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Sample records for activated t-cell nfat

  1. Dependence on nuclear factor of activated T-cells (NFAT) levels discriminates conventional T cells from Foxp3+ regulatory T cells.

    PubMed

    Vaeth, Martin; Schliesser, Ulrike; Müller, Gerd; Reissig, Sonja; Satoh, Kazuki; Tuettenberg, Andrea; Jonuleit, Helmut; Waisman, Ari; Müller, Martin R; Serfling, Edgar; Sawitzki, Birgit S; Berberich-Siebelt, Friederike

    2012-10-01

    Several lines of evidence suggest nuclear factor of activated T-cells (NFAT) to control regulatory T cells: thymus-derived naturally occurring regulatory T cells (nTreg) depend on calcium signals, the Foxp3 gene harbors several NFAT binding sites, and the Foxp3 (Fork head box P3) protein interacts with NFAT. Therefore, we investigated the impact of NFAT on Foxp3 expression. Indeed, the generation of peripherally induced Treg (iTreg) by TGF-β was highly dependent on NFAT expression because the ability of CD4(+) T cells to differentiate into iTreg diminished markedly with the number of NFAT family members missing. It can be concluded that the expression of Foxp3 in TGF-β-induced iTreg depends on the threshold value of NFAT rather than on an individual member present. This is specific for iTreg development, because frequency of nTreg remained unaltered in mice lacking NFAT1, NFAT2, or NFAT4 alone or in combination. Different from expectation, however, the function of both nTreg and iTreg was independent on robust NFAT levels, reflected by less nuclear NFAT in nTreg and iTreg. Accordingly, absence of one or two NFAT members did not alter suppressor activity in vitro or during colitis and transplantation in vivo. This scenario emphasizes an inhibition of high NFAT activity as treatment for autoimmune diseases and in transplantation, selectively targeting the proinflammatory conventional T cells, while keeping Treg functional.

  2. Nuclear factor of activated T-cells (NFAT) plays a role in SV40 infection

    SciTech Connect

    Manley, Kate; O'Hara, Bethany A.; Atwood, Walter J.

    2008-03-01

    Recent evidence highlighted a role for the transcription factor, nuclear factor of activated T-cells (NFAT), in the transcription of the human polyomavirus JCV. Here we show that NFAT is also important in the transcriptional control of the related polyomavirus, Simian Virus 40 (SV40). Inhibition of NFAT activity reduced SV40 infection of Vero, 293A, and HeLa cells, and this block occurred at the stage of viral transcription. Both NFAT3 and NFAT4 bound to the SV40 promoter through {kappa}B sites located within the 72 bp repeated enhancer region. In Vero cells, NFAT was involved in late transcription, but in HeLa and 293A cells both early and late viral transcription required NFAT activity. SV40 large T-Ag was found to increase NFAT activity and provided a positive feedback loop to transactivate the SV40 promoter.

  3. Selective NFAT targeting in T cells ameliorates GvHD while maintaining antitumor activity.

    PubMed

    Vaeth, Martin; Bäuerlein, Carina A; Pusch, Tobias; Findeis, Janina; Chopra, Martin; Mottok, Anja; Rosenwald, Andreas; Beilhack, Andreas; Berberich-Siebelt, Friederike

    2015-01-27

    Graft-versus-host disease (GvHD) is a life-threatening immunological complication after allogenic hematopoietic stem cell transplantation (allo-HCT). The intrinsic graft-versus-leukemia (GvL) effect, however, is the desirable curative benefit. Patients with acute GvHD are treated with cyclosporine A (CsA) or tacrolimus (FK506), which not only often causes severe adverse effects, but also interferes with the anticipated GvL. Both drugs inhibit calcineurin, thus at first suppressing activation of the nuclear factor of activated T cells (NFAT). Therefore, we explored the specific contribution of individual NFAT factors in donor T cells in animal models of GvHD and GvL. Ablation of NFAT1, NFAT2, or a combination of both resulted in ameliorated GvHD, due to reduced proliferation, target tissue homing, and impaired effector function of allogenic donor T cells. In contrast, the frequency of Foxp3(+) regulatory T (Treg) cells was increased and NFAT-deficient Tregs were fully protective in GvHD. CD8(+) T-cell recall response and, importantly, the beneficial antitumor activity were largely preserved in NFAT-deficient effector T cells. Thus, specific inhibition of NFAT opens an avenue for an advanced therapy of GvHD maintaining protective GvL.

  4. Constitutive nuclear localization of NFAT in Foxp3+ regulatory T cells independent of calcineurin activity.

    PubMed

    Li, Qiuxia; Shakya, Arvind; Guo, Xiaohua; Zhang, Hongbo; Tantin, Dean; Jensen, Peter E; Chen, Xinjian

    2012-05-01

    Foxp3 plays an essential role in conferring suppressive functionality to CD4(+)/Foxp3(+) regulatory T cells (Tregs). Although studies showed that Foxp3 has to form cooperative complexes with NFAT to bind to target genes, it remains unclear whether NFAT is available in the nucleus of primary Tregs for Foxp3 access. It is generally believed that NFAT in resting cells resides in the cytoplasm, and its nuclear translocation depends on calcineurin (CN) activation. We report that a fraction of NFAT protein constitutively localizes in the nucleus of primary Tregs, where it selectively binds to Foxp3 target genes. Treating Tregs with CN inhibitor does not induce export of NFAT from the nucleus, indicating that its nuclear translocation is independent of CN activity. Consistently, Tregs are resistant to CN inhibitors in the presence of IL-2 and continue to proliferate in response to anti-CD3 stimulation, whereas proliferation of non-Tregs is abrogated by CN inhibitors. In addition, PMA, which activates other transcription factors required for T cell activation but not NFAT, selectively induces Treg proliferation in the absence of ionomycin. TCR interaction with self-MHC class II is not required for PMA-induced Treg proliferation. Tregs expanded by PMA or in the presence of CN inhibitors maintain Treg phenotype and functionality. These findings shed light on Treg biology, paving the way for strategies to selectively activate Tregs.

  5. Nickel differentially regulates NFAT and NF-{kappa}B activation in T cell signaling

    SciTech Connect

    Saito, Rumiko; Hirakawa, Satoshi; Ohara, Hiroshi; Yasuda, Makoto; Yamazaki, Tomomi; Nishii, Shigeaki; Aiba, Setsuya

    2011-08-01

    Nickel is a potent hapten that induces contact hypersensitivity in human skin. While nickel induces the maturation of dendritic cells via NF-{kappa}B and p38 MAPK activation, it also exerts immunosuppressive effects on T cells through an unknown mechanism. To elucidate the molecular mechanisms of its effects on T cells, we examined the effects of NiCl{sub 2} on mRNA expression in human CD3+ T cells stimulated with CD3 and CD28 antibodies. Using a DNA microarray and Gene Ontology, we identified 70 up-regulated (including IL-1{beta}, IL-6 and IL-8) and 61 down-regulated (including IL-2, IL-4, IL-10 and IFN-{gamma}) immune responsive genes in NiCl{sub 2}-treated T cells. The DNA microarray results were verified using real-time PCR and a Bio-Plex{sup TM} suspension protein array. Suppression of IL-2 and IFN-{gamma} gene transcription by NiCl{sub 2} was also confirmed using Jurkat T cells transfected with IL-2 or IFN-{gamma} luciferase reporter genes. To explore the NiCl{sub 2}-regulated signaling pathway, we examined the binding activity of nuclear proteins to NFAT, AP-1, and NF-{kappa}B consensus sequences. NiCl{sub 2} significantly and dose-dependently suppressed NFAT- and AP-1-binding activity, but augmented NF-{kappa}B-binding activity. Moreover, NiCl{sub 2} decreased nuclear NFAT expression in stimulated T cells. Using Jurkat T cells stimulated with PMA/ionomycin, we demonstrated that NiCl{sub 2} significantly suppressed stimulation-evoked cytosolic Ca{sup 2+} increases, suggesting that NiCl{sub 2} regulates NFAT signals by acting as a blocker of Ca{sup 2+} release-activated Ca{sup 2+} (CRAC) channels. These data showed that NiCl{sub 2} decreases NFAT and increases NF-{kappa}B signaling in T cells. These results shed light on the effects of nickel on the molecular regulation of T cell signaling. - Graphical Abstract: Nickel suppresses stimulation-evoked cytosolic Ca{sup 2+} increase, which results in the suppression of NFAT signals. On the other hand, Ni rather

  6. Nuclear factor of activated T cells (NFAT) in pearl oyster Pinctada fucata: molecular cloning and functional characterization.

    PubMed

    Huang, Xian-De; Wei, Guo-jian; Zhang, Hua; He, Mao-Xian

    2015-01-01

    Nuclear factor of activated T cells (NFAT) plays an important role in nonimmune cells and also in T cells and many other cells of the immune system, by regulating the expression of a variety of genes involved in the immune response, organ development, developmental apoptosis and angiogenesis. In the present study, the NFAT homology gene, PfNFAT, from the pearl oyster Pinctada fucata was cloned and its genomic structure and promoter were analyzed. PfNFAT encodes a putative protein of 1226 amino acids, and contains a highly conserved Rel homology region (RHR) with DNA-binding specificity, and a regulatory domain (NFAT homology region, NHR) containing a potent transactivation domain (TAD). The PfNFAT gene consists of 12 exons and 11 introns, and its promoter contains potential binding sites for transcription factors such as NF-κB (Nuclear factor κB), STATx (signal transducer and activator of transcription), AP-1 (activator protein-1) and Sox-5/9 (SRY type HMG box-5/9), MyoD (Myogenic Differentiation Antigen) and IRF (Interferon regulatory factor). Comparison and phylogenetic analysis revealed that PfNFAT shows high identity with other invertebrate NFAT, and clusters with the NFAT5 subgroup. Furthermore, gene expression analysis revealed that PfNFAT is involved in the immune response to lipopolysaccharide (LPS) and Polyinosinic-polycytidylic acid (poly I:C) stimulation and in the nucleus inserting operation. The study of PfNFAT may increase understanding of molluscan innate immunity.

  7. Nuclear translocation of nuclear factor of activated T cells (NFAT) as a quantitative pharmacodynamic parameter for tacrolimus.

    PubMed

    Maguire, Orla; Tornatore, Kathleen M; O'Loughlin, Kieran L; Venuto, Rocco C; Minderman, Hans

    2013-12-01

    Nuclear factor of activated T cells (NFAT) is a family of transcription factors involved in regulating the immune response. The canonical NFAT pathway is calcium-dependent and upon activation, NFAT is dephosphorylated by the phosphatase, calcineurin. This results in its translocation from the cytoplasm to the nucleus and transcription of downstream target genes that include the cytokines IL-2, IL-10, and IFNγ. Calcineurin inhibitors including tacrolimus inhibit the NFAT pathway and are used as immunosuppressants in transplant settings to prevent graft rejection. There is, as yet, no direct means to monitor tacrolimus pharmacodynamics. In this study, a rapid, quantitative, image cytometry-based measurement of nuclear translocation of NFAT1 is used to evaluate NFAT activation in T cells and its tacrolimus-induced inhibition. A strong dose-dependent correlation between NFAT1 inhibition and tacrolimus dose is demonstrated in vitro. Time kinetic analysis of NFAT1 inhibition in plasma from stable renal transplant recipients before and after an in vivo dose with tacrolimus correlated with the expected pharmacokinetic profile of tacrolimus. This was further corroborated by analysis of patients' autologous CD4 and CD8 T cells. This is the first report to show that the measurement of NFAT1 activation potential by nuclear translocation can be used as a direct, sensitive, reproducible and quantitative pharmacodynamic readout for tacrolimus action. These results, and the rapid turnaround time for this assay, warrant its evaluation in a larger clinical setting to assess its role in therapeutic drug monitoring of calcineurin inhibitors.

  8. Virion encapsidated HIV-1 Vpr induces NFAT to prime non-activated T cells for productive infection.

    PubMed

    Höhne, Kristin; Businger, Ramona; van Nuffel, Anouk; Bolduan, Sebastian; Koppensteiner, Herwig; Baeyens, Ann; Vermeire, Jolien; Malatinkova, Eva; Verhasselt, Bruno; Schindler, Michael

    2016-07-01

    The majority of T cells encountered by HIV-1 are non-activated and do not readily allow productive infection. HIV-1 Vpr is highly abundant in progeny virions, and induces signalling and HIV-1 LTR transcription. We hence hypothesized that Vpr might be a determinant of non-activated T-cell infection. Virion-delivered Vpr activated nuclear factor of activated T cells (NFAT) through Ca(2+) influx and interference with the NFAT export kinase GSK3β. This leads to NFAT translocation and accumulation within the nucleus and was required for productive infection of unstimulated primary CD4(+) T cells. A mutagenesis approach revealed correlation of Vpr-mediated NFAT activation with its ability to enhance LTR transcription and mediate cell cycle arrest. Upon NFAT inhibition, Vpr did not augment resting T-cell infection, and showed reduced G2/M arrest and LTR transactivation. Altogether, Vpr renders unstimulated T cells more permissive for productive HIV-1 infection and stimulates activation of productively infected as well as virus-exposed T cells. Therefore, it could be involved in the establishment and reactivation of HIV-1 from viral reservoirs and might have an impact on the levels of immune activation, which are determinants of HIV-1 pathogenesis.

  9. Virion encapsidated HIV-1 Vpr induces NFAT to prime non-activated T cells for productive infection

    PubMed Central

    Höhne, Kristin; Businger, Ramona; van Nuffel, Anouk; Bolduan, Sebastian; Koppensteiner, Herwig; Baeyens, Ann; Vermeire, Jolien; Malatinkova, Eva; Verhasselt, Bruno; Schindler, Michael

    2016-01-01

    The majority of T cells encountered by HIV-1 are non-activated and do not readily allow productive infection. HIV-1 Vpr is highly abundant in progeny virions, and induces signalling and HIV-1 LTR transcription. We hence hypothesized that Vpr might be a determinant of non-activated T-cell infection. Virion-delivered Vpr activated nuclear factor of activated T cells (NFAT) through Ca2+ influx and interference with the NFAT export kinase GSK3β. This leads to NFAT translocation and accumulation within the nucleus and was required for productive infection of unstimulated primary CD4+ T cells. A mutagenesis approach revealed correlation of Vpr-mediated NFAT activation with its ability to enhance LTR transcription and mediate cell cycle arrest. Upon NFAT inhibition, Vpr did not augment resting T-cell infection, and showed reduced G2/M arrest and LTR transactivation. Altogether, Vpr renders unstimulated T cells more permissive for productive HIV-1 infection and stimulates activation of productively infected as well as virus-exposed T cells. Therefore, it could be involved in the establishment and reactivation of HIV-1 from viral reservoirs and might have an impact on the levels of immune activation, which are determinants of HIV-1 pathogenesis. PMID:27383627

  10. Virion encapsidated HIV-1 Vpr induces NFAT to prime non-activated T cells for productive infection.

    PubMed

    Höhne, Kristin; Businger, Ramona; van Nuffel, Anouk; Bolduan, Sebastian; Koppensteiner, Herwig; Baeyens, Ann; Vermeire, Jolien; Malatinkova, Eva; Verhasselt, Bruno; Schindler, Michael

    2016-07-01

    The majority of T cells encountered by HIV-1 are non-activated and do not readily allow productive infection. HIV-1 Vpr is highly abundant in progeny virions, and induces signalling and HIV-1 LTR transcription. We hence hypothesized that Vpr might be a determinant of non-activated T-cell infection. Virion-delivered Vpr activated nuclear factor of activated T cells (NFAT) through Ca(2+) influx and interference with the NFAT export kinase GSK3β. This leads to NFAT translocation and accumulation within the nucleus and was required for productive infection of unstimulated primary CD4(+) T cells. A mutagenesis approach revealed correlation of Vpr-mediated NFAT activation with its ability to enhance LTR transcription and mediate cell cycle arrest. Upon NFAT inhibition, Vpr did not augment resting T-cell infection, and showed reduced G2/M arrest and LTR transactivation. Altogether, Vpr renders unstimulated T cells more permissive for productive HIV-1 infection and stimulates activation of productively infected as well as virus-exposed T cells. Therefore, it could be involved in the establishment and reactivation of HIV-1 from viral reservoirs and might have an impact on the levels of immune activation, which are determinants of HIV-1 pathogenesis. PMID:27383627

  11. Interleukin-7 induces HIV replication in primary naive T cells through a nuclear factor of activated T cell (NFAT)-dependent pathway

    SciTech Connect

    Managlia, Elizabeth Z. . E-mail: lalharth@rush.edu

    2006-07-05

    Interleukin (IL)-7 plays several roles critical to T cell maturation, survival, and homeostasis. Because of these functions, IL-7 is under investigation as an immune-modulator for therapeutic use in lymphopenic clinical conditions, including HIV. We reported that naive T cells, typically not permissive to HIV, can be productively infected when pre-treated with IL-7. We evaluated the mechanism by which IL-7-mediates this effect. IL-7 potently up-regulated the transcriptional factor NFAT, but had no effect on NF{kappa}B. Blocking NFAT activity using a number of reagents, such as Cyclosporin A, FK-506, or the NFAT-specific inhibitor known as VIVIT peptide, all markedly reduced IL-7-mediated induction of HIV replication in naive T cells. Additional neutralization of cytokines present in IL-7-treated cultures and/or those that have NFAT-binding sequences within their promotors indicated that IL-10, IL-4, and most significantly IFN{gamma}, all contribute to IL-7-induction of HIV productive replication in naive T cells. These data clarify the mechanism by which IL-7 can overcome the block to HIV productive infection in naive T cells, despite their quiescent cell status. These findings are relevant to the treatment of HIV disease and understanding HIV pathogenesis in the naive CD4+ T cell compartment, especially in light of the vigorous pursuit of IL-7 as an in vivo immune modulator.

  12. Dephosphorylation of the nuclear factor of activated T cells (NFAT) transcription factor is regulated by an RNA-protein scaffold complex.

    PubMed

    Sharma, Sonia; Findlay, Gregory M; Bandukwala, Hozefa S; Oberdoerffer, Shalini; Baust, Beate; Li, Zhigang; Schmidt, Valentina; Hogan, Patrick G; Sacks, David B; Rao, Anjana

    2011-07-12

    Nuclear factor of activated T cells (NFAT) proteins are Ca(2+)-regulated transcription factors that control gene expression in many cell types. NFAT proteins are heavily phosphorylated and reside in the cytoplasm of resting cells; when cells are stimulated by a rise in intracellular Ca(2+), NFAT proteins are dephosphorylated by the Ca(2+)/calmodulin-dependent phosphatase calcineurin and translocate to the nucleus to activate target gene expression. Here we show that phosphorylated NFAT1 is present in a large cytoplasmic RNA-protein scaffold complex that contains a long intergenic noncoding RNA (lincRNA), NRON [noncoding (RNA) repressor of NFAT]; a scaffold protein, IQ motif containing GTPase activating protein (IQGAP); and three NFAT kinases, casein kinase 1, glycogen synthase kinase 3, and dual specificity tyrosine phosphorylation regulated kinase. Combined knockdown of NRON and IQGAP1 increased NFAT dephosphorylation and nuclear import exclusively after stimulation, without affecting the rate of NFAT rephosphorylation and nuclear export; and both NRON-depleted T cells and T cells from IQGAP1-deficient mice showed increased production of NFAT-dependent cytokines. Our results provide evidence that a complex of lincRNA and protein forms a scaffold for a latent transcription factor and its regulatory kinases, and support an emerging consensus that lincRNAs that bind transcriptional regulators have a similar scaffold function.

  13. Pseudoephedrine inhibits T-cell activation by targeting NF-κB, NFAT and AP-1 signaling pathways.

    PubMed

    Fiebich, Bernd L; Collado, Juan A; Stratz, Cristian; Valina, Christian; Hochholzer, Willibald; Muñoz, Eduardo; Bellido, Luz M

    2012-02-01

    Pseudoephedrine (PSE) is a stereoisomer of ephedrine that is commonly used as a nasal decongestant in combination with other anti-inflammatory drugs for the symptomatic treatment of some common pathologies such as common cold. Herein, we describe for the first time the effects of PSE on T-cell activation events. We found that PSE inhibits interleukin-2 (IL-2) and tumor necrosis factor (TNF) alpha-gene transcription in stimulated Jurkat cells, a human T-cell leukemia cell line. To further characterize the inhibitory mechanisms of PSE at the transcriptional level, we examined the transcriptional activities of nuclear factor kappa B (NF-κB), nuclear factor of activated T cells (NFAT), and activator protein-1 (AP-1) transcription factors and found that PSE inhibited NF-κB-dependent transcriptional activity without affecting either the phosphorylation, the degradation of the cytoplasmic NF-κB inhibitory protein, IκBα or the DNA-binding activity. However, phosphorylation of the p65/RelA subunit was clearly inhibited by PSE in stimulated cells. In addition, PSE inhibited the transcriptional activity of NFAT without interfering with the calcium-induced NFAT dephosphorylation event, which represents the major signaling pathway for its activation. NFAT cooperates with c-Jun, a compound of the AP-1 complex, to activate target genes, and we also found that PSE inhibited both JNK activation and AP-1 transcriptional activity. These findings provide new mechanistic insights into the potential immunomodulatory activities of PSE and highlight their potential in designing novel therapeutic strategies to manage inflammatory diseases.

  14. Prolonged CD154 Expression on Pediatric Lupus CD4 T Cells Correlates with Increased CD154 Transcription, Increased NFAT Activity, and Glomerulonephritis

    PubMed Central

    Mehta, Jay; Genin, Anna; Brunner, Michael; Scalzi, Lisabeth V; Mishra, Nilamadhab; Beukelman, Timothy; Cron, Randy Q

    2010-01-01

    Objective To assess CD154 expression in pediatric lupus and explore a transcriptional mechanism explaining dysregulated CD154 expression. Methods Cell surface CD154 expression was examined, pre- and post-activation, on peripheral blood CD4 T cells from 29 children with lupus and matched controls by flow cytometry. CD154 expression was correlated with clinical features, laboratory parameters, and treatments received. Increased CD154 expression on lupus CD4 T cells was correlated with CD154 message and transcription rates by real-time RT-PCR and nuclear run-on assays, respectively. NFAT transcriptional activity and NFAT mRNA levels in lupus CD4 T cells were explored by reporter gene analysis and real-time RT-PCR, respectively. Results CD154 surface protein levels were increased 1.44-fold on lupus CD4 T cells compared to controls at one day post-activation ex vivo. This increase correlated clinically with the presence of nephritis and elevated erythrocyte sedimentation rate. Increased CD154 protein also correlated with increased CD154 mRNA levels and rates of CD154 transcription, particularly at later time-points post-T cell activation. Reporter gene analyses revealed a trend for increased NFAT, but decreased AP-1 and similar NFκB, activity in lupus CD4 T cell compared to controls. Moreover, NFAT1 and, in particular, NFAT2 mRNA levels were notably increased in lupus CD4 T cells compared to controls. Conclusion Following activation, cell surface CD154 is increased on pediatric lupus CD4 T cells compared to controls, and this correlates with the presence of nephritis, increased CD154 transcription rates, and NFAT activity. These results suggest that NFAT/calcineurin inhibitors, such as tacrolimus and cyclosporine, may be beneficial in treating lupus nephritis. PMID:20506525

  15. Identification of two nuclear factor of activated T-cells (NFAT)-response elements in the 5'-upstream regulatory region of the ET-1 promoter.

    PubMed

    Strait, Kevin A; Stricklett, Peter K; Kohan, Rachel M; Kohan, Donald E

    2010-09-10

    Collecting duct-derived ET-1 regulates salt excretion and blood pressure. We have reported the presence of an inner medullary collecting duct (IMCD)-specific enhancer region in the 5'-upstream ET-1 promoter (Strait, K. A., Stricklett, P. K., Kohan, J. L., Miller, M. B., and Kohan, D. E. (2007) Am. J. Physiol. Renal Physiol. 293, F601-F606). The current studies provide further characterization of the ET-1 5'-upstream distal promoter to identify the IMCD-specific enhancer elements. Deletion studies identified two regions of the 5'-upstream ET-1 promoter, -1725 to -1319 bp and -1319 to -1026 bp, which were required for maximal promoter activity in transfected rat IMCD cells. Transcription factor binding site analysis of these regions identified two consensus nuclear factor of activated T-cells (NFAT) binding sites at -1263 and -1563. EMSA analysis using nuclear extracts from IMCD cells showed that both the -1263 and the -1563 NFAT sites in the ET-1 distal promoter competed for NFAT binding to previously identified NFAT sites in the IL-2 and TNF genes. Gel supershift analysis showed that each of the NFAT binding sites in the ET-1 promoter bound NFAT proteins derived from IMCD nuclear extracts, but they selectively bound different NFAT isoforms; ET-1263 bound NFATc1, whereas ET-1563 bound NFATc3. Site-directed mutagenesis of either the ET-1263 or the ET-1563 sites prevented NFAT binding and reduced ET-1 promoter activity. Thus, NFAT appears to be an important regulator of ET-1 transcription in IMCD cells, and thus, it may play a role in controlling blood pressure through ET-1 regulation of renal salt excretion.

  16. Distinct activation properties of the nuclear factor of activated T-cells (NFAT) isoforms NFATc3 and NFATc4 in neurons.

    PubMed

    Ulrich, Jason D; Kim, Man-Su; Houlihan, Patrick R; Shutov, Leonid P; Mohapatra, Durga P; Strack, Stefan; Usachev, Yuriy M

    2012-11-01

    The Ca(2+)/calcineurin-dependent transcription factor NFAT (nuclear factor of activated T-cells) is implicated in regulating dendritic and axonal development, synaptogenesis, and neuronal survival. Despite the increasing appreciation for the importance of NFAT-dependent transcription in the nervous system, the regulation and function of specific NFAT isoforms in neurons are poorly understood. Here, we compare the activation of NFATc3 and NFATc4 in hippocampal and dorsal root ganglion neurons following electrically evoked elevations of intracellular Ca(2+) concentration ([Ca(2+)](i)). We find that NFATc3 undergoes rapid dephosphorylation and nuclear translocation that are essentially complete within 20 min, although NFATc4 remains phosphorylated and localized to the cytosol, only exhibiting nuclear localization following prolonged (1-3 h) depolarization. Knocking down NFATc3, but not NFATc4, strongly diminished NFAT-mediated transcription induced by mild depolarization in neurons. By analyzing NFATc3/NFATc4 chimeras, we find that the region containing the serine-rich region-1 (SRR1) mildly affects initial NFAT translocation, although the region containing the serine-proline repeats is critical for determining the magnitude of NFAT activation and nuclear localization upon depolarization. Knockdown of glycogen synthase kinase 3β (GSK3β) significantly increased the depolarization-induced nuclear localization of NFATc4. In contrast, inhibition of p38 or mammalian target of rapamycin (mTOR) kinases had no significant effect on nuclear import of NFATc4. Thus, electrically evoked [Ca(2+)](i) elevation in neurons rapidly and strongly activates NFATc3, whereas activation of NFATc4 requires a coincident increase in [Ca(2+)](i) and suppression of GSK3β, with differences in the serine-proline-containing region giving rise to these distinct activation properties of NFATc3 and NFATc4.

  17. An Altered gp100 Peptide Ligand with Decreased Binding by TCR and CD8α Dissects T Cell Cytotoxicity from Production of Cytokines and Activation of NFAT.

    PubMed

    Schaft, Niels; Coccoris, Miriam; Drexhage, Joost; Knoop, Christiaan; de Vries, I Jolanda M; Adema, Gosse J; Debets, Reno

    2013-01-01

    Altered peptide ligands (APLs) provide useful tools to study T cell activation and potentially direct immune responses to improve treatment of cancer patients. To better understand and exploit APLs, we studied the relationship between APLs and T cell function in more detail. Here, we tested a broad panel of gp100280-288 APLs with respect to T cell cytotoxicity, production of cytokines, and activation of Nuclear Factor of Activated T cells (NFAT) by human T cells gene-engineered with a gp100-HLA-A2-specific TCRαβ. We demonstrated that gp100-specific cytotoxicity, production of cytokines, and activation of NFAT were not affected by APLs with single amino acid substitutions, except for an APL with an amino acid substitution at position 3 (APL A3), which did not elicit any T cell response. A gp100 peptide with a double amino acid mutation (APL S4S6) elicited T cell cytotoxicity and production of IFNγ, and to a lesser extent TNFα, IL-4, and IL-5, but not production of IL-2 and IL-10, or activation of NFAT. Notably, T cell receptor (TCR)-mediated functions showed decreases in sensitivities for S4S6 versus gp100 wild-type (wt) peptide, which were minor for cytotoxicity but at least a 1000-fold more prominent for the production of cytokines. TCR-engineered T cells did not bind A3-HLA-A2, but did bind S4S6-HLA-A2 although to a lowered extent compared to wt peptide-HLA-A2. Moreover, S4S6-induced T cell function demonstrated an enhanced dependency on CD8α. Taken together, most gp100 APLs functioned as agonists, but A3 and S4S6 peptides acted as a null ligand and partial agonist, respectively. Our results further suggest that TCR-mediated cytotoxicity can be dissected from production of cytokines and activation of NFAT, and that the agonist potential of peptide mutants relates to the extent of binding by TCR and CD8α. These findings may facilitate the design of APLs to advance the study of T cell activation and their use for therapeutic applications. PMID:24027572

  18. An Altered gp100 Peptide Ligand with Decreased Binding by TCR and CD8α Dissects T Cell Cytotoxicity from Production of Cytokines and Activation of NFAT

    PubMed Central

    Schaft, Niels; Coccoris, Miriam; Drexhage, Joost; Knoop, Christiaan; de Vries, I. Jolanda M.; Adema, Gosse J.; Debets, Reno

    2013-01-01

    Altered peptide ligands (APLs) provide useful tools to study T cell activation and potentially direct immune responses to improve treatment of cancer patients. To better understand and exploit APLs, we studied the relationship between APLs and T cell function in more detail. Here, we tested a broad panel of gp100280–288 APLs with respect to T cell cytotoxicity, production of cytokines, and activation of Nuclear Factor of Activated T cells (NFAT) by human T cells gene-engineered with a gp100-HLA-A2-specific TCRαβ. We demonstrated that gp100-specific cytotoxicity, production of cytokines, and activation of NFAT were not affected by APLs with single amino acid substitutions, except for an APL with an amino acid substitution at position 3 (APL A3), which did not elicit any T cell response. A gp100 peptide with a double amino acid mutation (APL S4S6) elicited T cell cytotoxicity and production of IFNγ, and to a lesser extent TNFα, IL-4, and IL-5, but not production of IL-2 and IL-10, or activation of NFAT. Notably, T cell receptor (TCR)-mediated functions showed decreases in sensitivities for S4S6 versus gp100 wild-type (wt) peptide, which were minor for cytotoxicity but at least a 1000-fold more prominent for the production of cytokines. TCR-engineered T cells did not bind A3-HLA-A2, but did bind S4S6-HLA-A2 although to a lowered extent compared to wt peptide-HLA-A2. Moreover, S4S6-induced T cell function demonstrated an enhanced dependency on CD8α. Taken together, most gp100 APLs functioned as agonists, but A3 and S4S6 peptides acted as a null ligand and partial agonist, respectively. Our results further suggest that TCR-mediated cytotoxicity can be dissected from production of cytokines and activation of NFAT, and that the agonist potential of peptide mutants relates to the extent of binding by TCR and CD8α. These findings may facilitate the design of APLs to advance the study of T cell activation and their use for therapeutic applications. PMID:24027572

  19. Inhibition of FOXP3/NFAT Interaction Enhances T Cell Function after TCR Stimulation.

    PubMed

    Lozano, Teresa; Villanueva, Lorea; Durántez, Maika; Gorraiz, Marta; Ruiz, Marta; Belsúe, Virginia; Riezu-Boj, José I; Hervás-Stubbs, Sandra; Oyarzábal, Julen; Bandukwala, Hozefa; Lourenço, Ana R; Coffer, Paul J; Sarobe, Pablo; Prieto, Jesús; Casares, Noelia; Lasarte, Juan J

    2015-10-01

    Regulatory T cell (Treg) activity is modulated by a cooperative complex between the transcription factor NFAT and FOXP3, a lineage specification factor for Tregs. FOXP3/NFAT interaction is required to repress expression of IL-2, upregulate expression of the Treg markers CTLA4 and CD25, and confer suppressor function to Tregs. However, FOXP3 is expressed transiently in conventional CD4(+) T cells upon TCR stimulation and may lead to T cell hyporesponsiveness. We found that a short synthetic peptide able to inhibit FOXP3/NFAT interaction impaired suppressor activity of conventional Tregs in vitro. Specific inhibition of FOXP3/NFAT interaction with this inhibitory peptide revealed that FOXP3 downregulates NFAT-driven promoter activity of CD40L and IL-17. Inhibition of FOXP3/NFAT interaction upregulated CD40L expression on effector T cells and enhanced T cell proliferation and IL-2, IFN-γ, IL-6, or IL-17 production in response to TCR stimulation. The inhibitory peptide impaired effector T cell conversion into induced Tregs in the presence of TGF-β. Moreover, in vivo peptide administration showed antitumor efficacy in mice bearing Hepa129 or TC1 tumor cells when combined with sorafenib or with an antitumor vaccine, respectively. Our results suggest that inhibition of NFAT/FOXP3 interaction might improve antitumor immunotherapies.

  20. Calcineurin Aβ regulates NADPH oxidase (Nox) expression and activity via nuclear factor of activated T cells (NFAT) in response to high glucose.

    PubMed

    Williams, Clintoria R; Gooch, Jennifer L

    2014-02-21

    Hypertrophy is an adaptive response that enables organs to appropriately meet increased functional demands. Previously, we reported that calcineurin (Cn) is required for glomerular and whole kidney hypertrophy in diabetic rodents (Gooch, J. L., Barnes, J. L., Garcia, S., and Abboud, H. E. (2003). Calcineurin is activated in diabetes and is required for glomerular hypertrophy and ECM accumulation. Am. J. Physiol. Renal Physiol. 284, F144-F154; Reddy, R. N., Knotts, T. L., Roberts, B. R., Molkentin, J. D., Price, S. R., and Gooch, J. L. (2011). Calcineurin Aβ is required for hypertrophy but not matrix expansion in the diabetic kidney. J. Cell Mol. Med. 15, 414-422). Because studies have also implicated the reactive oxygen species-generating enzymes NADPH oxidases (Nox) in diabetic kidney responses, we tested the hypothesis that Nox and Cn cooperate in a common signaling pathway. First, we examined the role of the two main isoforms of Cn in hypertrophic signaling. Using primary kidney cells lacking a catalytic subunit of Cn (CnAα(-/-) or CnAβ(-/-)), we found that high glucose selectively activates CnAβ, whereas CnAα is constitutively active. Furthermore, CnAβ but not CnAα mediates hypertrophy. Next, we found that chronic reactive oxygen species generation in response to high glucose is attenuated in CnAβ(-/-) cells, suggesting that Cn is upstream of Nox. Consistent with this, loss of CnAβ reduces basal expression and blocks high glucose induction of Nox2 and Nox4. Inhibition of nuclear factor of activated T cells (NFAT), a CnAβ-regulated transcription factor, decreases Nox2 and Nox4 expression, whereas NFAT overexpression increases Nox2 and Nox4, indicating that the CnAβ/NFAT pathway modulates Nox. These data reveal that the CnAβ/NFAT pathway regulates Nox and plays an important role in high glucose-mediated hypertrophic responses in the kidney.

  1. Inhibiting the Calcineurin-NFAT (Nuclear Factor of Activated T Cells) Signaling Pathway with a Regulator of Calcineurin-derived Peptide without Affecting General Calcineurin Phosphatase Activity*S⃞

    PubMed Central

    Mulero, Ma Carme; Aubareda, Anna; Orzáez, Mar; Messeguer, Joaquim; Serrano-Candelas, Eva; Martínez-Hoyer, Sergio; Messeguer, Àngel; Pérez-Payá, Enrique; Pérez-Riba, Mercè

    2009-01-01

    Calcineurin phosphatase plays a crucial role in T cell activation. Dephosphorylation of the nuclear factors of activated T cells (NFATs) by calcineurin is essential for activating cytokine gene expression and, consequently, the immune response. Current immunosuppressive protocols are based mainly on calcineurin inhibitors, cyclosporine A and FK506. Unfortunately, these drugs are associated with severe side effects. Therefore, immunosuppressive agents with higher selectivity and lower toxicity must be identified. The immunosuppressive role of the family of proteins regulators of calcineurin (RCAN, formerly known as DSCR1) which regulate the calcineurin-NFAT signaling pathway, has been described recently. Here, we identify and characterize the minimal RCAN sequence responsible for the inhibition of calcineurin-NFAT signaling in vivo. The RCAN-derived peptide spanning this sequence binds to calcineurin with high affinity. This interaction is competed by a peptide spanning the NFAT PXIXIT sequence, which binds to calcineurin and facilitates NFAT dephosphorylation and activation. Interestingly, the RCAN-derived peptide does not inhibit general calcineurin phosphatase activity, which suggests that it may have a specific immunosuppressive effect on the calcineurin-NFAT signaling pathway. As such, the RCAN-derived peptide could either be considered a highly selective immunosuppressive compound by itself or be used as a new tool for identifying innovative immunosuppressive agents. We developed a low throughput assay, based on the RCAN1-calcineurin interaction, which identifies dipyridamole as an efficient in vivo inhibitor of the calcineurin-NFAT pathway that does not affect calcineurin phosphatase activity. PMID:19189965

  2. Nerve growth factor (NGF) regulates activity of nuclear factor of activated T-cells (NFAT) in neurons via the phosphatidylinositol 3-kinase (PI3K)-Akt-glycogen synthase kinase 3β (GSK3β) pathway.

    PubMed

    Kim, Man-Su; Shutov, Leonid P; Gnanasekaran, Aswini; Lin, Zhihong; Rysted, Jacob E; Ulrich, Jason D; Usachev, Yuriy M

    2014-11-01

    The Ca(2+)/calcineurin-dependent transcription factor nuclear factor of activated T-cells (NFAT) plays an important role in regulating many neuronal functions, including excitability, axonal growth, synaptogenesis, and neuronal survival. NFAT can be activated by action potential firing or depolarization that leads to Ca(2+)/calcineurin-dependent dephosphorylation of NFAT and its translocation to the nucleus. Recent data suggest that NFAT and NFAT-dependent functions in neurons can also be potently regulated by NGF and other neurotrophins. However, the mechanisms of NFAT regulation by neurotrophins are not well understood. Here, we show that in dorsal root ganglion sensory neurons, NGF markedly facilitates NFAT-mediated gene expression induced by mild depolarization. The effects of NGF were not associated with changes in [Ca(2+)]i and were independent of phospholipase C activity. Instead, the facilitatory effect of NGF depended on activation of the PI3K/Akt pathway downstream of the TrkA receptor and on inhibition of glycogen synthase kinase 3β (GSK3β), a protein kinase known to phosphorylate NFAT and promote its nuclear export. Knockdown or knockout of NFATc3 eliminated this facilitatory effect. Simultaneous monitoring of EGFP-NFATc3 nuclear translocation and [Ca(2+)]i changes in dorsal root ganglion neurons indicated that NGF slowed the rate of NFATc3 nuclear export but did not affect its nuclear import rate. Collectively, our data suggest that NGF facilitates depolarization-induced NFAT activation by stimulating PI3K/Akt signaling, inactivating GSK3β, and thereby slowing NFATc3 export from the nucleus. We propose that NFAT serves as an integrator of neurotrophin action and depolarization-driven calcium signaling to regulate neuronal gene expression.

  3. The effect of nuclear factor of activated T-cells (NFAT) in kidney I/R mediated by C5a/C5aR.

    PubMed

    Zhang, Ze-Ying; Wu, Yang-Qian; Luo, Heng; Liu, Dong-Xu

    2015-01-01

    To investigate the relationship between NFAT and C5a/C5aR in C5a/C5aR-mediated kidney Ischemia/reperfusion (I/R) injury, the rats' NRK-52E cell line was used in this study and was distributed into 4 groups, I: the normal control (NC), II: the ischemia/reperfusion (I/R) injury cell model (MG), III: the ischemia/reperfusion (I/R) injury cell model treated with C5a (50 nmol/l) (MG + C5a), IV: the ischemia/reperfusion (I/R) injury cell model treated with C5aR antagonist (2.5 μmol/l) (MG + anti-C5aR). Reverse transcription polymerase chain reaction (RT-PCR), western blot, immunofluorescence and flow cytometry were performed. Nuclear Factor Activated T Cell (NFAT), tumor necrosis factor-α (TNF-α) and interleukin (IL-6) were detected in this study. The results of immunofluorescence showed that NFAT had a nuclear translocation phenomenon during the study. The RT-PCR and WB data indicated that the expression of TNF-α and IL-6 in group III were higher than any other groups. Apoptosis in group III was much serious than other groups. All the results in this study showed that NFAT plays an important role in ischemia/reperfusion injury, it can be induced to up-regulate the inflammatory factor TNF-α and IL-6 by the complement system member C5a/C5aR.

  4. Nuclear presence of nuclear factor of activated T cells (NFAT) c3 and c4 is required for Toll-like receptor-activated innate inflammatory response of monocytes/macrophages.

    PubMed

    Minematsu, Hiroshi; Shin, Mike J; Celil Aydemir, Ayse B; Kim, Kyung-Ok; Nizami, Saqib A; Chung, Gook-Jin; Lee, Francis Young-In

    2011-11-01

    Nuclear factor of activated T cells (NFATs) are crucial transcription factors that tightly control proinflammatory cytokine expression for adaptive immunity in T and B lymphocytes. However, little is known about the role of NFATs for innate immunity in macrophages. In this study, we report that NFAT is required for Toll-like receptor (TLR)-initiated innate immune responses in bone marrow-derived macrophages (BMMs). All TLR ligand stimulation including LPS, a TLR4 ligand, and Pam(3)CSK(4), a TLR1/2 ligand, induced expression of TNF which was inhibited by VIVIT, an NFAT-specific inhibitor peptide. BMMs from NFATc4 knock-out mouse expressed less TNF than wild type. Despite apparent association between NFAT and TNF, LPS did not directly activate NFAT based on NFAT-luciferase reporter assay, whereas NF-κB was inducibly activated by LPS. Instead, macrophage exhibited constitutive NFAT activity which was not increased by LPS and was decreased by VIVIT. Immunocytochemical examination of NFATc1-4 of BMMs exhibited nuclear localization of NFATc3/c4 regardless of LPS stimulation. LPS stimulation did not cause nuclear translocation of NFATc1/c2. Treatment with VIVIT resulted in nuclear export of NFATc3/c4 and inhibited TLR-activated TNF expression, suggesting that nuclear residence of NFATc is required for TLR-related innate immune response. Chromatin immunoprecipitation (ChIP) assay using anti-RNA polymerase II (PolII) antibody suggested that VIVIT decreased PolII binding to TNF gene locus, consistent with VIVIT inhibition of LPS-induced TNF mRNA expression. This study identifies a novel paradigm of innate immune regulation rendered by NFAT which is a well known family of adaptive immune regulatory proteins.

  5. Expression of nuclear factor of activated T cells (NFAT) and downstream muscle-specific proteins in ground squirrel skeletal and heart muscle during hibernation.

    PubMed

    Zhang, Yichi; Storey, Kenneth B

    2016-01-01

    The thirteen-lined ground squirrel (Ictidomys tridecemlineatus) undergoes remarkable adaptive changes during hibernation. Interestingly, skeletal muscle remodelling occurs during the torpor-arousal cycle of hibernation to prevent net muscle loss despite inactivity. Reversible cardiomyocyte hypertrophy occurs in cardiac muscle, allowing the heart to preserve cardiac output during hibernation, while avoiding chronic maladaptive hypertrophy post-hibernation. We propose that calcium signalling proteins [calcineurin (Cn), calmodulin (CaM), and calpain], the nuclear factor of activated T cell (NFAT) family of transcription factors, and the NFAT targets myoferlin and myomaker contribute significantly to adaptations taking place in skeletal and cardiac muscle during hibernation. Protein-level analyses were performed over several conditions: euthermic room temperature (ER), euthermic cold room (EC), entrance into (EN), early (ET), and late torpor (LT) time points, in addition to early (EA), interbout (IA), and late arousal (LA) time points using immunoblotting and DNA-protein interaction (DPI) enzyme-linked immunosorbent assay (ELISAs). In skeletal and cardiac muscle, NFATc2 protein levels were elevated during torpor. NFATc4 increased throughout the torpor-arousal cycle in both tissues, and NFATc1 showed this trend in cardiac muscle only. NFATc3 showed an elevation in DNA-binding activity but not expression during torpor. Myoferlin protein levels dramatically increased during torpor in both skeletal and cardiac muscle. Myomaker levels also increased significantly in cardiac muscle during torpor. Cardiac Cn levels remained stable, whereas CaM and calpain decreased throughout the torpor-arousal cycle. Activation and/or upregulation of NFATc2, c3, myoferlin, and myomaker at torpor could be part of a stress-response mechanism to preserve skeletal muscle mass, whereas CaM and calpain appear to initiate the rapid reversal of cardiac hypertrophy during arousal through

  6. Nuclear factor of activated T cell (NFAT) transcription proteins regulate genes involved in adipocyte metabolism and lipolysis

    SciTech Connect

    Holowachuk, Eugene W. . E-mail: geneh@telenet.net

    2007-09-21

    NFAT involvement in adipocyte physiological processes was examined by treatment with CsA and/or GSK3{beta} inhibitors (Li{sup +} or TZDZ-8), which prevent or increase NFAT nuclear translocation, respectively. CsA treatment reduced basal and TNF{alpha}-induced rates of lipolysis by 50%. Adipocytes preincubated with Li{sup +} or TZDZ-8 prior to CsA and/or TNF{alpha}, exhibited enhanced basal rates of lipolysis and complete inhibition of CsA-mediated decreased rates of lipolysis. CsA treatment dramatically reduced the mRNA levels of adipocyte-specific genes (aP2, HSL, PPAR{gamma}, ACS and Adn), compared with control or TNF{alpha}-treatment, whereas Li{sup +} pretreatment blocked the inhibitory effects of CsA, and mRNA levels of aP2, HSL, PPAR{gamma}, and ACS were found at or above control levels. NFAT nuclear localization, assessed by EMSA, confirmed that CsA or Li{sup +} treatments inhibited or increased NFAT nuclear translocation, respectively. These results show that NFAT proteins in mature adipocytes participate in the transcriptional control of genes involved in adipocyte metabolism and lipolysis.

