Pathological B Cells: Novel Strategies to Prevent and Treat Chronic GVHD

摘要 在这次更新中，我们继续努力开发能够使异基因造血干细胞 肝细胞移植（HCT）对患者的毒性较小。我们的主要目标是制定针对慢性移植物的策略， 抗宿主病（cGVHD）相关的免疫病理学，而不阻断关键的免疫效应 的HCT。B细胞在cGVHD中具有经证实的病理生物学作用，但区分疾病介导的B细胞 子集仍然难以捉摸。只针对异常的B细胞是重要的，因为我们已经发现， 用利妥昔单抗消除外周B细胞使所谓的改变的免疫稳态和cGVHD持续存在 无法恢复完整B细胞区室的患者。在上一个融资期，我们阐明了 病理性B细胞活化因子（BAFF）和NOTCH 2如何促进异常B细胞受体（BCR）- HCT后的信号。基于B细胞成熟过程中已知的BCR信号传导事件，我们假设， 抗原高反应性和SYK依赖性B细胞可被消除而不影响免疫稳态。在 在临床前研究和临床试验中，我们发现抑制BCR近端蛋白SYK， 异常激活B细胞并恢复B细胞稳态。我们也开始剖析小说 支持BCR高反应性的机制。已发表的证据和我们额外的初步数据， 包括单细胞RNA-Seq数据集，为这一提议奠定了坚实的科学基础，并为我们目前的研究提供了基础。 总体假设，即B细胞中的异常激活和效应子功能途径可以有效地 在cGVHD中。在目前的提案中，我们解决了我们对人类B细胞免疫的知识中的主要空白。 编程，其赋予cGVHD中B细胞耐受性持续丧失的倾向。我们的实验方法 需要在我们的离体B细胞测定系统和小鼠中的平行实验中研究cGVHD患者样品 使用B细胞条件性和诱导性敲除小鼠作为供体来验证分子机制。我们特别 将描绘促进病理性B细胞的分子途径，并确定不同的疾病介导 cGVHD B细胞的功能。我们将追求三个具体目标：1）BCR后SYK蛋白的持久性 参与，2）在II期临床试验中阻断SYK并恢复cGVHD患者的体液免疫的能力。 试验3）抗体和炎性细胞因子产生的分子基础。我们将定义为目标 我们将展示的B细胞亚群中的信号通路在cGVHD中具有病理性B效应细胞功能。我们 一项提案依赖于团队科学概念，并由杜克临床试验的合作研究者促成 团队（Horwitz和Rizzieri博士）和杜克癌症研究所统计组（Li和Owzar博士）。我们集团 仍然处于阐明异常人类B细胞信号传导和测试小分子 慢性GVHD患者中的抑制剂。

ABSTRACT In this renewal, we continue efforts to develop agents that will make allogeneic hematopoietic stem cell transplantation (HCT) less toxic for patients. Our major objective is to develop strategies that target chronic graft versus host disease (cGVHD)-related immune pathology without blocking the critical immunotherapeutic effects of HCT. B cells have a substantiated pathobiological role in cGVHD, but distinguishing disease-mediating B cells subsets remains elusive. Targeting only aberrant B cells is important because we have found that global abrogation of peripheral B cells with rituximab perpetuates so-called altered immune homeostasis and cGVHD in patients unable to recover a comprehensive B-cell compartment. In the previous funding period, we elucidated how pathological B Cell Activating Factor (BAFF) and NOTCH2 promote aberrant B Cell Receptor (BCR)- signaling after HCT. Based on known BCR signaling events during B cell maturation, we hypothesized that antigen-hyperresponsive and SYK-reliant B cells can be eliminated without affecting immune homeostasis. In preclinical studies and in the clinical trial, we showed that inhibition of the BCR-proximal protein, SYK, eliminated aberrantly activated B cells and afforded recovery of B-cell homeostasis. We have also begun to dissect novel mechanisms underpinning BCR hyper-responsiveness. Published evidence and our additional preliminary data, including a single cell RNA-Seq dataset, form the solid scientific basis for this proposal and afford our current overarching hypothesis that aberrant activation and effector function pathways in B cells can be effectively targeted in cGVHD. In the current proposal, we address major gaps in our knowledge regarding human B-cell programming that confer a propensity for ongoing loss of B cell tolerance in cGVHD. Our experimental approach entails study of cGVHD patient samples in our ex vivo B-cell assay system and parallel experiments in mice using B-cell conditional and inducible knockout mice as donors to verify molecular mechanisms. Specifically, we will delineate molecular pathways that promote pathological B cells and determine the distinct disease-mediating functions of cGVHD B cells. We will pursue three specific aims: 1) persistence of SYK protein after BCR engagement, 2) the ability to block SYK and restore humoral immunity in cGVHD patients in a Phase II clinical trial 3) molecular underpinnings of antibody and inflammatory cytokine production. We will define targetable signaling pathways in B cell subsets that we will show have pathological B-effector cell functions in cGVHD. Our proposal relies on a team science concept and is made possible by co-investigators on the Duke clinical trial team (Drs. Horwitz and Rizzieri) and Duke Cancer Institute statistical group (Drs. Li and Owzar). Our group remains at the forefront of efforts elucidating aberrant human B-cell signaling and testing small molecule inhibitors in chronic GVHD patients.