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: 8946531
• 负责人: Jinfang Zhu
• 金额: $ 80.29万
• 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/8946531
• 关键词: Address; Allergic; Animal Model; Animals; Area; Autoantigens; Autoimmune Diseases; Autoimmune Process; CD4 Positive T Lymphocytes; CD8B1 gene; Cell Differentiation process; Cells; Characteristics; China; Chronic; Citrobacter rodentium; Code; Collaborations; Color; Crossbreeding; Development; Disease; Failure; Functional RNA; Gene Expression Profile; Genes; Genomics; Helper-Inducer T-Lymphocyte; Hematopoietic; Heterogeneity; Human; Immune; Immune response; Immunity; In Vitro; Infection; Inflammatory; Injection of therapeutic agent; Interferon Type II; Interleukin 2 Receptor; Interleukin 2 Receptor Gamma; Interleukin-13; Interleukin-17; Interleukin-4; Interleukin-5; Interleukins; Knockout Mice; Lead; Lymphocyte; Lymphocyte Subset; Lymphoid; Lymphoid Cell; Lymphoid Tissue; Maintenance; Maps; Molecular; Mouse Strains; Mus; Natural Killer Cells; Organ; PPAR gamma; Pathogenesis; Pattern; Phenotype; Play; Population; Production; Proteins; RNA; Regulation; Regulatory T-Lymphocyte; Reporter; Reporting; Role; Sampling; Scientist; Signal Transduction; Structure; Structure of aggregated lymphoid follicle of small intestine; T cell differentiation; T-Cell Development; T-Cell Receptor; T-Lymphocyte; T-bet protein; Tamoxifen; Testing; Th1/Th2 Differentiation Pathway; Th2 Cells; The Sun; Tissues; Transcriptional Regulation; Transgenes; Up-Regulation; Work; cell motility; chemokine receptor; combinatorial; cytokine; genome wide association study; immunoregulation; in vivo; interleukin-22; lymph nodes; microorganism; mouse model; pathogen; peripheral tolerance; progenitor; research study; response; transcription factor

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. 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. There are innate counterparts of Th cells. A class of innate lymphoid cells (ILCs), whose development requires signaling through the IL-2 receptor (IL-2R) common gamma chain and IL-7Ralpha, has been recently discovered. Distinct subsets of ILCs are capable of producing similar sets of characteristic effector cytokines as produced by Th cells. Therefore, they 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. Like Th cells, ILCs are important for protective immune responses to infections and are responsible for the pathogenesis of many inflammatory diseases. Some ILCs such as lymphoid tissue inducers (LTis) are critical for lymphoid organ development. The activation, differentiation and expansion of Th cells are tightly regulated by specific transcription factors. Among the lineage-specific transcription factors, T-bet, GATA3, RORgammat and Foxp3 are deterministic for the differentiation of Th1, Th2, Th17 and Treg cells, respectively. These transcription factors have been referred as to master regulators. The differentiation of Th lineages is usually mutually exclusive, possibly due to the cross-regulation of the key transcription factors expressed by each lineage. However, recent reports indicate that the master regulator of one lineage may be expressed in other lineages. For example, among the Treg population, there are T-bet-expressing and GATA3-expressing Treg cells. It has been suggested that these cells may have unique functions in regulating tolerance and immune responses. In addition, RORgammat and T-bet co-expressing cells have been identified both in mice and in humans. How these master regulators function in a same cell is an intriguing question. The ILCs also express one or two of these master regulators, including T-bet, GATA3 and RORgammat, and these factors are critical for the development and functions of ILCs. During the past year, we reported that GATA3 plays an essential role in the development of all IL-7Ralpha-expressing ILCs including LTis, but not conventional NK cells and IL-7Ralpha-non-expressing ILCs. This mirrors the essential function of GATA3 during CD4 but not CD8 T cell development. Consequently, the mice lacking GATA3 in all hematopoietic cells do not develop lymph node structures and Peyers patches. These are also susceptible to Citrobacter rodentium infection (in collaboration with Yasmine Belkaid's group) due to the failure of ILC3 development. In addition, GATA3 is indispensable for maintaining the functions and