The transcription factor Sox4 is a downstream target of signaling by the cytokine TGF-ß and suppresses T(H)2 differentiation.

Sox4 is a transcription factor that regulates various developmental processes. Here we show that Sox4 was induced by TGF-ß 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-ß to inhibit GATA-3 function, T(H)2 differentiation and T(H)2 cell-mediated inflammation.

Membrane molecule bouncer regulates sperm binding activity in immature oocytes in the viviparous teleost species Poecilia reticulata (guppy).

Generally, in vertebrates, the first step toward fertilization is the ovulation of mature oocytes, followed by their binding to sperm cells outside of the ovary. Exceptionally, the oocytes of poeciliid fish are fertilized by sperm cells within the follicle, and the developmental embryo is subsequently released into the ovarian lumen before delivery. In the present study, we aimed to identify the factor(s) responsible for intrafollicular fertilization in a viviparous teleost species, Poecilia reticulata (guppy). Sperm tracking analysis in this regard indicated that in this species, sperm cells reached immature oocytes including the germinal vesicle, and the insemination assay indicated that the immature oocytes robustly adhered to the sperm cells; similar binding was not observed in Danio rerio (zebrafish) and Oryzias latipes (medaka). We also identified the Ly6/uPAR protein bouncer as the factor responsible for the observed sperm binding activity of the immature oocytes in this species. The recombinant bouncer peptide acted as an inhibitory decoy for the sperm-oocyte binding in guppy. On the other hand, ectopic expression of guppy bouncer in zebrafish oocytes resulted in interspecific sperm-oocyte binding. These results argue that bouncer is responsible for sperm-immature oocyte binding. Our findings highlight the unique reproductive strategies of guppy fish and enhance our understanding of the diverse reproductive mechanisms in vertebrates.

Giant impurity effect on anomalous Hall effect of Mn3Sn.

Mn3Sn is an anomalous Hall effect (AHE) antiferromagnet that exhibits the hysteretic AHE in antiferromagnetic (AFM) phase at room temperature. We report that whisker Mn3Sn crystals grown by the flux method exhibit a non-hysteretic AHE at mid-to-low temperatures when the whisker Mn3Sn is surrounded by a thin layer of ferromagnetic Mn2-xSn. These crystals exhibit a hysteretic AHE above 275 K due to the spin alignment of the inverse triangular lattice, which is similar to other crystals. However, upon cooling the crystal, it exhibits a non-hysteretic AHE with a spiral AFM spin structure at 100-200 K. We concluded that the non-hysteretic AHE is induced at the interface of Mn2-xSn/Mn3Sn. We believe that the scalar-spin chirality in the spiral AFM phase of Mn3Sn, modulated by Mn2-xSn through the magnetic proximity effect, produces the AHE. This discovery opens a new avenue for tailoring the AHE by magnetic layers.

The histone demethylase Utx controls CD8+ T-cell-dependent antitumor immunity via epigenetic regulation of the effector function.

CD8+ T cells play a central role in antitumor immune responses. Epigenetic gene regulation is essential to acquire the effector function of CD8+ T cells. However, the role of Utx, a demethylase of histone H3K27, in antitumor immunity remains unclear. In this study, we examined the roles of Utx in effector CD8+ T-cell differentiation and the antitumor immune response. In a murine tumor-bearing model, an increased tumor size and decreased survival rate were observed in T-cell-specific Utx KO (Utx KO) mice compared with wild-type (WT) mice. The number of CD8+ T cells in tumor-infiltrating lymphocytes (TILs) was significantly decreased in Utx KO mice. We found that the acquisition of effector function was delayed and attenuated in Utx KO CD8+ T cells. RNA sequencing revealed that the expression of effector signature genes was decreased in Utx KO effector CD8+ T cells, while the expression of naïve or memory signature genes was increased. Furthermore, the expression of Cxcr3, which is required for the migration of effector CD8+ T cells to tumor sites, was substantially decreased in Utx KO CD8+ T cells. These findings suggest that Utx promotes CD8+ T-cell-dependent antitumor immune responses partially through epigenetic regulation of the effector function.

Terahertz asymmetric metallic hole arrays with polarization-independent quasi-bound states in the continuum for membrane sensing.

