T-box家族转录因子Eomes和T-bet调控自然杀伤细胞发育和功能的机制研究

Natural killer (NK) cells development is sequentially regulated by several transcription factors, among which T-box transcription factors, mainly including Eomes and T-bet, are considered as key checkpoint for NK cell development and maturation. Germline deletion of Eomes or T-bet will lead to serious disruption of NK cell development. However, the mechanisms by which they regulate the development and function of NK cells, such as specific developmental stages and downstream target genes, remain controversial. Because NK cells can sequentially acquire stage-specific receptors along their maturation, we have generated two Cre mouse lines, CD122-Cre and Ncr1-Cre, which can delete certain genes at early and late stage of NK cell development, respectively. We preliminarily showed that Eomes and T-bet could co-regulate the NK cell survival in the early stage of NK cells, but played a different role in the terminal differentiation of NK cells, that is, T-bet promoted the maturation of NK cells, while Eomes inhibited this process. Through differential gene analysis, we found that Eomes and T-bet may differentially combine target genes (such as Myb and Zeb2) to promote cell survival or differentiation, and regulate the early development and differentiation of NK cells. The current application aims to further explore the role of Eomes and T-bet in the early development, terminal differentiation as well as function of NK cells. These studies will spatiotemporally reveal the molecular mechanism of the T-box family's regulation of NK cell development, and will provide the experimental basis for the basic biology of NK cells and tumor biotherapy.

自然杀伤（NK）细胞发育受到多种转录因子的时序性调控，其中T-box家族Eomes或T-bet胚系基因缺失会导致NK细胞发育严重受阻，但它们调节NK细胞发育和功能的机制（如具体发育阶段和下游靶基因）仍存在争议。我们前期通过制备两种NK细胞阶段特异性Cre小鼠（CD122-Cre和Ncr1-Cre），初步发现Eomes和T-bet联合调控早期NK细胞存活，但T-bet在分化末端促进NK细胞成熟，而Eomes则抑制该过程。通过差异基因分析，初步发现这两种转录因子可能通过靶向不同的影响细胞存活或分化的基因（如Myb和Zeb2），调节NK细胞早期发育和晚期分化。本课题拟进一步探讨该家族转录因子调控NK细胞早期发育、末端分化和功能的机制。通过这种时序性研究，有望在时空层面揭示T-box家族作为NK细胞发育稽查点蛋白的重要性及其分子机理，为NK细胞基础生物学和肿瘤生物治疗提供实验基础。

自然杀伤（NK）细胞是重要的固有免疫细胞，它们通过直接杀伤或分泌细胞因子的形式，清除恶性转化肿瘤细胞、病原体感染的细胞以及异基因骨髓移植细胞。在本课题中我们深入研究了T-box家族转录因子Eomes和T-bet对NK细胞发育和功能的时序性调控，阐明NK细胞发育分化过程中对转录因子有序取用的重要意义。同时我们进一步研究了T-box家族分子在限制性饮食调控的NK细胞抗肿瘤过程中作用；另外还增加了NK细胞中Eomes和T-bet活性调控的相关研究。实验结果证明T-box家族转录因子Eomes和T-bet在NK细胞发育的不同阶段发挥着不同的调控作用。在NK细胞发育的早期，Eomes的缺失导致未成熟的NK细胞比例升高，而在NK细胞末端缺失Eomes，则引起末端成熟的NK细胞比例升高。而T-bet在NK细胞末端缺失导致未成熟的NK细胞比例升高。说明Eomes和T-bet对NK细胞发育的调控作用主要是通过两者表达平衡的变化实现的。同时Eomes在NK细胞功能的执行过程中能够通过调控NK细胞与肿瘤细胞之间免疫突触的形成，从而影响NK细胞细胞因子的分泌以及杀伤作用。在限制性饮食调控的抗肿瘤过程中，NK细胞功能性亚群比例升高，NK细胞中Eomes的表达也升高，增强NK细胞的抗肿瘤能力。NK细胞中Eomes和T-bet的表达受到mTOR信号通路分子的调控。mTOR信号分子失活后，细胞因子不能很好的诱导NK细胞中Eomes的表达。该课题的顺利完成，系统的阐明了转录因子Eomes和T-bet对NK细胞发育和功能的调控作用。同时对利用限制性饮食的小鼠模型，将Eomes和T-bet对NK细胞发育和功能的调控作用进行了拓展。该研究将饮食调控跟机体固有免疫系统功能相联系，研究结果将会为饮食调控对免疫细胞尤其是固有免疫细胞功能影响的提供实验支持，对膳食结构调控对临床肿瘤治疗提供有效的实验线索。