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失调引发的，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，滤泡辅助性T（Tfh）和B细胞的扩增导致 在体液和细胞异常，并导致系统性自身免疫性疾病，如系统性红斑狼疮（SLE）的发展;然而，目前的知识，控制自身反应性B和T细胞功能的机制仍然不完整。因此，今天，人们越来越清楚，Tfh和B细胞生物学的理解和方法来操纵它们的功能可能被证明是无价的治疗自身抗体驱动的疾病。最近，我们产生并分析了GRAIL缺陷小鼠，发现老年GRAIL缺陷小鼠出现SLE样症状，其特征在于大量生发中心形成、Tfh和B细胞在淋巴组织中积累以及高滴度的抗dsDNA抗体。此外，与对照小鼠相比，GRAIL KO小鼠与狼疮易感B6.Faslpr/lpr小鼠杂交产生严重的狼疮表型。此外，GRAIL在活动性SLE患者和狼疮易感小鼠的T和B淋巴细胞中表达下调，进一步支持GRAIL在控制体液免疫应答中的作用。所有这些发现表明GRAIL在控制T和B细胞功能中是必不可少的，重要的是GRAIL下调可以作为SLE样疾病的标志物。然而，尚不清楚T和/或B细胞中缺乏GRAIL是否导致狼疮样自身免疫的发展。因此，我们推测GRAIL对B和Tfh细胞功能的调节可能是体液耐受中的重要检查点，这对于防止自身免疫的发展至关重要。 在Aim 1中，我们将通过利用基因敲低方法研究GRAIL在Tfh细胞发育和功能中的作用。我们将采用独特的Bcl 6-RFP和Bcl 6-RFP/Foxp 3GFP报告基因小鼠模型来确定GRAIL在正常人Tfh和T滤泡调节（Tfr）细胞发育中的功能。 和自身免疫系统此外，我们将通过使用基因表达的微阵列分析来确定GRAIL的靶点，通过该靶点，GRAIL促进Tfh细胞编程的控制。我们已经开发了一种新的方法，在体外产生的Tfh细胞，这将允许产生足够量的细胞用于所提出的分析。在Aim 2中，我们将评估GRAIL在B细胞活化和功能中的作用。重要的是，我们将采用尖端技术，包括双杂交筛选，以确定GRAIL的确切靶点，这些靶点决定了GRAIL在B细胞中的功能。我们的研究结果将为理解自身抗体介导的自身免疫性疾病提供机制上的见解，并可能导致识别新的治疗靶点，用于检测和预防。

 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.