survival of ILC2s similar to its functions in Th2 cells. Hundreds of GATA3-regulated genes in ILC2s including many critical genes that are involved in type 2 immune responses were identified through RNA-Seq. This work indicates that NK cells may represent innate version of CD8 T cells and that GATA3 plays parallel roles in establishing and regulating both adaptive and innate lymphocyte subsets. In collaboration with Dr. Yokoyamas group, we recently reported that CD49a+DX5- tissue-resident NK (trNK) cells are distinct from CD49a-DX5+ conventional NK (cNK) cells and thymic derived NK cells. While the development of thymic-derived NK cells requires GATA3, trNK cells can develop independent of GATA3 just as cNK cells. In collaboration with Dr. Keji Zhao's lab, we reported genome-wide identification of lineage-specific long intergenic non-coding RNAs (lincRNAs) and transcription factors during T cell differentiation. In this study, 1,524 long intergenic non-coding RNAs (lincRNAs) in 42 T cell samples from early T cell progenitors to terminally differentiated T helper subsets were identified. Genome wide analysis revealed highly dynamic and cell-specific expression patterns of lincRNAs during T cell differentiation. Importantly, these lincRNAs are located in genomic regions enriched for protein-coding genes with immune-regulatory functions. Unit scientists further demonstrated that the LincR-Ccr2-5'AS, a target of GATA-3, is important for Th2 cell migration by regulating the expression of many chemokine receptors. To study the heterogeneity and the relationship of Treg subsets, we created another T-bet reporter mouse strain, T-bet-AmCyan. This strain has been crossbred to Foxp3-RFP mice (made by Dr. Richard Flavell's group) and GATA3-GFP mice (made by Dr. Doug Engel's group) to generate a tri-color reporter mouse strain in which Th1- and Th2-like Treg cell subsets can be identified simultaneously in a same animal. By isolating Th1- or Th2- like Treg subsets from AmCyan/GATA3-GFP/Foxp3-RFP triple reporter mice, we found that T-bet and GATA3 were dynamically expressed by Treg cells. T-bet-expressing Treg cells can turn into GATA-3 expressing Treg cells and vice versa, both in vitro and in vivo. We then created a third mouse line carrying T-bet-ZsGreen-T2A-CreERT2 BAC transgene and crossed this mouse line to the ROSA26-loxp-STOP-loxp-tdTomato reporter strain to obtain a T-bet fate-mapping mouse model. Upon tamoxifen treatment, the cells expressing T-bet will express both ZsGreen and tdTomato. But, the cells previously expressed T-bet during tamoxifen treatment will only express tdTomato but not ZsGreen. Indeed, one week after tamoxifen injection, we observed that a large percentage of tdTomato+ Treg cells lost ZsGreen expression indicating that some T-bet-expressing Treg cells had turned off T-bet expression within one week. This result confirmed dynamic expression of T-bet in Treg cells. We then tested the functions of T-bet and GATA3 in Treg cells by deleting either Tbx21 or Gata3 gene specifically in Treg cells by Foxp3-Cre (kindly provided by Dr. Alexander Rudensky). Single deletion of either gene did not result in an obvious phenotype, however, combinatorial deletion of both Tbx21 and Gata3 in Treg cells allowed the development of autoimmune-like diseases in mice at the steady state. These results indicate that T-bet and GATA3 play a redundant role in maintaining Treg functions. Loss of suppressive functions in T-bet-GATA3 double-deficient Treg cells was associated with the upregulation of RORγt expression and IL-17 production in a cell-intrinsic manner.Overall, our results demonstrate that T-bet and GATA3-expressing Treg cells do not represent stable Treg subsets. At steady state, Treg cells can transiently upregulate either T-bet or GATA3 to antagonize RORgammat; such mechanism is critical for the maintenance of peripheral tolerance. In collaboration with Dr. Bing Sun's lab at the SIBCB, China, we are investigating the functions of several T-bet/GATA3-regulated molecules, such as PPARgamma, Bhlhb2 and ECM1, during T helper cell differentiation and migration in the context of several immune related diseases. We have prepared Pparg and Bhlhb2 conditional knockout mice. In vitro experiments indicate both Bhlhb2 and PPARgamma regulate Th1/Th2 cytokine production. We are now studying their functions using in vivo animal models.

