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BackgroundTET enzymes mediate DNA demethylation by oxidizing 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Since these oxidized methylcytosines (oxi-mCs) are not recognized by the maintenance methyltransferase DNMT1, DNA demethylation can occur through “passive,” replication-dependent dilution when cells divide. A distinct, replication-independent (“active”) mechanism of DNA demethylation involves excision of 5fC and 5caC by the DNA repair enzyme thymine DNA glycosylase (TDG), followed by base excision repair.ResultsHere by analyzing inducible gene-disrupted mice, we show that DNA demethylation during primary T cell differentiation occurs mainly through passive replication-dependent dilution of all three oxi-mCs, with only a negligible contribution from TDG. In addition, by pyridine borane sequencing (PB-seq), a simple recently developed method that directly maps 5fC/5caC at single-base resolution, we detect the accumulation of 5fC/5caC in TDG-deleted T cells. We also quantify the occurrence of concordant demethylation within and near enhancer regions in theIl4locus. In an independent system that does not involve cell division, macrophages treated with liposaccharide accumulate 5hmC at enhancers and show altered gene expression without DNA demethylation; loss of TET enzymes disrupts gene expression, but loss of TDG has no effect. We also observe that mice with long-term (1 year) deletion ofTdgare healthy and show normal survival and hematopoiesis.ConclusionsWe have quantified the relative contributions of TET and TDG to cell differentiation and DNA demethylation at representative loci in proliferating T cells. We find that TET enzymes regulate T cell differentiation and DNA demethylation primarily through passive dilution of oxi-mCs. In contrast, while we observe a low level of active, replication-independent DNA demethylation mediated by TDG, this process does not appear to be essential for immune cell activation or differentiation.

The Trithorax Cxxc1 molecule specifically regulates the chromatin accessibility of the genes that are downregulated during priming and reactivated in the later phase in differentiating CD4 T cells. The authors show Cxxc1 deletion induces CD4 T cell hyperactivation and enhanced allergic airway inflammation., Different dynamics of gene expression are observed during cell differentiation. In T cells, genes that are turned on early or turned off and stay off have been thoroughly studied. However, genes that are initially turned off but then turned on again after stimulation has ceased have not been defined; they are obviously important, especially in the context of acute versus chronic inflammation. Using the Th1/Th2 differentiation paradigm, we found that the Cxxc1 subunit of the Trithorax complex directs transcription of genes initially down-regulated by TCR stimulation but up-regulated again in a later phase. The late up-regulation of these genes was impaired either by prolonged TCR stimulation or Cxxc1 deficiency, which led to decreased expression of Trib3 and Klf2 in Th1 and Th2 cells, respectively. Loss of Cxxc1 resulted in enhanced pathogenicity in allergic airway inflammation in vivo. Thus, Cxxc1 plays essential roles in the establishment of a proper CD4+ T cell immune system via epigenetic control of a specific set of genes., Graphical Abstract

Memory helper T (Th) cells are crucial for secondary immune responses against infectious microorganisms but also drive the pathogenesis of chronic inflammatory diseases. Therefore, it is of fundamental importance to understand how memory T cells are generated. However, the molecular mechanisms governing memory Th cell generation remain incompletely understood. Here, we identified CD30 as a molecule heterogeneously expressed on effector Th1 and Th17 cells, and CD30(hi)effector Th1 and Th17 cells preferentially generated memory Th1 and Th17 cells. We found that CD30 mediated signal induced Transglutaminase-2 (TG2) expression, and that the TG2 expression in effector Th cells is essential for memory Th cell generation. In fact,Cd30-deficiency resulted in the impaired generation of memory Th1 and Th17 cells, which can be rescued by overexpression of TG2. Furthermore, transglutaminase-2(Tgm2)-deficient CD4 T cells failed to become memory Th cells. As a result, T cells fromTgm2-deficient mice displayed impaired antigen-specific antibody production and attenuated Th17-mediated allergic responses. Our data indicate that CD30-induced TG2 expression in effector Th cells is essential for the generation of memory Th1 and Th17 cells, and that CD30 can be a marker for precursors of memory Th1 and Th17 cells. Refereed/Peer-reviewed

