Basic and Translational Mechanisms of Alloimmunization to RBC Transfusion. Project 1

对红细胞上的同种异体抗原的体液免疫(RBC同种免疫)仍然是一个主要的并发症， 在某些情况下，还会阻碍各种疾病的输血治疗。然而，倾向于成为 同种异体免疫并不是均匀分布的；相反，患者倾向于对多种同种异体抗原进行同种免疫。 (应答者)或根本没有，尽管长期输血(无应答者)。然而，规范的因素 应答者和非应答者的状态仍然定义不清。红细胞同种异体免疫率在 患有自身免疫性疾病的患者，如系统性红斑狼疮(SLE)。我们最近做了 发现具有SLE样生物学(即产生抗核抗体(ANA))的小鼠有更高的发病率 红细胞同种异体免疫。这些小鼠由于一组特定的变异基因(NBA2)而患上ANA 包含与SLE相关的人类基因的多个同源基因。重要的是，NBA2小鼠没有表现出 与SLE相关的器官病理，导致假设ANA是SLE的独立组成部分 影响红细胞同种异体免疫。此外，在NBA2小鼠中，已知Toll样受体7(TLR7)和TLR9 通过相互拮抗来调节ANA的形成(TLR7促进，TLR9抑制)。这些发现 引出本项目的中心假说：中心假说：抗核抗体导致红细胞升高 同种异体免疫是由于TLR7和TLR9信号失控导致的B细胞活化。这 这一假设是通过一种新型的B细胞受体转基因小鼠来验证的，这种小鼠可以识别真实的人类血液 人Kel糖蛋白上的组抗原(KPB表位)。抗KPB小鼠与NBA2小鼠结合 然后将其与表达人Kel-K2变异体的转基因红细胞一起输注，该变异体含有 KPB抗原。这种方法使得对幼稚B的早期激活和分化的体内机制研究成为可能 输血红细胞上的RBC抗原特异性细胞。特异性靶点1和2测试ANA对RBC的作用 同种异体免疫，包括操纵TLR7和TLR9信号来严格测试中央 假设。特定目标3使用在项目4中研究的SCD患者的人类队列。 有很高的ANA率。患者血浆中ANA的存在与否将与模式相关 同种异体免疫。在存在或不存在含有ANA的情况下，患者的白细胞将在体外被刺激 血浆和/或TLR7和TLR9的激动性或拮抗性，使用一种测量B细胞激活和 分化反应对CD4+滤泡T细胞有帮助。项目之间的协同来自于项目之间的串扰 嘌呤能信号转导(方案2)和TLR7和TLR9，网织红细胞(方案3)为TLR7和TLR9提供激动剂 TLR9，并预测已知的使人类容易形成ANA的基因变异将出现为 SCD患者红细胞同种异体免疫的前瞻性研究中的相关性(项目4)。这个 拟议的研究既有能力确定红细胞同种异体免疫的新预测因素(即ANA)，也有能力 提供新的机制洞察力，可作为开发新疗法的基础。

Humoral immunization to alloantigens on red blood cells (RBC alloimmunization) remains a major complication, and in some cases barrier to transfusion therapy for myriad diseases. However, the tendency to become alloimmunized is not evenly distributed; rather, patients tend to become alloimmunized to multiple alloantigens (responders) or none at all despite chronic transfusion (nonresponders). However, the factors that regulate responder vs. nonresponder status remain poorly defined. Rates of RBC alloimmunization is increased in patients with autoimmune pathologies, such as systemic lupus erythematosus (SLE). We have recently discovered that mice with SLE-like biology (i.e. develop anti-nuclear antibodies (ANAs)) have an increased rate of RBC alloimmunization. These mice develop ANAs due to a specific cluster of variant genes (NBA2) that contains multiple orthologues to human genes associated with SLE. Importantly, the NBA2 mice do not display organ pathology associated with SLE, leading to the hypothesis that ANA are an isolated component of SLE that affects RBC alloimmunization. Moreover, in NBA2 mice, toll like receptor 7 (TLR7) and TLR9 are known to regulate ANA formation through mutual antagonism (TLR7 promotes and TLR9 suppresses). These findings lead to the central hypothesis of this project: Central Hypothesis: Anti-nuclear antibodies cause increased RBC alloimmunization through activation of B cells as a result of dysregulated TLR7 and TLR9 signaling. This hypothesis is tested using a novel B cell receptor transgenic mouse that recognizes an authentic human blood group antigen (Kpb epitope) on the human KEL glycoprotein. The anti-Kpb mouse is combined with NBA2 mice that are then transfused with transgenic RBCs expressing the KEL-K2 variant of human KEL, which contains the Kpb antigen. This approach allows an in vivo mechanistic study of early activation and differentiation of naïve B cells specific for an RBC antigen on transfused RBCs. Specific aims 1 and 2 test the role of ANAs on RBC alloimmunization, including the manipulation of TLR7 and TLR9 signaling to rigorously test the central hypothesis. Specific aim 3 uses the human cohort of SCD patients studied in project 4. Patients with SCD have high rates of ANAs. The presence or absence of ANAs in patient plasma will be correlated with patterns of alloimmunization. Patient leukocytes will be stimulated in vitro, in the presence or absence of ANA containing plasma and/or TLR7 and TLR9 agonism or antagonism, using an assay that measures B cell activation and differentiation in response to CD4+ follicular T cell help. Synergy between projects comes from crosstalk between purinergic signaling (project 2) and TLR7 and TLR9, that reticulocytes (project 3) provide agonists for TLR7 and TLR9, and the prediction that genetic variations known to predispose humans to ANA formation will emerge as associations in the prospective GWAS study on RBC alloimmunization in patients with SCD (project 4). The proposed studies have the capacity to both identify novel predictors of RBC alloimmunization (i.e. ANAs) and to provide novel mechanistic insight that can serve as the basis for development of new therapeutics.