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

V(D)J重组和CD45同种型表达在人类自然杀伤细胞发育中的作用

• 批准号: 531214878
• 负责人: Privatdozentin Dr. Kerstin Felgentreff
• 金额: --
• 依托单位: Universitätsklinik für Kinder- und Jugendmedizin
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/531214878?language=en
• 关键词: role; recombination; CD45; isotype; expression

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.

T和B淋巴细胞中V（D）J元件的重组是产生多样化T细胞（TCR）和免疫球蛋白（IG）受体的关键过程，与T和B淋巴细胞相反，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TCR和IG基因座上的非生产性遗传重排，表明淋巴前体的多谱系分化潜力。V（D）J重组的过程由RAG 1/2（“重组激活基因”）核酸内切酶启动，其靶向识别信号序列（RSS）以切割相邻的DNA。随后，DNA末端被加工并通过非同源末端连接（NHEJ）途径的因子连接。通过RAG 1/2-命运作图报告基因工程化的人诱导多能干细胞（hiPSC）在体外分化为NK细胞，揭示了在3-6%的NK细胞中RAG 1/2表达的个体发生，这通过IGH基因座中的基因组重排证实。具有RAG个体发生的NK细胞主要表达CD 45 dim/CD 45 RB，并显示出记忆样和终末分化的表型，具有受损的DNA损伤反应（DDR）。相反，发现没有可追踪的V（D）J重组的NK淋巴细胞由于CD 45 RA的主要表达而为CD 45 bright。同种型CD 45 RB已被报道在不同的记忆群体的CD 4+和CD 8 + T细胞，以及在B细胞。虽然CD 45 dim/CD 45 RO + NK细胞已在恶性肿瘤或严重感染个体的外周血中被描述，但迄今为止尚未研究CD 45同种型及其对NK细胞发育和功能的影响。我们打算在体外研究与V（D）J重组的发生和CD 45同种型表达相关的iPSC衍生的NK细胞前体的分化潜力。通过这些研究，我们旨在阐明V（D）J重组是否是NK细胞分化的可变道路上的随机过程，或者具有RAG-命运个体发生的NK细胞是否包含特定的功能特性。此外，还将研究从V（D）J重组缺陷导致的联合免疫缺陷患者、血癌患者和异基因干细胞移植后CMV感染患者中获得的NK细胞群的成熟、CD 45同种型表达和功能。