The Role of COTL1 in T cell development and activation

描述(由申请人提供)：T细胞激活是启动免疫反应的中心。T细胞抗原受体(TCR)结合共刺激信号，如通过CD28，导致肌动蛋白的改变，控制突触的形成，下游信号通路的激活和细胞因子的产生，包括IL-2。为了确定调节T细胞激活的新分子，我们建立了一个将遗传互补与Jurkat突变相结合的独特系统。建立Jurkat突变型T细胞系，其中TCR/CD28刺激不能激活含有IL-2启动子的RE/AP元件的报告基因。生化特征未能确定这些细胞系中T细胞激活缺陷的分子原因，这意味着新的分子或途径对此负有责任。因此，我们采取了一种遗传方法，通过逆转录病毒表达白细胞文库来挽救T细胞激活缺陷。在我们最初的筛选中，我们发现了一个细胞株，在该细胞系中，由于过表达了一种特征不佳的蛋白NKAP，T细胞活性得到了恢复。我们的工作证明了NKAP是T细胞发育和成熟所必需的转录抑制因子，验证了这种方法可以识别对T细胞激活至关重要的新蛋白。随后，我们从这个筛选中鉴定出第二个分子，COTL1(Coactosin-like Protein 1)。Coactosin最初在Dictyostelius中被鉴定为一种抑制 肌动蛋白封顶，但其在T细胞中的功能尚不清楚。利用shRNA敲除，我们证明了COTL1在T细胞激活过程中对肌动蛋白重塑至关重要。在没有COTL1的情况下，Jurkat T细胞在与超抗原冲击的Raji B细胞相互作用时，无法在CD3覆盖的盖片上扩散或形成片状脂膜，这与未能拉长F-肌动蛋白纤维一致。此外，COTL1的敲除阻断了TCR/CD28介导的IL-2启动子RE/AP元件的上调，表明它是TCR/CD28刺激下游的信号转导所必需的。因此，COTL1是一种新的肌动蛋白重塑和T细胞激活的调节因子。为了了解COTL1在活体中的作用，我们产生了带有COTL1等位基因的小鼠。本R21的目的是通过对COTL1条件性基因敲除小鼠的研究，确定COTL1在T细胞发育、激活、F-肌动蛋白重塑和迁移中的作用。

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.