Transcriptional Regulation of Immune Cell Development, Activation and Functions

免疫细胞发育、激活和功能的转录调控

• 批准号: 10692163
• 负责人: Jinfang Zhu
• 金额: $ 198.07万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10692163
• 关键词: Address; Adoptive Transfer; Allergic Disease; Antigen Receptors; Asthma; Autoimmune Diseases; Autoimmunity; Bacteria; Binding; Biology; CD4 Positive T Lymphocytes; Cell Differentiation process; Cell Lineage; Cells; Complex; Deoxyribonuclease I; Development; Disease; Disease Resistance; Elements; Experimental Autoimmune Encephalomyelitis; FOXP3 gene; GATA3 gene; Gene Expression Profile; Gene Expression Regulation; Generations; Genes; Genetic Enhancer Element; Goals; Harvest; Health; Helminths; Helper-Inducer T-Lymphocyte; Heterogeneity; Homeostasis; Host Defense; Hypersensitivity; Immune; Immune response; Immunity; Immunization; Impairment; In Vitro; Infection; Inflammatory; Inflammatory Bowel Diseases; Interferons; Interleukin 2 Receptor; Interleukin 2 Receptor Gamma; Interleukin-12; Interleukin-13; Interleukin-17; Interleukin-18; Interleukin-4; Interleukin-5; Interleukins; Investigation; Lead; Lymphocyte; Lymphoid; Lymphoid Cell; Lymphoid Tissue; Manuscripts; Mediating; Modeling; Molecular; Mouse Strains; Multiple Sclerosis; Mus; Natural Killer Cells; Organogenesis; Papain; Parasites; Pathogenesis; Pathologic; Pathway interactions; Peyer' s Patches; Phase; Play; Population; Process; Production; RUNX3 gene; Regulation; Reporter; Reporting; Research; Rheumatoid Arthritis; Role; Signal Transduction; Site; Spinal Cord Diseases; T cell differentiation; T-Cell Development; T-Cell Receptor; TCF Transcription Factor; TSLP gene; Testing; Th1 Cells; Th2 Cells; Tissues; Transcriptional Regulation; Tumor Necrosis Factor-Beta; Virus; Work; ZNF145 gene; adaptive immune response; cell type; chronic infection; cytokine; effector T cell; epigenome; epigenomics; extracellular; fungus; helminth infection; immunoregulation; in vivo; interleukin-18 receptor; interleukin-22; lymph nodes; microorganism; mouse model; novel; ovalbumin-alum; pathogen; pathogenic microbe; progenitor; receptor-mediated signaling; response; stem; transcription factor; transcription regulatory network; transcriptome; transcriptome sequencing; transcriptomics

CD4+ T lymphocytes play a central role in orchestrating adaptive immune responses. After activation 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)-g, interleukin (IL)-4 and IL-17, respectively, as their signature effector cytokines. Through the production of these distinct effector cytokines, specific Th subsets mediate crucial functions during different types of protective immune responses to various microorganisms. Th1 cells are important for host defense against intracellular bacteria and viruses; Th2 cells for expelling extracellular parasites such as helminths; and Th17 cells for controlling extracellular bacteria and fungi. Inappropriate Th responses to pathogens may lead to chronic infection and/or tissue damage to the host, whereas aberrant Th cell differentiation may result in many inflammatory allergic or autoimmune diseases including asthma, inflammatory bowel diseases (IBD), rheumatoid arthritis (RA) and