  7. A fluorescence-based assay to monitor transcriptional activity of NFAT in living cells.

    PubMed

    Rinne, Andreas; Blatter, Lothar A

    2010-09-01

    Ca(2+)-sensitive NFAT (nuclear factor of activated T-cells) transcription factors are implicated in many pathophysiological processes in different cell types. The precise control of activation varies with NFAT isoform and cell type. Here we present feasibility of an in vivo assay (NFAT-RFP) that reports transcriptional activity of NFAT via expression of red fluorescent protein (RFP) in individual cells. This new tool allows continuous monitoring of transcriptional activity of NFAT in a physiological context in living cells. Furthermore, NFAT-RFP can be used simultaneously with NFAT-GFP fusion proteins to monitor transcriptional activity and subcellular localization of NFAT in the same cell.

  8. HIV-1 gp120 induces NFAT nuclear translocation in resting CD4+ T-cells

    SciTech Connect

    Cicala, Claudia . E-mail: ccicala@nih.gov; Arthos, James; Censoplano, Nina; Cruz, Catherine; Chung, Eva; Martinelli, Elena; Lempicki, Richard A.; Natarajan, Ven; VanRyk, Donald; Daucher, Marybeth; Fauci, Anthony S.

    2006-02-05

    The replication of human immunodeficiency virus (HIV) in CD4+ T-cells is strongly dependent upon the state of activation of infected cells. Infection of sub-optimally activated cells is believed to play a critical role in both the transmission of virus and the persistence of CD4+ T-cell reservoirs. There is accumulating evidence that HIV can modulate signal-transduction pathways in a manner that may facilitate replication in such cells. We previously demonstrated that HIV gp120 induces virus replication in resting CD4+ T cells isolated from HIV-infected individuals. Here, we show that in resting CD4+ T-cells, gp120 activates NFATs and induces their translocation into the nucleus. The HIV LTR encodes NFAT recognition sites, and NFATs may play a critical role in promoting viral replication in sub-optimally activated cells. These observations provide insight into a potential mechanism by which HIV is able to establish infection in resting cells, which may have implications for both transmission of HIV and the persistence of viral reservoirs.

  9. Impaired NFAT and NFκB activation are involved in suppression of CD40 ligand expression by Δ{sup 9}-tetrahydrocannabinol in human CD4{sup +} T cells

    SciTech Connect

    Ngaotepprutaram, Thitirat; Kaplan, Barbara L.F.; Kaminski, Norbert E.

    2013-11-15

    We have previously reported that Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), the main psychoactive cannabinoid in marijuana, suppresses CD40 ligand (CD40L) expression by activated mouse CD4{sup +} T cells. CD40L is involved in pathogenesis of many autoimmune and inflammatory diseases. In the present study, we investigated the molecular mechanism of Δ{sup 9}-THC-mediated suppression of CD40L expression using peripheral blood human T cells. Pretreatment with Δ{sup 9}-THC attenuated CD40L expression in human CD4{sup +} T cells activated by anti-CD3/CD28 at both the protein and mRNA level, as determined by flow cytometry and quantitative real-time PCR, respectively. Electrophoretic mobility shift assays revealed that Δ{sup 9}-THC suppressed the DNA-binding activity of both NFAT and NFκB to their respective response elements within the CD40L promoter. An assessment of the effect of Δ{sup 9}-THC on proximal T cell-receptor (TCR) signaling induced by anti-CD3/CD28 showed significant impairment in the rise of intracellular calcium, but no significant effect on the phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β. Collectively, these findings identify perturbation of the calcium-NFAT and NFκB signaling cascade as a key mechanistic event by which Δ{sup 9}-THC suppresses human T cell function. - Highlights: • Δ{sup 9}-THC attenuated CD40L expression in activated human CD4+ T cells. • Δ{sup 9}-THC suppressed DNA-binding activity of NFAT and NFκB. • Δ{sup 9}-THC impaired elevation of intracellular Ca2+. • Δ{sup 9}-THC did not affect phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β.

  10. Transcription factor NFAT1 controls allergic contact hypersensitivity through regulation of activation induced cell death program

    PubMed Central

    Kwon, Ho-Keun; Kim, Gi-Cheon; Hwang, Ji Sun; Kim, Young; Chae, Chang-Suk; Nam, Jong Hee; Jun, Chang-Duk; Rudra, Dipayan; Surh, Charles D.; Im, Sin-Hyeog

    2016-01-01

    Allergic contact hypersensitivity (CHS) is an inflammatory skin disease mediated by allergen specific T cells. In this study, we investigated the role of transcription factor NFAT1 in the pathogenesis of contact hypersensitivity. NFAT1 knock out (KO) mice spontaneously developed CHS-like skin inflammation in old age. Healthy young NFAT1 KO mice displayed enhanced susceptibility to hapten-induced CHS. Both CD4+ and CD8+ T cells from NFAT1 KO mice displayed hyper-activated properties and produced significantly enhanced levels of inflammatory T helper 1(Th1)/Th17 type cytokines. NFAT1 KO T cells were more resistant to activation induced cell death (AICD), and regulatory T cells derived from these mice showed a partial defect in their suppressor activity. NFAT1 KO T cells displayed a reduced expression of apoptosis associated BCL-2/BH3 family members. Ectopic expression of NFAT1 restored the AICD defect in NFAT1 KO T cells and increased AICD in normal T cells. Recipient Rag2−/− mice transferred with NFAT1 KO T cells showed more severe CHS sensitivity due to a defect in activation induced hapten-reactive T cell apoptosis. Collectively, our results suggest the NFAT1 plays a pivotal role as a genetic switch in CD4+/CD8+ T cell tolerance by regulating AICD process in the T cell mediated skin inflammation. PMID:26777750

  11. PTD-mediated intracellular delivery of mutant NFAT minimum DNA binding domain inhibited the proliferation of T cells.

    PubMed

    Liu, Xia; Zhao, Qianqian; Peng, Xin; Xia, Sheng; Shen, Weihong; Zong, Yangyong; Cheng, Jing; Wu, Weijiang; Zhang, Miaomiao; Du, Fengyi; Xu, Wenrong; Qian, Hui; Shao, Qixiang

    2014-03-01

    The nuclear factor of activated T cell (NFAT) family of calcium-regulated transcription factors plays a key role in the development and function of the immune system. Calcineurin, a protein phosphatase, activates NFAT by dephosphorylation. The activated NFAT is translocated into the nucleus, where it up-regulates the expression of interleukin 2 (IL-2) and other target genes. Calcineurin inhibitors such as cyclosporine A (CsA) and FK506 are effective immunosuppressant drugs and dramatically increase the success rate of organ transplantation procedures. However, since calcineurin is expressed in most tissues in the body and calcineurin inhibition alters many cellular processes besides immune cell activation, the therapeutic use of calcineurin inhibitors is limited by serious side effects. Thus inhibiting NFAT by other mechanisms such as blocking its binding to DNA could be a more selective and safer approach to target NFAT for therapeutic applications. In peripheral T cells, productive immune responses are dependent upon the cooperative binding of the NFAT/AP-1 transcriptional complex to the promoter regions of genes such as interleukin-2 (IL-2), while NFAT in the absence of AP-1 leads to T cell anergy. Protein transduction domains (PTDs) are able to penetrate cell membranes and can be used to transport exogenous proteins across the cell and nuclear membranes. In this study, we constructed a fusion protein of PTD and a minimum DNA binding domain of human NFAT1 (PTD-ΔNFATminiDBD), which contains two mutations (R466A and T533G) in the AP-1 binding sites. The delivery and functions of this fusion protein in T cells were investigated. The results indicated that PTD-ΔNFATminiDBD could be effectively delivered into T cells and transported into the nucleus. PTD-ΔNFATminiDBD attenuated IL-2 production in T cells and then inhibited T cell proliferation, likely through competing against endogenous NFAT for binding to the IL-2 gene promoter. These results demonstrated that

  12. Genome-wide approaches reveal functional vascular endothelial growth factor (VEGF)-inducible nuclear factor of activated T cells (NFAT) c1 binding to angiogenesis-related genes in the endothelium.

    PubMed

    Suehiro, Jun-ichi; Kanki, Yasuharu; Makihara, Chihiro; Schadler, Keri; Miura, Mai; Manabe, Yuuka; Aburatani, Hiroyuki; Kodama, Tatsuhiko; Minami, Takashi

    2014-10-17

    VEGF is a key regulator of endothelial cell migration, proliferation, and inflammation, which leads to activation of several signaling cascades, including the calcineurin-nuclear factor of activated T cells (NFAT) pathway. NFAT is not only important for immune responses but also for cardiovascular development and the pathogenesis of Down syndrome. By using Down syndrome model mice and clinical patient samples, we showed recently that the VEGF-calcineurin-NFAT signaling axis regulates tumor angiogenesis and tumor metastasis. However, the connection between genome-wide views of NFAT-mediated gene regulation and downstream gene function in the endothelium has not been studied extensively. Here we performed comprehensive mapping of genome-wide NFATc1 binding in VEGF-stimulated primary cultured endothelial cells and elucidated the functional consequences of VEGF-NFATc1-mediated phenotypic changes. A comparison of the NFATc1 ChIP sequence profile and epigenetic histone marks revealed that predominant NFATc1-occupied peaks overlapped with promoter-associated histone marks. Moreover, we identified two novel NFATc1 regulated genes, CXCR7 and RND1. CXCR7 knockdown abrogated SDF-1- and VEGF-mediated cell migration and tube formation. siRNA treatment of RND1 impaired vascular barrier function, caused RhoA hyperactivation, and further stimulated VEGF-mediated vascular outgrowth from aortic rings. Taken together, these findings suggest that dynamic NFATc1 binding to target genes is critical for VEGF-mediated endothelial cell activation. CXCR7 and RND1 are NFATc1 target genes with multiple functions, including regulation of cell migration, tube formation, and barrier formation in endothelial cells.

  13. Glucocorticoid-induced TNF receptor expression by T cells is reciprocally regulated by NF-kappaB and NFAT.

    PubMed

    Zhan, Yifan; Gerondakis, Steve; Coghill, Elise; Bourges, Dorothee; Xu, Yuekang; Brady, Jamie L; Lew, Andrew M

    2008-10-15

    Although the transcription factor Foxp3 is implicated in regulating glucocorticoid-induced TNF receptor (GITR) expression in the T regulatory cell lineage, little is known about how GITR is transcriptionally regulated in conventional T cells. In this study, we provide evidence that TCR-mediated GITR expression depends on the ligand affinity and the maturity of conventional T cells. A genetic dissection of GITR transcriptional control revealed that of the three transcription factors downstream of the classical NF-kappaB pathway (RelA, cRel, and NF-kappaB1), RelA is a critical positive regulator of GITR expression, although cRel and NF-kappaB1 also play a positive regulatory role. Consistent with this finding, inhibiting NF-kappaB using Bay11-7082 reduces GITR up-regulation. In contrast, NFAT acts as a negative regulator of GITR expression. This was evidenced by our findings that agents suppressing NFAT activity (e.g., cyclosporin A and FK506) enhanced TCR-mediated GITR expression, whereas agents enhancing NFAT activity (e.g., lithium chloride) suppressed TCR-mediated GITR up-regulation. Critically, the induction of GITR was found to confer protection to conventional T cells from TCR-mediated apoptosis. We propose therefore that two major transcriptional factors activated downstream of the TCR, namely, NF-kappaB and NFAT, act reciprocally to balance TCR-mediated GITR expression in conventional T cells, an outcome that appears to influence cell survival.

  14. Regulation of different human NFAT isoforms by neuronal activity.

    PubMed

    Vihma, Hanna; Luhakooder, Mirjam; Pruunsild, Priit; Timmusk, Tõnis

    2016-05-01

    Nuclear factor of activated T-cells (NFAT) is a family of transcription factors comprising four calcium-regulated members: NFATc1, NFATc2, NFATc3, and NFATc4. Upon activation by the calcium-dependent phosphatase calcineurin (CaN), NFATs translocate from cytosol to the nucleus and regulate their target genes, which in the nervous system are involved in axon growth, synaptic plasticity, and neuronal survival. We have shown previously that there are a number of different splice variants of NFAT genes expressed in the brain. Here, we studied the subcellular localizations and transactivation capacities of alternative human NFAT isoforms in rat primary cortical or hippocampal neurons in response to membrane depolarization and compared the induced transactivation levels in neurons to those obtained from HEK293 cells in response to calcium signaling. We confirm that in neurons the translocation to the nucleus of all NFAT isoforms is reliant on the activity of CaN. However, our results suggest that both the regulation of subcellular localization and transcriptional activity of NFAT proteins in neurons is isoform specific. We show that in primary hippocampal neurons NFATc2 isoforms have very fast translocation kinetics, whereas NFATc4 isoforms translocate relatively slowly to the nucleus. Moreover, we demonstrate that the strongest transcriptional activators in HEK293 cells are NFATc1 and NFATc3, but in neurons NFATc3 and NFATc4 lead to the highest induction, and NFATc2 and NFATc1 display isoform-specific transcription activation capacities. Altogether, our results indicate that the effects of calcium signaling on the action of NFAT proteins are isoform-specific and can differ between cell types. We show that the effects of calcium signaling on the action of NFAT proteins are isoform-specific and differ between cell types. Although nuclear localization of all NFAT isoforms in neurons requires calcineurin, the subcellular distributions, neuronal activity-induced nuclear

  15. Immunosuppressive Activity of Daphnetin, One of Coumarin Derivatives, Is Mediated through Suppression of NF-κB and NFAT Signaling Pathways in Mouse T Cells

    PubMed Central

    Liu, Yan; Xu, Sisi; Huang, Guoren; Xiong, Ying; Zhang, Shuang; Xu, Linli; Deng, Xuming; Guan, Shuang

    2014-01-01

    Daphnetin, a plant-derived dihydroxylated derivative of coumarin, is an effective compound extracted from a plant called Daphne Korean Nakai. Coumarin derivates were known for their antithrombotic, anti-inflammatory, and antioxidant activities. The present study was aimed to determine the immunosuppressive effects and the underlying mechanisms of daphnetin on concanavalin A (ConA) induced T lymphocytes in mice. We showed that, in vitro, daphnetin suppressed ConA-induced splenocyte proliferation, influenced production of the cytokines and inhibited cell cycle progression through the G0/G1 transition. The data also revealed that daphnetin could down-regulate activation of ConA induced NF-κB and NFAT signal transduction pathways in mouse T lymphocyte. In vivo, daphnetin treatment significantly inhibited the 2, 4- dinitrofluorobenzene (DNFB) -induced delayed type hypersensitivity (DTH) reactions in mice. Collectively, daphnetin had strong immunosuppressive activity both in vitro and in vivo, suggesting a potential role for daphnetin as an immunosuppressive agent, and established the groundwork for further research on daphnetin. PMID:24800925

  16. Calcineurin-NFAT activation and DSCR-1 auto-inhibitory loop: how is homoeostasis regulated?

    PubMed

    Minami, Takashi

    2014-04-01

    Calcineurin-nuclear factor of activated T cells (NFAT) signalling plays a critical role not only in the immune and nervous systems, but also in cardiovascular development and pathological endothelial cell activation during angiogenesis or inflammation. Studies in NFAT-null mice demonstrated that there is high redundancy between functions of the different NFAT family members. Deletion of only one NFAT causes mild phenotypes, but compound deletions of multiple NFAT family members leads to severe abnormalities in multiple organ systems. Genome-wide transcription analysis revealed that many NFAT target genes are related to cell growth and inflammation, whereas the gene most strongly induced by NFAT in endothelial cells is an auto-inhibitory molecule, Down syndrome critical region (DSCR)-1. The NFAT-DSCR-1 signalling axis may vary depending on the cell-type or signal dosage level under the microenvironment. In the endothelium, stable expression of the DSCR-1 short isoform attenuates septic inflammatory shock, tumour growth and tumour metastasis to lung. Moreover, dysfunction of DSCR-1 and the NFAT priming kinase, DYRK1A, prevents NFAT nuclear occupancy. This change in NFAT nuclear localization is responsible for many of the features of Down syndrome. Thus, fine-tuning of the NFAT-DSCR-1 negative feedback loop may enable therapeutic manipulation in vasculopathic diseases.

  17. Calcineurin-NFAT activation and DSCR-1 auto-inhibitory loop: how is homoeostasis regulated?

    PubMed

    Minami, Takashi

    2014-04-01

    Calcineurin-nuclear factor of activated T cells (NFAT) signalling plays a critical role not only in the immune and nervous systems, but also in cardiovascular development and pathological endothelial cell activation during angiogenesis or inflammation. Studies in NFAT-null mice demonstrated that there is high redundancy between functions of the different NFAT family members. Deletion of only one NFAT causes mild phenotypes, but compound deletions of multiple NFAT family members leads to severe abnormalities in multiple organ systems. Genome-wide transcription analysis revealed that many NFAT target genes are related to cell growth and inflammation, whereas the gene most strongly induced by NFAT in endothelial cells is an auto-inhibitory molecule, Down syndrome critical region (DSCR)-1. The NFAT-DSCR-1 signalling axis may vary depending on the cell-type or signal dosage level under the microenvironment. In the endothelium, stable expression of the DSCR-1 short isoform attenuates septic inflammatory shock, tumour growth and tumour metastasis to lung. Moreover, dysfunction of DSCR-1 and the NFAT priming kinase, DYRK1A, prevents NFAT nuclear occupancy. This change in NFAT nuclear localization is responsible for many of the features of Down syndrome. Thus, fine-tuning of the NFAT-DSCR-1 negative feedback loop may enable therapeutic manipulation in vasculopathic diseases. PMID:24505143

  18. NFAT5 induction by the pre–T-cell receptor serves as a selective survival signal in T-lymphocyte development

    PubMed Central

    Berga-Bolaños, Rosa; Alberdi, Maria; Buxadé, Maria; Aramburu, José; López-Rodríguez, Cristina

    2013-01-01

    The Rel-like transcription factors nuclear factor kappa B (NF-κB) and the calcineurin-dependent nuclear factor of activated T cells (NFATc) control specific points of thymocyte maturation. Thymocytes also express a distinct member of the Rel family, the calcineurin-independent, osmostress response regulator NFAT5. Here we show that IKKβ regulates the expression of NFAT5 in thymocytes, which in turn contributes to the survival of T-cell receptor αβ thymocytes and the transition from the β-selection checkpoint to the double-positive stage in an osmostress-independent manner. NFAT5-deficient thymocytes had normal expression and proximal signaling of the pre–T-cell receptor but exhibited a partial defect in β-chain allelic exclusion and increased apoptosis. Further analysis showed that NFAT5 regulated the expression of the prosurvival factors A1 and Bcl2 and attenuated the proapoptotic p53/Noxa axis. These findings position NFAT5 as a target of the IKKβ/NF-κB pathway in thymocytes and as a downstream effector of the prosurvival role of the pre–T-cell receptor. PMID:24043824

  19. NFAT5 induction by the pre-T-cell receptor serves as a selective survival signal in T-lymphocyte development.

    PubMed

    Berga-Bolaños, Rosa; Alberdi, Maria; Buxadé, Maria; Aramburu, José; López-Rodríguez, Cristina

    2013-10-01

    The Rel-like transcription factors nuclear factor kappa B (NF-κB) and the calcineurin-dependent nuclear factor of activated T cells (NFATc) control specific points of thymocyte maturation. Thymocytes also express a distinct member of the Rel family, the calcineurin-independent, osmostress response regulator NFAT5. Here we show that IKKβ regulates the expression of NFAT5 in thymocytes, which in turn contributes to the survival of T-cell receptor αβ thymocytes and the transition from the β-selection checkpoint to the double-positive stage in an osmostress-independent manner. NFAT5-deficient thymocytes had normal expression and proximal signaling of the pre-T-cell receptor but exhibited a partial defect in β-chain allelic exclusion and increased apoptosis. Further analysis showed that NFAT5 regulated the expression of the prosurvival factors A1 and Bcl2 and attenuated the proapoptotic p53/Noxa axis. These findings position NFAT5 as a target of the IKKβ/NF-κB pathway in thymocytes and as a downstream effector of the prosurvival role of the pre-T-cell receptor.

  20. Assessment of costimulation and coinhibition in a triple parameter T cell reporter line: Simultaneous measurement of NF-κB, NFAT and AP-1.

    PubMed

    Jutz, Sabrina; Leitner, Judith; Schmetterer, Klaus; Doel-Perez, Iago; Majdic, Otto; Grabmeier-Pfistershammer, Katharina; Paster, Wolfgang; Huppa, Johannes B; Steinberger, Peter

    2016-03-01

    Engagement of the T cell receptor complex reprograms T cells for proliferation, cytokine production and differentiation towards effector cells. This process depends on activating costimulatory signals and is counteracted by coinhibitory molecules. Three transcription factors, namely NF-κB, NFAT and AP-1, have a major role in inducing the transcriptional program that is required for T cell activation and differentiation. Here we describe the generation of a triple parameter reporter based on the human Jurkat T cell line, where response elements for NF-κB, NFAT and AP-1 drive the expression of the fluorescent proteins CFP, eGFP and mCherry, respectively. The emission spectra of these proteins allow simultaneous assessment of NF-κB, NFAT and AP-1 activity in response to stimulation. Ligation of the TCR complex induced moderate reporter activity, which was strongly enhanced upon coengagement of the costimulatory receptors CD2 or CD28. Moreover, we have generated and tested triple parameter reporter cells that harbor costimulatory and inhibitory receptors not endogenously expressed in the Jurkat cells. In these experiments we could show that engagement of the costimulatory molecule 4-1BB enhances NF-κB and AP-1 activity, whereas coinhibition via PD-1 or BTLA strongly reduced the activation of NF-κB and NFAT. Engagement of BTLA significantly inhibited AP-1, whereas PD-1 had little effect on the activation of this transcription factor. Our triple parameter reporter T cell line is an excellent tool to assess the effect of costimulatory and coinhibitory receptors on NF-κB, NFAT and AP-1 activity and has a wide range of applications beyond the evaluation of costimulatory pathways.

  1. Oxytocin-stimulated NFAT transcriptional activation in human myometrial cells.

    PubMed

    Pont, Jason N A; McArdle, Craig A; López Bernal, Andrés

    2012-10-01

    Oxytocin (OXT) is a peptide hormone that binds the OXT receptor on myometrial cells, initiating an intracellular signaling cascade, resulting in accumulation of intracellular calcium and smooth muscle contraction. In other systems, an elevation of intracellular Ca(2+) stimulates nuclear translocation of the transcription factor, nuclear factor of activated T cells (NFAT), which is transcriptionally active in arterial and ileal smooth muscle. Here we have investigated the role of NFAT in the mechanism of action of OXT. Human myometrial cells expressed all five NFAT isoforms (NFATC1-C4 and -5). Myometrial cells were transduced with a recombinant adenovirus expressing a NFATC1-EFP reporter, and a semi-automated imaging system was used to monitor effects of OXT on reporter localization in live cells. OXT induced a concentration-dependent nuclear translocation of NFATC1-EFP in a reversible manner, which was inhibited by OXT antagonists and calcineurin inhibitors. Pulsatile stimulation with OXT caused intermittent, pulse-frequency-dependent, nuclear translocation of NFATC1-EFP, which was more efficient than sustained stimulation. OXT induced nuclear translocation of endogenous NFAT that was transcriptionally active, because OXT stimulated activity of a NFAT-response element-luciferase reporter and induced calcineurin-NFAT dependent expression of RGS2, RCAN1, and PTGS2 (COX2) mRNA. Furthermore, OXT-dependent transcription was dependent on protein neosynthesis; cycloheximide abolished RGS2 transcription but augmented RCAN1 and COX2 transcriptional readouts. This study identifies a novel signaling mechanism within the myometrium, whereby calcineurin-NFAT signaling mediates OXT-induced transcriptional activity. Furthermore, we show NFATC1-EFP is responsive to pulses of OXT, a mechanism by which myometrial cells could decode OXT pulse frequency.

  2. Expression, localisation and functional activation of NFAT-2 in normal human skin, psoriasis, and cultured keratocytes.

    PubMed

    Al-Daraji, Wael I; Malak, Tamer T; Prescott, Richard J; Abdellaoui, Adel; Ali, Mahmud M; Dabash, Tarek; Zelger, Bettina G; Zelger, Bernhard

    2009-06-18

    Ciclosporin A (CsA) is widely utilized for the treatment of inflammatory skin diseases such as psoriasis. The therapeutic effects of CsA are thought to be mediated via its immunosuppressive action on infiltrating lymphocytes in skin lesions. CsA and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor Nuclear Factor of Activated T cells (NFAT). As calcineurin and NFAT 1 have been shown to be functionally active in cultured human keratocytes, expression of other NFAT family members such as NFAT-2 and possible functional activation was investigated in human keratocytes. RT-PCR and Western Analysis were used to investigate the presence of NFAT-2 mRNA and protein in human keratocytes. Tissue culture of human keratocytes and immunostaining of cells on coverslips and confocal microscopy were used to assess the degree of nuclear localisation of NFAT-2 in cultured cells. Keratome biopsies were taken from patients with psoriasis (lesional and non-lesional skin) and normal skin and immunohistochemistry was used to assess the NFAT-2 localisation in these biopsies using a well characterized anti-NFAT-2 antibody. The NFAT-2 mRNA and protein expression was demonstrated using RT-PCR and Western blotting. Moreover, the expression of NFAT-2 in normal skin, non-lesional and lesional psoriasis showed a striking basal staining suggesting a role for NFAT-2 in keratocytes proliferation. A range of cell types in the skin express NFAT-2. The expression of NFAT-2 in human keratocytes and response to different agonists provides perhaps a unique opportunity to examine the regulation, subcellular localization and kinetics of translocation of different NFATs in primary cultured human cells. In these experiments the author assessed the expression, localization of NFAT-2 in cultured human keratocytes and measured the degree of nuclear localisaion of NFAT-2 using immunofluorescence

  3. Antisense-mediated loss of calcium homoeostasis endoplasmic reticulum protein (CHERP; ERPROT213-21) impairs Ca2+ mobilization, nuclear factor of activated T-cells (NFAT) activation and cell proliferation in Jurkat T-lymphocytes.

    PubMed Central

    O'Rourke, Flavia A; LaPlante, Janice M; Feinstein, Maurice B

    2003-01-01

    We recently discovered a novel gene on chromosome 19p13.1 and its product, an integral endoplasmic reticulum (ER) membrane protein, termed CHERP (calcium homoeostasis endoplasmic reticulum protein). A monoclonal antibody against its C-terminal domain inhibits Ins(1,4,5) P (3)-induced Ca(2+) release from ER membrane vesicles of many cell types, and an antisense-mediated knockdown of CHERP in human erythroleukemia (HEL) cells greatly impaired Ca(2+) mobilization by thrombin. In the present paper, we explore further CHERP's function in Jurkat T-lymphocytes. Confocal laser immunofluorescence microscopy showed that CHERP was co-localized with the Ins(1,4,5) P (3) receptor throughout the cytoplasmic and perinuclear region, as previously found in HEL cells. Transfection of Jurkat cells with a lac I-regulated mammalian expression vector containing CHERP antisense cDNA caused a knockdown of CHERP and impaired the rise of cytoplasmic Ca(2+) (measured by fura-2 acetoxymethyl ester fluorescence) caused by phytohaemagglutinin (PHA) and thrombin. A 50% fall of CHERP decreased the PHA-induced rise of the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)), but Ca(2+) influx was unaffected. Greater depletion of CHERP (>70%) did not affect the concentration of Ins(1,4,5) P (3) receptors, but diminished the rise of [Ca(2+)](i) in response to PHA to activation and translocation of the key transcription factor NFAT (nuclear factor of activated T-cells) from cytoplasm to nucleus was suppressed. Furthermore, cell proliferation was greatly slowed (as in HEL cells) along with a 60% decrease in cyclin D1, a key regulator of progression through the G(1) phase of the cell cycle. These findings

  4. Disparate effects of serum on basal and evoked NFAT activity in primary astrocyte cultures.

    PubMed

    Furman, Jennifer L; Artiushin, Irina A; Norris, Christopher M

    2010-01-29

    In astrocytes, the Ca(2+)-dependent protein phosphatase calcineurin (CN) strongly regulates neuro-immune/inflammatory cascades through activation of the transcription factor, nuclear factor of activated T cells (NFAT). While primary cell cultures provide a useful model system for investigating astrocytic CN/NFAT signaling, variable results may arise both within and across labs because of differences in culture conditions. Here, we determined the extent to which serum and cell confluency affect basal and evoked astrocytic NFAT activity in primary cortical astrocyte cultures. Cells were grown to either approximately 50% or >90% confluency, pre-loaded with an NFAT-luciferase reporter construct, and maintained for 16 h in medium with or without 10% fetal bovine serum (FBS). NFAT-dependent luciferase expression was then measured 5h after treatment with vehicle alone to assess basal NFAT activity, or with Ca(2+) mobilizers and IL-1 beta to assess evoked activity. The results revealed significantly higher levels of basal NFAT activity in FBS-containing medium, regardless of cell confluency. Conversely, evoked NFAT activation was significantly lower in serum-containing medium, with an even greater inhibition observed in confluent cultures. Application of 10% FBS to serum-free astrocyte cultures quickly evoked a roughly seven-fold increase in NFAT activity that was significantly reduced by co-delivery of neutralizing agents for IL-1 beta, TNFalpha, and/or IFN gamma, suggesting that serum occludes evoked NFAT activation through a cytokine-based mechanism. Together, the results demonstrate that the presence of serum and cell confluency have a major impact on CN/NFAT signaling in primary astrocyte cultures and therefore must be taken into consideration when using this model system.

  5. Dual effect of lithium on NFAT5 activity in kidney cells

    PubMed Central

    Küper, Christoph; Beck, Franz-Xaver; Neuhofer, Wolfgang

    2015-01-01

    Lithium salts are used widely for treatment of bipolar and other mental disorders. Lithium therapy is accompanied frequently by renal side effects, such as nephrogenic diabetes insipidus or chronic kidney disease (CKD), but the molecular mechanisms underlying these effects are still poorly understood. In the present study we examined the effect of lithium on the activity of the osmosensitive transcriptional activator nuclear factor of activated T cells 5 (NFAT5, also known as TonEBP), which plays a key role in renal cellular osmoprotection and urinary concentrating ability. Interestingly, we found different effects of lithium on NFAT5 activity, depending on medium osmolality and incubation time. When cells were exposed to lithium for a relative short period (24 h), NFAT5 activity was significantly increased, especially under isosmotic conditions, resulting in an enhanced expression of the NFAT5 target gene heat shock protein 70 (HSP70). Further analysis revealed that the increase of NFAT5 activity depended primarily on an enhanced activity of the c-terminal transactivation domain (TAD), while NFAT5 protein abundance was largely unaffected. Enhanced activity of the TAD is probably mediated by lithium-induced inhibitory phosphorylation of glycogen synthase kinase 3β (GSK-3β), which is in accordance with previous studies. When cells were exposed to lithium for a longer period (96 h), cellular NFAT5 activity and subsequently expression of HSP70 significantly decreased under hyperosmotic conditions, due to diminished NFAT5 protein abundance, also resulting from GSK-3β inhibition. Taken together, our results provide evidence that lithium has opposing effects on NFAT5 activity, depending on environmental osmolality and exposure duration. The potential impacts of these observations on the diverse effects of lithium on kidney function are discussed. PMID:26441681

  6. Dual effect of lithium on NFAT5 activity in kidney cells.

    PubMed

    Küper, Christoph; Beck, Franz-Xaver; Neuhofer, Wolfgang

    2015-01-01

    Lithium salts are used widely for treatment of bipolar and other mental disorders. Lithium therapy is accompanied frequently by renal side effects, such as nephrogenic diabetes insipidus or chronic kidney disease (CKD), but the molecular mechanisms underlying these effects are still poorly understood. In the present study we examined the effect of lithium on the activity of the osmosensitive transcriptional activator nuclear factor of activated T cells 5 (NFAT5, also known as TonEBP), which plays a key role in renal cellular osmoprotection and urinary concentrating ability. Interestingly, we found different effects of lithium on NFAT5 activity, depending on medium osmolality and incubation time. When cells were exposed to lithium for a relative short period (24 h), NFAT5 activity was significantly increased, especially under isosmotic conditions, resulting in an enhanced expression of the NFAT5 target gene heat shock protein 70 (HSP70). Further analysis revealed that the increase of NFAT5 activity depended primarily on an enhanced activity of the c-terminal transactivation domain (TAD), while NFAT5 protein abundance was largely unaffected. Enhanced activity of the TAD is probably mediated by lithium-induced inhibitory phosphorylation of glycogen synthase kinase 3β (GSK-3β), which is in accordance with previous studies. When cells were exposed to lithium for a longer period (96 h), cellular NFAT5 activity and subsequently expression of HSP70 significantly decreased under hyperosmotic conditions, due to diminished NFAT5 protein abundance, also resulting from GSK-3β inhibition. Taken together, our results provide evidence that lithium has opposing effects on NFAT5 activity, depending on environmental osmolality and exposure duration. The potential impacts of these observations on the diverse effects of lithium on kidney function are discussed. PMID:26441681

  7. Aberrant activation of nuclear factor of activated T cell 2 in lamina propria mononuclear cells in ulcerative colitis

    PubMed Central

    Shih, Tsung-Chieh; Hsieh, Sen-Yung; Hsieh, Yi-Yueh; Chen, Tse-Chin; Yeh, Chien-Yu; Lin, Chun-Jung; Lin, Deng-Yn; Chiu, Cheng-Tang

    2008-01-01

    AIM: To investigate the role of nuclear factor of activated T cell 2 (NFAT2), the major NFAT protein in peripheral T cells, in sustained T cell activation and intractable inflammation in human ulcerative colitis (UC). METHODS: We used two-dimensional gel-electrophoresis, immunohistochemistry, double immunohistochemical staining, and confocal microscopy to inspect the expression of NFAT2 in 107, 15, 48 and 5 cases of UC, Crohn’s disease (CD), non-specific colitis, and 5 healthy individuals, respectively. RESULTS: Up-regulation with profound nucleo-translocation/activation of NFAT2 of lamina propria mononuclear cells (LPMC) of colonic mucosa was found specifically in the affected colonic mucosa from patients with UC, as compared to CD or NC (P < 0.001, Kruskal-Wallis test). Nucleo-translocation/activation of NFAT2 primarily occurred in CD8+T, but was less prominent in CD4+ T cells or CD20+B cells. It was strongly associated with the disease activity, including endoscopic stage (τ = 0.2145, P = 0.0281) and histologic grade (τ = 0.4167, P < 0.001). CONCLUSION: We disclose for the first time the nucleo-translocation/activatin of NFAT2 in lamina propria mononuclear cells in ulcerative colitis. Activation of NFAT2 was specific for ulcerative colitis and highly associated with disease activity. Since activation of NFAT2 is implicated in an auto-regulatory positive feedback loop of sustained T-cell activation and NFAT proteins play key roles in the calcium/calcineurin signaling pathways, our results not only provide new insights into the mechanism for sustained intractable inflammation, but also suggest the calcium-calcineurin/NFAT pathway as a new therapeutic target for ulcerative colitis. PMID:18350607

  8. Angiotensin II and norepinephrine activate specific calcineurin-dependent NFAT transcription factor isoforms in cardiomyocytes.

    PubMed

    Lunde, Ida G; Kvaløy, Heidi; Austbø, Bjørg; Christensen, Geir; Carlson, Cathrine R

    2011-11-01

    Norepinephrine (NE) and angiotensin II (ANG II) are primary effectors of the sympathetic adrenergic and the renin-angiotensin-aldosterone systems, mediating hypertrophic, apoptotic, and fibrotic events in the myocardium. As NE and ANG II have been shown to affect intracellular calcium in cardiomyocytes, we hypothesized that they activate the calcium-sensitive, prohypertrophic calcineurin-nuclear factor of activated T-cell (NFATc) signaling pathway. More specifically, we have investigated isoform-specific activation of NFAT in NE- and ANG II-stimulated cardiomyocytes, as it is likely that each of the four calcineurin-dependent isoforms, c1-c4, play specific roles. We have stimulated neonatal ventriculocytes from C57/B6 and NFAT-luciferase reporter mice with ANG II or NE and quantified NFAT activity by luciferase activity and phospho-immunoblotting. ANG II and NE increased calcineurin-dependent NFAT activity 2.4- and 1.9-fold, measured as luciferase activity after 24 h of stimulation, and induced protein synthesis, measured by radioactive leucine incorporation after 24 and 72 h. To optimize measurements of NFAT isoforms, we examined the specificity of NFAT antibodies on peptide arrays and by immunoblotting with designed blocking peptides. Western analyses showed that both effectors activate NFATc1 and c4, while NFATc2 activity was regulated by NE only, as measured by phospho-NFAT levels. Neither ANG II nor NE activated NFATc3. As today's main therapies for heart failure aim at antagonizing the adrenergic and renin-angiotensin-aldosterone systems, understanding their intracellular actions is of importance, and our data, through validating a method for measuring myocardial NFATs, indicate that ANG II and NE activate specific NFATc isoforms in cardiomyocytes.

  9. Immune-suppressive activity of punicalagin via inhibition of NFAT activation

    SciTech Connect

    Lee, Sang-Ik; Kim, Byoung-Soo; Kim, Kyoung-Shin; Lee, Samkeun; Shin, Kwang-Soo; Lim, Jong-Soon

    2008-07-11

    Since T cell activation is central to the development of autoimmune diseases, we screened a natural product library comprising 1400 samples of medicinal herbal extracts, to identify compounds that suppress T cell activity. Punicalagin (PCG) isolated from the fruit of Punica granatum was identified as a potent immune suppressant, based on its inhibitory action on the activation of the nuclear factor of activated T cells (NFAT). PCG downregulated the mRNA and soluble protein expression of interleukin-2 from anti-CD3/anti-CD28-stimulated murine splenic CD4+ T cells and suppressed mixed leukocytes reaction (MLR) without exhibiting cytotoxicity to the cells. In vivo, the PCG treatment inhibited phorbol 12-myristate 13-acetate (PMA)-induced chronic ear edema in mice and decreased CD3+ T cell infiltration of the inflamed tissue. These results suggest that PCG could be a potential candidate for the therapeutics of various immune pathologies.

  10. The involvement of NFAT transcriptional activity suppression in SIRT1-mediated inhibition of COX-2 expression induced by PMA/Ionomycin.

    PubMed

    Jia, Yu-Yan; Lu, Jie; Huang, Yue; Liu, Guang; Gao, Peng; Wan, Yan-Zhen; Zhang, Ran; Zhang, Zhu-Qin; Yang, Rui-Feng; Tang, Xiaoqiang; Xu, Jing; Wang, Xu; Chen, Hou-Zao; Liu, De-Pei

    2014-01-01

    SIRT1, a class III histone deacetylase, acts as a negative regulator for many transcription factors, and plays protective roles in inflammation and atherosclerosis. Transcription factor nuclear factor of activated T cells (NFAT) has been previously shown to play pro-inflammatory roles in endothelial cells. Inhibition of NFAT signaling may be an attractive target to regulate inflammation in atherosclerosis. However, whether NFAT transcriptional activity is suppressed by SIRT1 remains unknown. In this study, we found that SIRT1 suppressed NFAT-mediated transcriptional activity. SIRT1 interacted with NFAT, and the NHR and RHR domains of NFAT mediated the interaction with SIRT1. Moreover, we found that SIRT1 primarily deacetylated NFATc3. Adenoviral over-expression of SIRT1 suppressed PMA and calcium ionophore Ionomycin (PMA/Io)-induced COX-2 expression in human umbilical vein endothelial cells (HUVECs), while SIRT1 RNAi reversed the effects in HUVECs. Moreover, inhibition of COX-2 expression by SIRT1 in PMA/Io-treated HUVECs was largely abrogated by inhibiting NFAT activation. Furthermore, SIRT1 inhibited NFAT-induced COX-2 promoter activity, and reduced NFAT binding to the COX-2 promoter in PMA/Io-treated HUVECs. These results suggest that suppression of NFAT transcriptional activity is involved in SIRT1-mediated inhibition of COX-2 expression induced by PMA/Io, and that the negative regulatory mechanisms of NFAT by SIRT1 may contribute to its anti-inflammatory effects in atherosclerosis.

  11. Activation of NFAT signaling in podocytes causes glomerulosclerosis.

    PubMed

    Wang, Yinqiu; Jarad, George; Tripathi, Piyush; Pan, Minggui; Cunningham, Jeanette; Martin, Daniel R; Liapis, Helen; Miner, Jeffrey H; Chen, Feng

    2010-10-01

    Mutant forms of TRPC6 can activate NFAT-dependent transcription in vitro via calcium influx and activation of calcineurin. The same TRPC6 mutants can cause FSGS, but whether this involves an NFAT-dependent mechanism is unknown. Here, we generated mice that allow conditional induction of NFATc1. Mice with NFAT activation in nascent podocytes in utero developed proteinuria and glomerulosclerosis postnatally, resembling FSGS. NFAT activation in adult mice also caused progressive proteinuria and FSGS. Ultrastructural studies revealed podocyte foot process effacement and deposition of extracellular matrix. NFAT activation did not initially affect expression of podocin, synaptopodin, and nephrin but reduced their expression as glomerular injury progressed. In contrast, we observed upregulation of Wnt6 and Fzd9 in the mutant glomeruli before the onset of significant proteinuria, suggesting a potential role for Wnt signaling in the pathogenesis of NFAT-induced podocyte injury and FSGS. These results provide in vivo evidence for the involvement of NFAT signaling in podocytes, proteinuria, and glomerulosclerosis. Furthermore, this study suggests that NFAT activation may be a key intermediate step in the pathogenesis of mutant TRPC6-mediated FSGS and that suppression of NFAT activity may contribute to the antiproteinuric effects of calcineurin inhibitors.

  12. Combination therapy for KIT-mutant mast cells: targeting constitutive NFAT and KIT activity.

    PubMed

    Macleod, Alison C; Klug, Lillian R; Patterson, Janice; Griffith, Diana J; Beadling, Carol; Town, Ajia; Heinrich, Michael C

    2014-12-01

    Resistant KIT mutations have hindered the development of KIT kinase inhibitors for treatment of patients with systemic mastocytosis. The goal of this research was to characterize the synergistic effects of a novel combination therapy involving inhibition of KIT and calcineurin phosphatase, a nuclear factor of activated T cells (NFAT) regulator, using a panel of KIT-mutant mast cell lines. The effects of monotherapy or combination therapy on the cellular viability/survival of KIT-mutant mast cells were evaluated. In addition, NFAT-dependent transcriptional activity was monitored in a representative cell line to evaluate the mechanisms responsible for the efficacy of combination therapy. Finally, shRNA was used to stably knockdown calcineurin expression to confirm the role of calcineurin in the observed synergy. The combination of a KIT inhibitor and a calcineurin phosphatase inhibitor (CNPI) synergized to reduce cell viability and induce apoptosis in six distinct KIT-mutant mast cell lines. Both KIT inhibitors and CNPIs were found to decrease NFAT-dependent transcriptional activity. NFAT-specific inhibitors induced similar synergistic apoptosis induction as CNPIs when combined with a KIT inhibitor. Notably, NFAT was constitutively active in each KIT-mutant cell line tested. Knockdown of calcineurin subunit PPP3R1 sensitized cells to KIT inhibition and increased NFAT phosphorylation and cytoplasmic localization. Constitutive activation of NFAT appears to represent a novel and targetable characteristic of KIT-mutant mast cell disease. Our studies suggest that combining KIT inhibition with NFAT inhibition might represent a new treatment strategy for mast cell disease.

  13. Effect Of Simulated Microgravity On Activated T Cell Gene Transcription

    NASA Technical Reports Server (NTRS)

    Morrow, Maureen A.

    2003-01-01

    Studies of T lymphocytes under the shear stress environment of clinorotation have demonstrated an inhibition of activation in response to TCR mediated signaling. These results mimic those observed during space flight. This work investigates the molecular signaling events of T lymphocyte activation with clinorotation. Purified human T lymphocytes and the T cell clone Jurkat exhibit an uncoupling of signaling as mediated through the TCR. Activation of the transcription factor AP-1 is inhibited while activation of NFAT occurs. NFAT dephosphorylation and activation is dependent on sustained Ca(++) influx. Alternatively, AP-1, which consists of two transcription factors, jun and fos, is activated by PKC and Ras mediated pathways. TCR signaling is known to be dependent on cytoskeletal rearrangements, in particular, raft aggregation is critical. Raft aggregation, as mediated through GM, crosslinking, overcomes the inhibition of T lymphocyte activation with clinorotation, indicating that the block is occurring upstream of raft aggregation. Clinorotation is shown to have an effect similar to a weak TCR signal.

  14. Herpesviral G protein-coupled receptors activate NFAT to induce tumor formation via inhibiting the SERCA calcium ATPase.

    PubMed

    Zhang, Junjie; He, Shanping; Wang, Yi; Brulois, Kevin; Lan, Ke; Jung, Jae U; Feng, Pinghui

    2015-03-01

    G protein-coupled receptors (GPCRs) constitute the largest family of proteins that transmit signal to regulate an array of fundamental biological processes. Viruses deploy diverse tactics to hijack and harness intracellular signaling events induced by GPCR. Herpesviruses encode multiple GPCR homologues that are implicated in viral pathogenesis. Cellular GPCRs are primarily regulated by their cognate ligands, while herpesviral GPCRs constitutively activate downstream signaling cascades, including the nuclear factor of activated T cells (NFAT) pathway. However, the roles of NFAT activation and mechanism thereof in viral GPCR tumorigenesis remain unknown. Here we report that GPCRs of human Kaposi's sarcoma-associated herpesvirus (kGPCR) and cytomegalovirus (US28) shortcut NFAT activation by inhibiting the sarcoplasmic reticulum calcium ATPase (SERCA), which is necessary for viral GPCR tumorigenesis. Biochemical approaches, entailing pharmacological inhibitors and protein purification, demonstrate that viral GPCRs target SERCA2 to increase cytosolic calcium concentration. As such, NFAT activation induced by vGPCRs was exceedingly sensitive to cyclosporine A that targets calcineurin, but resistant to inhibition upstream of ER calcium release. Gene expression profiling identified a signature of NFAT activation in endothelial cells expressing viral GPCRs. The expression of NFAT-dependent genes was up-regulated in tumors derived from tva-kGPCR mouse and human KS. Employing recombinant kGPCR-deficient KSHV, we showed that kGPCR was critical for NFAT-dependent gene expression in KSHV lytic replication. Finally, cyclosporine A treatment diminished NFAT-dependent gene expression and tumor formation induced by viral GPCRs. These findings reveal essential roles of NFAT activation in viral GPCR tumorigenesis and a mechanism of "constitutive" NFAT activation by viral GPCRs.