Mycobacterium bovis Bacille Calmette–Guérin (BCG) has been shown to possess potent anti-tumor activity particularly in various animal models, while the cellular and molecular mechanisms underlying its activity are not well understood. We found that lipomannan (BCG-LM), a lipophilic component of the mycobacterial cell envelope, specifically inhibits tumor growth and induces the infiltration of eosinophils at local tumor invasion sites. In contrast, neither lipoarabinomannan (BCG-LAM) nor the cell wall of Mycobacterium bovis BCG (BCG-CW) exerted anti-tumor immunity. BCG-LM enhances cytotoxic activity of eosinophils via the increased production of superoxide. Global transcriptomic analyses of BCG-LM-pulsed dendritic cells identified C-C motif ligand (CCL) 5 as a crucial chemokine for the anti-tumor immunity induced by BCG-LM, indicating that CCL5 plays an important role for the accumulation of eosinophils in the tumor microenvironment. Furthermore, BCG-LM and memory Th2 cells exerted a synergetic effect on tumor progression by cooperatively enhancing the eosinophil function. Thus, this study revealed an un-identified BCG-LM-mediated anti-tumor mechanism via superoxide produced by infiltrated eosinophils in the tumor microenvironment. Since BCG-LM activates this unique pathway, it may have potent therapeutic potential as immune cell therapy for cancer patients.

T cells expressing chimeric antigen receptors (CAR T cells) have shown impressive therapeutic efficacy against leukemias and lymphomas. However, they have not been as effective against solid tumors because they become hyporesponsive ("exhausted" or "dysfunctional") within the tumor microenvironment, with decreased cytokine production and increased expression of several inhibitory surface receptors. Here we define a transcriptional network that mediates CD8

Memory T helper (mTh) cells play important roles in the reinfection of pathogens and drive the pathogenesis of diseases. While recent studies have characterized the pathogenic mTh2 cell subpopulations driving allergic inflammation, those that induce immune responses against helminth infection remain unknown. We found that IL-5–producing CXCR6 + ST2 + CD44 + mTh2 cells play a crucial role in the IL-33–dependent inhibition of the fecundity of helminth, whereas other ST2 − mTh2 cells do not. Although both cell types induced the infiltration of granulocytes, especially eosinophils, into the lungs in response to helminth infection, the ST2 + mTh2 cell-induced eosinophils expressed higher levels of major basic protein (MBP), which is important for reducing the fecundity of Nippostrongylus brasiliensis (Nb), than ST2 − mTh2 cell-induced ones. Notably, we also found that ST2 + Treg cells but not ST2 − Treg cells suppressed CXCR6 + ST2 + mTh2 cell-mediated immune responses. Taken together, these findings show that we identified a mechanism against helminth elicited by a subpopulation of IL-5–producing mTh2 cells through the accumulation of eosinophils strongly expressing MBP in the lungs.

Immunological memory is central to adaptive immunity and protection from disease. Changing metabolic demands as antigen-specific T cells transition from effector to memory cells have been well documented, but the cell-specific pathways and molecules that govern this transition are poorly defined. Here we show that genetic deletion of ACC1, a rate-limiting enzyme in fatty acid biosynthesis, enhances the formation of CD4

ST2hi memory-type Th2 cells are identified as a pathogenic subpopulation in eosinophilic airway inflammation. These ST2hi pathogenic Th2 cells produce large amount of IL-5 upon T cell receptor stimulation, but not in response to IL-33 treatment. By contrast, IL-33 alone induces cytokine production in ST2+ group 2 innate lymphoid cells (ILC2). Here we show that a MAPK phosphatase Dusp10 is a key negative regulator of IL-33-induced cytokine production in Th2 cells. In this regard, Dusp10 is expressed by ST2hi pathogenic Th2 cells but not by ILC2, and Dusp10 expression inhibits IL-33-induced cytokine production. Mechanistically, this inhibition is mediated by DUSP10-mediated dephosphorylation and inactivation of p38 MAPK, resulting in reduced GATA3 activity. The deletion of Dusp10 renders ST2hi Th2 cells capable of producing IL-5 by IL-33 stimulation. Our data thus suggest that DUSP10 restricts IL-33-induced cytokine production in ST2hi pathogenic Th2 cells by controlling p38-GATA3 activity., T helper 2 (Th2) cells and type 2 innate lymphoid cells (ILC2) respond differently to interleukin-33 (IL-33) stimulation. Here the authors show that a phosphatase, Dusp10, is expressed in Th2, but not ILC2, to dephosphorylate p38 kinase, reduce GATA3 transcription factor activity, and suppress the induction of IL-5 in response to IL-33.

