Generation and characterization of CD40L-modified Mice

描述（由申请人提供）：历史上，CD40配体（CD40L, CD154）在活化的CD4+ T细胞和抗原呈递细胞（APC）上表达的CD40之间的相互作用在体液和细胞介导免疫中的功能意义得到了广泛的认识。然而，最近的发现强调了CD40-CD40L相互作用在炎症反应和自身免疫发病机制中的重要性。CD40L在适当和不适当的免疫反应中都起着核心作用，这一事实强调了全面了解调节CD40L表达的途径以及不同水平的CD40参与如何影响免疫反应的重要性。本提案的总体目标是通过在CD40L转录物（CD40L-¿5）的3'非翻译区（UTR）用缺乏RNA稳定元件的基因替换内源性CD40L基因来建立CD40L多样化表达的小鼠模型。一旦产生小鼠，实验将测试CD40L-¿5突变对体液免疫反应的影响。特别是，脾脏、淋巴结和骨髓中的B细胞群将被分析并与同窝对照中的这些细胞群进行比较。CD40L在突变小鼠中的表达也将在T滤泡辅助T （Tfh）细胞中进行分析，因为这些细胞是生发中心（GC）反应所需的T辅助细胞的关键亚群。我们实验室与当前提案相关的主要发现包括：1)确定了CD40L mRNA衰变的激活诱导途径；2)该途径是由一个含有ptb的复合体（复合体I）在激活后期与CD40L mRNA结合介导的；3)人类和小鼠CD40L转录本中稳定元件的表征；4)发现活化T细胞和非活化T细胞的CD40L RNA在细胞核和细胞质之间的适当分布需要PTB。拟议的研究通过在体内模型中表征CD40L mRNA稳定性的PTB途径，扩展了这些初步和已发表的发现。这些实验的结果将使我们更清楚地了解体液免疫中控制CD40L表达的因素，并将为治疗病原体相关疾病和自身免疫性炎症疾病开辟新的途径。

DESCRIPTION (provided by applicant): Historically, there has been a widespread appreciation of the functional significance of interactions between CD40 ligand (CD40L, CD154) on activated CD4+ T cells and CD40 expressed on antigen presenting cells (APC) in both humoral and cell-mediated immunity. However, more recent discoveries have underscored the significance of CD40-CD40L interactions in inflammatory responses and autoimmune pathogenesis. The fact that CD40L is central to both appropriate and inappropriate immune responses underscores the importance of having a comprehensive understanding of pathways that regulate CD40L expression and how different levels of CD40 engagement affect immune responses. The overall goal of this proposal is to develop a mouse model of variegated CD40L expression by replacing the endogenous CD40L gene with a gene lacking the RNA stability element in the 3' untranslated region (UTR) of the CD40L transcript (CD40L-¿5). Once the mouse is generated, experiments will test the effect of the CD40L-¿5 mutation on the humoral immune response. In particular, B cell populations in the spleen, lymph node and bone marrow will be analyzed and compared to these cell populations in littermate controls. The expression of CD40L in the mutant mouse will also be analyzed in the T follicular helper T (Tfh) cells since these cells are the critical subset of T helper cells required for the germinal center (GC) response. Key findings from our lab that are pertinent to the current proposal include, 1) the identification of an activation-induced pathway of CD40L mRNA decay; 2) the demonstration that this pathway is mediated by a PTB-containing complex (Complex I) binding to the CD40L mRNA at late times of activation; 3) the characterization of the stability element in both human and mouse CD40L transcripts; and 4) the finding that PTB is required for appropriate distribution of CD40L RNA between the nucleus and cytoplasm from both activated and na¿ve T cells. The proposed studies extend these preliminary and published findings by characterizing the PTB pathway of CD40L mRNA stability in an in vivo model. Results from these experiments will lead to a clearer understanding of factors controlling the expression of CD40L in humoral immunity and will uncover novel approaches for treating pathogen-related and autoimmune inflammatory diseases.