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

对红细胞上同种抗原的体液免疫（RBC同种免疫）仍然是一个主要的并发症， 并且在某些情况下阻碍了对多种疾病的输血治疗。然而， 同种免疫不是均匀分布的;相反，患者倾向于对多种同种抗原产生同种免疫 （应答者）或尽管慢性输血但根本没有（无应答者）。然而，调节的因素 应答者与非应答者状态仍然定义不清。RBC同种异体免疫率增加， 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 发现具有SLE样生物学特征（即产生抗核抗体（ANA））的小鼠 RBC同种异体免疫这些小鼠由于一组特定的变异基因（NBA 2）而产生ANA， 含有与SLE相关的人类基因的多个直系同源物。重要的是，NBA 2小鼠不显示 与SLE相关的器官病理学，导致ANA是SLE的一个孤立组分的假设， 影响RBC同种免疫。此外，在NBA 2小鼠中，已知Toll样受体7（TLR 7）和TLR 9与Toll样受体7（TLR 7）和Toll样受体9（TLR 9）结合。 通过相互拮抗作用（TLR 7促进和TLR 9抑制）调节ANA形成。这些发现 导致本项目的中心假设：中心假设：抗核抗体导致红细胞增加 由于TLR 7和TLR 9信号传导失调，通过激活B细胞的同种异体免疫。这 使用识别真实人类血液的新型B细胞受体转基因小鼠来检验假设 组抗原（Kpb表位）的人KEL糖蛋白。将抗Kpb小鼠与NBA 2小鼠组合 然后用表达人KEL的KEL-K2变体的转基因RBC输注，所述变体含有 Kpb抗原。这种方法允许对幼稚B细胞的早期激活和分化进行体内机制研究。 对输注红细胞上的红细胞抗原具有特异性的细胞。具体目标1和2测试ANA对RBC的作用 同种异体免疫，包括操纵TLR 7和TLR 9信号传导，以严格测试中枢免疫。 假说.具体目标3使用项目4中研究的SCD患者的人类队列。SCD患者 有很高的ANA率。患者血浆中是否存在ANA将与模式相关 同种免疫患者白细胞将在体外刺激，在存在或不存在ANA的情况下， 血浆和/或TLR 7和TLR 9激动或拮抗作用，使用测量B细胞活化的测定， 分化响应于CD 4+滤泡T细胞的帮助。项目之间的协同作用来自于 嘌呤能信号传导（项目2）和TLR 7和TLR 9，网织红细胞（项目3）提供TLR 7的激动剂， TLR 9，以及已知使人类易患ANA形成的遗传变异的预测将出现， SCD患者RBC同种异体免疫的前瞻性GWAS研究中的相关性（项目4）。的 提出的研究有能力鉴定RBC同种免疫的新预测因子（即ANA）， 提供了新的机制见解，可以作为新疗法开发的基础。

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.