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, lymphocytes from the secondary lymphoid organs of mice with GATA-3 single deletion specifically in Tregs were grossly normal in steady state. During the past year, we found that mice with Treg-specific Gata3 deletion exhibit a spontaneous Th2 and Th17 immunopathology in the lungs and in the intestine. The lungs of mice with GATA-3-null Treg cells exhibit eosinophilia and neutrophilia, which corresponds with Th2- and Th17-mediated inflammation. We also found that GATA-3 was expressed at different levels in Tregs cells depending on their activation status and tissue location. When we sub-optimally express GATA-3 in GATA-3-deficient Tregs, the Th2-mediated immunopathology was resolved and the lungs of these mice displayed a normal number of eosinophils. However, these mice had elevated levels Th17 cells and neutrophils. Therefore, we hypothesize that GATA-3 expressed at different levels in Tregs regulates several functional properties of these cells - while basal levels of GATA3 are required for controlling Th2 responses, optimal levels of GATA-3 expression are needed for restraining Th17 responses. Currently, we are investigating the molecular mechanism through which quantitative expression of GATA-3 may qualitatively regulate Treg functions. We also generated a ZsGreen-T2A-GATA3 knock-in mice with conditional knockout potential through Crispr/Cas9 technology to facilitate this study. Preliminary results suggest that this new reporter faithfully reflects GATA3 expression and it can be used to separate lymphocytes that express different levels of GATA3. 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). Through RNA-Seq analysis of ILC3 subsets (namely CCR6+ and NKp46+ ILC3) isolated from a novel T-bet-ZsGreen/RORgt-E2-Crimson dual-reporter mouse strain that we have generated, we identified hundreds of CCR6+ and NKp46+ ILC3 lineage specific genes. During the past year, we find that GATA3 may serve as a switch in determining the development of CCR6+ LTi cells versus other ILC lineages. Lymphoid tissue inducer (LTi) population is the founding member of ILCs, however, recent study has shown that these cells are not derived from a PLZF-expressing ILC common progenitor that generates other ILCs. The transcription factor(s) determining the fate of non-LTi progenitor versus LTi progenitor are unknown. Our unpublished new data indicate that 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 completed studies on other important transcription factors during the past year. We found that the transcription factor Bhlhe40 is required for optimal IFN-g production in Th1 cells through a mechanism independent of T-bet regulation. Bhlhe40 also represses IL-10 production by Th1 cells. Mice with conditional deletion of Bhlhe40 in T cells succumbed to Toxoplasma gondii infection and blockage of IL-10 signaling during infection rescued these mice from death. Thus, our results demonstrate that transcription factor Bhlhe40 is a molecular switch for determining the fate of inflammatory and anti-inflammatory Th1 cells. We also found that the transcription factor B cell lymphoma 11b (Bcl11b), a previously unknown component of the GATA3 transcriptional complex, is critically involved in GATA3-mediated gene regulation. Bcl11b binds to GATA3 through protein-protein interaction and they co-localize 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-mediated gene regulation (from RNA-Seq) and co-binding pattern (from ChIP-Seq) suggest that GATA3/Bcl11b complex is involved in limiting Th2 gene expression, as well as in inhibiting non-Th2 gene expression. Thus, Bcl11b controls both GATA3-mediated gene activation and repression. In collaboration with Dr. Keji Zhao's lab at the NHLBI, we reported that conditional deletion of histone demethylases UTX and JMJD3 by CD4-Cre leads to near complete loss of liver NKT cells, while conventional T cells are less affected (Cell Biosci. 7:25, 2017). Therefore, NKT cell development is sensitive to proper regulation of H3K27 methylation. The H3K27me3 demethylase enzymes, in particular UTX, promote NKT cell development, and are required for effective NKT function.
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