人Treg细胞FOXP3表达的不稳定性及其表观遗传学机制

The FOXP3+ regulatory T cells (Treg) constitute a subpopulation of CD4+ T cells, which possess suppressive activity and are pivotal for the maintenance of peripheral tolerance. Adoptive immunotherapy based on Treg cells is considered to be a promising immunosuppressive strategy for the induction of transplantation tolerance. The successful cell therapy using Treg cells, first of all, depends on how Treg cells are prepared as a pure population and how they can be maintained as a functionally stable population. However, the recent studies have showed that natural arising Treg cells was an unstable subpopulation and could down-regulate their lineage-de?ning marker FOXP3 and maybe covert to Th cells both in vivo and in vitro. We speculate that the losing of FOXP3 in Treg cells is resulted from the changes in the CpG methylation within FOXP3 gene and the modification of histones. Thus, in this study, we will isolate human FOXP3+ Treg cells with high purity and expand them in vitro to do some work as following. First, we will analyze the surface molecules, suppressive function and cytokine expression of FOXP3- Treg cells after losing FOXP3 expression to examine the instability of human natural Treg cells. Second, we will study the gene expression patterns in FOXP3-losing Treg cell to extend our understanding of this instability and reveal the underlying molecular mechanisms. Third, we will investigate epigenetic mechanisms of instability of Treg cells by assessing the degree of CpG methylation within FOXP3 gene and permissive histone modification H3K4me3 and repressive histone modification H3K27me3 as well as acetylation of histone H4. We will try to enucleate the instability of FOXP3 expression in Treg cells and the underlying epigenetic mechanism.

FOXP3+调节性T细胞（Treg）是一类具有免疫抑制功能的CD4+T细胞，Treg细胞的过继免疫治疗是诱导移植免疫耐受最具前景的手段之一。它的成功实施首先需获得足够、高纯度、功能稳定的Treg细胞。但新的研究显示Treg细胞在体内外会"自发"丢失FOXP3的表达，且存在向Th细胞转化的可能。我们推测这与逐渐发生的FOXP3基因的甲基化和组蛋白修饰的改变有关。为此，本课题拟：①分选人FOXP3+Treg细胞，分析FOXP3表达丢失后细胞表面标志分子、抑制功能和相关细胞因子的变化，阐明Treg细胞的不稳定性及FOXP3丢失后细胞的转化；②分析FOXP3丢失后基因表达谱的变化，筛选与FOXP3表达不稳定性相关基因；③分析基因甲基化、组蛋白H3K4me3、H3K27me3和H4Ac修饰在Treg细胞FOXP3表达丢失中的作用及机制。以此明确Treg细胞FOXP3表达的不稳定性及其表观遗传学机制。

FOXP3+调节性T细胞（Treg cells）在维持机体免疫耐受和免疫稳态中发挥着不可或缺的作用。Treg细胞的过继免疫治疗是诱导移植免疫耐受最具前景的手段之一。尽管这些研究结果引起了研究者极大的热情，但Treg细胞治疗方法仍面临着技术挑战，即如何在体外分离并扩增出功能稳定的Treg细胞群，并安全有效地用于体内治疗。为此，本项目探讨了人Treg细胞在体外扩增过程中的可塑性及表观遗传修饰在Treg细胞转化过程中的作用。结果发现，CD4+CD25highCD127low/- Treg细胞在体外培养中可转化为FOXP3+和FOXP3-两种亚群，数字基因表达谱分析结果显示体外扩增后人Treg细胞下调Treg特征基因表达，如FOXP3、CTLA4、ICOS、IKZF2和LRRC32，而上调了Th谱系相关基因，特别是Th2谱系相关基因，如GATA3、GFI1和IL13。通过ChIP-seq技术生成了FOXP3+和FOXP3-细胞的H3K4me3和H3K27me3修饰的全基因组图谱。结果显示，Treg细胞的转化与组蛋白修饰的改变有关，下调的Treg特征基因如FOXP3、CTLA4、LRRC32等上H3K4me3修饰水平降低，而上调的Th2谱系相关基因如IL4和IL5位点上H3K4me3修饰水平增高。然而，H3K27me3修饰在两群细胞间无明显差异。因此，本研究表明体外培养过程中人Treg细胞具有可塑性，FOXP3表达丢失可使其重塑为具有Th2谱系基因表达特征的Th样表型。同时，本研究首次生成了Treg细胞转化过程中的组蛋白修饰变化图谱，并发现组蛋白修饰可能介导了这一转化过程。这些结果为Treg细胞的可塑性提供了新的观点，对基于Treg的细胞免疫治疗研究具有重要意义。