  15. Activation of the Ca{sup 2+}/calcineurin/NFAT{sub 2} pathway controls smooth muscle cell differentiation

    SciTech Connect

    Larrieu, Daniel; Thiebaud, Pierre; Duplaa, Cecile; Sibon, Igor; Theze, Nadine; Lamaziere, Jean-Marie Daniel . E-mail: jean-marie.d-lamaziere@bordeaux.inserm.fr

    2005-10-15

    Cellular mechanisms controlling smooth muscle cells (SMCs) phenotypic modulation are largely unknown. Intracellular Ca{sup 2+} movements are essential to ensure SMC functions; one of the roles of Ca{sup 2+} is to regulate calcineurin, which in turn induces nuclear localization of the nuclear factor of activated T-cell (NFAT). In order to investigate, during phenotypic differentiation of SMCs, the effect of calcineurin inhibition on NFAT{sub 2} nuclear translocation, we used a culture model of SMC differentiation in serum-free conditions. We show that the treatment of cultured SMC with the calcineurin inhibitor cyclosporine A induced their dedifferentiation while preventing their differentiation. These findings suggest that nuclear translocation of NFAT{sub 2} is dependent of calcineurin activity during the in vitro SMC differentiation kinetic and that the nuclear presence of NFAT{sub 2} is critical in the acquisition and maintenance of SMC differentiation.

  16. Cyclic GMP/PKG-dependent inhibition of TRPC6 channel activity and expression negatively regulates cardiomyocyte NFAT activation Novel mechanism of cardiac stress modulation by PDE5 inhibition.

    PubMed

    Koitabashi, Norimichi; Aiba, Takeshi; Hesketh, Geoffrey G; Rowell, Janelle; Zhang, Manling; Takimoto, Eiki; Tomaselli, Gordon F; Kass, David A

    2010-04-01

    Increased cyclic GMP from enhanced synthesis or suppressed catabolism (e.g. PDE5 inhibition by sildenafil, SIL) activates protein kinase G (PKG) and blunts cardiac pathological hypertrophy. Suppressed calcineurin (Cn)-NFAT (nuclear factor of activated T-cells) signaling appears to be involved, though it remains unclear how this is achieved. One potential mechanism involves activation of Cn/NFAT by calcium entering via transient receptor potential canonical (TRPC) channels (notably TRPC6). Here, we tested the hypothesis that PKG blocks Cn/NFAT activation by modifying and thus inhibiting TRPC6 current to break the positive feedback loop involving NFAT and NFAT-dependent TRPC6 upregulation. TRPC6 expression rose with pressure-overload in vivo, and angiotensin (ATII) or endothelin (ET1) stimulation in neonatal and adult cardiomyocytes in vitro. 8Br-cGMP and SIL reduced ET1-stimulated TRPC6 expression and NFAT dephosphorylation (activity). TRPC6 upregulation was absent if its promoter was mutated with non-functional NFAT binding sites, whereas constitutively active NFAT triggered TRPC6 expression that was not inhibited by SIL. PKG phosphorylated TRPC6, and both T70 and S322 were targeted. Both sites were functionally relevant, as 8Br-cGMP strongly suppressed current in wild-type TRPC6 channels, but not in those with phospho-silencing mutations (T70A, S322A or S322Q). NFAT activation and increased protein synthesis stimulated by ATII or ET1 was blocked by 8Br-cGMP or SIL. However, transfection with T70A or S322Q TRPC6 mutants blocked this inhibitory effect, whereas phospho-mimetic mutants (T70E, S322E, and both combined) suppressed NFAT activation. Thus PDE5-inhibition blocks TRPC6 channel activation and associated Cn/NFAT activation signaling by PKG-dependent channel phosphorylation.

  17. Identification and evolution of an NFAT gene involving Branchiostoma belcheri innate immunity.

    PubMed

    Song, Xiaojun; Hu, Jing; Jin, Ping; Chen, Liming; Ma, Fei

    2013-10-01

    The Nuclear Factor of Activated T cells (NFAT) plays an important role in innate and adaptive immunity, but no NFAT genes have yet been identified in amphioxus species. Here we identified and characterized an NFAT-like gene from Branchiostoma belcheri, and also studied extensively the evolutionary history of NFAT family genes. We found that the amphioxus genome contains an AmphiNFAT gene encoding an NFAT homolog. The AmphiNFAT gene was found to be involved in the innate immune response to LPS stimulation in B. belcheri and was ubiquitously and differentially expressed in all investigated tissues. The NFAT family genes were present in a common ancestor with cnidaria, and NFAT1-4 paralogs were lost early in Branchiostoma and Strongylocentrotus genomes. We discovered that NFAT family genes underwent strong purifying selection. Taken together, our findings provide an insight into the innate immune response of amphioxus and the evolution of the NFAT gene family.

  18. Identification and evolution of an NFAT gene involving Branchiostoma belcheri innate immunity.

    PubMed

    Song, Xiaojun; Hu, Jing; Jin, Ping; Chen, Liming; Ma, Fei

    2013-10-01

    The Nuclear Factor of Activated T cells (NFAT) plays an important role in innate and adaptive immunity, but no NFAT genes have yet been identified in amphioxus species. Here we identified and characterized an NFAT-like gene from Branchiostoma belcheri, and also studied extensively the evolutionary history of NFAT family genes. We found that the amphioxus genome contains an AmphiNFAT gene encoding an NFAT homolog. The AmphiNFAT gene was found to be involved in the innate immune response to LPS stimulation in B. belcheri and was ubiquitously and differentially expressed in all investigated tissues. The NFAT family genes were present in a common ancestor with cnidaria, and NFAT1-4 paralogs were lost early in Branchiostoma and Strongylocentrotus genomes. We discovered that NFAT family genes underwent strong purifying selection. Taken together, our findings provide an insight into the innate immune response of amphioxus and the evolution of the NFAT gene family. PMID:23657135

  19. The calcineurin/NFAT pathway is activated in diagnostic breast cancer cases and is essential to survival and metastasis of mammary cancer cells.

    PubMed

    Quang, C Tran; Leboucher, S; Passaro, D; Fuhrmann, L; Nourieh, M; Vincent-Salomon, A; Ghysdael, J

    2015-02-26

    Nuclear factor of activated T cells 1 (NFAT1) expression has been associated with increased migratory/invasive properties of mammary tumor-derived cell lines in vitro. It is unknown, however, if NFAT activation actually occurs in breast cancer cases and whether the calcineurin/NFAT pathway is important to mammary tumorigenesis. Using a cohort of 321 diagnostic cases of the major subgroup of breast cancer, we found Cn/NFAT pathway activated in ER(-)PR(-)HER2(-) triple-negative breast cancer subtype, whereas its prevalence is less in other subgroups. Using a small hairpin RNA-based gene expression silencing approach in murine mammary tumor cell line (4T1), we show that not only NFAT1 but also NFAT2 and their upstream activator Cn are essential to the migratory and invasive properties of mammary tumor cells. We also demonstrate that Cn, NFAT1 and NFAT2 are essential to the tumorigenic and metastatic properties of these cells in mice, a phenotype which coincides with increased apoptosis in vivo. Finally, global gene expression analyses identified several NFAT-deregulated genes, many of them being previously associated with mammary tumorigenesis. In particular, we identified the gene encoding a disintegrin and metalloproteinase with thrombonspondin motifs 1, as being a potential direct target of NFAT1. Thus, our results show that the Cn/NFAT pathway is activated in diagnostic cases of breast cancers and is essential to the tumorigenic and metastatic potential of mammary tumor cell line. These results suggest that pharmacological inhibition of the Cn/NFAT pathway at different levels could be of therapeutical interest for breast cancer patients.

  20. NFAT2 Regulates Generation of Innate-Like CD8+ T Lymphocytes and CD8+ T Lymphocytes Responses

    PubMed Central

    Pachulec, Emilia; Neitzke-Montinelli, Vanessa; Viola, João P. B.

    2016-01-01

    Nuclear factor of activated T cells (NFAT) 2 null mutant mice die in utero of cardiac failure, precluding analysis of the role of NFAT2 in lymphocyte responses. Only the NFAT2−/−/Rag-1−/− chimeric mice model gave insight into the role of NFAT2 transcription factor in T lymphocyte development, activation, and differentiation. As reports are mainly focused on the role of NFAT2 in CD4+ T lymphocytes activation and differentiation, we decided to investigate NFAT2’s impact on CD8+ T lymphocyte responses. We report that NFAT2 is phosphorylated and inactive in the cytoplasm of naive CD8+ T cells, and upon TCR stimulation, it is dephosphorylated and translocated into the nucleus. To study the role of NFAT2 in CD8+ T responses, we employed NFAT2fl/flCD4-Cre mice with NFAT2 deletion specifically in T cells. Interestingly, the absence of NFAT2 in T cells resulted in increased percentage of non-conventional innate-like CD8+ T cells. These cells were CD122+, rapid producer of interferon gamma (IFN-γ) and had characteristics of conventional memory CD8+ T cells. We also observed an expansion of PLZF+ expressing CD3+ thymocyte population in the absence of NFAT2 and increased IL-4 production. Furthermore, we found that CD8+ T lymphocytes deficient in NFAT2 had reduced activation, proliferation, and IFN-γ and IL-2 production at suboptimal TCR strength. NFAT2 absence did not significantly influence differentiation of CD8+ T cells into cytotoxic effector cells but reduced their IFN-γ production. This work documents NFAT2 as a negative regulator of innate-like CD8+ T cells development. NFAT2 is required for complete CD8+ T cell responses at suboptimal TCR stimulation and regulates IFN-γ production by cytotoxic CD8+ T cells in vitro. PMID:27766099

  1. Na(+)/H(+) exchanger 1 directly binds to calcineurin A and activates downstream NFAT signaling, leading to cardiomyocyte hypertrophy.

    PubMed

    Hisamitsu, Takashi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

    2012-08-01

    The calcineurin A (CaNA) subunit was identified as a novel binding partner of plasma membrane Na(+)/H(+) exchanger 1 (NHE1). CaN is a Ca(2+)-dependent phosphatase involved in many cellular functions, including cardiac hypertrophy. Direct binding of CaN to the (715)PVITID(720) sequence of NHE1, which resembles the consensus CaN-binding motif (PXIXIT), was observed. Overexpression of NHE1 promoted serum-induced CaN/nuclear factor of activated T cells (NFAT) signaling in fibroblasts, as indicated by enhancement of NFAT promoter activity and nuclear translocation, which was attenuated by NHE1 inhibitor. In neonatal rat cardiomyocytes, NHE1 stimulated hypertrophic gene expression and the NFAT pathway, which were inhibited by a CaN inhibitor, FK506. Importantly, CaN activity was strongly enhanced with increasing pH, so NHE1 may promote CaN/NFAT signaling via increased intracellular pH. Indeed, Na(+)/H(+) exchange activity was required for NHE1-dependent NFAT signaling. Moreover, interaction of CaN with NHE1 and clustering of NHE1 to lipid rafts were also required for this response. Based on these results, we propose that NHE1 activity may generate a localized membrane microdomain with higher pH, thereby sensitizing CaN to activation and promoting NFAT signaling. In cardiomyocytes, such signaling can be a pathway of NHE1-dependent hypertrophy.

  2. Calcineurin/NFAT signalling inhibits myeloid haematopoiesis.

    PubMed

    Fric, Jan; Lim, Clarice X F; Koh, Esther G L; Hofmann, Benjamin; Chen, Jinmiao; Tay, Hock Soon; Mohammad Isa, Siti Aminah Bte; Mortellaro, Alessandra; Ruedl, Christiane; Ricciardi-Castagnoli, Paola

    2012-04-01

    Nuclear factor of activated T cells (NFAT) comprises a family of transcription factors that regulate T cell development, activation and differentiation. NFAT signalling can also mediate granulocyte and dendritic cell (DC) activation, but it is unknown whether NFAT influences their development from progenitors. Here, we report a novel role for calcineurin/NFAT signalling as a negative regulator of myeloid haematopoiesis. Reconstituting lethally irradiated mice with haematopoietic stem cells expressing an NFAT-inhibitory peptide resulted in enhanced development of the myeloid compartment. Culturing bone marrow cells in media supplemented with Flt3-L in the presence of the calcineurin/NFAT inhibitor Cyclosporin A increased numbers of differentiated DC. Global gene expression analysis of untreated DC and NFAT-inhibited DC revealed differential expression of transcripts that regulate cell cycle and apoptosis. In conclusion, these results provide evidence that calcineurin/NFAT signalling negatively regulates myeloid lineage development. The finding that inhibition of NFAT enhances myeloid development provides a novel insight into understanding how the treatment with drugs targeting calcineurin/NFAT signalling influence the homeostasis of the innate immune system.

  3. T cell Ig and mucin 1 (TIM-1) is expressed on in vivo-activated T cells and provides a costimulatory signal for T cell activation.

    PubMed

    de Souza, Anjali J; Oriss, Timothy B; O'malley, Katherine J; Ray, Anuradha; Kane, Lawrence P

    2005-11-22

    Polymorphisms in TIM-1, a member of the T cell Ig and mucin (TIM) domain family, are associated with relative susceptibility to the development of T helper 2-dominated immune responses such as in allergic asthma. Recent data have also suggested that ligation of TIM-1 can augment T cell activation. We have found that the TIM-1 protein is expressed on CD4(+) T cells in vivo after intranasal immunization. Ectopic expression of TIM-1 during T cell differentiation results in a significant increase in the number of cells producing IL-4 but not IFN-gamma. Furthermore, TIM-1 expression provides a costimulatory signal that increases transcription from the IL-4 promoter and from isolated nuclear factor of activated T cells/activating protein-1 (NFAT/AP-1) elements. Finally, we provide evidence that TIM-1 can be phosphorylated on tyrosine and that TIM-1 costimulation requires its cytoplasmic tail and the conserved tyrosine within that domain. These results constitute evidence that TIM-1 directly couples to phosphotyrosine-dependent intracellular signaling pathways.

  4. Cutting Edge: Localization of linker for activation of T cells to lipid rafts is not essential in T cell activation and development.

    PubMed

    Zhu, Minghua; Shen, Shudan; Liu, Yan; Granillo, Olivia; Zhang, Weiguo

    2005-01-01

    It has been proposed that upon T cell activation, linker for activation of T cells (LAT), a transmembrane adaptor protein localized to lipid rafts, orchestrates formation of multiprotein complexes and activates signaling cascades in lipid rafts. However, whether lipid rafts really exist or function remains controversial. To address the importance of lipid rafts in LAT function, we generated a fusion protein to target LAT to nonraft fractions using the transmembrane domain from a nonraft protein, linker for activation of X cells (LAX). Surprisingly, this fusion protein functioned well in TCR signaling. It restored MAPK activation, calcium flux, and NFAT activation in LAT-deficient cells. To further study the function of this fusion protein in vivo, we generated transgenic mice that express this protein. Analysis of these mice indicated that it was fully capable of replacing LAT in thymocyte development and T cell function. Our results demonstrate that LAT localization to lipid rafts is not essential during normal T cell activation and development.

  5. Angular-type furocoumarins from the roots of Angelica atropurpurea and their inhibitory activity on the NFAT signal transduction pathway.

    PubMed

    Nagasawa, Azumi; Sakasai, Mitsuyoshi; Sakaguchi, Daishi; Moriwaki, Shigeru; Nishizawa, Yoshinori; Kitahara, Takashi

    2014-12-01

    One new (1) and two known angular-type (2,3) furocoumarins, isoarchangelicin (1), archangelicin (2), and 2'-angeloyl-3'-isovaleryl vaginate (3), were isolated from the roots of Angelica atropurpurea. The structure of the new compound was established on the basis of one- and two-dimensional NMR spectra and other spectroscopic studies. The inhibitory activity of these three compounds and a deacylated form of archangelicin (4) on the nuclear factor of activated T cells (NFAT) signal transduction pathway was tested by NFAT-responsive luciferase reporter gene assay in cultured cells. Although 4 did not exhibit inhibitory activity on NFAT signaling, 1-3 exhibited dose-dependent inhibition with IC50 values of 16.5 (1), 9.0 (2), and 9.2 (3) μM.

  6. NFATc2 recruits cJun homodimers to an NFAT site to synergistically activate interleukin-2 transcription.

    PubMed

    Walters, Ryan D; Drullinger, Linda F; Kugel, Jennifer F; Goodrich, James A

    2013-11-01

    Transcription of interleukin-2 (IL-2), a pivotal cytokine in the mammalian immune response, is induced by NFAT and AP-1 transcriptional activators in stimulated T cells. NFATc2 and cJun drive high levels of synergistic human IL-2 transcription, which requires a unique interaction between the C-terminal activation domain of NFATc2 and cJun homodimers. Here we studied the mechanism by which this interaction contributes to synergistic activation of IL-2 transcription. We found that NFATc2 can recruit cJun homodimers to the -45 NFAT element, which lacks a neighboring AP-1 site. The bZip domain of cJun is sufficient to interact with the C-terminal activation domain of NFATc2 in the absence of DNA and this interaction is inhibited by AP-1 DNA. When the -45 NFAT site was replaced by either an NFAT/AP-1 composite site or a single AP-1 site the specificity for cJun homodimers in synergistically activating IL-2 transcription was lost, and cJun/cFos heterodimers strongly activated transcription. These studies support a model in which IL-2 transcriptional synergy is mediated by the unique recruitment of a cJun homodimer to the -45 NFAT site by NFATc2, where it acts as a co-activator for IL-2 transcription.

  7. Tracking and treating activated T cells

    PubMed Central

    Kim, N.H.; Nadithe, V.; Elsayed, M.; Merkel, O.M.

    2014-01-01

    Upon activation, T cells of various subsets are the most important mediators in cell-mediated immune responses. Activated T cells play an important role in immune system related diseases such as chronic inflammatory diseases, viral infections, autoimmune disease, transplant rejection, Crohn disease, diabetes, and many more. Therefore, efforts have been made to both visualize and treat activated T cells specifically. This review summarizes imaging approaches and selective therapeutics for activated T cells and gives an outlook on how tracking and treating can be combined into theragnositc agents for activated T cells. PMID:24660025

  8. Activation of NFAT signaling establishes a tumorigenic microenvironment through cell autonomous and non-cell autonomous mechanisms.

    PubMed

    Tripathi, P; Wang, Y; Coussens, M; Manda, K R; Casey, A M; Lin, C; Poyo, E; Pfeifer, J D; Basappa, N; Bates, C M; Ma, L; Zhang, H; Pan, M; Ding, L; Chen, F

    2014-04-01

    NFAT (the nuclear factor of activated T cells) upregulation has been linked to cellular transformation intrinsically, but it is unclear whether and how tissue cells with NFAT activation change the local environment for tumor initiation and progression. Direct evidence showing NFAT activation initiates primary tumor formation in vivo is also lacking. Using inducible transgenic mouse systems, we show that tumors form in a subset of, but not all, tissues with NFATc1 activation, indicating that NFAT oncogenic effects depend on cell types and tissue contexts. In NFATc1-induced skin and ovarian tumors, both cells with NFATc1 activation and neighboring cells without NFATc1 activation have significant upregulation of c-Myc and activation of Stat3. Besides known and suspected NFATc1 targets, such as Spp1 and Osm, we have revealed the early upregulation of a number of cytokines and cytokine receptors, as key molecular components of an inflammatory microenvironment that promotes both NFATc1(+) and NFATc1(-) cells to participate in tumor formation. Cultured cells derived from NFATc1-induced tumors were able to establish a tumorigenic microenvironment, similar to that of the primary tumors, in an NFATc1-dependent manner in nude mice with T-cell deficiency, revealing an addiction of these tumors to NFATc1 activation and downplaying a role for T cells in the NFATc1-induced tumorigenic microenvironment. These findings collectively suggest that beyond the cell autonomous effects on the upregulation of oncogenic proteins, NFATc1 activation has non-cell autonomous effects through the establishment of a promitogenic microenvironment for tumor growth. This study provides direct evidence for the ability of NFATc1 in inducing primary tumor formation in vivo and supports targeting NFAT signaling in anti-tumor therapy.

  9. NFAT5 Is Activated by Hypoxia: Role in Ischemia and Reperfusion in the Rat Kidney

    PubMed Central

    Villanueva, Sandra; Suazo, Cristian; Santapau, Daniela; Pérez, Francisco; Quiroz, Mariana; Carreño, Juan E.; Illanes, Sebastián; Lavandero, Sergio; Michea, Luis; Irarrazabal, Carlos E.

    2012-01-01

    The current hypothesis postulates that NFAT5 activation in the kidney's inner medulla is due to hypertonicity, resulting in cell protection. Additionally, the renal medulla is hypoxic (10–18 mmHg); however there is no information about the effect of hypoxia on NFAT5. Using in vivo and in vitro models, we evaluated the effect of reducing the partial pressure of oxygen (PO2) on NFAT5 activity. We found that 1) Anoxia increased NFAT5 expression and nuclear translocation in primary cultures of IMCD cells from rat kidney. 2) Anoxia increased transcriptional activity and nuclear translocation of NFAT5 in HEK293 cells. 3) The dose-response curve demonstrated that HIF-1α peaked at 2.5% and NFAT5 at 1% of O2. 4) At 2.5% of O2, the time-course curve of hypoxia demonstrated earlier induction of HIF-1α gene expression than NFAT5. 5) siRNA knockdown of NFAT5 increased the hypoxia-induced cell death. 6) siRNA knockdown of HIF-1α did not affect the NFAT5 induction by hypoxia. Additionally, HIF-1α was still induced by hypoxia even when NFAT5 was knocked down. 7) NFAT5 and HIF-1α expression were increased in kidney (cortex and medulla) from rats subjected to an experimental model of ischemia and reperfusion (I/R). 7) Experimental I/R increased the NFAT5-target gene aldose reductase (AR). 8) NFAT5 activators (ATM and PI3K) were induced in vitro (HEK293 cells) and in vivo (I/R kidneys) with the same timing of NFAT5. 8) Wortmannin, which inhibits ATM and PI3K, reduces hypoxia-induced NFAT5 transcriptional activation in HEK293 cells. These results demonstrate for the first time that NFAT5 is induced by hypoxia and could be a protective factor against ischemic damage. PMID:22768306

  10. Nuclear factor of activated T-cells 5 increases intestinal goblet cell differentiation through an mTOR/Notch signaling pathway

    PubMed Central

    Zhou, Yuning; Wang, Qingding; Weiss, Heidi L.; Evers, B. Mark

    2014-01-01

    The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis that is regulated by multiple signaling pathways. Previously, we have shown that the nuclear factor of activated T-cells 5 (NFAT5) is involved in the regulation of intestinal enterocyte differentiation. Here we show that treatment with sodium chloride (NaCl), which activates NFAT5 signaling, increased mTORC1 repressor regulated in development and DNA damage response 1 (REDD1) protein expression and inhibited mTOR signaling; these alterations were attenuated by knockdown of NFAT5. Knockdown of NFAT5 activated mammalian target of rapamycin (mTOR) signaling and significantly inhibited REDD1 mRNA expression and protein expression. Consistently, overexpression of NFAT5 increased REDD1 expression. In addition, knockdown of REDD1 activated mTOR and Notch signaling, whereas treatment with mTOR inhibitor rapamycin repressed Notch signaling and increased the expression of the goblet cell differentiation marker mucin 2 (MUC2). Moreover, knockdown of NFAT5 activated Notch signaling and decreased MUC2 expression, while overexpression of NFAT5 inhibited Notch signaling and increased MUC2 expression. Our results demonstrate a role for NFAT5 in the regulation of mTOR signaling in intestinal cells. Importantly, these data suggest that NFAT5 participates in the regulation of intestinal homeostasis via the suppression of mTORC1/Notch signaling pathway. PMID:25057011

  11. Transcriptional regulation of the c-Myc promoter by NFAT1 involves negative and positive NFAT-responsive elements.

    PubMed

    Mognol, Giuliana P; de Araujo-Souza, Patricia S; Robbs, Bruno K; Teixeira, Leonardo K; Viola, Joao P B

    2012-03-01

    A number of physiological processes in both normal and cancer cells are regulated by the proto-oncogene c-Myc. Among them, processes such as cell cycle regulation, apoptosis, angiogenesis and metastasis are also controlled by the nuclear factor of activated T cells (NFAT) family of transcription factors. It is already known that NFAT upregulates c-Myc expression by binding to an element located in the minimal c-Myc promoter. However, the importance of other NFAT sites in the context of the full promoter has not been evaluated. In this work, we demonstrate that the regulation of c-Myc by NFAT1 is more complex than previously conceived. In addition to the proximal site, NFAT1 directly binds to distal sites in the c-Myc promoter with different affinities. Promoter deletions and site-directed mutagenesis of NFAT binding sites in HEK293T cells suggest that in NFAT1-mediated transactivation, some NFAT elements are negative and dominant and others are positive and recessive. Furthermore, we demonstrate that cooperation with partner proteins, such as p300, enhances NFAT1-mediated transactivation of the c-Myc promoter. At last, the newly identified sites are also responsive to NFAT2 in HEK293T cells. However, in NIH3T3 cells, the regulation mediated by NFAT proteins is not dependent on the known NFAT sites, including the site previously described. Thus, our data suggest that the contribution of NFAT to the regulation of c-Myc expression may depend on a balance between the binding to positive and negative NFAT-responsive elements and cooperation with transcriptional cofactors, which may differ according to the context and/or cell type.

  12. Kinetic discrimination in T-cell activation.

    PubMed Central

    Rabinowitz, J D; Beeson, C; Lyons, D S; Davis, M M; McConnell, H M

    1996-01-01

    We propose a quantitative model for T-cell activation in which the rate of dissociation of ligand from T-cell receptors determines the agonist and antagonist properties of the ligand. The ligands are molecular complexes between antigenic peptides and proteins of the major histocompatibility complex on the surfaces of antigen-presenting cells. Binding of ligand to receptor triggers a series of biochemical reactions in the T cell. If the ligand dissociates after these reactions are complete, the T cell receives a positive activation signal. However, dissociation of ligand after completion of the first reaction but prior to generation of the final products results in partial T-cell activation, which acts to suppress a positive response. Such a negative signal is brought about by T-cell ligands containing the variants of antigenic peptides referred to as T-cell receptor antagonists. Results of recent experiments with altered peptide ligands compare favorably with T-cell responses predicted by this model. PMID:8643643

  13. Kinetic Discrimination in T-Cell Activation

    NASA Astrophysics Data System (ADS)

    Rabinowitz, Joshua D.; Beeson, Craig; Lyons, Daniel S.; Davis, Mark M.; McConnell, Harden M.

    1996-02-01

    We propose a quantitative model for T-cell activation in which the rate of dissociation of ligand from T-cell receptors determines the agonist and antagonist properties of the ligand. The ligands are molecular complexes between antigenic peptides and proteins of the major histocompatibility complex on the surfaces of antigen-presenting cells. Binding of ligand to receptor triggers a series of biochemical reactions in the T cell. If the ligand dissociates after these reactions are complete, the T cell receives a positive activation signal. However, dissociation of ligand after completion of the first reaction but prior to generation of the final products results in partial T-cell activation, which acts to suppress a positive response. Such a negative signal is brought about by T-cell ligands containing the variants of antigenic peptides referred to as T-cell receptor antagonists. Results of recent experiments with altered peptide ligands compare favorably with T-cell responses predicted by this model.

  14. SHP2-interacting Transmembrane Adaptor Protein (SIT), A Novel Disulfide-linked Dimer Regulating Human T Cell Activation

    PubMed Central

    Marie-Cardine, Anne; Kirchgessner, Henning; Bruyns, Eddy; Shevchenko, Andrej; Mann, Matthias; Autschbach, Frank; Ratnofsky, Sheldon; Meuer, Stefan; Schraven, Burkhart

    1999-01-01

    T lymphocytes express several low molecular weight transmembrane adaptor proteins that recruit src homology (SH)2 domain–containing intracellular molecules to the cell membrane via tyrosine-based signaling motifs. We describe here a novel molecule of this group termed SIT (SHP2 interacting transmembrane adaptor protein). SIT is a disulfide-linked homodimeric glycoprotein that is expressed in lymphocytes. After tyrosine phosphorylation by src and possibly syk protein tyrosine kinases SIT recruits the SH2 domain–containing tyrosine phosphatase SHP2 via an immunoreceptor tyrosine-based inhibition motif. Overexpression of SIT in Jurkat cells downmodulates T cell receptor– and phytohemagglutinin-mediated activation of the nuclear factor of activated T cells (NF-AT) by interfering with signaling processes that are probably located upstream of activation of phospholipase C. However, binding of SHP2 to SIT is not required for inhibition of NF-AT induction, suggesting that SIT not only regulates NF-AT activity but also controls NF-AT unrelated pathways of T cell activation involving SHP2. PMID:10209036

  15. NFAT-133 increases glucose uptake in L6 myotubes by activating AMPK pathway.

    PubMed

    Thakkar, Chandni S; Kate, Abhijeet S; Desai, Dattatraya C; Ghosh, Asit Ranjan; Kulkarni-Almeida, Asha A

    2015-12-15

    NFAT-133 is an aromatic compound with cinammyl alcohol moiety, isolated from streptomycetes strain PM0324667. We have earlier reported that NFAT-133 increases insulin stimulated glucose uptake in L6 myotubes using a PPARγ independent mechanism and reduces plasma or blood glucose levels in diabetic mice. Here we investigated the effects of NFAT-133 on cellular signaling pathways leading to glucose uptake in L6 myotubes. Our studies demonstrate that NFAT-133 increases glucose uptake in a dose- and time-dependent manner independent of the effects of insulin. Treatment with Akti-1/2, wortmannin and increasing concentrations of insulin had no effect on NFAT-133 mediated glucose uptake. NFAT-133 induced glucose uptake is completely mitigated by Compound C, an AMPK inhibitor. Further, the kinases upstream of AMPK activation namely; LKB-1 and CAMKKβ are not involved in NFAT-133 mediated AMPK activation nor does the compound NFAT-133 have any effect on AMPK enzyme activity. Further analysis confirmed that NFAT-133 indirectly activates AMPK by reducing the mitochondrial membrane potential and increasing the ratio of AMP:ATP.

  16. NFAT-133 increases glucose uptake in L6 myotubes by activating AMPK pathway.

    PubMed

    Thakkar, Chandni S; Kate, Abhijeet S; Desai, Dattatraya C; Ghosh, Asit Ranjan; Kulkarni-Almeida, Asha A

    2015-12-15

    NFAT-133 is an aromatic compound with cinammyl alcohol moiety, isolated from streptomycetes strain PM0324667. We have earlier reported that NFAT-133 increases insulin stimulated glucose uptake in L6 myotubes using a PPARγ independent mechanism and reduces plasma or blood glucose levels in diabetic mice. Here we investigated the effects of NFAT-133 on cellular signaling pathways leading to glucose uptake in L6 myotubes. Our studies demonstrate that NFAT-133 increases glucose uptake in a dose- and time-dependent manner independent of the effects of insulin. Treatment with Akti-1/2, wortmannin and increasing concentrations of insulin had no effect on NFAT-133 mediated glucose uptake. NFAT-133 induced glucose uptake is completely mitigated by Compound C, an AMPK inhibitor. Further, the kinases upstream of AMPK activation namely; LKB-1 and CAMKKβ are not involved in NFAT-133 mediated AMPK activation nor does the compound NFAT-133 have any effect on AMPK enzyme activity. Further analysis confirmed that NFAT-133 indirectly activates AMPK by reducing the mitochondrial membrane potential and increasing the ratio of AMP:ATP. PMID:26546724

  17. Recruitment of Slp-76 to the Membrane and Glycolipid-Enriched Membrane Microdomains Replaces the Requirement for Linker for Activation of T Cells in T Cell Receptor Signaling

    PubMed Central

    Boerth, Nancy J.; Sadler, Jeffrey J.; Bauer, Daniel E.; Clements, James L.; Gheith, Shereen M.; Koretzky, Gary A.

    2000-01-01

    Two hematopoietic-specific adapters, src homology 2 domain–containing leukocyte phosphoprotein of 76 kD (SLP-76) and linker for activation of T cells (LAT), are critical for T cell development and T cell receptor (TCR) signaling. Several studies have suggested that SLP-76 and LAT function coordinately to promote downstream signaling. In support of this hypothesis, we find that a fraction of SLP-76 localizes to glycolipid-enriched membrane microdomains (GEMs) after TCR stimulation. This recruitment of SLP-76 requires amino acids 224–244. The functional consequences of targeting SLP-76 to GEMs for TCR signaling are demonstrated using a LAT/SLP-76 chimeric protein. Expression of this construct reconstitutes TCR-inducted phospholipase Cγ1 phosphorylation, extracellular signal–regulated kinase activation, and nuclear factor of activated T cells (NFAT) promoter activity in LAT-deficient Jurkat T cells (J.CaM2). Mutation of the chimeric construct precluding its recruitment to GEMs diminishes but does not eliminate its ability to support TCR signaling. Expression of a chimera that lacks SLP-76 amino acids 224–244 restores NFAT promoter activity, suggesting that if localized, SLP-76 does not require an association with Gads to promote T cell activation. In contrast, mutation of the protein tyrosine kinase phosphorylation sites of SLP-76 in the context of the LAT/SLP-76 chimera abolishes reconstitution of TCR function. Collectively, these experiments show that optimal TCR signaling relies on the compartmentalization of SLP-76 and that one critical function of LAT is to bring SLP-76 and its associated proteins to the membrane. PMID:11015445

  18. Phenotypic models of T cell activation.

    PubMed

    Lever, Melissa; Maini, Philip K; van der Merwe, P Anton; Dushek, Omer

    2014-09-01

    T cell activation is a crucial checkpoint in adaptive immunity, and this activation depends on the binding parameters that govern the interactions between T cell receptors (TCRs) and peptide-MHC complexes (pMHC complexes). Despite extensive experimental studies, the relationship between the TCR-pMHC binding parameters and T cell activation remains controversial. To make sense of conflicting experimental data, a variety of verbal and mathematical models have been proposed. However, it is currently unclear which model or models are consistent or inconsistent with experimental data. A key problem is that a direct comparison between the models has not been carried out, in part because they have been formulated in different frameworks. For this Analysis article, we reformulated published models of T cell activation into phenotypic models, which allowed us to directly compare them. We find that a kinetic proofreading model that is modified to include limited signalling is consistent with the majority of published data. This model makes the intriguing prediction that the stimulation hierarchy of two different pMHC complexes (or two different TCRs that are specific for the same pMHC complex) may reverse at different pMHC concentrations.

  19. Band-pass processing in a GPCR signaling pathway selects for NFAT transcription factor activation.

    PubMed

    Sumit, M; Neubig, R R; Takayama, S; Linderman, J J

    2015-11-01

    Many biological processes are rhythmic and proper timing is increasingly appreciated as being critical for development and maintenance of physiological functions. To understand how temporal modulation of an input signal influences downstream responses, we employ microfluidic pulsatile stimulation of a G-protein coupled receptor, the muscarinic M3 receptor, in single cells with simultaneous real-time imaging of both intracellular calcium and NFAT nuclear localization. Interestingly, we find that reduced stimulation with pulses of ligand can give more efficient transcription factor activation, if stimuli are timed appropriately. Our experiments and computational analyses show that M3 receptor-induced calcium oscillations form a low pass filter while calcium-induced NFAT translocation forms a high pass filter. The combination acts as a band-pass filter optimized for intermediate frequencies of stimulation. We demonstrate that receptor desensitization and NFAT translocation rates determine critical features of the band-pass filter and that the band-pass may be shifted for different receptors or NFAT dynamics. As an example, we show that the two NFAT isoforms (NFAT4 and NFAT1) have shifted band-pass windows for the same receptor. While we focus specifically on the M3 muscarinic receptor and NFAT translocation, band-pass processing is expected to be a general theme that applies to multiple signaling pathways.

  20. The Helicobacter pylori Vacuolating Toxin Inhibits T Cell Activation by Two Independent Mechanisms

    PubMed Central

    Boncristiano, Marianna; Paccani, Silvia Rossi; Barone, Silvia; Ulivieri, Cristina; Patrussi, Laura; Ilver, Dag; Amedei, Amedeo; D'Elios, Mario Milco; Telford, John L.; Baldari, Cosima T.

    2003-01-01

    Helicobacter pylori toxin, VacA, damages the gastric epithelium by erosion and loosening of tight junctions. Here we report that VacA also interferes with T cell activation by two different mechanisms. Formation of anion-specific channels by VacA prevents calcium influx from the extracellular milieu. The transcription factor NF-AT thus fails to translocate to the nucleus and activate key cytokine genes. A second, channel-independent mechanism involves activation of intracellular signaling through the mitogen-activated protein kinases MKK3/6 and p38 and the Rac-specific nucleotide exchange factor, Vav. As a consequence of aberrant Rac activation, disordered actin polymerization is stimulated. The resulting defects in T cell activation may help H. pylori to prevent an effective immune response leading to chronic colonization of its gastric niche. PMID:14676300

  1. TIM-3 Suppresses Anti-CD3/CD28-Induced TCR Activation and IL-2 Expression through the NFAT Signaling Pathway.

    PubMed

    Tomkowicz, Brian; Walsh, Eileen; Cotty, Adam; Verona, Raluca; Sabins, Nina; Kaplan, Fred; Santulli-Marotto, Sandy; Chin, Chen-Ni; Mooney, Jill; Lingham, Russell B; Naso, Michael; McCabe, Timothy

    2015-01-01

    TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3) is a member of the TIM family of proteins that is preferentially expressed on Th1 polarized CD4+ and CD8+ T cells. Recent studies indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion). Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK), the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR)-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i) NF-kB/NFAT activation, ii) CD69 expression, and iii) suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8+ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation.

  2. TIM-3 Suppresses Anti-CD3/CD28-Induced TCR Activation and IL-2 Expression through the NFAT Signaling Pathway

    PubMed Central

    Tomkowicz, Brian; Walsh, Eileen; Cotty, Adam; Verona, Raluca; Sabins, Nina; Kaplan, Fred; Santulli-Marotto, Sandy; Chin, Chen-Ni; Mooney, Jill; Lingham, Russell B.; Naso, Michael; McCabe, Timothy

    2015-01-01

    TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3) is a member of the TIM family of proteins that is preferentially expressed on Th1 polarized CD4+ and CD8+ T cells. Recent studies indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion). Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK), the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR)-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i) NF-kB/NFAT activation, ii) CD69 expression, and iii) suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8+ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation. PMID:26492563

  3. Dynamic assembly of a membrane signaling complex enables selective activation of NFAT by Orai1.

    PubMed

    Kar, Pulak; Samanta, Krishna; Kramer, Holger; Morris, Otto; Bakowski, Daniel; Parekh, Anant B

    2014-06-16

    NFAT-dependent gene expression is essential for the development and function of the nervous, immune, and cardiovascular systems and kidney, bone, and skeletal muscle. Most NFAT protein resides in the cytoplasm because of extensive phosphorylation, which masks a nuclear localization sequence. Dephosphorylation by the Ca(2+)-calmodulin-activated protein phosphatase calcineurin triggers NFAT migration into the nucleus. In some cell types, NFAT can be activated by Ca(2+) nanodomains near open store-operated Orai1 and voltage-gated Ca(2+) channels in the plasma membrane. How local Ca(2+) near Orai1 is detected and whether other Orai channels utilize a similar mechanism remain unclear. Here, we report that the paralog Orai3 fails to activate NFAT. Orai1 is effective in activating gene expression via Ca(2+) nanodomains because it participates in a membrane-delimited signaling complex that forms after store depletion and brings calcineurin, via the scaffolding protein AKAP79, to calmodulin tethered to Orai1. By contrast, Orai3 interacts less well with AKAP79 after store depletion, rendering it ineffective in activating NFAT. A channel chimera of Orai3 with the N terminus of Orai1 was able to couple local Ca(2+) entry to NFAT activation, identifying the N-terminal domain of Orai1 as central to Ca(2+) nanodomain-transcription coupling. The formation of a store-dependent signaling complex at the plasma membrane provides for selective activation of a fundamental downstream response by Orai1.

  4. SH3BP2 is an activator of NFAT activity and osteoclastogenesis

    SciTech Connect

    Lietman, Steven A. Yin Lihong; Levine, Michael A.

    2008-07-11

    Heterozygous activating mutations in exon 9 of SH3BP2 have been found in most patients with cherubism, an unusual genetic syndrome characterized by excessive remodeling of the mandible and maxilla due to spontaneous and excessive osteoclastic bone resorption. Osteoclasts differentiate after binding of sRANKL to RANK induces a number of downstream signaling effects, including activation of the calcineurin/NFAT (nuclear factor of activated T cells) pathway. Here, we have investigated the functional significance of SH3BP2 protein on osteoclastogenesis in the presence of sRANKL. Our results indicate that SH3BP2 both increases nuclear NFATc1 in sRANKL treated RAW 264.7 preosteoclast cells and enhances expression of tartrate resistant acid phosphatase (TRAP), a specific marker of osteoclast differentiation. Moreover, overexpression of SH3BP2 in RAW 264.7 cells potentiates sRANKL-stimulated phosphorylation of PLC{gamma}1 and 2, thus providing a mechanistic pathway for the rapid translocation of NFATc1 into the nucleus and increased osteoclastogenesis in cherubism.

  5. γδ T Cells Support Pancreatic Oncogenesis by Restraining αβ T Cell Activation.

    PubMed

    Daley, Donnele; Zambirinis, Constantinos Pantelis; Seifert, Lena; Akkad, Neha; Mohan, Navyatha; Werba, Gregor; Barilla, Rocky; Torres-Hernandez, Alejandro; Hundeyin, Mautin; Mani, Vishnu Raj Kumar; Avanzi, Antonina; Tippens, Daniel; Narayanan, Rajkishen; Jang, Jung-Eun; Newman, Elliot; Pillarisetty, Venu Gopal; Dustin, Michael Loran; Bar-Sagi, Dafna; Hajdu, Cristina; Miller, George

    2016-09-01

    Inflammation is paramount in pancreatic oncogenesis. We identified a uniquely activated γδT cell population, which constituted ∼40% of tumor-infiltrating T cells in human pancreatic ductal adenocarcinoma (PDA). Recruitment and activation of γδT cells was contingent on diverse chemokine signals. Deletion, depletion, or blockade of γδT cell recruitment was protective against PDA and resulted in increased infiltration, activation, and Th1 polarization of αβT cells. Although αβT cells were dispensable to outcome in PDA, they became indispensable mediators of tumor protection upon γδT cell ablation. PDA-infiltrating γδT cells expressed high levels of exhaustion ligands and thereby negated adaptive anti-tumor immunity. Blockade of PD-L1 in γδT cells enhanced CD4(+) and CD8(+) T cell infiltration and immunogenicity and induced tumor protection suggesting that γδT cells are critical sources of immune-suppressive checkpoint ligands in PDA. We describe γδT cells as central regulators of effector T cell activation in cancer via novel cross-talk.

  6. γδ T Cells Support Pancreatic Oncogenesis by Restraining αβ T Cell Activation.

    PubMed

    Daley, Donnele; Zambirinis, Constantinos Pantelis; Seifert, Lena; Akkad, Neha; Mohan, Navyatha; Werba, Gregor; Barilla, Rocky; Torres-Hernandez, Alejandro; Hundeyin, Mautin; Mani, Vishnu Raj Kumar; Avanzi, Antonina; Tippens, Daniel; Narayanan, Rajkishen; Jang, Jung-Eun; Newman, Elliot; Pillarisetty, Venu Gopal; Dustin, Michael Loran; Bar-Sagi, Dafna; Hajdu, Cristina; Miller, George

    2016-09-01

    Inflammation is paramount in pancreatic oncogenesis. We identified a uniquely activated γδT cell population, which constituted ∼40% of tumor-infiltrating T cells in human pancreatic ductal adenocarcinoma (PDA). Recruitment and activation of γδT cells was contingent on diverse chemokine signals. Deletion, depletion, or blockade of γδT cell recruitment was protective against PDA and resulted in increased infiltration, activation, and Th1 polarization of αβT cells. Although αβT cells were dispensable to outcome in PDA, they became indispensable mediators of tumor protection upon γδT cell ablation. PDA-infiltrating γδT cells expressed high levels of exhaustion ligands and thereby negated adaptive anti-tumor immunity. Blockade of PD-L1 in γδT cells enhanced CD4(+) and CD8(+) T cell infiltration and immunogenicity and induced tumor protection suggesting that γδT cells are critical sources of immune-suppressive checkpoint ligands in PDA. We describe γδT cells as central regulators of effector T cell activation in cancer via novel cross-talk. PMID:27569912

  7. NFAT signaling in osteoblasts regulates the hematopoietic niche in the bone microenvironment.

    PubMed

    Sesler, Cheryl L; Zayzafoon, Majd

    2013-01-01

    Osteoblasts support hematopoietic cell development, including B lymphopoiesis. We have previously shown that the nuclear factor of activated T cells (NFAT) negatively regulates osteoblast differentiation and bone formation. Interestingly, in smooth muscle, NFAT has been shown to regulate the expression of vascular cellular adhesion molecule-1 (VCAM-1), a mediator of cell adhesion and signaling during leukocyte development. To examine whether NFAT signaling in osteoblasts regulates hematopoietic development in vivo, we generated a mouse model expressing dominant-negative NFAT driven by the 2.3 kb fragment of the collagen-αI promoter to disrupt NFAT activity in osteoblasts (dnNFAT(OB)). Bone histomorphometry showed that dnNFAT(OB) mice have significant increases in bone volume (44%) and mineral apposition rate (131%) and decreased trabecular thickness (18%). In the bone microenvironment, dnNFAT(OB) mice displayed a significant increase (87%) in Lineage(-)cKit(+)Sca-1(+) (LSK) cells and significant decreases in B220(+)CD19(-)IgM(-) pre-pro-B cells (41%) and B220(+)CD19(+)IgM(+) immature B cells (40%). Concurrent with these findings, LSK cell differentiation into B220(+) cells was inhibited when cocultured on differentiated primary osteoblasts harvested from dnNFAT(OB) mice. Gene expression and protein levels of VCAM-1 in osteoblasts decreased in dnNFAT(OB) mice compared to controls. These data suggest that osteoblast-specific NFAT activity mediates early B lymphopoiesis, possibly by regulating VCAM-1 expression on osteoblasts.

