Cytokine control of Red Blood Cell Alloimmunization

描述(申请人提供)：红细胞同种异体免疫仍然是输血医学中常见的临床问题。同种抗体可引起即时和延迟的溶血性输血反应；导致大量发病率和偶尔死亡。对于那些不幸制造出多种同种异体抗体的患者，提供相容的红细胞可能既耗费时间又耗费资源。在某些情况下，这可能导致无法找到原本可以挽救生命的疗法。尽管它在临床上很重要，但驱动红细胞免疫的基本分子信号仍然不清楚。与感染性病原体一样，针对红细胞的同种异体抗体的产生需要抗原特异性的CD4+T细胞和B细胞的激活，在这些细胞中，NAVE CD4+T细胞分化为T滤泡辅助细胞(TFH)，这是B细胞完全分化为抗体分泌细胞所必需的。然而，我们对NAOVE CD4+T细胞被诱导分化并在体内表达TFH遗传程序的分子机制的理解仍然不够深入。此外，相对较弱的红细胞输注刺激剂激活NAOVE T细胞和分化TFH的分子因素尚不清楚。如果我们要成功地干预这一致病过程，我们必须首先确定导致红细胞同种异体抗体产生的细胞靶点和分子信号。临床相关的小鼠红细胞储存和输注模型的发展为解决这一问题提供了一套新的工具。最近在这些模型中的工作表明，RBC的同种异体免疫通过RBC储存而增强，并且输注储存的RBC显著增加包括IL-6在内的多种细胞因子的系统产生。我们随后证明，i)IL-6缺陷小鼠在新鲜和储存的血液环境中对RBC同种异体免疫不那么敏感，ii)IL-6信号激活NAVE CD4+T细胞中的PI3K/AKT/mTOR信号通路，以及iii)PI3K/AKT/mTOR信号的诱导与原代NAOVE T细胞中BAF染色质重塑复合体的多个成员的磷酸化增加有关。因此，我们建议确定IL-6在RBC同种异体免疫中的特异性细胞靶点，并进一步确定IL-6诱导的NAVE CD4+T细胞的分子信号。我们研究计划的目的是验证特定的假设，即输注新鲜和储存的红细胞诱导的IL-6促进NAVE CD4+T细胞向TFH分化。此外，我们假设IL-6信号通过PI3K/AKT/mTOR信号通路诱导BAF染色质重塑复合体的磷酸化调节。我们将通过将已建立的RBC同种免疫和储存的小鼠模型与最近开发的表达IL-6受体(IL-6RA)的等位基因的小鼠品系相结合来验证我们的假设。随着时间的推移，我们预计，识别驱动RBC抗体产生的分子回路将为进一步的临床开发提供治疗和诊断靶点。 公共卫生相关性：红细胞(RBC)异基因免疫仍然是一个常见的临床问题，可能导致重大疾病和罕见的死亡。对于那些不幸制造出多种同种异体抗体的患者来说，提供相容的红细胞可能既耗时又耗费资源；有时会导致无法找到原本可以挽救生命的疗法。通过在小鼠的受控实验环境中识别驱动RBC抗体产生的分子电路，这一提议将为人类进一步的临床开发提供治疗和诊断靶点。

DESCRIPTION (provided by applicant): RBC alloimmunization remains a common clinical problem in transfusion medicine. Alloantibodies are responsible for both immediate and delayed hemolytic transfusion reactions; leading to substantial morbidity and occasional mortality. For those patients unfortunate enough to make multiple alloantibodies, provision of compatible RBCs can be both time and resource intensive. In some cases, this can result in an inability to locate an otherwise life-saving therapy. Despite its clinical importance, the fundamental molecular signals that drive RBC immunization remain unclear. Like infectious agents, the generation of alloantibodies to RBCs requires the activation of antigen specific CD4+ T cells and B cells, where naove CD4+ T cells differentiate into T Follicular Helper cells (TFH) that are essential for complete B cell differentiation into antibody secreting cells. However, our understanding of the molecular mechanisms by which naove CD4+ T cells are induced to differentiate and express a TFH genetic program "in vivo" remains partial. Furthermore, the molecular factors responsible for naove T cell activation and TFH differentiation by the relatively weak stimulant of RBC transfusion are unknown. If we are to ever successfully intervene in this pathogenic process, we must first identify the cellular targets and molecular signals responsible for RBC alloantibody generation. The development of clinically relevant mouse models of RBC storage and transfusion provides a novel set of tools to address this issue. Recent work in these models demonstrated that RBC alloimmunization is enhanced by RBC storage, and that transfusion of stored RBCs significantly increases the systemic production of multiple cytokines including IL-6. We have subsequently demonstrated that i) IL-6 deficient mice are less susceptible to RBC alloimmunization in both fresh and stored blood settings, ii) IL-6 signaling activates the PI3K/AKT/mTOR signaling pathway in naove CD4+ T cells, and iii) induction of PI3K/AKT/mTOR signals correlates with increased phosphorylation of multiple members of the BAF chromatin remodeling complex in primary naove T cells. We therefore propose to identify the specific cellular targets of IL-6 in RBC alloimmunization, and further determine the molecular signals in naove CD4+ T cells induced by IL-6. The objective of our research plan is to test the specific hypothesis that the IL-6 induced by transfusion of fresh and stored RBCs drives TFH differentiation from naove CD4+ T cells. Furthermore, we hypothesize that IL-6 signaling induces phospho-modulation of the BAF chromatin remodeling complex via the action of the PI3K/AKT/mTOR signaling pathway. We will test our hypothesis by combining a well established mouse model of RBC alloimmunization and storage with recently developed mouse strains expressing a floxed allele of IL-6 Receptor (IL-6RA). In the fullness of time, we anticipate that identification of the molecular circuitry driving RBC antibody production will provide therapeutic and diagnostic targets for further clinical development. PUBLIC HEALTH RELEVANCE: Red blood cell (RBC) alloimmunization remains a common clinical problem and can be responsible for significant illness and rare deaths. For those patients unfortunate enough to make multiple alloantibodies, provision of compatible RBC can be both time and resource intensive; at times leading to an inability to locate an otherwise life-saving therapy. By identifying the molecular circuitry driving RBC antibody production in the controlled experimental setting of mice, this proposal will provide therapeutic and diagnostic targets for further clinical development in humans.