Memory T cells provide long-lasting protective immunity, and distinct subpopulations of memory T cells drive chronic inflammatory diseases such as asthma. Asthma is a chronic allergic inflammatory disease with airway remodeling including fibrotic changes. The immunological mechanisms that induce airway fibrotic changes remain unknown. We found that interleukin-33 (IL-33) enhanced amphiregulin production by the IL-33 receptor, ST2hi memory T helper 2 (Th2) cells. Amphiregulin-epidermal growth factor receptor (EGFR)-mediated signaling directly reprogramed eosinophils to an inflammatory state with enhanced production of osteopontin, a key profibrotic immunomodulatory protein. IL-5-producing memory Th2 cells and amphiregulin-producing memory Th2 cells appeared to cooperate to establish lung fibrosis. The analysis of polyps from patients with eosinophilic chronic rhinosinusitis revealed fibrosis with accumulation of amphiregulin-producing CRTH2hiCD161hiCD45RO+CD4+ Th2 cells and osteopontin-producing eosinophils. Thus, the IL-33-amphiregulin-osteopontin axis directs fibrotic responses in eosinophilic airway inflammation and is a potential target for the treatment of fibrosis induced by chronic allergic disorders.

Helper T (Th) cell subsets direct immune responses by producing signature cytokines. Th2 cells produce IL-4, IL-5, and IL-13, which are important in humoral immunity and protection from helminth infection and are central to the pathogenesis of many allergic inflammatory diseases. Molecular analysis of Th2 cell differentiation and maintenance of function has led to recent discoveries that have refined our understanding of Th2 cell biology. Epigenetic regulation of Gata3 expression by chromatin remodeling complexes such as Polycomb and Trithorax is crucial for maintaining Th2 cell identity. In the context of allergic diseases, memory-type pathogenic Th2 cells have been identified in both mice and humans. To better understand these disease-driving cell populations, we have developed a model called the pathogenic Th population disease induction model. The concept of defined subsets of pathogenic Th cells may spur new, effective strategies for treating intractable chronic inflammatory disorders. Refereed/Peer-reviewed

A novel isoform of Sox5, Sox5t, and c-Maf activate RORγt to induce Th17 cells. Sox5−/− mice exhibit impaired Th17 differentiation and are thus resistant to EAE and delayed-type hypersensitivity., Stat3 signaling is essential for the induction of RORγt and subsequent Th17 cell differentiation. However, the downstream targets of Stat3 for RORγt expression remain largely unknown. We show here that a novel isoform of Sox5, named Sox5t, is induced in Th17 cells in a Stat3-dependent manner. In vivo, T cell–specific Sox5-deficient mice exhibit impaired Th17 cell differentiation and are resistant to experimental autoimmune encephalomyelitis and delayed-type hypersensitivity. Retrovirus-mediated induction of Sox5 together with c-Maf induces Th17 cell differentiation even in Stat3-deficient CD4+ T cells but not in RORγt-deficient CD4+ T cells, indicating that Sox5 and c-Maf induce Th17 cell differentiation as downstream effectors of Stat3 and as upstream inducers of RORγt. Moreover, Sox5 physically associates with c-Maf via the HMG domain of Sox5 and DNA-binding domain of c-Maf, and Sox5 together with c-Maf directly activates the promoter of RORγt in CD4+ T cells. Collectively, our results suggest that Sox5 and c-Maf cooperatively induce Th17 cell differentiation via the induction of RORγt as downstream targets of Stat3.