  8. Measurement of Ligand-Induced Activation in Single Viable T Cells Using the lacZ Reporter Gene

    NASA Astrophysics Data System (ADS)

    Karttunen, Jaana; Shastri, Nilabh

    1991-05-01

    We have used the bacterial β-galactosidase gene (lacZ) as a reporter gene for the rapid measurement of T-cell antigen receptor (TCR)-mediated activation of individual T cells. The reporter construct contained the lacZ gene under the control of the nuclear factor of activated T cells (NF-AT) element of the human interleukin 2 enhancer [Fiering, S., Northrop, J. P., Nolan, G. P., Matilla, P., Crabtree, G. R. & Herzenberg, L. A. (1990) Genes Dev. 4, 1823-1834]. The activity of the intracellular lacZ enzyme was analyzed by flow cytometric measurement of fluorescein accumulation in cells loaded with the fluorogenic β-galactosidase substrate fluorescein di-β-D-galactopyranoside. As a model system, the T-cell hybridoma BO4H9.1, which is specific for the lysozyme peptide (amino acids 74-88)/A^b complex, was transfected with the NF-AT-lacZ construct. lacZ activity was induced in 50-100% of the transfectant cells following exposure to pharmacological agents, to the physiological peptide/major histocompatibility complex ligand, or to other TCR-specific stimuli. Interestingly, increasing concentrations of the stimulus increased the fraction of lacZ^+ cells, but not the level of lacZ activity per cell. Even under widely varying levels of stimulus, the level of lacZ activity in individual lacZ^+ cells remained within a remarkably narrow range. These results demonstrate that TCR-mediated activation can be readily measured in single T cells and strongly suggest that, once committed to activation, the level of NF-AT transcriptional activity in individual T cells is independent of the form or concentration of stimulus. This assay is likely to prove useful for the study of early activation events in individual T cells and of TCR ligands.

  9. Sumoylation-independent activation of Calcineurin-NFAT-signaling via SUMO2 mediates cardiomyocyte hypertrophy

    PubMed Central

    Bernt, Alexander; Rangrez, Ashraf Y.; Eden, Matthias; Jungmann, Andreas; Katz, Sylvia; Rohr, Claudia; Müller, Oliver J.; Katus, Hugo A.; Sossalla, Samuel T.; Williams, Tatjana; Ritter, Oliver; Frank, Derk; Frey, Norbert

    2016-01-01

    The objective of this study was to identify unknown modulators of Calcineurin (Cn)-NFAT signaling. Measurement of NFAT reporter driven luciferase activity was therefore utilized to screen a human cardiac cDNA-library (~107 primary clones) in C2C12 cells through serial dilutions until single clones could be identified. This extensive screening strategy culminated in the identification of SUMO2 as a most efficient Cn-NFAT activator. SUMO2-mediated activation of Cn-NFAT signaling in cardiomyocytes translated into a hypertrophic phenotype. Prohypertrophic effects were also observed in mice expressing SUMO2 in the heart using AAV9 (Adeno-associated virus), complementing the in vitro findings. In addition, increased SUMO2-mediated sumoylation in human cardiomyopathy patients and in mouse models of cardiomyopathy were observed. To decipher the underlying mechanism, we generated a sumoylation-deficient SUMO2 mutant (ΔGG). Surprisingly, ΔGG replicated Cn-NFAT-activation and the prohypertrophic effects of native SUMO2, both in vitro and in vivo, suggesting a sumoylation-independent mechanism. Finally, we discerned a direct interaction between SUMO2 and CnA, which promotes CnA nuclear localization. In conclusion, we identified SUMO2 as a novel activator of Cn-NFAT signaling in cardiomyocytes. In broader terms, these findings reveal an unexpected role for SUMO2 in cardiac hypertrophy and cardiomyopathy, which may open the possibility for therapeutic manipulation of this pathway. PMID:27767176

  10. Syndecan-4 is essential for development of concentric myocardial hypertrophy via stretch-induced activation of the calcineurin-NFAT pathway.

    PubMed

    Finsen, Alexandra V; Lunde, Ida G; Sjaastad, Ivar; Østli, Even K; Lyngra, Marianne; Jarstadmarken, Hilde O; Hasic, Almira; Nygård, Ståle; Wilcox-Adelman, Sarah A; Goetinck, Paul F; Lyberg, Torstein; Skrbic, Biljana; Florholmen, Geir; Tønnessen, Theis; Louch, William E; Djurovic, Srdjan; Carlson, Cathrine R; Christensen, Geir

    2011-01-01

    Sustained pressure overload leads to compensatory myocardial hypertrophy and subsequent heart failure, a leading cause of morbidity and mortality. Further unraveling of the cellular processes involved is essential for development of new treatment strategies. We have investigated the hypothesis that the transmembrane Z-disc proteoglycan syndecan-4, a co-receptor for integrins, connecting extracellular matrix proteins to the cytoskeleton, is an important signal transducer in cardiomyocytes during development of concentric myocardial hypertrophy following pressure overload. Echocardiographic, histochemical and cardiomyocyte size measurements showed that syndecan-4(-/-) mice did not develop concentric myocardial hypertrophy as found in wild-type mice, but rather left ventricular dilatation and dysfunction following pressure overload. Protein and gene expression analyses revealed diminished activation of the central, pro-hypertrophic calcineurin-nuclear factor of activated T-cell (NFAT) signaling pathway. Cardiomyocytes from syndecan-4(-/-)-NFAT-luciferase reporter mice subjected to cyclic mechanical stretch, a hypertrophic stimulus, showed minimal activation of NFAT (1.6-fold) compared to 5.8-fold increase in NFAT-luciferase control cardiomyocytes. Accordingly, overexpression of syndecan-4 or introducing a cell-permeable membrane-targeted syndecan-4 polypeptide (gain of function) activated NFATc4 in vitro. Pull-down experiments demonstrated a direct intracellular syndecan-4-calcineurin interaction. This interaction and activation of NFAT were increased by dephosphorylation of serine 179 (pS179) in syndecan-4. During pressure overload, phosphorylation of syndecan-4 was decreased, and association between syndecan-4, calcineurin and its co-activator calmodulin increased. Moreover, calcineurin dephosphorylated pS179, indicating that calcineurin regulates its own binding and activation. Finally, patients with hypertrophic myocardium due to aortic stenosis had increased

  11. PKC-α contributes to high NaCl-induced activation of NFAT5 (TonEBP/OREBP) through MAPK ERK1/2.

    PubMed

    Wang, Hong; Ferraris, Joan D; Klein, Janet D; Sands, Jeff M; Burg, Maurice B; Zhou, Xiaoming

    2015-01-15

    High NaCl in the renal medullary interstitial fluid powers the concentration of urine but can damage cells. The transcription factor nuclear factor of activated T cells 5 (NFAT5) activates the expression of osmoprotective genes. We studied whether PKC-α contributes to the activation of NFAT5. PKC-α protein abundance was greater in the renal medulla than in the cortex. Knockout of PKC-α reduced NFAT5 protein abundance and expression of its target genes in the inner medulla. In human embryonic kidney (HEK)-293 cells, high NaCl increased PKC-α activity, and small interfering RNA-mediated knockdown of PKC-α attenuated high NaCl-induced NFAT5 transcriptional activity. Expression of ERK1/2 protein and phosphorylation of ERK1/2 were higher in the renal inner medulla than in the cortex. Knockout of PKC-α decreased ERK1/2 phosphorylation in the inner medulla, as did knockdown of PKC-α in HEK-293 cells. Also, knockdown of ERK2 reduced high NaCl-dependent NFAT5 transcriptional activity in HEK-293 cells. Combined knockdown of PKC-α and ERK2 had no greater effect than knockdown of either alone. Knockdown of either PKC-α or ERK2 reduced the high NaCl-induced increase of NFAT5 transactivating activity. We have previously found that the high NaCl-induced increase of phosphorylation of Ser(591) on Src homology 2 domain-containing phosphatase 1 (SHP-1-S591-P) contributes to the activation of NFAT5 in cell culture, and here we found high levels of SHP-1-S591-P in the inner medulla. PKC-α has been previously shown to increase SHP-1-S591-P, which raised the possibility that PKC-α might be acting through SHP-1. However, we did not find that knockout of PKC-α in the renal medulla or knockdown in HEK-293 cells affected SHP-1-S591-P. We conclude that PKC-α contributes to high NaCl-dependent activation of NFAT5 through ERK1/2 but not through SHP-1-S591. PMID:25391900

  12. Up-regulation of Store-operated Ca2+ Entry and Nuclear Factor of Activated T Cells Promote the Acinar Phenotype of the Primary Human Salivary Gland Cells.

    PubMed

    Jang, Shyh-Ing; Ong, Hwei Ling; Liu, Xibao; Alevizos, Ilias; Ambudkar, Indu S

    2016-04-15

    The signaling pathways involved in the generation and maintenance of exocrine gland acinar cells have not yet been established. Primary human salivary gland epithelial cells, derived from salivary gland biopsies, acquired an acinar-like phenotype when the [Ca(2+)] in the serum-free medium (keratinocyte growth medium, KGM) was increased from 0.05 mm (KGM-L) to 1.2 mm (KGM-H). Here we examined the mechanism underlying this Ca(2+)-dependent generation of the acinar cell phenotype. Compared with cells in KGM-L, those in KGM-H display enhancement of Orai1, STIM1, STIM2, and nuclear factor of activated T cells 1 (NFAT1) expression together with an increase in store-operated Ca(2+) entry (SOCE), SOCE-dependent nuclear translocation of pGFP-NFAT1, and NFAT-dependent but not NFκB-dependent gene expression. Importantly, AQP5, an acinar-specific protein critical for function, is up-regulated in KGM-H via SOCE/NFAT-dependent gene expression. We identified critical NFAT binding motifs in the AQP5 promoter that are involved in Ca(2+)-dependent up-regulation of AQP5. These important findings reveal that the Ca(2+)-induced switch of salivary epithelial cells to an acinar-like phenotype involves remodeling of SOCE and NFAT signaling, which together control the expression of proteins critically relevant for acinar cell function. Our data provide a novel strategy for generating and maintaining acinar cells in culture.

  13. NFAT5 promotes proliferation and migration of lung adenocarcinoma cells in part through regulating AQP5 expression

    SciTech Connect

    Guo, Kai; Jin, Faguang

    2015-09-25

    The osmoregulated transcription factor nuclear factor of activated T-cells 5(NFAT5), has been found to play important roles in the development of many kinds of human cancers, including breast cancer, colon carcinoma, renal cell carcinoma and melanoma. The aim of the present study was to determine whether NFAT5 is involved in the proliferation and migration of lung adenocarcinoma cells. We found that NFAT5 was upregulated in lung adenocarcinoma cells and knockdown of NFAT5 decreased proliferation and migration of the cells, accompanied by a significant reduction in the expression of AQP5. AQP5 was upregulated in lung adenocarcinoma cells and knockdown of AQP5 also inhibited proliferation and migration of the cells as knockdown of NFAT5 did. Moreover, overexpression of NFAT5 promoted proliferation and migration of lung adenocarcinoma cells, accompanied by a significant increase in the expression of AQP5. These results indicate that NFAT5 plays important roles in proliferation and migration of human lung adenocarcinoma cells through regulating AQP5 expression, providing a new therapeutic option for lung adenocarcinoma therapy. - Highlights: • NFAT5 expression is higher in lung adenocarcinoma cells compared with normal cells. • NFAT5 knockdown decreases proliferation and migration of lung adenocarcinoma cells. • Knockdown of NFAT5 reduces AQP5 expression in human lung adenocarcinoma cells. • Overexpression of NFAT5 promotes proliferation and migration of lung adenocarcinoma cells. • Overexpression of NFAT5 increases AQP5 expression in human lung adenocarcinoma cells.

  14. Potent Anti-Inflammatory Activity of Ursolic Acid, a Triterpenoid Antioxidant, Is Mediated through Suppression of NF-κB, AP-1 and NF-AT

    PubMed Central

    Checker, Rahul; Sandur, Santosh K.; Sharma, Deepak; Patwardhan, Raghavendra S.; Jayakumar, S.; Kohli, Vineet; Sethi, Gautam; Aggarwal, Bharat B.; Sainis, Krishna B.

    2012-01-01

    Background Ursolic acid (UA), a pentacyclic triterpenoid carboxylic acid, is the major component of many plants including apples, basil, cranberries, peppermint, rosemary, oregano and prunes and has been reported to possess antioxidant and anti-tumor properties. These properties of UA have been attributed to its ability to suppress NF-κB (nuclear factor kappa B) activation. Since NF-κB, in co-ordination with NF-AT (nuclear factor of activated T cells) and AP-1(activator protein-1), is known to regulate inflammatory genes, we hypothesized that UA might exhibit potent anti-inflammatory effects. Methodology/Principal Findings The anti-inflammatory effects of UA were assessed in activated T cells, B cells and macrophages. Effects of UA on ERK, JNK, NF-κB, AP-1 and NF-AT were studied to elucidate its mechanism of action. In vivo efficacy of UA was studied using mouse model of graft-versus-host disease. UA inhibited activation, proliferation and cytokine secretion in T cells, B cells and macrophages. UA inhibited mitogen-induced up-regulation of activation markers and co-stimulatory molecules in T and B cells. It inhibited mitogen-induced phosphorylation of ERK and JNK and suppressed the activation of immunoregulatory transcription factors NF-κB, NF-AT and AP-1 in lymphocytes. Treatment of cells with UA prior to allogenic transplantation significantly delayed induction of acute graft-versus-host disease in mice and also significantly reduced the serum levels of pro-inflammatory cytokines IL-6 and IFN-γ. UA treatment inhibited T cell activation even when added post-mitogenic stimulation demonstrating its therapeutic utility as an anti-inflammatory agent. Conclusions/Significance The present study describes the detailed mechanism of anti-inflammatory activity of UA. Further, UA may find application in the treatment of inflammatory disorders. PMID:22363615

  15. T-Cell Tumor Elimination as a Result of T-Cell Receptor-Mediated Activation

    NASA Astrophysics Data System (ADS)

    Ashwell, Jonathan D.; Longo, Dan L.; Bridges, Sandra H.

    1987-07-01

    It has recently been shown that activation of murine T-cell hybridomas with antigen inhibits their growth in vitro. The ``suicide'' of these neoplastic T cells upon stimulation with antigen suggested the possibility that activation via the antigen-specific receptor could also inhibit the growth of neoplastic T cells in vivo. To test this, mice were subcutaneously inoculated with antigen-specific T-cell hybridomas and then treated intraperitoneally with antigen. Administration of the appropriate antigen immediately after inoculation with the T-cell hybridoma abrogated tumor formation; antigen administered after tumors had become established decreased the tumor burden and, in a substantial fraction of animals, led to long-term survival. The efficacy of antigen therapy was due to both a direct inhibitory effect on tumor growth and the induction of host immunity. These studies demonstrate the utility of cellular activation as a means of inhibiting neoplastic T-cell growth in vivo and provide a rationale for studying the use of less selective reagents that can mimic the activating properties of antigen, such as monoclonal antibodies, in the treatment of T-cell neoplasms of unknown antigen specificity.

  16. NFAT activation by membrane potential follows a calcium pathway distinct from other activity-related transcription factors in skeletal muscle cells.

    PubMed

    Valdés, Juan Antonio; Gaggero, Eduardo; Hidalgo, Jorge; Leal, Nancy; Jaimovich, Enrique; Carrasco, M Angélica

    2008-03-01

    Depolarization of skeletal muscle cells triggers intracellular Ca2+ signals mediated by ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors. Previously, we have reported that K+-induced depolarization activates transcriptional regulators ERK, cAMP response element-binding protein, c-fos, c-jun, and egr-1 through IP3-dependent Ca2+ release, whereas NF-kappa B activation is elicited by both ryanodine and IP3 receptor-mediated Ca2+ signals. We have further shown that field stimulation with electrical pulses results in an NF-kappa B activation increase dependent of the amount of pulses and independent of their frequency. In this work, we report the results obtained for nuclear factor of activated T cells (NFAT)-mediated transcription and translocation generated by both K+ and electrical stimulation protocols in primary skeletal muscle cells and C2C12 cells. The Ca2+ source for NFAT activation is through release by ryanodine receptors and extracellular Ca2+ entry. We found this activation to be independent of the number of pulses within a physiological range of stimulus frequency and enhanced by long-lasting low-frequency stimulation. Therefore, activation of the NFAT signaling pathway differs from that of NF-kappa B and other transcription factors. Calcineurin enzyme activity correlated well with the relative activation of NFAT translocation and transcription using different stimulation protocols. Furthermore, both K+-induced depolarization and electrical stimulation increased mRNA levels of the type 1 IP3 receptor mediated by calcineurin activity, which suggests that depolarization may regulate IP3 receptor transcription. These results confirm the presence of at least two independent pathways for excitation-transcription coupling in skeletal muscle cells, both dependent on Ca2+ release and triggered by the same voltage sensor but activating different intracellular release channels. PMID:18184878

  17. Cell cycle and apoptosis regulation by NFAT transcription factors: new roles for an old player

    PubMed Central

    Mognol, G P; Carneiro, F R G; Robbs, B K; Faget, D V; Viola, J P B

    2016-01-01

    The NFAT (nuclear factor of activated T cells) family of transcription factors consists of four Ca2+-regulated members (NFAT1–NFAT4), which were first described in T lymphocytes. In addition to their well-documented role in T lymphocytes, where they control gene expression during cell activation and differentiation, NFAT proteins are also expressed in a wide range of cells and tissue types and regulate genes involved in cell cycle, apoptosis, angiogenesis and metastasis. The NFAT proteins share a highly conserved DNA-binding domain (DBD), which allows all NFAT members to bind to the same DNA sequence in enhancers or promoter regions. The same DNA-binding specificity suggests redundant roles for the NFAT proteins, which is true during the regulation of some genes such as IL-2 and p21. However, it has become increasingly clear that different NFAT proteins and even isoforms can have unique functions. In this review, we address the possible reasons for these distinct roles, particularly regarding N- and C-terminal transactivation regions (TADs) and the partner proteins that interact with these TADs. We also discuss the genes regulated by NFAT during cell cycle regulation and apoptosis and the role of NFAT during tumorigenesis. PMID:27100893

  18. Cell cycle and apoptosis regulation by NFAT transcription factors: new roles for an old player.

    PubMed

    Mognol, G P; Carneiro, F R G; Robbs, B K; Faget, D V; Viola, J P B

    2016-04-21

    The NFAT (nuclear factor of activated T cells) family of transcription factors consists of four Ca(2+)-regulated members (NFAT1-NFAT4), which were first described in T lymphocytes. In addition to their well-documented role in T lymphocytes, where they control gene expression during cell activation and differentiation, NFAT proteins are also expressed in a wide range of cells and tissue types and regulate genes involved in cell cycle, apoptosis, angiogenesis and metastasis. The NFAT proteins share a highly conserved DNA-binding domain (DBD), which allows all NFAT members to bind to the same DNA sequence in enhancers or promoter regions. The same DNA-binding specificity suggests redundant roles for the NFAT proteins, which is true during the regulation of some genes such as IL-2 and p21. However, it has become increasingly clear that different NFAT proteins and even isoforms can have unique functions. In this review, we address the possible reasons for these distinct roles, particularly regarding N- and C-terminal transactivation regions (TADs) and the partner proteins that interact with these TADs. We also discuss the genes regulated by NFAT during cell cycle regulation and apoptosis and the role of NFAT during tumorigenesis.

  19. Cell cycle and apoptosis regulation by NFAT transcription factors: new roles for an old player.

    PubMed

    Mognol, G P; Carneiro, F R G; Robbs, B K; Faget, D V; Viola, J P B

    2016-01-01

    The NFAT (nuclear factor of activated T cells) family of transcription factors consists of four Ca(2+)-regulated members (NFAT1-NFAT4), which were first described in T lymphocytes. In addition to their well-documented role in T lymphocytes, where they control gene expression during cell activation and differentiation, NFAT proteins are also expressed in a wide range of cells and tissue types and regulate genes involved in cell cycle, apoptosis, angiogenesis and metastasis. The NFAT proteins share a highly conserved DNA-binding domain (DBD), which allows all NFAT members to bind to the same DNA sequence in enhancers or promoter regions. The same DNA-binding specificity suggests redundant roles for the NFAT proteins, which is true during the regulation of some genes such as IL-2 and p21. However, it has become increasingly clear that different NFAT proteins and even isoforms can have unique functions. In this review, we address the possible reasons for these distinct roles, particularly regarding N- and C-terminal transactivation regions (TADs) and the partner proteins that interact with these TADs. We also discuss the genes regulated by NFAT during cell cycle regulation and apoptosis and the role of NFAT during tumorigenesis. PMID:27100893

  20. CD4 ligands inhibit the formation of multifunctional transduction complexes involved in T cell activation.

    PubMed

    Jabado, N; Pallier, A; Le Deist, F; Bernard, F; Fischer, A; Hivroz, C

    1997-01-01

    Ligands binding to the CD4 molecule can inhibit TCR-mediated T cell activation. We have previously reported that transcription factors regulating the expression of the IL-2 gene, NF-AT, NF-kappaB, and AP-1, are targets of this inhibitory effect in an in vitro model using peripheral human CD4+ T cells activated by a CD3 mAb. Two T cell activation pathways involved in the regulation of these transcription factors, calcium flux and the p21ras pathway, were investigated as potential targets. Binding of HIV envelope glycoprotein gp160/gp120 or a CD4 mAb to the CD4+ T cells, prior to TCR/CD3 activation, inhibited the intracellular calcium elevation. This event strongly suggested an inhibition of PLCgamma1 activity. Tyrosine phosphorylation of PLCgamma1, induced by CD3 activation, was not affected, but its association with tyrosine-phosphorylated proteins, including a 62-kDa protein, was disrupted. This PLCgamma1-associated p62 was found to be immunoreactive to p62-Sam68 Abs. The activation-induced phosphorylation of two p21ras effectors, Raf-1 and Erk2, was inhibited by the CD4 ligands, indirectly pointing to inhibition of the p21ras activation pathway. In addition, we demonstrate that TCR activation of normal CD4+ T cells induced the formation of p120GAP and PLCgamma1-containing complexes. These complexes also contain other unidentified proteins. CD4 ligand binding induced a defective formation of these transduction complexes. This may result in inefficient signaling, partially accounting for the inhibitory effects of the CD4 ligands on both p21ras and calcium-activation pathways.

  1. Hyperoxia Inhibits T Cell Activation in Mice

    NASA Astrophysics Data System (ADS)

    Hughes-Fulford, M.; Meissler, J.; Aguayo, E. T.; Globus, R.; Aguado, J.; Candelario, T.

    2013-02-01

    , spleens were removed and the splenocytes were isolated and kept as individual biological samples. We have also examined transcription factors (JASPAR) and pathways of the immune system to help us understand the mechanism of regulation. Results: Our recent mouse immunology experiment aboard STS-131 suggests that the early T cell immune response was inhibited in animals that have been exposed to spaceflight, even 24 hours after return to earth. Moreover, recent experiments in hyperoxic mice show that many of the same genes involved in early T cell activation were altered. Specifically, expression of IL-2Rα, Cxcl2, TNFα, FGF2, LTA and BCL2 genes are dysregulated in mice exposed to hyperoxia. Conclusions: If these hyperoxia-induced changes of gene expression in early T cell activation are additive to the changes seen in the microgravity of spaceflight, there could be an increased infection risk to EVA astronauts, which should be addressed prior to conducting a Mars or other long-term mission.

  2. Modulation of Cytokine Production and Transcription Factors Activities in Human Jurkat T Cells by Thymol and Carvacrol

    PubMed Central

    Gholijani, Nasser; Gharagozloo, Marjan; Kalantar, Fathollah; Ramezani, Amin; Amirghofran, Zahra

    2015-01-01

    Purpose: Thymol and carvacrol, two main components of thyme, have shown anti-inflammatory effects. The aim of this study was to assess the effects of these components on Jurkat leukemia cells as an in vitro T cell model and their molecular mechanisms of activity. Methods: Cells were cultured in the presence of components and subsequently stimulated with phorbol-12-myristate-13-acetate (PMA)/calcium ionophore for evaluating interleukin (IL)-2 and interferon (IFN)-γ production. The activation of T cell transcription factors that included nuclear factors of activated T cells (NFATs), activator protein-1 (AP-1; c-Jun/c-Fos), and nuclear factor (NF)-κB were examined by Western blot analysis. Results: Thymol and carvacrol at 25 µg/ml significantly reduced IL-2 levels from 119.4 ± 8pg/ml in control cells treated only with PMA/Calcium ionophore and the solvent to 66.9 ± 6.4pg/ml (thymol) and 32.3 ± 3.6pg/ml (carvacrol) and IFN-γ from 423.7 ± 19.7pg/ml in control cells to 311.9 ± 11.6pg/ml (thymol) and 293.5 ± 16.7pg/ml (carvacrol). Western blot analyses of nuclear extracts showed that the same concentrations of components significantly reduced NFAT-2 to 44.2 ± 2.7% (thymol) and 91.4 ± 2.3% (carvacrol) of the control (p<0.05), and c-Fos to 31.2 ± 6.2% (thymol) and 27.6 ± 3.1% (carvacrol) of the control (p<0.01). No effects on NFAT-1, c-Jun and phospho-NF-κBp65 levels were observed. Conclusion: Thymol and carvacrol could contribute to modulation of T cell activity by reducing IL-2 and IFN-γ production possibly through down regulation of AP-1 and NFAT-2 transcription factors suggesting their potential usefulness for reduction of T cell overactivity in immune-mediated diseases. PMID:26793612

  3. A CB2-Selective Cannabinoid Suppresses T-cell Activities and Increases Tregs and IL-10

    PubMed Central

    Robinson, Rebecca H.; Meissler, Joseph J.; Fan, Xiaoxuan; Yu, Daohai; Adler, Martin W.; Eisenstein, Toby K.

    2015-01-01

    We have previously shown that agonists selective for the cannabinoid receptor 2 (CB2), including O-1966, inhibit the Mixed Lymphocyte Reaction (MLR), an in vitro correlate of organ graft rejection, predominantly through effects on T-cells. Current studies explored the mechanism of this immunosuppression by O-1966 using mouse spleen cells. Treatment with O-1966 dose-relatedly decreased levels of the active nuclear forms of the transcription factors NF-κB and NFAT in wild-type T-cells, but not T-cells from CB2 knockout (CB2R k/o) mice. Additionally, a gene expression profile of purified T-cells from MLR cultures generated using a PCR T-cell activation array showed that O-1966 decreased mRNA expression of CD40 ligand and CyclinD3, and increased mRNA expression of Src-like-adaptor 2 (SLA2), Suppressor of Cytokine Signaling 5 (SOCS5), and IL-10. The increase in IL-10 was confirmed by measuring IL-10 protein levels in MLR culture supernatants. Further, an increase in the percentage of regulatory T-cells (Tregs) was observed in MLR cultures. Pretreatment with anti-IL-10 resulted in a partial reversal of the inhibition of proliferation and blocked the increase of Tregs. Additionally, O-1966 treatment caused a dose-related decrease in the expression of CD4 in MLR cultures from wild-type, but not CB2R k/o, mice. These data support the potential of CB2-selective agonists as useful therapeutic agents to prolong graft survival in transplant patients, and strengthens their potential as a new class of immunosuppressive agents with broader applicability. PMID:25980325

  4. NFAT targets signaling molecules to gene promoters in pancreatic β-cells.

    PubMed

    Lawrence, Michael C; Borenstein-Auerbach, Nofit; McGlynn, Kathleen; Kunnathodi, Faisal; Shahbazov, Rauf; Syed, Ilham; Kanak, Mazhar; Takita, Morihito; Levy, Marlon F; Naziruddin, Bashoo

    2015-02-01

    Nuclear factor of activated T cells (NFAT) is activated by calcineurin in response to calcium signals derived by metabolic and inflammatory stress to regulate genes in pancreatic islets. Here, we show that NFAT targets MAPKs, histone acetyltransferase p300, and histone deacetylases (HDACs) to gene promoters to differentially regulate insulin and TNF-α genes. NFAT and ERK associated with the insulin gene promoter in response to glucagon-like peptide 1, whereas NFAT formed complexes with p38 MAPK (p38) and Jun N-terminal kinase (JNK) upon promoters of the TNF-α gene in response to IL-1β. Translocation of NFAT and MAPKs to gene promoters was calcineurin/NFAT dependent, and complex stability required MAPK activity. Knocking down NFATc2 expression, eliminating NFAT DNA binding sites, or interfering with NFAT nuclear import prevented association of MAPKs with gene promoters. Inhibiting p38 and JNK activity increased NFAT-ERK association with promoters, which repressed TNF-α and enhanced insulin gene expression. Moreover, inhibiting p38 and JNK induced a switch from NFAT-p38/JNK-histone acetyltransferase p300 to NFAT-ERK-HDAC3 complex formation upon the TNF-α promoter, which resulted in gene repression. Histone acetyltransferase/HDAC exchange was reversed on the insulin gene by p38/JNK inhibition in the presence of glucagon-like peptide 1, which enhanced gene expression. Overall, these data indicate that NFAT directs signaling enzymes to gene promoters in islets, which contribute to protein-DNA complex stability and promoter regulation. Furthermore, the data suggest that TNF-α can be repressed and insulin production can be enhanced by selectively targeting signaling components of NFAT-MAPK transcriptional/signaling complex formation in pancreatic β-cells. These findings have therapeutic potential for suppressing islet inflammation while preserving islet function in diabetes and islet transplantation.

  5. Role of nuclear factor of activated T-cells 5 in regulating hypertonic-mediated secretin receptor expression in kidney collecting duct cells.

    PubMed

    Chua, Oscar W H; Wong, Kenneth K L; Ko, Ben C; Chung, Sookja K; Chow, Billy K C; Lee, Leo T O

    2016-07-01

    A growing body of evidence suggests that secretin (SCT) is an important element in the osmoregulatory pathway. It is interesting to note that both SCT and its receptor (SCTR) gene are activated upon hyperosmolality in the kidney. However, the precise molecular mechanisms underlying the induction of the SCTR gene expression in response to changes in osmolality have yet to be clarified. Detailed DNA sequence analysis of the promoter regions of the SCTR gene reveals the presence of multiple osmotic response elements (ORE). The ORE is the binding site of a key osmosensitive transactivator, namely, the nuclear factor of activated T-cells 5 (NFAT5). SCTR and NFAT5 are co-expressed in the kidney cortex and medulla collecting duct cells. We therefore hypothesize that NFAT5 is responsible for modulating SCTR expression in hypertonic environments. In this study, we found hypertonicity stimulates the promoter activities and endogenous gene expression of SCTR in mouse kidney cortex collecting duct cells (M1) and inner medulla collecting duct cells (mIMCD3). The overexpression and silencing of NFAT5 further confirmed it to be responsible for the up-regulation of the SCTR gene under hypertonic conditions. A significant increase in the interaction between NFAT5 and the SCTR promoter was also observed following chromatin immunoprecipitation assay. In vivo, osmotic stress up-regulates the SCTR gene in the kidney cortex and medulla of wild-type mice, but does not do so in NFAT5(+/-) animals. Hence, this study provides comprehensive information on how NFAT5 regulates SCTR expression in different osmotic environments.

  6. Synergistic inhibition of T-cell activation by a cell-permeable ZAP-70 mutant and ctCTLA-4

    SciTech Connect

    Kim, Kyun-Do; Choi, Je-Min; Chae, Wook-Jin; Lee, Sang-Kyou

    2009-04-10

    T-cell activation requires TcR-mediated and co-stimulatory signals. ZAP-70 participates in the initial step of TcR signal transduction, while a co-receptor, CTLA-4, inhibits T-cell activation. In previous studies, the overexpression of a ZAP-70 mutant (ZAP-70-Y319F) inhibited the TcR-induced activation of NFAT and IL-2 production, while Hph-1-ctCTLA-4 prevented allergic inflammation. To develop an effective immunosuppressive protein drug that blocks both TcR-mediated and co-stimulatory signaling pathways, a fusion protein of ZAP-70-Y319F and the Hph-1 protein transduction domain was generated. Hph-1-ZAP-70-Y319F inhibited the phosphorylation of ZAP-70-Tyr{sup 319}, LAT-Tyr{sup 191}, and p44/42 MAPK induced by TcR stimulation, NFAT- and AP-1-mediated gene transcription, and the induction of CD69 expression and IL-2 secretion. Hph-1-ZAP-70-Y319F and Hph-1-ctCTLA-4 synergistically inhibited signaling events during T-cell activation. This is the first report to demonstrate the synergistic inhibition of signals transmitted via TcR and its co-stimulatory receptor by cell-permeable forms of intracellular signal mediators.

  7. GrpL, a Grb2-related Adaptor Protein, Interacts with SLP-76 to Regulate Nuclear Factor of Activated T Cell Activation

    PubMed Central

    Law, Che-Leung; Ewings, Maria K.; Chaudhary, Preet M.; Solow, Sasha A.; Yun, Theodore J.; Marshall, Aaron J.; Hood, Leroy; Clark, Edward A.

    1999-01-01

    Propagation of signals from the T cell antigen receptor (TCR) involves a number of adaptor molecules. SH2 domain–containing protein 76 (SLP-76) interacts with the guanine nucleotide exchange factor Vav to activate the nuclear factor of activated cells (NF-AT), and its expression is required for normal T cell development. We report the cloning and characterization of a novel Grb2-like adaptor molecule designated as Grb2-related protein of the lymphoid system (GrpL). Expression of GrpL is restricted to hematopoietic tissues, and it is distinguished from Grb2 by having a proline-rich region. GrpL can be coimmunoprecipitated with SLP-76 but not with Sos1 or Sos2 from Jurkat cell lysates. In contrast, Grb2 can be coimmunoprecipitated with Sos1 and Sos2 but not with SLP-76. Moreover, tyrosine-phosphorylated LAT/pp36/38 in detergent lysates prepared from anti-CD3 stimulated T cells associated with Grb2 but not GrpL. These data reveal the presence of distinct complexes involving GrpL and Grb2 in T cells. A functional role of the GrpL–SLP-76 complex is suggested by the ability of GrpL to act alone or in concert with SLP-76 to augment NF-AT activation in Jurkat T cells. PMID:10209041

  8. Doxorubicin-mediated Apoptosis in Glioma Cells Requires NFAT3

    PubMed Central

    Gopinath, Sreelatha; Vanamala, Sravan K.; Gujrati, Meena; Klopfenstein, Jeffrey D.; Dinh, Dzung H.; Rao, Jasti S.

    2009-01-01

    Nuclear Factor of Activated T cells (NFAT), a family of transcription factors, has been implicated in many cellular processes, including some cancers. For the first time, the present study characterizes the role of NFAT3 in doxorubicin (DOX) mediated apoptosis, migration, and invasion in SNB19 and U87 glioma cells. This study demonstrates specific knockdown of NFAT3 results in a dramatic inhibition of the apoptotic effect, induced by DOX, and favors cell survival. Inhibition of NFAT3 activation by shNFAT3 (shNF3) significantly downregulated TNF-α induction, its receptor TNFR1, caspase 10, caspase 3 and PARP, abrogating DOX-mediated apoptosis in glioma cells. DOX treatment resulted in NFAT3 translocation to the nucleus. Similarly, shNF3 treatment in SNB19 and U87 cells reversed DOX-induced inhibition of cell migration and invasion as determined by wound healing and matrigel invasion assays. Taken together, these results indicate that NFAT3 is a prerequisite for the induction of DOX-mediated apoptosis in glioma cells. PMID:19784808

  9. Focal adhesion kinase regulates the activity of the osmosensitive transcription factor TonEBP/NFAT5 under hypertonic conditions

    PubMed Central

    Neuhofer, Wolfgang; Küper, Christoph; Lichtnekert, Julia; Holzapfel, Konstantin; Rupanagudi, Khader V.; Fraek, Maria-Luisa; Bartels, Helmut; Beck, Franz-Xaver

    2014-01-01

    TonEBP/NFAT5 is a major regulator of the urinary concentrating process and is essential for the osmoadaptation of renal medullary cells. Focal adhesion kinase (FAK) is a mechanosensitive non-receptor protein tyrosine kinase expressed abundantly in the renal medulla. Since osmotic stress causes cell shrinkage, the present study investigated the contribution of FAK on TonEBP/NFAT5 activation. Osmotic stress induced time-dependent activation of FAK as evidenced by phosphorylation at Tyr-397, and furosemide reduces FAK Tyr-397 phosphorylation in the rat renal medulla. Both pharmacological inhibition of FAK and siRNA-mediated knockdown of FAK drastically reduced TonEBP/NFAT5 transcriptional activity and target gene expression in HEK293 cells. This effect was not mediated by impaired nuclear translocation or by reduced transactivating activity of TonEBP/NFAT5. However, TonEBP/NFAT5 abundance under hypertonic conditions was diminished by 50% by FAK inhibition or siRNA knockdown of FAK. FAK inhibition only marginally reduced transcription of the TonEBP/NFAT5 gene. Rather, TonEBP/NFAT5 mRNA stability was diminished significantly by FAK inhibition, which correlated with reduced reporter activity of the TonEBP/NFAT5 mRNA 3′ untranslated region (3′-UTR). In conclusion, FAK is a major regulator of TonEBP/NFAT5 activity by increasing its abundance via stabilization of the mRNA. This in turn, depends on the presence of the TonEBP/NFAT5 3′-UTR. PMID:24772088

  10. Magnetic Field-Induced T Cell Receptor Clustering by Nanoparticles Enhances T Cell Activation and Stimulates Antitumor Activity

    PubMed Central

    2015-01-01

    Iron–dextran nanoparticles functionalized with T cell activating proteins have been used to study T cell receptor (TCR) signaling. However, nanoparticle triggering of membrane receptors is poorly understood and may be sensitive to physiologically regulated changes in TCR clustering that occur after T cell activation. Nano-aAPC bound 2-fold more TCR on activated T cells, which have clustered TCR, than on naive T cells, resulting in a lower threshold for activation. To enhance T cell activation, a magnetic field was used to drive aggregation of paramagnetic nano-aAPC, resulting in a doubling of TCR cluster size and increased T cell expansion in vitro and after adoptive transfer in vivo. T cells activated by nano-aAPC in a magnetic field inhibited growth of B16 melanoma, showing that this novel approach, using magnetic field-enhanced nano-aAPC stimulation, can generate large numbers of activated antigen-specific T cells and has clinically relevant applications for adoptive immunotherapy. PMID:24564881

  11. Expression, fermentation and purification of a predicted intrinsically disordered region of the transcription factor, NFAT5.

    PubMed

    DuMond, Jenna F; He, Yi; Burg, Maurice B; Ferraris, Joan D

    2015-11-01

    Hypertonicity stimulates Nuclear Factor of Activated T-cells 5 (NFAT5) nuclear localization and transactivating activity. Many transcription factors are known to contain intrinsically disordered regions (IDRs) which become more structured with local environmental changes such as osmolality, temperature and tonicity. The transactivating domain of NFAT5 is predicted to be intrinsically disordered under normal tonicity, and under high NaCl, the activity of this domain is increased. To study the binding of co-regulatory proteins at IDRs a cDNA construct expressing the NFAT5 TAD was created and transformed into Escherichia coli cells. Transformed E. coli cells were mass produced by fermentation and extracted by cell lysis to release the NFAT5 TAD. The NFAT5 TAD was subsequently purified using a His-tag column, cation exchange chromatography as well as hydrophobic interaction chromatography and then characterized by mass spectrometry (MS). PMID:26256058

  12. NFAT1 transcription factor regulates cell cycle progression and cyclin E expression in B lymphocytes.

    PubMed

    Teixeira, Leonardo K; Carrossini, Nina; Sécca, Cristiane; Kroll, José E; DaCunha, Déborah C; Faget, Douglas V; Carvalho, Lilian D S; de Souza, Sandro J; Viola, João P B

    2016-09-01

    The NFAT family of transcription factors has been primarily related to T cell development, activation, and differentiation. Further studies have shown that these ubiquitous proteins are observed in many cell types inside and outside the immune system, and are involved in several biological processes, including tumor growth, angiogenesis, and invasiveness. However, the specific role of the NFAT1 family member in naive B cell proliferation remains elusive. Here, we demonstrate that NFAT1 transcription factor controls Cyclin E expression, cell proliferation, and tumor growth in vivo. Specifically, we show that inducible expression of NFAT1 inhibits cell cycle progression, reduces colony formation, and controls tumor growth in nude mice. We also demonstrate that NFAT1-deficient naive B lymphocytes show a hyperproliferative phenotype and high levels of Cyclin E1 and E2 upon BCR stimulation when compared to wild-type B lymphocytes. NFAT1 transcription factor directly regulates Cyclin E expression in B cells, inhibiting the G1/S cell cycle phase transition. Bioinformatics analysis indicates that low levels of NFAT1 correlate with high expression of Cyclin E1 in different human cancers, including Diffuse Large B-cell Lymphomas (DLBCL). Together, our results demonstrate a repressor role for NFAT1 in cell cycle progression and Cyclin E expression in B lymphocytes, and suggest a potential function for NFAT1 protein in B cell malignancies.

  13. Clustering of T cell ligands on artificial APC membranes influences T cell activation and protein kinase C theta translocation to the T cell plasma membrane.

    PubMed

    Giannoni, Francesca; Barnett, Joellen; Bi, Kun; Samodal, Rodrigo; Lanza, Paola; Marchese, Patrizia; Billetta, Rosario; Vita, Randi; Klein, Mark R; Prakken, Berent; Kwok, William W; Sercarz, Eli; Altman, Amnon; Albani, Salvatore

    2005-03-15

    T cell activation is associated with active clustering of relevant molecules in membrane microdomains defined as the supramolecular activation cluster. The contact area between these regions on the surface of T cells and APC is defined as the immunological synapse. It has been recently shown that preclustering of MHC-peptide complexes in membrane microdomains on the APC surface affects the efficiency of immune synapse formation and the related T cell activation. Disruption of such clusters may reduce the efficiency of stimulation. We describe here an entirely artificial system for Ag-specific, ex vivo stimulation of human polyclonal T cells (artificial APC (aAPC)). aAPC are based on artificial membrane bilayers containing discrete membrane microdomains encompassing T cell ligands (i.e., appropriate MHC-peptide complexes in association with costimulatory molecules). We show here that preclustering of T cell ligands triggered a degree of T cell activation significantly higher than the one achieved when we used either soluble tetramers or aAPC in which MHC-peptide complexes were uniformly distributed within artificial bilayer membranes. This increased efficiency in stimulation was mirrored by increased translocation from the cytoplasm to the membrane of protein kinase theta, a T cell signaling molecule that colocalizes with the TCR within the supramolecular activation cluster, thus indicating efficient engagement of T cell activation pathways. Engineered aAPC may have immediate application for basic and clinical immunology studies pertaining to modulation of T cells ex vivo.

  14. Fermentation, Isolation, Structure, and antidiabetic activity of NFAT-133 produced by Streptomyces strain PM0324667

    PubMed Central

    2011-01-01

    Type-2 diabetes is mediated by defects in either insulin secretion or insulin action. In an effort to identify extracts that may stimulate glucose uptake, similar to insulin, a high throughput-screening assay for measuring glucose uptake in skeletal muscle cells was established. During the screening studies to discover novel antidiabetic compounds from microbial resources a Streptomyces strain PM0324667 (MTCC 5543, the Strain accession number at Institute of Microbial Technology, Chandigarh, India), an isolate from arid soil was identified which expressed a secondary metabolite that induced glucose uptake in L6 skeletal muscle cells. By employing bioactivity guided fractionation techniques, a tri-substituted simple aromatic compound with anti-diabetic potential was isolated. It was characterized based on MS and 2D NMR spectral data and identified as NFAT-133 which is a known immunosuppressive agent that inhibits NFAT-dependent transcription in vitro. Our investigations revealed the antidiabetic potential of NFAT-133. The compound induced glucose uptake in differentiated L6 myotubes with an EC50 of 6.3 ± 1.8 μM without activating the peroxisome proliferator-activated receptor-γ. Further, NFAT-133 was also efficacious in vivo in diabetic animals and reduced systemic glucose levels. Thus it is a potential lead compound which can be considered for development as a therapeutic for the treatment of type-2 diabetes. We have reported herewith the isolation of the producer microbe, fermentation, purification, in vitro, and in vivo antidiabetic activity of the compound. PMID:22104600

  15. Fermentation, Isolation, Structure, and antidiabetic activity of NFAT-133 produced by Streptomyces strain PM0324667.

    PubMed

    Kulkarni-Almeida, Asha A; Brahma, Manoja K; Padmanabhan, Prabhu; Mishra, Prabhu D; Parab, Rajashri R; Gaikwad, Nitin V; Thakkar, Chandni S; Tokdar, Pradipta; Ranadive, Prafull V; Nair, Amrutha S; Damre, Anagha A; Bahirat, Umakant A; Deshmukh, Nitin J; Doshi, Lalit S; Dixit, Amol V; George, Saji D; Vishwakarma, Ram A; Nemmani, Kumar Vs; Mahajan, Girish B

    2011-11-21

    Type-2 diabetes is mediated by defects in either insulin secretion or insulin action. In an effort to identify extracts that may stimulate glucose uptake, similar to insulin, a high throughput-screening assay for measuring glucose uptake in skeletal muscle cells was established. During the screening studies to discover novel antidiabetic compounds from microbial resources a Streptomyces strain PM0324667 (MTCC 5543, the Strain accession number at Institute of Microbial Technology, Chandigarh, India), an isolate from arid soil was identified which expressed a secondary metabolite that induced glucose uptake in L6 skeletal muscle cells. By employing bioactivity guided fractionation techniques, a tri-substituted simple aromatic compound with anti-diabetic potential was isolated. It was characterized based on MS and 2D NMR spectral data and identified as NFAT-133 which is a known immunosuppressive agent that inhibits NFAT-dependent transcription in vitro. Our investigations revealed the antidiabetic potential of NFAT-133. The compound induced glucose uptake in differentiated L6 myotubes with an EC50 of 6.3 ± 1.8 μM without activating the peroxisome proliferator-activated receptor-γ. Further, NFAT-133 was also efficacious in vivo in diabetic animals and reduced systemic glucose levels. Thus it is a potential lead compound which can be considered for development as a therapeutic for the treatment of type-2 diabetes. We have reported herewith the isolation of the producer microbe, fermentation, purification, in vitro, and in vivo antidiabetic activity of the compound.

