Transcriptional Regulation of Immune Cell Development, Activation and Functions

CD4+ T淋巴细胞在协调适应性免疫反应中发挥核心作用。在特定的细胞因子环境中通过其T细胞受体（TCR）激活后，初始CD4+ T细胞分化为不同的T辅助细胞（Th）谱系，包括Th1、Th2和Th17细胞，它们分别产生干扰素(IFN)-、白细胞介素(IL)-4和IL-17，作为它们的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中介导关键功能。Th1细胞在宿主防御细胞内细菌和病毒中起重要作用；Th2细胞，用于驱逐细胞外寄生虫，如蠕虫；以及控制胞外细菌和真菌的Th17细胞。不适当的Th对病原体的反应可能导致宿主的慢性感染和/或组织损伤，而异常的Th细胞分化可能导致许多炎症性过敏或自身免疫性疾病，包括哮喘、炎症性肠病、类风湿关节炎和多发性硬化症。

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)-, 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, rheumatoid arthritis and multiple sclerosis. Innate lymphoid cells (ILCs), which lack expression of antigen receptors, require signaling through the IL-2 receptor (IL-2R) common chain and IL-7R, 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 IFN, 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 T-bet, GATA3 and RORt, because we hypothesize that they are the major nodes in these networks. By focusing on the regulation and actions of T-bet, GATA3 and RORt 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 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. Previously, we have generated reporter mouse strains for T-bet expression, T-bet-ZsGreen (Immunity 37:660-673, 2012) and T-bet-AmCyan (Nat. Immunol. 16:197-206, 2015), in which GFP or BFP faithfully reflects T-bet expression, respectively. We have also generated a RORt-E2Crimson reporter strain (Nat. Immunol. 17: 169-178, 2016). By generating T-bet-ZsGreen (or AmCyan)-RORt-E2Crimson-Foxp3-RFP triple reporter mice through cross-breeding, we studied the differentiation of T-bet and RORt single- or dual-expressing effector (Foxp3-RFP negative) CD4+ T cells and their relationships and functions in EAE during past year. By using these novel reporter mice, we were able to demonstrate that RORt-expressing cells can generate T-bet/RORt dual-expressors as well as cells only expressing T-bet after adoptive transfer. RNA-Seq analysis of subsets harvested from the spinal cord of the disease mice indicates that T-bet and RORt each regulate (induce or inhibit) distinct and shared sets of genes.By generating other novel mouse models, we also demonstrated that a transition from expressing only RORt to expressing both RORt and T-bet, and finally to expressing only T-bet is a natural process in autoimmunity and all three cell subsets play unique roles in disease induction. We have previously reported that GATA3 regulates the balance between T-bet and RORt during the development of NKp46+ ILC3s which co-express T-bet and RORt (Nat. Immunol. 17: 169-178, 2016). Since T-bet and RORt co-expression is also found in differentiating CD4 T cells during EAE induction, during the past year, we asked whether GATA3 also modulates the balance between T-bet and RORt in CD4 T cells. Interestingly, GATA3 expression is transiently upregulated in RORt-expressing Th17 cells. More importantly, early deletion of GATA3 in nave CD4 T cells results in diminished disease induction and a failure to generate the T-bet/RORt dual expressors in EAE. In addition, GATA3 plays a critical role in the regulation of GM-CSF expression after the development of T-bet/RORt dual expressors. CD4 T cells generated in vivo upon immunization fail to induce EAE after a late inducible deletion of Gata3 possibly because of a lack of GM-CSF expression by these cells. We have previously reported that GATA3 plays an essential role in the development of all IL-7R-expressing ILCs but not conventional natural killer (NK) cells (Immunity 40: 378-88, 2014). During the past year, we have reported that 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. Indeed, all the residual ILCs in GATA3 conditional knockout mice are LTi cells and conventional ILC1s, ILC2s and ILC3s that derive from the PLZF/PD-1-expressing non-LTi progenitors are completely absent. The levels of GATA3 expression are positively correlated with the expression levels of non-LTi progenitor-associated genes, but negatively correlated with the expression levels of LTi progenitor-associated genes. These results indicate that GATA3 serves as a switch in determining the development of CCR6+ LTi cells versus other ILC lineages. So far, GATA3 is the only known transcription factor that plays a critical role in this lineage bifurcation process. Interestingly, while LTi cells are present in the GATA3 deficient mice, these mice do not develop lymph nodes, suggesting that low levels of GATA3 expression by LTi progenitors are critical for the generation of functional LTi cells. Indeed, a GATA3 transgene with low levels of GATA3 expression is able to rescue LTi functions and the generation of lymph nodes without inducing the development of PLZF-expressing ILC progenitors. Thus, quantitative expression of GATA3 determines the functions of LTi cells and the bifurcation of LTi and non-LTi ILC progenitors (Immunity. 52: 83-95, 2020).