Resonances with both high-quality factor and polarization-independent characteristics are highly desirable for terahertz (THz) sensing. Here, THz sensors based on asymmetric metallic hole arrays (AMHAs) are experimentally demonstrated. Such sensors consisting of four-hole arrays support polarization-independent quasi-bound states in the continuum (BICs). The induced quasi-BIC presents a quality factor exceeding 2000, which enables enhanced sensing for thin membranes. Results show that the frequency shift is 97.5 GHz for the 25-µm thick polyimide (PI), corresponding to a sensitivity of 147.7 GHz/RIU. The sensing performance strongly relates to the enhanced field originating from sharp quasi-BICs. A maximum field enhancement of 15.88 in contrast to the incident field is achieved. When the PI thickness is large than the decay length of enhanced fields, the interaction strength of field-PI becomes weak, resulting in a saturation effect for the shift of quasi-BICs. The proposed sensor possessing polarization-independent quasi-BICs has great potential for practical sensing applications in real-time chemical and biomolecular.

The Loss of H3K27 Histone Demethylase Utx in T Cells Aggravates Allergic Contact Dermatitis.

The pathogenesis of allergic contact dermatitis (ACD) requires the activation of Ag-specific T cells, including effector and regulatory T cells. The differentiation and function of these T cells is epigenetically regulated through DNA methylation and histone modifications. However, the roles of altered histone H3K27 methylation in T cells in the development of ACD remain unknown. Two types of histone H3K27 demethylases, Utx and Jmjd3, have been reported in mammals. To determine the role of the histone H3K27 demethylase expression of T cells in the development of ACD, we generated T cell-specific, Utx-deficient (Utx KO) mice or Jmjd3-deficient (Jmjd3 KO) mice. Unlike control mice, Utx KO mice had severer symptoms of ACD, whereas Jmjd3 KO mice showed symptoms identical to those in control mice. In Utx KO mice with ACD, the massive infiltration of myeloid cells, including neutrophils and dendritic cells, has been observed. In addition, the expression of proinflammatory cytokines in CD4+ T cells of the draining lymph nodes (LNs) and in CD8+ T cells of the skin was increased in Utx KO mice, whereas the ratio of Foxp3+ regulatory CD4+ T cells to Foxp3- conventional CD4+ T cells was decreased in both the draining LNs and the skin of Utx KO mice with ACD. Furthermore, Foxp3+ regulatory CD4+ T cells of Utx KO mice with ACD expressed a decreased level of CCR4 (a skin-tropic chemokine receptor) in comparison with control. Thus, in CD4+ T cells, Utx could potentially be involved in the regulation of the pathogenesis of ACD.

Glucocorticoid imprints a low glucose metabolism onto CD8 T cells and induces the persistent suppression of the immune response.

Glucocorticoids (GCs), immunosuppressive, and anti-inflammatory agents have various effects on T cells. However, the long-term influence of GCs on the T cell-mediated immune response remain to be elucidated. We demonstrated that the administration of GC during the TCR-mediated activation phase induced long-lasting suppression of glycolysis, even after the withdrawal of GC. The acquisition of the effector functions was inhibited, while the expression of PD-1 was increased in CD8 T cells activated in the presence of GC. Furthermore, adoptive transfer experiments revealed that GC-treated CD8 T cells reduced memory T cell formation and anti-tumor activity. These findings reveal that GCs have long-lasting influence on the T cell-mediated immune response via modulation of T cell metabolism.

Intensity Interference in a Coherent Spin-Polarized Electron Beam.

We investigate the intensity interference between pairs of electrons using a spin-polarized electron beam having a high polarization and a narrow energy width. We observe spin-dependent antibunching on the basis of coincident counts of electron pairs performed with a spin-polarized transmission electron microscope, which could control the spin-polarization without any changes in the electron optics. The experimental results show that the time correlation was only affected by the spin polarization, demonstrating that the antibunching is associated with fermionic statistics. The coherent spin-polarized electron beam facilitates the extraction of intrinsic quantum interference.

BATF-Interacting Proteins Dictate Specificity in Th Subset Activity.

