Molecular analysis of thymic negative selection

胸腺负选择的分子分析

• 批准号: 6721399
• 负责人: LINDA KATHLEEN CLAYTON
• 金额: $ 36.7万
• 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/6721399
• 关键词: T cell receptor; T lymphocyte; autoimmunity; binding proteins; genetic recombination; genetic transcription; genetically modified animals; laboratory mouse; messenger RNA; nuclear factor kappa beta; organ culture; protein structure function; proteomics; thymus

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（申请人提供）：T细胞库是通过严格调节的发育选择程序产生的，其中具有免疫学上期望的T细胞受体（TCR）特异性的胸腺细胞被保留，而那些表达有害特异性的胸腺细胞通过称为负选择的凋亡机制被清除。其在TCR触发但不是地塞米松或γ辐射刺激的胸腺细胞死亡时快速表达。预测的蛋白质含有7个锚蛋白重复序列并且与已知的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的转录被触发负选择的肽刺激而不是被诱导正选择的肽刺激（即，存活）或非选择肽IkBNS阻断NF-κ B报告基因的转录，改变NF-κ B电泳迁移率变化，并在TCR刺激后与胸腺核裂解物中的NF-κ B蛋白相互作用。使用抗IkBNS单克隆抗体，已经确定了胸腺中IkappaBNS蛋白表达的动力学，并且与IkappaB α的植物群非常不同。此外，IkappaBNS定位于细胞核。在胎儿胸腺器官培养中使用逆转录病毒转导过表达IkappaBNS改变胸腺细胞的发育并增强抗CD 3 ε诱导的细胞死亡在这个提议中，我描述了进一步表征这种胸腺NF-κ B抑制剂的计划。将分析改变的肽配体对IkappaBNS mRNA诱导的影响。将在使用突变型和野生型IkappaBNS的胎儿胸腺器官培养物中、在表达组织特异性IkBNS的转基因小鼠中以及在IkBNS基因缺失的动物中分析IkappaBNS对体内阴性选择的影响。我还将确定哪些蛋白质与IkBNS（NF-κ B二聚体或其他蛋白质）相关。这些研究将阐明IkBNS在胸腺发育过程中的负选择过程中的作用。对负选择机制的理解可以形成操纵免疫应答的基础，从而允许控制自身反应性T细胞的消除和增强具有医学相关特异性的T细胞的产生。