CD4 T 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)-4 和 IL-17 分别作为其特征效应细胞因子。这些细胞对于针对各种微生物的不同类型的免疫是不可或缺的。对病原体的不当 Th 反应可能会导致宿主慢性感染和/或组织损伤。同样，无害的环境抗原或自身抗原对 Th1、Th17 或 Th2 细胞的不必要激活可能导致器官特异性自身免疫性疾病或过敏性炎症性疾病。 Th 细胞有先天的对应物。最近发现了一类先天淋巴细胞 (ILC)，其发育需要通过 IL-2 受体 (IL-2R) 共同伽马链和 IL-7Rα 进行信号传导。 ILC 的不同亚群能够产生与 Th 细胞相似的特征性效应细胞因子组。因此，它们被分为产生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。与 Th 细胞一样，ILC 对于感染的保护性免疫反应很重要，并且是许多炎症性疾病的发病机制。一些 ILC，例如淋巴组织诱导剂 (LTis)，对于淋巴器官的发育至关重要。 Th 细胞的激活、分化和扩增受到特定转录因子的严格调控。在谱系特异性转录因子中，T-bet、GATA3、RORgammat 和 Foxp3 分别对 Th1、Th2、Th17 和 Treg 细胞的分化具有决定性作用。这些转录因子被称为主调节因子。 Th谱系的分化通常是相互排斥的，可能是由于每个谱系表达的关键转录因子的交叉调节。然而，最近的报告表明，一个谱系的主要调节因子可能在其他谱系中表达。例如，在Treg细胞群中，存在表达T-bet和表达GATA3的Treg细胞。有人认为这些细胞在调节耐受性和免疫反应方面可能具有独特的功能。此外，RORgammat 和 T-bet 共表达细胞已在小鼠和人类中被发现。这些主调节因子如何在同一细胞中发挥作用是一个有趣的问题。 ILC 还表达一种或两种主调节因子，包括 T-bet、GATA3 和 RORgammat，这些因子对于 ILC 的发育和功能至关重要。 在过去的一年中，我们报道了 GATA3 在包括 LTis 在内的所有表达 IL-7Rα 的 ILC 的发育中发挥着重要作用，但在传统 NK 细胞和不表达 IL-7Rα 的 ILC 中则不然。这反映了 GATA3 在 CD4 而非 CD8 T 细胞发育过程中的基本功能。因此，所有造血细胞中缺乏 GATA3 的小鼠不会形成淋巴结结构和派耶斯集结。由于 ILC3 发育失败，它们也容易受到啮齿类柠檬酸杆菌感染（与 Yasmine Belkaid 小组合作）。 此外，GATA3 对于维持 ILC2 的功能和生存是不可或缺的，类似于其在 Th2 细胞中的功能。通过 RNA-Seq 鉴定了 ILC2 中数百个 GATA3 调节的基因，包括许多参与 2 型免疫反应的关键基因。这项工作表明 NK 细胞可能代表 CD8 T 细胞的先天版本，而 GATA3 在建立和调节适应性和先天淋巴细胞亚群方面发挥着平行作用。我们与 Yokoyamas 博士小组合作，最近报道了 CD49a+DX5- 组织驻留 NK (trNK) 细胞不同于 CD49a-DX5+ 传统 NK (cNK) 细胞和胸腺来源的 NK 细胞。虽然胸腺来源的 NK 细胞的发育需要 GATA3，但 trNK 细胞可以像 cNK 细胞一样独立于 GATA3 发育。 我们与赵克吉博士的实验室合作，报告了 T 细胞分化过程中谱系特异性长基因间非编码 RNA (lincRNA) 和转录因子的全基因组鉴定。