Genetic complementation screen to identify novel regulators of T cell activation

遗传互补筛选以确定 T 细胞激活的新型调节因子

• 批准号: 7677470
• 负责人: Virginia Smith Shapiro
• 金额: $ 22.67万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-20 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7677470
• 关键词: Activation Analysis; Antigens; Autoimmune Diseases; Biochemical; Biochemical Pathway; CD28 gene; Candidate Disease Gene; Cell Line; Cell surface; Complementary DNA; Complex; Coupling; Defect; Elements; Gene Activation; Generations; Genes; Genetic; Genetic Screening; Immune response; Immune system; Immunotherapy; Interleukin-2; Investigation; Lead; Leukocytes; Libraries; Mediating; Mining; Modeling; Molecular; Mutagenesis; Pathway interactions; Production; Proteins; Receptor Activation; Regulation; Reporter; Signal Pathway; Signal Transduction; Silicon Dioxide; System; T Cell Receptor Signaling Pathway; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Therapeutic; Therapeutic Intervention; Up-Regulation; Work; anergy; gene function; improved; insight; mutant; notch protein; novel; overexpression; pathogen; protein expression; public health relevance; receptor-mediated signaling; response; retroviral transduction; small hairpin RNA

DESCRIPTION (provided by applicant): T cell activation is central to initiating an immune response. Minimally, two signals are required: specific recognition of antigen through the T cell receptor (TCR) and a co-stimulatory signal, primarily provided by CD28 in na¿ve T cells. TCR activation without appropriate co-stimulation not only fails to activate T cells but also leads to a state of unresponsiveness, or anergy. Therapeutic inhibition of inappropriate T cell activation by co-stimulatory modulation is currently under investigation for several autoimmune diseases. Although the importance of CD28 and co-stimulatory signals in T cell activation and the immune response is understood, the biochemical signals resulting from CD28 engagement on the cell surface are poorly characterized. Therefore, to identify novel molecules that regulate T cell activation, but which may function outside of well-characterized TCR signaling pathways, we established a unique system coupling genetic complementation with Jurkat mutagenesis. Using the RE/AP reporter element as a model of signal integration, we generated mutant clones in the Jurkat T cell line in which TCR-mediated signaling leading to NFAT activation is normal, while co-stimulation leading to IL-2 upregulation is abrogated. Biochemical characterization of these cell lines failed to pinpoint the molecular cause for the defects in T cell activation in these cell lines, implying that novel molecules or pathways may be responsible. Therefore, we took a genetic approach to rescue the T cell activation defect by retroviral expression of a leukocyte library. From our initial screen, we identified one cell line in which T cell activation was restored due to overexpression of a poorly characterized protein, NKAP. Subsequent work has identified NKAP as a component of the Notch co-repressor complex. Since Notch signaling has been shown to regulate T cell activation and IL-2 production, it demonstrates a proof-of-principal that this genetic screen is capable of identifying novel regulators of T cell activation. Thus, we have established a functional screen that will lead to novel insights into the biochemical regulation of T cell activation, and mining this system will likely lead to additional molecules critical to generating an immune response. Our specific aims are: Specific Aim #1. Genetic complementation of Jurkat mutant cell lines via retroviral transduction of a cDNA utilizing a second-generation dual readout. Specific Aim #2. Biochemical characterization of the function of novel regulators of T cell activation identified by the complementation screen. PUBLIC HEALTH RELEVANCE: Biochemical pathways initiated upon T cell activation regulate the immune system responses to either a foreign pathogen, or to itself as occurs in autoimmune disease. Manipulation of these pathways is currently under investigation for their use in immunotherapy. This proposal focuses on identifying novel molecules that regulate T cell activation, which may become good targeted for therapeutic intervention.

描述（由申请方提供）：T细胞活化是启动免疫应答的关键。最低限度，需要两个信号：通过T细胞受体（TCR）特异性识别抗原和共刺激信号，主要由幼稚T细胞中的CD 28提供。没有适当的共刺激的TCR活化不仅不能活化T细胞，而且还导致无反应性或无反应性的状态。目前正在研究通过共刺激调节对不适当的T细胞活化的治疗性抑制用于几种自身免疫性疾病。虽然CD 28和共刺激信号在T细胞活化和免疫应答中的重要性已被理解，但由细胞表面上的CD 28接合产生的生物化学信号的特征很差。因此，为了鉴定调节T细胞活化但可能在充分表征的TCR信号传导途径之外起作用的新分子，我们建立了将遗传互补与Jurkat诱变偶联的独特系统。使用RE/AP报告元件作为信号整合的模型，我们在Jurkat T细胞系中产生突变克隆，其中导致NFAT活化的TCR介导的信号传导是正常的，而导致IL-2上调的共刺激被废除。这些细胞系的生物化学表征未能查明这些细胞系中T细胞活化缺陷的分子原因，这意味着新的分子或途径可能是负责的。因此，我们采取了一种遗传方法，通过白细胞文库的逆转录病毒表达来挽救T细胞活化缺陷。从我们的初步筛选，我们确定了一个细胞系，其中T细胞活化恢复由于过度表达的一个不好的特点蛋白，NKAP。随后的工作已经将NKAP鉴定为Notch共阻遏物复合物的组分。由于Notch信号传导已被证明可调节T细胞活化和IL-2产生，因此证明了这种遗传筛选能够鉴定T细胞活化的新型调节剂的原理证明。因此，我们已经建立了一个功能性筛选，这将导致对T细胞活化的生化调节的新见解，并且挖掘这个系统可能会导致对产生免疫应答至关重要的其他分子。我们的目标是：具体目标#1。利用第二代双读出器通过逆转录病毒转导cDNA对Jurkat突变细胞系进行遗传互补。具体目标#2通过互补筛选鉴定的T细胞活化的新型调节剂的功能的生物化学表征。 公共卫生相关性：在T细胞活化时启动的生化途径调节免疫系统对外来病原体或自身免疫性疾病中发生的自身免疫系统应答。目前正在研究这些途径的操纵以用于免疫治疗。该提案的重点是确定调节T细胞活化的新分子，这可能成为治疗干预的良好靶向。