Dissecting Hem-1 functions in B lymphocyte Development and Primary Immunodeficiency Disease

项目总结 原发免疫缺陷疾病是一组遗传性疾病，其免疫成分 信号通路要么缺失，要么功能失调，导致严重的反复感染，通常 自身免疫性疾病或癌症的进展。在迄今已确定的300多个ID中， 相当数量(~100)的PID的分子基础尚未确定。最近，有9个人(现在有4个 来自4个独立家庭的)被鉴定为与NCKAP1L突变有关的ID， 它编码一种保守的造血细胞特异性肌动蛋白调节蛋白，称为造血学 蛋白质-1(Hem-1)。受影响的儿童出现反复的呼吸道和皮肤感染，中耳炎， 肺炎球菌免疫的抗体反应受损(B细胞免疫缺陷的特征)， 细胞因子产生失调和自身免疫。尽管HEM-1的细胞和分子功能 苍蝇和蠕虫中的同源同源基因的特征相对较好，但关于 Hem-1在原代哺乳动物免疫细胞中的细胞特异性功能。我们的长期目标是克服这一点 通过剖析Hem-1在发育过程中细胞特异性细胞和分子功能的知识差距 适应性免疫细胞和先天免疫细胞的功能。这项建议的目标是在小学以HEM-1为目标 以B细胞特异性方式确定Hem-1在B细胞中的作用 发育、信号和保护性免疫。我们的具体目标是：(1)利用条件CRE-loxP 在小鼠中介导基因打靶以确定Hem-1中B细胞特异性功能丧失突变的改变 肺炎球菌和甲型流感的外周B细胞发育、抗体反应和保护性免疫 病毒，两种重要的社区获得性感染；(2)确定Hem-1突变对B细胞的影响 (3)在人源化小鼠中利用CRISPR/Cas9基因编辑来评估 Hem-1功能缺失突变对原代人B淋巴细胞动态发育的影响至 证明了可行性，我们已经在hem1(hem1pt/pt)、hem1中产生了非编码点突变的小鼠 空(hem1-/-)小鼠，hem1fl/fl(hem1fl/fl)小鼠，以及缺乏hem1的原代人造血干细胞 细胞。利用这些创新的老鼠和人体模型系统，我们将测试我们的总体假设B Hem-1的细胞特异性表达对边缘区和B1 B细胞、T细胞的发育至关重要。 独立的抗体反应和保护性B细胞免疫。这些研究非常有意义，因为 他们将利用创新的方法首次定义细胞和分子机制如何 NCLAP1L的非编码突变扰乱了B细胞的发育、信号传递和保护性体液免疫， 导致产后出血和潜在的自身免疫力。因为这4个人的PID具有广泛的遗传异质性 家庭，有限的患者数量，以及合并感染，细胞类型特异性的hem1的中断 方式对于剖析Hem-1突变如何导致PID和自身免疫的机制至关重要。

PROJECT SUMMARY Primary immunodeficiency diseases (PID) are group of inherited conditions where components of immune signaling pathways are either missing or are dysfunctional, resulting in severe recurring infections that often progress to autoimmune disease or cancer. Of the greater than 300 PIDs that have been identified so far, the molecular basis of a significant number (~100) of PIDs have yet to be defined. Recently, 9 individuals (4 now deceased) from 4 independent families were identified with PIDs that were linked to mutations in NCKAP1L, which encodes for a conserved hematopoietic cell-specific actin regulatory protein called Hematopoietic protein-1 (Hem-1). Affected children presented with recurring respiratory and skin infections, otitis media, impaired 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 primary mammalian immune cells. Our longterm goal is to overcome this knowledge gap by dissecting the cell-specific cellular and molecular functions of Hem-1 in the development and functions of adaptive and innate immune cells. The objective of this proposal is to target Hem-1 in primary murine and human B lymphocytes in a B cell-specific manner to define the roles of Hem-1 in B cell development, signaling, and protective immunity. Our Specific Aims are to: (1) utilize conditional Cre-LoxP mediated gene targeting in mice to define how B cell specific loss-of-function mutations in Hem-1 alter peripheral B cell development, antibody responses, and protective immunity to Pneumococcus and Influenza A virus, two important community acquired infections; (2) determine the impact of Hem-1 mutations on B cell signaling and transcription; (3) utilize CRISPR/Cas9 gene editing in “humanized mice” to assess the impact of loss-of-function mutations in Hem-1 on the dynamic development of primary human B lymphocytes. To demonstrate feasibility, we have generated mice with a non-coding point mutation in Hem1 (Hem1pt/pt), Hem1 null (Hem1-/-) mice, Hem1floxed (Hem1fl/fl) mice, as well as Hem1 deficient primary human hematopoietic stem cells. Utilizing these innovative mouse and human model systems, we will test our overall hypothesis that B cell specific expression of Hem-1 is essential for the development of marginal zone and B1 B cells, T- independent antibody responses, and protective B cell immunity. These studies are highly significant because they will utilize innovative approaches to define for the first time, the cellular and molecular mechanisms of how non-coding mutations in NCLAP1L disrupt B cell development, signaling, and protective humoral immunity, resulting in PID and potentially autoimmunity. Because of extensive genetic heterogeneity of the 4 human PID families, limited number of patients, and concurrent infections, the disruption of Hem1 in cell-type specific manner is critical for dissecting the mechanisms of how mutations in Hem-1 result in PID and autoimmunity.