Transcriptome and epigenome analysis of helper T cell specification and plasticit

描述（由申请人提供）：CD4+辅助性T （Th）细胞是自身免疫性疾病的关键介质，如关节炎、牛皮癣和狼疮。激活后，nave t细胞分化为效应细胞亚群，其特点是具有不同的基因表达和免疫调节功能。在过去的几年里，许多新的Th亚群被鉴定出来，包括Foxp3+调节性T细胞、促炎Th17细胞和T滤泡辅助（Tfh）细胞。这些细胞不仅在自身免疫性疾病中具有重要的正调控或负调控作用，而且还具有相互的发育调控作用。激活T细胞中的TGF2信号可诱导Foxp3的表达，而TGF2与IL-6或IL-21的结合可驱动Th17细胞分化。另一方面，我们最近报道了IL-6或IL-21在缺乏TGF2信号的情况下驱动Tfh细胞分化。STAT3是IL-6和IL-21下游的一种常见转录因子，如何与tgf2激活的SMAD转录因子在T细胞命运决定中相互作用尚不清楚。我们的数据表明，它们可能介导谱系特异性转录因子的上调，如Treg细胞中的Foxp3， Th17细胞中的ROR1/3和Tfh细胞中的Bcl6。虽然这些细胞特异性转录因子已经被其他人和我们证明在细胞谱系规范中是必不可少的，但它们的确切作用机制也不清楚，它们的交叉抑制功能也不清楚。建议的工作是基于我们在过去五年中做出重要贡献的成熟的多学科合作团队。该项目的中心目标是对Treg、Th17和Tfh细胞进行大规模的全基因组转录组和表观基因组分析，以确定关键转录因子的直接功能及其可能的激动和拮抗相互作用。我们的中心假设是，关键转录因子诱导的转录和表观遗传机制强调了新型Th谱系细胞的特异性和可塑性。为了验证这一假设，我们将对缺乏TGF2和STAT3信号通路或Foxp3、ROR1/3和Bcl6转录因子的T细胞进行转录组分析。此外，我们将对TGF2和STAT3信号通路或Foxp3、ROR1/3和Bcl6转录因子缺乏的T细胞进行全基因组表观基因组分析。第三，我们将对SMAD、STAT3、ROR3/1和Bcl6靶点进行全基因组染色质免疫沉淀测序分析。这些实验的结果将受到生物信息学分析，以了解谱系规范和可塑性。该项目是高度可行的，可以在两年的时间框架内完成，因为所有必要的试剂都在手中，方法也很好地建立了。拟议的项目将提供系统的见解，细胞谱系承诺和细胞可塑性。该研究通过分析积极或消极调节皮肤和风湿性自身免疫性疾病发病机制的关键细胞类型，符合NIAMS的使命。

DESCRIPTION (provided by applicant): CD4+ helper T (Th) cells are critical mediators in autoimmune diseases, such as arthritis, psoriasis and lupus. Upon activation, naove Th cells differentiate into effector cell subsets that are characterized by their distinct gene expression and immunoregulatory function. In the past several years, a number of novel Th subsets have been identified, including Foxp3+ regulatory T cells, pro-inflammatory Th17 cells and T follicular helper (Tfh) cells. These cells not only have been shown to be important positive or negative regulators in autoimmune diseases, but also share reciprocal developmental regulation. TGF2 signaling in activated T cells induces Foxp3 expression, while the combination of TGF2 and IL-6 or IL-21 drives Th17 cell differentiation. On the other hand, we recently reported that IL-6 or IL-21, in the absence of TGF2 signaling, drives Tfh cell differentiation. How STAT3, a common transcription factor downstream of IL-6 and IL-21, and TGF2-activated SMAD transcription factors interplay in T cell fate decision has not been understood. Our data suggest that they may mediate the upregulation of lineage-specific transcription factors, such as Foxp3 in Treg cells, ROR1/3 in Th17 cells and Bcl6 in Tfh cells. Although these cell-specific transcription factors have been shown by others and us to be essential in Th cell lineage specification, the exact mechanisms of their action have not been understood either, nor their cross-inhibitory functions. The proposed work is based on our well-established, multi-disciplinary collaborative team that has made important contributions in the past five years. The central goal of this project is to perform large-scale, genome-wide transcriptome and epigenome analysis of Treg, Th17 and Tfh cells to determine the direct functions of key transcription factors and their likely agonistic and antagonistic interactions. Our central hypothesis is that transcriptional and epigenetic mechanisms induced by key transcription factors underscore the specification and plasticity of novel Th lineage cells. To address this hypothesis, we will perform transcriptome analysis of T cells deficient in TGF2 and STAT3 signaling pathways or Foxp3, ROR1/3 and Bcl6 transcription factors. Moreover, we will perform genome-wide epigenome analysis of T cells deficient in TGF2 and STAT3 signaling pathways or Foxp3, ROR1/3 and Bcl6 transcription factors. Thirdly, we will perform genome-wide chromatin immunoprecipitation-sequencing analysis on the targets of SMAD, STAT3, ROR3/1 and Bcl6. The results of these experiments will be subject to bioinformatic analysis to understand Th lineage specification and plasticity. The project is highly feasible and can be accomplished within the two-year time frame as all the necessary reagents are in hand and methods are well established. The proposed project will provide systemic insights into Th cell lineage commitment and cellular plasticity. The study suits the mission of NIAMS by analyzing the critical cell types that positively or negatively regulate the pathogenesis of skin and rheumatic autoimmune diseases.