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. There are innate counterparts of Th cells. A class of innate lymphoid cells (ILCs), whose development requires signaling through the IL-2 receptor (IL-2R) common gamma chain and IL-7Ralpha, has been recently discovered. 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 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. 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 lymphoid tissue inducers (LTis) are critical for lymphoid organ development. The activation, differentiation and expansion of Th cells are tightly regulated by specific transcription factors. Among the lineage-specific transcription factors, T-bet, GATA3, RORgammat and Foxp3 are deterministic for the differentiation of Th1, Th2, Th17 and Treg cells, respectively. These transcription factors have been referred as to master regulators. The differentiation of Th lineages is usually mutually exclusive, possibly due to the cross-regulation of the key transcription factors expressed by each lineage. However, recent reports indicate that the master regulator of one lineage may be expressed in other lineages. For example, among the Treg population, there are T-bet-expressing and GATA3-expressing Treg cells. It has been suggested that these cells may have unique functions in regulating tolerance and immune responses. In addition, RORgammat and T-bet co-expressing cells have been identified both in mice and in humans. How these master regulators function in a same cell is an intriguing question. The ILCs also express one or two of these master regulators, including T-bet, GATA3 and RORgammat, and these factors are critical for the development and functions of ILCs. During the past year, we reported that GATA3 plays an essential role in the development of all IL-7Ralpha-expressing ILCs including LTis, but not conventional NK cells and IL-7Ralpha-non-expressing ILCs. This mirrors the essential function of GATA3 during CD4 but not CD8 T cell development. Consequently, the mice lacking GATA3 in all hematopoietic cells do not develop lymph node structures and Peyers patches. These are also susceptible to Citrobacter rodentium infection (in collaboration with Yasmine Belkaid's group) due to the failure of ILC3 development. In addition, GATA3 is indispensable for maintaining the functions and survival of ILC2s similar to its functions in Th2 cells. Hundreds of GATA3-regulated genes in ILC2s including many critical genes that are involved in type 2 immune responses were identified through RNA-Seq. This work indicates that NK cells may represent innate version of CD8 T cells and that GATA3 plays parallel roles in establishing and regulating both adaptive and innate lymphocyte subsets. In collaboration with Dr. Yokoyamas group, we recently reported that CD49a+DX5- tissue-resident NK (trNK) cells are distinct from CD49a-DX5+ conventional NK (cNK) cells and thymic derived NK cells. While the development of thymic-derived NK cells requires GATA3, trNK cells can develop independent of GATA3 just as cNK cells. In collaboration with Dr. Keji Zhao's lab, we reported genome-wide identification of lineage-specific long intergenic non-coding RNAs (lincRNAs) and transcription factors during T cell differentiation. In this study, 1,524 long intergenic non-coding RNAs (lincRNAs) in 42 T cell samples from early T cell progenitors to terminally differentiated T helper subsets were identified. Genome wide analysis revealed highly dynamic and cell-specific expression patterns of lincRNAs during T cell differentiation. Importantly, these lincRNAs are located in genomic regions enriched for protein-coding genes with immune-regulatory functions. Unit scientists further demonstrated that the LincR-Ccr2-5'AS, a target of GATA-3, is important for Th2 cell migration by regulating the expression of many chemokine receptors. To study the heterogeneity and the relationship of Treg subsets, we created another T-bet reporter mouse strain, T-bet-AmCyan. This strain has been crossbred to Foxp3-RFP mice (made by Dr. Richard Flavell's group) and GATA3-GFP mice (made by Dr. Doug Engel's group) to generate a tri-color reporter mouse strain in which Th1- and Th2-like Treg cell subsets can be identified simultaneously in a same animal. By isolating Th1- or Th2- like Treg subsets from AmCyan/GATA3-GFP/Foxp3-RFP triple reporter mice, we found that T-bet and GATA3 were dynamically expressed by Treg cells. T-bet-expressing Treg cells can turn into GATA-3 expressing Treg cells and vice versa, both in vitro and in vivo. We then created a third mouse line carrying T-bet-ZsGreen-T2A-CreERT2 BAC transgene and crossed this mouse line to the ROSA26-loxp-STOP-loxp-tdTomato reporter strain to obtain a T-bet fate-mapping mouse model. Upon tamoxifen treatment, the cells expressing T-bet will express both ZsGreen and tdTomato. But, the cells previously expressed T-bet during tamoxifen treatment will only express tdTomato but not ZsGreen. Indeed, one week after tamoxifen injection, we observed that a large percentage of tdTomato+ Treg cells lost ZsGreen expression indicating that some T-bet-expressing Treg cells had turned off T-bet expression within one week. This result confirmed dynamic expression of T-bet in Treg cells. We then tested the functions of T-bet and GATA3 in Treg cells by deleting either Tbx21 or Gata3 gene specifically in Treg cells by Foxp3-Cre (kindly provided by Dr. Alexander Rudensky). Single deletion of either gene did not result in an obvious phenotype, however, combinatorial deletion of both Tbx21 and Gata3 in Treg cells allowed the development of autoimmune-like diseases in mice at the steady state. These results indicate that T-bet and GATA3 play a redundant role in maintaining Treg functions. Loss of suppressive functions in T-bet-GATA3 double-deficient Treg cells was associated with the upregulation of RORγt expression and IL-17 production in a cell-intrinsic manner.Overall, our results demonstrate that T-bet and GATA3-expressing Treg cells do not represent stable Treg subsets. At steady state, Treg cells can transiently upregulate either T-bet or GATA3 to antagonize RORgammat;such mechanism is critical for the maintenance of peripheral tolerance. In collaboration with Dr. Bing Sun's lab at the SIBCB, China, we are investigating the functions of several T-bet/GATA3-regulated molecules, such as PPARgamma, Bhlhb2 and ECM1, during T helper cell differentiation and migration in the context of several immune related diseases. We have prepared Pparg and Bhlhb2 conditional knockout mice. In vitro experiments indicate both Bhlhb2 and PPARgamma regulate Th1/Th2 cytokine production. We are now studying their functions using in vivo animal models.
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