WAVE Regulatory Complex in Primary Immunodeficiency Disease and autoimmunity

项目总结 在迄今已确定的350多种人类原发免疫缺陷疾病中， 相当数量(~100)的PID的分子基础尚未确定。最近，有9个孩子(现在有4个 来自4个独立家庭的)被鉴定出患有严重的ID，这些ID与 NCKAP1L基因编码一种保守的造血细胞特异性造血蛋白-1(Hem-1) 组成波肌动蛋白调节复合体(WRC)。受影响的儿童出现严重的复发 呼吸道和皮肤感染，肺炎球菌免疫抗体反应失败(B 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同源基因在苍蝇和蠕虫中的分子功能得到了较好的表征，存在一个关键的 关于Hem-1在原发肿瘤的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定的角色来克服这种知识鸿沟 HEM-1在适应性和先天免疫细胞的发育和功能中的作用。这项提议的目标是 以B细胞特异性方式干扰原代小鼠和人B淋巴细胞Hem-1的表达 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 目的是在小鼠中利用可诱导的B细胞特异性基因打靶，并在小鼠中利用CRISPR/Cas9介导的hem1缺失 “人源化小鼠”，以验证我们的中心假设，即HEM1中B细胞特异性破坏会导致：(1)受损 B细胞发育的部分原因是发育中的B细胞前体细胞归巢和滞留减少 淋巴壁龛；(2)缺乏T细胞非依赖性抗体反应，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；以及 (3)高反应性B细胞信号和T-bet驱动的转录组，导致自身抗体增加 制作。为了证明可行性，我们生成了创新的鼠标模型来模拟HEM1 PID 患者包括在hem1(hem1pt/pt)、hem1缺失(hem1-/-)小鼠中具有非编码点突变的小鼠， Hem1开花(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。根据我们的初步结果，这有力地支持了我们的假设， 我们预计这些研究的结果将是非常重要的，将产生很大的影响，因为它们 将首次定义细胞和分子机制的功能丧失的变体如何在 NCLAP1L干扰B细胞发育、信号转导和保护性抗体介导的免疫导致PID 和自身免疫力。由于4个人类PID家族广泛的遗传异质性，有限数量的 患者和并发感染，这些创新的小鼠模型系统的开发对于 剖析Hem-1基因突变如何导致前列腺癌和自身免疫的细胞和分子机制， 并提供急需的平台，以开发和测试治疗和治愈HEM1缺乏症儿童的疗法。

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.