Posttranslational modifications of histones are well-established epigenetic modifications that play an important role in gene expression and regulation. These modifications are partly mediated by the Trithorax group (TrxG) complex, which regulates the induction or maintenance of gene transcription. We investigated the role of Menin, a component of the TrxG complex, in the acquisition and maintenance of Th2 cell identity using T cell–specific Menin-deficient mice. Our gene expression analysis revealed that Menin was involved in the maintenance of the high expression of the previously identified Th2-specific genes rather than the induction of these genes. This result suggests that Menin plays a role in the maintenance of Th2 cell identity. Menin directly bound to the Gata3 gene locus, and this Menin-Gata3 axis appeared to form a core unit of the Th2-specific gene regulatory network. Consistent with the phenotype of Menin-deficient Th2 cells observed in vitro, Menin deficiency resulted in the attenuation of effector Th2 cell–induced airway inflammation. In addition, in memory Th2 (mTh2) cells, Menin was found to play an important role in the maintenance of the expression of Th2-specific genes, including Gata3, Il4, and Il13. Consequently, Menin-deficient mTh2 cells showed an impaired ability to recruit eosinophils to the lung, resulting in the attenuation of mTh2 cell–induced airway inflammation. This study confirmed the critical role of Menin in Th2 cell–mediated immune responses.

Background: Natural killer T (NKT) cells express a T-cell receptor that recognizes endogenous and environmental glycolipid antigens. Several subsets of NKT cells have been identified, including IFN-γ–producing NKT1 cells, IL-4–producing NKT2 cells, and IL-17–producing NKT17 cells. However, little is known about the factors that regulate their differentiation and respective functions within the immune system. Objective: We sought to determine whether the polycomb repressive complex 2 protein enhancer of zeste homolog 2 (Ezh2) restrains pathogenicity of NKT cells in the context of asthma-like lung disease. Methods: Numbers of invariant natural killer T (iNKT) 1, iNKT2, and iNKT17 cells and tissue distribution, cytokine production, lymphoid tissue localization, and transcriptional profiles of iNKT cells from wild-type and Ezh2 knockout (KO) iNKT mice were determined. The contribution of NKT cells to development of spontaneous and house dust mite–induced airways pathology, including airways hyperreactivity (AHR) to methacholine, was also assessed in wild-type, Ezh2 KO, and Ezh2 KO mice lacking NKT cells. Results: Ezh2 restrains development of pathogenic NKT cells, which induce spontaneous asthma-like disease in mice. Deletion of Ezh2 increased production of IL-4 and IL-13 and induced spontaneous AHR, lung inflammation, mucus production, and IgE. Increased IL-4 and IL-13 levels, AHR, lung inflammation, and IgE levels were all dependent on iNKT cells. In house dust mite–exposed animals Ezh2 KO resulted in enhanced AHR that was also dependent on iNKT cells. Conclusion: Ezh2 is a central regulator of iNKT pathogenicity and suppresses the ability of iNKT cells to induce asthma-like pathology. Refereed/Peer-reviewed

Sox4 is a transcription factor that regulates various developmental processes. Here we show that Sox4 was induced by TGF-β and negatively regulated the transcription factor GATA-3, the master regulator of function of T helper type 2 (T H 2) cells, by two distinct mechanisms. First, Sox4 bound directly to GATA-3, preventing its binding to GATA-3 consensus DNA sequences. Second, Sox4 bound to the promoter region of the gene encoding interleukin 5 (IL-5), a T H 2 cytokine, and prevented binding of GATA-3 to this promoter. T H 2 cell-driven airway inflammation was modulated by alterations in Sox4 expression. Thus, Sox4 acted as a downstream target of TGF-β to inhibit GATA-3 function, T H 2 differentiation and T H 2 cell-mediated inflammation. Refereed/Peer-reviewed

Trithorax group complex is needed for initiation as well as maintenance of GATA3 gene expression in Th2 cells., Polycomb group (PcG) and trithorax group (TrxG) complexes exert opposing effects on the maintenance of the transcriptional status of the developmentally regulated Hox genes. In this study, we show that activation of STAT6 induces displacement of the PcG complex by the TrxG complex at the upstream region of the gene encoding GATA3, a transcription factor essential for T helper type 2 (Th2) cell differentiation. Once Th2 cells differentiate, TrxG complex associated with the TrxG component Menin binds to the whole GATA3 gene locus, and this binding is required for the long-term maintenance of expression of GATA3 and Th2 cytokine. Thus, STAT6-mediated displacement of PcG by the TrxG complex establishes subsequent STAT6-independent maintenance of GATA3 expression in Th2 cells via the recruitment of the Menin–TrxG complex.

