Role of Grail in maintaining humoral immune tolerance

 描述(由申请人提供)：自身免疫是由中枢或外周耐受性的失调启动的，导致自身反应性T和B细胞逃避正常选择。越来越多的证据表明，卵泡辅助性T细胞(TFH)和B细胞的扩张导致 在体液和细胞异常中，并导致系统性自身免疫性疾病的发展，如系统性红斑狼疮(SLE)；然而，目前对控制自身反应性B细胞和T细胞功能的机制的了解仍然不完整。因此，今天越来越清楚的是，了解转铁蛋白和B细胞的生物学和操纵它们的功能的方法可能被证明对自身抗体驱动的疾病的治疗非常有价值。最近，我们建立并分析了GRAIL缺乏的小鼠，发现老年GRAIL缺乏的小鼠出现了类似SLE的症状，其特征是大量生发中心的形成，淋巴组织中Tfh和B细胞的聚集，以及高滴度的抗dsDNA抗体。此外，GRAIL KO小鼠与狼疮易感小鼠B6杂交，Faslpr/LPR小鼠与对照小鼠相比出现了严重的狼疮表型。此外，活动期SLE患者和狼疮易感小鼠的T和B淋巴细胞中GRAIL的表达下调，进一步支持了GRAIL在体液免疫反应中的调控作用。所有这些发现表明，GRAIL在控制T和B细胞功能方面可能是必不可少的，重要的是，GRAIL下调可以作为SLE样疾病的标志。然而，目前尚不清楚T和/或B细胞中缺乏GRAIL是否与狼疮样自身免疫的发生有关。因此，我们推测GRAIL对B细胞和TfH细胞功能的调节可能是体液耐受的一个重要关卡，而体液耐受对防止自身免疫的发展至关重要。在Aim1中，我们将利用基因敲除方法研究GRAIL在TFH细胞发育和功能中的作用。我们将使用独特的Bcl6-RFP和Bcl6-RFP/Foxp3GFP报告小鼠模型来确定GRAIL在正常Tfh和T滤泡调节(TFR)细胞发育中的作用 和自身免疫设置。此外，我们将确定GRAIL的靶点，通过GRAIL通过使用基因表达的微阵列分析来促进对TFH细胞编程的控制。我们已经开发了一种新的体外培养TFH细胞的方法，这将允许产生足够数量的细胞来进行拟议的分析。在AIM2中，我们将评估GRAIL在B细胞激活和功能中的作用。重要的是，我们将使用包括双杂交筛选在内的尖端技术来确定GRAIL的确切靶点(S)，该靶点决定了GRAIL在B细胞中的功能。我们的结果将为理解自身抗体介导的自身免疫性疾病提供机械性的见解，并可能导致确定检测和预防这些疾病的新的治疗靶点。

 DESCRIPTION (provided by applicant): Autoimmunity is initiated by the dysregulation of central or peripheral tolerance, resulting in the escape of autoreactive T and B cells from normal selection. There is rising evidence that expansion of follicular helper T (Tfh) and B cells results in humoral and cellular abnormalities and leads to the development of systemic autoimmune diseases such as Systemic lupus erythematosus (SLE); however, current knowledge of the mechanisms that control autoreactive B and T cell function remains incomplete. Thus, today it is becoming clear that understanding of Tfh and B cell biology and ways to manipulate their function may prove to be invaluable for the treatment of autoantibody-driven diseases. Recently, we generated and analyzed mice deficient in GRAIL and found that aged GRAIL deficient mice developed SLE-like symptoms characterized by massive germinal center formation, accumulation of Tfh and B cells in the lymphoid tissues and high titers of anti-dsDNA antibodies. In addition, GRAIL KO mice crossed with lupus prone B6.Faslpr/lpr mice developed severe lupus phenotype compared to control mice. Moreover, GRAIL expression was down regulated in T and B lymphocytes of patients with active SLE and lupus prone mice, further supporting the role of GRAIL in controlling humoral immune responses. All these findings suggest that GRAIL could be essential in controlling T and B cell functions, and importantly GRAIL down-regulation could serve as a marker for SLE-like disorders. However, it is not clear whether lack of GRAIL in T and/or B cells is responsible for development of lupus-like autoimmunity. We therefore hypothesize that regulation of B and Tfh cell function by GRAIL may be an important checkpoint in humoral tolerance, which is crucial to prevent development of autoimmunity. In Aim1, we will investigate the role of GRAIL in Tfh cell development and function by utilizing gene knockdown approaches. We will employ unique Bcl6-RFP and Bcl6-RFP/Foxp3GFP reporter mouse models to define the function of GRAIL in Tfh and T follicular regulatory (Tfr) cell development in normal and autoimmune settings. In addition, we will identify targets of GRAIL through which it facilitates control of Tfh cell programming by using microarray analysis of gene expression. We have developed a new method of generation of Tfh cells in vitro, which will allow generation of sufficient amount of cells for the proposed analysis. In Aim2, we will assess the role of GRAIL in B cell activation and function. Importantly, we will employ cutting edge technologies, including two-hybrid screening, to identify the exact target(s) of GRAIL that determines its function in B cells. Our results will provide mechanistic insights into the understanding of autoantibody-mediated autoimmune diseases and may lead to identify new therapeutic targets for their detection and prevention.