A BTB-ZF protein, ZNF131, is required for early B cell development.

Members of the BTB-ZF transcription factor family play important roles in lymphocyte development. During T cell development, ZNF131, a BTB-ZF protein, is critical for the double-negative (DN) to double-positive (DP) transition and is also involved in cell proliferation. Here, we report that knockout of Znf131 at the pre-pro-B cell stage in mb1-Cre knock-in mouse resulted in defect of pro-B to pre-B cell transition. ZNF131 was shown to be required for efficient pro-B cell proliferation as well as for immunoglobulin heavy chain gene rearrangement that occurs in the proliferating pro-B cells. We speculate that inefficient gene rearrangement may be due to loss of cell proliferation, since cell cycle progression and immunoglobulin gene rearrangement, which would occur in a mutually exclusive manner, may be interconnected or coupled to avoid occurrence of genomic instability. ZNF131 suppresses expression of Cdk inhibitor, p21cip1, and that of pro-apoptotic factors, Bax and Puma, targets of p53, to facilitate cell cycle progression and suppress unnecessary apoptosis, respectively, of pro-B cells. There results demonstrate the essential roles of ZNF131 in coordinating the B cell differentiation and proliferation.

BTB-ZF Protein Znf131 Regulates Cell Growth of Developing and Mature T Cells.

Many members of the BTB-ZF family have been shown to play important roles in lymphocyte development and function. The role of zinc finger Znf131 (also known as Zbtb35) in T cell lineage was elucidated through the production of mice with floxed allele to disrupt at different stages of development. In this article, we present that Znf131 is critical for T cell development during double-negative to double-positive stage, with which significant cell expansion triggered by the pre-TCR signal is coupled. In mature T cells, Znf131 is required for the activation of effector genes, as well as robust proliferation induced upon TCR signal. One of the cyclin-dependent kinase inhibitors, p21(Cip1) encoded by cdkn1a gene, is one of the targets of Znf131. The regulation of T cell proliferation by Znf131 is in part attributed to its suppression on the expression of p21(Cip1).

Blockade of TLR3 protects mice from lethal radiation-induced gastrointestinal syndrome.

High-dose ionizing radiation induces severe DNA damage in the epithelial stem cells in small intestinal crypts and causes gastrointestinal syndrome (GIS). Although the tumour suppressor p53 is a primary factor inducing death of crypt cells with DNA damage, its essential role in maintaining genome stability means inhibiting p53 to prevent GIS is not a viable strategy. Here we show that the innate immune receptor Toll-like receptor 3 (TLR3) is critical for the pathogenesis of GIS. Tlr3(-/-) mice show substantial resistance to GIS owing to significantly reduced radiation-induced crypt cell death. Despite showing reduced crypt cell death, p53-dependent crypt cell death is not impaired in Tlr3(-/-) mice. p53-dependent crypt cell death causes leakage of cellular RNA, which induces extensive cell death via TLR3. An inhibitor of TLR3-RNA binding ameliorates GIS by reducing crypt cell death. Thus, we propose blocking TLR3 activation as a novel approach to treat GIS.

The enhancer HS2 critically regulates GATA-3-mediated Il4 transcription in T(H)2 cells.

GATA-3 is a master regulator of T helper type 2 (T(H)2) differentiation. However, the molecular basis of GATA-3-mediated T(H)2 lineage commitment is poorly understood. Here we identify the DNase I-hypersensitive site 2 (HS2) element located in the second intron of the interleukin 4 locus (Il4) as a critical enhancer strictly controlled by GATA-3 binding. Mice lacking HS2 showed substantial impairment in their asthmatic responses and their production of IL-4 but not of other T(H)2 cytokines. Overexpression of Gata3 in HS2-deficient T cells failed to restore Il4 expression. HS2 deletion impaired the trimethylation of histone H3 at Lys4 and acetylation of histone H3 at Lys9 and Lys14 in the Il4 locus. Our results indicate that HS2 is the target of GATA-3 in regulating chromosomal modification of the Il4 locus and is independent of the Il5 and Il13 loci.

Selective down-regulation of Th2 cytokines by C-terminal binding protein 2 in human T cells.

