Mechanisms of Natural Killer cell expansion and neoplastic transformation

适应性淋巴细胞的典型特征之一是在遇到抗原时进行克隆扩增。自然杀伤细胞（NK）是先天淋巴细胞，缺乏重排受体，在响应病原体/危险信号或细胞因子时迅速激活。值得注意的是，人巨细胞病毒（CMV）感染诱导了肽特异性适应性NKG2C+ NK细胞亚群的扩增，这些细胞经历了全局转录和表观遗传重塑，类似于记忆细胞。在我们的初步数据中，我们执行了scRNAseq， scATACseq， CITE-seq以及线粒体DNA （mtDNA）突变分析，以跟踪CMV+和CMV-健康供者体外NK细胞的克隆性。该分析显示，与传统NK细胞池相比，CMV+供体中的人类记忆NKG2C+ NK细胞由稳定的克隆扩增组成，显示两种主要类型的开放染色质结构域：第一，CMV+供体中所有适应性NK细胞的共同特征（“公共记忆”）；其次，一组独特的开放染色质区域与个体的急剧扩张有关，稳定的NK细胞克隆（“私人记忆”）。基于这一意想不到的发现，我们假设克隆和与之相关的表观遗传特征可能有利于NK细胞的扩增，但可能使它们面临肿瘤转化的风险。有趣的是，NK细胞的异常增殖，最近被归类为慢性NK细胞增生性疾病（CLPD-NK），占成熟大颗粒淋巴细胞（LGL）疾病的15%，包括从淋巴细胞增多症到显性白血病的一系列疾病。CLPD-NK细胞扩增的病理生理机制、克隆程度和表观遗传重塑尚不清楚。在这个项目中，我们将结合scRNAseq， scATACseq， CITE-seq和mtDNA突变分析来研究健康CMV+和CMV-个体以及诊断为CLPD-NK的不同临床谱患者的NK细胞，以确定可能的共同表观遗传特征以及与来自健康供体和/或患者的常规NK细胞和记忆NK细胞的克隆关系。总之，该项目将揭示NK细胞生物学的新方面，以及CLPD-NK疾病的病理生理学，并为识别与疾病相关的新诊断和预后标志物开辟了新的视角。

One of the paradigmatic feature of adaptive lymphocytes is to undergo clonal expansion upon antigen encounter. Natural Killer (NK) cells are innate lymphocytes lacking rearranged receptors that are rapidly activated in response to pathogen/danger signals or cytokines. Remarkably, infection with human cytomegalovirus (CMV) induces the expansion of a subset of peptide-specific adaptive NKG2C+ NK cells undergoing global transcriptional and epigenetic remodeling, similar to memory cells. In our preliminary data, we have performed scRNAseq, scATACseq, CITE-seq as well as analysis of mitochondrial DNA (mtDNA) mutations to track clonality within human NK cells from CMV+ and CMV- healthy donors ex vivo. This analysis revealed that, in contrast to the conventional NK cell pool, human memory NKG2C+ NK cells in CMV+ donors comprise of stable clonal expansions, displaying two major types of open chromatin domains: first, a shared signature featured by all adaptive NK cells across CMV+ donors (“public memory”); second, a diverse set of unique open chromatin regions associated with the drastic expansions of individual, stable NK cell clones (“private memory”). Based on this unexpected finding, we hypothesize that clonality and the epigenetic features associated to it might be advantageous for NK cell expansion but might put them at risk of neoplastic transformation. Interestingly, abnormal proliferations of NK cells, more recently classified as chronic lymphoproliferative disease of NK cells (CLPD-NK), comprise 15% of mature large granular lymphocytes (LGL) disorders, a spectrum of conditions ranging from lymphocytosis to overt leukemia. The pathophysiological mechanisms underlying CLPD-NK cell expansions, the degree of their clonality and epigenetic remodeling remain unclear. In this project, we will combine scRNAseq, scATACseq, CITE-seq and mtDNA mutation analysis to study NK cells from healthy CMV+ and CMV- individuals as well as from patients with diagnosed CLPD-NK with different clinical spectra in order to identify possible common epigenetic features and clonal relationships with conventional and memory NK cells from healthy donors and/or within patients. Altogether, this project will shed light on new aspects of NK cell biology, as well as into the pathophysiology of CLPD-NK disorders, and opens a perspective on the identification of new diagnostic and prognostic markers associated to disease.