Basic and Translational Mechanisms of Alloimmunization to RBC Transfusion. Project 3

在美国，红细胞(RBC)输注是患者最常见的治疗方式， 每年大约每70个美国人中就有1人输血。尽管输注红细胞可以挽救生命， 一些患者会产生针对供体红细胞血型抗原的同种异体抗体。临床意义 同种异体抗体在输血、妊娠和移植环境中有不良影响。只有几个 有效的治疗干预措施，防止红细胞同种异体免疫，和/或消除 已经致敏的患者中的同种异体抗体。因此，有一种尚未得到满足的临床需要来了解这种风险。 可以识别(1)哪些RBC单位是免疫原性的和(2)哪些患者有高风险的因素 同种免疫；这一知识将导致预防和/或减轻这些不良事件的新战略。 异体免疫风险可能受到以下因素的影响：(1)捐献者的红细胞单位和(2)受血者的免疫力 系统。观察到，重复献血者的红细胞单位中网织红细胞计数较高。 因为代偿性网织红细胞增多症持续的时间比两次捐献之间的间隔时间更长。另外， 异体免疫风险最高的输血接受者也有持续的网织红细胞增多症(例如， 溶血性贫血)。因此，我们建立了易于处理的小鼠模型来检验网织红细胞 调节异体免疫率。在这里，我们提供了可靠的初步数据，确认网织红细胞是一种 捐赠者和接受者的红细胞同种免疫的重要风险因素：较高的同种免疫 RBC供体单位中含有高网织红细胞计数的RBC供体单位输注后，Rate/同种抗体水平可见如下 以及向表现出网织红细胞增多症的受者输注同种异体红细胞。因为网织红细胞不同 从成熟的红细胞(例如，网织红细胞有线粒体)，我们假设网织红细胞含有和/或 表达激活免疫细胞和启动同种异体免疫反应的配体。我们使用两种临床前模型 和来自人类的样本来阐明网织红细胞介导的增强的机制 同种异体免疫。为此，我们开发了4种小鼠模型来研究反映人类环境的网织红细胞 (例如，溶血性贫血、重复静脉切开术)，因为不同类型的应激性红细胞生成可能导致 网织红细胞差异。为了阐明网织红细胞如何增强同种异体抗体，我们将利用复杂的 识别、跟踪和测量RBC单位和/或其上对网织红细胞的免疫反应的方法 对输血接受者的诱导。为了将我们的发现应用于人类，我们还将研究网织红细胞如何 调节网织红细胞增多症(如溶血性贫血)患者B细胞的功能 与健康对照组相比。阐明网织红细胞如何增强同种异体免疫将(1)有助于识别 潜在的免疫原性供者红细胞单位和(2)预测哪些输血接受者有更高的风险 开发同种异体抗体；这些共同作用将防止未来的同种免疫事件。此外，这一点 知识将提供对预防和/或治疗同种免疫的潜在治疗目标的洞察。

Red blood cell (RBC) transfusion is the most common therapeutic modality given to patients in America, with approximately 1 out of every 70 Americans being transfused each year. Although RBC transfusions save lives, some patients develop alloantibodies against donor RBC blood group antigens. Clinically significant alloantibodies have adverse effects in transfusion, pregnancy, and transplant settings. There are very few effective therapeutic interventions to prevent RBC alloimmunization, in general, and/or to eliminate alloantibodies in already sensitized patients. Thus, there is an unmet clinical need to understand the risk factors that can identify (1) which RBC units are immunogenic and (2) which patients are at high risk for alloimmunization; this knowledge will lead to novel strategies to prevent and/or alleviate these adverse events. Alloimmunization risk may be impacted by (1) the donor RBC unit and (2) the transfusion recipient’s immune system. Observationally, higher reticulocyte counts are observed in RBC units from repeat blood donors because compensatory reticulocytosis lasts longer than the interval between donations. Additionally, transfusion recipients at highest risk of alloimmunization also have ongoing reticulocytosis (e.g., patients with hemolytic anemias). Thus, we generated tractable murine models to test the hypothesis that reticulocytes modulate alloimmunization rates. Herein, we provide robust preliminary data that identify reticulocytes as a significant risk factor for RBC alloimmunization at both the donor and recipient levels: higher alloimmunization rates/alloantibody levels are seen upon transfusion of RBC donor units containing high reticulocyte counts as well as upon allogeneic RBC transfusion into recipients exhibiting reticulocytosis. Because reticulocytes differ from mature RBCs (e.g., reticulocytes have mitochondria), we hypothesize that reticulocytes contain and/or express ligands that activate immune cells and initiate alloimmune responses. We use both pre-clinical models and samples from humans to elucidate the mechanisms underlying reticulocyte-mediated enhanced alloimmunization. To that end, we developed 4 murine models to study reticulocytes that reflect human settings (e.g., hemolytic anemia, repetitive phlebotomy) as different types of stress erythropoiesis may result in reticulocyte differences. To elucidate how reticulocytes enhance alloantibodies, we will leverage sophisticated methods to identify, track, and measure immune responses to reticulocytes in an RBC unit and/or upon their induction in a transfusion recipient. To translate our findings to humans, we will also study how reticulocytes modulate the function of B cells collected from patients with reticulocytosis (e.g., hemolytic anemia) as compared to healthy controls. Elucidating how reticulocytes enhance alloimmunization will (1) aid in identifying potentially immunogenic donor RBC units and (2) predict which transfusion recipients are at higher risk of developing alloantibodies; together these will prevent future alloimmunization events. Additionally, this knowledge will provide insights into potential therapeutic targets to prevent and/or treat alloimmunization.