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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.

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.)

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.

Schnurri (Shn)-2 is a large zinc finger-containing protein, which plays a critical role in cell growth, signal transduction and lymphocyte development. In Shn-2-deficient (Shn-2(-/-)) CD4 T cells, the activation of nuclear factor-kappaB is up-regulated and their ability to differentiate into Th2 is enhanced. Here, we extend our investigation and demonstrate that Shn-2 regulates Th2 responses in vivo using an ovalbumin-induced allergic asthma model. Eosinophilic inflammation, mucus hyperproduction and airway hyperresponsiveness (AHR) were all enhanced in Shn-2(-/-) mice. Moreover, eosinophilic infiltration and AHR were enhanced in mice given a transfer of Shn-2(-/-) effector Th2. Shn-2 in Th2 is thus considered to play an important role as a negative regulator in allergic airway inflammation.

Schnurri-2 (Shn-2) is a large zinc-finger containing protein, and it plays a critical role in cell growth, signal transduction and lymphocyte development. In Shn-2-deficient CD4 T cells, the activation of NF-kappaB was up-regulated and their ability to differentiate into Th2 cells was enhanced. We herein demonstrate that Th1 and Th2 memory cells are not properly generated from Shn-2-deficient effector Th1/Th2 cells. Even a week after the transfer of effector Th1/Th2 cells into syngeneic mice, a dramatic decrease in the number of Shn-2-deficient donor T cells was detected particularly in the lymphoid organs. The transferred Shn-2-deficient Th1/Th2 cells express higher levels of the activation marker CD69. No significant defect in the BrdU incorporation in the Shn-2-deficient transferred CD4 T cells was observed. The numbers of apoptotic cells were selectively higher in Shn-2-deficient donor Th1/Th2 cell population. Moreover, Shn-2-deficient effector Th1 and Th2 cells showed an increased susceptibility to cell death in in vitro cultures with increased expression of FasL. Transfer of Th2 effector cells over-expressing the p65 subunit of NF-kappaB resulted in a decreased number of p65-expressing cells in the lymphoid organs. As expected, T cell-dependent Ab responses after in vivo immunization of Shn-2-deficient mice were significantly reduced. Thus, Shn-2 appears to control the generation of memory Th1/Th2 cells through a change in their susceptibility to cell death.

The differentiation of mouse naïve CD4 T cells into type 2 helper (Th2) cells is accompanied by chromatin remodeling at the nucleosomes associated with the IL-4, IL-13 and IL-5 genes. However, little is known about how chromatin remodeling of these Th2 cytokine gene loci occurs in human Th2 cells. We herein established an in vitro culture system in which both Th1 and Th2 cells are efficiently differentiated from human peripheral blood naïve CD4 T cells. This system allowed us to investigate the chromatin status at the Th2 cytokine gene loci and the IFNgamma locus in human Th2 and Th1 cells, respectively. In typical individuals, the chromatin remodeling indicated by the induction of hyper-acetylation of histone H3 lysine 9 and hyper-methylation of histone H3 lysine 4 was induced at the whole Th2 cytokine gene loci in developing Th2 cells. We more precisely assessed the methylation status of histone H3 lysine 4 at the Th2 cytokine gene loci (IL-5 exon 3, IL-5 promoter, IL-5/RAD50 intergenic region, RAD50 promoter, CGRE, CNS1, IL-13 promoter, IL-4 promoter, and VA enhancer regions) and the IFNgamma locus in developing Th1 and Th2 cells prepared from 20 healthy volunteers. Th2-cell specific chromatin remodeling was induced at most of the Th2 cytokine gene loci. In parallel with the induction of chromatin remodeling, GATA3 mRNA was preferentially expressed in developing Th2 cells, whereas T-bet, HLX and ROG mRNA was selectively expressed in developing Th1 cells.

