesis; Mutant Strains Mice; Mutate; Mutation; Nature; PTPN6 gene; Pathogenesis; Pathway interactions; Phase; Phosphoric Monoester Hydrolases; Play; Process; Reaction; Relative (related person); Role; Signal Pathway; Signal Transduction; Specific qualifier value; Specificity; Structure of germinal center of lymph node; System; Tyrosine; Work; base; crosslink; in vivo; loss of function; mutant; recombinase; research study; response; vector

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.

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细胞的产生、维持和反应。此外，虽然我们希望在第一个资助期内完成对BCR特异性在B1和B2亚集形成和维持中的作用的分析，但我们预计为延长期提出的实验将通过确定关键的细胞内信号级联来进一步有助于理解这一问题。 我们在第一个资助期的工作表明，成熟B细胞的维持除了需要通过BAFF-R的NFicB介导的信号(Sasaki等人，2006年)，还需要通过BCR的维持信号，涉及Igo/p细胞质尾巴(Kraus等人，2004年)。然而，这一信号的分子性质仍未确定。 我们关于延长期的提议的目标1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。我们在这个资助时期之前和现在的工作(Kraus等人，2001；Patterson等人，2006)已经确定，保守的ITAM和IGA细胞质尾部的非ITAM酪氨酸在T依赖和非依赖的B细胞激活中发挥着不同的作用。虽然我们对非ITAM酪氨酸的分析已经得出了结论，但ITAM酪氨酸似乎扮演着一个神秘的双重角色，因为它们与Ig结合在一起(3 ITAM)，它们似乎对BCR信号传导是至关重要的(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IGA ITAM酪氨酸对BCR信号的启动和终止是必不可少的，并且前者的功能与Ig P的ITAM酪氨酸是多余的。因此，我们的实验旨在利用生化方法和随后的遗传学验证来识别BCR信号终止中针对IGA ITAM酪氨酸的负反馈环。我们还将评估IGA ITAM在抗原呈递中的可能作用，因为携带突变的IGA ITAM的B细胞的T依赖反应减少(Kraus等人，2001年)。利用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细胞的维持和对抗原挑战的反应性中的作用，这一问题在过去很大程度上仍未被探索。我们将使用我们最近建立的细胞转移系统来解决这个问题，该系统允许在体内选择性地追踪带有基因标记的记忆B细胞，以及我们在过去建立的BCR组件的条件等位基因和可诱导基因打靶系统。在此基础上，我们将评估BCR的表达和信号转导以及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带有延伸的细胞质尾巴，而不是幼稚的B细胞表达的BCR，因此特别关注IGA/p异源二聚体的相对贡献和y亚类的IgH链的细胞质尾巴。我们已经构建了表达Igyl链或IGN链的小鼠突变体，其尾部为y2b细胞质，而不是IgM链，并正在产生一种条件等位基因，允许Cre重组酶介导的lygl链被基因标记的无尾突变体取代。最后，如果记忆B细胞的维持最终被证明是依赖于BCR的，我们将使用AIMS 1和2中描述的功能增益和功能损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。