  16. Exposure of Jurkat cells to bis (tri-n-butyltin) oxide (TBTO) induces transcriptomics changes indicative for ER- and oxidative stress, T cell activation and apoptosis

    SciTech Connect

    Katika, Madhumohan R.; Hendriksen, Peter J.M.; Loveren, Henk van; Peijnenburg, Ad

    2011-08-01

    Tributyltin oxide (TBTO) is an organotin compound that is widely used as a biocide in agriculture and as an antifouling agent in paints. TBTO is toxic for many cell types, particularly immune cells. The present study aimed to identify the effects of TBTO on the human T lymphocyte cell line Jurkat. Cells were treated with 0.2 and 0.5 {mu}M TBTO for 3, 6, 12 and 24 h and then subjected to whole genome gene expression microarray analysis. The biological interpretation of the gene expression profiles revealed that endoplasmic reticulum (ER) stress is among the earliest effects of TBTO. Simultaneously or shortly thereafter, oxidative stress, activation of NFKB and NFAT, T cell activation, and apoptosis are induced. The effects of TBTO on genes involved in ER stress, NFAT pathway, T cell activation and apoptosis were confirmed by qRT-PCR. Activation and nuclear translocation of NFATC1 and the oxidative stress response proteins NRF2 and KEAP1 were confirmed by immunocytology. Taking advantage of previously published microarray data, we demonstrated that the induction of ER stress, oxidative stress, T cell activation and apoptosis by TBTO is not unique for Jurkat cells but does also occur in mouse thymocytes both ex vivo and in vivo and rat thymocytes ex vivo. We propose that the induction of ER stress leading to a T cell activation response is a major factor in the higher sensitivity of immune cells above other types of cells for TBTO. - Research Highlights: > The human T lymphocyte cell line Jurkat was exposed to TBTO. > Whole-genome microarray experiments were performed. > Data analysis revealed the induction of ER stress and activation of NFAT and NFKB. > Exposure to TBTO also led to T cell activation, oxidative stress and apoptosis.

  17. The calcineurin-NFAT pathway negatively regulates megakaryopoiesis.

    PubMed

    Zaslavsky, Alexander; Chou, Stella T; Schadler, Keri; Lieberman, Allyson; Pimkin, Maxim; Kim, Yeo Jung; Baek, Kwan-Hyuck; Aird, William C; Weiss, Mitchell J; Ryeom, Sandra

    2013-04-18

    The calcium regulated calcineurin-nuclear factor of activated T cells (NFAT) pathway modulates the physiology of numerous cell types, including hematopoietic. Upon activation, calcineurin dephosphorylates NFAT family transcription factors, triggering their nuclear entry and activation or repression of target genes. NFATc1 and c2 isoforms are expressed in megakaryocytes. Moreover, human chromosome 21 (Hsa21) encodes several negative regulators of calcineurin-NFAT, candidates in the pathogenesis of Down syndrome (trisomy 21)-associated transient myeloproliferative disorder and acute megakaryoblastic leukemia. To investigate the role of calcineurin-NFAT in megakaryopoiesis, we examined wild-type mice treated with the calcineurin inhibitor cyclosporin A and transgenic mice expressing a targeted single extra copy of Dscr1, an Hsa21-encoded calcineurin inhibitor. Both murine models exhibited thrombocytosis with increased megakaryocytes and megakaryocyte progenitors. Pharmacological or genetic inhibition of calcineurin in mice caused accumulation of megakaryocytes exhibiting enhanced 5-bromo-2'-deoxyuridine uptake and increased expression of messenger RNAs encoding CDK4 and G1 cyclins, which promote cell division. Additionally, human megakaryocytes with trisomy 21 show increased proliferation and decreased NFAT activation compared with euploid controls. Our data indicate that inhibition of calcineurin-NFAT drives proliferation of megakaryocyte precursors by de-repressing genes that drive cell division, providing insights into mechanisms of normal megakaryopoiesis and megakaryocytic abnormalities that accompany Down syndrome.

  18. Multivalent Antigens for Promoting B and T Cell Activation

    PubMed Central

    Bennett, Nitasha R.; Zwick, Daniel B.; Courtney, Adam H.; Kiessling, Laura L.

    2015-01-01

    Efficacious vaccines require antigens that elicit productive immune system activation. Antigens that afford robust antibody production activate both B and T cells. Elucidating the antigen properties that enhance B–T cell communication is difficult with traditional antigens. We therefore used ring-opening metathesis polymerization to access chemically defined, multivalent antigens containing both B and T cell epitopes to explore how antigen structure impacts B cell and T cell activation and communication. The bifunctional antigens were designed so that the backbone substitution level of each antigenic epitope could be quantified using 19F NMR. The T cell peptide epitope was appended so that it could be liberated in B cells via the action of the endosomal protease cathepsin D, and this design feature was critical for T cell activation. Antigens with high BCR epitope valency induce greater BCR-mediated internalization and T cell activation than did low valency antigens, and these high-valency polymeric antigens were superior to protein antigens. We anticipate that these findings can guide the design of more effective vaccines. PMID:25970017

  19. Decrease in T Cell Activation and Calcium Flux during Clinorotation

    NASA Technical Reports Server (NTRS)

    Sams, Clarence; Holtzclaw, J. David

    2006-01-01

    We investigated the effect of altered gravitational environments on T cell activation. We isolated human, naive T cells (CD3+CD14-CD19-CD16-CD56-CD25-CD69-CD45RA-) following IRB approved protocols. These purified T cells were then incubated with 6 mm polystyrene beads coated with OKT3 (Ortho Biotech, Raritan, NJ) and antiCD28 (Becton Dickinson (BD), San Jose, CA) at 37 C for 24 hours. Antibodies were at a 1:1 ratio and the bead-to-cell ratio was 2:1. Four incubation conditions existed: 1) static or "1g"; 2) centrifugation at 10 relative centrifugal force (RCF) or "10g"; 3) clinorotation at 25 RPM (functional weightlessness or "0g"); and 4) clinorotation at 80 RPM ("1g" plus net shear force approx.30 dynes/sq cm). Following incubation, T cells were stained for CD25 expression (BD) and intracellular calcium (ratio of Fluo4 to Fura Red, Molecular Probes, Eugene, OR) and analyzed by flow cytometry (Coulter EPICS XL, Miami, FL). Results: Static or "1g" T cells had the highest level of CD25 expression and intracellular calcium. T cells centrifuged at 10 RCF ("10g") had lower CD25 expression and calcium levels compared to the static control. However, cells centrifuged at 10 RCF had higher CD25 expression and calcium levels than those exposed to 24 RPM clinorotation ("0g"). T cells exposed to 24 RPM clinorotation had lower CD25 expression, but the approximately the same calcium levels than T cells exposed to 80 RPM clinorotation. These data suggest that stress-activated calcium channel exist in T cells and may play a role during T cell activation.

  20. Remote Control of T Cell Activation Using Magnetic Janus Particles.

    PubMed

    Lee, Kwahun; Yi, Yi; Yu, Yan

    2016-06-20

    We report a strategy for using magnetic Janus microparticles to control the stimulation of T cell signaling with single-cell precision. To achieve this, we designed Janus particles that are magnetically responsive on one hemisphere and stimulatory to T cells on the other side. By manipulating the rotation and locomotion of Janus particles under an external magnetic field, we could control the orientation of the particle-cell recognition and thereby the initiation of T cell activation. This study demonstrates a step towards employing anisotropic material properties of Janus particles to control single-cell activities without the need of complex magnetic manipulation devices.

  1. PLZF+ Innate T Cells Support the TGF-β-Dependent Generation of Activated/Memory-Like Regulatory T Cells

    PubMed Central

    Kang, Byung Hyun; Park, Hyo Jin; Park, Hi Jung; Lee, Jae-II; Park, Seong Hoe; Jung, Kyeong Cheon

    2016-01-01

    PLZF-expressing invariant natural killer T cells and CD4 T cells are unique subsets of innate T cells. Both are selected via thymocyte-thymocyte interaction, and they contribute to the generation of activated/memory-like CD4 and CD8 T cells in the thymus via the production of IL-4. Here, we investigated whether PLZF+ innate T cells also affect the development and function of Foxp3+ regulatory CD4 T cells. Flow cytometry analysis of the thymus and spleen from both CIITA transgenic C57BL/6 and wild-type BALB/c mice, which have abundant PLZF+ CD4 T cells and invariant natural killer T cells, respectively, revealed that Foxp3+ T cells in these mice exhibited a CD103+ activated/memory-like phenotype. The frequency of CD103+ regulatory T cells was considerably decreased in PLZF+ cell-deficient CIITATgPlzflu/lu and BALB/c.CD1d−/− mice as well as in an IL-4-deficient background, such as in CIITATgIL-4−/− and BALB/c.lL-4−/− mice, indicating that the acquisition of an activated/memory-like phenotype was dependent on PLZF+ innate T cells and IL-4. Using fetal thymic organ culture, we further demonstrated that IL-4 in concert with TGF-β enhanced the acquisition of the activated/memory-like phenotype of regulatory T cells. In functional aspects, the activated/memory-like phenotype of Treg cells was directly related to their suppressive function; regulatory T cells of CIITATgPIV−/− mice more efficiently suppressed ovalbumin-induced allergic airway inflammation compared with their counterparts from wild-type mice. All of these findings suggest that PLZF+ innate T cells also augmented the generation of activated/memory-like regulation via IL-4 production. PMID:27101876

  2. Malignant T cells express lymphotoxin α and drive endothelial activation in cutaneous T cell lymphoma

    PubMed Central

    Lauenborg, Britt; Christensen, Louise; Ralfkiaer, Ulrik; Kopp, Katharina L.; Jønson, Lars; Dabelsteen, Sally; Bonefeld, Charlotte M.; Geisler, Carsten; Gjerdrum, Lise Mette R.; Zhang, Qian; Wasik, Mariusz A.; Ralfkiaer, Elisabeth; Ødum, Niels; Woetmann, Anders

    2015-01-01

    Lymphotoxin α (LTα) plays a key role in the formation of lymphatic vasculature and secondary lymphoid structures. Cutaneous T cell lymphoma (CTCL) is the most common primary lymphoma of the skin and in advanced stages, malignant T cells spreads through the lymphatic to regional lymph nodes to internal organs and blood. Yet, little is known about the mechanism of the CTCL dissemination. Here, we show that CTCL cells express LTα in situ and that LTα expression is driven by aberrantly activated JAK3/STAT5 pathway. Importantly, via TNF receptor 2, LTα functions as an autocrine factor by stimulating expression of IL-6 in the malignant cells. LTα and IL-6, together with VEGF promote angiogenesis by inducing endothelial cell sprouting and tube formation. Thus, we propose that LTα plays a role in malignant angiogenesis and disease progression in CTCL and may serve as a therapeutic target in this disease. PMID:25915535

  3. NFAT2 mediates high glucose-induced glomerular podocyte apoptosis through increased Bax expression

    SciTech Connect

    Li, Ruizhao; Zhang, Li; Shi, Wei; Zhang, Bin; Liang, Xinling; Liu, Shuangxin; Wang, Wenjian

    2013-04-15

    Background: Hyperglycemia promotes podocyte apoptosis and plays a key role in the pathogenesis of diabetic nephropathy. However, the mechanisms that mediate hyperglycemia-induced podocyte apoptosis is still far from being fully understood. Recent studies reported that high glucose activate nuclear factor of activated T cells (NFAT) in vascular smooth muscle or pancreatic β-cells. Here, we sought to determine if hyperglycemia activates NFAT2 in cultured podocyte and whether this leads to podocyte apoptosis. Meanwhile, we also further explore the mechanisms of NFAT2 activation and NFAT2 mediates high glucose-induced podocyte apoptosis. Methods: Immortalized mouse podocytes were cultured in media containing normal glucose (NG), or high glucose (HG) or HG plus cyclosporine A (a pharmacological inhibitor of calcinerin) or 11R-VIVIT (a special inhibitor of NFAT2). The activation of NFAT2 in podocytes was detected by western blotting and immunofluorescence assay. The role of NFAT2 in hyperglycemia-induced podocyte apoptosis was further evaluated by observing the inhibition of NFAT2 activation by 11R-VIVIT using flow cytometer. Intracellular Ca{sup 2+} was monitored in HG-treated podcocytes using Fluo-3/AM. The mRNA and protein expression of apoptosis gene Bax were measured by real time-qPCR and western blotting. Results: HG stimulation activated NFAT2 in a time- and dose-dependent manner in cultured podocytes. Pretreatment with cyclosporine A (500 nM) or 11R-VIVIT (100 nM) completely blocked NFAT2 nuclear accumulation. Meanwhile, the apoptosis effects induced by HG were also abrogated by concomitant treatment with 11R-VIVIT in cultured podocytes. We further found that HG also increased [Ca{sup 2+}]i, leading to activation of calcineurin, and subsequent increased nuclear accumulation of NFAT2 and Bax expression in cultured podocytes. Conclusion: Our results identify a new finding that HG-induced podocyte apoptosis is mediated by calcineurin/NFAT2/Bax signaling pathway

  4. Isoform- and tissue-specific regulation of the Ca(2+)-sensitive transcription factor NFAT in cardiac myocytes and heart failure.

    PubMed

    Rinne, Andreas; Kapur, Nidhi; Molkentin, Jeffery D; Pogwizd, Steven M; Bers, Donald M; Banach, Kathrin; Blatter, Lothar A

    2010-06-01

    Nuclear factors of activated T cells (NFATs) are Ca(2+)-sensitive transcription factors that have been implicated in hypertrophy, heart failure (HF), and arrhythmias. Cytosolic NFAT is activated by dephosphorylation by the Ca(2+)-sensitive phosphatase calcineurin, resulting in translocation to the nucleus, which is opposed by kinase activity, rephosphorylation, and nuclear export. Four different NFAT isoforms are expressed in the heart. The activation and regulation of NFAT in adult cardiac myocytes, which may depend on the NFAT isoform and cell type, are not fully understood. This study compared basal localization, import, and export of NFATc1 and NFATc3 in adult atrial and ventricular myocytes to identify isoform- and tissue-specific regulatory mechanisms of NFAT activation under physiological conditions and in HF. NFAT-green fluorescent protein fusion proteins and NFAT immunocytochemistry were used to analyze NFAT regulation in adult cat and rabbit myocytes. NFATc1 displayed basal nuclear localization in atrial and ventricular myocytes, an effect that was attenuated by reducing intracellular Ca(2+) concentration and inhibiting calcineurin, and enhanced by the inhibition of nuclear export. In contrast, NFATc3 was localized to the cytoplasm but could be driven to the nucleus by angiotensin II and endothelin-1 stimulation in atrial, but not ventricular, cells. Inhibition of nuclear export (by leptomycin B) facilitated nuclear localization in both cell types. Ventricular myocytes from HF rabbits showed increased basal nuclear localization of endogenous NFATc3 and reduced responsiveness of NFAT translocation to phenylephrine stimulation. In control myocytes, Ca(2+) overload, leading to spontaneous Ca(2+) waves, induced substantial translocation of NFATc3 to the nucleus. We conclude that the activation of NFAT in adult cardiomyocytes is isoform and tissue specific and is tightly controlled by nuclear export. NFAT is activated in myocytes from HF animals and may be

  5. A cell permeable peptide inhibitor of NFAT inhibits macrophage cytokine expression and ameliorates experimental colitis.

    PubMed

    Elloumi, Houda Z; Maharshak, Nitsan; Rao, Kavitha N; Kobayashi, Taku; Ryu, Hyungjin S; Mühlbauer, Marcus; Li, Fengling; Jobin, Christian; Plevy, Scott E

    2012-01-01

    Nuclear factor of activated T cells (NFAT) plays a critical role in the development and function of immune and non-immune cells. Although NFAT is a central transcriptional regulator of T cell cytokines, its role in macrophage specific gene expression is less defined. Previous work from our group demonstrated that NFAT regulates Il12b gene expression in macrophages. Here, we further investigate NFAT function in murine macrophages and determined the effects of a cell permeable NFAT inhibitor peptide 11R-VIVIT on experimental colitis in mice. Treatment of bone marrow derived macrophages (BMDMs) with tacrolimus or 11R-VIVIT significantly inhibited LPS and LPS plus IFN-γ induced IL-12 p40 mRNA and protein expression. IL-12 p70 and IL-23 secretion were also decreased. NFAT nuclear translocation and binding to the IL-12 p40 promoter was reduced by NFAT inhibition. Experiments in BMDMs from IL-10 deficient (Il10(-/-)) mice demonstrate that inhibition of IL-12 expression by 11R-VIVIT was independent of IL-10 expression. To test its therapeutic potential, 11R-VIVIT was administered systemically to Il10(-/-) mice with piroxicam-induced colitis. 11R-VIVIT treated mice demonstrated significant improvement in colitis compared to mice treated with an inactive peptide. Moreover, decreased spontaneous secretion of IL-12 p40 and TNF in supernatants from colon explant cultures was demonstrated. In summary, NFAT, widely recognized for its role in T cell biology, also regulates important innate inflammatory pathways in macrophages. Selective blocking of NFAT via a cell permeable inhibitory peptide is a promising therapeutic strategy for the treatment of inflammatory bowel diseases.

  6. Nuclear Factor of Activated T Cells Is Activated in the Endothelium of Retinal Microvessels in Diabetic Mice

    PubMed Central

    Zetterqvist, Anna V.; Blanco, Fabiana; Öhman, Jenny; Kotova, Olga; Berglund, Lisa M.; de Frutos Garcia, Sergio; Al-Naemi, Raed; Wigren, Maria; McGuire, Paul G.; Gonzalez Bosc, Laura V.; Gomez, Maria F.

    2015-01-01

    The pathogenesis of diabetic retinopathy (DR) remains unclear but hyperglycemia is an established risk factor. Endothelial dysfunction and changes in Ca2+ signaling have been shown to precede the onset of DR. We recently demonstrated that high extracellular glucose activates the Ca2+/calcineurin-dependent transcription factor NFAT in cerebral arteries and aorta, promoting the expression of inflammatory markers. Here we show, using confocal immunofluorescence, that NFAT is expressed in the endothelium of retinal microvessels and is readily activated by high glucose. This was inhibited by the NFAT blocker A-285222 as well as by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. Acute hyperglycemia induced by an IP-GTT (intraperitoneal glucose tolerance test) resulted in increased NFATc3 nuclear accumulation and NFAT-dependent transcriptional activity in retinal vessels of NFAT-luciferase reporter mice. In both Akita (Ins2+/−) and streptozotocin- (STZ-) induced diabetic mice, NFAT transcriptional activity was elevated in retinal vessels. In vivo inhibition of NFAT with A-285222 decreased the expression of OPN and ICAM-1 mRNA in retinal vessels, prevented a diabetes driven downregulation of anti-inflammatory IL-10 in retina, and abrogated the increased vascular permeability observed in diabetic mice. Results identify NFAT signaling as a putative target for treatment of microvascular complications in diabetes. PMID:25918731

  7. Ets-1 facilitates nuclear entry of NFAT proteins and their recruitment to the IL-2 promoter.

    PubMed

    Tsao, Hsiao-Wei; Tai, Tzong-Shyuan; Tseng, William; Chang, Hui-Hsin; Grenningloh, Roland; Miaw, Shi-Chuen; Ho, I-Cheng

    2013-09-24

    E26 transformation-specific sequence 1 (Ets-1), the prototype of the ETS family of transcription factors, is critical for the expression of IL-2 by murine Th cells; however, its mechanism of action is still unclear. Here we show that Ets-1 is also essential for optimal production of IL-2 by primary human Th cells. Although Ets-1 negatively regulates the expression of Blimp1, a known suppressor of IL-2 expression, ablation of B lymphocyte-induced maturation protein 1 (Blimp1) does not rescue the expression of IL-2 by Ets-1-deficient Th cells. Instead, Ets-1 physically and functionally interacts with the nuclear factor of activated T-cells (NFAT) and is required for the recruitment of NFAT to the IL-2 promoter. In addition, Ets-1 is located in both the nucleus and cytoplasm of resting Th cells. Nuclear Ets-1 quickly exits the nucleus in response to calcium-dependent signals and competes with NFAT proteins for binding to protein components of noncoding RNA repressor of NFAT complex (NRON), which serves as a cytoplasmic trap for phosphorylated NFAT proteins. This nuclear exit of Ets-1 precedes rapid nuclear entry of NFAT and Ets-1 deficiency results in impaired nuclear entry, but not dephosphorylation, of NFAT proteins. Thus, Ets-1 promotes the expression of IL-2 by modulating the activity of NFAT.

  8. Transgelin-2 in B-Cells Controls T-Cell Activation by Stabilizing T Cell - B Cell Conjugates

    PubMed Central

    Chae, Myoung-Won; Kim, Hye-Ran; Kim, Chang-Hyun; Jun, Chang-Duk; Park, Zee-Yong

    2016-01-01

    The immunological synapse (IS), a dynamic and organized junction between T-cells and antigen presenting cells (APCs), is critical for initiating adaptive immunity. The actin cytoskeleton plays a major role in T-cell reorganization during IS formation, and we previously reported that transgelin-2, an actin-binding protein expressed in T-cells, stabilizes cortical F-actin, promoting T-cell activation in response to antigen stimulation. Transgelin-2 is also highly expressed in B-cells, although no specific function has been reported. In this study, we found that deficiency in transgelin-2 (TAGLN2-/-) in B-cells had little effect on B-cell development and activation, as measured by the expression of CD69, MHC class II molecules, and CD80/86. Nevertheless, in B-cells, transgelin-2 accumulated in the IS during the interaction with T-cells. These results led us to hypothesize that transgelin-2 may also be involved in IS stability in B-cells, thereby influencing T-cell function. Notably, we found that transgelin-2 deficiency in B-cells reduced T-cell activation, as determined by the release of IL-2 and interferon-γ and the expression of CD69. Furthermore, the reduced T-cell activation was correlated with reduced B-cell–T-cell conjugate formation. Collectively, these results suggest that actin stability in B-cells during IS formation is critical for the initiation of adaptive T-cell immunity. PMID:27232882

  9. T cell-activating protein on murine lymphocytes.

    PubMed

    Yeh, E T; Reiser, H; Benacerraf, B; Rock, K L

    1986-12-01

    A functional T cell surface molecule, T cell-activating protein (TAP), has been identified on murine lymphocytes. TAP is a protein with an apparent molecular mass of 10-12 kilodaltons (kDa) nonreduced, 16-17 kDa reduced. Cross-linking of TAP can result in profound activation of T lymphocytes to produce lymphokines and to enter the cell cycle. Furthermore, antibody binding to TAP can modulate antigen-driven T cell stimulation. Current data suggest that TAP is physically distinct from the T cell receptor complex. On unstimulated lymphocytes, TAP is expressed on T cells and defines heterogeneity within the major T cell subsets. Its profile of expression is rapidly altered on cell activation. Ontologically, it is first detected in the thymus, where it is found on both the most immature and the most mature cell subsets, and it is functional on both. Together, these TAP+ cells constitute a small fraction of thymocytes. TAP expression, however, defines the immunocompetent compartment of the thymus, and thus could be involved in functional maturation. Finally, the gene controlling TAP expression has been mapped, and is tightly linked to a locus of hematopoietic antigens (Ly-6). TAP is molecularly distinct from these antigens. Furthermore, all of these proteins show a markedly distinct developmental regulation in their cell surface expression.

  10. T Cells

    MedlinePlus

    ... or turn off the immune response. Cytotoxic or “killer” T cells directly attack and destroy cells bearing ... involve selective activation of helper T cells and killer T cells, with a corresponding decrease in regulatory ...

  11. Regulation of T cell-dendritic cell interactions by IL-7 governs T-cell activation and homeostasis

    PubMed Central

    Saini, Manoj; Pearson, Claire

    2009-01-01

    Interleukin-7 (IL-7) plays a central role in the homeostasis of the T-cell compartment by regulating T-cell survival and proliferation. Whether IL-7 can influence T-cell receptor (TCR) signaling in T cells remains controversial. Here, using IL-7–deficient hosts and TCR-transgenic T cells that conditionally express IL-7R, we examined antigen-specific T-cell responses in vitro and in vivo to viral infection and lymphopenia to determine whether IL-7 signaling influences TCR-triggered cell division events. In vitro, we could find no evidence that IL-7 signaling could costimulate T-cell activation over a broad range of conditions, suggesting that IL-7 does not directly tune TCR signaling. In vivo, however, we found an acute requirement for IL-7 signaling for efficiently triggering T-cell responses to influenza A virus challenge. Furthermore, we found that IL-7 was required for the enhanced homeostatic TCR signaling that drives lymphopenia-induced proliferation by a mechanism involving efficient contacts of T cells with dendritic cells. Consistent with this, saturating antigen-presenting capacity in vivo overcame the triggering defect in response to cognate peptide. Thus, we demonstrate a novel role for IL-7 in regulating T cell–dendritic cell interactions that is essential for both T-cell homeostasis and activation in vivo. PMID:19357399

  12. TRESK channel as a potential target to treat T-cell mediated immune dysfunction

    SciTech Connect

    Han, Jaehee; Kang, Dawon

    2009-12-25

    In this review, we propose that TRESK background K{sup +} channel could serve as a potential therapeutic target for T-cell mediated immune dysfunction. TRESK has many immune function-related properties. TRESK is abundantly expressed in the thymus, the spleen, and human leukemic T-lymphocytes. TRESK is highly activated by Ca{sup 2+}, calcineurin, acetylcholine, and histamine which induce hypertrophy, whereas TRESK is inhibited by immunosuppressants, such as cyclosporin A and FK506. Cyclosporine A and FK506 target the binding site of nuclear factor of activated T-cells (NFAT) to inhibit calcineurin. Interestingly, TRESK possesses an NFAT-like docking site that is present at its intracellular loop. Calcineurin has been found to interact with TRESK via specific NFAT-like docking site. When the T-cell is activated, calcineurin can bind to the NFAT-docking site of TRESK. The activation of both TRESK and NFAT via Ca{sup 2+}-calcineurin-NFAT/TRESK pathway could modulate the transcription of new genes in addition to regulating several aspects of T-cell function.

  13. NFAT5 regulates transcription of the mouse telomerase reverse transcriptase gene

    SciTech Connect

    Fujiki, Tsukasa; Udono, Miyako; Kotake, Yojiro; Yamashita, Makiko; Shirahata, Sanetaka; Katakura, Yoshinori

    2010-12-10

    We aimed to clarify the transcription-regulation mechanisms of the mouse telomerase reverse transcriptase gene (mTERT). First, we searched for the promoter region required for transcriptional activation of mTERT and identified an enhancer cis-element (named mTERT-EE) located between - 200 and - 179 bp of the mouse TERT gene (mTERT). EMSA results suggested that nuclear factor of activated T cells (NFAT) member proteins bind to mTERT-EE. We then identified NFAT5 as the factor binding to mTERT-EE and found that it activates the transcription of the mTERT core promoter. The results that siRNA directed against NFAT5 significantly reduced mTERT expression and mTERT core promoter activity and that the expressions of NFAT5 and mTERT were well correlated in various mouse tissues except liver suggest that NFAT5 dominantly and directly regulates mTERT expression. To clarify their functionality further, we investigated the effect of hypertonic stress, a known stimulus affecting the expression and transcriptional activity of NFAT5, on mTERT expression. The result indicated that hypertonic stress activates mTERT transcription via the activation and recruitment of NFAT5 to the mTERT promoter. These results provide useful information about the transcription-regulation mechanisms of mTERT.

  14. NFAT regulates calcium-sensing receptor-mediated TNF production

    SciTech Connect

    abdullah, huda ismail; Pedraza, Paulina L.; Hao, Shoujin; Rodland, Karin D.; McGiff, John C.; Ferreri, Nicholas R.

    2006-05-01

    Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca2+ (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca2+ were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.

  15. T Cell Activation Thresholds are Affected by Gravitational

    NASA Technical Reports Server (NTRS)

    Adams, Charley; Gonzalez, M.; Nelman-Gonzalez, M.

    1999-01-01

    T cells stimulated in space flight by various mitogenic signals show a dramatic reduction in proliferation and expression of early activation markers. Similar results are also obtained in a ground based model of microgravity, clinorotation, which provides a vector-averaged reduction of the apparent gravity on cells without significant shear force. Here we demonstrate that T cell inhibition is due to an increase in the required threshold for activation. Dose response curves indicate that cells activated during clinorotation require higher stimulation to achieve the same level of activation, as measured by CD69 expression. Interleukin 2 receptor expression, and DNA synthesis. The amount of stimulation necessary for 50% activation is 5 fold in the clinostat relative to static. Correlation of TCR internalization with activation also exhibit a dramatic right shift in clinorotation, demonstrating unequivocally that signal transduction mechanism independent of TCR triggering account for the increased activation threshold. Previous results from space flight experiments are consistent with the dose response curves obtained for clinorotation. Activation thresholds are important aspects of T cell memory, autoimmunity and tolerance Clinorotation is a useful, noninvasive tool for the study of cellular and biochemical event regulating T cell activation threshold and the effects of gravitation forces on these systems.

  16. NFAT transcription factors regulate survival, proliferation, migration, and differentiation of neural precursor cells.

    PubMed

    Serrano-Pérez, María C; Fernández, Miriam; Neria, Fernando; Berjón-Otero, Mónica; Doncel-Pérez, Ernesto; Cano, Eva; Tranque, Pedro

    2015-06-01

    The study of factors that regulate the survival, proliferation, and differentiation of neural precursor cells (NPCs) is essential to understand neural development as well as brain regeneration. The Nuclear Factor of Activated T Cells (NFAT) is a family of transcription factors that can affect these processes besides playing key roles during development, such as stimulating axonal growth in neurons, maturation of immune system cells, heart valve formation, and differentiation of skeletal muscle and bone. Interestingly, NFAT signaling can also promote cell differentiation in adults, participating in tissue regeneration. The goal of the present study is to evaluate the expression of NFAT isoforms in NPCs, and to investigate its possible role in NPC survival, proliferation, migration, and differentiation. Our findings indicate that NFAT proteins are active not only in neurogenic brain regions such as hippocampus and subventricular zone (SVZ), but also in cultured NPCs. The inhibition of NFAT activation with the peptide VIVIT reduced neurosphere size and cell density in NPC cultures by decreasing proliferation and increasing cell death. VIVIT also decreased NPC migration and differentiation of astrocytes and neurons from NPCs. In addition, we identified NFATc3 as a predominant NFAT isoform in NPC cultures, finding that a constitutively-active form of NFATc3 expressed by adenoviral infection reduces NPC proliferation, stimulates migration, and is a potent inducer of NPC differentiation into astrocytes and neurons. In summary, our work uncovers active roles for NFAT signaling in NPC survival, proliferation and differentiation, and highlights its therapeutic potential for tissue regeneration.

  17. Up-regulation of interleukin-4 production via NF-AT/AP-1 activation in T cells by biochanin A, a phytoestrogen and its metabolites

    SciTech Connect

    Park, Jin; Chung, Su Wol; Kim, Seung Hyun; Kim, Tae Sung . E-mail: tskim@korea.ac.kr

    2006-05-01

    Phytoestrogens are naturally occurring compounds derived from plants. Although phytoestrogens exhibit many biological functions including estrogen agonist/antagonist properties, the effect on allergic responses remains unclear. In this study, we investigated whether biochanin A, a phytoestrogen and its metabolites, genistein, p-ethylphenol and phenolic acid, affect production of IL-4, a pro-inflammatory cytokine closely associated with allergic immune responses, in primary CD4{sup +} T cells and EL4 T lymphoma cells. Biochanin A, genistein and p-ethylphenol significantly enhanced IL-4 production from both CD4{sup +} T cells and EL4 cells in a dose-dependent manner, while phenolic acid did not. Biochanin A, genistein and p-ethylphenol also enhanced IL-4 gene promoter activity in EL4 cells transiently transfected with IL-4 promoter constructs, but this effect was impaired in EL4 cells transfected with an IL-4 promoter construct deleted of a P4 site carrying NF-AT and AP-1 binding sites. In addition, biochanin A, genistein and p-ethylphenol increased both NF-AT and AP-1 DNA binding activities, indicating that they might enhance IL-4 production via NF-AT/AP-1 activation. Furthermore, biochanin A, genistein and p-ethylphenol increased p38 MAPK phosphorylation and PKC activity, while they did not affect ERK phosphorylation. The enhanced NF-AT DNA binding activities were suppressed by inhibitors for PI3-K and PKC, but not by p38 MAPK inhibitors. In contrast, the enhanced AP-1 DNA binding activities and p38 MAPK phosphorylation were significantly suppressed by specific inhibitors for PKC and p38 MAPK, but not by PI3-K inhibitors. These results demonstrate, for the first time, that biochanin A, genistein and p-ethylphenol enhance IL-4 production in activated T cells by two independent pathways, PI3-K/PKC/NF-AT and PKC/p38 MAPK/AP-1.

  18. NFAT5 in cellular adaptation to hypertonic stress - regulations and functional significance.

    PubMed

    Cheung, Chris Yk; Ko, Ben Cb

    2013-01-01

    The Nuclear Factor of Activated T Cells-5 (NFAT5), also known as OREBP or TonEBP, is a member of the nuclear factors of the activated T cells family of transcription factors. It is also the only known tonicity-regulated transcription factor in mammals. NFAT5 was initially known for its role in the hypertonic kidney inner medulla for orchestrating a genetic program to restore the cellular homeostasis. Emerging evidence, however, suggests that NFAT5 might play a more diverse functional role, including a pivotal role in blood pressure regulation and the development of autoimmune diseases. Despite the growing significance of NFAT5 in physiology and diseases, our understanding of how its activity is regulated remains very limited. Furthermore, how changes in tonicities are converted into functional outputs via NFAT5 remains elusive. Therefore, this review aims to summarize our current knowledge on the functional roles of NFAT5 in osmotic stress adaptation and the signaling pathways that regulate its activity. PMID:23618372

  19. Integrative genomics identifies DSCR1 (RCAN1) as a novel NFAT-dependent mediator of phenotypic modulation in vascular smooth muscle cells.

    PubMed

    Lee, Monica Y; Garvey, Sean M; Baras, Alex S; Lemmon, Julia A; Gomez, Maria F; Schoppee Bortz, Pamela D; Daum, Guenter; LeBoeuf, Renee C; Wamhoff, Brian R

    2010-02-01

    Vascular smooth muscle cells (SMCs) display remarkable phenotypic plasticity in response to environmental cues. The nuclear factor of activated T-cells (NFAT) family of transcription factors plays a critical role in vascular pathology. However, known functional NFAT gene targets in vascular SMCs are currently limited. Publicly available whole-genome expression array data sets were analyzed to identify differentially expressed genes in human, mouse and rat SMCs. Comparison between vehicle and phenotypic modulatory stimuli identified 63 species-conserved, upregulated genes. Integration of the 63 upregulated genes with an in silico NFAT-ome (a species-conserved list of gene promoters containing at least one NFAT binding site) identified 18 putative NFAT-dependent genes. Further intersection of these 18 potential NFAT target genes with a mouse in vivo vascular injury microarray identified four putative NFAT-dependent, injury-responsive genes. In vitro validations substantiated the NFAT-dependent role of Cyclooxygenase 2 (COX2/PTGS2) in SMC phenotypic modulation and uncovered Down Syndrome Candidate Region 1 (DSCR1/RCAN1) as a novel NFAT target gene in SMCs. We show that induction of DSCR1 inhibits calcineurin/NFAT signaling through a negative feedback mechanism; DSCR1 overexpression attenuates NFAT transcriptional activity and COX2 protein expression, whereas knockdown of endogenous DSCR1 enhances NFAT transcriptional activity. Our integrative genomics approach illustrates how the combination of publicly available gene expression arrays, computational databases and empirical research methods can answer specific questions in any cell type for a transcriptional network of interest. Herein, we report DSCR1 as a novel NFAT-dependent, injury-inducible, early gene that may serve to negatively regulate SMC phenotypic switching.

  20. TRPC6 channel activation promotes neonatal glomerular mesangial cell apoptosis via calcineurin/NFAT and FasL/Fas signaling pathways

    PubMed Central

    Soni, Hitesh; Adebiyi, Adebowale

    2016-01-01

    Glomerular mesangial cell (GMC) proliferation and death are involved in the pathogenesis of glomerular disorders. The mechanisms that control GMC survival are poorly understood, but may include signal transduction pathways that are modulated by changes in intracellular Ca2+ ([Ca2+]i) concentration. In this study, we investigated whether activation of the canonical transient receptor potential (TRPC) 6 channels and successive [Ca2+]i elevation alter neonatal GMC survival. Hyperforin (HF)-induced TRPC6 channel activation increased [Ca2+]i concentration, inhibited proliferation, and triggered apoptotic cell death in primary neonatal pig GMCs. HF-induced neonatal GMC apoptosis was not associated with oxidative stress. However, HF-induced TRPC6 channel activation stimulated nuclear translocation of the nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1). HF also increased cell death surface receptor Fas ligand (FasL) level and caspase-8 activity in the cells; effects mitigated by [Ca2+]i chelator BAPTA, calcineurin/NFAT inhibitor VIVIT, and TRPC6 channel knockdown. Accordingly, HF-induced neonatal GMC apoptosis was attenuated by BAPTA, VIVIT, Fas blocking antibody, and a caspase-3/7 inhibitor. These findings suggest that TRPC6 channel-dependent [Ca2+]i elevation and the ensuing induction of the calcineurin/NFAT, FasL/Fas, and caspase signaling cascades promote neonatal pig GMC apoptosis. PMID:27383564

  1. The adaptor protein 3BP2 associates with VAV guanine nucleotide exchange factors to regulate NFAT activation by the B-cell antigen receptor.

    PubMed

    Foucault, Isabelle; Le Bras, Séverine; Charvet, Céline; Moon, Chéol; Altman, Amnon; Deckert, Marcel

    2005-02-01

    Engagement of the B-cell antigen receptor (BCR) activates kinases of the Src and Syk families and signaling complexes assembled by adaptor proteins, which dictate B-cell fate and function. The adaptor 3BP2/SH3BP2, an Abl Src homology domain 3 (SH3)-binding and Syk-kinases interacting protein, exhibits positive regulatory roles in T, natural killer (NK), and basophilic cells. However, its involvement in BCR signaling is completely unknown. Here we show that 3BP2 is tyrosine phosphorylated following BCR aggregation on B lymphoma cells, and that 3BP2 is a substrate for Syk and Fyn, but not Btk. To further explore the function of 3BP2 in B cells, we screened a yeast 2-hybrid B-lymphocyte library and found 3BP2 as a binding partner of Vav proteins. The interaction between 3BP2 and Vav proteins involved both constitutive and inducible mechanisms. 3BP2 also interacted with other components of the BCR signaling pathway, including Syk and phospholipase C gamma (PLC-gamma). Furthermore, overexpression and RNAi blocking experiments showed that 3BP2 regulated BCR-mediated activation of nuclear factor of activated T cells (NFATs). Finally, evidence was provided that 3BP2 functionally cooperates with Vav proteins and Rho GTPases to activate NFATs. Our results show that 3BP2 may regulate BCR-mediated gene activation through Vav proteins.

  2. Activation requirements and responses to TLR ligands in human CD4+ T cells: comparison of two T cell isolation techniques.

    PubMed

    Lancioni, Christina L; Thomas, Jeremy J; Rojas, Roxana E

    2009-05-15

    Direct regulation of T cell function by microbial ligands through Toll-like receptors (TLR) is an emerging area of T cell biology. Currently either immunomagnetic cell sorting (IMACS) or fluorescence-activated cell sorting (FACS), are utilized to isolate T-cell subsets for such studies. However, it is unknown to what extent differences in T cell purity between these isolation techniques influence T cell functional assays. We compared the purity, response to mitogen, activation requirements, and response to TLR ligands between human CD4(+) T cells isolated either by IMACS (IMACS-CD4(+)) or by IMACS followed by FACS (IMACS/FACS-CD4(+)). As expected, IMACS-CD4(+) were less pure than IMACS/FACS-CD4(+) (92.5%+/-1.4% versus 99.7%+/-0.2%, respectively). Consequently, IMACS-CD4(+) proliferated and produced cytokines in response to mitogen alone and had lower activation requirements compared to IMACS/FACS-CD4(+). In addition IMACS-CD4(+) but not IMACS/FACS-CD4(+) responses were upregulated by the TLR-4 ligand lipopolysaccharide (LPS). On the other hand, TLR-2 and TLR-5 engagement induced costimulation in both IMACS-CD4(+) and highly purified IMACS-/FACS-CD4(+). Altogether these results indicate that small differences in cell purity can significantly alter T cell responses to TLR ligands. This study stresses the importance of a stringent purification method when investigating the role of microbial ligands in T cell function. PMID:19272393

  3. Combination of nifedipine and subtherapeutic dose of cyclosporin additively suppresses mononuclear cells activation of patients with rheumatoid arthritis and normal individuals via Ca2+–calcineurin–nuclear factor of activated T cells pathway

    PubMed Central

    Lai, N-S; Yu, C-L; Yin, W-Y; Yu, H-C; Huang, H-B; Tung, C-H; Lu, M-C

    2012-01-01

    Abnormal Ca2+-mediated signalling contributes to the pathogenesis of rheumatoid arthritis (RA). However, the potential implication of calcium channel blocker in RA remained unknown. We hypothesized that nifedipine, an L-type calcium channel blocker, combined with a calcineurin inhibitor, could suppress T cell activation via targeting different level of the Ca2+ signalling pathway. The percentage of activated T cells and the apoptotic rate of mononuclear cells (MNCs) was measured by flow cytometry. The MNC viability, cytokine production, cytosolic Ca2+ level and activity of the nuclear factor of activated T cells (NFAT) were measured by enzyme-linked immunosorbent assay (ELISA). The NFAT-regulated gene expression, including interleukin (IL)-2, interferon (IFN)-γ and granulocyte–macrophage colony-stimulating factor (GM-CSF), was measured by real-time polymerase chain reaction (PCR). We found that the percentage of activated T cells in anti-CD3 + anti-CD28-activated MNC was higher in RA patients. High doses of nifedipine (50 µM) increased MNCs apoptosis, inhibited T cell activation and decreased T helper type 2 (Th1) (IFN-γ)/Th2 (IL-10) cytokine production in both groups. The Ca2+ influx was lower in anti-CD3 + anti-CD28-activated MNC from RA patients than healthy volunteers and suppressed by nifedipine. When combined with a subtherapeutic dose (50 ng/ml) of cyclosporin, 1 µM nifedipine suppressed the percentage of activated T cells in both groups. Moreover, this combination suppressed more IFN-γ secretion and NFAT-regulated gene (GM-CSF and IFN-γ) expression in RA-MNCs than normal MNCs via decreasing the activity of NFATc1. In conclusion, we found that L-type Ca2+ channel blockers and subtherapeutic doses of cyclosporin act additively to suppress the Ca2+-calcineurin-NFAT signalling pathway, leading to inhibition of T cell activity. We propose that this combination may become a potential treatment of RA. PMID:22385242

  4. Oligomeric Procyanidins Interfere with Glycolysis of Activated T Cells. A Novel Mechanism for Inhibition of T Cell Function.

    PubMed

    Goto, Masao; Wakagi, Manabu; Shoji, Toshihiko; Takano-Ishikawa, Yuko

    2015-01-01

    Procyanidins, which are flavonoids that are found in a variety of plant species, reduce or prevent immune disorders, such as allergy and autoimmune diseases, through an unknown mechanism. In the present study, we investigated the effects of procyanidins on the T cell receptor (TCR)-mediated responses of CD4⁺ T cells in vitro. Apple procyanidins strongly suppressed the proliferation of splenic CD4⁺ T cells that were stimulated by an anti-CD3ε antibody, as well as splenocytes stimulated by antigen, but did not alter interleukin (IL)-2 secretion from these cells. Furthermore, we found that oligomeric procyanidins strongly suppressed, in a degree of polymerization dependent manner, the proliferation of activated CD4⁺ T cells, as well as their production of effector cytokines, including glycolysis associated-cytokines, without affecting IL-2 secretion. Additionally, we investigated the inhibitory effects of oligomeric procyanidins on the glycolytic activity of activated CD4⁺ T cells. We show that pentameric procyanidin suppressed L-lactate production and glucose uptake in activated CD4⁺ T cells. These results suggest that oligomeric procyanidins suppress the functions of activated CD4⁺ T cells by interfering with glycolysis. PMID:26492229

  5. Activated CD8+ T cells induce expansion of Vβ5+ regulatory T cells via TNFR2 signaling

    PubMed Central

    Joedicke, Jara J; Myers, Lara; Carmody, Aaron B; Messer, Ronald J; Wajant, Harald; Lang, Karl S; Lang, Philipp A; Mak, Tak W; Hasenkrug, Kim J; Dittmer, Ulf

    2014-01-01

    Vβ5+ regulatory T cells (Tregs), which are specific for a mouse endogenous retroviral superantigen, become activated and proliferate in response to Friend retrovirus (FV) infection. We previously reported that FV-induced expansion of this Treg subset was dependent on CD8+ T cells and TNFα, but independent of IL-2. We now show that the inflammatory milieu associated with FV infection is not necessary for induction of Vβ5+ Treg expansion. Rather, it is the presence of activated CD8+ T cells that is critical for their expansion. The data indicate that the mechanism involves signaling between the membrane-bound form of TNFα (memTNFα) on activated CD8+ T cells and TNF receptor 2 (TNFR2) on Tregs. CD8+ T cells expressing memTNFα but no soluble TNFα (solTNFα) remained competent to induce strong Vβ5+ Treg expansion in vivo. In addition, Vβ5+ Tregs expressing only TNFR2 but no TNFR1 were still responsive to expansion. Finally, treatment of naïve mice with solTNFα did not induce Vβ5+ Treg expansion, but treatment with a TNFR2-specific agonist did. These results reveal a new mechanism of intercellular communication between activated CD8+ T cell effectors and Tregs that results in the activation and expansion of a Treg subset that subsequently suppresses CD8+ T cell functions. PMID:25098294

  6. The calcineurin-NFAT pathway controls activity-dependent circadian gene expression in slow skeletal muscle

    PubMed Central

    Dyar, Kenneth A.; Ciciliot, Stefano; Tagliazucchi, Guidantonio Malagoli; Pallafacchina, Giorgia; Tothova, Jana; Argentini, Carla; Agatea, Lisa; Abraham, Reimar; Ahdesmäki, Miika; Forcato, Mattia; Bicciato, Silvio; Schiaffino, Stefano; Blaauw, Bert

    2015-01-01

    Objective Physical activity and circadian rhythms are well-established determinants of human health and disease, but the relationship between muscle activity and the circadian regulation of muscle genes is a relatively new area of research. It is unknown whether muscle activity and muscle clock rhythms are coupled together, nor whether activity rhythms can drive circadian gene expression in skeletal muscle. Methods We compared the circadian transcriptomes of two mouse hindlimb muscles with vastly different circadian activity patterns, the continuously active slow soleus and the sporadically active fast tibialis anterior, in the presence or absence of a functional skeletal muscle clock (skeletal muscle-specific Bmal1 KO). In addition, we compared the effect of denervation on muscle circadian gene expression. Results We found that different skeletal muscles exhibit major differences in their circadian transcriptomes, yet core clock gene oscillations were essentially identical in fast and slow muscles. Furthermore, denervation caused relatively minor changes in circadian expression of most core clock genes, yet major differences in expression level, phase and amplitude of many muscle circadian genes. Conclusions We report that activity controls the oscillation of around 15% of skeletal muscle circadian genes independently of the core muscle clock, and we have identified the Ca2+-dependent calcineurin-NFAT pathway as an important mediator of activity-dependent circadian gene expression, showing that circadian locomotor activity rhythms drive circadian rhythms of NFAT nuclear translocation and target gene expression. PMID:26629406

  7. Electrical field stimulation induces cardiac fibroblast proliferation through the calcineurin-NFAT pathway.

    PubMed

    Chen, Qing-Qing; Zhang, Wei; Chen, Xiang-Fan; Bao, Yun-Jian; Wang, Jing; Zhu, Wei-Zhong

    2012-12-01

    Most cardiac diseases are associated with fibrosis. Calcineurin (CaN) is regulated by Ca(2+)/calmodulin (CaM). The CaN-NFAT (nuclear factor of activated T cell) pathway is involved in the process of cardiac diseases, such as cardiac hypertrophy, but its effect on myocardial fibrosis remains unclear. The present study investigates whether the CaN-NFAT pathway is involved in cardiac fibroblast (CF) proliferation induced by electrical field stimulation (EFS), which recently became a popular treatment for heart failure and cardiac tissue engineering. CF proliferation was evaluated by a cell survival assay (MTT) and cell counts. Myocardial fibrosis was assessed by collagen I and collagen III protein expression. Green fluorescent protein (GFP)-tagged NFAT was used to detect NFAT nuclear translocation. CF proliferation, myocardial fibrosis, CaN activity, and NFAT nuclear translocation were enhanced by EFS. More importantly, these effects were abolished by CaN inhibitors, dominant negative CaN (DN-CaN), and CaN gene silenced with siRNA. Furthermore, buffering intracellular Ca(2+) with BAPTA-AM and blocking Ca(2+) influx with nifedipine suppressed EFS-induced increase in intracellular Ca(2+) and CF proliferation. These results suggested that the CaN-NFAT pathway mediates CF proliferation, and that the CaN-NFAT pathway might be a possible therapeutic target for EFS-induced myocardial fibrosis and cardiac tissue engineering.

