Basic and Translational Mechanisms of Alloimmunization to RBC Transfusion. Project 2

当患者对同种异体供体上表达的外源抗原产生抗体时，发生RBC同种免疫 红细胞，并且仍然是慢性输血患者发病率和死亡率的重要原因。 虽然绝大多数输注的红细胞表达外源抗原，但只有一部分输注患者 会产生同种抗体事实上，患者倾向于分成那些产生多种同种抗体的人， （应答者）和尽管多次输血但从未产生同种抗体的人（无应答者）。 然而，调节应答者与非应答者状态的分子因素定义不清。RBCs 长期以来，人们都知道它能分泌ATP，并通过嘌呤能调节内皮细胞功能 信号转导，但嘌呤能信号转导对RBC同种免疫的影响以前尚未解决。 我们假设红细胞驱动的嘌呤能信号倾向于抑制抗体对输血的反应。 红细胞，并进一步指出，患者之间嘌呤能信号传导敏感性的固有差异可能影响其免疫应答。 应答者状态。使用RBC同种异体免疫的小鼠模型，我们的初步数据表明， 嘌呤能信号通路中的多个不同步骤（P2X7R、CD73和ADORA2b）确实调节了 抗RBC同种抗体产生。这项提议旨在确定细胞和分子的确切位置， 嘌呤能信号调节红细胞同种免疫的机制，并进一步确定 已知的嘌呤能受体的功能获得和丧失多态性可以决定给定的患者是否将 产生抗红细胞IgG同种抗体。具体来说，我们提案的Aim 1将测试B细胞结合的假设 输注的红细胞诱导PANX 1介导的ATP分泌，这种细胞外ATP驱动P2X7R activation.我们进一步假设P2X7R激活抑制IgG类别转换和Germination中心。 通过诱导关键B细胞表面蛋白的胞外域脱落形成。我们提案的目标2将测试 假设B细胞上的CD 73表达调节AMP和腺苷信号传导的平衡， 应答B细胞，最终通过调节细胞内环AMP信号传导改变B细胞应答。 最后，本提案的Aim3将检验P2X7R、ADORA1和ADORA2中已知多态性的假设。 ADORA2b基因可以解释在应答者和非应答者中观察到的一些表型差异。 应答患者人群。项目之间的协同作用来自于我们对 嘌呤能信号传导和（a）P2X7R信号传导和免疫复合物信号传导之间的已知相互作用 通过TLR7和TLR9（项目1），（B）P2X7 R在RBC前体上高度表达， 随着红细胞成熟而降低（项目3），以及（c）已知可溶性CD73的增强和水平的增加 镰状细胞病患者的ATP、ADP、AMP和腺苷（项目4）。

RBC alloimmunization occurs when patients make antibodies to foreign antigens expressed on allogenic donor RBCs, and remains a significant cause of morbidity and mortality among chronically transfused patients. Though the vast majority of RBC transfusions express foreign antigens, only a subset of transfused patients will make alloantibodies. Indeed, patients tend to stratify into those who make multiple alloantibodies (responders) and those who never make an alloantibody despite multiple transfusions (non-responders). However, the molecular factors that regulate responder vs. non-responder status are poorly defined. RBCs have long been known to secrete of ATP and regulate endothelial cell function via resultant purinergic signaling, but the impact of purinergic signaling on RBC alloimmunization has not been previously addressed. We hypothesize that RBC driven purinergic signaling tends to suppress the antibody response to transfused RBCs, and further that inherent differences in purinergic signaling sensitivities among patients can impact their responder status. Using a mouse model of RBC alloimmunization, our preliminary data demonstrate that multiple different steps in the purinergic signaling pathway (P2X7R, CD73, and ADORA2b) do indeed regulate anti-RBC alloantibody production. This proposal sets out to determine the exact cellular and molecular mechanisms by which purinergic signaling regulates RBC alloimmunization, and further determine whether the known gain and loss of function polymorphisms of purinergic receptors can dictate whether a given patient will make anti-RBC IgG alloantibodies. Specifically, Aim1 of our proposal will test the hypothesis that B cell binding to transfused RBCs induces PANX1 mediated ATP secretion, and this extracellular ATP drives P2X7R activation. We further hypothesize that P2X7R activation suppresses IgG class-switching and germinal center formation by inducing ectodomain shedding of key B cell surface proteins. Aim2 of our proposal will test the hypothesis that CD73 expression on B cells regulates the balance of AMP and Adenosine signaling on responding B cells, ultimately altering B cells responses by regulating intracellular cyclic-AMP signaling. Finally, Aim3 of this proposal will test the hypothesis that known polymorphisms in the P2X7R, ADORA1 and ADORA2b genes can account for some of the phenotypic differences observed among responder and non- responder patient populations. Synergy between projects comes from crosstalk between our focus on purinergic signaling and (a) the known interactions between P2X7R signaling and immune complex signaling through TLR7 and TLR9 (Project 1), (b) the fact that P2X7R is highly expresses on RBC precursors but decreases as RBCs mature (Project 3), and (c) the known enhancement of soluble CD73 and increased levels of ATP, ADP, AMP and Adenosine in patients with sickle cell disease (Project 4).