Human B cell infiltration into allografts: mechanisms and molecules

人 B 细胞浸润同种异体移植物：机制和分子

• 批准号: 10043269
• 负责人: Thomas David Manes
• 金额: $ 25.13万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-21 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10043269
• 关键词: Ablation; Acute; Adhesives; Alloantigen; Allografting; Animal Model; Antibodies; Antigen Presentation; Antigens; Archives; B-Lymphocytes; Blood Circulation; Blood Vessels; CD3 Antigens; Cell Adhesion Molecules; Cell physiology; Chronic; Clinical Trials; Cloning; Complement; Data; Endothelial Cells; Event; Goals; Human; Immune; Immune system; Immunocompromised Host; Immunofluorescence Immunologic; Immunofluorescence Microscopy; Immunologics; Immunosuppression; In Vitro; Individual; Infiltration; Intervention; Kidney Transplantation; Lasers; Light; Mediating; Memory; Microfluidics; Microscopy; Migration Assay; Molecular Target; Mus; Organ Transplantation; Pathogenesis; Pathway interactions; Pattern; Periodicity; Pharmaceutical Preparations; Plasma Cells; Plasmablast; Population; Process; Receptor Signaling; Receptors, Antigen, B-Cell; Risk; Sampling; Signal Transduction; Source; Specificity; T cell response; T memory cell; T-Cell Receptor; T-Lymphocyte; Techniques; Testing; Therapeutic; Tissues; Work; adhesion process; adhesion receptor; cell type; chemokine; chemokine receptor; crosslink; cytokine; effective therapy; high dimensionality; human migration; in vitro Assay; in vitro Model; insight; kappa opioid receptors; kidney allograft; migration; monolayer; novel therapeutic intervention; outcome forecast; peripheral blood; prevent; receptor; recruit; response; tertiary lymphoid organ

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.

7.项目总结/摘要 在过去的三年中，由于慢性排斥反应导致的晚期肾移植物丢失率没有显著变化。 尽管在控制急性排斥反应方面取得了显著的进步，但仍有数十年的时间。虽然免疫发病机制是 目前还不完全清楚，相当大比例的同种异体移植物含有浸润的宿主B细胞， B细胞的缺乏与预后不良相关，这可能与供体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，并且可以由趋化因子或抗原启动 由于B细胞在血管腔内的存在，对B细胞募集的了解要少得多。我们建议研究 这些过程的目的是鉴定可能抑制B细胞浸润的途径和分子靶点， 移植物为了研究趋化因子是否以及如何诱导这一过程，我们将使用高维 人同种异体移植组织的循环组织免疫荧光显微镜检查和跨内皮细胞的体外测定 通过人外周血B细胞的TEM迁移。组织分析将提供关于以下亚型的信息： 在移植物浸润中发现的B细胞，它们表达的趋化因子和粘附受体，以及它们与 激活的内皮细胞可能是入侵点体外试验将使用人微血管 微流控室中的内皮细胞单层，以表征和操纵能够 使用我们在T细胞研究中开发的方法进行TEM。研究这个过程是否以及如何 通过抗原诱导，我们将使用我们的高维IF方法来确定移植物中的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 我们将从中克隆并表达抗原结合区以产生抗体， 对不同类型移植物抗原的特异性。我们还将修改我们的体外TEM试验， 细胞通过展示抗人κ抗体交联并通过抗原的B细胞受体发出信号， 再次采用我们用来用抗人CD 3模拟T细胞受体信号传导的技术。我们预计 这些研究将导致识别目标，可能是目前批准的药物， 可以限制B细胞募集到同种异体移植物中，这是一种可以减少晚期移植物丢失而没有 免疫抑制患者介导的总B细胞消融。