GENETIC, IMMUNOLOGIC AND MECHANISTIC BASIS OF HUMAN NK CELL DEFICIENCY

 描述(由申请人提供)：自然杀伤(NK)细胞缺陷(NKD)是最近认识到的原发免疫缺陷(PID)的一个子集，其中NK细胞缺陷代表主要的免疫学缺陷。这些疾病通常是严重的；患者对病毒感染和癌症的易感性极高，往往是致命的。到目前为止，只有三个NKD基因被鉴定出来，对它们干扰NK细胞数量和/或功能的生物学机制知之甚少。与此同时，对于那些受影响的人来说，没有好的治疗选择。十多年来，我们的实验室一直在为这些罕见但致命的疾病寻求遗传、免疫学和治疗方面的解决方案。在这一过程中，我们建立了强大的全球转诊基础，组建了一支由遗传学家、免疫学家和临床医生组成的专家团队，现在准备极大地促进我们对NKD以及NK细胞在健康和疾病中的作用的了解。通过两个目标，我们将定义与人类NKD有关的基因(目标1)，并通过密集的生物学特征确定它们的相关性(目标2)。在目标1中，我们使用整个外显子组测序和串联染色体微阵列来鉴定世界上最大的NKD患者队列中的NKD突变。我们已经使用这种方法确定了7个新的候选基因。在目标2中，我们将严格评估我们的候选NKD基因，利用精心设计的前沿免疫学实验进展，确定它们在NK细胞杀伤功能、发育成熟、受体谱系以及免疫调节和炎症功能中的作用。将使用的工具包括“金标准”51Cr释放杀伤试验、Western blots、shRNA敲除、使用CRISPR/CAS技术的基因敲除、基于扩展的多参数流式细胞术的表型和功能分析、裂解免疫突触的高分辨率和超分辨率定量、转录因子激活和裂解突触动力学的成像流式细胞术定量、以及CD34+造血干细胞的体外NK细胞分化。具体地说，我们将证明这些基因在体外NKD患者细胞、体外健康供体NK细胞和NK细胞系(“敲除”和“敲入”)以及患者来源的诱导多能干细胞(IPSC)和重建的人源化小鼠中导致NKD。此外，还将评估临床上可用的细胞因子对每个候选患者的潜在治疗益处。总体而言，我们的方法代表了基因发现、确认、生物验证、 和免疫学洞察力。我们相信，这些创新的方法与我们成熟的NK细胞和遗传专业知识以及特权的NKD患者管道相结合，将推动这一新兴领域取得有意义的诊断、治疗和基础科学进步。我们提供了我们发现的两个例子--ATP6V0A2和MCM10--的初步数据，作为拟议中的工作将如何实质性推进该领域的具体例子。

 DESCRIPTION (provided by applicant): Natural Killer (NK) cell deficiency (NKD) is a recently appreciated subset of primary immunodeficiency (PID) in which NK cell deficits represent the main immunological defect. These diseases are typically severe; patients suffer from profound and often lethal susceptibility to viral infection and cancer. To date, only three NKD genes have been identified and little is known of the biological mechanisms by which they interfere with NK cell numbers and/or function. At the same time, no good therapeutic options are available for those affected. For over a decade, our laboratory has sought genetic, immunologic, and therapeutic solutions to these rare, but deadly diseases. In the process, we have established a robust worldwide referral base, assembled an expert team of geneticists, immunologists and clinicians, and are now poised to dramatically advance our understanding of NKD and the role of NK cells in health and disease. Through two Aims, we will define genes responsible for human NKD (Aim 1), and determine their relevance through intensive biological characterization (Aim 2). In Aim 1, we use whole exome sequencing and tandem chromosomal microarray to identify NKD mutations in the world's largest cohort of NKD patients. We have already identified 7 novel gene candidates using this approach. In Aim 2, we will rigorously evaluate our candidate NKD genes, defining their roles in NK cell cytolytic functions, developmental maturation, receptor repertoire, and immunoregulatory and inflammatory function using a carefully thought out progression of cutting-edge immunologic experiments. Tools to be used include "gold-standard" 51Cr-release killing assays, Western blots, shRNA knockdown, gene knockout using CRISPR/Cas technology, extended multiparameter flow cytometry-based phenotypic and functional analysis, high- and super-resolution quantification of the lytic immunological synapse, imaging flow cytometry quantification of transcription factor activation and lytic synapse dynamics, and in vitro NK cell differentiation from CD34+ hematopoetic stem cells. Specifically, we will prove that these genes cause NKD in ex vivo NKD patient cells, ex vivo healthy donor NK cells and NK cell lines ("knock-outs" and "knock-ins"), and patient-derived induced pluripotent stem cells (iPSC) and reconstituted humanized mice. Additionally, the potential therapeutic benefit of clinically available cytokines will be evaluated for each candidate. Overall our approach represents iterative cycles of genetic discovery, confirmation, biological validation, and immunologic insight. We are confident that these innovative approaches, combined with our established NK cell and genetic expertise, and privileged NKD patient pipeline, will drive meaningful diagnostic, therapeutic, and basic science progress in this burgeoning field. We provide preliminary data from two examples of our discoveries, ATP6V0A2 and MCM10, as concrete examples of how the proposed work will substantively advance the field.