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: 10927869
• 负责人: Jinfang Zhu
• 金额: $ 211.9万
• 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/10927869
• 关键词: Address; Adoptive Transfer; Affect; Alleles; Allergic Disease; Antigen Receptors; Asthma; Autoimmune Diseases; Autoimmunity; Bacteria; Biology; CD4 Positive T Lymphocytes; Cell Differentiation process; Cell Lineage; Cells; Classification; Collaborations; Communities; Complex; Data; Defect; Development; Disease; EGR2 gene; Elements; Exons; Experimental Autoimmune Encephalomyelitis; Failure; GATA3 gene; Gene Expression Profile; Gene Expression Regulation; Generations; Goals; Granulocyte-Macrophage Colony-Stimulating Factor; Health; Helminths; Helper-Inducer T-Lymphocyte; Heterogeneity; Homeostasis; Host Defense; Hypersensitivity; IL17 gene; IL27RA gene; Immune; Immune response; Immunity; Immunization; Immunology; Impairment; In Vitro; Infection; Inflammatory; Inflammatory Bowel Diseases; Interferon Type II; Interleukin 2 Receptor; Interleukin 2 Receptor Gamma; Interleukin-13; Interleukin-4; Interleukin-5; Interleukins; Lymphocyte; Lymphoid; Lymphoid Cell; Lymphoid Tissue; Maintenance; Mediating; Modeling; Molecular; Monitor; Mouse Strains; Multiple Sclerosis; Mus; Organogenesis; Ovalbumin; Papain; Paper; Parasites; Pathogenesis; Pathogenicity; Pathologic; Pathway interactions; Peyer' s Patches; Phase; Play; Population; Process; Production; Publishing; Regulation; Reporter; Reporting; Research; Rheumatoid Arthritis; Role; Signal Transduction; Site; Symptoms; System; T cell differentiation; T-Cell Development; T-Cell Receptor; TCF Transcription Factor; TSLP gene; Tamoxifen; Th1 Cells; Th2 Cells; Tissues; Transcriptional Regulation; Translation Initiation; Tumor Necrosis Factor-Beta; Virus; Visualization; Work; adaptive immune response; aluminum sulfate; cell type; chronic infection; cytokine; epigenomics; extracellular; frontier; fungus; helminth infection; immunoregulation; in vivo; inducible Cre; interleukin-22; lymph nodes; microorganism; mouse model; novel; pathogen; pathogenic microbe; protein expression; receptor-mediated signaling; response; tool; 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)-gamma, interleukin (IL)-4 and IL-17, respectively. Through the production of these distinct signature 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-7Ralpa, 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 IFNgamma, 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 RORgammat (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 RORgammat 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 published a paper entitled: Crosstalk between ILC2s and Th2 cells varies among mouse models (Cell Reports. 42:112073, 2023). In this study, we addressed the relative importance and crosstalk between Th2 cells and ILC2s during a variety of type 2 immune responses. By generating and utilizing mouse strains that are deficient in either ILC2s or Th2 cells, we report that IL-33-mediated ILC2 activation promotes the Th2 cell response to papain; however, the Th2 cell response to ovalbumin (OVA)/alum immunization is thymic stromal lymphopoietin (TSLP) dependent but independent of ILC2s. During helminth infection, ILC2s and Th2 cells collaborate at different phases of the immune responses. Th2 cells, mainly through IL-4 production, induce the expression of IL-25, IL-33, and TSLP, among which IL-25 and IL-33 redundantly promote ILC2 expansion. Thus, while Th2 cell differentiation can occur independently of ILC2s, activation of ILC2s may promote Th2 responses, and Th2 cells can expand ILC2s by inducing type 2 alarmins. 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). In the past fiscal year, we further reported that the transcription factor TCF-1, just as GATA3, was indispensable for the development of non-LTi ILC subsets (Cell Reports. 42: 112924, 2023). While LTi cells are still present in TCF-1-deficient mice, the organogenesis of Peyer's patches (PPs), but not of lymph nodes, is impaired in these mice. LTi cells from different tissues have distinct gene expression patterns, and TCF-1 regulates the expression of lymphotoxin specifically in PP LTi cells. Mechanistically, TCF-1 may directly and/or indirectly regulate Lta, including 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. GATA3 regulates the development of NKp46+ ILC3s which co-express T-bet and RORgammat (Nat. Immunol. 17: 169-178, 2016). Since T-bet and RORgammat co-expression is also found in differentiating CD4 T cells during EAE induction, we have also been studying the role of GATA3 during this process. As a result, during the past fiscal year, we reported that GATA3 induces the pathogenicity of Th17 cells via regulating GM-CSF expression (Frontiers in Immunology. 14:1186580, 2023). In this study, we found that Th17 cells dynamically expressed GATA3 during their differentiation both in vitro and in vivo. An early deletion of Gata3 limited the pathogenicity of Th17 cells during EAE, which was correlated with a defect in generating pathogenic T-bet-expressing Th17 cells; a late deletion of Gata3 in the adoptive transfer EAE model resulted in a cell intrinsic failure to induce EAE symptoms which was correlated with a substantial reduction in GM-CSF production without affecting the generation and/or maintenance of T-bet-expressing Th17 cells. RNA-Seq analysis revealed that GATA3 regulates the expression of Egr2, Bhlhe40, and Csf2. Thus, our data highlights a novel role for GATA3 in promoting and maintaining the pathogenicity of T-bet-expressing Th17 cells in EAE, via putative regulation of Egr2, Bhlhe40, and GM-CSF expression. To facilitate the research on ILC2s and Th2 cells in vivo, we generated novel GATA3 reporter mouse strains carrying the Gata3ZsG or Gata3ZsG-fl allele (Frontiers in Immunology. 13:975958, 2023). This was achieved by inserting a ZsGreen-T2A cassette at the translation initiation site of either the wild type Gata3 allele or the modified Gata3 allele which carries two loxP sites flanking the exon 4. ZsGreen faithfully reflected the endogenous GATA3 protein expression in Th2 cells and ILC2s both in vitro and in vivo. These reporter mice also allowed us to visualize Th2 cells and ILC2s in vivo. An inducible Gata3 deletion system was created by crossing Gata3ZsG-fl/fl mice with a tamoxifen-inducible Cre. Continuous expression of ZsGreen even after the Gata3 exon 4 deletion was noted, which allows us to isolate and monitor GATA3-deficient "Th2" cells and "ILC2s" during in vivo immune responses. These novel GATA3 reporters will provide valuable research tools to the scientific community in investigating type 2 immune responses in vivo.

