Immunodeficiency caused by mutations in LRRC8

描述(申请人提供)：亮氨酸富含重复(LRR)蛋白是许多生物过程的关键，包括天然免疫，但对它们在适应性免疫中的作用知之甚少。1例反复感染，无丙种球蛋白血症，无循环B细胞，但T细胞数量正常的患者，被发现携带截短的LRRC8(LRR包含8)细胞表面受体(7)。由于平衡易位，外显子3和4之间的断裂点导致了截短的LRRC8？91/35蛋白，其中91C-末端A.A.的胞外区被35A.A.取代。表达LRRC8-91/35的小鼠CD34祖细胞不能重建受者的B细胞室。我们的初步数据显示，LRRC8-/-小鼠的死亡率增加，生长发育迟缓，许多组织中出现异常。胸腺发育严重受损，在DN2期和DN3期之间的主要障碍，外周T细胞无法增殖或分泌IL-2对抗CD3的反应。我们推测LRRC8激活一条途径，例如Notch1依赖的信号转导，促进T细胞发育，但关闭B细胞发育。我们将验证LRRC8对T细胞发育和T细胞自主功能至关重要的假设。我们还将检验这样一种假设，即91 A.A.的缺失。从LRRC8的EC结构域或/和向LRRC8的EC结构域添加35A.A会导致突变体产生结构性信号，从而主要抑制B细胞的发育。我们将1.通过对LRRC8-/-小鼠的T细胞和胸腺上皮细胞(TECs)进行详细的表型表征，来检验LRRC8-/-小鼠中LRRC8对T细胞发育至关重要的假设。2.验证LRRC8-/-T细胞在发育和功能上存在先天缺陷的假设。我们将评估LRRC8-/-胸腺细胞前体细胞的发育潜力，检查结构性活性Notch1是否拯救LRRC8-/-T细胞成熟块，分析LRRC8-/-TECs支持T细胞发育的能力，并确定在T细胞成熟的特定阶段有条件地切除LRRC8对T细胞发育和功能的影响。3.验证LRRC8-/-T细胞在发育和功能上存在先天缺陷的假设。我们将比较LRRC8、91/35、LRRC8、91和LRRC8-35突变体对细胞信号和骨髓祖细胞T、B细胞发育的影响，并分析LRRC8、91/35基因敲除小鼠的淋巴细胞发育和功能。这些研究的结果将加强我们对LRRC8在正常免疫功能中的作用以及LRCC8突变患者无丙种球蛋白血症的机制的理解。这些结果将对原发免疫缺陷、宿主防御自身免疫和变态反应具有重要意义。公共卫生相关性：富含亮氨酸重复序列(LRR)蛋白在多种生物学过程中起着关键作用，如细胞黏附、细胞转运、激素受体相互作用和细胞凋亡，但对LRR蛋白在适应性免疫中的作用几乎一无所知。这一建议试图为我们的总体假设提供证据，即LRRC8激活了一条促进T细胞发育但关闭B细胞发育的途径

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