NK细胞功能性罕见突变的筛选、鉴定及其在SLE中的作用机制研究

NK cells are important components of innate immunity, that serve as the first defense line of virus and tumor defense. Through the cytotoxic activity and the release of cytokines, NK cells have also been implicated in the modulation of T-cell and B-cell-mediated autoimmune disorders. Systemic Lupus Erythematosus (SLE) is a chronic, heterogenous autoimmune disease with multifactorial etiology. Diminished NK cell number and impaired NK cell cytotoxicity were shown in SLE patients, and correlated with clinical manifestations of SLE. However, the mechanism by which NK cell function was impaired remains unclear. At China-Australia Centre for Personalized Immunology (CACPI), we have identified 12 patient cohorts with rare or novel mutations in NK cell function-associated genes including CD226, NKTR and FASLG. In this project, we will further validate those identified variants using in vitro phenotypic or functional assays, as well as in vivo animal models through introducing those variants into mouse modes using the CRISPR/Cas 9 technique. Elucidating the genetic predisposition to NK cell disability allows a better understanding of NK cells to the development of SLE and in turn potentiates clinical significance.

NK细胞是固有免疫系统的核心成员，是宿主抵御病毒感染及细胞恶性转化的重要防线。通过细胞毒活性及细胞因子分泌，NK细胞也参与调节适应性免疫应答，在自身免疫病领域愈来愈受到关注。SLE是一种慢性、异质性自身免疫疾病，患者常出现NK细胞功能降低及数量下降，但机制尚未明晰。通过全基因组外显子测序技术，我们在12位中国SLE患者中发现了数个潜在的NK细胞功能性罕见突变，涉及NKTR、CD226、FASLG等基因，主要集中在细胞识别和杀伤通路。本项目将进一步利用CRISPR/Cas9基因编辑技术将患者突变或同源突变导入细胞系以及基因工程小鼠，利用FACS、细胞杀伤、抗体阻断、细胞清除及细胞过继转输等实验方法验证这些突变是否为致病性突变，研究这些突变通过何种机制影响NK细胞功能进而参与疾病发生。该项目的最终目的是阐明SLE与NK细胞的遗传相关性，为SLE机制研究及治疗方案提供新思路。

SLE是一种慢性、异质性自身免疫性疾病，患者常出现NK细胞功能降低及数量下降，但机制尚未明晰。在三年的研究时间里，课题组完成了近千例自身免疫病患者和家属的全基因外显子测序，并发现了数个潜在的NK细胞功能性罕见突变，涉及NKTR、CD226、FASLG、P2RY8等基因。我们进一步利用基因定点突变、过表达技术和CRISPR/Cas9编辑技术将患者的突变或同源突变导入细胞系及工程小鼠，通过FACS、荧光素酶报告法、蛋白磷酸化分析、免疫荧光、细胞迁移、细胞过继转输等实验方法，从蛋白的表达、稳定性和功能的发挥等层面筛选到三种P2RY8基因功能性罕见变异。项目还通过人群验证性实验，对患者PBMCs进行表型和功能分析，发现狼疮肾炎P2RY8蛋白表达水平较非肾炎病人显著降低，提示P2RY8通路缺陷广泛参与了狼疮疾病的发生发展。这一系列研究结果增强了人们对自身免疫性疾病风险遗传变异及致病机理的认知，为自身免疫性疾病的治疗提供了新的靶点和方向。

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