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)-γ、白细胞介素 (IL) 的 Th1、Th2 和 Th17 细胞分别为-4和IL-17。通过产生这些独特的效应细胞因子，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) 共同伽玛链和 IL-7Ralpa 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IFNγ的1型先天淋巴细胞（ILC1）、产生IL-5和IL-13的2型先天淋巴细胞（ILC2）和产生IL-17的3型先天淋巴细胞（ILC3）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 RORgammat（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 RORgammat 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。 在上一财年，我们发表了一篇题为：ILC2s 和 Th2 细胞之间的串扰因小鼠模型而异的论文 (Cell Reports. 42:112073, 2023)。在这项研究中，我们探讨了各种 2 型免疫反应期间 Th2 细胞和 ILC2 之间的相对重要性和串扰。通过生成和利用 ILC2 或 Th2 细胞缺陷的小鼠品系，我们报告 IL-33 介导的 ILC2 激活促进 Th2 细胞对木瓜蛋白酶的反应；然而，Th2 细胞对卵清蛋白 (OVA)/明矾免疫的反应依赖于胸腺基质淋巴细胞生成素 (TSLP)，但独立于 ILC2。在蠕虫感染期间，ILC2 和 Th2 细胞在免疫反应的不同阶段进行协作。 Th2细胞主要通过IL-4的产生，诱导IL-25、IL-33和TSLP的表达，其中IL-25和IL-33冗余地促进ILC2扩增。因此，虽然 Th2 细胞分化可以独立于 ILC2 发生，但 ILC2 的激活可能会促进 Th2 反应，并且 Th2 细胞可以通过诱导 2 型警报素来扩增 ILC2。 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)。在上一财年，我们进一步报道了转录因子TCF-1，就像GATA3一样，对于非LTi ILC亚群的发育是不可或缺的（Cell Reports. 42: 112924, 2023）。虽然 LTi 细胞仍然存在于 TCF-1 缺陷小鼠中，但这些小鼠中派尔氏淋巴结 (PP) 的器官发生受到损害，但淋巴结的器官发生不受损害。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。从机制上讲，TCF-1可能直接和/或间接调节Lta，包括通过促进GATA3的表达。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。 GATA3 调节共表达 T-bet 和 RORgammat 的 NKp46+ ILC3 的发育（Nat.Immunol.17:169-178, 2016）。由于 T-bet 和 RORgammat 共表达也存在于 EAE 诱导期间分化 CD4 T 细胞中，因此我们也一直在研究 GATA3 在此过程中的作用。因此，在上一财年，我们报道了 GATA3 通过调节 GM-CSF 表达诱导 Th17 细胞的致病性（Frontiers in Immunology. 14:1186580, 2023）。在这项研究中，我们发现 Th17 细胞在体外和体内分化过程中动态表达 GATA3。 Gata3 的早期缺失限制了 EAE 期间 Th17 细胞的致病性，这与生成表达致病性 T-bet 的 Th17 细胞的缺陷相关；过继转移 EAE 模型中 Gata3 的后期缺失导致细胞内在无法诱导 EAE 症状，这与 GM-CSF 产生的大幅减少相关，但不影响表达 T-bet 的 Th17 细胞的产生和/或维持。 RNA-Seq 分析表明，GATA3 调节 Egr2、Bhlhe40 和 Csf2 的表达。因此，我们的数据强调了 GATA3 通过假定的 Egr2、Bhlhe40 和 GM-CSF 表达调节，在促进和维持 EAE 中表达 T-bet 的 Th17 细胞致病性方面的新作用。 为了促进体内 ILC2 和 Th2 细胞的研究，我们生成了携带 Gata3ZsG 或 Gata3ZsG-fl 等位基因的新型 GATA3 报告小鼠品系 (Frontiers in Immunology. 13:975958, 2023)。这是通过在野生型 Gata3 等位基因或修饰的 Gata3 等位基因的翻译起始位点插入 ZsGreen-T2A 盒来实现的，修饰的 Gata3 等位基因在外显子 4 侧翼带有两个 loxP 位点。ZsGreen 忠实地反映了 Th2 细胞和 ILC2 中的内源 GATA3 蛋白表达体外和体内。这些报告小鼠还使我们能够在体内观察 Th2 细胞和 ILC2。通过将 Gata3ZsG-fl/fl 小鼠与他莫昔芬诱导型 Cre 杂交，创建了诱导型 Gata3 缺失系统。即使在 Gata3 外显子 4 缺失后，ZsGreen 仍持续表达，这使我们能够在体内免疫反应期间分离和监测 GATA3 缺陷的“Th2”细胞和“ILC2”。这些新型 GATA3 报告基因将为科学界研究体内 2 型免疫反应提供有价值的研究工具。