The basic leucine zipper (bZIP) transcription factor BATF is expressed in multiple Th subsets and cooperates with other factors to regulate gene transcription. BATF activates lineage-specific cytokines in Th subsets, activating IL-9 in Th9 cells and IL-17 in Th17 cells, but not IL-9 or IL-17 in the reciprocal subset. The mechanism for this restricted activity is unclear. In this report, we define BATF binding partners that contribute to Th subset-specific functions. Although BATF and IRF4 are expressed in greater amounts in Th9 than Th17, increased expression of both factors is not sufficient to induce IL-9 in Th17 cells. BATF also requires heterodimer formation with Jun family members to bind DNA and induce gene expression. Using primary mouse T cell culture, we observed that JunB and c-Jun, but not JunD, promote IL-9 production in Th9 cells. Ectopic expression of BATF with either JunB or c-Jun generates modest, but significant, increases in IL-9 production in Th17 cells, suggesting that the low expression of Jun family members is one factor limiting the ability of BATF to induce IL-9 in Th17 cells. We further identified that Bach2 positively regulates IL-9 production by directly binding to the Il9 gene and by increasing transcription factor expression in Th9 cells. Strikingly, cotransduction of Bach2 and BATF significantly induces IL-9 production in both Th9 and Th17 cells. Taken together, our results reveal that JunB, c-Jun, and Bach2 cooperate with BATF to contribute to the specificity of BATF-dependent cytokine induction in Th subsets.

Glycolysis and subsequent mevalonate biosynthesis play an important role in Th2 cell differentiation.

Th2 cytokine such as IL-4, IL -5 and IL-13 are important therapeutic targets for Th2-type chronic inflammation. Several biologics targeting Th2 cytokine and its receptors are effective in clinical practice; however, the development of small-molecule compounds that inhibit Th2 cytokine productions is awaited. We found that an inhibitor for pyruvate dehydrogenase kinase (PDHK) suppresses the differentiation of IL-5/IL-13-producing Th2 cells. The expression of the Th2-related transcriptional factors Pparγ was decreased by treatment with inhibitor, whereas Gata3, a master regulator of Th2 cell differentiation, remained unchanged. The oxygen consumption rate was unaffected, whereas the level of farnesylated proteins was decreased by the PDHK inhibitor. Furthermore, the inhibitors for farnesyltransferase and hydroxymethylglutaryl-CoA reductase showed an inhibitory effect similar to that of the PDHK inhibitor. These results suggest that the mevalonate biosynthesis and subsequent protein prenylation may be novel therapeutic target for Th2 cell-dependent immune dysregulation, such as in allergic diseases.

Eomesodermin controls interleukin-5 production in memory T helper 2 cells through inhibition of activity of the transcription factor GATA3.

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.

The critical role of Bach2 in regulating type 2 chronic airway inflammation.

Although Bach2 (broad complex-tramtrack-bric a brac and Cap'n'collar homology 2) plays an important role in regulating Th2 cell differentiation and type 2 immune responses, the underlying molecular mechanisms remain unclear. Our current studies demonstrate that Bach2 associates with Batf (basic leucine zipper transcription factor ATF-like) family transcription factors and binds to the regulatory regions of the Th2 cytokine gene loci. The Bach2-Batf complex antagonizes the recruitment of the interferon regulatory factor 4 (Irf4)-containing Batf complex to activator protein 1 (AP-1) motifs in the Th2 cytokine gene locus and suppresses Th2 cytokine production and/or Th2 cell differentiation. The deletion of Batf ameliorated the spontaneous development of type 2 airway inflammation that is found in mice with Bach2 deficiency specifically in T cells. Interestingly, Bach2 regulates Batf and Batf3 expression via two distinct pathways. First, the Bach2-Batf complex directly binds to the Batf and Batf3 gene loci and reduces transcription by interfering with the Batf-Irf4 complex. Second, Bach2 suppresses interleukin 4 (IL-4)-induced augmentation of Batf and Batf3 expression through the regulation of IL-4 production. These findings suggest that IL-4 and Batf family transcription factors form a positive feedback amplification loop to induce Th2 cell differentiation and that Bach2-Batf interactions block the formation of this amplification loop. Furthermore, we found that reductions in Bach2 confer an innate immunological function on CD4 T cells to induce antigen-independent cytokine production. Some Bach2-deficient lung CD4 T cells showed characteristic features similar to pathogenic Th2 cells, including IL-33 receptor expression and IL-33-dependent Th2 cytokine production. These results suggest a critical role for Bach2 in regulating Th2 cell differentiation and the subsequent onset of chronic type 2 inflammation.