BACKGROUND: Among several C-terminal binding proteins (CtBPs), friend of GATA (FOG) has been implicated in the down-regulation of GATA-3-mediated Th2 cell differentiation. Here we investigated the role of CtBP2 in Th1 and Th2 cytokine expression in human T cells. METHODS: CtBP2 was introduced into human peripheral CD4+ T cells by a lentiviral transduction system. Subsequently, the expression of Th1 and Th2 cytokine mRNA was determined by quantitative real-time RT-PCR. RESULTS: CtBP2 significantly suppressed stimulation-induced expression of IL-4, IL-5 and IL-13 in human T cells. However, IFN-gamma expression was not affected by the introduction of CtBP2. CONCLUSION: CtBP2 selectively down-regulates Th2 cytokines, therefore it is a potential target for the treatment of allergic diseases.

Regulation of DNA demethylation during maturation of CD4+ naive T cells by the conserved noncoding sequence 1.

Demethylation of transcriptional regulatory elements and gene coding regions is an important step in the epigenetic regulation of gene expression. Several noncoding conserved regions are required for the efficient transcription of cytokine genes. In this paper, we show that the deletion of one such sequence, conserved noncoding sequence 1 (CNS-1), interferes with the efficient demethylation of Th2 cytokine genes but has little effect on histone modifications in the area. Th2 cells derived from CD4 single-positive (SP) mature thymocytes exhibit more rapid demethylation of CNS-1 and Th2-specific cytokine genes and produce more Th2 cytokines than do Th2 cells derived from CD4-positive peripheral naive T cells. De-repression of the Th1 cytokine IFN-gamma was also detected in Th2-primed CD4 SP thymocytes but not in naive T cells. Our results indicate that susceptibility to demethylation determines the efficiency and kinetics of cytokine gene transcription. The extrathymic maturation step undergone by naive T cells suppresses robust and rapid cytokine expression, whereas mature CD4 SP thymocytes maintain a rapid and less-specific cytokine expression profile. Finally, we detected the methyl cytosine binding protein MBD2 at CNS-1 in mature thymocytes, suggesting that this protein may regulate the demethylation of this region.

T-box 21 transcription factor is responsible for distorted T(H)2 differentiation in human peripheral CD4+ T cells.

BACKGROUND: Regardless of T(H)1/T(H)2 theory, CD4(+) T cells of patients with allergic asthma, a typical T(H)2 disease, and those of healthy subjects expressed equivalent levels of IFN-gamma, even though T(H)2 cytokines were significantly upregulated in asthmatic patients. OBJECTIVE: The mechanisms underlying distorted T(H)2 cell polarization in human T cells were elucidated. METHODS: Cytokine-producing activity and the expression of T(H)1/T(H)2-specific transcription factors in naïve, T(H)1/T(H)2, or both CD4(+) T cells derived from human peripheral and cord blood were comparatively analyzed. The mechanisms of the differential expression of T-box 21 transcription factor (T-bet) in the cells were assessed by determining the chromatin accessibility at the TBX21 gene. The functional roles of T-bet and other transcription factors in human T(H)1/T(H)2 differentiation were further investigated. RESULTS: T(H)2 cells derived from naive CD4(+) T cells in peripheral blood but not in cord blood produced IFN-gamma. T-bet was expressed in peripheral, but not cord blood, resting naive T cells. Consistently, the accessibility at the proximal TBX21 gene promoter in peripheral naive T cells was higher than that in cord blood naive T cells. IFN-gamma-producing activity was induced in T(H)2-differentiated cord blood T cells by means of ectopic expression of T-bet. In addition, a reduction of T-bet in peripheral T cells suppressed IFN-gamma production. T-bet not only upregulated IFN-gamma but also downregulated IL-4 and IL-13 gene transcription, independently of the modification of T(H)1/T(H)2 balance. CONCLUSION: The expression of T-bet at a naive stage is crucial for the development of IFN-gamma-producing T cells in human peripheral blood, even in T(H)2-related diseases.

Ly49Q ligand expressed by activated B cells induces plasmacytoid DC maturation.

Ly49Q, a type II C-type lectin expressed on mouse plasmacytoid DC (pDC), contains a single carbohydrate recognition domain in its extracellular region and an ITIM in its cytoplasmic domain. We have identified the MHC class I molecule H-2K(b) as a Ly49Q ligand, confirming prior reports. Although H-2K(b) is expressed on essentially all hematopoietic cells, we found that only CpG-stimulated B cells were able to activate Ly49Q. This discovery correlated with our finding that although H-2K(b) forms clusters on CpG-activated B cells, it is diffusely expressed on resting B cells. Furthermore, CpG-stimulated, but not resting, B cells up-regulated co-stimulatory molecules on pDC. This finding was confirmed by the fact that binding by anti-Ly49Q mAb to Ly49Q led to pDC maturation in vitro. Our results suggest that clustered H-2K(b) on activated B cells act as ligands for Ly49Q and induce pDC maturation in vitro.