multiple sclerosis (MS). Innate lymphoid cells (ILCs), which lack expression of antigen receptors, require signaling through the IL-2 receptor (IL-2R) common gamma chain and IL-7Ra, for their development, maturation or homeostasis. Distinct ILC subsets mirror different Th cell subsets in their cytokine production. Therefore, ILCs are classified into type 1 innate lymphoid cells (ILC1s) that produce IFNg, type 2 innate lymphoid cells (ILC2s) that produce IL-5 and IL-13, and type 3 innate lymphoid cells (ILC3s) that produce IL-17 and IL-22. Although some ILCs, such as lymphoid tissue inducer (LTi) cells, are specifically critical for lymphoid organogenesis, most ILCs, like Th cells, are important for protective immune responses to infections and contribute to the pathogenesis of many 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 and TCR-mediate signaling. Our major research goal is to better understand the transcriptional regulatory networks and mechanisms that control differentiation processes leading to the distinct Th and ILC lineages. We have chosen to focus on the master regulators (also known as lineage-determining transcription factors, LDTFs) including T-bet, GATA3, and RORgt (for type 1, type 2 and type 3 lymphocytes, respectively), because we hypothesize that they are the major nodes in these networks. By comparing the regulation and actions of T-bet, GATA3 and RORgt in distinct Th and ILC lineages, we aim to identify new components and/or connections of these complex networks controlling Th cell differentiation and ILC development. Comparing gene regulation at the transcriptomic and epigenomic level between these two cell types will allow us to identify the core elements that determine their shared functionality and unique molecules/pathways that control their specialized functions. During the past fiscal year, we have reported a representative study focusing on the regulation of T-bet expression in type 1 lymphocytes to elucidate the similarities and differences in the induction of LDTFs in innate and adaptive lymphocytes (Immunity 55:639-655, 2022). In this manuscript, we showed that Th1 cells and NK cells displayed distinct epigenomes at the Tbx21 locus, which encodes T-bet, the LDTF for type 1 lymphocytes. The initial induction of T-bet in NK precursors was dependent on the NK-specific DNase I hypersensitive site Tbx21-CNS-3 and the expression of IL-18 receptor; IL-18 induced T-bet expression through RUNX3, which bound to Tbx21-CNS-3. By contrast, STAT-binding motifs within Tbx21-CNS-12 were critical for IL-12-induced T-bet expression during Th1 cell differentiation both in vitro and in vivo. Thus, type 1 innate and adaptive lymphocytes utilize distinct enhancer elements for their development and differentiation. In previous years, we have used T-bet-ZsGreen (or AmCyan)-RORgt-E2Crimson-Foxp3-RFP triple reporter mice to investigate the dynamic expression of T-bet and RORgt in EAE, a mouse model for MS. We found that RORgt-expressing cells can generate T-bet/RORgt dual-expressors as well as cells only expressing T-bet after adoptive transfer. RNA-Seq analysis of these subsets harvested from the spinal cord of the disease mice