Inflammatory responses induce an identity crisis of alveolar macrophages, leading to pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is a severe respiratory disease characterized by dyspnea caused by accumulation of surfactant protein. Dysfunction of alveolar macrophages (AMs), which regulate the homeostasis of surfactant protein, leads to the development of PAP; for example, in mice lacking BTB and CNC homology 2 (Bach2). However, how Bach2 helps prevent PAP is unknown, and the cell-specific effects of Bach2 are undefined. Using mice lacking Bach2 in specific cell types, we found that the PAP phenotype of Bach2-deficient mice is due to Bach2 deficiency in more than two types of immune cells. Depletion of hyperactivated T cells in Bach2-deficient mice restored normal function of AMs and ameliorated PAP. We also found that, in Bach2-deficient mice, hyperactivated T cells induced gene expression patterns that are specific to other tissue-resident macrophages and dendritic cells. Moreover, Bach2-deficient AMs exhibited a reduction in cell cycle progression. IFN-γ released from T cells induced Bach2 expression in AMs, in which Bach2 then bound to regulatory regions of inflammation-associated genes in myeloid cells. Of note, in AMs, Bach2 restricted aberrant responses to excessive T cell-induced inflammation, whereas, in T cells, Bach2 puts a brake on T cell activation. Moreover, Bach2 stimulated the expression of multiple histone genes in AMs, suggesting a role of Bach2 in proper histone expression. We conclude that Bach2 is critical for the maintenance of AM identity and self-renewal in inflammatory environments. Treatments targeting T cells may offer new therapeutic strategies for managing secondary PAP.

Reinforce the antitumor activity of CD8+ T cells via glutamine restriction.

The antitumor activity of activated CD8+ T cells in the tumor microenvironment seems to be limited due to their being metabolically unfit. This metabolic unfitness is closely associated with T-cell exhaustion and impairment of memory formation, which are barriers to successful antitumor adoptive immunotherapy. We therefore assessed the role of glutamine metabolism in the antitumor activity of CD8+ T cells using a tumor-inoculated mouse model. The adoptive transfer of tumor-specific CD8+ T cells cultured under glutamine-restricted (dGln) conditions or CD8+ T cells treated with specific inhibitors of glutamine metabolism efficiently eliminated tumors and led to better survival of tumor-inoculated mice than with cells cultured under control (Ctrl) conditions. The decreased expression of PD-1 and increased Ki67 positivity among tumor-infiltrating CD8+ T cells cultured under dGln conditions suggested that the inhibition of glutamine metabolism prevents CD8+ T-cell exhaustion in vivo. Furthermore, the transferred CD8+ T cells cultured under dGln conditions expanded more efficiently against secondary OVA stimulation than did CD8+ T cells under Ctrl conditions. We found that the expression of a pro-survival factor and memory T cell-related transcription factors was significantly higher in CD8+ T cells cultured under dGln conditions than in those cultured under Ctrl conditions. Given these findings, our study uncovered an important role of glutamine metabolism in the antitumor activity of CD8+ T cells. The novel adoptive transfer of tumor-specific CD8+ T cells cultured in glutamine-restricted conditions may be a promising approach to improve the efficacy of cell-based adoptive immunotherapy.

Menin Plays a Critical Role in the Regulation of the Antigen-Specific CD8+ T Cell Response upon Listeria Infection.

Menin, a tumor suppressor protein, is encoded by the MEN1 gene in humans. Certain germinal mutations of MEN1 induce an autosomal-dominant syndrome that is characterized by concurrent parathyroid adenomas and several other tumor types. Although menin is also expressed in hematopoietic lineages, its role in CD8+ T cells remains unclear. We generated Meninflox/flox CD4-Cre (Menin-KO) mice by crossing Meninflox/flox mice with CD4-Cre transgenic (Tg) mice to determine the role of menin in CD8+ T cells. Wild-type (WT) and Menin-KO mice were infected with Listeria monocytogenes expressing OVA to analyze the immune response of Ag-specific CD8+ T cells. Menin deficiency resulted in an impaired primary immune response by CD8+ T cells. On day 7, there were fewer Menin-KO OVA-specific CD8+ T cells compared with WT cells. Next, we adoptively transferred WT and Menin-KO OT-1 Tg CD8+ T cells into congenic recipient mice and infected them with L. monocytogenes expressing OVA to determine the CD8+ T cell-intrinsic effect. Menin-KO OT-1 Tg CD8+ T cells were outcompeted by the WT cells upon infection. Increased expression of Blimp-1 and T-bet, cell cycle inhibitors, and proapoptotic genes was observed in the Menin-KO OT-1 Tg CD8+ T cells upon infection. These data suggest that menin inhibits differentiation into terminal effectors and positively controls proliferation and survival of Ag-specific CD8+ T cells that are activated upon infection. Collectively, our study uncovered an important role for menin in the immune response of CD8+ T cells to infection.