Suppressive role of C-terminal binding protein 1 in IL-4 synthesis in human T cells.

The functional role of C-terminal binding protein (CtBP)1, a transcriptional corepressor, in Th1 and Th2 cytokine expression in human T cells was investigated. Upon introduction of CtBP1 by lentiviral transduction system, IL-4 synthesis was suppressed but IFN-gamma was weakly up-regulated in human CD4(+) T cells. In contrast, a reduction of endogenously expressed CtBP1 in Jurkat T cells using RNAi technology selectively augmented IL-4 expression. The down-regulation of IL-4 by CtBP1 was achieved at the level of gene transcription. Deletion mutation analysis revealed that N-terminal approximately 200 amino acid and C-terminal approximately 50 amino acid residues are participated in CtBP1-mediated suppression of IL-4 expression. CtBP1 expressed in human CD4(+) T cells crucially contribute to Th1/Th2 differentiation via selective down-regulation of IL-4 synthesis.

Cyclic AMP-induced chromatin changes support the NFATc-mediated recruitment of GATA-3 to the interleukin 5 promoter.

Elevated intracellular cyclic AMP levels, which suppress the proliferation of naive T cells and type 1 T helper (Th1) cells are a property of T helper 2 (Th2) cells and regulatory T cells. While cyclic AMP signals interfere with the IL-2 promoter induction, they support the induction of Th2-type genes, in particular of il-5 gene. We show here that cyclic AMP signals support the generation of three inducible DNase I hypersensitive chromatin sites over the il-5 locus, including its promoter region. In addition, cyclic AMP signals enhance histone H3 acetylation at the IL-5 promoter and the concerted binding of GATA-3 and NFATc to the promoter. This is facilitated by direct protein-protein interactions involving the C-terminal Zn(2+)-finger of GATA-3 and the C-terminal region of the NFATc1 DNA binding domain. Because inhibition of NFATc binding to the IL-5 promoter in vivo also affects the binding of GATA-3, one may conclude that upon induction of Th2 effector cells NFATc recruits GATA-3 to Th2-type genes. These data demonstrate the functional importance of cyclic AMP signals for the interplay between GATA-3 and NFATc factors in the transcriptional control of lymphokine expression in Th2 effector cells.

The role of CD11b in phagocytosis and dendritic cell development.

Activation of resting T cells is highly dependent on dendritic cells (DCs), which take up antigens and present antigenic peptides to T cells in the context of the major histocompatibility complex (MHC). In this study, we generated a monoclonal antibody, which we call 1C4 that recognizes integrin alpha(M)beta(2) (CD11b/CD18) on the surface of conventional DCs (cDCs) and is internalized after binding. Addition of 1C4 inhibited the ability of immature DCs to phagocytose apoptotic cells. 1C4 treatment also partially inhibited the generation of cDCs from bone marrow in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF). Our findings suggest that not only CD11b is involved in the phagocytosis of apoptotic cells, but also that mAb such as 1C4 may be a useful tool for the delivery of specific proteins into the cytoplasm of immature DCs.

Airway eosinophilic inflammation is attenuated in conserved noncoding sequence-1-deficient mice.

BACKGROUND: Conserved noncoding sequence-1 (CNS-1) is an important regulatory element for T helper 2 cytokine expression. IL-4, IL-5 and IL-13 expression as well as serum IgE level were attenuated in CNS-1-/- mice. METHOD: CNS-1-/- and CNS-1+/+ mice were sensitized with ovalbumin (OVA) followed by antigen challenge. The number of eosinophils and T helper 2 cytokine concentration in the bronchoalveolar lavage fluid, OVA-specific IgE antibody (Ab) in the serum and bronchial responsiveness to methacholine were examined. RESULTS: Bronchoalveolar lavage fluid eosinophilia was significantly attenuated in CNS-1-/- mice compared to CNS-1+/+ mice, which were sensitized with OVA/aluminum once. OVA-specific IgE Ab was also attenuated. When mice were sensitized with OVA/aluminum twice, induction of eosinophilia and OVA-specific IgE Ab was not significantly different between CNS-1-/- and CNS-1+/+ mice. CONCLUSION: CNS-1 locus regulates eosinophilic inflammation in vivo.

Downregulation of IL-13 gene transcription by T-bet in human T cells.

