Role and mechanisms of the PI3K pathway in B cell tolerance

项目摘要 B细胞耐受性是发展对自身免疫耐受的免疫系统的关键，但是， 这个过程的执行还没有被清楚地理解。我们研究的首要目标是阐明 在未成熟和成熟之间的发育检查点调节B细胞选择的分子途径， 过渡细胞阶段，加强自身反应性骨髓B细胞的耐受过程，同时促进 非自身反应性细胞的分化及其骨髓排出。通过研究IG基因敲入小鼠模型， 我们记录了中心B细胞耐受的RAS、ERK和PI3 K信号分子，它们是 GTP酶、MAP激酶和脂质激酶分别在非自身反应性心肌病中活性达到较高的基础水平。 自身反应性未成熟B细胞。我们的研究发现，虽然ERK和PI3K都是必要的， 体外未成熟B细胞向过渡型B细胞的分化，仅体内B细胞中PI3 K的活化 1）消除中枢B细胞耐受性，包括受体编辑和克隆缺失，2） 支持自身反应性B细胞从骨髓中排出，和3）促进其进一步成熟， activation.综上所述，这些发现表明PI3K通路可以覆盖中枢神经系统， 小鼠自身反应性B细胞的外周耐受性，即使这些细胞对自身免疫应答具有高亲和力， 抗原的此授权的目标是定义PI3 K控制B的中心检查点的机制 小鼠中的细胞选择以及它是否在人B细胞中具有类似的功能。目标1和 2将利用小鼠耐受模型来研究FOXO 1，CXCR 4和ERK是否以及如何翻译 PI3 K在未成熟B细胞中的功能。非自身反应性B细胞和自身反应性B细胞的RNAseq分析， 断裂或闭合耐受性将有助于在该过程中发现另外的PI3K介体。患者 最近发现了PI3K β的功能获得性突变，它们经常表现出自身抗体， 其他自身免疫表现。目标3中的实验将采用两种互补且新颖的方法 确定PI3 κ B功能获得性突变破坏人B细胞中枢耐受性的能力。 总的来说，这些研究是重要的，因为它们将产生一个更深层次的机械理解， 原代B细胞库的形成，以及B细胞耐受性如何在肿瘤发生过程中被破坏， 自身反应性B细胞，提高原代B细胞库的自身反应能力，因此， 自身免疫风险。

PROJECT SUMMARY B cell tolerance is key to the development of an immune system tolerant to self, but the mechanisms that enforce this process are not yet clearly understood. The overarching goal of our studies is to elucidate the molecular pathways that regulate B cell selection at the developmental checkpoint between the immature and transitional cell stages, enforcing the process of tolerance in autoreactive bone marrow B cells while promoting the differentiation of nonautoreactive cells and their bone marrow exit. By studying Ig knock-in mouse models of central B cell tolerance we documented that the RAS, ERK, and PI3K signaling molecules, which are GTPases, MAP kinases, and lipid kinases respectively, are active to a higher basal level in nonautoreactive than autoreactive immature B cells. Our studies have found that while both ERK and PI3K are necessary for the in vitro differentiation of immature B cells into transitional B cells, only the in vivo activation of PI3K in B cells, but not that of ERK 1) abrogates central B cell tolerance, including receptor editing and clonal deletion, 2) supports the exit of autoreactive B cells from the bone marrow, and 3) promotes their further maturation and activation. Taken together, these findings establish that the PI3K pathway can override central and early peripheral tolerance in mouse autoreactive B cells, even when these cells possess high avidity for the self- antigen. The goal of this grant is to define the mechanisms by which PI3K controls this central checkpoint of B cell selection in mice and whether it similarly functions in human B cells. Experiments described in Aims 1 and 2 will utilize mouse models of tolerance to investigate whether and how FOXO1, CXCR4, and ERK translate PI3K function in immature B cells. RNAseq analyses of nonautoreactive B cells and of autoreactive B cells that either break or make tolerance will aid the discovery of additional PI3K mediators in this process. Patients with gain-of-function mutations in PI3K have been recently discovered, and they often exhibit autoantibodies and other autoimmune manifestations. Experiments in Aim 3 will utilize two complementary and novel approaches to determine the ability of gain-of-function mutations in PI3K to break central tolerance in human B cells. Overall, these studies are significant because they will generate a deeper mechanistic understanding of how the primary B cell repertoire is formed and how B cell tolerance can be breached during the development of autoreactive B cells, raising the autoreactive capacity of the primary B cell repertoire and, consequently, the risk for autoimmunity.