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Uncontrolled type 2 immunity by type 2 helper T (Th2) cells causes intractable allergic diseases; however, whether the interaction of CD4 + T cells shapes the pathophysiology of allergic diseases remains unclear. We identified a subset of Th2 cells that produced the serine proteases granzyme A and B early in differentiation. Granzymes cleave protease-activated receptor (Par)-1 and induce phosphorylation of p38 mitogen-activated protein kinase (MAPK), resulting in the enhanced production of IL-5 and IL-13 in both mouse and human Th2 cells. Ubiquitin-specific protease 7 (USP7) regulates IL-4-induced phosphorylation of STAT3, resulting in granzyme production during Th2 cell differentiation. Genetic deletion of Usp7 or Gzma and pharmacological blockade of granzyme B ameliorated allergic airway inflammation. Furthermore, PAR-1 + and granzyme + Th2 cells were colocalized in nasal polyps from patients with eosinophilic chronic rhinosinusitis. Thus, the USP7-STAT3-granzymes-Par-1 pathway is a potential therapeutic target for intractable allergic diseases., Competing Interests: Competing interests statement:The authors declare no competing interest.

Type 2 helper T (Th2) cells, a subset of CD4 + T cells, play an important role in the host defense against pathogens and allergens by producing Th2 cytokines, such as interleukin-4 (IL-4), IL-5, and IL-13, to trigger inflammatory responses. Emerging evidence reveals that Th2 cells also contribute to the repair of injured tissues after inflammatory reactions. However, when the tissue repair process becomes chronic, excessive, or uncontrolled, pathological fibrosis is induced, leading to organ failure and death. Thus, proper control of Th2 cells is needed for complete tissue repair without the induction of fibrosis. Recently, the existence of pathogenic Th2 (Tpath2) cells has been revealed. Tpath2 cells produce large amounts of Th2 cytokines and induce type 2 inflammation when activated by antigen exposure or tissue injury. In recent studies, Tpath2 cells are suggested to play a central role in the induction of type 2 inflammation whereas the role of Tpath2 cells in tissue repair and fibrosis has been less reported in comparison to conventional Th2 cells. In this review, we discuss the roles of conventional Th2 cells and pathogenic Th2 cells in the sequence of tissue inflammation, repair, and fibrosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kokubo, Onodera, Kiuchi, Tsuji, Hirahara and Nakayama.)

T cells possess distinguishing effector functions and drive inflammatory disorders. We have previously identified IL-5-producing Th2 cells as the pathogenic population predominantly involved in the pathology of allergic inflammation. However, the cell-intrinsic signaling pathways that control the pathogenic Th2 cell function are still unclear. We herein report the high expression of acetyl-CoA carboxylase 1 (ACC1) in the pathogenic CD4+ T cell population in the lung and skin. The genetic deletion of CD4+ T cell-intrinsic ACC1 dampened eosinophilic and basophilic inflammation in the lung and skin by constraining IL-5 or IL-3 production. Mechanistically, ACC1-dependent fatty acid biosynthesis induces the pathogenic cytokine production of CD4+ T cells via metabolic reprogramming and the availability of acetyl-CoA for epigenetic regulation. We thus identified a distinct phenotype of the pathogenic T cell population in the lung and skin, and ACC1 was shown to be an essential regulator controlling the pathogenic function of these populations to promote type 2 inflammation., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Nakajima et al.)

While IFNγ is a well-known cytokine that actively promotes the type I immune response, it is also known to suppress the type II response by inhibiting the differentiation and proliferation of Th2 cells. However, the mechanism by which IFNγ suppresses Th2 cell proliferation is still not fully understood. We found that IFNγ decreases the expression of growth factor independent-1 transcriptional repressor (GFI1) in Th2 cells, resulting in the inhibition of Th2 cell proliferation. The deletion of the Gfi1 gene in Th2 cells results in the failure of their proliferation, accompanied by an impaired cell cycle progression. In contrast, the enforced expression of GFI1 restores the defective Th2 cell proliferation, even in the presence of IFNγ. These results demonstrate that GFI1 is a key molecule in the IFNγ-mediated inhibition of Th2 cell proliferation., Competing Interests: The authors declare no conflicts of interest in association with the present study.

