Functional and Epigenetic Analysis of Th2 Heterogeneity

T细胞的一个亚群，称为Th2细胞，是产生过敏免疫反应所必需的。Th2细胞表达三种标志性细胞因子，IL-4、IL-5和IL-13，每种细胞因子在过敏病理中都有特定的作用。IL-4促进Th2细胞分化，在抗体类向IgE转化过程中起主导作用，IL-5是主要的嗜酸性粒细胞活性细胞因子，IL-13通过上调黏附分子和趋化因子来驱动富含嗜酸性粒细胞的组织炎症。 我们假设Th2谱系内的异质性将产生具有不同细胞因子表达、效应器功能和在疾病发病机制中作用的Th2亚群。我们用流式细胞术检测IL-4、IL-5和IL-13，发现人Th2细胞主要由两个亚群组成：少数IL-5+(IL-5+、IL-4+、IL-13+)和多数IL-5-(IL-5-、IL-4+、IL-13+)Th2群。使用多种培养系统、抗原、检测系统和受试者群体，这些亚群在各种实验方法中的存在证明了这些发现对所有人类Th2反应的概括性。 分选的IL-5+和IL-5-Th2细胞在培养一周后保留了各自的IL-5偏向，表明这种不同的IL-5表达是这些Th2亚群的一种可遗传和持久的特性。通过在Th2极化条件下进行一系列的分化来检验这些亚群与Th2分化的关系。体外分化最初产生IL-5-Th2细胞，但需要多轮分化才能产生IL-5+Th2细胞。与IL-5-Th2表型相比，IL-5+的获得有一致且明显延迟。此外，IL-5+Th2细胞表达与其高度分化的T细胞一致的表型标志(PD-1+，CD27-)。这提示以慢性Ag暴露为特征的Th2显性疾病可能优先驱动IL-5+Th2细胞分化和嗜酸性炎症。 IL-5+表型在体外的持久性和延迟获得性表明，表观遗传学变化是导致IL-5表达差异的原因。IL-5+Th2细胞表达Th2系特异性转录因子GATA-3，提示IL-5基因表达受表观遗传控制。通过对纯化的IL-5+和IL-5-Th2亚群进行染色质免疫沉淀证实了这一点，与IL-5-Th2细胞相比，IL-5+中的GATA-3和H3K4me3与IL-5启动子的结合更强。相反，在IL-5-Th2细胞中，H3K27me3与IL5启动子的结合更强。综上所述，这些发现证明了IL5启动子中开放的染色质结构，这与IL-5+Th2细胞的较大IL-5表达是一致的。 综上所述，这些发现表明，Th2谱系的异质性是人类免疫反应的一个可概括和持久的特征。这些发现表明，IL-5+和IL-5-Th2细胞分别代表更多和更少的高度分化的Th2细胞亚群，每个亚群都具有不同的表型和表观遗传学特征。这种异质性的一个潜在后果是，特定的Th2亚群可能对Th2驱动的免疫病理有不同的贡献，因此可能代表不同的治疗靶点。 该项目已经完成，将在本财政年度(2012年9月30日)结束时终止。

A subset of T cells, termed Th2 cells, is required for the generation of allergic immune responses. Th2 cells express three signature cytokines, IL-4, IL-5, and IL-13, each of which makes a specific contribution to allergic pathology. IL-4 drives Th2 differentiation and plays a dominant role in antibody class switching to IgE, IL-5 is the major eosinophil active cytokine, and IL-13 drives eosinophil-rich tissue inflammation through its upregulation of adhesion molecules and chemokines. We hypothesized heterogeneity within the Th2 lineage would yield Th2 subpopulations with different cytokine expression, effector functions and roles in disease pathogenesis. We used polychromatic flow cytometry for IL-4, IL-5 and IL-13 to demonstrate that human Th2 cells are composed of two major subpopulations: a minority IL-5+ (IL-5+, IL-4+, IL-13+) and majority IL-5- (IL-5-, IL-4+, IL-13+) Th2 population. The generalizability of these findings to all human Th2 responses was demonstrated by the presence of these subpopulations across a wide variety of experimental approaches, using multiple culture systems, antigens, assay systems, and subject populations. Sorted IL-5+ and IL-5- Th2 cells retained their respective IL-5 bias after one week of culture, demonstrating that this differential IL-5 expression is a hereditable and durable property of these Th2 subpopulations. The relationship between these subpopulations and Th2 differentiation was examined by performing serial rounds of differentiation under Th2 polarizing conditions. In vitro differentiation initially yielded IL-5- Th2 cells, but required multiple rounds of differentiation to generate IL-5+ Th2 cells. There was a consistent and markedly delayed acquisition of the IL-5+ relative to the IL-5- Th2 phenotype. Additionally, IL-5+ Th2 cells expressed phenotypic markers (PD-1+, CD27-) consistent with their being highly differentiated T cells. This suggests the possibility that Th2 dominant diseases characterized by chronic Ag exposure may preferentially drive IL-5+ Th2 cell differentiation and eosinophilic inflammation. The durability and delayed acquisition of the IL-5+phenotype in vitro suggests that epigenetic changes underlie the differences in IL-5 expression. IL-5+ Th2 cells expressed greater GATA-3, the Th2 lineage specific transcription factor, suggesting epigenetic control of IL5 gene expression. This was confirmed by performing chromatin immunoprecipitation on purified IL-5+ and IL-5- Th2 subpopulations which demonstrated greater GATA-3 and H3K4me3 binding to the IL5 promoter in IL-5+ relative to IL-5- Th2 cells. Conversely, H3K27me3 binding to the IL5 promoter was greater in IL-5- Th2 cells. In sum, these findings demonstrate an open chromatin configuration in the IL5 promoter specific to IL-5+ Th2 cells, which is consistent with the greater IL-5 expression by this subpopulation. In summary, these findings demonstrate that Th2 lineage heterogeneity is a generalizable and durable feature of human immune responses. These findings establish that IL-5+ and IL-5- Th2 cells respectively represent more and less highly differentiated Th2 cell subpopulations, with each having distinct phenotypic and epigenetic features. A potential consequence of this heterogeneity is that specific Th2 subpopulations may differentially contribute to Th2 driven immunopathology, and as such may represent distinct therapeutic targets. This project has been completed and will be terminated at the end of the current fiscal year (September 30th, 2012).