Allergic asthma is an airway inflammatory disease characterized by chronically increased expression of multiple inflammatory proteins, including cytokines, chemokines, adhesion molecules, enzymes, and receptors. Several transcription factors are known to play a crucial role in the pathogenesis of chronic inflammatory diseases such as asthma, including signal transducer and activator of transcription factors, nuclear factor-kappaB, nuclear factor of activated T cells, activator protein-1 family proteins, and Th2 cellrelated transcription factors including GATA3, JunB, and c-Maf. Modulation of the activity of certain transcription factors has been shown to result in the inhibition of inflammation during asthma. Therefore, both agonists and inhibitors of transcription factors may be potential tools for the treatment of asthma. In this review, we summarize the current knowledge of the role of well-established transcription factors in asthma.

SummaryInterleukin-6 (IL-6) and IL-27 signal through a shared receptor subunit and employ the same downstream STAT transcription proteins, but yet are ascribed unique and overlapping functions. To evaluate the specificity and redundancy for these cytokines, we quantified their global transcriptomic changes and determined the relative contributions of STAT1 and STAT3 using genetic models and chromatin immunoprecipitation-sequencing (ChIP-seq) approaches. We found an extensive overlap of the transcriptomes induced by IL-6 and IL-27 and few examples in which the cytokines acted in opposition. Using STAT-deficient cells and T cells from patients with gain-of-function STAT1 mutations, we demonstrated that STAT3 is responsible for the overall transcriptional output driven by both cytokines, whereas STAT1 is the principal driver of specificity. STAT1 cannot compensate in the absence of STAT3 and, in fact, much of STAT1 binding to chromatin is STAT3 dependent. Thus, STAT1 shapes the specific cytokine signature superimposed upon STAT3’s action.

Background Nishiki-nezumi Cinnamon/Nagoya (NC/Nga) mice raised in nonair-controlled conventional circumstances spontaneously develop atopic dermatitis-like skin lesions; however, the underlying mechanisms remain unclear. Objective We wanted to identify the critical intracellular signaling molecules in T cells that induce atopic dermatitis-like skin legions in NC/Nga mice. Methods We examined the levels of signal transduction and cytokine production in T cells, particularly those in atopic dermatitis-like lesions induced by the topical injection of mite antigens in NC/Nga mice under specific pathogen-free conditions. Results In NC/Nga mice maintained under specific pathogen-free conditions, the capability of T H 1/T H 2 and T cytotoxic 1/T cytotoxic 2 (Tc1/Tc2) cell differentiation increased significantly. T-cell antigen receptor–mediated activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase cascade and nuclear factor-κB (NF-κB) signaling were enhanced in NC/Nga T cells. The expression of T H 2 cytokines (IL-4, IL-13, and IL-5) and that of GATA-binding protein 3 (GATA3), avian musculoaponeurotic fibrosarcoma (c-Maf), NF-κB, and activator protein 1 (AP1) selectively increased in draining lymph node T cells of NC/Nga mice. Moreover, the cell transfer of inhibitory NF-κB mutant-infected T H 2 cells reduced ear thickness in the mite antigen-induced skin lesion of NC/Nga mice. Conclusion Hyperresponsive T H 2 cells with an enhanced activity of NF-κB and AP1 play a crucial role in the pathogenesis of atopic dermatitis-like skin lesions in NC/Nga mice. Clinical implications These results indicate potential therapeutic usefulness of developing selective inhibitors for NF-κB in the treatment of human atopic dermatitis.

During T-cell development, Cd8 expression is controlled via dynamic regulation of its cis-regulatory enhancer elements. Insufficiency of enhancer activity causes variegated Cd8 expression in CD4(+)CD8(+) double-positive (DP) thymocytes. Brd1 is a subunit of the Hbo1 histone acetyltransferase (HAT) complex responsible for acetylation of histone H3 at lysine 14 (H3K14). Here we show that deletion of Brd1 in haematopoietic progenitors causes variegated expression of Cd8, resulting in the appearance of CD4(+)CD8(-)TCRβ(-/low) thymocytes indistinguishable from DP thymocytes in their properties. Biochemical analysis confirms that Brd1 forms a HAT complex with Hbo1 in thymocytes. ChIP analysis demonstrates that Brd1 localizes at the known enhancers in the Cd8 genes and is responsible for acetylation at H3K14. These findings indicate that the Brd1-mediated HAT activity is crucial for efficient activation of Cd8 expression via acetylation at H3K14, which serves as an epigenetic mark that promotes the recruitment of transcription machinery to the Cd8 enhancers.

