Transcriptional Regulation of Immune Cell Development, Activation and Functions

CD 4 T细胞在协调适应性免疫应答中发挥核心作用。在特定细胞因子环境中通过其T细胞受体（TCR）活化后，初始CD 4 T细胞分化成不同的T辅助（Th）谱系，包括分别产生干扰素（IFN）-γ、白细胞介素（IL）-4和IL-17作为其特征效应细胞因子的Th 1、Th 2和Th 17细胞。这些细胞对于针对各种微生物的不同类型的免疫是必不可少的：Th 1细胞对于针对细胞内病毒和细菌感染的保护性免疫应答是重要的; Th 2细胞用于驱逐细胞外寄生虫，如蠕虫;和Th 17细胞用于控制细胞外细菌和真菌。另外的CD 4 T细胞包括滤泡T细胞（Tfh）和调节性T细胞（Treg）。Tfh细胞对于促进抗体应答是至关重要的，而Treg细胞由天然存在的调节性T细胞（nT细胞）和诱导性调节性T细胞（iT细胞）组成，负责维持免疫耐受和淋巴细胞稳态。不适当的Th对病原体的反应可能导致慢性感染和/或对宿主的组织损伤。类似地，无害的环境抗原或自身抗原对Th 1、Th 17或Th 2细胞的不必要的激活可引起器官特异性自身免疫性疾病或过敏性炎性疾病。 Th细胞的激活、分化和扩增受到细胞因子组合诱导和/或激活的特定转录因子的严格调节。在Th 1细胞分化过程中，IFN γ和IL-12分别负责诱导T-bet基因表达和Stat 4磷酸化; T-bet和Stat 4对于Th 1细胞产生IFN γ至关重要。类似地，分别由IL-4和IL-2诱导的加塔-3表达和Stat 5活化是Th 2细胞分化和功能的两个关键要素。ROR γ mat/Stat 3、Foxp 3/Stat 5和Bcl-6/Stat 3分别是Th 17、Treg和Tfh细胞分化过程中的关键对应物。然而，这些所谓的主调节剂，虽然必要的，可能不足以诱导特定的Th细胞表型。与这些关键因子形成网络的许多其他转录因子也在T细胞分化过程中起作用。此外，对于这些已知的转录因子，它们如何通过与顺式调节元件结合来调节谱系特异性基因表达和/或表观遗传修饰仍然难以捉摸。因此，确定转录调控网络的组成部分，并调查其作用机制是必不可少的理解分化过程中不同的Th细胞谱系。 在过去的一年中，Unit科学家报告了一种T-bet-ZsGreen报告基因（TBGR）小鼠品系，其中GFP忠实地反映了T-bet的表达。在将TBGR与一些缺乏关键Th 1因子（如Stat 4、IFN γ RI和T-bet）的小鼠品系杂交后，我们研究了T-bet在体内对T.弓形虫感染这些小鼠也被用于研究T-bet在其他免疫细胞（如先天淋巴细胞）中的调节和功能。 为了根据关键转录因子的表达分离和研究T细胞和ILC亚群，我们最近产生了一种新的小鼠模型，其中T-bet表达由蓝色荧光蛋白（AmCyan）指示。该小鼠品系已与其他转录因子报告小鼠一起繁殖。这些小鼠也被转移到我们的合作者那里，用于研究其他免疫细胞亚群。 为了更好地理解T细胞分化中的转录调控网络，我们通过RNAseq（与Keji Zhao的小组合作）在体外分析了不同T细胞亚群在不同分化时间点的基因表达模式。这些数据集不仅指导我们未来的研究，而且也是整个免疫学研究界的有用资源。我们已经分析了专注于长基因间非编码RNA（LincRNA）的数据集;所有数据都已存入GEO并准备出版。我们希望未来对我们研究中鉴定的lincRNA的分析将揭示它们在T细胞发育、分化和免疫应答中的重要功能。此外，我们的数据集将作为研究T细胞发育和分化过程中转录调控网络的重要资源，通过比较蛋白质编码基因的动态表达，包括转录因子，细胞因子，趋化因子，细胞因子受体，趋化因子受体，其他细胞表面分子和信号分子。 先天性淋巴样细胞（inate lymphoid cells，ILC）在机体对病原体的先天性免疫应答和淋巴器官发育过程中起着重要作用。IL-7 Ra + ILC亚群，类似于T辅助（Th）细胞亚群，产生独特的效应细胞因子。然而，IL-7 Ra + ILC发展和维持的分子控制尚未被剖析。我们发现GATA 3对于所有IL-7 Ra + ILC亚群的发育是不可或缺的。与该缺陷一致，Gata 3条件性缺陷小鼠没有淋巴结并且对Citrobactor rodentium感染敏感（感染是与Yasmine Belkaid的小组合作进行的）。全基因组基因分析表明，GATA 3调节ILC 2和Th 2细胞中类似的一组细胞因子和受体，并且对ILC 2的维持至关重要。因此，GATA 3在建立和调节适应性淋巴细胞和先天性淋巴细胞中起着平行的作用。这一发现揭示了ILC和T细胞发育受转录因子GATA 3控制的共同基本原理。

CD4 T cells play a central role in orchestrating adaptive immune responses. After being activated through their T cell receptor (TCR) in a particular cytokine milieu, naive CD4 T cells differentiate into distinct T helper (Th) lineages, including Th1, Th2 and Th17 cells that produce interferon (IFN)-gamma;, interleukin (IL)-4 and IL-17, respectively, as their signature effector cytokines. These cells are indispensable for different types of immunity to various microorganisms: Th1 cells are important for protective immune responses to intracellular viral and bacterial infection; Th2 cells for expelling extracellular parasites such as helminths; and Th17 cells for controlling extracellular bacteria and fungi. Additional CD4 T cells include follicular T cells (Tfh) and regulatory T cells (Treg). Tfh cells are critical for promoting antibody responses, whereas Treg cells, which consist of natural-occurring regulatory T cells (nTregs) and inducible regulatory T cells (iTregs), are responsible for maintaining immune tolerance and lymphocyte homeostasis. Inappropriate Th responses to pathogens may lead to chronic infection and/or tissue damage to the host. Similarly, unnecessary activation of Th1, Th17 or Th2 cells by harmless environmental- or self-antigens can cause organ-specific autoimmune diseases or allergic inflammatory diseases. The activation, differentiation and expansion of Th cells are tightly regulated by specific transcription factors that are induced and/or activated by a combination of cytokines. During Th1 cell differentiation, IFNgamma and IL-12 are responsible for inducing T-bet gene expression and Stat4 phosphorylation, respectively; both T-bet and Stat4 are critical for Th1 cells to produce IFNgamma. Similarly, GATA-3 expression and Stat5 activation induced by IL-4 and IL-2, respectively, are the two key elements for Th2 cell