Molecular analysis of thymic negative selection

胸腺负选择的分子分析

• 批准号: 7214706
• 负责人: LINDA KATHLEEN CLAYTON
• 金额: $ 34.8万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7214706
• 关键词: 6-bromo-2-naphthyl sulfate; Animals; Ankyrin Repeat; Apoptotic; Avidity; Cell Death; Cell Nucleus; Cessation of life; Class; Development; Dexamethasone; Family member; Fetal Thymic Organ Culture; Generations; Genes; Genetic Transcription; I Kappa B-Alpha; Immune response; Kinetics; Ligands; Localized; MHC Class II Genes; Messenger RNA; Modeling; Molecular Analysis; Mus; NF-kappa B; Nuclear; Peptide/MHC Complex; Peptides; Process; Proteins; Reporter; Role; Signal Transduction; Specificity; T-Cell Antigen Receptor Specificity; T-Cell Receptor; T-Lymphocyte; Thymocyte Development; Thymus Gland; Tissues; Transgenic Mice; Ubiquitination; autoreactive T cell; base; dimer; homologous recombination; in vivo; inhibitor/antagonist; irradiation; mRNA Expression; mutant; novel; programs; protein expression; representational difference analysis; retroviral transduction; retroviral-mediated; thymocyte

DESCRIPTION (provided by the applicant): The T-cell repertoire is generated through a tightly regulated developmental program of selection in which thymocytes bearing immunologically desirable T-cell receptor (TCR) specificities are preserved and those expressing harmful specificities are eliminated via an apoptotic mechanism termed negative selection Using representational difference analysis, we have cloned a novel inhibitor of NF-KB, IkBNS, which is rapidly expressed upon TCR-triggered but not dexamethasone- or gamma irradiation-stimulated thymocyte death The predicted protein contains 7 ankyrin repeats and is homologous to known IkB family members but lacks ubiquitination-based degradation signals In Class I and Class II MHC-restricted TCR transgenic mice, transcription of IkBNS is stimulated by peptides that trigger negative selection but not by those inducing positive selection (i.e., survival) or nonselecting peptides IkBNS blocks transcription from NF-kappaB reporters, alters NF-kappaB electrophoretic mobility shifts and interacts with NF-KappaB proteins in thymic nuclear lysates following TCR stimulation. Using an anti-IkBNS mAb, the kinetics of IkappaBNS protein expression in the thymus have been determined and are very different flora those of IkappaBalpha. In addition, IkappaBNS localizes to the nucleus. Over-expression of IkappaBNS using retroviral transduction in fetal thymic organ culture alters the development of thymocytes and enhances anti-CD3epsilon-induced cell death. In this proposal, I describe plans to further characterize this thymic NF-kappaB inhibitor. The effect of altered peptide ligands on IkappaBNS mRNA induction will be analyzed. The effects of IkappaBNS on negative selection in vivo will be analyzed in fetal thymic organ culture using mutant and wild type IkappaBNS, in transgenic mice expressing tissue specific IkBNS as well as in IkBNS gene deleted animals. I will also determine what proteins associate with IkBNS (NF-kB dimers or other proteins,). These studies will elucidate the role of IkBNS in the process of negative selection during thymic development. An understanding of the mechanism of negative selection may form the basis for manipulating the immune response allowing controlled elimination of autoreactive T cells and enhanced generation of T cells with medically relevant specificities.

描述（由申请人提供）：T 细胞库是通过严格调控的选择发育程序产生的，其中保留了具有免疫学所需 T 细胞受体（TCR）特异性的胸腺细胞，并通过称为负选择的细胞凋亡机制消除了表达有害特异性的胸腺细胞。使用代表性差异分析，我们克隆了一种新型 NF-κB 抑制剂 IkBNS，该抑制剂可快速 在 TCR 触发而非地塞米松或伽马射线照射刺激的胸腺细胞死亡时表达 预测的蛋白包含 7 个锚蛋白重复序列，与已知的 IkB 家族成员同源，但缺乏基于泛素化的降解信号 在 I 类和 II 类 MHC 限制性 TCR 转基因小鼠中，IkBNS 的转录受到触发负选择的肽刺激，但不刺激 那些诱导正选择（即生存）或非选择肽 IkBNS 阻断 NF-kappaB 报告基因的转录，改变 NF-kappaB 电泳迁移率变化，并在 TCR 刺激后与胸腺核裂解物中的 NF-KappaB 蛋白相互作用。使用抗 IkBNS mAb，胸腺中 IkappaBNS 蛋白表达的动力学已被确定，并且与 IkappaBalpha 的菌群非常不同。此外，IkappaBNS 定位于细胞核。在胎儿胸腺器官培养物中使用逆转录病毒转导过度表达 IkappaBNS 会改变胸腺细胞的发育并增强抗 CD3ε 诱导的细胞死亡。在本提案中，我描述了进一步表征这种胸腺 NF-kappaB 抑制剂的计划。将分析改变的肽配体对 IkappaBNS mRNA 诱导的影响。将使用突变型和野生型 IkappaBNS 在胎儿胸腺器官培养物中、在表达组织特异性 IkBNS 的转基因小鼠以及 IkBNS 基因缺失的动物中分析 IkappaBNS 对体内负选择的影响。我还将确定哪些蛋白质与 IkBNS（NF-kB 二聚体或其他蛋白质）相关。这些研究将阐明 IkBNS 在胸腺发育过程中负选择过程中的作用。对负选择机制的理解可能构成操纵免疫反应的基础，从而控制自身反应性 T 细胞的消除并增强具有医学相关特异性的 T 细胞的生成。