Gfi1, a transcriptional repressor, inhibits the induction of the T helper type 1 programme in activated CD4 T cells.

A transcriptional repressor Gfi1 promotes T helper type 2 (Th2) cell development and inhibits Th17 and inducible regulatory T-cell differentiation. However, the role of Gfi1 in regulating Th1 cell differentiation and the Th1-type immune response remains to be investigated. We herein demonstrate that Gfi1 inhibits the induction of the Th1 programme in activated CD4 T cells. The activated Gfi1-deficient CD4 T cells spontaneously develop into Th1 cells in an interleukin-12- and interferon-γ-independent manner. The increase of Th1-type immune responses was confirmed in vivo in Gfi1-deficient mice using a murine model of nickel allergy and delayed-type hypersensitivity (DTH). The expression levels of Th1-related transcription factors were found to increase in Gfi1-deficient activated CD4 T cells. Tbx21, Eomes and Runx2 were identified as possible direct targets of Gfi1. Gfi1 binds to the Tbx21, Eomes and Runx2 gene loci and reduces the histone H3K4 methylation levels in part by modulating Lsd1 recruitment. Together, these findings demonstrate a novel regulatory role of Gfi1 in the regulation of the Th1-type immune response.

A novel function of interferon regulatory factor-1: inhibition of Th2 cells by down-regulating the Il4 gene during Listeria infection.

Infection with certain pathogens induces a shift of the Th subset balance to a Th1 dominant state. This, in turn, results in the suppression of Th2 responses. We focused on the involvement of interferon regulatory factor-1 (IRF-1) in the suppression of Th2 cells during Listeria infection. We found that the inhibition of IL-4 production by Th2 cells is mediated by a soluble factor (LmSN) produced by Listeria-infected antigen-presenting cells. The inhibition is not observed with T cells from Irf1 gene-targeted mice. IRF-1 suppresses transcription of the Il4 gene in Th2 cells. Under the influence of the LmSN, IRF-1 binds to the 3' untranslated region (UTR) region of the Il4 gene and down-regulates Il4 gene transcription. Finally, we identified IL-1α and IL-1ß as the mediator of the LmSN activity. Signaling through IL-1R induces the stabilization and/or nuclear translocation of IRF-1. We propose that IRF-1 functions to induce the T-cell subset shift via a novel mechanism. Under the influence of IL-1, IRF-1 translocates into the nucleus and acts on the 3'UTR region of the Il4 gene, thus inhibiting its transcription in Th2 cells. As a result, the immune system shifts predominantly to a Th1 response during Listeria infection, resulting in effective protection of the host.

Identification of a new pathway for Th1 cell development induced by cooperative stimulation with IL-4 and TGF-ß.

IL-4 plays an important role in the induction of Th2 and Th9 cells, as well as in the inhibition of Th1 cell generation. We show that a combination of IL-4 and TGF-ß augments the development of Th1 cells that express CD103 (CD103(+) Th1 cells) if IFN-γ is present. The T-box-containing transcription factor eomesodermin (Eomes) is preferentially expressed in CD103(+) Th1 cells and is involved in IFN-γ production. The induction of T-bet during early T cell activation is essential for the formation of the active chromatin at both the Eomes and IFN-γ gene loci. TGF-ß is required for the induction of Eomes and CD103, as well as the inhibition of Th2 cytokine expression. In addition, IL-4 induces Eomes transcription through activation of the Stat6-signaling pathway. IFN-γ-producing CD103(+) Th1 cells are detected in the intraepithelial lymphocytes of normal mice, and their numbers significantly decrease in Tbet- and Stat6-deficient mice. To our knowledge, these results represent the first molecular mechanism of IL-4/TGF-ß-dependent augmentation of Th1 cell generation and raise the possibility that IL-4 and TGF-ß simultaneously enhance the Th1 cell-mediated immune responses under certain cytokine conditions.

Development of silicon-on-insulator direct electron detector with analog memories in pixels for sub-microsecond imaging.

We have developed a high-speed recordable direct electron detector based on silicon-on-insulator technology. The detector has sixteen analog memories in each pixel to record sixteen images with sub-microsecond temporal resolution. A dedicated data acquisition system has also been developed to display and record the results on a personal computer. The performance of the direct electron detector as an image sensor is evaluated under electron irradiation with an energy of 30 keV in a low-voltage transmission electron microscope equipped with a photocathode electron gun. We demonstrate that the detector can record images at an exposure time of 100 ns and an interval of 900 ns.