Transcriptional Regulation of Immune Cell Development, Activation and Functions

CD4T细胞在协调适应性免疫反应中起着核心作用。初始的CD4T细胞在特定的细胞因子环境中通过T细胞受体(TCR)被激活后，分化为不同的辅助性T细胞(Th)，包括Th1、Th2和Th17细胞，它们分别产生干扰素(IFN)-γ、白介素4(IL)-4和白介素17(IL-17)作为其标志性的效应细胞因子。这些细胞对于不同类型的微生物的免疫是必不可少的：Th1细胞对细胞内病毒和细菌感染的保护性免疫反应很重要；Th2细胞用于驱逐蠕虫等细胞外寄生虫；Th17细胞用于控制细胞外细菌和真菌。其他的CD4T细胞包括滤泡T细胞(TFH)和调节性T细胞(Treg)。TFH细胞是促进抗体应答的关键细胞，而Treg细胞由自然产生的调节性T细胞(NTregs)和诱导性调节性T细胞(ITregs)组成，负责维持免疫耐受和淋巴细胞动态平衡。对病原体不适当的Th反应可能会导致慢性感染和/或对宿主的组织损伤。同样，无害的环境或自身抗原不必要地激活Th1、Th17或Th2细胞会导致器官特异性自身免疫性疾病或过敏性炎症性疾病。 Th细胞的激活、分化和扩增受到特定转录因子的严格调控，这些转录因子是由细胞因子组合诱导和/或激活的。在Th1细胞分化过程中，IFNGamma和IL-12分别负责诱导T-bet基因表达和STAT4磷酸化；T-bet和STAT4对Th1细胞产生IFNGamma起关键作用。同样，IL-4和IL-2诱导的GATA-3表达和Stat5激活分别是Th2细胞分化和功能的两个关键因素。RORGammat/STAT3、Foxp3/Stat5和Bcl6/STAT3分别是Th17、Treg和Tfh细胞分化过程中的关键调控因子。然而，这些所谓的主要调节因子虽然是必要的，但可能不足以诱导特定的Th细胞表型。许多其他转录因子与这些关键因子形成网络，也在T细胞分化过程中发挥作用。此外，对于这些已知的转录因子，它们如何通过与顺式调控元件的结合来调节谱系特异性的基因表达和/或表观遗传修饰仍然是未知的。因此，识别转录调控网络的组成并研究它们的作用机制对于理解向不同Th细胞系分化的过程是至关重要的。 在过去的一年里，该单位的科学家报告了T-bet-ZsGreen Report(TBGR)小鼠品系，其中GFP忠实地反映了T-bet的表达。本研究通过与一些缺乏Th1关键因子的小鼠品系，如STAT4、IFNGammaRI和T-bet单独或联合杂交，研究了T-bet在体内对弓形虫感染的调节和作用。这些小鼠也被用于研究T-bet在其他免疫细胞中的调节和功能，如先天性淋巴细胞。 为了根据T细胞和ILC亚群的关键转录因子的表达来分离和研究它们，我们最近建立了一种新的小鼠模型，其中T-bet的表达由一个蓝色荧光蛋白(AmCyan)来指示。这个小鼠品系已经与其他转录因子报告小鼠杂交。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。 为了更好地了解T细胞分化中的转录调控网络，我们通过RNAseq(与赵克基的团队合作)在体外绘制了不同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社区的有用资源。我们已经分析了以长基因间非编码RNA(LincRNAs)为重点的数据集；所有数据都已存入GEO并准备发布。我们期待未来对我们研究中发现的lincRNAs的分析将揭示它们在T细胞发育、分化和免疫反应中的重要功能。此外，我们的数据集将成为研究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(ILCs)在对病原体的先天免疫反应和淋巴器官发育过程中起着关键作用。IL-7Ra+ILC亚群与T辅助(Th)细胞亚群相似，能产生独特的效应细胞因子。然而，IL-7ra+ILC发生和维持的分子调控尚未被剖析。我们发现GATA3对于所有IL-7Ra+ILC亚群的发育都是不可或缺的。与这一缺陷一致的是，GATA3条件性缺陷小鼠没有淋巴结，容易感染轮状柠檬酸杆菌(感染是与Yasmine Belkaid的小组合作进行的)。全基因组基因分析表明，GATA3在ILC2s和Th2细胞中调节一组相似的细胞因子和受体，对ILC2s的维持至关重要。因此，GATA3在建立和调节适应性和先天淋巴细胞方面起着平行的作用。这一发现揭示了转录因子GATA3控制的ILC和T细胞发育的共同基本原理。

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.