项目成果

Tet2: breaking down barriers to T cell cytokine expression.

Tet2：打破 T 细胞细胞因子表达的障碍。

• 发表时间: 2015-04-21
• 影响因子: 32.4
• 作者: Zhong, Chao;Zhu, Jinfang
• 通讯作者: Zhu, Jinfang

Histone demethylases UTX and JMJD3 are required for NKT cell development in mice.

小鼠 NKT 细胞发育需要组蛋白去甲基酶 UTX 和 JMJD3。

• 发表时间: 2017
• 作者: Northrup, Daniel;Yagi, Ryoji;Cui, Kairong;Proctor, William R;Wang, Chaochen;Placek, Katarzyna;Pohl, Lance R;Wang, Rongfu;Ge, Kai;Zhu, Jinfang;Zhao, Keji
• 通讯作者: Zhao, Keji

T-bet-dependent NKp46+ innate lymphoid cells regulate the onset of TH17-induced neuroinflammation.

T-bet 依赖性 NKp46 先天淋巴细胞调节 TH17 诱导的神经炎症的发作。

• 发表时间: 2017-10
• 影响因子: 30.5
• 作者: Kwong, Brandon;Rua, Rejane;Gao, Yuanyuan;Flickinger Jr, John;Wang, Yan;Kruhlak, Michael J;Zhu, Jinfang;Vivier, Eric;McGavern, Dorian B;Lazarevic, Vanja
• 通讯作者: Lazarevic, Vanja

Seventeen-Year Journey Working With a Master.

与大师共事十七年。

• 发表时间: 2018
• 作者: Zhu; Jinfang
• 通讯作者: Jinfang

Redefining the Foreign Antigen and Self-Driven Memory CD4+ T-Cell Compartments via Transcriptomic, Phenotypic, and Functional Analyses.

通过转录组学、表型和功能分析重新定义外源抗原和自驱动记忆 CD4 T 细胞区室。

• 发表时间: 2022
• 作者: Kawabe, Takeshi;Ciucci, Thomas;Kim, Kwang Soon;Tayama, Shunichi;Kawajiri, Akihisa;Suzuki, Takumi;Tanaka, Riou;Ishii, Naoto;Jankovic, Dragana;Zhu, Jinfang;Sprent, Jonathan;Bosselut, Rémy;Sher, Alan
• 通讯作者: Sher, Alan