Human B cell infiltration into allografts: mechanisms and molecules

7.项目摘要/摘要 在过去的三年中，由于慢性排斥反应而导致的晚期肾移植失败率没有显著变化。 几十年，尽管在控制急性排斥反应方面有了明显的进步。而免疫机制是 尚不完全了解，相当大比例的同种异体移植物含有浸润性宿主B细胞和存在 B细胞的缺失与预后不良有关，这可能与供者特异性抗体的存在无关。 虽然T细胞向同种异体移植物的募集是众所周知的，并且可能是由趋化因子或抗原启动的 B细胞在血管腔内的表现，对B细胞的募集知之甚少。我们建议研究 这些过程的目的是确定可能抑制B细胞侵袭的途径和分子靶点 嫁接。为了研究趋化因子是否以及如何诱导这一过程，我们将使用两个高维 人同种异体移植组织循环免疫荧光显微镜及体外跨内皮细胞检测 人外周血B细胞的迁移(TEM)。组织分析将提供有关亚型的信息 移植物浸润液中发现的B细胞，它们表达的趋化因子和黏附受体，以及它们与 激活的内皮细胞是可能的入口点。体外试验将使用人体微血管 微流体室内皮细胞单层用于表征和操纵B细胞 使用我们在T细胞研究中开发的方法进行透射电子显微镜检查。研究这一过程是否可以以及如何进行 由抗原诱导，我们将使用我们的高维IF方法来确定移植物内的B细胞是否 与相关的T细胞群相互作用，我们将使用激光捕获显微镜分离单个B细胞 我们将从其中克隆和表达抗原结合区以产生抗体，然后检测 不同类型移植物抗原的特异性。我们还将修改我们的体外透射电子显微镜分析，以允许内皮细胞 通过展示抗人kappa抗体，使细胞通过B细胞受体对抗原进行交联和信号传递， 再一次使用了我们用来用抗人CD3模拟T细胞受体信号的技术。我们期待着 这些研究将导致识别靶点，可能是目前批准的药物所能抑制的 可以限制B细胞在同种异体移植物中的募集，这一干预措施可能会减少晚期移植物丢失，而不会有 全B细胞消融介导的免疫抑制患者。

7. PROJECT SUMMARY/ABSTRACT Rates of late renal allograft loss due to chronic rejection have not significantly changed over the past three decades despite marked improvements in controlling acute rejection. While the immune pathogenesis is incompletely understood, a significant proportion of allografts contain infiltrated host B cells and the presence of B cells correlates with poor prognosis that may be independent of the presence of a donor specific antibody. While T cell recruitment to allografts is well understood and may be initiated by either chemokines or antigen presentation within the vascular lumen, much less is known about B cell recruitment. We propose to study these processes with the goal of identifying pathways and molecular targets that may inhibit B cell infiltration of grafts. To study if and how this process can be induced by chemokines, we will employ both high dimensional cyclic tissue immunofluorescence microscopy of human allograft tissue and in vitro assays of transendothelial migration (TEM) by human peripheral blood B cells. Tissue analyses will provide information re the subtypes of B cells found within graft infiltrates, the chemokine and adhesion receptors they express, and their proximity to activated endothelial cells that are the likely point of entry. In vitro assays will use human microvascular endothelial cell monolayers in microfluidic chambers to characterize and manipulate B cells capable of undergoing TEM using approaches we developed in our T cell studies. To study if and how this process can be induced by antigen, we will use our high dimensional IF approach to determine if B cells within the graft are interacting with relevant T cell populations and we will use laser capture microscopy to isolate individual B cells from which we will clone and express the antigen combining regions to generate antibodies and then test specificity for different types of graft antigens. We will also modify our in vitro TEM assays to allow endothelial cells to cross-link and signal through the B cell receptor for antigen by display of anti-human kappa antibody, again employing a technique we used to mimic T cell receptor signaling with anti-human CD3. We anticipate that these studies will lead to identification of targets, possibly inhibitable by currently approved agents, that can limit B cell recruitment into allografts, an intervention that may reduce late graft loss without the risk to immunosuppressed patients mediated by total B cell ablation.