Spatiotemporal Regulation of KLF2 and T-bet by Eomesodermin During NK Cell Development

I. Research Background

Natural Killer (NK) cells are important cells in the immune system, capable of recognizing and killing tumor cells and pathogen-infected cells. The generation and development of NK cells depend on the gradual acquisition of a series of specific receptors, a process strictly regulated by multiple transcription factors, including Eomesodermin (Eomes) and T-bet from the T-box transcription factor family. However, the specific spatiotemporal regulatory mechanism of Eomes in NK cell development remains unclear.

Eomes and T-bet are important regulatory factors for NK cell development and function. Eomes is mainly expressed in immature NK cells, affecting the early lineage differentiation of NK cells, while T-bet plays a dominant role in mature NK cells. Existing studies suggest that Eomes and T-bet may play different roles at different developmental stages, but the specific regulatory mechanisms still need further investigation.

II. Paper Source

This paper was collaboratively completed by Junming He, Donglin Chen, Wei Xiong, Xinlei Hou, Yuhe Quan, Meixiang Yang, and Zhongjun Dong, from the First Affiliated Hospital of Anhui Medical University and Institute of Clinical Immunology, Tsinghua University School of Medicine, Jinan University Zhuhai Institute of Translational Medicine, and other top domestic laboratories. The paper was published in the journal “Cellular & Molecular Immunology”, with an online publication date of May 13, 2024.

III. Research Process

1. Research Methods and Procedures

1.1 Construction of in vivo Eomes deficiency models

The study established Eomes-deficient mouse models to investigate the effects of Eomes on different stages of NK cell development. Specifically: - Using CD122-Cre transgenic mice to construct mice with NK progenitor (NKP) stage-specific Eomes deficiency (Eomes^f/f/CD122^Cre/+). - Using NCR1-Cre transgenic mice to construct mice with mature NK cell stage-specific Eomes deficiency (Eomes^f/f/NCR1^Cre/+).

1.2 NK cell sorting and analysis

Flow cytometry was used to sort NK cells at different developmental stages and analyze cell differentiation, proliferation, and receptor expression. Chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-seq technologies were used to analyze Eomes binding sites on chromosomes with target genes (such as Klf2 and T-bet) and their effects on transcriptional levels.

1.3 Viral infection and bone marrow reconstruction experiments

Using CRISPR technology, specific regions of Klf2 were edited, and a mouse model expressing Klf2 was constructed using a retroviral system to evaluate its compensatory effect on NK cell development under Eomes-deficient conditions.

2. Main Results and Data Support

2.1 Effects of Eomes on NK cell development at different stages

The study found that Eomes deficiency at the NKP stage leads to impaired NK cell maturation, while Eomes deficiency at the mature NK cell stage promotes terminal maturation of NK cells. Specific findings include: - High expression of Eomes in NKP and CD27+CD11b- (CD27 SP) immature NK cells, and low expression in CD27+CD11b+ (DP) and CD27-CD11b+ (CD11b SP) mature NK cells. - After in vivo specific deletion of Eomes, the proportion and absolute number of NK cells in Eomes^f/f/CD122^Cre/+ mice decreased significantly, with reduced numbers of CD3-NK1.1+ NK cells in tissues such as spleen and bone marrow. - The developmental arrest of NK cells caused by Eomes deficiency mainly manifests in the transition from NKP to immature NK cells.

2.2 Role of Eomes in regulating Klf2 during early NK cell development

RNA-seq and ChIP-seq data showed that Eomes regulates Klf2 expression by directly binding to its promoter region. Klf2 deficiency leads to NK cell developmental defects similar to Eomes deficiency: - Clonal immune editing (CHIME) and CRISPR technology verified that specific site editing of Klf2 produces phenotypic effects similar to Eomes deficiency. - Quantitative RT-PCR (qRT-PCR) results showed significantly reduced Klf2 expression in NK cells of Eomes-deficient mice.

2.3 Antagonistic regulation of Eomes and T-bet in NK cell terminal maturation

In the terminal maturation stage of NK cells, Eomes and T-bet antagonistically regulate each other: - ChIP-seq data showed that Eomes directly binds to and inhibits the T-bet gene, while T-bet promotes terminal differentiation of NK cells by regulating downstream effector Zeb2. - In the Eomes^f/f/NCR1^Cre/+ mouse model, Eomes deficiency led to upregulation of T-bet and Zeb2 expression in NK cells, reflecting the release of Eomes inhibition on T-bet.

3. Research Value and Significance

3.1 Scientific Value

This study reveals the spatiotemporal regulatory mechanism of Eomes in NK cell development, comprehensively analyzing the role of Eomes at different developmental stages through two genetic mouse models. This discovery fills a gap in understanding the interaction mechanisms of important transcription factors in NK cell development.

3.2 Application Value

Through a deeper understanding of the mechanism by which Eomes regulates NK cell development, new ideas can be provided for immunotherapy and treatment of NK cell-related diseases. For example, intervening in the Eomes-Klf2 or Eomes-T-bet pathways could become a method to promote NK cell function or expansion.

3.3 Research Highlights and Innovation

• Used multiple genetic models, including CD122-Cre and NCR1-Cre transgenic mice, as well as CRISPR technology, to interpret in detail the function of Eomes at different developmental stages.
• Systematically analyzed Eomes’ direct target genes and its transcriptional regulatory network by combining multiple technical approaches such as ChIP-seq and RNA-seq.

IV. Conclusion

Through the in-depth analysis of this study, we not only revealed the complex and crucial role Eomes plays in NK cell development but also provided new perspectives for research on immune system development and function. The spatiotemporal regulation and interaction mechanisms of Eomes and T-bet suggest that we can explore new targets and methods in future immunotherapy strategies to improve the precision and effectiveness of treatments.