miRNA对γδT细胞发育关键调节因子的精细调控机制研究

As the first line of defense, innate-like lymphocytes γδ T cells critically involve in the tumor surveillance and defense, in which their unique epithelial tissue distribution is essential for proper functions. However, mechanisms regulating their tissue-specific development are poorly understood. Our previous research had elucidated that a TCR-strength dependent selection plays an important role in promoting acquisition of tissue-specific property, and the homing potential is intrinsically programmed even before the selection. Based on our observation that microRNA-185 (miR-185) negatively regulates the γδ T cell lineage transcriptional factor Sox13, we hypothesize that miR-185/Sox13 axis regulates the acquisition of unique tissue-specific homing properties for different sub-populations of γδ T cells during their intra-thymic development. The γδ T pregenitor cells with the genes of interest manipulated by lentivirus system would be reconstituted with fetal thymic lobes (FTOC assay) to study: 1. the contribution of miR-185/Sox13 axis to the γδ T cell development, 2. explore the regulating mechanism of miR-185/Sox13 axis. Combined with mining the downstream targets of miR-185/Sox13 axis from the high throughput databases, we would further dissect the acquisition mechanisms of unique tissue distribution in detail. All results generated as previously described would be confirmed in miR-185 overexpression and knockdown mouse models. This project would uncover the molecular regulation mechanisms of γδ T cells development and the acquisition of unique tissue specific homing properties, which would shed light on how to maneuver the tissue specific targeting of γδ T cells as clinical treatments.

类先天免疫细胞γδT细胞在脊椎动物多种免疫反应中起不可替代的作用，特定组织定位对其功能行使至关重要。γδT细胞如何获得组织特异定位能力尤其是miRNA参与的调控仍属未知。Sox13是γδT细胞标志性重要转录因子。我们通过对不同发育状态γδT细胞miRNA库的分析发现Sox13和miR-185负相关，结合定位不同组织γδT细胞亚群中miR-185的差异表达，提出"miR-185/Sox13轴精细调控γδ T细胞发育及组织定位"的假说。本课题拟采用慢病毒感染及胚胎胸腺器官培养系统，结合miR-185转基因及基因沉默小鼠，阐明该轴在γδT细胞发育中的关键地位；研究对该轴进行调控的上游网络及其下游调控的靶标，揭示不同信号强度下组织特异性定位的获得机制。同时将阐明包括miR-185在内的miRNA通过调控γδT细胞中的关键因子实现细胞发育迁移定位及功能精细调控的机制，为临床γδT细胞治疗提供新线索。

本项目以Vγ3+细胞和Vγ2+细胞为γδ T细胞胸腺发育研究模型，对发育成熟前后两种状态进行miRNA-seq分析，结果表明伴随成熟γδ T细胞中miRNA的种类增多； miR-21与Vγ3+细胞亚群特征相关； miR-150与miR-181与Vγ2+细胞的成熟相关；在比较得到γδ T细胞亚群相关、成熟相关和特异表达的miRNA特征谱的同时发现，相对mRNA 表达谱而言，miRNA在γδ T细胞两个亚群的胸腺成熟过程中表现出更为特异和显著的改变。