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. The activation, differentiation and expansion of Th cells are tightly regulated by specific transcription factors. Among the lineage-specific transcription factors, T-bet, GATA3, RORgt and Foxp3 are deterministic for the differentiation and functions of Th1, Th2, Th17 and Treg cells, respectively. These transcription factors have been referred as to master regulators. Innate counterparts of Th cells are innate lymphoid cells (ILCs), whose development requires signaling through the IL-2 receptor (IL-2R) common gamma chain and IL-7R alpha chain. 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 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. The ILCs also express one or two or even three of the master regulators T-bet, GATA3 and RORgt, in a single cell level, and these factors are critical for the development and functions of ILC subsets. Within the ILC3s all of which express RORgt, there are two subsets -- CCR6+ (mainly lymphoid tissue inducers, LTis) and CCR6- ILC3s -- with the latter having the potential to develop into NKp46+ ILC3s that express both RORgt and T-bet. CCR6+ and NKp46+ ILC3s seem to have distinct biological functions and develop from different precursors. 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 LTis are critical for lymphoid organ development. We have previously reported that T-bet and GATA3 are dynamically expressed by Foxp3-expressing regulatory T cells and such dynamic expression is critical for maintaining immune tolerance (Nature Immunology, 16: 197-206, 2015). However, using a T-bet fate mapping reporter mouse strain, we found that T-bet was also dynamically expressed during the differentiation of non-treg cells including a T follicular helper (Tfh) cell subset. Tfh cells, which reside in the B cell follicles and help Ig class switching, express the master transcription factor BCL-6 and signature cytokine IL-21. Tfh cells are also capable of expressing IFNg, however, whether IFN-g-producing Tfh population represents a unique Tfh subset and how they develop are unknown. While T-bet is the master regulator for IFN-g production in Th1 cells, Tfh cells express no or very low levels of T-bet. During the past year, by using the T-bet-fate-mapping mouse strain, we found that all the IFN-g-producing Tfh cells identified after immunization have previously expressed T-bet. DNase I hypersensitivity analysis indicates that the Ifng gene locus is partially accessible in this ex-T-bet population but inaccessible in Tfh cells without a history of T-bet expression. Furthermore, multi-color tissue imaging shows that the ex-T-bet Tfh cells found in germinal centers express IFN-g in situ. Finally, IFN-g-expressing Tfh cells are absent in T-bet-deficient mice. Thus, transient expression of T-bet epigenetically imprints the Ifng locus for cytokine production in Tfh cells. Our results also indicate that Tfh cells are composed of different subsets and they may use genetic programs similar to those of classical non-Tfh T helper cells in order to acquire unique cytokine producing capacity. Th1 cells play a critical role in host defense against intracellular pathogens and in autoimmune diseases by producing a key inflammatory cytokine IFN-g; some Th1 cells can also be anti-inflammatory through producing IL-10. However, the molecular switch for regulating the differentiation of inflammatory and anti-inflammatory Th1 cells is still elusive. During the past year, we reported that that transcription factor Bhlhe40 is a molecular switch for determining the fate of inflammatory and antiinflammatory Th1 cells (J Exp Med. 215: 1813-1821, 2018). Bhlhe40-deficient CD4 Th1 cells produced less IFN-g but substantially more IL-10 than wild-type Th1 cells both in vitro and in vivo. Bhlhe40-mediated IFN-g production was independent of transcription factor T-bet regulation. Mice with conditional deletion of Bhlhe40 in T cells succumbed to Toxoplasma gondii infection, and blockade of IL-10 signaling during infection rescued these mice from death. GATA3 acts as the master transcription factor for Th2 cell differentiation and function. However, it is still elusive how GATA3 function is precisely regulated in Th2 cells. During the past year, we reported that the transcription factor B cell lymphoma 11b (Bcl11b), a previously unknown component of GATA3 transcriptional complex, is involved in GATA3-mediated gene activation and repression (J Exp Med. 215: 1449-1462, 2018). Bcl11b binds to GATA3 through protein-protein interaction, and they colocalize at many important cis-regulatory elements in Th2 cells. The expression of type 2 cytokines, including IL-4, IL-5, and IL-13, is up-regulated in Bcl11b-deficient Th2 cells both in vitro and in vivo; such up-regulation is completely GATA3 dependent. Genome-wide analyses of Bcl11b- and GATA3-regulated genes (from RNA sequencing), cobinding patterns (from chromatin immunoprecipitation sequencing), and Bcl11b-modulated epigenetic modification and gene accessibility suggest that GATA3/Bcl11b complex is involved in limiting Th2 gene expression, as well as in inhibiting non-Th2 gene expression. For ILC studies, we have previously reported that GATA3 plays an essential role in the development of all IL-7Ra-expressing ILCs but not conventional NK cells (Immunity, 40: 378-88, 2014). We further reported that despite its low expression level in mature ILC3s, GATA3 has important functions in regulating homeostasis, further maturation and functions of distinct ILC3 subsets (Nat. Immunol., 17:169-78, 2016). During the past year, we performed additional experiments to confirm that GATA3 serves as a switch in determining the development of CCR6+ LTi cells versus other ILC lineages. GATA3 is absolutely required for the generation of PLZF-expressing non-LTi progenitors, which express high level of GATA3, but not for the generation of RORgammat-expressing LTi progenitors consistent with low levels of GATA3 expression in these progenitors. Nevertheless, low level of GATA3 expression by LTi progenitors is critical for the generation of functional LTi cells. Thus, quantitative expression of GATA3 functionally determines the fates and functions of distinct ILC progenitors. We have also generated a ZsGreen-T2A-GATA3 knock-in mice with conditional knockout potential through Crispr-Cas9 technology, This new reporter strain allows us to identify and separate GATA3-expressing lymphocyte subsets. Furthermore, by crossing this new line to Cre-ERT2 line, we were able to obtain GATA3-deficient Th2 and ILC2s based on high levels of reporter expression. We are currently use this model to study GATA3-mediated gene regulation in vivo.
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