Cytokine control of Red Blood Cell Alloimmunization

描述（由申请人提供）：红细胞异体免疫仍然是输血医学中常见的临床问题。同种异体抗体可引起即时和延迟性溶血性输血反应；导致大量的发病率和偶尔的死亡率。对于那些不幸产生多种同种异体抗体的患者，提供相容的红细胞可能既耗时又耗费资源。在某些情况下，这可能导致无法找到原本可以挽救生命的治疗方法。尽管其临床重要性，驱动红细胞免疫的基本分子信号仍不清楚。与感染因子一样，产生针对红细胞的同种抗体需要激活抗原特异性CD4+ T细胞和B细胞，其中原始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，我们对诱导幼稚CD4+ T细胞在体内分化和表达TFH遗传程序的分子机制的理解仍然是片面的。此外，在相对较弱的红细胞输血刺激下，负责幼稚T细胞活化和TFH分化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。临床相关小鼠红细胞储存和输血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研究表明，红细胞储存增强了红细胞异体免疫，并且输入储存的红细胞显著增加了包括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缺陷小鼠在新鲜血液和储存血液环境中对红细胞异体免疫的敏感性较低，ii) IL-6信号通路激活新生CD4+ T细胞中的PI3K/AKT/mTOR信号通路，iii) PI3K/AKT/mTOR信号的诱导与原发性新生T细胞中BAF染色质重塑复合体的多个成员磷酸化增加相关。因此，我们建议确定IL-6在红细胞异体免疫中的特异性细胞靶点，并进一步确定IL-6诱导的幼稚CD4+ T细胞中的分子信号。我们的研究计划的目的是验证由新鲜和储存的红细胞输血诱导的IL-6驱动原始CD4+ T细胞的TFH分化的特定假设。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。我们将通过将建立良好的小鼠红细胞同种异体免疫和储存模型与最近开发的表达IL-6受体（IL-6RA）的floxed等位基因的小鼠菌株相结合来验证我们的假设。随着时间的推移，我们预计驱动红细胞抗体产生的分子电路的鉴定将为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.