Mechanisms of CD40 signaling in B lympocytes in vivo

体内 B 淋巴细胞 CD40 信号传导机制

• 批准号: 6633442
• 负责人: RANDOLPH J. NOELLE
• 金额: $ 31.6万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6633442
• 关键词: B lymphocyte; CD40 molecule; autoimmunity; binding sites; biological signal transduction; cell differentiation; cell proliferation; cytokine receptors; gene expression; gene targeting; genetically modified animals; humoral immunity; laboratory mouse; leukocyte activation /transformation; protein structure function; proteomics; systemic lupus erythematosus; tissue /cell culture; tumor necrosis factor alpha

DESCRIPTION (provided by applicant): Humoral immune and autoimmune responses are governed by the interactions of CD154 expressed on helper T cells and CD40 on B cells. Extensive efforts have been made to unravel how CD40 engagement transduces the diversity of signals to trigger B cell expansion and differentiation. Like many of the other TNF Receptor family members, it is believed that the recruitment of TNF Receptor Associate Factors (TRAFs) to the cytoplasmic domain of CD40 plays a critical role in regulating CD40 biology. This proposal focuses on the role that TRAFs, and other signaling elements play in regulating B cell function in vivo. We present a unique cohort of Tg/knock-out mice that express mutations in the cytoplasmic tail of CD40 which disrupt the interaction of the cytoplasmic domain with specific TRAFs. B cells from these mice will be studied in vitro (Specific Aim #1) and in vivo (Specific Aim #2) to address the causal relationships between signaling domains of the cytoplasmic tail of CD40 and the induction of in vitro and in vivo B cell biochemistry and function. Preliminary data shows that the loss in TRAF recruitment in B cells exerts minimal or no impact on early IgM and lgG responses in vivo, however, a major effect of TRAF recruitment is observed on the durability of humoral immune responses in vivo. While loss in TRAF recruitment results in defined lesions in humoral immunity, many aspects of the humoral immune response are intact in the absence of TRAF recruitment. We propose to identify (Specific Aim #3) non-TRAF binding domains in CD40 that are critical for early B cell activation. Genetic and proteonomic approaches are presented that will identify novel, functionally significant sites in the CD40 tail and the factors they bind. Enhanced TRAF recruitment and chronic CD40 signaling in the B cell compartment is believed to contribute to the development of lupus. We propose that the expression of a constitutively active form of CD40 (CD40zip; a leucine-zippered, myristoylated CD40 tail), within the B cell compartment will drive B cell expansion and differentiation in vivo and result in the development of a lupus-like syndrome (Specific Aim#4). Assuming the development of autoimmunity in Tg CD40zip mice, mutations in the TRAF binding sites of CD40zip, will be engineered to evaluate the contribution of specific TRAFs to the development of autoimmunity.

描述（由申请人提供）：体液免疫和自身免疫反应由辅助 T 细胞上表达的 CD154 和 B 细胞上表达的 CD40 的相互作用控制。人们付出了大量努力来阐明 CD40 参与如何转导信号多样性以触发 B 细胞扩增和分化。与许多其他 TNF 受体家族成员一样，人们相信 TNF 受体相关因子 (TRAF) 募集到 CD40 的细胞质结构域在调节 CD40 生物学方面发挥着关键作用。该提案重点关注 TRAF 和其他信号元件在体内调节 B 细胞功能中所发挥的作用。我们提出了一组独特的 Tg/敲除小鼠，它们在 CD40 的细胞质尾部表达突变，从而破坏细胞质结构域与特定 TRAF 的相互作用。来自这些小鼠的 B 细胞将在体外（具体目标 #1）和体内（具体目标 #2）进行研究，以解决 CD40 胞质尾部信号传导域与体外和体内 B 细胞诱导之间的因果关系生物化学和功能。初步数据表明，B 细胞中 TRAF 募集的丧失对体内早期 IgM 和 IgG 反应影响极小或没有影响，然而，观察到 TRAF 募集对体内体液免疫反应的持久性有重大影响。虽然 TRAF 募集的丧失导致体液免疫的明确损伤，但在没有 TRAF 募集的情况下，体液免疫反应的许多方面是完整的。我们建议鉴定（具体目标#3）CD40 中对于早期 B 细胞激活至关重要的非 TRAF 结合域。提出的遗传和蛋白质组学方法将鉴定 CD40 尾部中新颖的、功能上重要的位点及其结合的因子。 B 细胞区室中增强的 TRAF 募集和慢性 CD40 信号传导被认为有助于狼疮的发展。我们认为，B 细胞区室中 CD40 的组成型活性形式（CD40zip；亮氨酸拉链、肉豆蔻酰化 CD40 尾部）的表达将驱动 B 细胞体内扩增和分化，并导致狼疮样综合征的发生（具体目标#4）。假设 Tg CD40zip 小鼠中出现自身免疫，将设计 CD40zip TRAF 结合位点的突变，以评估特定 TRAF 对自身免疫发展的贡献。