Antigen-primed T cells respond to restimulation much faster than naïve T cells and form the cellular basis of immunological memory. The formation of memory Th2 cells starts when naïve CD4 T cells are transformed into effector Th2 cells and is completed after antigen clearance and a long-term resting phase accompanied by epigenetic changes in the Th2 signature genes. Memory Th2 cells maintain their functions and acquired heterogeneity through epigenetic machinery, on which the recall-response of memory Th2 cells is also dependent. We provide an overview of the epigenetics in the whole Th2 cell cycle, mainly focusing on two different histone lysine methyltransferase complexes: the Polycomb and Trithorax groups. We finally discuss the pathophysiology and potential therapeutic strategies for the treatment of Th2-mediated inflammatory diseases in mice and humans.

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., Competing Interests: The authors declare no conflict of interest.

ST2 hi memory-type Th2 cells are identified as a pathogenic subpopulation in eosinophilic airway inflammation. These ST2 hi 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 ST2 hi 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 ST2 hi Th2 cells capable of producing IL-5 by IL-33 stimulation. Our data thus suggest that DUSP10 restricts IL-33-induced cytokine production in ST2 hi pathogenic Th2 cells by controlling p38-GATA3 activity.

Background: Although Th2 cells are well known to play important roles in allergic diseases including allergic rhinitis (AR), the factors that induce and sustain the pathogenesis of AR remain unclear. The recent development of sublingual immunotherapy (SLIT) is expected to allow changes to the underlying pathogenesis of AR. However, which Th2 cell subsets are important in house dust mite-induced AR (HDM-AR), the influence of SLIT on the pathogenic Th2 cells, and the association of Th2 cell subsets with SLIT efficacy have not been clarified., Methods: The cytokine production and frequency of HDM-reactive T-cell subsets in peripheral blood mononuclear cells (PBMCs) were evaluated using flow cytometry in 89 HDM-AR patients (placebo [n = 43] and HDM 300 IR [n = 46]) who participated in a placebo-controlled study of SLIT with HDM tablets. All patients provided samples both before treatment as a baseline and at the end of the 52-week study. The PBMCs were stained with CellTrace™ Violet (CTV) before culture with HDM extract, and HDM-reactive T cells were detected as the proliferated cells with diminished CTV., Results: HDM-reactive IL-5 + IL-13 + CD27 - CD161 + CD4 + cells and ST2 + CD45RO + CD4 + cells were observed in the peripheral blood from each patient with HDM-AR; these cells significantly decreased after SLIT in the group treated with active tablets. HDM-reactive ST2 + CD45RO + CD4 + cells were significantly lower in active-responders., Conclusion: Allergen-reactive ST2 + CD45RO + CD4 + cells or those combined with IL-5 + IL-13 + CD27 - CD161 + CD4 + cells may be useful as markers indicating the successful treatment of SLIT. These cells may play a crucial role in the pathogenesis of AR as pathogenic memory Th2 cells., (© 2018 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Atopic dermatitis is a chronic inflammatory skin disease involving T-helper (Th) 2 cells, eosinophils, and mast cells. Although CCR4 is a major chemokine receptor expressed on Th2 cells and regarded as a potential therapeutic target for allergic diseases, its role in atopic dermatitis remains unclear. Here, by using a hydrogel patch as a transcutaneous delivery device for ovalbumin (an antigen) and Staphylococcus aureus δ-toxin (a mast cell activator), we efficiently induced acute atopic dermatitis-like skin lesions in BALB/c mice, a strain prone to Th2 responses, which were characterized by increased numbers of eosinophils, mast cells, and CCR4-expressing Th2 cells in the skin lesions; elevated levels of total and ovalbumin-specific IgE in the sera; and increased expression of IL-4, IL-17A, IL-22, CCL17, CCL22, and CCR4 in the skin lesions. Of note, the same model was less efficient in C57BL/6 mice, a strain prone to Th1 responses. Using this atopic dermatitis model in BALB/c mice, we demonstrated that CCR4-deficiency or a CCR4 antagonist ameliorated the allergic responses. Collectively, these results demonstrate that CCR4 plays a pivotal role in skin allergic inflammation of BALB/c mice by recruiting CCR4-expressing Th2 cells and Th17 cells., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

