Transcriptional Regulation of Immune Cell Development, Activation and Functions

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

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.