在这项研究中，从早期 T 细胞祖细胞到终末分化 T 辅助亚群的 42 个 T 细胞样本中鉴定出了 1,524 个长基因间非编码 RNA (lincRNA)。全基因组分析揭示了 T 细胞分化过程中 lincRNA 的高度动态和细胞特异性表达模式。重要的是，这些 lincRNA 位于富含具有免疫调节功能的蛋白质编码基因的基因组区域。该单位科学家进一步证明，GATA-3 的靶点 LincR-Ccr2-5'AS 通过调节许多趋化因子受体的表达，对 Th2 细胞迁移非常重要。 为了研究 Treg 亚群的异质性和关系，我们创建了另一种 T-bet 报告小鼠品系，T-bet-AmCyan。 该品系已与Foxp3-RFP小鼠（由Richard Flavell博士团队制造）和GATA3-GFP小鼠（由Doug Engel博士团队制造）杂交，产生三色报告小鼠品系，其中Th1-和Th2-like Treg 细胞亚群可以在同一动物中同时鉴定。通过从AmCyan/GATA3-GFP/Foxp3-RFP三重报告小鼠中分离Th1或Th2样Treg亚群，我们发现T-bet和GATA3由Treg细胞动态表达。在体外和体内，表达 T-bet 的 Treg 细胞可以转化为表达 GATA-3 的 Treg 细胞，反之亦然。然后，我们创建了第三个携带 T-bet-ZsGreen-T2A-CreERT2 BAC 转基因的小鼠品系，并将该小鼠品系与 ROSA26-loxp-STOP-loxp-tdTomato 报告菌株杂交，以获得 T-bet 命运图谱小鼠模型。在他莫昔芬治疗后，表达 T-bet 的细胞将表达 ZsGreen 和 tdTomato。但是，之前在他莫昔芬治疗期间表达 T-bet 的细胞将仅表达 tdTomato，而不表达 ZsGreen。事实上，注射他莫昔芬一周后，我们观察到很大比例的 tdTomato+ Treg 细胞失去了 ZsGreen 表达，这表明一些表达 T-bet 的 Treg 细胞在一周内关闭了 T-bet 表达。该结果证实了T-bet在Treg细胞中的动态表达。然后，我们通过 Foxp3-Cre（由 Alexander Rudensky 博士友情提供）在 Treg 细胞中特异删除 Tbx21 或 Gata3 基因，测试了 T-bet 和 GATA3 在 Treg 细胞中的功能。任一基因的单一缺失都不会导致明显的表型，然而，Treg 细胞中 Tbx21 和 Gata3 的组合缺失使得小鼠在稳定状态下发生自身免疫样疾病。这些结果表明T-bet和GATA3在维持Treg功能方面发挥着冗余作用。 T-bet-GATA3 双缺陷 Treg 细胞中抑制功能的丧失与细胞固有方式的 RORγt 表达和 IL-17 产生上调有关。总体而言，我们的结果表明 T-bet 和表达 GATA3 的 Treg 细胞不代表稳定的 Treg 子集。在稳定状态下，Treg 细胞可以短暂上调 T-bet 或 GATA3 以拮抗 RORgammat；这种机制对于维持外周耐受性至关重要。 我们与中国 SIBCB 的 Bing Sun 博士实验室合作，研究了几种 T-bet/GATA3 调节分子（例如 PPARgamma、Bhlhb2 和 ECM1）在 T 辅助细胞分化和迁移过程中的功能。免疫相关疾病。我们制备了Pparg和Bhlhb2条件敲除小鼠。体外实验表明 Bhlhb2 和 PPARgamma 均可调节 Th1/Th2 细胞因子的产生。我们现在正在使用体内动物模型研究它们的功能。