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内或周围以及肌内膜组织中的B细胞浸润相关（quilty效应）。假设这些细胞参与排斥过程，尽管仍然缺乏这种贡献的正式证明。总的来说，浸润性B细胞界限不清。它们的反应表型、抗原特异性和可能的功能目前尚不清楚。了解B细胞如何参与移植排斥反应的机制无疑将促进靶向它们并治疗排斥反应的治疗剂的开发。由于样本来源有限以及难以建立研究直接从组织中提取的原代B细胞的技术，人体研究具有挑战性。由于这些原因，评估人移植物浸润 迄今为止，B细胞受到限制，大多数研究都集中在使用血液样本的体液免疫上。我们已经开始收集因CAV而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进行再次移植。使用深度测序原位分析重排的免疫球蛋白重链库的初步实验证实了4种心脏同种异体移植物中B细胞克隆的大量扩增和体细胞突变。我们也已经开始直接从这些心脏移植物标本中克隆永生化B细胞。我们提出的研究将表征这些细胞，揭示它们的主要功能，并评估它们在CAV病理生理学中的参与。目标1。为了表征CAV期间移植物浸润B细胞的表型、克隆性和特异性：我们将首先评估排斥移植物组织中B细胞的分布和表型。然后，B细胞库分析将鉴定原位扩增的主要B细胞克隆。平行地，将从移植物中分离的B细胞永生化并在体外培养。将进一步表征对应于原位扩增的B细胞的所选克隆。目标2.鉴定移植物浸润B细胞分泌的抗体的功能 CAV期间：将评估在目的1中发现原位扩增的永生化克隆分泌的单克隆抗体形成免疫复合物和诱导巨噬细胞原位分泌细胞因子以及促进抗原呈递给T细胞的能力。目标3。为了确定移植物浸润B细胞在CAV期间的功能：除了它们分泌致病性抗体的能力之外，我们将检查移植物浸润B细胞在CAV期间是否可以摄取并将抗原呈递给T细胞。我们还将研究B细胞是否能原位抑制T细胞反应。最后，我们还将研究补体调节这些反应的作用。

 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.