indicates that T-bet and RORgt each regulate (induce or inhibit) distinct and shared sets of genes, however, we did not observe any synergistic effect between T-bet and RORgt in gene regulation. Nevertheless, mice with conditional deletion of Rorc gene by T-bet-driven Cre were completely resistant to the disease induction. By generating other novel mouse models, we also demonstrated that a transition from expressing only RORgt to expressing both RORgt and T-bet, and finally to expressing only T-bet is a natural process in autoimmunity. In the past fiscal year, we have further demonstrated that the cells expressing only RORgt contain a stem-like population and have a better capacity in populating T-bet-expressing effector cells than T-bet-expressing cells themselves upon transfer. Thus, all three cell subsets (RORgt and T-bet single and dual expressors) play unique roles in the disease induction in EAE. We are now also testing this in IBD. We have previously reported that GATA3 serves as a switch in determining the development of LTi cells versus other ILC lineages (Immunity. 52: 83-95, 2020). While GATA3 is absolutely required for the generation of PLZF-expressing non-LTi progenitors, which express high level of GATA3, it is not necessary for the generation of RORt-expressing LTi progenitors consistent with low levels of GATA3 expression in these progenitors. In the past fiscal year, we further found that the transcription factor TCF-1, just as GATA3, was indispensable for the development of non-LTi ILC subsets. While LTi cells were still present in the TCF-1-deficient mice, the organogenesis of Peyers patches (PPs) but not of lymph nodes was impaired in these mice. LTi cells from different tissues had distinct gene expression patterns and TCF-1 regulated the expression of lymphotoxin specifically in PP LTi cells. Mechanistically, TCF-1 indirectly regulated Lta through promoting the expression of GATA3. Thus, the TCF-1-GATA3 axis, which plays an important role during T cell development, also critically regulates the development of non-LTi cells and tissue-specific functions of LTi cells. We continued our investigation on the relative importance and crosstalk between ILC2s and Th2 cells during type 2 immune responses by utilizing unique mouse strains that are specifically deficient in either ILC2s or Th2 cells. In previous years, we have found that IL-33-mediated ILC2 activation promoted Th2 cell differentiation in the papain model, however, Th2 cell differentiation was completely independent of ILC2s in the OVA/alum immunization model. On the other hand, Th2 cells induced the expression of IL-25, IL-33, and TSLP, which played a redundant role in promoting ILC2 expansion. During a helminth infection, ILC2s and Th2 cells participated in different phases of host defense and collaborated in promoting each others expansion. During the past fiscal year, we have further demonstrated that IL-4 produced by Th2 cells played a critical role in inducing type 2 alarmin expression. While IL-25 and IL-33 played a redundant role in activating ILC2s, TSLP was important for ILC2-independent Th2 cell response induced by OVA/alum immunization. In addition, pre-activation of ILC2s by either IL-33 or IL-25 in the ILC2-independent OVA model further promoted Th2 cell differentiation. Thus, alarmin- and IL-4-mediated crosstalk between ILC2s and Th2 cells varies in different type 2 immune responses in mice