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, ST2 hi 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 CRTH2 hi CD161 hi CD45RO + 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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Allergic rhinitis (AR) consists of three developmental stages that are based on the presence/absence of antigen-specific IgE and symptoms. The pathogenic Th2 (Tpath2) cells constitute a population of Th2 cells with additional potentially pathogenic characteristics. We examined the relationship between Tpath2 cells and the stages of allergic rhinitis by focusing on ST2, which is an IL-33 receptor., Methods: Patients with Japanese cedar pollen-induced AR (JCP-AR) and healthy volunteers were divided into "nonsensitized," "asymptomatic sensitized (AS)," and "JCP-AR" groups. We analyzed the ST2 expression and the Th2 function of cultured CD4 + T cells. Next, we observed the progress of patients in the AS stage around the time of seasonal pollen dispersal, with the characteristics of Th2 cells., Results: The ST2 expression of T cells was only upregulated in the AR group. The production of IL-4 and IL-13 was found in CD4 + T cells obtained from AS by stimulation with JCP, but reactivity to IL-33 was not observed. Although IL-33 did not induce the elevation of IL-4 production in the JCP-AR group, IL-33 substantially increased the production of IL-5 and IL-13 in comparison with antigen stimulation alone. In newly afflicted patients, the increased expression of ST2 and elevated reactivity to IL-33 was observed, even before the pollen dispersal season., Conclusions: Our study demonstrated that the pathogenicity of memory Th2 cells is linked to sensitization and the stage of allergic rhinitis. Therefore, Tpath2 cells may provide useful insights into the mechanism of the onset and progression of allergic rhinitis., (© 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

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., (© The Japanese Society for Immunology. 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Immunological memory is an important protective mechanism that enables host organisms to respond rapidly and vigorously to pathogens that have been previously encountered. In addition to the protective function, memory CD4 + T helper (Th) cells play a central role in the pathogenesis of chronic inflammatory disorders, including asthma. Recently, several investigators have identified phenotypically and functionally distinct memory Th2 cell subsets that produce IL-5. These memory Th2 cell subsets play an important role in the pathology of allergic inflammation and function as memory-type "pathogenic Th2 (Tpath2) cells" both in mice and humans. We review the role of lung Tpath2 cells in the development of allergic inflammation and, in the context of recent findings, propose a mechanism by which Tpath2 cells not only survive but also continue to function at the sites where antigens were encountered. A greater understanding of the functional molecules or signaling pathways that regulate the inflammatory niche for Tpath2 cells may aid in the design of more effective treatments for chronic inflammatory disorders., (Copyright © 2017 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.)

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.

Although Bach2 has an important role in regulating the Th2-type immune response, the underlying molecular mechanisms remain unclear. We herein demonstrate that Bach2 associates with Batf and binds to the regulatory regions of the Th2 cytokine gene loci. The Bach2-Batf complex antagonizes the recruitment of the Batf-Irf4 complex to AP-1 motifs and suppresses Th2 cytokine production. Furthermore, we find that Bach2 regulates the Batf and Batf3 expressions via two distinct pathways. First, Bach2 suppresses the maintenance of the Batf and Batf3 expression through the inhibition of IL-4 production. Second, the Bach2-Batf complex directly binds to the Batf and Batf3 gene loci and reduces transcription by interfering with the Batf-Irf4 complex. These findings suggest that IL-4 and Batf form a positive feedback amplification loop to induce Th2 cell differentiation and the subsequent Th2-type immune response, and Bach2-Batf interactions are required to prevent an excessive Th2 response.

Th2 cells produce Th2 cytokines such as IL-4, IL-5 and IL-13, but repress Th1 cytokine IFNγ. Recent studies have revealed various distinct memory-type Th2 cell subsets, one of which produces a substantial amount of IFNγ in addition to Th2 cytokines, however it remains unclear precisely how these Th2 cells produce IFNγ. We herein show that phosphorylation of Gata3 at Ser308, Thr315 and Ser316 induces dissociation of a histone deacetylase Hdac2 from the Gata3/Chd4 repressive complex in Th2 cells. We also identify Akt1 as a Gata3-phosphorylating kinase, and the activation of Akt1 induces derepression of Tbx21 and Ifng expression in Th2 cells. Moreover, T-bet-dependent IFNγ expression in IFNγ-producing memory Th2 cells appears to be controlled by the phosphorylation status of Gata3 in human and murine systems. Thus, this study highlights the molecular basis for posttranslational modifications of Gata3 that control the regulation of IFNγ expression in memory Th2 cells.