  8. The C-Type Lectin OCILRP2 Costimulates EL4 T Cell Activation via the DAP12-Raf-MAP Kinase Pathway

    PubMed Central

    Lou, Qiang; Zhang, Wei; Liu, Guangchao; Ma, Yuanfang

    2014-01-01

    OCILRP2 is a typical Type-II transmembrane protein that is selectively expressed in activated T lymphocytes, dendritic cells, and B cells and functions as a novel co-stimulator of T cell activation. However, the signaling pathways underlying OCILRP2 in T cell activation are still not completely understood. In this study, we found that the knockdown of OCILRP2 expression with shRNA or the blockage of its activity by an anti-OCILRP2 antagonist antibody reduced CD3/CD28-costimulated EL4 T cell viability and IL-2 production, inhibit Raf1, MAPK3, and MAPK8 activation, and impair NFAT and NF-κB transcriptional activities. Furthermore, immunoprecipitation results indicated that OCILRP2 could interact with the DAP12 protein, an adaptor containing an intracellular ITAM motif that can transduce signals to induce MAP kinase activation for T cell activation. Our data reveal that after binding with DAP12, OCILRP2 activates the Raf-MAP kinase pathways, resulting in T cell activation. PMID:25411776

  9. Annexin A7 deficiency potentiates cardiac NFAT activity promoting hypertrophic signaling

    SciTech Connect

    Voelkl, Jakob; Alesutan, Ioana; Pakladok, Tatsiana; Viereck, Robert; Feger, Martina; Mia, Sobuj; Schönberger, Tanja; Noegel, Angelika A.; Gawaz, Meinrad; Lang, Florian

    2014-02-28

    Highlights: • Cardiac Anxa7 expression was up-regulated following TAC. • The hypertrophic response following TAC was augmented in Anxa7-deficient mice. • Silencing of Anxa7 increased indicators of HL-1 cardiomyocytes hypertrophy. • Silencing of Anxa7 induced Nfatc1 nuclear translocation. • Silencing of Anxa7 enhanced NFAT-dependent transcriptional activity. - Abstract: Annexin A7 (Anxa7) is a cytoskeletal protein interacting with Ca{sup 2+} signaling which in turn is a crucial factor for cardiac remodeling following cardiac injury. The present study explored whether Anxa7 participates in the regulation of cardiac stress signaling. To this end, mice lacking functional Anxa7 (anxa7{sup −/−}) and wild-type mice (anxa7{sup +/+}) were investigated following pressure overload by transverse aortic constriction (TAC). In addition, HL-1 cardiomyocytes were silenced with Anxa7 siRNA and treated with isoproterenol. Transcript levels were determined by quantitative RT-PCR, transcriptional activity by luciferase reporter assay and protein abundance by Western blotting and confocal microscopy. As a result, TAC treatment increased the mRNA and protein levels of Anxa7 in wild-type mice. Moreover, TAC increased heart weight to body weight ratio and the cardiac mRNA levels of αSka, Nppb, Col1a1, Col3a1 and Rcan1, effects more pronounced in anxa7{sup −/−} mice than in anxa7{sup +/+} mice. Silencing of Anxa7 in HL-1 cardiomyocytes significantly increased nuclear localization of Nfatc1. Furthermore, Anxa7 silencing increased NFAT-dependent transcriptional activity as well as αSka, Nppb, and Rcan1 mRNA levels both, under control conditions and following β-adrenergic stimulation by isoproterenol. These observations point to an important role of annexin A7 in the regulation of cardiac NFAT activity and hypertrophic response following cardiac stress conditions.

  10. Effect of age on proteasomal activity of T cells and macrophages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    T cell function is impaired with aging. Proteasome activity in T cells is important for T cell activation and its activity in macrophages is required for processing antigens in order to be presented via class I major histocompatibility complex to CD8+ T cells. Since studies have demonstrated that pr...

  11. Annexin A7 deficiency potentiates cardiac NFAT activity promoting hypertrophic signaling.

    PubMed

    Voelkl, Jakob; Alesutan, Ioana; Pakladok, Tatsiana; Viereck, Robert; Feger, Martina; Mia, Sobuj; Schönberger, Tanja; Noegel, Angelika A; Gawaz, Meinrad; Lang, Florian

    2014-02-28

    Annexin A7 (Anxa7) is a cytoskeletal protein interacting with Ca(2+) signaling which in turn is a crucial factor for cardiac remodeling following cardiac injury. The present study explored whether Anxa7 participates in the regulation of cardiac stress signaling. To this end, mice lacking functional Anxa7 (anxa7(-/-)) and wild-type mice (anxa7(+/+)) were investigated following pressure overload by transverse aortic constriction (TAC). In addition, HL-1 cardiomyocytes were silenced with Anxa7 siRNA and treated with isoproterenol. Transcript levels were determined by quantitative RT-PCR, transcriptional activity by luciferase reporter assay and protein abundance by Western blotting and confocal microscopy. As a result, TAC treatment increased the mRNA and protein levels of Anxa7 in wild-type mice. Moreover, TAC increased heart weight to body weight ratio and the cardiac mRNA levels of αSka, Nppb, Col1a1, Col3a1 and Rcan1, effects more pronounced in anxa7(-/-) mice than in anxa7(+/+) mice. Silencing of Anxa7 in HL-1 cardiomyocytes significantly increased nuclear localization of Nfatc1. Furthermore, Anxa7 silencing increased NFAT-dependent transcriptional activity as well as αSka, Nppb, and Rcan1 mRNA levels both, under control conditions and following β-adrenergic stimulation by isoproterenol. These observations point to an important role of annexin A7 in the regulation of cardiac NFAT activity and hypertrophic response following cardiac stress conditions. PMID:24508799

  12. Anabolic steroids activate calcineurin-NFAT signaling and thereby increase myotube size and reduce denervation atrophy.

    PubMed

    Qin, Weiping; Pan, Jiangping; Wu, Yong; Bauman, William A; Cardozo, Christopher

    2015-01-01

    Anabolic androgens have been shown to reduce muscle loss due to immobilization, paralysis and many other medical conditions, but the molecular basis for these actions is poorly understood. We have recently demonstrated that nandrolone, a synthetic androgen, slows muscle atrophy after nerve transection associated with down-regulation of regulator of calcineurin 2 (RCAN2), a calcineurin inhibitor, suggesting a possible role of calcineurin-NFAT signaling. To test this possibility, rat gastrocnemius muscle was analyzed at 56 days after denervation. In denervated muscle, calcineurin activity declined and NFATc4 was excluded from the nucleus and these effects were reversed by nandrolone. Similarly, nandrolone increased calcineurin activity and nuclear NFATc4 levels in cultured L6 myotubes. Nandrolone also induced cell hypertrophy that was blocked by cyclosporin A or overexpression of RCAN2. Finally protection against denervation atrophy by nandrolone in rats was blocked by cyclosporin A. These results demonstrate for the first time that nandrolone activates calcineurin-NFAT signaling, and that such signaling is important in nandrolone-induced cell hypertrophy and protection against paralysis-induced muscle atrophy.

  13. Parathyroid hormone induction of cyclooxygenase-2 in murine osteoblasts: role of the calcium-calcineurin-NFAT pathway.

    PubMed

    Huang, Hechang; Chikazu, Daichi; Voznesensky, Olga S; Herschman, Harvey R; Kream, Barbara E; Drissi, Hicham; Pilbeam, Carol C

    2010-04-01

    Murine MC3T3-E1 and MC-4 cells were stably transfected with -371/+70 bp of the murine cyclooxygenase-2 (COX-2) promoter fused to a luciferase reporter (Pluc371) or with Pluc371 carrying site-directed mutations. Mutations were made in (1) the cAMP response element (CRE) at -57/-52 bp, (2) the activating protein-1 (AP-1)-binding site at -69/-63 bp, (3) the nuclear factor of activated T-cells (NFAT)-binding site at -77/-73 bp, and (4) both the AP-1 and NFAT sites, which comprise a composite consensus sequence for NFAT/AP-1. Single mutation of CRE, AP-1, or NFAT sites decreased parathyroid hormone (PTH)-stimulated COX-2 promoter activity 40% to 60%, whereas joint mutation of NFAT and AP-1 abrogated the induction. On electrophoretic mobility shift analysis, PTH stimulated binding of phosphorylated CREB to an oligonucleotide spanning the CRE and binding of NFATc1, c-Fos, and c-Jun to an oligonucleotide spanning the NFAT/AP-1 composite site. Mutation of the NFAT site was less effective than mutation of the AP-1 site in competing binding to the composite element, suggesting that cooperative interactions of NFATc1 and AP-1 are more dependent on NFAT than on AP-1. Both PTH and forskolin, an activator of adenylyl cyclase, stimulated NFATc1 nuclear translocation. PTH- and forskolin-stimulated COX-2 promoter activity was inhibited 56% to 80% by calcium chelation or calcineurin inhibitors and 60% to 98% by protein kinase A (PKA) inhibitors. These results indicate an important role for the calcium-calcineurin-NFAT signaling pathway in the PTH induction of COX-2 and suggest that cross-talk between the cAMP/PKA pathway and the calcium-calcineurin-NFAT pathway may play a role in other functions of PTH in osteoblasts.

  14. Glycogen synthase kinase 3{beta} regulation of nuclear factor of activated T-cells isoform c1 in the vascular smooth muscle cell response to injury

    SciTech Connect

    Chow Winsion; Hou Guangpei; Bendeck, Michelle P.

    2008-10-01

    The migration and proliferation of vascular smooth muscle cells (vSMCs) are critical events in neointima formation during atherosclerosis and restenosis. The transcription factor nuclear factor of activated T-cells-isoform c1 (NFATc1) is regulated by atherogenic cytokines, and has been implicated in the migratory and proliferative responses of vSMCs through the regulation of gene expression. In T-cells, calcineurin de-phosphorylates NFATc1, leading to its nuclear import, while glycogen synthase kinase 3 {beta} (GSK3{beta}) phosphorylates NFATc1 and promotes its nuclear export. However, the relationship between NFATc1 and GSK3{beta} has not been studied during SMC migration and proliferation. We investigated this by scrape wounding vSMCs in vitro, and studying wound repair. NFATc1 protein was transiently increased, reaching a peak at 8 h after wounding. Cell fractionation and immunocytochemistry revealed that NFATc1 accumulation in the nucleus was maximal at 4 h after injury, and this was coincident with a significant 9 fold increase in transcriptional activity. Silencing NFATc1 expression with siRNA or inhibition of NFAT with cyclosporin A (CsA) attenuated wound closure by vSMCs. Phospho-GSK3{beta} (inactive) increased to a peak at 30 min after injury, preceding the nuclear accumulation of NFATc1. Overexpression of a constitutively active mutant of GSK3{beta} delayed the nuclear accumulation of NFATc1, caused a 50% decrease in NFAT transcriptional activity, and attenuated vSMC wound repair. We conclude that NFATc1 promotes the vSMC response to injury, and that inhibition of GSK3{beta} is required for the activation of NFAT during wound repair.

  15. A novel T cell receptor single-chain signaling complex mediates antigen-specific T cell activity and tumor control

    PubMed Central

    Stone, Jennifer D.; Harris, Daniel T.; Soto, Carolina M.; Chervin, Adam S.; Aggen, David H.; Roy, Edward J.; Kranz, David M.

    2014-01-01

    Adoptive transfer of genetically modified T cells to treat cancer has shown promise in several clinical trials. Two main strategies have been applied to redirect T cells against cancer: 1) introduction of a full-length T cell receptor (TCR) specific for a tumor-associated peptide-MHC, or 2) introduction of a chimeric antigen receptor (CAR), including an antibody fragment specific for a tumor cell surface antigen, linked intracellularly to T cell signaling domains. Each strategy has advantages and disadvantages for clinical applications. Here, we present data on the in vitro and in vivo effectiveness of a single-chain signaling receptor incorporating a TCR variable fragment as the targeting element (referred to as TCR-SCS). This receptor contained a single-chain TCR (Vβ-linker-Vα) from a high-affinity TCR called m33, linked to the intracellular signaling domains of CD28 and CD3ζ. This format avoided mispairing with endogenous TCR chains, and mediated specific T cell activity when expressed in either CD4 or CD8 T cells. TCR-SCS-transduced CD8-negative cells showed an intriguing sensitivity, compared to full-length TCRs, to higher densities of less stable pepMHC targets. T cells that expressed this peptide-specific receptor persisted in vivo, and exhibited polyfunctional responses. Growth of metastatic antigen-positive tumors was significantly inhibited by T cells that expressed this receptor, and tumor cells that escaped were antigen loss variants. TCR-SCS receptors represent an alternative targeting receptor strategy that combines the advantages of single-chain expression, avoidance of TCR chain mispairing, and targeting of intracellular antigens presented in complex with MHC proteins. PMID:25082071

  16. O-GlcNAc signaling is essential for NFAT-mediated transcriptional reprogramming during cardiomyocyte hypertrophy.

    PubMed

    Facundo, Heberty T; Brainard, Robert E; Watson, Lewis J; Ngoh, Gladys A; Hamid, Tariq; Prabhu, Sumanth D; Jones, Steven P

    2012-05-15

    The regulation of cardiomyocyte hypertrophy is a complex interplay among many known and unknown processes. One specific pathway involves the phosphatase calcineurin, which regulates nuclear translocation of the essential cardiac hypertrophy transcription factor, nuclear factor of activated T-cells (NFAT). Although metabolic dysregulation is frequently described during cardiac hypertrophy, limited insights exist regarding various accessory pathways. One metabolically derived signal, beta-O-linked N-acetylglucosamine (O-GlcNAc), has emerged as a highly dynamic posttranslational modification of serine and threonine residues regulating physiological and stress processes. Given the metabolic dysregulation during hypertrophy, we hypothesized that NFAT activation is dependent on O-GlcNAc signaling. Pressure overload-induced hypertrophy (via transverse aortic constriction) in mice or treatment of neonatal rat cardiac myocytes with phenylephrine significantly enhanced global O-GlcNAc signaling. NFAT-luciferase reporter activity revealed O-GlcNAc-dependent NFAT activation during hypertrophy. Reversal of enhanced O-GlcNAc signaling blunted cardiomyocyte NFAT-induced changes during hypertrophy. Taken together, these results demonstrate a critical role of O-GlcNAc signaling in NFAT activation during hypertrophy and provide evidence that O-GlcNAc signaling is coordinated with the onset and progression of cardiac hypertrophy. This represents a potentially significant and novel mechanism of cardiac hypertrophy, which may be of particular interest in future in vivo studies of hypertrophy.

  17. In situ activation of helper T cells in the lung.

    PubMed

    Raju, B; Tung, C F; Cheng, D; Yousefzadeh, N; Condos, R; Rom, W N; Tse, D B

    2001-08-01

    To better understand the lung and systemic responses of helper T cells mediating memory immunity to Mycobacterium tuberculosis, we used three- and four-color flow cytometry to study the surface phenotype of CD4(+) lymphocytes. Bronchoalveolar lavage (BAL) fluid and peripheral blood (PB) samples were obtained from a total of 25 subjects, including 10 tuberculosis (TB)-infected subjects, 8 purified-protein-derivative-negative subjects, and 7 purified-protein-derivative-positive subjects. In marked contrast to CD4(+) lymphocytes from PB (9% +/- 5% expressing CD45RA and CD29), the majority (55% +/- 16%) of CD4(+) lymphocytes in BAL (ALs) simultaneously expressed CD45RA, a naïve T-cell marker, and CD29, members of the very late activation family. Further evaluation revealed that CD4(+) ALs expressed both CD45RA and CD45RO, a memory T-cell marker. In addition, the proportion of CD4(+) lymphocytes expressing CD69, an early activation marker, was drastically increased in BAL fluid (83% +/- 9%) compared to PB (1% +/- 1%), whereas no significant difference was seen in the expression of CD25, the low-affinity interleukin 2 receptor (34% +/- 15% versus 40% +/- 16%). More importantly, we identified a minor population of CD69(bright) CD25(bright) CD4(+) lymphocytes in BAL (10% +/- 6%) that were consistently absent from PB (1% +/- 1%). Thus, CD4(+) lymphocytes in the lung paradoxically coexpress surface molecules characteristic of naïve and memory helper T cells as well as surface molecules commonly associated with early and late stages of activation. No difference was observed for ALs obtained from TB-infected and uninfected lung segments in this regard. It remains to be determined if these surface molecules are induced by the alveolar environment or if CD4(+) lymphocytes coexpressing this unusual combination of surface molecules are selectively recruited from the circulation. Our data suggest that ex vivo experiments on helper T-cell subsets that display distinctive

  18. T-cell activation and early gene response in dogs.

    PubMed

    Mortlock, Sally-Anne; Wei, Jerry; Williamson, Peter

    2015-01-01

    T-cells play a crucial role in canine immunoregulation and defence against invading pathogens. Proliferation is fundamental to T-cell differentiation, homeostasis and immune response. Initiation of proliferation following receptor mediated stimuli requires a temporally programmed gene response that can be identified as immediate-early, mid- and late phases. The immediate-early response genes in T-cell activation engage the cell cycle machinery and promote subsequent gene activation events. Genes involved in this immediate-early response in dogs are yet to be identified. The present study was undertaken to characterise the early T-cell gene response in dogs to improve understanding of the genetic mechanisms regulating immune function. Gene expression profiles were characterised using canine gene expression microarrays and quantitative reverse transcription PCR (qRT-PCR), and paired samples from eleven dogs. Significant functional annotation clusters were identified following stimulation with phytohemagluttinin (PHA) (5μg/ml), including the Toll-like receptor signaling pathway and phosphorylation pathways. Using strict statistical criteria, 13 individual genes were found to be differentially expressed, nine of which have ontologies that relate to proliferation and cell cycle control. These included, prostaglandin-endoperoxide synthase 2 (PTGS2/COX2), early growth response 1 (EGR1), growth arrest and DNA damage-inducible gene (GADD45B), phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), V-FOS FBJ murine osteosarcoma viral oncogene homolog (FOS), early growth response 2 (EGR2), hemogen (HEMGN), polo-like kinase 2 (PLK2) and polo-like kinase 3 (PLK3). Differential gene expression was re-examined using qRT-PCR, which confirmed that EGR1, EGR2, PMAIP1, PTGS2, FOS and GADD45B were significantly upregulated in stimulated cells and ALAS2 downregulated. PTGS2 and EGR1 showed the highest levels of response in these dogs. Both of these genes are involved in cell cycle

  19. Generation of a conditional knockout allele for the NFAT5 gene in mice.

    PubMed

    Küper, Christoph; Beck, Franz-Xaver; Neuhofer, Wolfgang

    2014-01-01

    The osmosensitive transcription factor nuclear factor of activated T-cells 5 (NFAT5), also known as tonicity enhancer element binding protein (TonEBP) plays a crucial role in protection of renal medullary cells against hyperosmotic stress, urinary concentration, the adaptive immune response, and other physiological systems. Since it is also important for development, conventional homozygous-null mutations result in perinatal death, which hinders the analysis of NFAT5 function in specific tissues in vivo. Here we describe the generation of mice with a conditional-null allele, in which loxP sites are inserted around exon 4. Mice harboring the floxed allele (NFAT5(flx) ) were mated to a strain expressing a tamoxifen-inducible derivative of the Cre-recombinase (Cre (+)) under the control of the ubiqitinC promoter. The resultant homozygous conditional knockout mice (Cre (+) NFAT5 (flx/flx) ) are viable, fertile, and show normal expression of NFAT5 and NFAT5 target genes, indicating that the conditional alleles retain their wild-type function. Induction of Cre-mediated recombination by administration of tamoxifen in 8-week-old mice resulted in a decrease in NFAT5 expression of about 70-90% in all tested tissues (renal cortex, renal outer medulla, renal inner medulla, heart, lung, spleen, skeletal muscle). Accordingly, the expression of the NFAT5 target genes aldose reductase and heat shock protein 70 in the renal medulla was also significantly decreased. Mice harboring this conditional knockout allele should be useful in future studies for gaining a better understanding of tissue and cell-type specific functions of NFAT5 in adult animals under physiological and pathophysiological conditions. PMID:25601839

  20. NFAT as cancer target: Mission possible?

    PubMed Central

    Qin, Jiang-Jiang; Nag, Subhasree; Wang, Wei; Zhou, Jianwei; Zhang, Wei-Dong; Wang, Hui; Zhang, Ruiwen

    2014-01-01

    The NFAT signaling pathway regulates various aspects of cellular functions; NFAT acts as a calcium sensor, integrating calcium signaling with other pathways involved in development and growth, immune response, and inflammatory response. The NFAT family of transcription factors regulates diverse cellular functions such as cell survival, proliferation, migration, invasion, and angiogenesis. The NFAT isoforms are constitutively activated and overexpressed in several cancer types wherein they transactivate downstream targets that play important roles in cancer development and progression. Though the NFAT family has been conclusively proved to be pivotal in cancer progression, the different isoforms play distinct roles in different cellular contexts. In this review, our discussion is focused on the mechanisms that drive activation of the various NFAT isoforms in cancer. Additionally, we analyze the potential of NFAT as a valid target for cancer prevention and therapy. PMID:25072963

  1. NFAT as cancer target: mission possible?

    PubMed

    Qin, Jiang-Jiang; Nag, Subhasree; Wang, Wei; Zhou, Jianwei; Zhang, Wei-Dong; Wang, Hui; Zhang, Ruiwen

    2014-12-01

    The NFAT signaling pathway regulates various aspects of cellular functions; NFAT acts as a calcium sensor, integrating calcium signaling with other pathways involved in development and growth, immune response, and inflammatory response. The NFAT family of transcription factors regulates diverse cellular functions such as cell survival, proliferation, migration, invasion, and angiogenesis. The NFAT isoforms are constitutively activated and overexpressed in several cancer types wherein they transactivate downstream targets that play important roles in cancer development and progression. Though the NFAT family has been conclusively proved to be pivotal in cancer progression, the different isoforms play distinct roles in different cellular contexts. In this review, our discussion is focused on the mechanisms that drive the activation of various NFAT isoforms in cancer. Additionally, we analyze the potential of NFAT as a valid target for cancer prevention and therapy.

  2. 5-HT3 receptor-channels coupled with Na+ influx in human T cells: role in T cell activation.

    PubMed

    Khan, N A; Poisson, J P

    1999-09-01

    The study was conducted on a human (Jurkat) T cell line, loaded with a Na+ fluorescent probe, SBFI/AM. Serotonin and an agonist of 5-HT3 receptor-channels, 2-methyl-5HT, evoked Na+ influx, whereas the agonists of other serotonergic receptor subtypes, i.e., 5-HT1A and 5-HT1B receptors, failed to induce Na+ influx in these cells. By using 3H-BRL43694, an agonist of 5-HT3 receptor-channels, we characterized 5-HT3 lymphocyte receptors which exhibited a density (Bmax) of 300 +/- 20 fmol/10(6) cells and a Kd of 30 nM in Jurkat T cells. The T-cell 5-HT3 receptor-channel is not regulated either by the protein kinase C or by the free intracellular calcium concentrations as the agents known to activate the PKC and to induce increases in intracellular free calcium concentrations failed to influence the free intracellular Na+ concentrations, [Na+]i, in these cells. Furthermore, an increase in [Na+]i, induced by 2-methyl-5HT, via 5-HT3 receptor-channels seems to stimulate T-cell activation by facilitating the progression of T cells from S to G2/M phase of the cell cycle.

  3. Regulation of HIV-1 Latency by T-cell Activation

    PubMed Central

    Williams, Samuel A.; Greene, Warner C.

    2007-01-01

    HIV infected patients harbor ~105–106 memory CD4 T-cells that contain fully integrated but transcriptionally silent HIV proviruses. While small in number, these latently infected cells form a drug-insensitive reservoir that importantly contributes to the life-long persistence of HIV despite highly effective antiviral therapy. In tissue culture, latent HIV proviruses can be activated when their cellular hosts are exposed to select proinflammatory cytokines or their T-cell receptors are ligated. However, due to a lack of potency and/or dose-limiting toxicity, attempts to purge virus from this latent reservoir in vivo with immune-activating agents including anti-CD3 antibodies and IL-2 have failed. A deeper understanding of the molecular underpinnings of HIV latency is clearly required, including determining whether viral latency is actively reinforced by transcriptional repressors, defining which inducible host transcription factors most effectively antagonize latency, and elucidating the role of chromatin in viral latency. Only through such an improved understanding will it be possible to identify combination therapies that might allow complete purging of the latent reservoir and realization of the difficult and elusive goal of complete eradication of HIV in infected patients. PMID:17643313

  4. Leishmania infantum-chagasi activates SHP-1 and reduces NFAT5/TonEBP activity in the mouse kidney inner medulla.

    PubMed

    Zhou, Xiaoming; Wang, Hong; Koles, Nancy L; Zhang, Aihong; Aronson, Naomi E

    2014-09-01

    Visceral leishmaniasis patients have been reported to have a urine concentration defect. Concentration of urine by the renal inner medulla is essentially dependent on a transcription factor, NFAT5/TonEBP, because it activates expression of osmoprotective genes betaine/glycine transporter 1 (BGT1) and sodium/myo-inositol transporter (SMIT), and water channel aquaporin-2, all of which are imperative for concentrating urine. Leishmania parasites evade macrophage immune defenses by activating protein tyrosine phosphatases, among which SHP-1 is critical. We previously demonstrated that SHP-1 inhibits tonicity-dependent activation of NFAT5/TonEBP in HEK293 cells through screening a genome-wide small interfering (si) RNA library against phosphatases (Zhou X, Gallazzini M, Burg MB, Ferraris JD. Proc Natl Acad Sci USA 107: 7072-7077, 2010). We sought to examine whether Leishmania can activate SHP-1 and inhibit NFAT5/TonEBP activity in the renal inner medulla in a murine model of visceral leishmaniasis by injection of female BALB/c mice with a single intravenous dose of 5 × 10(5) L. chagasi metacyclic promastigotes. We found that SHP-1 is expressed in the kidney inner medulla. L. chagasi activates SHP-1 with an increase in stimulatory phosphorylation of SHP-1-Y536 in the region. L. chagasi reduces expression of NFAT5/TonEBP mRNA and protein as well as expression of its targeted genes: BGT1, SMIT, and aquaporin-2. The culture supernatant from L. chagasi metacyclic promastigotes increases SHP-1 protein abundance and potently inhibits NFAT5 transcriptional activity in mIMCD3 cells. However, L. chagasi in our animal model has no significant effect on urinary concentration. We conclude that L. chagasi, most likely through its secreted virulence factors, activates SHP-1 and reduces NFAT5/TonEBP gene expression, which leads to reduced NFAT5/TonEBP transcriptional activity in the kidney inner medulla. PMID:24990897

  5. Cutting edge: tubulin α functions as an adaptor in NFAT-importin β interaction.

    PubMed

    Ishiguro, Kazuhiro; Ando, Takafumi; Maeda, Osamu; Watanabe, Osamu; Goto, Hidemi

    2011-03-01

    Upon T cell stimulation, NFAT is dephosphorylated by calcineurin, leading to nuclear translocation via NFAT-importin β interaction. Whereas the process of NFAT dephosphorylation has been well researched, the molecular mechanism of NFAT-importin β interaction remains unknown. In contrast to NF-κB and STAT, no importin α family members have been reported as adaptor proteins for NFAT. Our study shows that tubulin α, but not tubulin β, binds to the N-terminal region of NFAT containing the regulatory and Rel homology domains. Importin β interacts with the NFAT-tubulin α complex rather than NFAT or tubulin α alone, resulting in cotranslocation of NFAT and tubulin α into the nucleus. Furthermore, the interaction is suppressed by acetate-induced tubulin α acetylation at lysine 40. In conclusion, tubulin α functions as an adaptor in NFAT-importin β interaction, and this function is regulated by acetate-induced acetylation.

  6. Calcineurin/NFAT signaling and innate host defence: a role for NOD1-mediated phagocytic functions

    PubMed Central

    2014-01-01

    The calcineurin/nuclear factor of activated T cells (NFATs) signaling pathway plays a central role in T cell mediated adaptive immune responses, but a number of recent studies demonstrated that calcineurin/NFAT signaling also plays a key role in the control of the innate immune response by myeloid cells. Calcineurin inhibitors, such as cyclosporine A (CsA) and tacrolimus (FK506), are commonly used in organ transplantation to prevent graft rejection and in a variety of immune diseases. These immunosuppressive drugs have adverse effects and significantly increase host’s susceptibility towards bacterial or fungal infections. Recent studies highlighted the role of NFAT signaling in fungal infection and in the control of the pattern recognition receptor nucleotide-binding oligomerization domain-containing protein 1 (NOD1), which predominantly senses invasive Gram-negative bacteria and mediates neutrophil phagocytic functions. This review summarises some of the current knowledge concerning the role of NFAT signaling in the innate immune response and the recent advances on NFAT-dependent inhibition of NOD1-mediated innate immune response caused by CsA, which may contribute to sensitizing transplant recipients to bacterial infection. PMID:24479879

  7. NFAT5 promotes proliferation and migration of lung adenocarcinoma cells in part through regulating AQP5 expression.

    PubMed

    Guo, Kai; Jin, Faguang

    2015-09-25

    The osmoregulated transcription factor nuclear factor of activated T-cells 5(NFAT5), has been found to play important roles in the development of many kinds of human cancers, including breast cancer, colon carcinoma, renal cell carcinoma and melanoma. The aim of the present study was to determine whether NFAT5 is involved in the proliferation and migration of lung adenocarcinoma cells. We found that NFAT5 was upregulated in lung adenocarcinoma cells and knockdown of NFAT5 decreased proliferation and migration of the cells, accompanied by a significant reduction in the expression of AQP5. AQP5 was upregulated in lung adenocarcinoma cells and knockdown of AQP5 also inhibited proliferation and migration of the cells as knockdown of NFAT5 did. Moreover, overexpression of NFAT5 promoted proliferation and migration of lung adenocarcinoma cells, accompanied by a significant increase in the expression of AQP5. These results indicate that NFAT5 plays important roles in proliferation and migration of human lung adenocarcinoma cells through regulating AQP5 expression, providing a new therapeutic option for lung adenocarcinoma therapy.

  8. Phosphatidylinositol-3-kinase regulates PKCtheta activity in cytotoxic T cells.

    PubMed

    Puente, Lawrence G; Mireau, Laura R; Lysechko, Tara L; Ostergaard, Hanne L

    2005-06-01

    Protein kinase C (PKC) theta plays a crucial role in T cell activation. We, therefore, examined the regulation of PKCtheta activity in cytotoxic T lymphocytes (CTL). We demonstrated that PMA did not stimulate PKCtheta activation and phospholipase C inhibition did not block anti-CD3-stimulated PKCtheta activation in a CTL clone. This suggests that diacylglycerol is neither sufficient nor required for PKCtheta activation. Furthermore, PKCtheta was only activated in a CTL clone stimulated with plate-bound anti-CD3 but not soluble anti-CD3. However, PMA or cross-linked anti-CD3 stimulated phosphorylation of PKCtheta as measured by a migratory shift, suggesting that phosphorylation was not sufficient for activity. Phosphatidylinositol 3-kinase activity was required for anti-CD3, but not PMA, stimulated phosphorylation and for immobilized anti-CD3-triggered PKCtheta activity. A substantial fraction of PKCtheta was constitutively membrane associated and PMA or CD3 stimulation did not significantly increase membrane association. Our data indicate that phosphorylation of PKCtheta is not a suitable surrogate measurement for PKCtheta activity and that additional, yet to be defined steps, are required for the regulation of PKCtheta enzymatic activity in CTL.

  9. The potential role of NFAT5 and osmolarity in peritoneal injury.

    PubMed

    Seeger, Harald; Kitterer, Daniel; Latus, Joerg; Alscher, Mark Dominik; Braun, Niko; Segerer, Stephan

    2015-01-01

    A rise in osmotic concentration (osmolarity) activates the transcription factor Nuclear Factor of Activated T Cells 5 (NFAT5, also known as Tonicity-responsive Enhancer Binding Protein, TonEBP). This is part of a regulatory mechanism of cells adjusting to environments of high osmolarity. Under physiological conditions these are particularly important in the kidney. Activation of NFAT5 results in the modulation of various genes including some which promote inflammation. The osmolarity increases in patients with renal failure. Additionally, in peritoneal dialysis the cells of the peritoneal cavity are repeatedly exposed to a rise and fall in osmotic concentrations. Here we review the current information about NFAT5 activation in uremic patients and patients on peritoneal dialysis. We suggest that high osmolarity promotes injury in the "uremic" milieu, which results in inflammation locally in the peritoneal membrane, but most likely also in the systemic circulation. PMID:26495302

  10. CD1 and mycobacterial lipids activate human T cells

    PubMed Central

    Van Rhijn, Ildiko; Moody, D. Branch

    2014-01-01

    Summary For decades, proteins were thought to be the sole or at least the dominant source of antigens for T cells. Studies in the 1990s demonstrated that CD1 proteins and mycobacterial lipids form specific targets of human αβ T cells. The molecular basis by which T-cell receptors (TCRs) recognize CD1-lipid complexes is now well understood. Many types of mycobacterial lipids function as antigens in the CD1 system, and new studies done with CD1 tetramers identify T-cell populations in the blood of tuberculosis patients. In human populations, a fundamental difference between the CD1 and major histocompatibility complex systems is that all humans express nearly identical CD1 proteins. Correspondingly, human CD1 responsive T cells show evidence of conserved TCRs. In addition to natural killer T cells and mucosal-associated invariant T (MAIT cells), conserved TCRs define other subsets of human T cells, including germline-encoded mycolyl-reactive (GEM) T cells. The simple immunogenetics of the CD1 system and new investigative tools to measure T-cell responses in humans now creates a situation in which known lipid antigens can be developed as immunodiagnostic and immunotherapeutic reagents for tuberculosis disease. PMID:25703557

  11. Placental TonEBP/NFAT5 osmolyte regulation in an ovine model of intrauterine growth restriction.

    PubMed

    Arroyo, Juan A; Garcia-Jones, Pastora; Graham, Amanda; Teng, Cecilia C; Battaglia, Frederick C; Galan, Henry L

    2012-03-01

    TonEBP/NFAT5 (the tonicity-responsive enhancer binding protein/nuclear factor of activated T cells) modulates cellular response to osmotic changes by accumulating inositol and sorbitol inside the cells. Our objective was to assess placental osmolytes, TonEBP/NFAT5 RNA and protein expression, and signaling molecules across gestation between control and intrauterine growth restriction (IUGR) ovine pregnancies. Pregnant sheep were placed in hyperthermic conditions to induce IUGR. Placental tissues were collected at 55, 95, and 130 days gestational age (dGA) to measure inositol, sorbitol, TonEBP/NFAT5 (NFAT5), sodium-dependent myo-inositol transporter (SMIT; official symbol SLC5A3), aldose reductase (AR), and NADPH (official symbol DE-CR1). Placental weight was reduced in IUGR compared to controls at 95 and 130 dGA. Osmolyte concentrations were similar between control and IUGR placentas, but both groups demonstrated a significant decrease in inositol concentration and an increase in sorbitol concentration with advancing gestation. Cytosolic NFAT5 protein decreased significantly from 55 to 95 dGA in both groups, and nuclear NFAT5 protein increased only at 130 dGA in the IUGR group, but no differences were seen between groups for either cytosolic or nuclear NFAT5 protein concentrations. DE-CR1 concentrations were similar between groups and increased significantly with advancing gestational age. AR was lowest at 55dGA, and SLC5A3 increased with advancing gestational age. We conclude that both placental osmolytes inositol and sorbitol (and their corresponding proteins SLC5A3 and AR) change with gestational age and are regulated, at least in part, by NFAT5 and DE-CR1 (NADPH). The inverse relationship between each osmolyte across gestation (e.g., inositol higher in early gestation and sorbitol higher in late gestation) may reflect nutritional needs that change across gestation. PMID:22190709

  12. NFAT5-mediated expression of S100A4 contributes to proliferation and migration of renal carcinoma cells.

    PubMed

    Küper, Christoph; Beck, Franz-Xaver; Neuhofer, Wolfgang

    2014-01-01

    The osmosensitive transcription factor nuclear factor of activated T-cells (NFAT) 5, also known as tonicity enhancer binding protein (TonEBP), has been associated with the development of a variety of tumor entities, among them breast cancer, colon carcinoma, and melanoma. The aim of the present study was to determine whether NFAT5 is also involved in the development of renal cell carcinoma (RCC). The most common type of RCC, the clear cell RCC, originates from the proximal convoluted tubule. We tested our hypothesis in the clear cell RCC cell line CaKi-1 and the non-cancerous proximal tubule cell line HK-2, as control. Basal expression of NFAT5 and NFAT5 activity in CaKi-1 cells was several times higher than in HK-2 cells. Osmotic stress induced an increased NFAT5 activity in both CaKi-1 and HK-2 cells, again with significantly higher activities in CaKi-1 cells. Analysis of NFAT5-regulating signaling pathways in CaKi-1 cells revealed that inhibition of the MAP kinases p38, c-Jun-terminal kinase (JNK) and extracellular regulated kinase (ERK) and of the focal adhesion kinase (FAK) partially blunted NFAT5 activity. FAK and ERK were both constitutively active, even under isotonic conditions, which may contribute to the high basal expression and activity of NFAT5 in CaKi-1 cells. In contrast, the MAP kinases p38 and JNK were inactive under isotonic conditions and became activated under osmotic stress conditions, indicating that p38 and JNK mediate upregulation of NFAT5 activity under these conditions. siRNA-mediated knockdown of NFAT5 in CaKi-1 cells reduced the expression of S100A4, a member of the S100 family of proteins, which promotes metastasis. Knockdown of NFAT5 was accompanied by a significant decrease in proliferation and migration activity. Taken together, our results indicate that NFAT5 induces S100A4 expression in CaKi-1 cells, thereby playing an important role in RCC proliferation and migration. PMID:25152734

  13. CD8+CD122+CD49dlow regulatory T cells maintain T-cell homeostasis by killing activated T cells via Fas/FasL-mediated cytotoxicity.

    PubMed

    Akane, Kazuyuki; Kojima, Seiji; Mak, Tak W; Shiku, Hiroshi; Suzuki, Haruhiko

    2016-03-01

    The Fas/FasL (CD95/CD178) system is required for immune regulation; however, it is unclear in which cells, when, and where Fas/FasL molecules act in the immune system. We found that CD8(+)CD122(+) cells, which are mostly composed of memory T cells in comparison with naïve cells in the CD8(+)CD122(-) population, were previously shown to include cells with regulatory activity and could be separated into CD49d(low) cells and CD49d(high) cells. We established in vitro and in vivo experimental systems to evaluate the regulatory activity of CD122(+) cells. Regulatory activity was observed in CD8(+)CD122(+)CD49d(low) but not in CD8(+)CD122(+)CD49d(high) cells, indicating that the regulatory cells in the CD8(+)CD122(+) population could be narrowed down to CD49d(low) cells. CD8(+)CD122(-) cells taken from lymphoproliferation (lpr) mice were resistant to regulation by normal CD122(+) Tregs. CD122(+) Tregs taken from generalized lymphoproliferative disease (gld) mice did not regulate wild-type CD8(+)CD122(-) cells, indicating that the regulation by CD122(+) Tregs is Fas/FasL-dependent. CD122(+) Tregs taken from IL-10-deficient mice could regulate CD8(+)CD122(-) cells as equally as wild-type CD122(+) Tregs both in vitro and in vivo. MHC class I-missing T cells were not regulated by CD122(+) Tregs in vitro. CD122(+) Tregs also regulated CD4(+) cells in a Fas/FasL-dependent manner in vitro. These results suggest an essential role of Fas/FasL as a terminal effector of the CD122(+) Tregs that kill activated T cells to maintain immune homeostasis. PMID:26869716

  14. Calcineurin/nuclear factor of activated T cells-coupled vanilliod transient receptor potential channel 4 ca2+ sparklets stimulate airway smooth muscle cell proliferation.

    PubMed

    Zhao, Limin; Sullivan, Michelle N; Chase, Marlee; Gonzales, Albert L; Earley, Scott

    2014-06-01

    Proliferation of airway smooth muscle cells (ASMCs) contributes to the remodeling and irreversible obstruction of airways during severe asthma, but the mechanisms underlying this disease process are poorly understood. Here we tested the hypothesis that Ca(2+) influx through the vanilliod transient receptor potential channel (TRPV) 4 stimulates ASMC proliferation. We found that synthetic and endogenous TRPV4 agonists increase proliferation of primary ASMCs. Furthermore, we demonstrate that Ca(2+) influx through individual TRPV4 channels produces Ca(2+) microdomains in ASMCs, called "TRPV4 Ca(2+) sparklets." We also show that TRPV4 channels colocalize with the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin in ASMCs. Activated calcineurin dephosphorylates nuclear factor of activated T cells (NFAT) transcription factors cytosolic (c) to allow nuclear translocation and activation of synthetic transcriptional pathways. We show that ASMC proliferation in response to TRPV4 activity is associated with calcineurin-dependent nuclear translocation of the NFATc3 isoform tagged with green florescent protein. Our findings suggest that Ca(2+) microdomains created by TRPV4 Ca(2+) sparklets activate calcineurin to stimulate nuclear translocation of NFAT and ASMC proliferation. These findings further suggest that inhibition of TRPV4 could diminish asthma-induced airway remodeling.

