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

在T淋巴细胞和B淋巴细胞中，V(D)J元素的重组是生成多样化T细胞（TCR）和免疫球蛋白（Ig）受体的关键过程，而自然杀伤细胞（NK）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TCR和Ig位点的非生产性基因重排，这表明淋巴样前体具有多谱系分化潜力。V(D)J重组过程是由RAG1/2（“重组激活基因”）内切酶靶向识别信号序列（RSS）裂解邻近DNA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命运定位报告基因介导的人诱导多能干细胞（hiPSCs）在体外向NK细胞分化的过程中发现，3-6%的NK细胞中RAG1/2的表达在个体发生，IGH位点的基因组重排证实了这一点。具有RAG个体发生的NK细胞主要表达CD45dim/CD45RB，表现出记忆样和终末分化表型，DNA损伤反应（DDR）受损。相比之下，由于CD45RA的主要表达，没有可追溯的V(D)J重组的NK淋巴细胞被发现是CD45bright的。同种型CD45RB已被报道存在于CD4+和CD8+ T细胞以及B细胞的不同记忆群体中。虽然CD45dim/CD45RO+ NK细胞已在恶性肿瘤或严重感染个体的外周血中被描述，但CD45同型及其对NK细胞发育和功能的影响迄今尚未被研究。我们打算在体外研究ipsc衍生的NK细胞前体与V(D)J重组和CD45同型表达相关的分化潜力。通过这些研究，我们旨在阐明V(D)J重组是NK细胞分化道路上的随机过程，还是具有RAG-fate个体发生的NK细胞具有特定的功能特性。此外，从V(D)J重组缺陷引起的联合免疫缺陷患者、血癌患者和同种异体干细胞移植后的巨细胞病毒感染患者中获得的NK细胞群将在成熟、CD45同型表达和功能方面进行研究。

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.