Gata3 acts as a master regulator for T helper 2 (Th2) cell differentiation by inducing chromatin remodeling of the Th2 cytokine loci, accelerating Th2 cell proliferation, and repressing Th1 cell differentiation. Gata3 also directly transactivates the interleukin-5 (Il5) gene via additional mechanisms that have not been fully elucidated. We herein identified a mechanism whereby the methylation of Gata3 at Arg-261 regulates the transcriptional activation of the Il5 gene in Th2 cells. Although the methylation-mimicking Gata3 mutant retained the ability to induce IL-4 and repress IFNγ production, the IL-5 production was selectively impaired. We also demonstrated that heat shock protein (Hsp) 60 strongly associates with the methylation-mimicking Gata3 mutant and negatively regulates elongation of the Il5 transcript by RNA polymerase II. Thus, arginine methylation appears to play a pivotal role in the organization of Gata3 complexes and the target gene specificity of Gata3., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Memory CD4(+) T helper (Th) cells provide long-term protection against pathogens and are essential for the development of vaccines; however, some antigen-specific memory Th cells also drive immune-related pathology, including asthma. The mechanisms regulating the pathogenicity of memory Th cells remain poorly understood. We found that interleukin-33 (IL-33)-ST2 signals selectively licensed memory Th2 cells to induce allergic airway inflammation via production of IL-5 and that the p38 MAP kinase pathway was a central downstream target of IL-33-ST2 in memory Th2 cells. In addition, we found that IL-33 induced upregulation of IL-5 by memory CD4(+) T cells isolated from nasal polyps of patients with eosinophilic chronic rhinosinusitis. Thus, IL-33-ST2-p38 signaling appears to directly instruct pathogenic memory Th2 cells to produce IL-5 and induce eosinophilic inflammation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Dendritic cells (DCs) play critical roles in determining the fate of CD4⁺ T cells. Among DC sub-populations, monocyte-derived inflammatory DCs (iDCs) have been shown to play an important role in the induction of adaptive immune responses under inflammatory conditions. Although previous studies have shown that DCs have an indispensable role in the induction of allergic airway inflammation and airway hyperreactivity (AHR) in murine asthma models, the precise roles of iDCs in the asthmatic responses remain largely unknown. We show here that T(h)2 cell-mediated inflammation in murine asthma models induces the expression of some markers of alternatively activated macrophage such as arginase 1 and resistin-like molecule-α in iDCs by a mechanism depending on the intrinsic expression of STAT6. In contrast, T(h)1 cell-mediated inflammation induces iDCs to express TNF-α and inducible nitric oxide synthase (iNOS), markers of TNF-α- and iNOS-producing DCs. Moreover, we show that iDCs under a T(h)2 environment play an important role in the induction of AHR, independently of allergic airway inflammation. Our results thus indicate the importance of iDCs in the induction of AHR as downstream effector cells in T(h)2 cell-mediated asthmatic responses.

Immunological memory is a hallmark of adaptive immunity. Memory CD4 T helper (Th) cells are central to acquired immunity, and vaccines for infectious diseases are developed based on this concept. However, memory Th cells also play a critical role in the pathogenesis of various chronic inflammatory diseases, including asthma. We refer to these populations as 'pathogenic memory Th cells.' Here, we review recent developments highlighting the functions and characteristics of several pathogenic memory type Th2 cell subsets in allergic inflammation. Also discussed are the similarities and differences between pathogenic memory Th2 cells and recently identified type 2 innate lymphoid cells (ILC2), focusing on cytokine production and phenotypic profiles., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

After antigen encounter by CD4(+) T cells, 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. In vivo, 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(+) T cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

GATA-binding protein 3 (Gata3) controls the differentiation of naive CD4 T cells into T helper 2 (Th2) cells by induction of chromatin remodeling of the Th2 cytokine gene loci, direct transactivation of Il5 and Il13 genes, and inhibition of Ifng. Gata3 also facilitates Th2 cell proliferation via additional mechanisms that are far less well understood. We herein found that Gata3 associates with RuvB-like protein 2 (Ruvbl2) and represses the expression of a CDK inhibitor, cyclin-dependent kinase inhibitor 2c (Cdkn2c) to facilitate the proliferation of Th2 cells. Gata3 directly bound to the Cdkn2c locus in an Ruvbl2-dependent manner. The defect in the proliferation of Gata3-deficient Th2 cells is rescued by the knockdown of Cdkn2c, indicating that Cdkn2c is a key molecule involved in the Gata3-mediated induction of Th2 cell proliferation. Ruvbl2-knockdown Th2 cells showed decreased antigen-induced expansion and caused less airway inflammation in vivo. We therefore have identified a functional Gata3/Ruvbl2 complex that regulates the proliferation of differentiating Th2 cells through the repression of a CDK inhibitor, Cdkn2c.