  15. Peroxisome Proliferator-Activated Receptor γ Deficiency in T Cells Accelerates Chronic Rejection by Influencing the Differentiation of CD4+ T Cells and Alternatively Activated Macrophages

    PubMed Central

    Ye, Ping; Cheng, Chao; Wu, Jie; Wang, Sihua; Sun, Yuan; Liu, Zheng; Xie, Aini; Xia, Jiahong

    2014-01-01

    Background In a previous study, activation of the peroxisome proliferator–activated receptor γ (PPARγ) inhibited chronic cardiac rejection. However, because of the complexity of chronic rejection and the fact that PPARγ is widely expressed in immune cells, the mechanism of the PPARγ - induced protective effect was unclear. Materials and Methods A chronic rejection model was established using B6.C-H-2bm12KhEg (H-2bm12) mice as donors, and MHC II-mismatched T-cell-specific PPARγ knockout mice or wild type (WT) littermates as recipients. The allograft lesion was assessed by histology and immunohistochemistry. T cells infiltrates in the allograft were isolated, and cytokines and subpopulations were detected using cytokine arrays and flow cytometry. Transcription levels in the allograft were measured by RT-PCR. In vitro, the T cell subset differentiation was investigated after culture in various polarizing conditions. PPARγ-deficient regularory T cells (Treg) were cocultured with monocytes to test their ability to induce alternatively activated macrophages (AAM). Results T cell-specific PPARγ knockout recipients displayed reduced cardiac allograft survival and an increased degree of pathology compared with WT littermates. T cell-specific PPARγ knockout resulted in more CD4+ T cells infiltrating into the allograft and altered the Th1/Th2 and Th17/Treg ratios. The polarization of AAM was also reduced by PPARγ deficiency in T cells through the action of Th2 and Treg. PPARγ-deficient T cells eliminated the pioglitazone-induced polarization of AAM and reduced allograft survival. Conclusions PPARγ-deficient T cells influenced the T cell subset and AAM polarization in chronic allograft rejection. The mechanism of PPARγ activation in transplantation tolerance could yield a novel treatment without side effects. PMID:25383620

  16. Cutting Edge: Engineering Active IKKβ in T Cells Drives Tumor Rejection.

    PubMed

    Evaristo, César; Spranger, Stefani; Barnes, Sarah E; Miller, Michelle L; Molinero, Luciana L; Locke, Frederick L; Gajewski, Thomas F; Alegre, Maria-Luisa

    2016-04-01

    Acquired dysfunction of tumor-reactive T cells is one mechanism by which tumors can evade the immune system. Identifying and correcting pathways that contribute to such dysfunction should enable novel anticancer therapy design. During cancer growth, T cells show reduced NF-κB activity, which is required for tumor rejection. Impaired T cell-intrinsic NF-κB may create a vicious cycle conducive to tumor progression and further T cell dysfunction. We hypothesized that forcing T cell-intrinsic NF-κB activation might break this cycle and induce tumor elimination. NF-κB was activated in T cells by inducing the expression of a constitutively active form of the upstream activator IκB kinase β (IKKβ). T cell-restricted constitutively active IKKβ augmented the frequency of functional tumor-specific CD8(+) T cells and improved tumor control. Transfer of constitutively active IKKβ-transduced T cells also boosted endogenous T cell responses that controlled pre-established tumors. Our results demonstrate that driving T cell-intrinsic NF-κB can result in tumor control, thus identifying a pathway with potential clinical applicability. PMID:26903482

  17. The antihistamine olopatadine regulates T cell activation in palladium allergy.

    PubMed

    Iguchi, Naohiko; Takeda, Yuri; Sato, Naoki; Ukichi, Kenichirou; Katakura, Akira; Ueda, Kyosuke; Narushima, Takayuki; Higuchi, Shigehito; Ogasawara, Kouetsu

    2016-06-01

    Because of its corrosion resistance palladium (Pd) has been widely used in many consumer products ranging from fashion accessories to dental materials. Recently, however, an increase in Pd allergy cases has been reported. Metal allergy is categorized as a Type IV allergy, which is characterized as a delayed-type hypersensitivity reaction in which T cells are known to play an important role; however, the precise mechanism of their action remains unclear. Here we defined the relationship between histamine and the Pd allergic reaction specifically with respect to T cell responses. To verify the effects of histamine on T cells, we examined whether there is a change in IFN-γ production following stimulation of histamine or the antihistamine, olopatadine hydrochloride (OLP), in vitro. In addition, we assessed whether OLP administration affected the degree of footpad swelling or IFN-γ production during the Pd allergy response in mice. We found that histamine stimulation increased IFN-γ production in T cells, specifically enhancing IFN-γ production in CD8(+) T cells compared with CD4(+) T cells. Interestingly, OLP suppressed the production of IFN-γ in CD8(+) T cells, and this compound inhibited footpad swelling and IFN-γ production in mice with Pd allergy. These results suggest that histamine promotes the Type IV allergic reaction and thus, the histamine 1 receptor (H1R) might be useful therapeutic target for treatment of metal allergy.

  18. The ion channel TRPV1 regulates the activation and proinflammatory properties of CD4+ T cells

    PubMed Central

    Bertin, Samuel; Aoki-Nonaka, Yukari; de Jong, Petrus Rudolf; Stanwood, Shawna R.; Srikanth, Sonal; Lee, Jihyung; To, Keith; Abramson, Lior; Yu, Timothy; Han, Tiffany; Touma, Ranim; Li, Xiangli; González-Navajas, José M.; Herdman, Scott; Corr, Maripat; Fu, Guo; Dong, Hui; Gwack, Yousang; Franco, Alessandra; Jefferies, Wilfred A.; Raz, Eyal

    2016-01-01

    TRPV1 is a Ca2+-permeable channel mostly studied as a pain receptor in sensory neurons. However, its role in other cell types is poorly understood. Here, we demonstrate that TRPV1 is functionally expressed in CD4+ T cells where it acts as a non-store-operated Ca2+ channel and contributes to T cell receptor (TCR)-induced Ca2+ influx, TCR signaling and T cell activation. In models of T cell-mediated colitis, TRPV1 promotes colitogenic T cell responses and intestinal inflammation. Furthermore, genetic and pharmacological inhibition of TRPV1 in human CD4+ T cells recapitulates the phenotype of murine Trpv1−/− CD4+ T cells. These findings suggest that TRPV1 inhibition could represent a new therapeutic strategy to restrain proinflammatory T cell responses. PMID:25282159

  19. Expression of CD39 on Activated T Cells Impairs their Survival in Older Individuals.

    PubMed

    Fang, Fengqin; Yu, Mingcan; Cavanagh, Mary M; Hutter Saunders, Jessica; Qi, Qian; Ye, Zhongde; Le Saux, Sabine; Sultan, William; Turgano, Emerson; Dekker, Cornelia L; Tian, Lu; Weyand, Cornelia M; Goronzy, Jörg J

    2016-02-01

    In an immune response, CD4(+) T cells expand into effector T cells and then contract to survive as long-lived memory cells. To identify age-associated defects in memory cell formation, we profiled activated CD4(+) T cells and found an increased induction of the ATPase CD39 with age. CD39(+) CD4(+) T cells resembled effector T cells with signs of metabolic stress and high susceptibility to undergo apoptosis. Pharmacological inhibition of ATPase activity dampened effector cell differentiation and improved survival, suggesting that CD39 activity influences T cell fate. Individuals carrying a low-expressing CD39 variant responded better to vaccination with an increase in vaccine-specific memory T cells. Increased inducibility of CD39 after activation may contribute to the impaired vaccine response with age. PMID:26832412

  20. Stephanthraniline A suppressed CD4(+) T cell-mediated immunological hepatitis through impairing PKCθ function.

    PubMed

    Chen, Feng-Yang; Zhou, Li-Fei; Li, Xiao-Yu; Zhao, Jia-Wen; Xu, Shi-Fang; Huang, Wen-Hai; Gao, Li-Juan; Hao, Shu-Juan; Ye, Yi-Ping; Sun, Hong-Xiang

    2016-10-15

    Stephanthraniline A (STA), a C21 steroid isolated from Stephanotis mucronata (Blanco) Merr., was previously shown to inhibit T cells activation and proliferation in vitro and in vivo. The purpose of this study was to further evaluate the in vivo immunosuppressive activity of STA and to elucidate its potential mechanisms. The results showed that pretreatment with STA significantly attenuated concanavalin A (Con A)-induced hepatitis and reduced CD4(+) T cells activation and aggregation in hepatic tissue in mice. STA directly suppressed the activation and proliferation of Con A-induced CD4(+) T cells, and inhibited NFAT, NFκB and MAPK signaling cascades in activated CD4(+) T cells in vitro. Moreover, it was proved that STA inhibited T cells activation and proliferation through proximal T cell-receptor (TCR) signaling- and Ca(2+) signaling-independent way. The molecular docking studies predicted that STA could tight bind to PKCθ via five hydrogen. The further findings indicated STA directly inhibited PKCθ kinase activity, and its phosphorylation in activated CD4(+) T cells in vitro. Collectively, the present study indicated that STA could protect against CD4(+) T cell-mediated immunological hepatitis in mice through PKCθ and its downstream NFAT, NFκB and MAPK signaling cascades. These results highlight the potential of STA as an effective leading compound for use in the treatment of CD4(+) T cell-mediated inflammatory and autoimmune diseases.

  1. TNF-α contributes to caspase-3 independent apoptosis in neuroblastoma cells: role of NFAT.

    PubMed

    Alvarez, Susana; Blanco, Almudena; Fresno, Manuel; Muñoz-Fernández, Ma Ángeles

    2011-01-27

    There is increasing evidence that soluble factors in inflammatory central nervous system diseases not only regulate the inflammatory process but also directly influence electrophysiological membrane properties of neurons and astrocytes. In this context, the cytokine TNF-α (tumor necrosis factor-α) has complex injury promoting, as well as protective, effects on neuronal viability. Up-regulated TNF-α expression has also been found in various neurodegenerative diseases such as cerebral malaria, AIDS dementia, Alzheimer's disease, multiple sclerosis, and stroke, suggesting a potential pathogenic role of TNF-α in these diseases as well. We used the neuroblastoma cells SK-N-MC. Transcriptional activity was measured using luciferase reporter gene assays by using lipofectin. We performed cotransfection experiments of NFAT (nuclear factor of activated T cells) promoter constructed with a dominant negative version of NFAT (dn-NFAT). Cell death was performed by MTT (3-(4,5-dimethylthiazol-2-yl)5,5-diphenyltetrazolium bromide) and TUNEL assays. NFAT translocation was confirmed by Western blot. Involvement of NFAT in cell death was assessed by using VIVIT. P53, Fas-L, caspase-3, and caspase-9 expressions were carried out by Western blot. The mechanisms involved in TNF-α-induced cell death were assessed by using microarray analysis. TNF-α causes neuronal cell death in the absence of glia. TNF-α treatment results in nuclear translocation of NFAT through activation of calcineurin in a Ca(2+) independent manner. We demonstrated the involvement of FasL/Fas, cytochrome c, and caspase-9 but the lack of caspase-3 activation. NB cell death was absolutely reverted in the presence of VIVIT, and partially diminished by anti-Fas treatment. These data demonstrate that TNF-α promotes FasL expression through NFAT activation in neuroblastoma cells and this event leads to increased apoptosis through independent caspase-3 activation.

  2. Chronic activation of the kinase IKKβ impairs T cell function and survival

    PubMed Central

    Krishna, Sruti; Xie, Danli; Gorentla, Balachandra; Shin, Jinwook; Gao, Jimin; Zhong, Xiao-Ping

    2012-01-01

    Activation of the transcription factor NF-κB is critical for cytokine production and T cell survival after T cell receptor (TCR) engagement. The effects of persistent NF-κB activity on T cell function and survival are poorly understood. In this study, using a murine model that expresses a constitutively active form of inhibitor of κB kinase β(caIKKβ) in a T-cell specific manner, we demonstrate that chronic IKKβ signaling promotes T cell apoptosis, attenuates responsiveness to TCR-mediated stimulation in vitro, and impairs T cell responses to bacterial infection in vivo. CaIKKβ T cells showed increased FasL expression and caspase-8 activation, and blocking Fas/FasL interactions enhanced cell survival. T cell unresponsiveness was associated with defects in TCR proximal signaling, and elevated levels of Blimp1, a transcriptional repressor that promotes T cell exhaustion. CaIKKβ T cells also showed a defect in IL-2 production, and addition of exogenous IL-2 enhanced their survival and proliferation. Conditional deletion of Blimp1 partially rescued sensitivity of caIKKβ T cells to TCR triggering. Furthermore, adoptively transferred caIKKβ T cells showed diminished expansion and increased contraction in response to infection with Listeria monocytogenes expressing a cognate antigen. Despite their functional defects, caIKKβ T cells readily produced pro-inflammatory cytokines and mice developed autoimmunity. In contrast to NF-κB's critical role in T cell activation and survival, our study demonstrates that persistent IKK-NF-κB signaling is sufficient to impair both T cell function and survival. PMID:22753932

  3. Transcriptomic analysis of mouse EL4 T cells upon T cell activation and in response to protein synthesis inhibition via cycloheximide treatment.

    PubMed

    Lim, Pek Siew; Hardy, Kristine; Peng, Kaiman; Shannon, Frances M

    2016-03-01

    T cell activation involves the recognition of a foreign antigen complexed to the major histocompatibility complex on the antigen presenting T cell to the T cell receptor. This leads to activation of signaling pathways, which ultimately leads to induction of key cytokine genes responsible for eradication of foreign antigens. We used the mouse EL4 T cell as a model system to study genes that are induced as a result of T cell activation using phorbol myristate acetate (PMA) and calcium ionomycin (I) as stimuli. We were also interested to examine the importance of new protein synthesis in regulating the expression of genes involved in T cell activation. Thus we have pre-treated mouse EL4 T cells with cycloheximide, a protein synthesis inhibitor, and left the cells unstimulated or stimulated with PMA/I for 4 h. We performed microarray expression profiling of these cells to correlate the gene expression with chromatin state of T cells upon T cell activation [1]. Here, we detail further information and analysis of the microarray data, which shows that T cell activation leads to differential expression of genes and inducible genes can be further classified as primary and secondary response genes based on their protein synthesis dependency. The data is available in the Gene Expression Omnibus under accession number GSE13278. PMID:26981393

  4. A role for Peroxisome Proliferator-Activated Receptor Beta in T cell development

    PubMed Central

    Mothe-Satney, Isabelle; Murdaca, Joseph; Sibille, Brigitte; Rousseau, Anne-Sophie; Squillace, Raphaëlle; Le Menn, Gwenaëlle; Rekima, Akila; Larbret, Frederic; Pelé, Juline; Verhasselt, Valérie; Grimaldi, Paul A.; Neels, Jaap G.

    2016-01-01

    Metabolism plays an important role in T cell biology and changes in metabolism drive T cell differentiation and fate. Most research on the role of metabolism in T lymphocytes focuses on mature T cells while only few studies have investigated the role of metabolism in T cell development. In this study, we report that activation or overexpression of the transcription factor Peroxisome Proliferator-Activated Receptor β (PPARβ) increases fatty acid oxidation in T cells. Furthermore, using both in vivo and in vitro models, we demonstrate that PPARβ activation/overexpression inhibits thymic T cell development by decreasing proliferation of CD4−CD8− double-negative stage 4 (DN4) thymocytes. These results support a model where PPARβ activation/overexpression favours fatty acid- instead of glucose-oxidation in developing T cells, thereby hampering the proliferative burst normally occurring at the DN4 stage of T cell development. As a consequence, the αβ T cells that are derived from DN4 thymocytes are dramatically decreased in peripheral lymphoid tissues, while the γδ T cell population remains untouched. This is the first report of a direct role for a member of the PPAR family of nuclear receptors in the development of T cells. PMID:27680392

  5. Calcineurin/NFAT Activation-Dependence of Leptin Synthesis and Vascular Growth in Response to Mechanical Stretch

    PubMed Central

    Soudani, Nadia; Ghantous, Crystal M.; Farhat, Zein; Shebaby, Wassim N.; Zibara, Kazem; Zeidan, Asad

    2016-01-01

    Background and Aims: Hypertension and obesity are important risk factors of cardiovascular disease. They are both associated with high leptin levels and have been shown to promote vascular hypertrophy, through the RhoA/ROCK and ERK1/2 phosphorylation. Calcineurin/NFAT activation also induces vascular hypertrophy by upregulating various genes. This study aimed to decipher whether a crosstalk exists between the RhoA/ROCK pathway, Ca2+/calcineurin/NFAT pathway, and ERK1/2 phosphorylation in the process of mechanical stretch-induced vascular smooth muscle cell (VSMC) hypertrophy and leptin synthesis. Methods and Results: Rat portal vein (RPV) organ culture was used to investigate the effect of mechanical stretch and exogenous leptin (3.1 nM) on VSMC hypertrophy and leptin synthesis. Results showed that stretching the RPV significantly upregulated leptin secretion, mRNA, and protein expression, which were inhibited by the calcium channel blocker nifedipine (10 μM), the selective calcineurin inhibitor FK506 (1 nM), and the ERK1/2 inhibitor PD98059 (1 μM). The transcription inhibitor actinomycin D (0.1 μM) and the translation inhibitor cycloheximide (1 mM) significantly decreased stretch-induced leptin protein expression. Mechanical stretch or leptin caused an increase in wet weight changes and protein synthesis, considered as hypertrophic markers, while they were inhibited by FK506 (0.1 nM; 1 nM). In addition, stretch or exogenous leptin significantly increased calcineurin activity and MCIP1 expression whereas leptin induced NFAT nuclear translocation in VSMCs. Moreover, in response to stretch or exogenous leptin, the Rho inhibitor C3 exoenzyme (30 ng/mL), the ROCK inhibitor Y-27632 (10 μM), and the actin depolymerization agents Latrunculin B (50 nM) and cytochalasin D (1 μM) reduced calcineurin activation and NFAT nuclear translocation. ERK1/2 phosphorylation was inhibited by FK506 and C3. Conclusions: Mechanical stretch-induced VSMC hypertrophy and leptin

  6. Calcium and calcineurin-NFAT signaling regulate granulocyte-monocyte progenitor cell cycle via Flt3-L.

    PubMed

    Fric, Jan; Lim, Clarice X F; Mertes, Alexandra; Lee, Bernett T K; Viganò, Elena; Chen, Jinmiao; Zolezzi, Francesca; Poidinger, Michael; Larbi, Anis; Strobl, Herbert; Zelante, Teresa; Ricciardi-Castagnoli, Paola

    2014-12-01

    Maintenance of myeloid progenitor cells is controlled by complex regulatory mechanisms and is orchestrated by multiple different transcription factors. Here, we report that the activation of the transcription factor nuclear factor of activated T cells (NFAT) by calcium-sensing protein calcineurin inhibits the proliferation of myeloid granulocyte-monocyte progenitors (GMPs). Myeloid progenitor subtypes exhibit variable sensitivity to induced Ca(2+) entry and consequently display differential engagement of the calcineurin-NFAT pathway. This study shows that inhibition of the calcineurin-NFAT pathway enhances the proliferation of GMPs both in vitro and in vivo and demonstrates that calcineurin-NFAT signaling in GMPs is initiated by Flt3-L. Inhibition of the calcineurin-NFAT pathway modified expression of the cell cycle regulation genes Cdk4, Cdk6, and Cdkn1a (p21), thus enabling rapid cell cycle progression specifically in GMPs. NFAT inhibitor drugs are extensively used in the clinic to restrict the pathological activation of lymphoid cells, and our data reveal for the first time that these therapies also exert potent effects on maintenance of the myeloid cell compartment through specific regulation of GMP proliferation.

  7. Calcium and Calcineurin-NFAT Signaling Regulate Granulocyte-Monocyte Progenitor Cell Cycle via Flt3-L

    PubMed Central

    Fric, Jan; Lim, Clarice XF; Mertes, Alexandra; Lee, Bernett TK; Viganò, Elena; Chen, Jinmiao; Zolezzi, Francesca; Poidinger, Michael; Larbi, Anis; Strobl, Herbert; Zelante, Teresa; Ricciardi-Castagnoli, Paola

    2014-01-01

    Abstract Maintenance of myeloid progenitor cells is controlled by complex regulatory mechanisms and is orchestrated by multiple different transcription factors. Here, we report that the activation of the transcription factor nuclear factor of activated T cells (NFAT) by calcium-sensing protein calcineurin inhibits the proliferation of myeloid granulocyte–monocyte progenitors (GMPs). Myeloid progenitor subtypes exhibit variable sensitivity to induced Ca2+ entry and consequently display differential engagement of the calcineurin-NFAT pathway. This study shows that inhibition of the calcineurin-NFAT pathway enhances the proliferation of GMPs both in vitro and in vivo and demonstrates that calcineurin-NFAT signaling in GMPs is initiated by Flt3-L. Inhibition of the calcineurin-NFAT pathway modified expression of the cell cycle regulation genes Cdk4, Cdk6, and Cdkn1a (p21), thus enabling rapid cell cycle progression specifically in GMPs. NFAT inhibitor drugs are extensively used in the clinic to restrict the pathological activation of lymphoid cells, and our data reveal for the first time that these therapies also exert potent effects on maintenance of the myeloid cell compartment through specific regulation of GMP proliferation. Stem Cells 2014;32:3232–3244 PMID:25100642

  8. Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

    PubMed Central

    Lund, Peder J.; Elias, Joshua E.

    2016-01-01

    T cell activation in response to Ag is largely regulated by protein posttranslational modifications. Although phosphorylation has been extensively characterized in T cells, much less is known about the glycosylation of serine/threonine residues by O-linked N-acetylglucosamine (O-GlcNAc). Given that O-GlcNAc appears to regulate cell signaling pathways and protein activity similarly to phosphorylation, we performed a comprehensive analysis of O-GlcNAc during T cell activation to address the functional importance of this modification and to identify the modified proteins. Activation of T cells through the TCR resulted in a global elevation of O-GlcNAc levels and in the absence of O-GlcNAc, IL-2 production and proliferation were compromised. T cell activation also led to changes in the relative expression of O-GlcNAc transferase (OGT) isoforms and accumulation of OGT at the immunological synapse of murine T cells. Using a glycoproteomics approach, we identified >200 O-GlcNAc proteins in human T cells. Many of the identified proteins had a functional relationship to RNA metabolism, and consistent with a connection between O-GlcNAc and RNA, inhibition of OGT impaired nascent RNA synthesis upon T cell activation. Overall, our studies provide a global analysis of O-GlcNAc dynamics during T cell activation and the first characterization, to our knowledge, of the O-GlcNAc glycoproteome in human T cells. PMID:27655845

  9. Activity-dependent Transcriptional Regulation of M-type (Kv7) K+ Channels by AKAP79/150-mediated NFAT Actions

    PubMed Central

    Zhang, Jie; Shapiro, Mark S.

    2013-01-01

    Summary M-type K+ channels, encoded by the KCNQ2-5 (Kv7) gene family, play key roles in regulation of neuronal excitability; however, less is known about the mechanisms controlling their transcriptional expression. Here, we discovered a novel mechanism regulating KCNQ2/3 transcriptional expression by neuronal activity in rodent neurons, involving activation of calcineurin and Nuclear Factor of Activated T-cells (NFAT) transcription factors, orchestrated by A-kinase-anchoring protein (AKAP)79/150. The signal requires Ca2+ influx through L-type Ca2+ channels and both local and global Ca2+ elevations. We postulate increased M-channel expression to act as a negative-feedback to suppress hyper-excitability of neurons, demonstrated by profoundly up-regulated KCNQ2/3 transcription in hippocampi from wild-type mice after drug-induced seizures, an effect nearly eliminated in AKAP150−/− mice. Thus, we suggest a distinct role of AKAP79/150 and the complex it organizes in activity-dependent M-channel transcription, which may potentially serve throughout the nervous system to limit over-excitability associated with disease states such as epilepsy. PMID:23259949

  10. Differential requirement of RasGRP1 for γδ T cell development and activation

    PubMed Central

    Chen, Yong; Ci, Xinxin; Gorentla, Balachandra; Sullivan, Sarah A.; Stone, James C.; Zhang, Weiguo; Pereira, Pablo; Lu, Jianxin; Zhong, Xiao-Ping

    2012-01-01

    γδ T cells (γδT) belong to a distinct T cell lineage that performs immune functions different from αβ T cells (αβT). Previous studies have established that Erk1/2 MAPKs are critical for positive selection of αβT cells. Additional evidence also suggests that elevated Erk1/2 activity promotes γδT cell generation. RasGRP1, a guanine nucleotide releasing factor for Ras, plays an important role in positive selection of αβT cells by activating the Ras-Erk1/2 pathway. In this report, we demonstrate that RasGRP1 is critical for TCR-induced Erk1/2 activation in γδT cells but exerts different roles for γδT cell generation and activation. Deficiency of RasGRP1 does not obviously affect γδT cell numbers in the thymus but leads to increased γδT cells, particularly CD4−CD8+ γδT cells, in the peripheral lymphoid organs. The virtually unhindered γδT cell development in the RasGRP1−/− thymus proved to be cell intrinsic, while the increase in CD8+ γδT cells is caused by non-cell-intrinsic mechanisms. Our data provides genetic evidence that decreased Erk1/2 activation in the absence of RasGRP1 is compatible for γδT cell generation. Although RasGRP1 is dispensable for γδT cell generation, RasGRP1-deficient γδT cells are defective in proliferation following TCR stimulation. Additionally, RasGRP1-deficient γδT cells are impaired to produce IL-17 but not IFNγ. Together, these observations have revealed that RasGRP1 plays differential roles for γδ and αβ T cell development but is critical for γδT cell proliferation and production of IL-17. PMID:22623331

  11. T cell-expressed CD40L potentiates the bone anabolic activity of intermittent PTH treatment.

    PubMed

    Robinson, Jerid W; Li, Jau-Yi; Walker, Lindsey D; Tyagi, Abdul Malik; Reott, Michael A; Yu, Mingcan; Adams, Jonathan; Weitzmann, M Neale; Pacifici, Roberto

    2015-04-01

    T cells are known to potentiate the bone anabolic activity of intermittent parathyroid hormone (iPTH) treatment. One of the involved mechanisms is increased T cell secretion of Wnt10b, a potent osteogenic Wnt ligand that activates Wnt signaling in stromal cells (SCs). However, additional mechanisms might play a role, including direct interactions between surface receptors expressed by T cells and SCs. Here we show that iPTH failed to promote SC proliferation and differentiation into osteoblasts (OBs) and activate Wnt signaling in SCs of mice with a global or T cell-specific deletion of the T cell costimulatory molecule CD40 ligand (CD40L). Attesting to the relevance of T cell-expressed CD40L, iPTH induced a blunted increase in bone formation and failed to increase trabecular bone volume in CD40L(-/-) mice and mice with a T cell-specific deletion of CD40L. CD40L null mice exhibited a blunted increase in T cell production of Wnt10b and abrogated CD40 signaling in SCs in response to iPTH treatment. Therefore, expression of the T cell surface receptor CD40L enables iPTH to exert its bone anabolic activity by activating CD40 signaling in SCs and maximally stimulating T cell production of Wnt10b.

  12. Alternative splicing of MALT1 controls signalling and activation of CD4+ T cells

    PubMed Central

    Meininger, Isabel; Griesbach, Richard A.; Hu, Desheng; Gehring, Torben; Seeholzer, Thomas; Bertossi, Arianna; Kranich, Jan; Oeckinghaus, Andrea; Eitelhuber, Andrea C.; Greczmiel, Ute; Gewies, Andreas; Schmidt-Supprian, Marc; Ruland, Jürgen; Brocker, Thomas; Heissmeyer, Vigo; Heyd, Florian; Krappmann, Daniel

    2016-01-01

    MALT1 channels proximal T-cell receptor (TCR) signalling to downstream signalling pathways. With MALT1A and MALT1B two conserved splice variants exist and we demonstrate here that MALT1 alternative splicing supports optimal T-cell activation. Inclusion of exon7 in MALT1A facilitates the recruitment of TRAF6, which augments MALT1 scaffolding function, but not protease activity. Naive CD4+ T cells express almost exclusively MALT1B and MALT1A expression is induced by TCR stimulation. We identify hnRNP U as a suppressor of exon7 inclusion. Whereas selective depletion of MALT1A impairs T-cell signalling and activation, downregulation of hnRNP U enhances MALT1A expression and T-cell activation. Thus, TCR-induced alternative splicing augments MALT1 scaffolding to enhance downstream signalling and to promote optimal T-cell activation. PMID:27068814

  13. Cytokine treatment of macrophage suppression of T cell activation.

    PubMed

    Silberman, Daniel; Bucknum, Amanda; Kozlowski, Megan; Matlack, Robin; Riggs, James

    2010-01-01

    High Mphi:T cell ratios suppress the immune response to the retroviral superantigen Mls by IFNgamma-triggered production of the arg- and trp-consuming enzymes iNOS and IDO. Attempts to reverse suppression by treatment with pro-inflammatory cytokines revealed that IL-6 improved the T cell response to Mls and the pro-hematopoietic cyokines IL-3 and GM-CSF increased suppression. GM-CSF treatment increased Mphi expression of CD80, a ligand for the immune suppressive B7H1 and CTLA-4 receptors. These results illustrate potential strategies for reversing the suppression of cell-mediated immunity characteristic of the high Mphi:T cell ratios found in many tumors.

  14. Activated human T cells accomplish MHC class II expression through T cell-specific occupation of class II transactivator promoter III.

    PubMed

    Holling, Tjadine M; van der Stoep, Nienke; Quinten, Edwin; van den Elsen, Peter J

    2002-01-15

    Activated human T cells express HLA-DR, HLA-DQ, and HLA-DP on their surface, but the regulation and functioning of MHC class II molecules in T lymphocytes are poorly understood. Because the MHC class II transactivator (CIITA) is essential for MHC class II expression, we have investigated transcriptional activation of CIITA in activated T cells. In this study, we show that in human activated CD4(+) T cells, CIITA promoter III (CIITA-PIII) drives the expression of CIITA. The in vivo genomic footprint analysis revealed activated T cell-specific occupation of CIITA-PIII. Subsequent EMSA analysis of several promoter regions showed differences in banding pattern among activated T cells, naive T cells, primary B cells, and Raji B cells. Activating response element (ARE)-1 is shown to interact with the acute myeloid leukemia 2 transcription factor in nuclear extracts derived from both T and B cells. Interestingly, the acute myeloid leukemia 3 transcription factor was bound in nuclear extracts of T cells only. The ARE-2 sequence is able to bind CREB/activating transcription factor family members in both T and B cells. In addition, a yet unidentified Ets family member was found to interact with site C in activated T cells, whereas in B cells site C was bound by PU.1 and Pip/IFN regulatory factor 4/IFN consensus sequence binding protein for activated T cells. In Jurkat T cells, both ARE-1 and ARE-2 are crucial for CIITA-PIII activity, similar to Raji B cells. The differential banding pattern in in vivo genomic footprinting and transcription factor binding at the ARE-1 and site C between T cells and B cells probably reflects differences in CIITA-PIII activation pathways employed by these cell types. PMID:11777970

  15. T Cells Expressing Constitutively Active Akt Resist Multiple Tumor-associated Inhibitory Mechanisms

    PubMed Central

    Sun, Jiali; Dotti, Gianpietro; Huye, Leslie E; Foster, Aaron E; Savoldo, Barbara; Gramatges, Maria M; Spencer, David M; Rooney, Cliona M

    2010-01-01

    Adoptive transfer of antigen-specific cytotoxic T lymphocytes has shown promise for the therapy of cancer. However, tumor-specific T cells are susceptible to diverse inhibitory signals from the tumor microenvironment. The Akt/protein kinase B plays a central role in T-cell proliferation, function, and survival and we hypothesized that expression of constitutively active Akt (caAkt) in T cells could provide resistance to many of these tumor-associated inhibitory mechanisms. caAkt expression in activated human T cells increased proliferation and cytokine production, a likely result of their sustained expression of nuclear factor-κB (NF-κB) and provided resistance to apoptosis by upregulating antiapoptotic molecules. caAkt expressing T cells (caAkt-T-cells) were also relatively resistant to suppression by and conversion into regulatory T cells (Tregs). These characteristics provided a survival advantage to T cells cocultured with tumor cells in vitro; CD3/28-stimulated T cells expressing a chimeric antigen receptor (CAR) specific for disialoganglioside (GD2) that redirected their activity to the immunosuppressive, GD2-expressing neuroblastoma cell line, LAN-1, resisted tumor-induced apoptosis when co-expressing transgenic caAkt. In conclusion, caAkt-transduced T cells showed resistance to several evasion strategies employed by tumors and may therefore enhance the antitumor activity of adoptively transferred T lymphocytes. PMID:20842106

  16. Selective activation of T cells in newly diagnosed insulin-dependent diabetic patients: evidence for heterogeneity of T cell receptor usage.

    PubMed Central

    Kontiainen, S; Toomath, R; Lowder, J; Feldmann, M

    1991-01-01

    Cell surface phenotyping of 58 newly diagnosed diabetic children and 25 controls confirmed the presence of activated T cells, expressing HLA class II antigens or receptors for interleukin-2 (IL-2R, CD25) in the majority of the patients. Some of these cells putatively include those involved in islet cell destruction, as reported previously. Monoclonal antibodies recognizing three families of the variable regions of the beta chain (V beta) of the T cell receptor were used to determine the percentage of peripheral blood cells expressing those specific gene segment products. The number of the activated T cells from each V beta family was compared with that of the resting T cells of the same family in the patients and the controls. In 18 out of 58 (31%) of these patients there was evidence of oligoclonal proliferation of activated T cells as judged by marked increases in cells expressing a V beta family in the IL-2R+ T cell pool, compared with the total T cell pool. However, different V beta families were augmented in individual patients, indicating considerable heterogeneity of T cell activation in different patients. These results are in contrast to murine models of autoimmunity, where virtually monoclonal T cell activation, restricted to a single V beta family has been reported. PMID:1825939

  17. Estrogen-related receptor gamma regulates dopaminergic neuronal phenotype by activating GSK3β/NFAT signaling in SH-SY5Y cells.

    PubMed

    Lim, Juhee; Choi, Hueng-Sik; Choi, Hyun Jin

    2015-05-01

    The orphan nuclear receptor estrogen-related receptor gamma (ERRγ) is highly expressed in the nervous system during embryogenesis and in adult brains, but its physiological role in neuronal development remains unknown. In this study, we evaluated the relevance of ERRγ in regulating dopaminergic (DAergic) phenotype and the corresponding signaling pathway. We used retinoic acid (RA) to differentiate human neuroblastoma SH-SY5Y cells. RA induced neurite outgrowth of SH-SY5Y cells with an increase in DAergic neuron-like properties, including up-regulation of tyrosine hydroxylase, dopamine transporter, and vesicular monoamine transporter 2. ERRγ, but not ERRα, was up-regulated by RA, and participated in RA effect on SH-SY5Y cells. ERRγ over-expression enhanced mature DAergic neuronal phenotype with neurite outgrowth as with RA treatment; and RA-induced increase in DAergic phenotype was attenuated by silencing ERRγ expression. ERRγ appears to have a crucial role in morphological and functional regulation of cells that is selective for DAergic neurons. Polo-like kinase 2 was up-regulated in ERRγ-over-expressing SH-SY5Y cells, which was involved in phosphorylation of glycogen synthase kinase 3β and resulting downstream activation of nuclear factor of activated T cells. The likely involvement of ERRγ in regulating the DAergic neuronal phenotype makes this orphan nuclear receptor a novel target for understanding DAergic neuronal differentiation. We propose the relevance of estrogen-related receptor gamma (ERRγ) in regulating dopaminergic neuronal phenotype: ERRγ is up-regulated by retinoic acid in SH-SY5Y cells, and enhances dopaminergic phenotypes and induces neurite outgrowth; Polo-like kinase 2 (PLK2) and glycogen synthase kinase 3 beta/nuclear factor of activated T cells (GSK3β/NFAT) signaling are responsible for the ERRγ effect. Our findings provide the first insights into the role of ERRγ in the brain, as a novel approach toward understanding

  18. T-plastin expression downstream to the calcineurin/NFAT pathway is involved in keratinocyte migration.

    PubMed

    Brun, Cécilia; Demeaux, Agathe; Guaddachi, Frédéric; Jean-Louis, Francette; Oddos, Thierry; Bagot, Martine; Bensussan, Armand; Jauliac, Sébastien; Michel, Laurence

    2014-01-01

    Cutaneous wound healing requires keratinocyte proliferation, migration and differentiation to restore the barrier function of the skin. The calcineurin/nuclear factor of activated-T-cell (NFAT) signaling pathway has been recently shown to be involved in keratinocyte growth, differentiation and migration. It is induced by an increased intracellular calcium rate and its inhibition results in decreased capacities of keratinocytes to migrate. Nevertheless, the link between calcineurin activation and keratinocyte migration remains unknown. Recently, Orai1, a pore subunit of a store-operated calcium channel that favors calcium influx, was shown to play a critical role to control proliferation and migration of basal keratinocytes. Of interest, the actin-bundling T-plastin is crucial in cell motility through cross-linking to actin filament and its synthesis was shown to be induced by calcium influx and regulated by the calcineurin/NFAT pathway in tumor Sezary cells. We investigated herein the role of the calcineurin/NFAT pathway-dependent T-plastin in keratinocyte migration, by quantifying T-plastin expression in keratinocytes and by analyzing their migration under calcineurin inhibition or knockdown of NFAT2 or T-plastin. We did confirm the role of the calcineurin/NFAT pathway in keratinocyte migration as shown by their decreased capacities to migrate after FK506 treatment or siNFAT2 transfection in both scratching and Boyden assays. The expression of NFAT2 and T-plastin in keratinocytes was decreased under FK506 treatment, suggesting that T-plastin plays a role in keratinocyte migration downstream to the calcineurin/NFAT pathway. Accordingly, siRNA knockdown of T-plastin expression also decreased their migration capacities. Actin lamellipodia formation as well as FAK and β6-integrin expression were also significantly decreased after treatment with FK506 or siRNA, reinforcing that NFAT2-dependent T-plastin expression plays a role in keratinocyte migration. These results

  19. NFAT is required for spontaneous pulmonary hypertension in superoxide dismutase 1 knockout mice.

    PubMed

    Ramiro-Diaz, Juan Manuel; Nitta, Carlos H; Maston, Levi D; Codianni, Simon; Giermakowska, Wieslawa; Resta, Thomas C; Gonzalez Bosc, Laura V

    2013-05-01

    Elevated reactive oxygen species are implicated in pulmonary hypertension (PH). Superoxide dismutase (SOD) limits superoxide bioavailability, and decreased SOD activity is associated with PH. A decrease in SOD activity is expected to increase superoxide and reduce hydrogen peroxide levels. Such an imbalance of superoxide/hydrogen peroxide has been implicated as a mediator of nuclear factor of activated T cells (NFAT) activation in epidermal cells. We have shown that NFATc3 is required for chronic hypoxia-induced PH. However, it is unknown whether NFATc3 is activated in the pulmonary circulation in a mouse model of decreased SOD1 activity and whether this leads to PH. Therefore, we hypothesized that an elevated pulmonary arterial superoxide/hydrogen peroxide ratio activates NFATc3, leading to PH. We found that SOD1 knockout (KO) mice have elevated pulmonary arterial wall superoxide and decreased hydrogen peroxide levels compared with wild-type (WT) littermates. Right ventricular systolic pressure (RVSP) was elevated in SOD1 KO and was associated with pulmonary arterial remodeling. Vasoreactivity to endothelin-1 was also greater in SOD1 KO vs. WT mice. NFAT activity and NFATc3 nuclear localization were increased in pulmonary arteries from SOD1 KO vs. WT mice. Administration of A-285222 (selective NFAT inhibitor) decreased RVSP, arterial wall thickness, vasoreactivity, and NFAT activity in SOD1 KO mice to WT levels. The SOD mimetic, tempol, also reduced NFAT activity, NFATc3 nuclear localization, and RVSP to WT levels. These findings suggest that an elevated superoxide/hydrogen peroxide ratio activates NFAT in pulmonary arteries, which induces vascular remodeling and increases vascular reactivity leading to PH.

  20. Activation of Human T Cells in Hypertension: Studies of Humanized Mice and Hypertensive Humans.

    PubMed

    Itani, Hana A; McMaster, William G; Saleh, Mohamed A; Nazarewicz, Rafal R; Mikolajczyk, Tomasz P; Kaszuba, Anna M; Konior, Anna; Prejbisz, Aleksander; Januszewicz, Andrzej; Norlander, Allison E; Chen, Wei; Bonami, Rachel H; Marshall, Andrew F; Poffenberger, Greg; Weyand, Cornelia M; Madhur, Meena S; Moore, Daniel J; Harrison, David G; Guzik, Tomasz J

    2016-07-01

    Emerging evidence supports an important role for T cells in the genesis of hypertension. Because this work has predominantly been performed in experimental animals, we sought to determine whether human T cells are activated in hypertension. We used a humanized mouse model in which the murine immune system is replaced by the human immune system. Angiotensin II increased systolic pressure to 162 versus 116 mm Hg for sham-treated animals. Flow cytometry of thoracic lymph nodes, thoracic aorta, and kidney revealed increased infiltration of human leukocytes (CD45(+)) and T lymphocytes (CD3(+) and CD4(+)) in response to angiotensin II infusion. Interestingly, there was also an increase in the memory T cells (CD3(+)/CD45RO(+)) in the aortas and lymph nodes. Prevention of hypertension using hydralazine and hydrochlorothiazide prevented the accumulation of T cells in these tissues. Studies of isolated human T cells and monocytes indicated that angiotensin II had no direct effect on cytokine production by T cells or the ability of dendritic cells to drive T-cell proliferation. We also observed an increase in circulating interleukin-17A producing CD4(+) T cells and both CD4(+) and CD8(+) T cells that produce interferon-γ in hypertensive compared with normotensive humans. Thus, human T cells become activated and invade critical end-organ tissues in response to hypertension in a humanized mouse model. This response likely reflects the hypertensive milieu encountered in vivo and is not a direct effect of the hormone angiotensin II.

  1. Activation of Human T Cells in Hypertension: Studies of Humanized Mice and Hypertensive Humans.

    PubMed

    Itani, Hana A; McMaster, William G; Saleh, Mohamed A; Nazarewicz, Rafal R; Mikolajczyk, Tomasz P; Kaszuba, Anna M; Konior, Anna; Prejbisz, Aleksander; Januszewicz, Andrzej; Norlander, Allison E; Chen, Wei; Bonami, Rachel H; Marshall, Andrew F; Poffenberger, Greg; Weyand, Cornelia M; Madhur, Meena S; Moore, Daniel J; Harrison, David G; Guzik, Tomasz J

    2016-07-01

    Emerging evidence supports an important role for T cells in the genesis of hypertension. Because this work has predominantly been performed in experimental animals, we sought to determine whether human T cells are activated in hypertension. We used a humanized mouse model in which the murine immune system is replaced by the human immune system. Angiotensin II increased systolic pressure to 162 versus 116 mm Hg for sham-treated animals. Flow cytometry of thoracic lymph nodes, thoracic aorta, and kidney revealed increased infiltration of human leukocytes (CD45(+)) and T lymphocytes (CD3(+) and CD4(+)) in response to angiotensin II infusion. Interestingly, there was also an increase in the memory T cells (CD3(+)/CD45RO(+)) in the aortas and lymph nodes. Prevention of hypertension using hydralazine and hydrochlorothiazide prevented the accumulation of T cells in these tissues. Studies of isolated human T cells and monocytes indicated that angiotensin II had no direct effect on cytokine production by T cells or the ability of dendritic cells to drive T-cell proliferation. We also observed an increase in circulating interleukin-17A producing CD4(+) T cells and both CD4(+) and CD8(+) T cells that produce interferon-γ in hypertensive compared with normotensive humans. Thus, human T cells become activated and invade critical end-organ tissues in response to hypertension in a humanized mouse model. This response likely reflects the hypertensive milieu encountered in vivo and is not a direct effect of the hormone angiotensin II. PMID:27217403

  2. IFT20 controls LAT recruitment to the immune synapse and T-cell activation in vivo.

    PubMed

    Vivar, Omar I; Masi, Giulia; Carpier, Jean-Marie; Magalhaes, Joao G; Galgano, Donatella; Pazour, Gregory J; Amigorena, Sebastian; Hivroz, Claire; Baldari, Cosima T

    2016-01-12

    Biogenesis of the immune synapse at the interface between antigen-presenting cells and T cells assembles and organizes a large number of membrane proteins required for effective signaling through the T-cell receptor. We showed previously that the intraflagellar transport protein 20 (IFT20), a component of the intraflagellar transport system, controls polarized traffic during immune synapse assembly. To investigate the role of IFT20 in primary CD4(+) T cells in vitro and in vivo, we generated mice bearing a conditional defect of IFT20 expression in T cells. We show that in the absence of IFT20, although cell spreading and the polarization of the centrosome were unaffected, T-cell receptor (TCR)-mediated signaling and recruitment of the signaling adaptor LAT (linker for activation of T cells) at the immune synapse were reduced. As a consequence, CD4(+) T-cell activation and proliferation were also defective. In vivo, conditional IFT20-deficient mice failed to mount effective antigen-specific T-cell responses, and their T cells failed to induce colitis after adoptive transfer to Rag(-/-) mice. IFT20 is therefore required for the delivery of the intracellular pool of LAT to the immune synapse in naive primary T lymphocytes and for effective T-cell responses in vivo. PMID:26715756

  3. IFT20 controls LAT recruitment to the immune synapse and T-cell activation in vivo

    PubMed Central

    Vivar, Omar I.; Masi, Giulia; Carpier, Jean-Marie; Magalhaes, Joao G.; Galgano, Donatella; Pazour, Gregory J.; Amigorena, Sebastian; Hivroz, Claire; Baldari, Cosima T.

    2016-01-01

    Biogenesis of the immune synapse at the interface between antigen-presenting cells and T cells assembles and organizes a large number of membrane proteins required for effective signaling through the T-cell receptor. We showed previously that the intraflagellar transport protein 20 (IFT20), a component of the intraflagellar transport system, controls polarized traffic during immune synapse assembly. To investigate the role of IFT20 in primary CD4+ T cells in vitro and in vivo, we generated mice bearing a conditional defect of IFT20 expression in T cells. We show that in the absence of IFT20, although cell spreading and the polarization of the centrosome were unaffected, T-cell receptor (TCR)-mediated signaling and recruitment of the signaling adaptor LAT (linker for activation of T cells) at the immune synapse were reduced. As a consequence, CD4+ T-cell activation and proliferation were also defective. In vivo, conditional IFT20-deficient mice failed to mount effective antigen-specific T-cell responses, and their T cells failed to induce colitis after adoptive transfer to Rag−/− mice. IFT20 is therefore required for the delivery of the intracellular pool of LAT to the immune synapse in naive primary T lymphocytes and for effective T-cell responses in vivo. PMID:26715756

  4. Bystander Activation and Anti-Tumor Effects of CD8+ T Cells Following Interleukin-2 Based Immunotherapy Is Independent of CD4+ T Cell Help

    PubMed Central

    Grossenbacher, Steven K.; Hsiao, Hui-Hua; Zamora, Anthony E.; Mirsoian, Annie; Koehn, Brent; Blazar, Bruce R.; Weiss, Jonathan M.; Wiltrout, Robert H.; Sckisel, Gail D.; Murphy, William J.

    2014-01-01

    We have previously demonstrated that immunotherapy combining agonistic anti-CD40 and IL-2 (IT) results in synergistic anti-tumor effects. IT induces expansion of highly cytolytic, antigen-independent “bystander-activated” (CD8+CD44high) T cells displaying a CD25−NKG2D+ phenotype in a cytokine dependent manner, which were responsible for the anti-tumor effects. While much attention has focused on CD4+ T cell help for antigen-specific CD8+ T cell expansion, little is known regarding the role of CD4+ T cells in antigen-nonspecific bystander-memory CD8+ T cell expansion. Utilizing CD4 deficient mouse models, we observed a significant expansion of bystander-memory T cells following IT which was similar to the non-CD4 depleted mice. Expanded bystander-memory CD8+ T cells upregulated PD-1 in the absence of CD4+ T cells which has been published as a hallmark of exhaustion and dysfunction in helpless CD8+ T cells. Interestingly, compared to CD8+ T cells from CD4 replete hosts, these bystander expanded cells displayed comparable (or enhanced) cytokine production, lytic ability, and in vivo anti-tumor effects suggesting no functional impairment or exhaustion and were enriched in an effector phenotype. There was no acceleration of the post-IT contraction phase of the bystander memory CD8+ response in CD4-depleted mice. The response was independent of IL-21 signaling. These results suggest that, in contrast to antigen-specific CD8+ T cell expansion, CD4+ T cell help is not necessary for expansion and activation of antigen-nonspecific bystander-memory CD8+ T cells following IT, but may play a role in regulating conversion of these cells from a central memory to effector phenotype. Additionally, the expression of PD-1 in this model appears to be a marker of effector function and not exhaustion. PMID:25119341

  5. The Us3 Protein of Herpes Simplex Virus 1 Inhibits T Cell Signaling by Confining Linker for Activation of T Cells (LAT) Activation via TRAF6 Protein*

    PubMed Central

    Yang, Yin; Wu, Songfang; Wang, Yu; Pan, Shuang; Lan, Bei; Liu, Yaohui; Zhang, Liming; Leng, Qianli; Chen, Da; Zhang, Cuizhu; He, Bin; Cao, Youjia

    2015-01-01

    Herpes simplex virus 1 (HSV-1) is the most prevalent human virus and causes global morbidity because the virus is able to infect multiple cell types. Remarkably, HSV infection switches between lytic and latent cycles, where T cells play a critical role. However, the precise way of virus-host interactions is incompletely understood. Here we report that HSV-1 productively infected Jurkat T-cells and inhibited antigen-induced T cell receptor activation. We discovered that HSV-1-encoded Us3 protein interrupted TCR signaling and interleukin-2 production by inactivation of the linker for activation of T cells. This study unveils a mechanism by which HSV-1 intrudes into early events of TCR-mediated cell signaling and may provide novel insights into HSV infection, during which the virus escapes from host immune surveillance. PMID:25907557

  6. Homocysteine activates T cells by enhancing endoplasmic reticulum-mitochondria coupling and increasing mitochondrial respiration.

