WAVE Regulatory Complex in Primary Immunodeficiency Disease and autoimmunity

项目概要 迄今为止已发现的超过 350 种人类原发性免疫缺陷病 (PID) 中， 大量（~100）PID 的分子基础尚未确定。最近，9 个孩子（现在 4 个） 来自 4 个独立家庭的死者）被鉴定患有严重的 PID，这些疾病与基因突变有关 NCKAP1L 基因编码造血蛋白-1 (Hem-1)，一种保守的造血细胞特异性蛋白 WAVE 肌动蛋白调节复合体 (WRC) 的组成部分。受影响的儿童出现严重的反复发作 呼吸道和皮肤感染、肺炎球菌免疫抗体反应失败（B 型病毒的特征） 细胞免疫缺陷）、细胞因子产生失调和自身免疫。尽管蜂窝和 Hem-1 直系同源物在果蝇和蠕虫中的分子功能已相对较好地表征，有一个关键的 关于 Hem-1 在初级细胞发育和功能中的细胞特异性功能的知识差距 免疫细胞。我们的长期目标是通过剖析细胞特异性作用来克服这一知识差距 Hem-1 在适应性和先天免疫细胞的发育和功能中的作用。该提案的目标是 以 B 细胞特异性方式破坏原代小鼠和人 B 淋巴细胞中的 Hem-1 表达，以定义 Hem-1 在 B 细胞发育、保护性体液免疫和自身免疫中的作用。我们的具体目标 将在小鼠中利用诱导性 B 细胞特异性基因靶向，并在小鼠中利用 CRISPR/Cas9 介导的 Hem1 缺失 “人源化小鼠”，以测试我们的中心假设，即 Hem1 中 B 细胞特异性破坏会导致：(1) 受损 B 细胞发育部分归因于发育中 B 细胞祖细胞归巢和保留的减少 淋巴生态位；(2) 缺乏 T 细胞独立抗体反应，导致保护性免疫受损 甲型流感病毒和肺炎链球菌是重要的社区获得性呼吸道病原体；和 (3) 高反应性 B 细胞信号传导和 T-bet 驱动的转录组，导致自身抗体增加 生产。为了证明可行性，我们生成了创新的小鼠模型来模拟 Hem1 PID 患者包括 Hem1 非编码点突变小鼠 (Hem1pt/pt)、Hem1 缺失 (Hem1-/-) 小鼠、 Hem1floxed (Hem1fl/fl) 小鼠，以及含有 Hem1 缺陷的 Hem1 缺陷“人源化小鼠” 原代人类造血细胞。基于我们的初步结果有力地支持了我们的假设， 我们预计这些研究的结果将非常重要并产生重大影响，因为它们 将首次定义功能丧失变异如何发生的细胞和分子机制 NCLAP1L 破坏 B 细胞发育、信号传导和保护性抗体介导的免疫，导致 PID 和自身免疫。由于 4 个人类 PID 家族存在广泛的遗传异质性，因此数量有限 患者和并发感染，这些创新小鼠模型系统的开发对于 剖析 Hem-1 突变如何导致 PID 和自身免疫的细胞和分子机制， 并提供急需的平台来开发和测试治疗和治愈 Hem1 缺陷儿童的疗法。

PROJECT SUMMARY Of the greater than 350 Primary Immunodeficiency Diseases (PID) in humans that have been identified to date, the molecular basis of a significant number (~100) of PIDs have yet to be defined. Recently, 9 children (4 now deceased) from 4 independent families were identified with severe PIDs that were linked to mutations in the NCKAP1L gene encoding for Hematopoietic protein-1 (Hem-1), a conserved hematopoietic cell-specific component the WAVE actin regulatory complex (WRC). Affected children presented with severe recurring respiratory and skin infections, failed antibody responses to pneumococcal immunization (characteristic of B cell immunodeficiency), dysregulated cytokine production, and autoimmunity. Although the cellular and molecular functions of Hem-1 orthologues in flies and worms are relatively well characterized, there is a critical knowledge gap regarding the cell specific functions of Hem-1 in the development and functions of primary immune cells. Our longterm goal is to overcome this knowledge gap by dissecting the cell-specific roles of Hem-1 in the development and functions of adaptive and innate immune cells. The objective of this proposal is to disrupt Hem-1 expression in primary murine and human B lymphocytes in a B cell-specific manner to define the roles of Hem-1 in B cell development, protective humoral immunity, and autoimmunity. Our Specific Aims are to utilize inducible B cell specific gene targeting in mice, and CRISPR/Cas9 mediated Hem1 deletion in “humanized mice”, to test our central hypotheses that B cell specific disruption in Hem1 results in: (1) impaired B cell development in part due to reduced homing and retention of developing B cell progenitors in essential lymphoid niches;(2) absent T cell independent antibody responses resulting in crippled protective immunity to influenza A virus and Streptococcus pneumoniae, important community acquired respiratory pathogens; and (3) hyper-responsive B cell signaling and T-bet driven transcriptome, resulting in increased autoantibody production. To demonstrate feasibility, we have generated innovative mouse models to emulate Hem1 PID patients including mice with a non-coding point mutation in Hem1 (Hem1pt/pt), Hem1 null (Hem1-/-) mice, Hem1floxed (Hem1fl/fl) mice, as well as Hem1 deficient ”humanized mice” which contain Hem1 deficient primary human hematopoietic cells. Based on our preliminary results which strongly support our hypotheses, we expect that the results of these studies will be highly significant and will have a high impact because they will define for the first time, the cellular and molecular mechanisms of how loss-of-function variants in NCLAP1L disrupt B cell development, signaling, and protective antibody-mediated immunity resulting in PID and autoimmunity. Because of extensive genetic heterogeneity of the 4 human PID families, limited number of patients, and concurrent infections, the development of these innovative mouse model systems are critical for dissecting the cellular and molecular mechanisms of how mutations in Hem-1 result in PID and autoimmunity, and to provide much needed platforms to develop and test therapies to treat and cure Hem1 deficient children.