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.

IL-5 is a key cytokine that plays an important role in the development of pathological conditions in allergic inflammation. Identifying strategies to inhibit IL-5 production is important in order to establish new therapies for treating allergic inflammation. We found that SH-2251, a novel thioamide-related small compound, selectively inhibits the differentiation of IL-5-producing Th2 cells. SH-2251 inhibited the induction of active histone marks at the Il5 gene locus during Th2 cell differentiation. The recruitment of RNA polymerase II, and following expression of the Th2 cell-specific intergenic transcripts around the Il5 gene locus was also inhibited. Furthermore, Th2 cell-dependent airway inflammation in mice was suppressed by the oral administration of SH-2251. Gfi1, a transcriptional repressor, was identified as a downstream target molecule of SH-2251 using a DNA microarray analysis. The Gfi1 expression dramatically decreased in SH-2251-treated Th2 cells, and the SH-2251-mediated inhibition of IL-5-producing Th2 cell differentiation was restored by transduction of Gfi1. Therefore, our study unearthed SH-2251 as a novel therapeutic candidate for allergic inflammation that selectively inhibits active histone marks at the Il5 gene locus.

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.

The transcriptional repressor Bcl6 is a critical arbiter of Th cell fate, promoting the follicular Th lineage while repressing other Th cell lineages. Bcl6-deficient (Bcl6(-/-)) mice develop a spontaneous and severe Th2-type inflammatory disease, thus warranting assessment of Bcl6 in regulatory T cell (Treg) function. Bcl6(-/-) Tregs were competent at suppressing T cell proliferation in vitro and Th1-type colitogenic T cell responses in vivo. In contrast, Bcl6(-/-) Tregs strongly exacerbated lung inflammation in a model of allergic airway disease and promoted higher Th2 responses, including systemic upregulation of microRNA-21. Further, Bcl6(-/-) Tregs were selectively impaired at controlling Th2 responses, but not Th1 and Th17 responses, in mixed chimeras of Bcl6(-/-) bone marrow with Foxp3(-/-) bone marrow. Bcl6(-/-) Tregs displayed increased levels of the Th2 transcription factor Gata3 and other Th2 and Treg genes. Bcl6 potently repressed Gata3 transcriptional transactivation, providing a mechanism for the increased expression of Th2 genes by Bcl6(-/-) Tregs. Gata3 has a critical role in regulating Foxp3 expression and functional fitness of Tregs; however, the signal that regulates Gata3 and restricts its transactivation of Th2 cytokines in Tregs has remained unexplored. Our results identify Bcl6 as an essential transcription factor regulating Gata3 activity in Tregs. Thus, Bcl6 represents a crucial regulatory layer in the Treg functional program that is required for specific suppression of Gata3 and Th2 effector responses by Tregs.

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.

