Functional and Epigenetic Analysis of Th2 Heterogeneity

T细胞的一个子集，称为Th 2细胞，是产生过敏性免疫反应所必需的。Th 2细胞表达三种标志性细胞因子，IL-4、IL-5和IL-13，每种细胞因子对过敏性病理都有特异性贡献。IL-4驱动Th 2分化并在抗体类别转换为IgE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通过其上调粘附分子和趋化因子驱动富含嗜酸性粒细胞的组织炎症。 我们假设Th 2谱系内的异质性将产生具有不同细胞因子表达、效应子功能和在疾病发病机制中的作用的Th 2亚群。我们使用IL-4、IL-5和IL-13的多色流式细胞术证明人Th 2细胞由两个主要亚群组成：少数IL-5+（IL-5+、IL-4+、IL-13+）和多数IL-5-（IL-5-、IL-4+、IL-13+）Th 2群体。这些发现对所有人Th 2应答的普遍性通过使用多种培养系统、抗原、测定系统和受试者群体在各种实验方法中存在这些亚群来证明。 分选的IL-5+和IL-5-Th 2细胞在培养一周后保留了它们各自的IL-5偏好，证明这种差异性IL-5表达是这些Th 2亚群的可遗传和持久的性质。通过在Th 2极化条件下进行连续轮分化来检查这些亚群与Th 2分化之间的关系。体外分化最初产生IL-5-Th 2细胞，但需要多轮分化以产生IL-5+ Th 2细胞。存在相对于IL-5-Th 2表型的IL-5+的一致且显著延迟的获得。此外，IL-5+ Th 2细胞表达表型标志物（PD-1+，CD 27-），这与它们是高度分化的T细胞一致。这表明以慢性Ag暴露为特征的Th 2优势疾病可能优先驱动IL-5+ Th 2细胞分化和嗜酸性粒细胞炎症。 体外IL-5+表型的持久性和延迟获得表明表观遗传变化是IL-5表达差异的基础。IL-5+ Th 2细胞表达更高的加塔-3，Th 2谱系特异性转录因子，表明IL 5基因表达的表观遗传控制。通过对纯化的IL-5+和IL-5-Th 2亚群进行染色质免疫沉淀证实了这一点，相对于IL-5-Th 2细胞，纯化的IL-5+和IL-5-Th 2亚群显示出更大的加塔-3和H3 K4 me 3与IL-5启动子的结合。相反，在IL-5-Th 2细胞中，H3 K27 me 3与IL 5启动子的结合更大。总之，这些发现证明了IL-5+ Th 2细胞特异性的IL-5启动子中的开放染色质构型，这与该亚群更高的IL-5表达一致。 总之，这些发现表明，Th 2谱系异质性是人类免疫应答的普遍和持久的特征。这些发现证实IL-5+和IL-5-Th 2细胞分别代表了分化程度较高和较低的Th 2细胞亚群，每个亚群具有不同的表型和表观遗传特征。这种异质性的一个潜在后果是，特定的Th 2亚群可能对Th 2驱动的免疫病理学有不同的贡献，因此可能代表不同的治疗靶点。

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.