Immunodeficiency caused by mutations in LRRC8

描述（由申请人提供）：Leucine Rich Repeat （LRR）蛋白对包括先天免疫在内的许多生物过程至关重要，但对其在适应性免疫中的作用知之甚少。一个反复感染，无球蛋白血症，缺乏循环B细胞，但T细胞数量正常的患者，被发现携带截断的LRRC8 （LRR Containing 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断LRRC8？在91/+35蛋白中，细胞外结构域的91个c端a.a.被可读内含子3编码的35个a.a.所取代。小鼠CD34+祖细胞表达LRRC8？91/+35不能重建受者的B细胞区室。我们的初步数据显示，LRRC8-/-小鼠死亡率增加，生长发育迟缓，许多组织出现异常。胸腺发育严重受损，主要是DN2和DN3阶段之间的阻滞，外周T细胞不能增殖或分泌IL-2以响应抗cd3。我们假设LRRC8激活了一个通路，例如Notch1依赖的信号转导，促进T细胞的发育，但关闭B细胞的发育。我们将验证LRRC8在T细胞自主方式下对T细胞发育和功能至关重要的假设。我们还将验证一个假设，即从LRRC8的EC结构域删除91个a.a或/和添加35个a.a导致突变体的组成信号传导，这主要抑制B细胞的发育。我们会的。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，验证LRRC8对LRRC8-/-小鼠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内在缺陷的假设。我们将评估LRRC8-/-胸腺细胞祖细胞的发育潜力，检查组成性活性Notch1是否能挽救LRRC8-/- T细胞成熟阻滞，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条件消融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内在缺陷的假设。我们将比较LRRC8？91 / + 35 LRRC8吗?91和LRRC8+35突变体对BM祖细胞信号传导和T细胞和B细胞发育的影响，我们将分析LRRC8？91/+35敲入小鼠。这些研究结果将有助于我们理解LRRC8在正常免疫功能中的作用，以及LRCC8突变体患者无球蛋白血症的机制。这些结果将对原发性免疫缺陷、宿主防御自身免疫和过敏具有重要意义。公共卫生相关性：亮氨酸富重复（LRR）蛋白对多种生物过程至关重要，例如细胞粘附、细胞运输、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。该建议旨在为我们的总体假设提供证据，即LRRC8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