Lactic acid bacteria have potential in immunomodulation therapy, but their clinical efficacy and underlying mechanisms are unclear. We aimed to clarify the anti-allergic immune responses induced by intragastric and sublingual administration of heat-killed Lactobacillus paracasei KW3110 and Lactobacillus acidophilus L-92. The KW3110 strain (but not the L-92 strain) enhanced ovalbumin (OVA)-induced expression of CCR-7 and PD-L2 in murine dendritic cells (DCs), and strongly inhibited IL-5 and IL-13 production in vitro in co-cultures with Th2-skewed CD4(+) T cells from DO11.10 transgenic mice. Sublingual administration of low-dose KW3110 (but not L-92) to OVA-sensitized mice selectively suppressed serum IgE production and Th2 cytokine expression in cervical lymph nodes, and significantly improved symptoms after OVA provocation in vivo. KW3110 probably accelerates DC migration into the regional lymph nodes and inhibits Th2 cytokine production through enhanced CCR-7 and PD-L2 expression. Thus, sublingual KW3110 administration may be effective in reducing allergic inflammation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The regulation of memory CD4(+) helper T (Th) cell function, such as polarized cytokine production, remains unclear. Here we show that memory T helper 2 (Th2) cells are divided into four subpopulations by CD62L and CXCR3 expression. All four subpopulations produced interleukin-4 (IL-4) and IL-13, whereas only the CD62L(lo)CXCR3(lo) population produced IL-5 accompanied by increased H3-K4 methylation at the Il5 gene locus. The transcription factor Eomesodermin (encoded by Eomes) was highly expressed in memory Th2 cells, whereas its expression was selectively downregulated in the IL-5-producing cells. Il5 expression was enhanced in Eomes-deficient cells, and Eomesodermin was shown to interact with the transcription factor GATA3, preventing GATA3 binding to the Il5 promoter. Memory Th2 cell-dependent airway inflammation was attenuated in the absence of the CD62L(lo)CXCR3(lo) population but was enhanced by Eomes-deficient memory Th2 cells. Thus, IL-5 production in memory Th2 cells is regulated by Eomesodermin via the inhibition of GATA3 activity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (©2011 AACR.)

The cytokine thymic stromal lymphopoietin (TSLP) has been implicated in the development and progression of allergic inflammation in both humans and mice. TSLP has been shown to promote a Th2-type response through upregulation of OX40L on dendritic cells, and through direct induction of IL-4 production in naïve CD4+ T cells. However, its direct effect on effector Th cells has not been extensively investigated. In this study, we show that the level of TSLP receptor (TSLPR) expression on mouse effector Th2 cells is higher than on Th1 and Th17 cells, and that TSLP induced proliferation of effector Th2, but not Th1 nor Th17 cells. TSLP also induced the phosphorylation of signal transducer and activator of transcription (Stat) 5, and expression of the anti-apoptotic factor Bcl-2 in Th2 cells. Finally, TSLP-mediated proliferation on Th2 cells was enhanced by TCR stimulation, through IL-4-mediated induction of TSLPR expression. Taken together, these results indicate that TSLP is involved in exacerbation of mouse Th2-mediated allergic inflammation in a Th2 environment through direct stimulation of Th2 effector cells., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Repressor of GATA (ROG) inhibits Th2 cell differentiation and allergic airway inflammation in the lung. To determine the role of ROG in the pathogenesis of contact hypersensitivity (CHS), a hapten-induced mouse model of CHS using ROG Tg and ROG-deficient (ROG(-/-)) was used. ROG Tg mice showed little ear swelling, while ROG(-/-) mice showed enhanced ear swelling in comparison to wild type mice. Interstitial edema and mast cell degranulation at the local inflammation sites were mild in ROG Tg mice and exacerbated in ROG(-/-) mice. In addition, the serum total IgE and hapten-specific IgG1 levels were increased in ROG(-/-) mice. Adoptive transfer of ROG(-/-) CD4(+) T cells exacerbated CHS in wild type mice, while transfer of ROG Tg CD4(+) T cells resulted in the attenuation of CHS. These results indicate ROG negatively regulates the induction of CHS by controlling the CD4(+) T cell-mediated allergic responses, including IgE generation and mast cell degranulation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

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.

Polycomb group (PcG) gene products regulate the maintenance of homeobox gene expression in Drosophila and vertebrates. In the immune system, PcG molecules control cell cycle progression of thymocytes, Th2 cell differentiation, and the generation of memory CD4 T cells. In this paper, we extended the study of PcG molecules to the regulation of in vivo Th2 responses, especially allergic airway inflammation, by using conditional Ring1B-deficient mice with a CD4 T cell-specific deletion of the Ring1B gene (Ring1B(-/-) mice). In Ring1B(-/-) mice, CD4 T cell development appeared to be normal, whereas the differentiation of Th2 cells but not Th1 cells was moderately impaired. In an Ag-induced Th2-driven allergic airway inflammation model, eosinophilic inflammation was attenuated in Ring1B(-/-) mice. Interestingly, Ring1B(-/-) effector Th2 cells were highly susceptible to apoptosis in comparison with wild-type effector Th2 cells in vivo and in vitro. The in vitro experiments revealed that the expression of Bim was increased at both the transcriptional and protein levels in Ring1B(-/-) effector Th2 cells, and the enhanced apoptosis in Ring1B(-/-) Th2 cells was rescued by the knockdown of Bim but not the other proapoptotic genes, such as Perp, Noxa, or Bax. The enhanced apoptosis detected in the transferred Ring1B(-/-) Th2 cells in the lung of the recipient mice was also rescued by knockdown of Bim. Therefore, these results indicate that Ring1B plays an important role in Th2-driven allergic airway inflammation through the control of Bim-dependent apoptosis of effector Th2 cells in vivo.