differentiation and functions. RORgammat/Stat3, Foxp3/Stat5, and Bcl-6/Stat3 are the crucial counterparts during differentiation of Th17, Treg and Tfh cells, respectively. However, these so-called master regulators, although necessary, may not be sufficient to induce a specific Th cell phenotype. Many other transcription factors, forming a network with these key factors, also act in the process of T cell differentiation. In addition, for these known transcription factors, how they regulate lineage-specific gene expression and/or epigenetic modifications through their binding to cis-regulatory elements are still elusive. Therefore, identifying the components of transcriptional regulatory network and investigating mechanisms of their actions are essential for understanding the differentiation processes into distinct Th cell lineages. During the past year, Unit scientists reported a T-bet-ZsGreen reporter (TBGR) mouse strain, in which GFP faithfully reflects T-bet expression. After cross-breeding TBGR with a number of mouse strains deficient in key Th1 factors, such as Stat4, IFNgammaRI and T-bet, alone or in combination, we studied the regulation and functions of T-bet in vivo in responses to T. gondii infection. These mice are also being used for investigating T-bet regulation and functions in other immune cells such as innate lymphoid cells. In order to isolate and study T cell and ILC subsets based on their expression of key transcription factor, we have recently generated a new mouse model in which T-bet expression is indicated by a blue fluorescent protein (AmCyan). This mouse strain has been bred with other transcription factor reporter mice. The mice have also been transferred to our collaborators for studying other immune cell subsets. To better understand the transcriptional regulatory network in T cell differentiation, we have profiled the gene expression patterns of distinct T cell subsets at various differentiating time points in vitro via RNAseq (in collaboration with Keji Zhao's group). The datasets not only guide us in our future studies but also serve as a useful resource for the whole immunology research community. We have analyzed the datasets focusing on long intergenic non-coding RNAs (LincRNAs); all the data have been deposited to GEO and ready for publication. We expect that future analysis of the lincRNAs that are identified in our study will reveal their important functions in T cell development, differentiation and immune response. In addition, our dataset will serve as an important resource for studying transcriptional regulatory networks during T cell development and differentiation by comparing the dynamic expression of protein-coding genes including transcription factors, cytokines, chemokines, cytokine receptors, chemokine receptors, other cell surface molecules, and signaling molecules. Innate lymphoid cells (ILCs) play critical roles during innate immune responses to pathogens and lymphoid organ development. IL-7Ra+ ILC subsets, similar to T helper (Th) cell subsets, produce distinctive effector cytokines. However, the molecular control of IL-7Ra+ ILC development and maintenance has yet to be dissected. We found that GATA3 is indispensable for the development of all IL-7Ra+ ILC subsets. Consistent with this defect, Gata3 conditional deficient mice have no lymph nodes and are susceptible to Citrobactor rodentium infection (infection was done in collaboration with Yasmine Belkaid's group). Genome-wide gene analyses indicate that GATA3 regulates a similar set of cytokines and receptors in ILC2s and Th2 cells and is critical for the maintenance of ILC2s. Thus, GATA3 plays parallel roles in establishing and regulating both adaptive and innate lymphocytes. This finding reveals a common basic principle of ILC and T cell development controlled by transcription factor GATA3.