CD4+ T 淋巴细胞在协调适应性免疫反应中发挥着核心作用。在特定细胞因子环境中通过 T 细胞受体 (TCR) 激活后，幼稚 CD4+ T 细胞分化成不同的 T 辅助细胞 (Th) 谱系，包括产生干扰素 (IFN)-g、白细胞介素 (IL) 的 Th1、Th2 和 Th17 细胞-4 和 IL-17 分别作为其标志性效应细胞因子。通过产生这些不同的效应细胞因子，特定的 Th 亚群在针对各种微生物的不同类型的保护性免疫反应期间介导关键功能。 Th1 细胞对于宿主防御细胞内细菌和病毒非常重要； Th2细胞用于排出细胞外寄生虫，如蠕虫； Th17细胞用于控制细胞外细菌和真菌。对病原体的不适当的 Th 反应可能会导致宿主的慢性感染和/或组织损伤，而异常的 Th 细胞分化可能会导致许多炎症过敏或自身免疫性疾病，包括哮喘、炎症性肠病 (IBD)、类风湿性关节炎 (RA) 和多发性关节炎 (RA) 等。硬化症（MS）。 先天性淋巴样细胞 (ILC) 缺乏抗原受体的表达，需要通过 IL-2 受体 (IL-2R) 共同伽马链和 IL-7Ra 进行信号传导，以实现其发育、成熟或体内平衡。不同的 ILC 子集反映了不同 Th 细胞子集细胞因子产生的情况。因此，ILC分为产生IFNg的1型先天淋巴细胞（ILC1s）、产生IL-5和IL-13的2型先天淋巴细胞（ILC2s）和产生IL-17的3型先天淋巴细胞（ILC3s）和IL-22。虽然一些 ILC，例如淋巴组织诱导 (LTi) 细胞，对于淋巴器官发生特别重要，但大多数 ILC，例如 Th 细胞，对于感染的保护性免疫反应非常重要，并有助于许多炎症性疾病的发病机制。 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和 TCR 介导的信号传导组合诱导和/或激活的。我们的主要研究目标是更好地了解控制分化过程的转录调控网络和机制，从而产生不同的 Th 和 ILC 谱系。我们选择关注主调节因子（也称为谱系决定转录因子，LDTF），包括 T-bet、GATA3 和 RORgt（分别针对 1 型、2 型和 3 型淋巴细胞），因为我们假设它们是这些网络中的主要节点。通过比较 T-bet、GATA3 和 RORgt 在不同 Th 和 ILC 谱系中的调节和作用，我们的目标是确定这些控制 Th 细胞分化和 ILC 发育的复杂网络的新组件和/或连接。比较这两种细胞类型在转录组和表观基因组水平上的基因调控将使我们能够确定决定它们共同功能的核心元件以及控制它们特殊功能的独特分子/途径。 在过去的财年中，我们报告了一项代表性研究，重点关注 1 型淋巴细胞中 T-bet 表达的调节，以阐明先天性和适应性淋巴细胞中 LDTF 诱导的异同（Immunity 55:639-655, 2022） ）。在这份手稿中，我们表明 Th1 细胞和 NK 细胞在 Tbx21 位点显示出不同的表观基因组，该位点编码 T-bet，即 1 型淋巴细胞的 LDTF。 NK 前体中 T-bet 的初始诱导依赖于 NK 特异性 DNase I 超敏位点 Tbx21-CNS-3 和 IL-18 受体的表达； IL-18 通过 RUNX3 诱导 T-bet 表达，RUNX3 与 Tbx21-CNS-3 结合。相比之下，Tbx21-CNS-12 内的 STAT 结合基序对于 Th1 细胞体外和体内分化过程中 IL-12 诱导的 T-bet 表达至关重要。因此，1 型先天性和适应性淋巴细胞利用不同的增强子元件进行发育和分化。 前几年，我们使用T-bet-ZsGreen（或AmCyan）-RORgt-E2Crimson-Foxp3-RFP三重报告小鼠来研究T-bet和RORgt在EAE（MS小鼠模型）中的动态表达。我们发现表达 RORgt 的细胞可以产生 T-bet/RORgt 双表达细胞，以及过继转移后仅表达 T-bet 的细胞。对从患病小鼠脊髓中收获的这些子集进行的 RNA-Seq 分析表明，T-bet 和 RORgt 各自调节（诱导或抑制）不同和共享的基因组，但是，我们没有观察到 T-bet 之间有任何协同效应和RORgt在基因调控中的作用。然而，通过 T-bet 驱动的 Cre 有条件删除 Rorc 基因的小鼠对疾病诱导完全具有抵抗力。通过生成其他新型小鼠模型，我们还证明从仅表达 RORgt 到同时表达 RORgt 和 T-bet，最后到仅表达 T-bet 的转变是自身免疫的自然过程。在上一财年，我们进一步证明，仅表达 RORgt 的细胞包含干细胞样群体，并且在转移后比表达 T-bet 的细胞本身具有更好的填充表达 T-bet 的效应细胞的能力。因此，所有三个细胞亚群（RORgt 和 T-bet 单表达和双表达）在 EAE 疾病诱导中发挥独特的作用。我们现在也在 IBD 中对此进行测试。 我们之前曾报道过，GATA3 作为决定 LTi 细胞与其他 ILC 谱系发育的开关 (Immunity. 52: 83-95, 2020)。虽然 GATA3 对于产生表达高水平 GATA3 的表达 PLZF 的非 LTi 祖细胞来说是绝对必需的，但对于产生与这些祖细胞中低水平 GATA3 表达一致的表达 RORt 的 LTi 祖细胞来说，GATA3 并不是必需的。在上一财年，我们进一步发现转录因子TCF-1和GATA3一样，对于非LTi ILC亚群的发育是不可或缺的。虽然 LTi 细胞仍然存在于 TCF-1 缺陷小鼠中，但这些小鼠中派耶氏淋巴结 (PP) 的器官发生受损，但淋巴结的器官发生并未受损。来自不同组织的LTi细胞具有不同的基因表达模式，TCF-1在PP LTi细胞中特异性调节淋巴毒素的表达。从机制上来说，TCF-1通过促进GATA3的表达间接调控Lta。因此，在T细胞发育过程中发挥重要作用的TCF-1-GATA3轴也关键调节非LTi细胞的发育和LTi细胞的组织特异性功能。 我们利用特别缺乏 ILC2 或 Th2 细胞的独特小鼠品系，继续研究 2 型免疫反应期间 ILC2 和 Th2 细胞之间的相对重要性和串扰。前几年我们发现木瓜蛋白酶模型中IL-33介导的ILC2激活促进Th2细胞分化，而OVA/明矾免疫模型中Th2细胞分化完全独立于ILC2。另一方面，Th2细胞诱导IL-25、IL-33和TSLP的表达，这在促进ILC2扩增中发挥了冗余作用。在蠕虫感染期间，ILC2 和 Th2 细胞参与宿主防御的不同阶段，并协作促进彼此的扩张。在过去的财年中，我们进一步证明了Th2细胞产生的IL-4在诱导2型警报素表达中发挥着关键作用。虽然 IL-25 和 IL-33 在激活 ILC2 中发挥着多余的作用，但 TSLP 对于 OVA/明矾免疫诱导的不依赖于 ILC2 的 Th2 细胞反应非常重要。此外，在不依赖ILC2的OVA模型中，IL-33或IL-25对ILC2的预激活进一步促进了Th2细胞分化。因此，警报蛋白和 IL-4 介导的 ILC2 和 Th2 细胞之间的串扰在小鼠的不同 2 型免疫反应中有所不同