Genetic complementation screen to identify novel regulators of T cell activation

描述（由申请人提供）：T细胞激活是启动免疫反应的核心。至少需要两个信号：通过T细胞受体（TCR）特异性识别抗原和主要由na - ve T细胞中的CD28提供的共刺激信号。没有适当的共刺激激活TCR不仅不能激活T细胞，而且会导致无反应或能量状态。目前正在研究通过共刺激调节抑制不适当T细胞活化治疗几种自身免疫性疾病。虽然CD28和共刺激信号在T细胞活化和免疫应答中的重要性已经被理解，但CD28参与细胞表面产生的生化信号却很少被表征。因此，为了确定调节T细胞活化的新分子，但可能在已知的TCR信号通路之外起作用，我们建立了一个独特的系统，将遗传互补与Jurkat突变耦合在一起。使用RE/AP报告元件作为信号整合模型，我们在Jurkat T细胞系中生成突变克隆，其中tcr介导的导致NFAT激活的信号是正常的，而导致IL-2上调的共刺激被取消。这些细胞系的生化表征未能确定这些细胞系中T细胞激活缺陷的分子原因，这意味着可能是新的分子或途径起作用。因此，我们采用了一种遗传方法，通过白细胞文库的逆转录病毒表达来挽救T细胞激活缺陷。从我们最初的筛选中，我们确定了一种细胞系，其中T细胞激活由于过表达一种特征不佳的蛋白NKAP而恢复。随后的工作已经确定NKAP是Notch共抑制因子复合物的一个组成部分。由于Notch信号已被证明调节T细胞激活和IL-2的产生，它证明了这种基因筛选能够识别T细胞激活的新调节因子。因此，我们已经建立了一个功能筛选，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产生免疫反应的其他关键分子。我们的具体目标是：利用第二代双读出技术通过逆转录病毒转导cDNA实现Jurkat突变细胞系的遗传互补。具体目标2。通过互补筛选鉴定的新型T细胞活化调节因子功能的生化表征。

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.