Schnurri (Shn) is a large zinc-finger containingprotein, which plays a critical role in cell growth, signal transduction and lymphocyte development. There are three orthologues (Shn-1, Shn-2 and Shn-3) in vertebrates. In Shn-2-deficient mice, the activation of NF-kappaB in CD4 T cells is upregulated and their ability to differentiate into Th2 cells is enhanced in part through the increased expression of GATA3. Shn-2 is found to compete with p50 NF-kappaB for binding to a consensus NF-kappaB motif and inhibit the NF-kappaB-driven promoter activity. In addition, Th2-driven allergic airway inflammation was enhanced in Shn-2-deficient mice. Therefore, Shn-2 appears to negatively control the differentiation of Th2 cells and Th2 responses through the repression of NF-kappaB function. Memory Th1/Th2 cells are not properly generated from Shn-2-deficient effector Th1/Th2 cells. The expression levels of CD69 and the number ofapoptotic cells are selectively increased in Shn-2-deficient Thl/Th2 cells when they are transferred into syngeneic host animals, in which memoryh Th1/Th2 cells are generated within a month. In addition, an increased susceptibility to apoptotic cell death is also observed in vitro accompanied with the increased expression of FasL, one of the NF-kappaB-dependent genes. Th2 effector cells overexpressing the p65 subunit of NF-kappaB demonstrate a decreased cell survival particularly in the lymph node. These results indicate that Shn-2-mediated repression of NF-kappaB is required for cell survival and the successful generation of memory Th1/Th2 cells. This may point to the possibility that after antigen clearance the recovery of the quiescent state in effector Th cells is required for the generation of memory Th cells. A repressor molecule Shn-2 plays an important role in this process.

Recently, it was reported that the expression of Runt-related transcription factor 3 (Runx3) is up-regulated in CD4(+) helper T cells during Th1 cell differentiation, and that Runx3 functions in a positive feed-forward manner with the T-box family transcription factor, T-bet, which is a master regulator of Th1 cell differentiation. The relative expression levels of IFN-gamma and IL-4 are also regulated by the Th2-associated transcription factor, GATA3. Here, we demonstrate that Runx3 was induced in Th2 as well as Th1 cells and that Runx3 interacted with GATA3 and attenuated GATA3 transcriptional activity. Ectopic expression of Runx3 in vitro in cultured cells or transgenic expression of Runx3 in mice accelerated CD4(+) cells to a Th1-biased population or down-modulated Th2 responses, in part by neutralizing GATA3. Our results suggest that the balance of Runx3 and GATA3 is one factor that influences the manifestation of CD4(+) cells as the Th1 or Th2 phenotypes.

Studies of human asthma and of animal models of allergic airway inflammation revealed a crucial role for Th2 cells in the pathogenesis of allergic asthma. Kruppel-type zinc finger proteins are the largest family of a regulatory transcription factor for cellular development and function. Zinc finger protein (Zfp) 35 is an 18-zinc finger motif-containing Kruppel-type zinc finger protein, while its function remains largely unknown. The aim of this study was to clarify the role of Zfp35 in the pathogenesis of Th2-dependent allergic inflammation, such as allergic asthma. We examined airway eosinophilic inflammation and hyperresponsiveness in two mouse models, which use our newly generated Zfp35-deficient (Zfp35(-/-)) mice and adoptive transfer of cells. In Zfp35(-/-) mice, Th2 cell differentiation, Th2 cytokine production, eosinophilic inflammation, and airway hyperresponsiveness were substantially enhanced. Furthermore, adoptive transfer of Ag-sensitized Zfp35(-/-) CD4 T cells into the asthmatic mice resulted in enhanced airway inflammation and airway hyperresponsiveness. These results indicate that Zfp35 controls Th2 cell differentiation, allergic airway inflammation, and airway hyperresponsiveness in a negative manner. Thus, Zfp35 may control Th2-dependent diseases, such as allergic asthma.

