Generation and characterization of CD40L-modified Mice

CD40L 修饰小鼠的生成和表征

• 批准号: 8660640
• 负责人: LORI Ruth COVEY
• 金额: $ 7.46万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-15 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8660640
• 关键词: 3' Untranslated Regions; Affect; Antibodies; Antibody Affinity; Antibody Formation; Antigen-Presenting Cells; Area; Autoimmune Process; B cell differentiation; B-Cell Neoplasm; B-Lymphocytes; Binding; Bone Marrow; CD4 Positive T Lymphocytes; CD40 Ligand; Cell Nucleus; Cells; Cellular Immunity; Complex; Cytoplasm; Data; Disease; Elements; Generations; Genes; Goals; Helper-Inducer T-Lymphocyte; Human; Humoral Immunities; Immune response; Indium; Inflammatory; Inflammatory Response; Lead; Link; Lymphocyte; Lymphoid; Lymphoid Tissue; Mediating; Messenger RNA; Mouse Strains; Mus; Mutant Strains Mice; Mutation; Organ; Pathogenesis; Pathway interactions; Peptide/MHC Complex; Play; Polypyrimidine Tract-Binding Protein; Population; Positioning Attribute; Process; Production; Publishing; RNA; RNA Binding; RNA Stability; Reagent; Regulation; Reporting; Research; Role; Signal Transduction; Spleen; Staging; Structure of germinal center of lymph node; T-Cell Activation; T-Lymphocyte; TNFRSF5 gene; TNFSF5 gene; Testing; Time; Transcript; Vaccines; base; blastocyst; defined contribution; design; embryonic stem cell; experience; in vivo Model; interest; lymph nodes; mRNA Decay; mRNA Stability; macrophage; malignant phenotype; mouse model; novel; novel strategies; pathogen; public health relevance; research study; response; screening

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.

描述（由申请人提供）：历史上，人们普遍认识到活化的 CD4+ T 细胞上的 CD40 配体（CD40L、CD154）与抗原呈递细胞（APC）上表达的 CD40 之间的相互作用在体液和细胞介导的免疫中的功能意义。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 对于适当和不适当的免疫反应都至关重要，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。该提案的总体目标是通过用 CD40L 转录物 (CD40L-¿5) 3' 非翻译区 (UTR) 中缺乏 RNA 稳定性元件的基因替换内源 CD40L 基因，开发杂色 CD40L 表达的小鼠模型。一旦小鼠产生，实验将测试CD40L-¿5突变对体液免疫反应的影响。特别是，将分析脾脏、淋巴结和骨髓中的 B 细胞群，并与同窝对照中的这些细胞群进行比较。突变小鼠中 CD40L 的表达也将在滤泡辅助性 T (Tfh) 细胞中进行分析，因为这些细胞是生发中心 (GC) 反应所需的辅助性 T 细胞的关键子集。我们实验室与当前提案相关的主要发现包括：1）鉴定出激活诱导的 CD40L mRNA 衰变途径； 2) 证明该途径是由在激活后期与 CD40L mRNA 结合的含有 PTB 的复合物（复合物 I）介导的； 3) 人和小鼠 CD40L 转录物中稳定性元件的表征； 4)发现PTB是活化T细胞和幼稚T细胞的CD40L RNA在细胞核和细胞质之间适当分布所必需的。拟议的研究通过表征体内模型中 CD40L mRNA 稳定性的 PTB 途径，扩展了这些初步和已发表的发现。这些实验的结果将有助于更清楚地了解体液免疫中控制 CD40L 表达的因素，并将揭示治疗病原体相关和自身免疫炎症性疾病的新方法。

项目成果

A Posttranscriptional Pathway of CD40 Ligand mRNA Stability Is Required for the Development of an Optimal Humoral Immune Response.

• DOI: 10.4049/jimmunol.2001074
• 发表时间: 2021-06-01
• 期刊: Journal of immunology (Baltimore, Md. : 1950)
• 作者: Narayanan B;Prado de Maio D;La Porta J;Voskoboynik Y;Ganapathi U;Xie P;Covey LR
• 通讯作者: Covey LR