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)-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, 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 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 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 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 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 further studied the dynamic expression of T-bet and RORgt in EAE. We used T-bet-ZsGreen (or AmCyan)-RORgt-E2Crimson-Foxp3-RFP triple reporter mice to demonstrate 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 observed 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 and all three cell subsets play unique roles in disease induction. GATA3 regulates the development of NKp46+ ILC3s which co-express T-bet and RORgt (Nat. Immunol. 17: 169-178, 2016). Since T-bet and RORgt co-expression is also found in differentiating CD4 T cells during EAE induction, we studied the role of GATA3 during this process. Interestingly, GATA3 expression was transiently upregulated in RORgt-expressing Th17 cells. More importantly, early deletion of GATA3 in naive CD4 T cells resulted in diminished disease induction and a failure to generate the T-bet/RORgt dual expressors in EAE. In addition, GATA3 plays a critical role in the regulation of GM-CSF expression after the development of T-bet/RORgt 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 are currently studying whether GATA3 can directly bind to the Csf2 gene that encodes GM-CSF to regulate its expression or GATA3-mediated GM-CSF induction requires other transcription factors such as Bhlhe40. Immunoglobulin A (IgA)-producing plasma cells derived from conventional B cells in the gut play an important role in maintaining the homeostasis of gut flora. Both T cell-dependent and T cell-independent IgA class switching occurs in the lymphoid structures in the gut, whose formation depends on lymphoid tissue inducers (LTis), a subset of ILCs. However, our knowledge on the functions of non-LTi helper-like ILCs in promoting IgA production is still limited. By cell adoptive transfer and utilizing a unique mouse strain, we demonstrated that the generation of IgA-producing plasma cells from B cells in the gut occurred efficiently in the absence of both T cells and helper-like ILCs and without engaging TGF signaling. Nevertheless, B cell recruitment and/or retention in the gut required functional NKp46-CCR6+ LTis. Therefore, while CCR6+ LTis contribute to the accumulation of B cells in the gut through inducing lymphoid structure formation, helper-like ILCs are not essential for the T cell-independent generation of IgA-producing plasma cells. This study has been recently published in PNAS (Proc Natl Acad Sci U S A. 2021 Jul 6;118(27):e2106754118. doi: 10.1073/pnas.2106754118). While CD4 T helper cells and their innate counterparts, innate lymphoid cells, utilize an identical set of lineage-determining transcription factors (LDTFs) for their differentiation and functions, the similarities and differences in the induction of LDTFs in these lymphocytes are still elusive. During the past year, we studied the regulation of T-bet in type 1 lymphocytes. We show 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. We also investigated 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. We 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.