Genetic Regulation of Natural Killer cell Homeostasis and Function

自然杀伤（NK）细胞是一群细胞毒性先天淋巴细胞，在保护免受肿瘤和病毒感染方面起着至关重要的作用。对罕见患者和具有导致NK细胞缺陷的生殖系突变的小鼠突变体的研究已经阐明了NK细胞发育和功能的基本机制。值得注意的是，某些NK细胞耗竭突变影响每个细胞中存在的细胞机制，如DNA复制或核糖体组装。后者被称为核糖体病，这是由核糖体结构蛋白或成熟因子突变引起的遗传性疾病。核糖体病表现出特有的组织特异性效应，部分原因是p53介导的细胞死亡途径可以被游离核糖体蛋白激活。这项发现补助金将面临的问题是，为什么NK细胞似乎比其他淋巴细胞更容易受到核糖体缺陷的影响，希望阐明NK细胞发育和稳态要求的新分子决定因素。在最近的全基因组化学（ENU）屏幕改变小鼠巨细胞病毒（MCMV）感染的反应，我们已经确定了一个家系显示严重的易感性MCMV由于NK细胞特异性缺陷。翻译型GTP酶鸟嘌呤核苷酸结合蛋白样1（Gnl 1）基因中的隐性停止增益突变（C567 X）决定了NK细胞特异性表型，其特征为1）体内缺乏稳态增殖和细胞存活率降低; 2）对离体稳态细胞因子IL-15的响应阻断细胞周期; 3）MCMV感染期间的炎性增殖。GNL 1被预测为编码核糖体组装因子，其功能障碍导致p53依赖性细胞死亡。p53和Gnl 1双缺陷小鼠的免疫区室的表征显示了Gnl 1（C367 X）在NK细胞数量和存活率方面的缺陷的完全拯救。基于这些结果，我们假设p53激活是Gnl 1缺陷小鼠体内稳态和存活中NK细胞异常的驱动机制。为了验证这一假设，我们提出了一种多管齐下的方法，包括1）使用骨髓嵌合小鼠，和过继转移细胞分选的细胞群，以确定受Gnl 1缺陷影响的免疫区室; 2）通过使用RNA测序检查胞质和多核糖体相关转录物，鉴定Gnl 1（C367 X）NK细胞中被破坏的转录网络; 3）筛选和验证GNL 1结合配偶体以最终在生化途径中抑制GNL 1。 这项研究计划将进一步加深我们对NK细胞发育和稳态的理解，同时产生关于核糖体相关蛋白组织特异性功能的尖端结果。该提案将使一名研究生和一名博士后研究员能够学习先进的免疫学、基因组学和生物化学技术，从而为培养高素质人才提供理想的环境。

Natural Killer (NK) cells are a population of cytotoxic innate lymphocytes with a crucial role in protection against tumors and virus infections. Studies of rare patients and mouse mutants with germ line mutations leading to NK cell deficiencies have illuminated fundamental mechanisms of NK cell development and function. It is notable that certain NK cell depleting mutations impact on cell mechanisms present in every cell such as DNA replication or ribosome assembly. The latter are known as ribosomopathies, which are genetic diseases caused by mutation in ribosome structural proteins or maturation factors. Ribosomopathies show characteristically tissue specific effects, in part due to the p53-mediated cell-death pathway that can be activated by free ribosomal proteins. This discovery grant will confront the issue of why NK cells appear to be more susceptible than other lymphocytes to defects in ribosomes hoping to illuminate new molecular determinants of NK cell development and homeostatic requirements. In a recent genome-wide chemical (ENU) screen for altered response to mouse cytomegalovirus (MCMV)infection, we have identified a pedigree showing severe susceptibility to MCMV due to NK cell-specific deficiency. A recessive stop-gain mutation (C567X) in the translational GTPase guanine nucleotide binding protein-like 1 (Gnl1) gene dictated an NK cell-specific phenotype characterized by 1) lack of homeostatic proliferation and decreased cell survival in vivo; 2) blockade of cell cycle in response to the homeostatic cytokine IL-15 ex vivo; 3) inflammatory proliferation during MCMV infection. GNL1 is predicted to encode a ribosome assembly factor the dysfunction of which results in p53-dependent cell death. Characterization of the immune compartment in p53 and Gnl1 doubly-deficient mice showed a full rescue of Gnl1(C367X) deficits in NK cell numbers and survival. Based on these results we hypothesized that p53 activation is a driving mechanism for NK cell abnormalities in homeostasis and survival in Gnl1-deficient mice. To test this hypothesis, we propose a multipronged approach involving 1) the use of bone marrow chimera mice, and adoptive transfer of cell sorted cell populations, to determine the immunological compartments affected by Gnl1 deficiency; 2) the identification of transcriptional networks disrupted in Gnl1(C367X) NK cells by examining cytosolic and polysome-associated transcripts, using RNA sequencing; 3) the screening and validating GNL1 binding partners to ultimately situate GNL1 in a biochemical pathway. This research program will further our understanding of NK cell development and homeostasis while producing cutting-edge results about tissue-specific functions of ribosome-associated proteins. The proposal will enable a graduate student and a post-doctoral fellow to learn advanced immunological, genomic and biochemical techniques, thus providing an ideal setting for training of highly qualified personnel.