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 shown previously that T-bet expression by some Foxp3-expressing regulatory T cells are transient (Nature Immunology, 16: 197-206, 2015). In the past year, we have reported that T-bet is also dynamically expressed during the differentiation of a T follicular helper (Tfh) cell subset (J. Exp. Med, 215: 2705-2714, 2018). These Tfh cells are capable of producing IFNg without expressing T-bet, the master regulator for IFN-g production in Th1 cells. By using the T-bet-fate-mapping mouse strain, we demonstrated 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, however, T-bet expression is no longer required for IFN-g production after these IFN-g-expressing Tfh cells are generated. 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. We have previously reported that the transcription factor Bhlhe40 is a molecular switch for determining the fate of inflammatory and anti-inflammatory 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 is also highly upregulated during the differentiation of other T helper cells, however, the functions of Bhlhe40 during the differentiation of these cells are still elusive. In the past year, we have compared gene expression patterns between wild type and Bhlhe40-deficient cells cultured under distinct culture conditions through RNA-Seq. In addition, by generating V5-tagged Bhlhe40 knock-in mice through CRISPR/Cas9, we have also performed anti-V5 ChIP-Seq to identify Bhlhe40 direct targets in different T helper cells. Many direct common targets of Bhlhe40 including several genes encoding transcription factors have been identified. We are now actively investigating the functions of these transcription factors during T cell differentiation through loss-of-function and gain-of-function 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). During the past year, we have further confirmed 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. This study has been submitted for publication and it is now in revision. It has been reported that type 2 immune responses involve the cooperative actions of Th2 cells and ILC2s, however, the crosstalk between these cells is still controversial. To assess the crosstalk between Th2 cells and ILC2s in type 2 immune responses, we have generated and utilized Th2-deficient and ILC2-deficient mice models to assess type 2 immune responses in these animals. In a chronic papain challenge model, ILC2s were less activated in the absence of Th2 cells, suggesting a partial requirement of Th2 cells for optimal ILC2 activation. Whereas, in ILC2-deficient mice, Th2 cells were only able to partially expand, suggesting that ILC2s may aid Th2 responses. By contrast, in an OVA-induced model of allergy, ILC2-deficient mice exhibited a normal Th2 response. Thus, the crosstalk between ILC2s and Th2 cells may depend on the route and/or type of allergen exposure. Currently we are investigating the mechanism(s) through which Th2 cells may help ILC2 cell expansion and activation as well as the physiological importance of Th2-mediated ILC2 expansion/activation during type 2 immune responses.
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