Genetic Regulation of Natural Killer cell Homeostasis and Function

自然杀伤(Natural Killer，NK)细胞是一类具有细胞毒性的天然淋巴细胞，对肿瘤和病毒感染具有重要的保护作用。对罕见的患者和具有生殖系突变导致NK细胞缺陷的小鼠突变的研究已经阐明了NK细胞发育和功能的基本机制。值得注意的是，某些NK细胞耗竭突变会影响存在于每个细胞中的细胞机制，如DNA复制或核糖体组装。后者被称为核糖病，是由核糖体结构蛋白或成熟因子突变引起的遗传性疾病。核病症表现出典型的组织特异性效应，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。这项发现拨款将面临为什么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的问题，希望能阐明NK细胞发育和体内平衡要求的新分子决定因素。在最近对小鼠巨细胞病毒(MCMV)感染反应改变的全基因组化学(ENU)筛查中，我们发现了一个由于NK细胞特异性缺陷而对MCMV严重易感的家系。翻译GTP酶鸟嘌呤核苷酸结合蛋白样蛋白1(Gnl1)基因的一个隐性止盈突变(C567X)决定了NK细胞的特异性表型，其特征是：1)体内缺乏稳态增殖，细胞存活率降低；2)体外对稳态细胞因子IL-15的反应导致细胞周期受阻；3)在MCMV感染过程中出现炎性增殖。GNL1被预测编码一种核糖体组装因子，其功能障碍导致P53依赖的细胞死亡。对P53和Gnl1双缺陷小鼠的免疫隔间的特征表明，GnL1(C367X)在NK细胞数量和存活率方面的缺陷得到了充分的修复。基于这些结果，我们推测P53的激活是Gnl1基因缺陷小鼠体内NK细胞动态平衡异常和存活的驱动机制。为了验证这一假说，我们提出了一种多管齐下的方法，涉及1)使用骨髓嵌合体小鼠，并过继转移细胞分选细胞群，以确定Gnl1缺乏影响的免疫区段；2)通过检查胞浆和多聚体相关的转录产物，鉴定GnL1(C367X)NK细胞中被破坏的转录网络；3)筛选和验证GNL1结合伙伴，最终将GNL1定位在生化途径中。这项研究计划将加深我们对NK细胞发育和动态平衡的理解，同时产生关于核糖体相关蛋白组织特定功能的尖端结果。该建议将使研究生和博士后能够学习先进的免疫学、基因组和生化技术，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.