Host defense requires the specification of CD4+ helper T (Th) cells into distinct fates, including Th1 cells that preferentially produce interferon-γ (IFN-γ). IFN-γ, a member of a large family of anti-pathogenic and anti-tumor IFNs, induces T-bet, a lineage-defining transcription factor for Th1 cells, which in turn supports IFN-γ production in a feed-forward manner. Herein, we show that a cell-intrinsic role of T-bet influences how T cells perceive their secreted product in the environment. In the absence of T-bet, IFN-γ aberrantly induced a type I IFN transcriptomic program. T-bet preferentially repressed genes and pathways ordinarily activated by type I IFNs to ensure that its transcriptional response did not evoke an aberrant amplification of type I IFN signaling circuitry, otherwise triggered by its own product. Thus, in addition to promoting Th1 effector commitment, T-bet acts as a repressor in differentiated Th1 cells to prevent abberant autocrine type I IFN and downstream signaling.

Epigenetic modifications, such as posttranslational modifications of histones, play an important role in gene expression and regulation. These modifications are in part mediated by the Trithorax group (TrxG) complex and the Polycomb group (PcG) complex, which activate and repress transcription, respectively. We herein investigate the role of Menin, a component of the TrxG complex in T helper (Th) cell differentiation and show a critical role for Menin in differentiation and maintenance of Th17 cells. Menin -/- T cells do not efficiently differentiate into Th17 cells, leaving Th1 and Th2 cell differentiation intact in in vitro cultures. Menin deficiency resulted in the attenuation of Th17-induced airway inflammation. In differentiating Th17 cells, Menin directly bound to the Il17a gene locus and was required for the deposition of permissive histone modifications and recruitment of the RNA polymerase II transcriptional complex. Interestingly, although Menin bound to the Rorc locus, Menin was dispensable for the induction of Rorc expression and permissive histone modifications in differentiating Th17 cells. In contrast, Menin was required to maintain expression of Rorc in differentiated Th17 cells, indicating that Menin is essential to stabilize expression of the Rorc gene. Thus, Menin orchestrates Th17 cell differentiation and function by regulating both the induction and maintenance of target gene expression. Refereed/Peer-reviewed

GATA binding protein 3 (Gata3) is a GATA family transcription factor that controls differentiation of naïve CD4 T cells into T helper 2 (Th2) cells. However, it is unknown how Gata3 simultaneously activates Th2-specific genes while repressing those of other Th lineages. Here we show that chromodomain helicase DNA-binding protein 4 (Chd4) forms a complex with Gata3 in Th2 cells that both activates Th2 cytokine transcription and represses the Th1 cytokine IFN-γ. We define a Gata3/Chd4/p300 transcriptional activation complex at the Th2 cytokine loci and a Gata3/Chd4–nucleosome remodeling histone deacetylase repression complex at the Tbx21 locus in Th2 cells. We also demonstrate a physiological role for Chd4 in Th2-dependent inflammation in an in vivo model of asthmatic inflammation. Thus, Gata3/Chd4 forms functionally distinct complexes, which mediate both positive and negative gene regulation to facilitate Th2 cell differentiation.

After antigen encounter by CD4 + Tcells, polarizing cytokines induce the expression of master regulators that control differentiation. Inactivation of the histone methyltransferase Ezh2 was found to specifically enhance T helper 1 (Th1) and Th2 cell differentiation and plasticity. Ezh2 directly bound and facilitated correct expression of Tbx21 and Gata3 in differentiating Th1 and Th2 cells, accompanied by substantial trimethylation at lysine 27 of histone 3 (H3K27me3). In addition, Ezh2 deficiency resulted in spontaneous generation of discrete IFN-γ and Th2 cytokine-producing populations in nonpolarizing cultures, and under these conditions IFN-γ expression was largely dependent on enhanced expression of the transcription factor Eomesodermin. Invivo, loss of Ezh2 caused increased pathology in a model of allergic asthma and resulted in progressive accumulation of memory phenotype Th2 cells. This study establishes a functional link between Ezh2 and transcriptional regulation of lineage-specifying genes in terminally differentiated CD4 + Tcells. Refereed/Peer-reviewed