The Polycomb group (PcG) gene products regulate the maintenance of the homeobox gene expression in Drosophila and vertebrates and also the cell cycle progression in thymocytes and Th2 cell differentiation in mature T cells. We herein studied the role of PcG gene bmi-1 product in Th1/Th2 cell differentiation and found that Bmi-1 facilitates Th2 cell differentiation in a Ring finger-dependent manner. Biochemical studies indicate that Bmi-1 interacts with GATA3 in T cells, which is dependent on the Ring finger of Bmi-1. The overexpression of Bmi-1 resulted in a decreased ubiquitination and an increased protein stability of GATA3. In bmi-1-deficient Th cells, the levels of Th2 cell differentiation decreased as the degradation and ubiquitination on GATA3 increased. Therefore, Bmi-1 plays a crucial role in the control of Th2 cell differentiation in a Ring finger-dependent manner by regulating GATA3 protein stability.

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-kappaB (NF-kappaB) 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-kappaB, and activator protein 1 (AP1) selectively increased in draining lymph node T cells of NC/Nga mice. Moreover, the cell transfer of inhibitory NF-kappaB 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-kappaB 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-kappaB in the treatment of human atopic dermatitis.

The Mixed-Lineage Leukemia (MLL) gene, a mammalian homolog of the Drosophila trithorax, is implicated in regulating the maintenance of Hox gene expression and hematopoiesis. The physiological functions of MLL in the immune system remain largely unknown. Although MLL(+/-) CD4 T cells differentiate normally into antigen-specific effector Th1/Th2 cells in vitro, the ability of memory Th2 cells to produce Th2 cytokines was selectively reduced. Furthermore, histone modifications at the Th2 cytokine gene loci were not properly maintained in MLL(+/-) memory Th2 cells. The reduced expression of MLL in memory Th2 cells resulted in decreased GATA3 expression accompanied with impaired GATA3 locus histone modifications. The direct association of MLL with the GATA3 locus and the Th2 cytokine gene loci was demonstrated. Memory Th2 cell-dependent allergic airway inflammation was decreased in MLL(+/-) Th2 cell-transferred mice. Thus, a crucial role for MLL in the maintenance of memory Th2 cell function is indicated.

Age-related changes in lymphocytes are most prominent in the T cell compartment. There have been substantial numbers of reports on T cell function in aged mice and humans, such as on the production of Th1 and Th2 cytokines, but the results show considerable variation and contradictions. In the present study, we used 8- to 12-mo-old aging mice and a well-established in vitro Th1/Th2 cell differentiation culture system to identify molecular defects in Th1/Th2 cell differentiation that can be detected in the relatively early stages of aging. The capability to differentiate into Th2 cells is reduced in aging mouse CD4(+) T cells. Decreased activation of the ERK MAPK cascade upon TCR stimulation, but normal intracellular-free calcium ion concentration mobilization and normal IL-4-induced STAT6 activation were observed in aging mouse CD4(+) T cells. In addition, reduced expression of GATA3 was detected in developing Th2 cells. Chromatin remodeling of the Th2 cytokine gene locus was found to be impaired. Th2-dependent allergic airway inflammation was milder in aging mice compared with in young adult mice. These results suggest that the levels of Th2 cell differentiation and resulting Th2-dependent immune responses, including allergic airway inflammation, decline during aging through defects in the activation of the ERK MAPK cascade, expression of GATA3 protein and GATA3-dependent chromatin remodeling of the Th2 cytokine gene locus. In the present study, we provide the first evidence indicating that a chromatin-remodeling event in T cells is impaired by aging.

Schnurri (Shn) is a large zinc finger protein implicated in cell growth, signal transduction, and lymphocyte development. Vertebrates possess at least three Shn orthologues (Shn-1, Shn-2, and Shn-3), which appear to act within the bone morphogenetic protein, transforming growth factor beta, and activin signaling pathways. However, the physiological functions of the Shn proteins remain largely unknown. In Shn-2-deficient mice, mature peripheral T cells exhibited normal anti-T cell receptor-induced proliferation, although there was dramatic enhancement in the differentiation into T helper type (Th)2 cells and a marginal effect on Th1 cell differentiation. Shn-2-deficient developing Th2 cells showed constitutive activation of nuclear factor kappaB (NF-kappaB) and enhanced GATA3 induction. Shn-2 was able to compete with p50 NF-kappaB for binding to a consensus NF-kappaB motif and inhibit NF-kappaB-driven promoter activity. Thus, Shn-2 plays a crucial role in the control of Th2 cell differentiation by regulating NF-kappaB function.