The Role of COTL1 in T cell development and activation

描述（由申请方提供）：T细胞活化是启动免疫应答的关键。T细胞抗原受体（TCR）结合与共刺激信号（例如通过CD 28）相结合，导致肌动蛋白的改变，所述肌动蛋白控制突触形成、下游信号传导途径的激活和细胞因子（包括IL-2）的产生。为了鉴定调节T细胞活化的新分子，我们建立了一个独特的系统，该系统将遗传互补与Jurkat诱变偶联。产生了Jurkat突变T细胞系，其中TCR/CD 28刺激不能激活含有来自IL-2启动子的RE/AP元件的报道基因。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负责。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。我们的工作已经证明，NKAP是T细胞发育和成熟所需的转录抑制因子，验证了这种方法可以识别对T细胞活化至关重要的新型蛋白质。随后，我们从该筛选中鉴定了第二种分子，COTL 1（类辅肌动蛋白1）。Coactosin最初在网骨藻中被鉴定为 肌动蛋白加帽，但其在T细胞中的功能尚不清楚。使用shRNA敲除，我们证明了COTL 1是T细胞活化过程中肌动蛋白重塑的关键。在不存在COTL 1的情况下，Jurkat T细胞在与超抗原脉冲的Raji B细胞相互作用时不能在CD 3包被的盖玻片上扩散或形成板状伪足，这与不能延长F-肌动蛋白纤维一致。此外，COTL 1的敲低阻断了TCR/CD 28介导的RE/AP元件从IL-2启动子的上调，表明它是TCR/CD 28刺激下游信号转导所必需的。因此，COTL 1是肌动蛋白重塑和T细胞活化的新调节剂。为了了解COTL 1在体内的作用，我们产生了具有floxed COTL 1等位基因的小鼠。本R21的目的是通过检测COTL 1条件性敲除小鼠来确定COTL 1在T细胞发育、活化、F-actin重塑和迁移中的功能。

DESCRIPTION (provided by applicant): T cell activation is central to initiating an immune response. T cell antigen receptor (TCR) engagement, in concert with costimulatory signals such as through CD28, leads to alterations in actin that control synapse formation, activation of downstream signaling pathways and cytokine production, including IL-2. To identify novel molecules that regulate T cell activation, we established a unique system coupling genetic complementation with Jurkat mutagenesis. Jurkat mutant T cell lines were generated in which TCR/CD28 stimulation failed to activate a reporter containing the RE/AP element from the IL-2 promoter. Biochemical characterization failed to pinpoint the molecular cause for the defects in T cell activation in these cell lines, implying that novel molecules or pathways were 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. Our work has demonstrated that NKAP is a transcriptional repressor that is required for T cell development and maturation, validating that this approach can identify novel proteins critical for T cell activation. Subsequently, we have identified a second molecule from this screen, COTL1 (coactosin-like protein 1). Coactosin was initially identified in Dictyostelium, as an inhibitor of actin capping, but its function in T cells was unknown. Using shRNA knockdown, we demonstrate that COTL1 is critical for actin remodeling during T cell activation. In the absence of COTL1, Jurkat T cells fail to spread on CD3- coated coverslips or form a lamellipodia when interacting with superantigen-pulsed Raji B cells, consistent with a failure to elongate F-actin fibers. In addition, knockdown of COTL1 blocked TCR/CD28-mediated upregulation of the RE/AP element from the IL-2 promoter, demonstrating that it is required for signal transduction downstream of TCR/CD28 stimulation. Therefore, COTL1 is a novel regulator of actin remodeling and T cell activation. To understand the role of COTL1 in vivo, we have generated mice with a floxed COTL1 allele. The purpose of this R21 is to determine the function of COTL1 in T cell development, activation, F-actin remodeling and migration through the examination of COTL1 conditional knockout mice.