BACKGROUND: Downregulation of a Th2 cytokine, IL-4, by a Th1-specific transcription factor, T-bet, has been demonstrated. However, the regulatory role of T-bet in another Th2 cytokine, IL-13, is not fully delineated. METHODS: IL-13 mRNA expression in Jurkat cells was examined by quantitative RT-PCR, while the transcriptional activity of 5'-flanking region in the IL-13 gene encompassing -1077 to +49 was investigated by fluorescence-based promoter reporter assay. The effect of T-bet was investigated by transfection of the cells with the T-bet expression vector. RESULTS: Stimulation with phorbol ester plus Ca2+ ionophore clearly induced IL-13 gene transcription in Jurkat cells. Ectopically expressed T-bet significantly suppressed the inducible mRNA expression and promoter activity of IL-13. CONCLUSION: IL-13 expression was downregulated by T-bet at the level of gene transcription, independently of the modulation of Th1/Th2 balance. T-bet is the potential key factor in the development of Th1/Th2-related diseases.

Differential contribution of NFATc2 and NFATc1 to TNF-alpha gene expression in T cells.

The NFAT family transcription factors play crucial roles in immunological and other biological events; however, the functional differences among NFAT members have not been fully elucidated. This study investigated the relative contribution of NFATc2 and NFATc1 to the transactivation of cytokine genes in T cells. Ectopic expression of NFATc2 but not NFATc1, especially its short isoform, enhanced TNF-alpha synthesis in human T cells at the gene transcription level, whereas both NFATs augmented IL-2 expression. In addition, a reduction of the shortest NFATc1 isoform using RNA interference technology failed to suppress TNF-alpha expression. The promoter/enhancer activity of the NFAT-binding site in the TNF-alpha gene was up-regulated by NFATc2 but not by NFATc1, whereas both NFATs associated similarly with this region. A study of mRNA expression using NFATc2/NFATc1 chimeric molecules revealed that the enhancing activity of NFAT on the TNF-alpha gene was lost by truncation of its C-terminal transactivation domain. In addition, this domain derived from NFATc2 behaved as a dominant negative against the NFAT site in TNF-alpha promoter-dependent transcriptional activity in T cells. We conclude that the C-terminal transactivation domain in NFAT is crucial for TNF-alpha gene expression in human T cells.

Type I interferon regulates pDC maturation and Ly49Q expression.

Ly49Q is expressed on peripheral mouse plasmacytoid dendritic cells (pDC). Immature Ly49Q-negative pDC precursors acquire Ly49Q in the bone marrow and then migrate into the periphery. While searching for molecules that regulate pDC maturation, we found that type I interferon (IFN) inhibited Ly49Q acquisition in vitro. Infections that induce type I IFN production by cells other than pDC (a condition mimicked by poly(I:C) injection in vivo) increase the prevalence of Ly49Q(-) pDC in the bone marrow and peripheral lymphoid organs in wild-type but not IFN-alpha/beta receptor knockout BALB/c mice. Moreover, in vivo exposure to type I IFN causes some Ly49Q(-), but not Ly49Q(+), pDC to convert to conventional DC, defined as B220(-) CD11c(+) CD11b(+) cells. These data suggest that type I IFN regulates pDC development and affects their distribution in the body.

IL-4 gene transcription in human T cells is suppressed by T-bet.

UNLABELLED: T-bet is crucially implicated in Th1 differentiation due to its strong promoting activity for IFN-gamma gene transcription. However, the regulatory role of T-bet in Th2 cytokines is not fully delineated. METHODS: The effect of T-bet on mRNA expression as well as the promoter activity of IL-4 in human T cells was investigated by employing quantitative RT-PCR and fluorescence-based promoter reporter assay procedures. RESULTS: IL-4 mRNA expression as well as the transcriptional activity of 5'-flanking region in the IL-4 gene encompassing -1105 to +4 in Jurkat cells was clearly upregulated upon stimulation. The inducible mRNA expression and the promoter activity of IL-4 were significantly diminished by ectopic expression of T-bet. CONCLUSION: IL-4 gene transcription is inhibited by T-bet via the suppression of its promoter activity, independently of IFN-gamma. T-bet facilitates Th1 differentiation through not only upregulation of IFN-gamma, but also downregulation of IL-4 gene transcription.

Characterization of binding activity between nuclear factor of activated T cells and calcineurin by amplified luminescent proximity homogeneous assay.