Differentiation of naive CD4 T cells into Th2 cells is accompanied by chromatin remodeling and increased expression of a set of Th2-specific genes, including those encoding Th2 cytokines. IL-4–mediated STAT6 activation induces high levels of transcription of GATA3, a master regulator of Th2 cell differentiation, and enforced expression of GATA3 induces Th2 cytokine expression. However, it remains unclear whether the expression of other Th2-specific genes is induced directly by GATA3. A genome-wide unbiased chromatin immunoprecipitation assay coupled with massive parallel sequencing analysis revealed that GATA3 bound to 1279 genes selectively in Th2 cells, and 101 genes in both Th1 and Th2 cells. Simultaneously, we identified 26 highly Th2-specific STAT6-dependent inducible genes by DNA microarray analysis-based three-step selection processes, and among them 17 genes showed GATA3 binding. We assessed dependency on GATA3 for the transcription of these 26 Th2-specific genes, and 10 genes showed increased transcription in a GATA3-dependent manner, whereas 16 genes showed no significant responses. The transcription of the 16 GATA3-nonresponding genes was clearly increased by the introduction of an active form of STAT6, STAT6VT. Therefore, although GATA3 has been recognized as a master regulator of Th2 cell differentiation, many Th2-specific genes are not regulated by GATA3 itself, but in collaboration with STAT6.

Functionally polarized helper T cells (Th cells) play crucial roles in the induction of tumor immunity. There is considerable knowledge about the contributions of IFN-producing Th1 cells that supports the role of cytotoxic cluster of differentiation (CD8) T cells and natural killer (NK) cells, but much less is known about how IL-4–producing Th2 cells contribute to tumor immunity. In this study, we investigated the cellular and molecular mechanisms employed by memory Th2 cells in sustaining tumor immunity by using a mouse model system wherein ovalbumin (OVA) is used as a specific tumor antigen. In this model, we found that OVA-specific memory Th2 cells exerted potent and long-lasting antitumor effects against NK-sensitive OVA-expressing tumor cells, wherein antitumor effects were mediated by NK cells. Specifically, NK cell cytotoxic activity and expression of perforin and granzyme B were dramatically enhanced by the activation of memory Th2 cells. Interleukin 4 (IL-4) produced by memory Th2 cells in vivo was critical for the antitumor effects of the NK cells, which IL-4 directly stimulated to induce their perforin- and granzyme-B–dependent cytotoxic activity. Our findings show that memory Th2 cells can induce potent antitumor immunity through IL-4–induced activation of NK cells, suggesting potential applications in cellular therapy for cancer patients. Cancer Res; 71(14); 4790–8. ©2011 AACR.

Functionally polarized CD4+ T helper (Th) cells such as Th1, Th2 and Th17 cells are central to the regulation of acquired immunity. However, the molecular mechanisms governing the maintenance of the polarized functions of Th cells remain unclear. GATA3, a master regulator of Th2 cell differentiation, initiates the expressions of Th2 cytokine genes and other Th2-specific genes. GATA3 also plays important roles in maintaining Th2 cell function and in continuous chromatin remodeling of Th2 cytokine gene loci. However, it is unclear whether continuous expression of GATA3 is required to maintain the expression of various other Th2-specific genes. In this report, genome-wide DNA gene expression profiling revealed that GATA3 expression is critical for the expression of a certain set of Th2-specific genes. We demonstrated that GATA3 dependency is reduced for some Th2-specific genes in fully developed Th2 cells compared to that observed in effector Th2 cells, whereas it is unchanged for other genes. Moreover, effects of a loss of GATA3 expression in Th2 cells on the expression of cytokine and cytokine receptor genes were examined in detail. A critical role of GATA3 in the regulation of Th2-specific gene expression is confirmed in in vivo generated antigen-specific memory Th2 cells. Therefore, GATA3 is required for the continuous expression of the majority of Th2-specific genes involved in maintaining the Th2 cell identity.