The role of V(D)J recombination and CD45 isotype expression in human natural killer cell development

不同于T和B淋巴细胞中V(D)J元件的重组是产生各种T细胞(TCR)和免疫球蛋白(Ig)受体的关键过程，自然杀伤(NK)细胞被认为是没有克隆型受体的先天淋巴细胞。然而，在NK细胞中也观察到了TCR和Ig基因上的非生产性基因重排，这表明淋巴前体具有多系分化的潜力。V(D)J重组过程是由RAG1/2(“重组激活基因”)核酸内切酶靶向识别信号序列(RSS)切割相邻DNA而启动的。随后，DNA末端被非同源末端连接(NHEJ)途径的因子加工和连接。RAG1/2-FATE基因定位报告构建的人诱导多能干细胞(HiPSCs)体外诱导分化为NK细胞后，有3-6%的NK细胞表达RAG1/2，这一结果被IgH基因重排证实。具有RAG个体发育的NK细胞主要表达CD45dim/CD45RB，并表现为记忆样和终末分化的表型，并伴有DNA损伤反应(DDR)受损。而未见V(D)J重组的NK细胞由于CD45RA的优势表达而表达CD45bright。CD45RB亚型不仅存在于B细胞上，也存在于不同的记忆群体中的CD4+和CD8+T细胞上。虽然CD45dim/CD45RO+NK细胞在恶性肿瘤或严重感染患者的外周血中已有报道，但CD45亚型及其对NK细胞发育和功能的影响尚未被研究。我们打算在体外研究IPSC来源的NK细胞前体细胞的分化潜能与V(D)J重组和CD45亚型表达的关系。通过这些研究，我们旨在阐明V(D)J重组是否是NK细胞分化过程中的一个随机过程，或者具有碎屑命运个体发育的NK细胞是否具有特定的功能特性。此外，将对V(D)J重组缺陷引起的联合免疫缺陷患者、血癌患者以及异基因干细胞移植后CMV感染患者的NK细胞群进行成熟度、CD45亚型表达和功能的研究。

In contrast to T and B lymphocytes in which recombination of V(D)J elements is a crucial process for the generation of diversified T cell (TCR) and immunoglobulin (Ig) receptors, natural killer (NK) cells are considered as innate lymphocytes without clonotypic receptors. However, non-productive genetic rearrangements on TCR and Ig loci have also been observed in NK cells suggesting a multilineage differentiation potential of lymphoid precursors. The process of V(D)J recombination is initiated by RAG1/2 (“recombination activation genes”) endonucleases targeting recognition signal sequences (RSS) to cleave adjacent DNA. Subsequently, DNA ends are processed and joined by factors of the non-homologous end-joining (NHEJ) pathway. Differentiation of human induced pluripotent stem cells (hiPSCs) engineered by RAG1/2-fate mapping reporter into NK cells in vitro revealed an ontogeny of RAG1/2 expression in 3-6% of NK cells, which was confirmed by genomic rearrangements in the IGH locus. NK cells with RAG ontogeny expressed predominantly CD45dim/CD45RB and displayed a memory-like and terminally differentiated phenotype with impaired DNA damage response (DDR). In contrast, NK lymphocytes without traceable V(D)J recombination were found to be CD45bright due to the predominant expression of CD45RA. The isotype CD45RB has been reported on distinct memory populations of CD4+ and CD8+ T cells, as well as on B cells. Although CD45dim/CD45RO+ NK cells have been described in peripheral blood of individuals with malignancies or severe infections, CD45 isotypes and their impact on development and function have not been investigated on NK cells so far. We intend to study the differentiation potential of iPSC-derived NK cell precursors related to the occurrence of V(D)J recombination and CD45 isotype expression in vitro. With these investigations we aim to elucidate whether V(D)J recombination is a random process on the rather variable road of NK cell differentiation, or if NK cells with RAG-fate ontogeny contain specific functional properties. In addition, NK cell populations obtained from patients with combined immunodeficiencies due to V(D)J recombination defects, patients with blood cancers, and patients with CMV infection following allogeneic stem cell transplantation will be investigated in terms of maturation, CD45 isotype expression and function.