Immunodeficiency caused by mutations in LRRC8

描述（由申请人提供）：富含亮氨酸重复序列（LRR）蛋白对包括先天免疫在内的许多生物学过程至关重要，但对其在适应性免疫中的作用知之甚少。发现一名复发性感染、无丙种球蛋白血症和缺乏循环B细胞但T细胞数量正常的患者携带截短的LRRC 8（含LRR 8）细胞表面受体（7）。外显子3和4之间的断点，由于平衡易位导致截短LRRC 8？91/+35蛋白，其中91 C-末端a.a.的胞外结构域被35 a. a.由读通内含子3编码。小鼠CD 34+祖细胞表达LRRC 8？91/+35不能重建受照者的B细胞室。我们的初步数据显示，LRRC 8-/-小鼠的死亡率增加，生长发育迟缓，许多组织异常。胸腺发育严重受损，在DN 2和DN 3阶段之间出现主要阻滞，外周T细胞无法增殖或分泌IL-2以响应抗CD 3。我们假设LRRC 8激活促进T细胞发育的途径，例如Notch 1依赖性信号转导，但关闭B细胞发育。我们将测试LRRC 8对T细胞自主方式的T细胞发育和功能至关重要的假设。我们还将检验91 a.a.来自或/和添加35 a.a至LRRC 8的EC结构域导致突变体的组成型信号传导，其主要抑制B细胞发育。我们将1。通过对LRRC 8-/-小鼠中的T细胞和胸腺上皮细胞（TEC）进行详细的表型表征，检验LRRC 8对LRRC 8-/-小鼠中的T细胞发育至关重要的假设。2.测试LRRC 8-/- T细胞在发育和功能方面存在内在缺陷的假设。我们将评估LRRC 8-/-胸腺祖细胞的发育潜力，检查组成型活性Notch 1是否挽救LRRC 8-/- T细胞成熟阻滞，分析LRRC 8-/-TEC支持T细胞发育的能力，并确定在T细胞成熟的特定阶段条件性消融LRRC 8对T细胞发育和功能的影响。3.测试LRRC 8-/- T细胞在发育和功能方面存在内在缺陷的假设。我们将比较LRRC 8？91/+35 LRRC8？91和LRRC 8 +35突变体对细胞信号传导和T和B细胞从BM祖细胞发育的影响，我们将分析LRRC 8？91/+35敲入小鼠。这些研究的结果应加强我们对LRRC 8在正常免疫功能中的作用以及LRCC 8突变患者无丙种球蛋白血症的机制的理解。这些结果对原发性免疫缺陷、宿主防御性自身免疫和变态反应的研究具有重要意义。公共卫生相关性：富含亮氨酸重复序列（Leucine Rich Repeat，LRR）蛋白在多种生物学过程中起关键作用，例如细胞粘附、细胞运输、受体相互作用和细胞凋亡，但关于LRR蛋白在获得性免疫中的作用几乎一无所知。这项提议试图为我们的总体假设提供证据，即LRRC 8激活促进T细胞发育的途径，但关闭B细胞发育

DESCRIPTION (provided by applicant): Leucine Rich Repeat (LRR) proteins are pivotal to many biological processes including innate immunity, but little is known about their role in adaptive immunity. A patient with recurrent infections, agammaglobulinemia, and absence of circulating B cells, but normal numbers of T cells, was found to carry a truncated LRRC8 (LRR Containing 8) cell surface receptor (7). A breakpoint between exons 3 and 4 due to a balanced translocation resulted in a truncated LRRC8?91/+35 protein in which the 91 C-terminal a.a. of the extra-cellular domain were replaced by 35 a.a. encoded by read though intron 3. Mouse CD34+ progenitors that express LRRC8?91/+35 failed to reconstitute the B cell compartment in irradiated recipients. Our preliminary data show that LRRC8-/- mice have increased mortality, stunted growth and abnormalities in a number of tissues. Thymic development was severely impaired with a major block between DN2 and DN3 stage and peripheral T cells failed to proliferate or secrete IL-2 in response to anti-CD3. We postulate that LRRC8 activates a pathway, e.g. Notch1 dependent signal transduction, that promotes T cell development, but shuts off B cell development. We will test the hypothesis that LRRC8 is critical for T cell development and function in a T cell autonomous fashion. We will also test the hypothesis that the deletion of 91 a.a. from or/and addition of 35 a.a to the EC domain of LRRC8 results in constitutive signaling by the mutant, which dominantly inhibits B cell development. We will 1. Test the hypothesis that LRRC8 is critical for T cell development in LRRC8-/- mice by performing detailed phenotypic characterization of T cells and thymic epithelial cells (TECs) in LRRC8-/- mice. 2. Test the hypothesis that LRRC8-/- T cells have an intrinsic defect in development and function. We will assess the developmental potential of LRRC8-/- thymocyte progenitors, examine if constitutively active Notch1 rescues the LRRC8-/- T cell maturation block, analyze the ability of LRRC8-/- TECs to support T cell development and determine the effect of conditional ablation of LRRC8 at defined stages of T cell maturation on T cell development and function. 3. Test the hypothesis that LRRC8-/- T cells have an intrinsic defect in development and function. We will compare the effect of LRRC8?91/+35 LRRC8?91 and LRRC8+35 mutants on cell signaling and on T and B cell development from BM progenitors, and we will analyze lymphocyte development and function in LRRC8?91/+35 knock-in mice. The results of these studies should enhance our understanding of the role of LRRC8 in normal immune function and the mechanisms of agammaglobulinemia in the patient with the LRCC8 mutant. These results will have important implications for primary immunedeficiencies, host defense autoimmunity and allergy. PUBLIC HEALTH RELEVANCE: Leucine Rich Repeat (LRR) proteins are pivotal to a diversity of biological processes, e.g. cell adhesion, cellular trafficking, hormone-receptor interactions and apoptosis but almost nothing is known about the role of LRR proteins in adaptive immunity. This proposal seeks to provide evidence for our overall hypothesis that LRRC8 activates a pathway that promotes T cell development, but shuts off B cell development