Contribution of B cells to human cardiac allograft vasculopathy

 描述(由申请人提供)：CAV几乎总是与冠状动脉或冠状动脉周围以及心内膜心肌组织中的B细胞浸润有关(Quilty效应)。它认为S假设这些细胞参与了排斥过程，尽管仍然缺乏对这种贡献的正式证明。总体而言，浸润性B细胞的定义很宽松。它们的抗原表型、抗原特异性和可能的功能目前尚不清楚。了解B细胞如何参与移植物排斥反应的机制，无疑将有助于开发针对B细胞的治疗药物并治疗排斥反应。人体研究具有挑战性，因为样本来源有限，而且很难建立技术来研究直接从组织中提取的初级B细胞。出于这些原因，对人体移植物渗透的评估 到目前为止，B细胞有限，大多数研究都集中在利用血液样本进行体液免疫方面。我们已经开始收集因CAV而被排斥的新鲜心脏移植标本，并通过美国和加拿大的移植中心合作网络移植到再次移植。通过对重排的免疫球蛋白重链进行原位分析，初步证实了4例心脏移植物中B细胞克隆的大量扩增和体细胞突变。我们还开始直接从这些心脏移植标本中克隆出不朽的B细胞。我们提出的研究将描述这些细胞的特征，揭示它们的主要功能，并评估它们参与CAV的病理生理学。目的1.研究CAV过程中移植物浸润性B细胞的表型、克隆性和特异性：首先评估排斥移植物组织中B细胞的分布和表型。然后，B细胞谱系分析将确定主要的原位扩增的B细胞克隆。同时，从移植的移植物中分离出的B细胞将被永生并在体外培养。与原位扩增的B细胞相对应的选定克隆将进一步鉴定。目的2.鉴定移植物浸润性B细胞分泌抗体的功能 在CAV期间：在AIM 1中发现原位扩增的永生化克隆分泌的单抗将被评估其形成免疫复合体和诱导巨噬细胞原位分泌细胞因子的能力以及促进抗原呈递给T细胞的能力。目的3.确定CAV过程中移植物浸润性B细胞的功能：除了分泌致病抗体的能力外，我们还将检测CAV过程中移植物浸润性B细胞是否能够摄取抗原并将抗原呈递给T细胞。我们还将研究B细胞是否能在原位极化T细胞反应。最后，我们还将研究补体在调节这些反应方面的作用。

 DESCRIPTION (provided by applicant): CAV is almost invariably associated with B cell infiltrates in or around coronary arteries as well as in endomyocardial tissue (quilty effect). It s assumed that these cells participate in the rejection process, although a formal demonstration of such contribution is still lacking. On the whole, infiltrating B cells are loosely defined. Their eact phenotype, antigen specificity and possible function are currently unknown. Understanding how B cells contribute to mechanisms of graft rejection would undoubtedly facilitate the development of therapeutic agents to target them and treat rejection. Studies in humans are challenging due to the limited source of samples and the difficulty of setting up techniques to study primary B cells retrieved directly from tissue. For these reasons, the assessment of human graft infiltrating B cells has been limited thus far and most studies have focused on humoral immunity using blood samples. We have begun collecting fresh cardiac graft specimens rejected because of CAV and explanted for re-transplantation through a collaborative network of transplant centers in the USA and Canada. Preliminary experiments using deep sequencing to analyze rearranged immunoglobulin heavy chain repertoire in situ demonstrated the massive expansion and somatic mutation of B cell clones in 4 cardiac allografts. We have also started immortalizing B cell clones directly from these cardiac allograft specimens. Our proposed research will characterize these cells, uncover their main function and evaluate their participation in the pathophysiology of CAV. Aim 1. To characterize the phenotype, clonality and specificity of graft infiltrating B cells during CAV: We will first assess the distribution and phenotype of B cells within the rejected graft tissue. B cell repertoire analyses will then identify predominant B cell clones expanded in situ. In parallel, B cells isolated from explanted grafts will be immortalized and cultured in vitro. Selected clones corresponding to B cells expanded in situ will be further characterized. Aim 2. To identify the function of antibodies secreted by graft infiltrating B cells during CAV: Monoclonal antibodies secreted by immortalized clones found to be expanded in situ in aim 1, will be assessed for their capacity to form immune complexes and induce cytokine secretion by macrophages in situ as well as facilitate antigen presentation to T cells. Aim 3. To determine the function of graft infiltrating B cells during CAV: Aside from their capacity to secrete pathogenic antibodies, we will examine whether graft-infiltrating B cells during CAV can uptake and present antigens to T cells. We will also investigate whether B cells can polarize T cell responses in situ. Lastly, we will also examine the role of the complement to modulate these responses.