The maintenance of memory CD4 T cells is crucial for the establishment of immunological memory. The Polycomb (PcG) and Trithorax group (TrxG) genes control key developmental regulators such as the homeobox genes, and these two antagonize each other in the same developmental processes. Recently, PcG gene Bmi1 has been found to control memory Th1/Th2 cell survival and TrxG gene MLL is to control the maintenance of memory Th2 cell function selectively. Therefore, in memory CD4 T cells, PcG and TrxG genes appear to control distinct processes in a distinct manner, which indicates a novel regulatory feature of the PcG/TrxG genes.

GATA-3 is the master transcription factor for T helper 2 (Th2) cell differentiation and is critical for the expression of Th2 cytokines. Little is known, however, about the nature of the functional molecular complexes of GATA-3. We identified a high-mobility group (HMG)-box type transcription factor, lymphoid enhancer factor 1 (LEF-1), in the GATA-3 complex present in Th2 cells using a Flag-calmodulin-binding peptide (CBP)-tag based proteomics method. The interaction between GATA-3 and LEF-1 was confirmed by co-immunoprecipitation experiments using LEF-1-introduced T-cell lineage TG40 cells. The HMG-box domain of LEF-1 and two zinc finger domains of GATA-3 were found to be important for the physical association. The introduction of LEF-1 into developing Th2 cells resulted in the suppression of Th2 cytokine production. The suppression was significantly lower in the cells into which a HMG-box-deleted LEF-1 mutant was introduced. Moreover, LEF-1 inhibited the binding activity of GATA-3 to the interleukin (IL)-5 promoter. These results suggest that LEF-1 is involved in the GATA-3 complex, while also regulating the GATA-3 function, such as the induction of Th2 cytokine expression via the inhibition of the DNA-binding activity of GATA-3.

The differentiation of naive CD4 T cells into Th2 cells requires the T cell receptor-mediated activation of the ERK MAPK cascade. Little is known, however, in regard to how the ERK MAPK cascade regulates Th2 cell differentiation. We herein identified Gfi1 (growth factor independent-1) as a downstream target of the ERK MAPK cascade for Th2 cell differentiation. In the absence of Gfi1, interleukin-5 production and the change of histone modification at the interleukin-5 gene locus were severely impaired. Furthermore, the interferon gamma gene showed a striking activation in the Gfi1(-/-) Th2 cells. An enhanced ubiquitin/proteasome-dependent degradation of GATA3 protein was observed in Gfi1(-/-) Th2 cells, and the overexpression of GATA3 eliminated the defect of Th2 cell function in Gfi1-deficient Th2 cells. These data suggest that the T cell receptor-mediated induction of Gfi1 controls Th2 cell differentiation through the regulation of GATA3 protein stability.

Background: Studies of human asthma and of animal models of allergic inflammation/asthma highlight a crucial role for T(H)2 cells in the pathogenesis of allergic asthma. Repressor of GATA (ROG) is a POZ (BTB) domain-containing Kruppel-type zinc finger family (or POK family) repressor. A repressive function to GATA3, a master transcription factor for T(H)2 cell differentiation, is indicated., Objective: The aim of this study was to clarify the regulatory roles of ROG in the pathogenesis of T(H)2-driven allergic diseases, such as allergic asthma., Methods: We examined allergic airway inflammation and airway hyperresponsiveness (AHR) in 3 different mouse models, which use either ROG-deficient (ROG(-/-)) mice, ROG transgenic mice, or adoptive transfer of cells., Results: In ROG(-/-) mice T(H)2 cell differentiation, T(H)2 responses, eosinophilic airway inflammation, and AHR were enhanced. In ROG transgenic mice the levels of eosinophilic airway inflammation and AHR were dramatically reduced. Furthermore, adoptive transfer of T(H)2 cells with increased or decreased levels of ROG expression into the asthmatic mice resulted in reduced or enhanced airway inflammation, respectively., Conclusion: These results indicate that ROG regulates allergic airway inflammation and AHR in a negative manner, and thus ROG might represent another potential therapeutic target for the treatment of asthmatic patients.