For quantitative evaluation of the relationship between biological binding partners, including protein-protein interactions, a novel analyzing system, amplified luminescent proximity homogeneous assay (ALPHA), has been developed. We here employed ALPHA for accurate assessment of the binding properties between nuclear factor of activated T cells 1 (NFAT1) and calcineurin (CN), which is essential for Ca2+-dependent regulation of immune responses. A recombinant protein of the Ca2+ regulatory domain (CRD) of NFAT1 was prepared and its binding activity with biotinylated CN was determined by ALPHA (Kd = 0.20 microM). The contribution of each CN-binding component involved in the CRD of NFAT1 to CN/NFAT1 binding was next examined by competitive assay. Not only the whole CRD but also the N- and C-terminal CN-binding regions (CNBR1 and CNBR2, respectively) dose-dependently blocked CN/NFAT1 binding and their potency was CRD >> CNBR2 > or = CNBR1. CN/NFAT1-binding properties were further characterized using short inhibitory peptides derived from NFAT1-CNBR1 as well as NFAT4-CNBR2. In conclusion, ALPHA is a useful system to analyze biological signaling cascades, due to its capability of quantitative evaluation of protein-protein interactions.

Replication initiation from a novel origin identified in the Th2 cytokine cluster locus requires a distant conserved noncoding sequence.

Lineage commitment of Th cells is associated with the establishment of specific transcriptional programs of cytokines. However, how Th cell differentiation affects the program of DNA replication has not been addressed. To gain insight into interplays between differentiation-induced transcription regulation and initiation of DNA replication, we took advantage of an in vitro differentiation system of naive T cells, in which one can manipulate their differentiation into Th1 or Th2 cells. We searched for replication origins in the murine IL-4/IL-13 locus and compared their profiles in the two Th cell lineages which were derived in vitro from the same precursor T cells. We identified a replication origin (ori(IL-13)) downstream from exon 4 of IL-13 and showed that this origin functions in both Th2 and Th1 cells. A distant regulatory element called CNS-1 (conserved noncoding sequence 1) in the IL-4/IL-13 intergenic region coincides with a Th2-specific DNase I-hypersensitive site and is required for efficient, coordinated expression of Th2 cytokines. Replication initiation from ori(IL-13) is significantly reduced in Th1 and Th2 cells derived from CNS-1-deficient mice. However, the replication timing of this locus is consistently early during S phase in both Th1 and Th2 cells under either the wild-type or CNS-1 deletion background. Thus, the conserved noncoding element in the intergenic region regulates replication initiation from a distant replication origin in a manner independent from its effect on lineage-specific transcription but not the replication timing of the segment surrounding this origin.

Correlation between mRNA expression of Th1/Th2 cytokines and their specific transcription factors in human helper T-cell clones.

The mechanisms that underlie Th1/Th2 differentiation of human T cells are incompletely defined. In the present study, a panel of human T-cell clones was used to elucidate the relationship between Th1/Th2-specific transcription factors and cytokine production in human helper T cells. The mRNA expression level of T-bet, a Th1-specific transcription factor, was higher in Th1 clones than in Th2 clones. In contrast, inducible expression of Th2-specific transcription factors (GATA-3 and c-Maf) in Th2 clones was higher than that in Th1 clones. The expression level of T-bet in various T-cell clones was positively correlated with that of IFN-gamma and negatively correlated with that of Th2 cytokines, particularly IL-4. Interestingly, the expression of IL-3 and IL-13, but not of other Th2 cytokines IL-4 and IL-5, was strongly correlated with GATA-3 mRNA levels. A reduction of GATA-3 using RNA interference technology suppressed, whereas overexpression of GATA-3 enhanced, the expression of IL-3 and IL-13. In conclusion, the level of T-bet expression is correlated with Th1/Th2 polarization status, whereas GATA-3 is a crucial factor in determining the IL-3 and IL-13 producing capacity of human T cells.

[Inhibitors of NFAT-calcineurin pathway].

Cyclosporin A(CsA) and tacrolimus (FK506) are important immunosuppressants to inhibit rejection of transplanted organs and to treat various immunological disorders, however those drugs produce major side effects. Those drugs form complexes with cellular proteins, immunophilins (cyclophilin for CsA and FKBP for tacrolimus) and inhibit Ca-calmodulin dependent phosphatase calcineurin through direct binding. Calcineurin dephosphorylates various substrates including NFAT family proteins required for the expression of immunoregulatory molecules especially cytokines. NFAT-calcineurin pathway offers a good model system to apply new technology to develop drugs. Enzyme-substrate interaction could be an important target to develop drugs with high specificity accompanied with less side effects.