WAVE Regulatory Complex in Primary Immunodeficiency Disease and autoimmunity

项目摘要 在迄今为止已确定的超过350种人类原发性免疫缺陷病（PID）中， 大量（约100种）PID的分子基础还有待确定。最近，9名儿童（目前有4名 来自4个独立家庭的10名患者（已死亡）被确定患有严重的PID，这些PID与基因突变有关。 NCKAP 1 L基因编码造血蛋白-1（Hem-1），一种保守的造血细胞特异性转录因子， WAVE actin regulatory complex（WRC）。受影响的儿童出现严重的复发性 呼吸道和皮肤感染，对肺炎球菌免疫的抗体应答失败（B的特征 细胞免疫缺陷）、失调的细胞因子产生和自身免疫。虽然细胞和 Hem-1直向同源物在蝇和蠕虫中的分子功能相对较好地表征， 关于Hem-1在原发性肝癌的发展和功能中的细胞特异性功能的知识缺口 免疫细胞我们的长期目标是通过解剖细胞特异性作用来克服这一知识差距， Hem-1在适应性和先天性免疫细胞的发育和功能中的作用。本提案的目的是 以B细胞特异性方式破坏原代鼠和人B淋巴细胞中的Hem-1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 在小鼠中利用诱导型B细胞特异性基因靶向，在小鼠中利用CRISPR/Cas9介导的Hem 1缺失。 “人源化小鼠”，以测试我们的中心假设，即Hem 1中的B细胞特异性破坏导致：（1）受损 B细胞发育的部分原因是由于发育中的B细胞祖细胞的归巢和滞留减少， （2）缺乏T细胞非依赖性抗体应答，导致对 甲型流感病毒和肺炎链球菌，重要的社区获得性呼吸道病原体;以及 (3)高反应性B细胞信号传导和T-bet驱动转录组，导致自身抗体增加 生产为了证明可行性，我们已经生成了创新的小鼠模型来模拟Hem 1 PID 患者，包括Hem 1非编码点突变小鼠（Hem 1 pt/pt），Hem 1缺失小鼠（Hem 1-/-）， Hem 1floxed（Hem 1fl/fl）小鼠，以及含有Hem 1缺陷的Hem 1缺陷“人源化小鼠”， 原代人类造血细胞根据我们的初步结果，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，并将产生很大的影响，因为它们 将首次定义功能丧失变异体的细胞和分子机制， NCLAP 1 L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家族的广泛遗传异质性， 患者和并发感染，这些创新的小鼠模型系统的发展是至关重要的 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Hem 1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.