    PubMed

    Feng, Juan; Lü, Silin; Ding, Yanhong; Zheng, Ming; Wang, Xian

    2016-06-01

    Hyperhomocysteinemia (HHcy) accelerates atherosclerosis by increasing proliferation and stimulating cytokine secretion in T cells. However, whether homocysteine (Hcy)-mediated T cell activation is associated with metabolic reprogramming is unclear. Here, our in vivo and in vitro studies showed that Hcy-stimulated splenic T-cell activation in mice was accompanied by increased levels of mitochondrial reactive oxygen species (ROS) and calcium, mitochondrial mass and respiration. Inhibiting mitochondrial ROS production and calcium signals or blocking mitochondrial respiration largely blunted Hcy-induced T-cell interferon γ (IFN-γ) secretion and proliferation. Hcy also enhanced endoplasmic reticulum (ER) stress in T cells, and inhibition of ER stress with 4-phenylbutyric acid blocked Hcy-induced T-cell activation. Mechanistically, Hcy increased ER-mitochondria coupling, and uncoupling ER-mitochondria by the microtubule inhibitor nocodazole attenuated Hcy-stimulated mitochondrial reprogramming, IFN-γ secretion and proliferation in T cells, suggesting that juxtaposition of ER and mitochondria is required for Hcy-promoted mitochondrial function and T-cell activation. In conclusion, Hcy promotes T-cell activation by increasing ER-mitochondria coupling and regulating metabolic reprogramming.

  7. Diacylglycerol Kinases (DGKs): Novel Targets for Improving T Cell Activity in Cancer

    PubMed Central

    Riese, Matthew J.; Moon, Edmund K.; Johnson, Bryon D.; Albelda, Steven M.

    2016-01-01

    Diacylglycerol kinases (DGKs) are a family of enzymes that catalyze the metabolism of diacylglycerol (DAG). Two isoforms of DGK, DGKα, and DGKζ, specifically regulate the pool of DAG that is generated as a second messenger after stimulation of the T cell receptor (TCR). Deletion of either isoform in mouse models results in T cells bearing a hyperresponsive phenotype and enhanced T cell activity against malignancy. Whereas, DGKζ appears to be the dominant isoform in T cells, rationale exists for targeting both isoforms individually or coordinately. Additional work is needed to rigorously identify the molecular changes that result from deletion of DGKs in order to understand how DAG contributes to T cell activation, the effect of DGK inhibition in human T cells, and to rationally develop combined immunotherapeutic strategies that target DGKs. PMID:27800476

  8. The effects of Cyclosporine A and azathioprine on human T cells activated by different costimulatory signals

    PubMed Central

    Leitner, Judith; Drobits, Karin; Pickl, Winfried F.; Majdic, Otto; Zlabinger, Gerhard; Steinberger, Peter

    2011-01-01

    Immunosuppression is an important treatment modality in transplantation and human diseases that are associated with aberrant T cell activation. There are considerable differences regarding the cellular processes targeted by the immunosuppressive drugs that are in clinical use. Drugs like azathioprine (Aza) mainly act by halting proliferation of fast dividing cells, whereas others like cyclosporine A (CsA) specifically target signaling pathways in T cells. Since the outcome of T cell responses critically depends on the quality and strength of costimulatory signals, this study has addressed the interplay between costimulation and the immunosuppressive agents CsA and Aza during the in vitro activation of human T cells. We used an experimental system that allows analyzing T cells activated in the presence of selected costimulatory ligands to study T cells stimulated via CD28, CD2, LFA-1, ICOS or 4-1BB. The mean inhibitory concentrations (IC50) for Aza and CsA were determined for the proliferation of T cells receiving different costimulatory signals as well as for T cells activated in the absence of costimulation. CD28 signals but not costimulation via CD2, 4-1BB, ICOS or LFA-1 greatly increased the IC50 for CsA. By contrast, the inhibitory effects of Aza were not influenced by T cell costimulatory signals. Our results might have implications for combining standard immunosuppressive drugs with CTLA-4Ig fusion proteins, which act by blocking CD28 costimulation. PMID:21756939

  9. The BMP Pathway Participates in Human Naive CD4+ T Cell Activation and Homeostasis

    PubMed Central

    Martínez, Víctor G.; Sacedón, Rosa; Hidalgo, Laura; Valencia, Jaris; Fernández-Sevilla, Lidia M.; Hernández-López, Carmen

    2015-01-01

    Bone Morphogenetic Proteins (BMPs) form a group of secreted factors that belongs to the TGF-β superfamily. Among different roles in a number of immune cell types, BMPs are known to regulate T cell development within the thymus, although the role of BMP signaling in human mature T cells remains elusive. In this study, we demonstrate that canonical BMP signaling is necessary during two critical events that regulate the size and function of human naive CD4+ T cell population: activation and homeostasis. Upon stimulation via TCR, naive CD4+ T cells upregulate the expression of BMP ligands triggering canonical BMP signaling in CD25+ cells. Blockade of BMP signaling severely impairs CD4+ T cell proliferation after activation mainly through regulation of IL-2, since the addition of this cytokine recuperates normal T cell expansion after inhibition of BMP signaling. Similarly, activation of canonical BMP pathway is required for both the maintenance of cell survival and the homeostatic proliferation induced by IL-7, a key factor for T cell homeostasis. Moreover, upregulation of two critical receptors for T cell homeostasis, CXCR4 and CCR9, triggered by IL-7 is also abrogated in the absence of BMP signaling. Collectively, we describe important roles of the canonical BMP signaling in human naive CD4+ T cell activation and homeostasis that could be valuable for clinical application. PMID:26110906

  10. Immune activation induces immortalization of HTLV-1 LTR-Tax transgenic CD4+ T cells.

    PubMed

    Swaims, Alison Y; Khani, Francesca; Zhang, Yingyu; Roberts, Arthur I; Devadas, Satish; Shi, Yufang; Rabson, Arnold B

    2010-10-21

    Infection with the human T-cell leukemia virus-1 (HTLV-1) results in a variety of diseases including adult T-cell leukemia/lymphoma (ATL). Although the pathogenesis of these disorders is poorly understood, it involves complex interactions with the host immune system. Activation of infected T cells may play an important role in disease pathogenesis through induction of the oncogenic HTLV-1 Tax transactivator protein. To test this hypothesis, we employed transgenic mice in which Tax is regulated by the HTLV-1 LTR. T-cell receptor stimulation of LTR-Tax CD4(+) T cells induced Tax expression, hyper-proliferation, and immortalization in culture. The transition to cellular immortalization was accompanied by markedly increased expression of the antiapoptotic gene, mcl-1, previously implicated as important in T-cell survival. Immortalized cells exhibited a CD4(+)CD25(+)CD3(-) phenotype commonly observed in ATL. Engraftment of activated LTR-Tax CD4(+) T cells into NOD/Shi-scid/IL-2Rγ null mice resulted in a leukemia-like phenotype with expansion and tissue infiltration of Tax(+), CD4(+) lymphocytes. We suggest that immune activation of infected CD4(+) T cells plays an important role in the induction of Tax expression, T-cell proliferation, and pathogenesis of ATL in HTLV-1-infected individuals. PMID:20634377

  11. IL-15 promotes activation and expansion of CD8+ T cells in HIV-1 infection

    PubMed Central

    Younes, Souheil-Antoine; Freeman, Michael L.; Mudd, Joseph C.; Shive, Carey L.; Reynaldi, Arnold; Estes, Jacob D.; Deleage, Claire; Lucero, Carissa; Anderson, Jodi; Schacker, Timothy W.; Davenport, Miles P.; McCune, Joseph M.; Hunt, Peter W.; Lee, Sulggi A.; Debernardo, Robert L.; Jacobson, Jeffrey M.; Canaday, David H.; Sekaly, Rafick-Pierre; Sieg, Scott F.; Lederman, Michael M.

    2016-01-01

    In HIV-1–infected patients, increased numbers of circulating CD8+ T cells are linked to increased risk of morbidity and mortality. Here, we identified a bystander mechanism that promotes CD8 T cell activation and expansion in untreated HIV-1–infected patients. Compared with healthy controls, untreated HIV-1–infected patients have an increased population of proliferating, granzyme B+, CD8+ T cells in circulation. Vβ expression and deep sequencing of CDR3 revealed that in untreated HIV-1 infection, cycling memory CD8 T cells possess a broad T cell repertoire that reflects the repertoire of the resting population. This suggests that cycling is driven by bystander activation, rather than specific antigen exposure. Treatment of peripheral blood mononuclear cells with IL-15 induced a cycling, granzyme B+ phenotype in CD8+ T cells. Moreover, elevated IL-15 expression in the lymph nodes of untreated HIV-1–infected patients correlated with circulating CD8+ T cell counts and was normalized in these patients following antiretroviral therapy. Together, these results suggest that IL-15 drives bystander activation of CD8+ T cells, which predicts disease progression in untreated HIV-1–infected patients and suggests that elevated IL-15 may also drive CD8+ T cell expansion that is linked to increased morbidity and mortality in treated patients. PMID:27322062

  12. Kindlin-3 regulates integrin activation and adhesion reinforcement of effector T cells.

    PubMed

    Moretti, Federico A; Moser, Markus; Lyck, Ruth; Abadier, Michael; Ruppert, Raphael; Engelhardt, Britta; Fässler, Reinhard

    2013-10-15

    Activated T cells use very late antigen-4/α4β1 integrin for capture, rolling on, and firm adhesion to endothelial cells, and use leukocyte function-associated antigen-1/αLβ2 integrin for subsequent crawling and extravasation. Inhibition of α4β1 is sufficient to prevent extravasation of activated T cells and is successfully used to combat autoimmune diseases, such as multiple sclerosis. Here we show that effector T cells lacking the integrin activator Kindlin-3 extravasate and induce experimental autoimmune encephalomyelitis in mice immunized with autoantigen. In sharp contrast, adoptively transferred autoreactive T cells from Kindlin-3-deficient mice fail to extravasate into the naïve CNS. Mechanistically, autoreactive Kindlin-3-null T cells extravasate when the CNS is inflamed and the brain microvasculature expresses high levels of integrin ligands. Flow chamber assays under physiological shear conditions confirmed that Kindlin-3-null effector T cells adhere to high concentrations of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, albeit less efficiently than WT T cells. Although these arrested T cells polarize and start crawling, only few remain firmly adherent over time. Our data demonstrate that the requirement of Kindlin-3 for effector T cells to induce α4β1 and αLβ2 integrin ligand binding and stabilization of integrin-ligand bonds is critical when integrin ligand levels are low, but of less importance when integrin ligand levels are high. PMID:24089451

  13. Essential role of CD11a in CD8+ T-cell accumulation and activation in adipose tissue

    Technology Transfer Automated Retrieval System (TEKTRAN)

    T-cells, particularly CD8+ T-cells, are major participants in obesity-linked adipose tissue inflammation. We examined the mechanisms of CD8+ T-cell accumulation and activation in adipose tissue and the role of CD11a, a beta2 integrin. CD8+ T-cells in adipose tissue of obese mice showed activated phe...

  14. Antigen conformation determines processing requirements for T-cell activation.

    PubMed Central

    Streicher, H Z; Berkower, I J; Busch, M; Gurd, F R; Berzofsky, J A

    1984-01-01

    We studied the difference in requirements for processing and presentation to a single T-cell clone of four different forms of the same epitope of sperm whale myoglobin--namely, on the native protein, on two conformationally altered forms of the protein, or as a 22-residue antigenic peptide fragment. The T-cell clone was I-Ed-restricted and specific for an epitope on the CNBr fragment 132-153 involving Lys-140. As inhibitors of macrophage processing of antigen, we used several agents that inhibit lysosomal function: the weak bases chloroquine and NH4Cl, the cationic ionophore monensin, and the competitive protease inhibitor leupeptin. When these agents were used to inhibit processing of antigen by presenting cells and then washed out before T cells were added to culture, they inhibited the presentation of native antigen but not of fragment 132-153. To our surprise, the intact but denatured form, S-methylmyoglobin, behaved like the fragment not like the native protein. Apomyoglobin was intermediate in susceptibility to inhibition. Thus, native myoglobin requires a processing step that appears to involve lysosomal proteolysis, which is not required by fragment 132-153 or the denatured unfolded forms. For an antigen the size of myoglobin (Mr 17,800), it appears that unfolding of the native conformation, rather than further reduction in size, is the critical parameter determining the need for processing. Since a major difference between native myoglobin and the other forms is the greater accessibility in the latter of sites, such as hydrophobic residues, buried in the native protein, we propose that processing may be necessary to expose these sites, perhaps for interaction with the cell membrane or the Ia of the antigen-presenting cell. PMID:6333686

  15. Complement receptor type 1 (CR1/CD35) expressed on activated human CD4+ T cells contributes to generation of regulatory T cells.

    PubMed

    Török, Katalin; Dezső, Balázs; Bencsik, András; Uzonyi, Barbara; Erdei, Anna

    2015-04-01

    The role of complement in the regulation of T cell immunity has been highlighted recently by several groups. We were prompted to reinvestigate the role of complement receptor type 1 (CR1, CD35) [corrected] in human T cells based on our earlier data showing that activated human T cells produce C3 (Torok et al. (2012) [48]) and also by results demonstrating that engagement of Membrane Cofactor Protein (MCP, CD46) induces a switch of anti-CD35-activated [corrected] helper T cells into regulatory T cells (Kemper et al. (2003) [17]). We demonstrate here that co-ligation of CD46 and CD35, [corrected] the two C3b-binding structures present on activated CD4+ human T cells significantly enhances CD25 expression, elevates granzyme B production and synergistically augments cell proliferation. The role of CR1 in the development of the Treg phenotype was further confirmed by demonstrating that its engagement enhances IL-10 production and reduces IFNγ release by the activated CD4+ T cells in the presence of excess IL-2. The functional in vivo relevance of our findings was highlighted by the immunohistochemical staining of tonsils, revealing the presence of CD4/CD35 [corrected] double positive lymphocytes mainly in the inter-follicular regions where direct contact between CD4+ T cells and B lymphocytes occurs. Regarding the in vivo relevance of the complement-dependent generation of regulatory T cells in secondary lymphoid organs we propose a scenario shown in the figure. The depicted process involves the sequential binding of locally produced C3 fragments to CD46 and CD35 [corrected] expressed on activated T cells, which - in the presence of excess IL-2 - leads to the development of Treg cells.

  16. Transmission of survival signals through Delta-like 1 on activated CD4+ T cells

    PubMed Central

    Furukawa, Takahiro; Ishifune, Chieko; Tsukumo, Shin-ichi; Hozumi, Katsuto; Maekawa, Yoichi; Matsui, Naoko; Kaji, Ryuji; Yasutomo, Koji

    2016-01-01

    Notch expressed on CD4+ T cells transduces signals that mediate their effector functions and survival. Although Notch signaling is known to be cis-inhibited by Notch ligands expressed on the same cells, the role of Notch ligands on T cells remains unclear. In this report we demonstrate that the CD4+ T cell Notch ligand Dll1 transduces signals required for their survival. Co-transfer of CD4+ T cells from Dll1−/− and control mice into recipient mice followed by immunization revealed a rapid decline of CD4+ T cells from Dll1−/− mice compared with control cells. Dll1−/− mice exhibited lower clinical scores of experimental autoimmune encephalitis than control mice. The expression of Notch target genes in CD4+ T cells from Dll1−/− mice was not affected, suggesting that Dll1 deficiency in T cells does not affect cis Notch signaling. Overexpression of the intracellular domain of Dll1 in Dll1-deficient CD4+ T cells partially rescued impaired survival. Our data demonstrate that Dll1 is an independent regulator of Notch-signaling important for the survival of activated CD4+ T cells, and provide new insight into the physiological roles of Notch ligands as well as a regulatory mechanism important for maintaining adaptive immune responses. PMID:27659682

  17. CD4+ T cell activation in multiple sclerosis.

    PubMed

    Verselis, S J; Goust, J M

    1987-02-01

    Interleukin-2 (IL-2) production by CD4-enriched T cells from multiple sclerosis (MS) patients and normal individuals stimulated with concanavalin A (conA) and/or autologous and allogeneic B lymphoid cell lines (B-LCL) was evaluated 24, 48 and 96 h after stimulation. ConA-stimulated CD4+ cells from MS patients did not produce significantly more IL-2 than normal CD4+ cells. In contrast, autologous B-LCL-induced IL-2 production by MS CD4+ cells significantly (P = 0.026) exceeded that produced by normal CD4+ cells identically stimulated after 24 h in culture. Differences in IL-2 production by CD4+ cells from MS patients reached highest significance using allogeneic B-LCL, whose stimulatory capacity was similar, whether established from normal individuals or MS patients. This increased IL-2 production in response to B-LCL may represent a supranormal response of CD4+ cells from MS patients to class II major histocompatibility (MHC)-associated stimuli. It suggests that the deficiency of suppressor T cell functions postulated to play a role in MS does not arise from a lack of IL-2 induction and might indicate that bursts of IL-2 production could play a role in MS. PMID:3492511

  18. NFAT5 Is Up-Regulated by Hypoxia: Possible Implications in Preeclampsia and Intrauterine Growth Restriction.

    PubMed

    Dobierzewska, Aneta; Palominos, Macarena; Irarrazabal, Carlos E; Sanchez, Marianela; Lozano, Mauricio; Perez-Sepulveda, Alejandra; Monteiro, Lara J; Burmeister, Yara; Figueroa-Diesel, Horacio; Rice, Gregory E; Illanes, Sebastian E

    2015-07-01

    During gestation, low oxygen environment is a major determinant of early placentation process, while persistent placental hypoxia leads to pregnancy-related complications such as preeclampsia (PE) and intrauterine growth restriction (IUGR). PE affects 5%-8% of all pregnancies worldwide and is a cause of maternal and fetal morbidity and mortality. During placental development, persistent hypoxia due to poor trophoblast invasion and reduced uteroplacental perfusion leads to maternal endothelial dysfunction and clinical manifestation of PE. Here we hypothesized that nuclear factor of activated T cells-5 (NFAT5), a well-known osmosensitive renal factor and recently characterized hypoxia-inducible protein, is also activated in vivo in placentas of PE and IUGR complications as well as in the in vitro model of trophoblast hypoxia. In JAR cells, low oxygen tension (1% O2) induced NFAT5 mRNA and increased its nuclear abundance, peaking at 16 h. This increase did not occur in parallel with the earlier HIF1A induction. Real-time PCR and Western blot analysis confirmed up-regulation of NFAT5 mRNA and NFAT5 nuclear content in human preeclamptic placentas and in rabbit placentas of an experimentally induced IUGR model, as compared with the control groups. In vitro lambda protein phosphatase (lambda PPase) treatment revealed that increased abundance of NFAT5 protein in nuclei of either JAR cells (16 h of hypoxia) or PE and IUGR placentas is at least partially due to NFAT5 phosphorylation. NFAT5 downstream targets aldose reductase (AR) and sodium-myo-inositol cotransporter (SMIT; official symbol SLC5A3) were not significantly up-regulated either in JAR cells exposed to hypoxia or in placentas of PE- and IUGR-complicated pregnancies, suggesting that hypoxia-dependent activation of NFAT5 serves as a separate function to its tonicity-dependent stimulation. In conclusion, we propose that NFAT5 may serve as a novel marker of placental hypoxia and ischemia independently of HIF1A. PMID

  19. Activated human B lymphocytes express three CTLA-4 counterreceptors that costimulate T-cell activation.

    PubMed Central

    Boussiotis, V A; Freeman, G J; Gribben, J G; Daley, J; Gray, G; Nadler, L M

    1993-01-01

    Signaling via the T-cell receptor complex is necessary but not sufficient to induce antigen-specific T lymphocytes to expand clonally. To proliferate, T cells must receive one or more costimulatory signals provided by antigen presenting cells (APCs). One such critical costimulatory signal is delivered by the CD28/CTLA-4 counterreceptor, B7, expressed on APCs. B7 costimulation induces CD28 signaling, resulting in interleukin 2 (IL-2) secretion, and T-cell proliferation. Conversely, T-cell receptor signaling in the absence of B7 costimulation results in induction of antigen-specific tolerance. Here, we show that activated human B lymphocytes express two additional CTLA-4 counterreceptors also capable of providing T-cell costimulation. At 24 hr postactivation, B cells express a CTLA-4 counterreceptor not recognized by anti-B7 or -BB-1 monoclonal antibodies (mAbs), which induces detectable IL-2 secretion and T-cell proliferation. At 48 and 72 hr postactivation, B cells express both B7 and a third CTLA-4 counterreceptor identified by the anti-BB-1 mAb. BB-1 appears to be a molecule distinct from B7 by its expression on B7- cells and its capacity to induce T cells to proliferate without significant accumulation of IL-2. As observed for B7, costimulatory signals mediated by these alternative CTLA-4/CD28 counterreceptors are likely to be essential for generation of an immune response and their absence may result in antigen-specific tolerance. We propose the following terminology for these CTLA-4 counterreceptors: (i) B7, B7-1; (ii) early CTLA-4 binding counterreceptor, B7-2; and (iii) BB-1, B7-3. PMID:7504293

  20. Versatile strategy for controlling the specificity and activity of engineered T cells.

    PubMed

    Ma, Jennifer S Y; Kim, Ji Young; Kazane, Stephanie A; Choi, Sei-Hyun; Yun, Hwa Young; Kim, Min Soo; Rodgers, David T; Pugh, Holly M; Singer, Oded; Sun, Sophie B; Fonslow, Bryan R; Kochenderfer, James N; Wright, Timothy M; Schultz, Peter G; Young, Travis S; Kim, Chan Hyuk; Cao, Yu

    2016-01-26

    The adoptive transfer of autologous T cells engineered to express a chimeric antigen receptor (CAR) has emerged as a promising cancer therapy. Despite impressive clinical efficacy, the general application of current CAR-T--cell therapy is limited by serious treatment-related toxicities. One approach to improve the safety of CAR-T cells involves making their activation and proliferation dependent upon adaptor molecules that mediate formation of the immunological synapse between the target cancer cell and T-cell. Here, we describe the design and synthesis of structurally defined semisynthetic adaptors we refer to as "switch" molecules, in which anti-CD19 and anti-CD22 antibody fragments are site-specifically modified with FITC using genetically encoded noncanonical amino acids. This approach allows the precise control over the geometry and stoichiometry of complex formation between CD19- or CD22-expressing cancer cells and a "universal" anti-FITC-directed CAR-T cell. Optimization of this CAR-switch combination results in potent, dose-dependent in vivo antitumor activity in xenograft models. The advantage of being able to titrate CAR-T-cell in vivo activity was further evidenced by reduced in vivo toxicity and the elimination of persistent B-cell aplasia in immune-competent mice. The ability to control CAR-T cell and cancer cell interactions using intermediate switch molecules may expand the scope of engineered T-cell therapy to solid tumors, as well as indications beyond cancer therapy. PMID:26759368

  1. Interleukin-13 interferes with activation-induced t-cell apoptosis by repressing p53 expression

    PubMed Central

    Yang, Li; Xu, Ling-Zhi; Liu, Zhi-Qiang; Yang, Gui; Geng, Xiao-Rui; Mo, Li-Hua; Liu, Zhi-Gang; Zheng, Peng-Yuan; Yang, Ping-Chang

    2016-01-01

    The etiology and the underlying mechanism of CD4+ T-cell polarization are unclear. This study sought to investigate the mechanism by which interleukin (IL)-13 prevents the activation-induced apoptosis of CD4+ T cells. Here we report that CD4+ T cells expressed IL-13 receptor α2 in the intestine of sensitized mice. IL-13 suppressed both the activation-induced apoptosis of CD4+ T cells and the expression of p53 and FasL. Exposure to recombinant IL-13 inhibited activation-induced cell death (AICD) along with the expression of p53, caspase 3, and tumor necrosis factor-α in CD4+ T cells. Administration of an anti-IL-13 antibody enhanced the effect of specific immunotherapy on allergic inflammation in the mouse intestine, enforced the expression of p53 in intestinal CD4+ T cells, and enhanced the frequency of CD4+ T-cell apoptosis upon challenge with specific antigens. In summary, blocking IL-13 enhances the therapeutic effect of antigen-specific immunotherapy by regulating apoptosis and thereby enforcing AICD in CD4+ T cells. PMID:26189367

  2. Form and pattern of MUC1 expression on T cells activated in vivo or in vitro suggests a function in T-cell migration

    PubMed Central

    Correa, Isabel; Plunkett, Tim; Vlad, Anda; Mungul, Arron; Candelora-Kettel, Jessica; Burchell, Joy M; Taylor–papadimitriou, Joyce; Finn, Olivera J

    2003-01-01

    MUC1 is a transmembrane mucin that is expressed on ductal epithelial cells and epithelial malignancies and has been proposed as a target antigen for immunotherapy. The expression of MUC1 has recently been reported on T and B cells. In this study we demonstrate that following activation in vivo or activation by different stimuli in vitro, human T cells expressed MUC1 at the cell surface. However, the level of expression in activated human T cells was significantly lower than that seen on normal epithelial cells or on breast cancer cells. In contrast, resting T cells did not bind MUC1-specific monoclonal antibodies (mAbs), nor was MUC1 mRNA detectable by reverse transcription–polymerase chain reaction (RT–PCR) or Northern blot analysis in these cells. The profile of activated T-cell reactivity with different MUC1-specific antibodies suggested that the glycoform of MUC1 expressed by the activated T cells carried core 2-based O-glycans, as opposed to the core 1 structures that dominate in the cancer-associated mucin. Confocal microscopy revealed that MUC1 was uniformly distributed on the surface of activated T cells. However, when the cells were polarized in response to a migratory chemokine, MUC1 was found on the leading edge rather than on the uropod, where other large mucin-like molecules on T cells are trafficked. The concentration of MUC1 at the leading edge of polarized activated human T cells suggests that MUC1 could be involved in early interactions between T cells and endothelial cells at inflammatory sites. PMID:12519300

  3. The lncRNA NRON modulates HIV-1 replication in a NFAT-dependent manner and is differentially regulated by early and late viral proteins.

    PubMed

    Imam, Hasan; Bano, Aalia Shahr; Patel, Paresh; Holla, Prasida; Jameel, Shahid

    2015-03-02

    A majority of the human genome is transcribed into noncoding RNAs, of which the functions of long noncoding RNAs (lncRNAs) are poorly understood. Many host proteins and RNAs have been characterized for their roles in HIV/AIDS pathogenesis, but there is only one lncRNA, NEAT1, which is shown to affect the HIV-1 life cycle. We profiled 90 disease-related lncRNAs and found NRON (noncoding repressor of Nuclear Factor of Activated T cells [NFAT]) to be one of several lncRNAs whose expression was significantly altered following HIV-1 infection. The regulation of NRON expression during the HIV-1 life cycle was complex; its levels were reduced by the early viral accessory protein Nef and increased by the late protein Vpu. Consequently, Nef and Vpu also modulated activity of the transcription factor NFAT. The knockdown of NRON enhanced HIV-1 replication through increased activity of NFAT and the viral LTR. Using siRNA-mediated NFAT knockdown, we show the effects of NRON on HIV-1 replication to be mediated by NFAT, and the viral Nef and Vpu proteins to modulate NFAT activity through their effects on NRON. These findings add the lncRNA, NRON to the vast repertoire of host factors utilized by HIV for infection and persistence.

  4. Toward immunotherapy with redirected T cells in a large animal model: ex vivo activation, expansion, and genetic modification of canine T cells.

    PubMed

    Mata, Melinda; Vera, Juan F; Gerken, Claudia; Rooney, Cliona M; Miller, Tasha; Pfent, Catherine; Wang, Lisa L; Wilson-Robles, Heather M; Gottschalk, Stephen

    2014-10-01

    Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) has shown promising antitumor activity in early phase clinical studies, especially for hematological malignancies. However, most preclinical models do not reliably mimic human disease. We reasoned that developing an adoptive T-cell therapy approach for spontaneous osteosarcoma (OS) occurring in dogs would more closely reproduce the condition in human cancer. To generate CAR-expressing canine T cells, we developed expansion and transduction protocols that allow for the generation of sufficient numbers of CAR-expressing canine T cells for future clinical studies in dogs within 2 weeks of ex vivo culture. To evaluate the functionality of CAR-expressing canine T cells, we targeted HER2(+) OS. We demonstrate that canine OS is positive for HER2, and that canine T cells expressing a HER2-specific CAR with human-derived transmembrane and CD28.ζ signaling domains recognize and kill HER2(+) canine OS cell lines in an antigen-dependent manner. To reduce the potential immunogenicity of the CAR, we evaluated a CAR with canine-derived transmembrane and signaling domains, and found no functional difference between human and canine CARs. Hence, we have successfully developed a strategy to generate CAR-expressing canine T cells for future preclinical studies in dogs. Testing T-cell therapies in an immunocompetent, outbred animal model may improve our ability to predict their safety and efficacy before conducting studies in humans.

  5. Halofuginone inhibits NF-kappaB and p38 MAPK in activated T cells.

    PubMed

    Leiba, M; Cahalon, L; Shimoni, A; Lider, O; Zanin-Zhorov, A; Hecht, I; Sela, U; Vlodavsky, I; Nagler, A

    2006-08-01

    Halofuginone, a low molecular weight plant alkaloid, inhibits collagen alpha1 (I) gene expression in several animal models and in patients with fibrotic disease, including scleroderma and graft-versus-host disease. In addition, halofuginone has been shown to inhibit angiogenesis and tumor progression. It was demonstrated recently that halofuginone inhibits transforming growth factor-beta (TGF-beta), an important immunomodulator. The present study was undertaken to explore the effects of halofuginone on activated T cells. Peripheral blood T cells were activated by anti-CD3 monoclonal antibodies in the absence and presence of halofuginone and assessed for nuclear factor (NF)-kappaB activity, production of tumor necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma), T cell apoptosis, chemotaxis, and phosphorylation of p38 mitogen-activated protein kinase (MAPK). A delayed-type hypersensitivity (DTH) model was applied to investigate the effect of halofuginone on T cells in vivo. Preincubation of activated peripheral blood T cells with 10-40 ng/ml halofuginone resulted in a significant dose-dependent decrease in NF-kappaB activity (80% inhibition following incubation with 40 ng halofuginone, P = 0.002). In addition, 40 ng/ml halofuginone inhibited secretion of TNF-alpha, IFN-gamma, interleukin (IL)-4, IL-13, and TGF-beta (P < 0.005). Similarly, halofuginone inhibited the phosphorylation of p38 MAPK and apoptosis in activated T cells (P = 0.0001 and 0.005, respectively). In contrast, T cell chemotaxis was not affected. Halofuginone inhibited DTH response in mice, indicating suppression of T cell-mediated inflammation in vivo. Halofuginone inhibits activated peripheral blood T cell functions and proinflammatory cytokine production through inhibition of NF-kappaB activation and p38 MAPK phosphorylation. It also inhibited DTH response in vivo, making it an attractive immunomodulator and anti-inflammatory agent. PMID:16769768

  6. Signals involved in T cell activation. I. Phorbol esters enhance responsiveness but cannot replace intact accessory cells in the induction of mitogen-stimulated T cell proliferation

    SciTech Connect

    Davis, L.; Lipsky, P.E.

    1985-11-01

    The role of accessory cells (AC) in the initiation of mitogen-induced T cell proliferation was examined by comparing the effect of intact macrophages (M phi) with that of 4-..beta..-phorbol 12-myristate 13-acetate (PMA). In high-density cultures, purified guinea pig T cells failed to proliferate in response to stimulation with phytohemagglutinin (PHA), concanavalin A (Con A), or PMA alone. The addition of M phi to PHA or Con A but not PMA-stimulated cultures restored T cell proliferation. The addition of PMA to high-density T cell cultures stimulated with PHA or Con A also permitted (/sup 3/H)thymidine incorporation, but was less effective than intact M phi in this regard. This action of PMA was dependent on the small number of Ac contaminating the T cell cultures as evidenced by the finding that PMA could not support mitogen responsiveness of T cells that had been depleted of Ia-bearing cells by panning, even when these cells were cultured at high density. A low-density culture system was used to examine in greater detail the possibility that PMA could completely substitute for M phi in promoting T cells activation. In low-density cultures, mitogen-induced T cell proliferation required intact M phi. These results support a model of T cell activation in which AC play at least two distinct roles. The initiation of the response requires a signal conveyed by an intact M phi, which cannot be provided by either a M phi supernatant factor or PMA. The response can be amplified by additional M phi or M phi supernatant factors. PMA can substitute for M phi in this regard and can provide the signal necessary for amplification of T cell proliferation supported by small numbers of intact AC.

  7. Pomegranate Polyphenols and Extract Inhibit Nuclear Factor of Activated T-Cell Activity and Microglial Activation In Vitro and in a Transgenic Mouse Model of Alzheimer Disease123

    PubMed Central

    Rojanathammanee, Lalida; Puig, Kendra L.; Combs, Colin K.

    2013-01-01

    Alzheimer disease (AD) brain is characterized by extracellular plaques of amyloid β (Aβ) peptide with reactive microglia. This study aimed to determine whether a dietary intervention could attenuate microgliosis. Memory was assessed in 12-mo-old male amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice via Barnes maze testing followed by division into either a control-fed group provided free access to normal chow and water or a treatment group provided free access to normal chow and drinking water supplemented with pomegranate extract (6.25 mL/L) for 3 mo followed by repeat Barnes maze testing for both groups. Three months of pomegranate feeding decreased the path length to escape of mice compared with their initial 12-mo values (P < 0.05) and their control-fed counterparts (P < 0.05). Brains of the 3-mo study pomegranate-fed mice had lower tumor necrosis factor α (TNF-α) concentrations (P < 0.05) and lower nuclear factor of activated T-cell (NFAT) transcriptional activity (P < 0.05) compared with controls. Brains of the 3-mo pomegranate or control mice were also compared with an additional control group of 12-mo-old mice for histologic analysis. Immunocytochemistry showed that pomegranate- but not control-fed mice had attenuated microgliosis (P < 0.05) and Aβ plaque deposition (P < 0.05) compared with 12-mo-old mice. An additional behavioral study again used 12-mo-old male APP/PS1 mice tested by T-maze followed by division into a control group provided with free access to normal chow and sugar supplemented drinking water or a treatment group provided with normal chow and pomegranate extract–supplemented drinking water (6.25 mL/L) for 1 mo followed by repeat T-maze testing in both groups. One month of pomegranate feeding increased spontaneous alternations versus control-fed mice (P < 0.05). Cell culture experiments verified that 2 polyphenol components of pomegranate extract, punicalagin and ellagic acid, attenuated NFAT activity in a reporter

  8. CD4(+) T cells from lupus-prone mice are hyperresponsive to T cell receptor engagement with low and high affinity peptide antigens: a model to explain spontaneous T cell activation in lupus.

    PubMed

    Vratsanos, G S; Jung, S; Park, Y M; Craft, J

    2001-02-01

    Polyclonal CD4(+) T cell activation is characteristic of spontaneous lupus. As a potential explanation for this phenotype, we hypothesized that T cells from lupus-prone mice are intrinsically hyperresponsive to stimulation with antigen, particularly to those peptide ligands having a low affinity for the T cell receptor (TCR). To test this hypothesis, we backcrossed the alpha and beta chain genes of the AND TCR specific for amino acids 88-104 of pigeon cytochrome C (PCC) to the Fas-intact MRL/Mp(+)(Fas-lpr) and to the H-2(k)-matched control backgrounds B10.BR and CBA/CaJ (MRL.AND, B10.AND, and CBA.AND, respectively), and assessed naive CD4(+) TCR transgenic T cell activation in vitro after its encounter with cognate antigen and lower affinity altered peptide ligands (APLs). MRL.AND T cells, compared with control B10.AND and CBA.AND cells, proliferated more when stimulated with agonist antigen. More strikingly, MRL.AND T cells proliferated significantly more and produced more interleukin 2 when stimulated with the APLs of PCC 88-104, having lower affinity for the transgenic TCR. These results imply that one of the forces driving polyclonal activation of alpha/beta T cells in lupus is an intrinsically heightened response to peptide antigen, particularly those with low affinity for the TCR, independent of the nature of the antigen-presenting cell and degree of costimulation.

  9. Role of monocyte fucose-receptors in T-cell fibronectin activity.

    PubMed Central

    Donson, J; Mandy, K; Feng, Z H; Mandy, S; Brown, E J; Godfrey, H P

    1991-01-01

    T-cell fibronectin (FN) is a lymphokine produced by antigen- and mitogen-activated T cells that agglutinates human monocytes at femtomolar concentrations. This extreme degree of activity derives from co-operative interactions between multiple FN domains and multiple monocyte integrin protein receptors. T-cell FN, like other FN, is a glycoprotein. The role interactions between T-cell FN carbohydrate and lectin-like monocyte surface receptors play in mediating T-cell FN activity was studied by determining the ability of monosaccharides to inhibit T-cell FN activity. L-Fucose and L-rhamnose significantly inhibited T-cell FN-mediated monocyte agglutination at concentrations as low as 0.01 mM; D-glucose, D- or L-galactose, D- or L-mannose and D-fucose were not inhibitory at 10-100 mM. This inhibition appeared to be due to interference with the binding of T-cell FN fucose residues to monocyte fucose receptors since: (i) treatment of T-cell FN with alpha-L-fucosidase abolished its agglutinating activity for human monocytes, while treatment with beta-D-galactosidase or with alpha-L-fucosidase in the presence of L-fucose had no effect; (ii) treatment of monocytes with alpha-L-fucosidase did not affect their response to T-cell FN; and (iii) L-fucose or L-rhamnose did not alter the expression of monocyte integrin FN receptors under conditions where T-cell FN-mediated monocyte agglutination was completely inhibited. In vivo, 1 mumol intracutaneous L-fucose inhibited expression of delayed hypersensitivity by 30% (P much less than 0.001); similar doses of L-rhamnose inhibited responses by 10% (P less than 0.02). These data implicate a fucose receptor in monocyte response to T-cell FN, and suggest that T-cell FN is only one of the mediators involved in initiating delayed hypersensitivity reactions in vivo. PMID:1769694

  10. Expression of T-cell-activating protein in peripheral lymphocyte subsets.

    PubMed Central

    Yeh, E T; Reiser, H; Benacerraf, B; Rock, K L

    1986-01-01

    T-cell-activating protein (TAP) is an allelic 12-kDa membrane protein that participates in T-cell activation. Soluble anti-TAP monoclonal antibodies can trigger antigen-specific, major histocompatibility complex-restricted T-cell hybridomas to produce interleukin 2 and are mitogenic for normal T cells and thymocytes. TAP is expressed on 10% of thymocytes, which are mainly cortisone-resistant and mature. In the periphery, TAP is expressed on 70% of resting T cells but not on resting B cells. In this report, we analyze in detail the nature of TAP expression on peripheral lymphocyte subsets by immunofluorescence techniques. We show that all inducer (L3T4+) T cells are TAP+. In contrast, only 50% of Lyt-2+ T cells express detectable TAP. Functional studies demonstrated that at least part of the heterogeneity of TAP expression is present in the Lyt-2+ cytolytic T-cell (CTL) subset. Unstimulated CTL precursors are TAP- but are induced to express TAP in the effector state. Furthermore, this reflects actual synthesis of TAP, as TAP is detectable on activated Lyt-2+ CTLs passaged in vitro under conditions where passive acquisition can be ruled out. To extend this observation, we have studied the expression of TAP on activated T and B cells. Upon activation, all T and B cells became TAP+. Furthermore, the TAP molecules on B and T cells are indistinguishable by NaDodSO4/polyacrylamide gel electrophoresis. This suggests that TAP expression defines further heterogeneity of lymphocytes, with activation being one parameter influencing its expression. Images PMID:3020545

  11. T Cell Receptor-induced Nuclear Factor κB (NF-κB) Signaling and Transcriptional Activation Are Regulated by STIM1- and Orai1-mediated Calcium Entry.

    PubMed

    Liu, Xiaohong; Berry, Corbett T; Ruthel, Gordon; Madara, Jonathan J; MacGillivray, Katelyn; Gray, Carolyn M; Madge, Lisa A; McCorkell, Kelly A; Beiting, Daniel P; Hershberg, Uri; May, Michael J; Freedman, Bruce D

    2016-04-15

    T cell activation following antigen binding to the T cell receptor (TCR) involves the mobilization of intracellular Ca(2+) to activate the key transcription factors nuclear factor of activated T lymphocytes (NFAT) and NF-κB. The mechanism of NFAT activation by Ca(2+) has been determined. However, the role of Ca(2+) in controlling NF-κB signaling is poorly understood, and the source of Ca(2+) required for NF-κB activation is unknown. We demonstrate that TCR- but not TNF-induced NF-κB signaling upstream of IκB kinase activation absolutely requires the influx of extracellular Ca(2+) via STIM1-dependent Ca(2+) release-activated Ca(2+)/Orai channels. We further show that Ca(2+) influx controls phosphorylation of the NF-κB protein p65 on Ser-536 and that this posttranslational modification controls its nuclear localization and transcriptional activation. Notably, our data reveal that this role for Ca(2+) is entirely separate from its upstream control of IκBα degradation, thereby identifying a novel Ca(2+)-dependent distal step in TCR-induced NF-κB activation. Finally, we demonstrate that this control of distal signaling occurs via Ca(2+)-dependent PKCα-mediated phosphorylation of p65. Thus, we establish the source of Ca(2+) required for TCR-induced NF-κB activation and define a new distal Ca(2+)-dependent checkpoint in TCR-induced NF-κB signaling that has broad implications for the control of immune cell development and T cell functional specificity.

  12. Bam32: a novel mediator of Erk activation in T cells.

    PubMed

    Sommers, Connie L; Gurson, Jordan M; Surana, Rishi; Barda-Saad, Mira; Lee, Jan; Kishor, Aparna; Li, Wenmei; Gasser, Adam J; Barr, Valarie A; Miyaji, Michihiko; Love, Paul E; Samelson, Lawrence E

    2008-07-01

    Bam32 (B lymphocyte adapter molecule of 32 kDa) is an adapter protein expressed in some hematopoietic cells including B and T lymphocytes. It was previously shown that Bam32-deficient mice have defects in various aspects of B cell activation including B cell receptor (BCR)-induced Erk activation, BCR-induced proliferation and T-independent antibody responses. In this study, we have examined the role of Bam32 in T cell activation using Bam32-deficient mice. By comparing CD4(+) T cells from lymph nodes of wild-type and Bam32-deficient mice, we found that Bam32 was required for optimal TCR-induced Erk activation, cytokine production, proliferation and actin-mediated spreading of CD4(+) T cells. These results indicate a novel pathway to Erk activation in T cells involving the adapter protein Bam32.

  13. Bam32: a novel mediator of Erk activation in T cells

    PubMed Central

    Sommers, Connie L.; Gurson, Jordan M.; Surana, Rishi; Barda-Saad, Mira; Lee, Jan; Kishor, Aparna; Li, WenMei; Gasser, Adam J.; Barr, Valarie A.; Miyaji, Michihiko; Love, Paul E.; Samelson, Lawrence E.

    2009-01-01

    Bam32 (B lymphocyte adapter molecule of 32 kDa) is an adapter protein expressed in some hematopoietic cells including B and T lymphocytes. It was previously shown that Bam32-deficient mice have defects in various aspects of B cell activation including B cell receptor (BCR)-induced Erk activation, BCR-induced proliferation and T-independent antibody responses. In this study, we have examined the role of Bam32 in T cell activation using Bam32-deficient mice. By comparing CD4+ T cells from lymph nodes of wild-type and Bam32-deficient mice, we found that Bam32 was required for optimal TCR-induced Erk activation, cytokine production, proliferation and actin-mediated spreading of CD4+ T cells. These results indicate a novel pathway to Erk activation in T cells involving the adapter protein Bam32. PMID:18448454

  14. A Combined Omics Approach to Generate the Surface Atlas of Human Naive CD4+ T Cells during Early T-Cell Receptor Activation*

    PubMed Central

    Graessel, Anke; Hauck, Stefanie M.; von Toerne, Christine; Kloppmann, Edda; Goldberg, Tatyana; Koppensteiner, Herwig; Schindler, Michael; Knapp, Bettina; Krause, Linda; Dietz, Katharina; Schmidt-Weber, Carsten B.; Suttner, Kathrin

    2015-01-01

    Naive CD4+ T cells are the common precursors of multiple effector and memory T-cell subsets and possess a high plasticity in terms of differentiation potential. This stem-cell-like character is important for cell therapies aiming at regeneration of specific immunity. Cell surface proteins are crucial for recognition and response to signals mediated by other cells or environmental changes. Knowledge of cell surface proteins of human naive CD4+ T cells and their changes during the early phase of T-cell activation is urgently needed for a guided differentiation of naive T cells and may support the selection of pluripotent cells for cell therapy. Periodate oxidation and aniline-catalyzed oxime ligation technology was applied with subsequent quantitative liquid chromatography-tandem MS to generate a data set describing the surface proteome of primary human naive CD4+ T cells and to monitor dynamic changes during the early phase of activation. This led to the identification of 173 N-glycosylated surface proteins. To independently confirm the proteomic data set and to analyze the cell surface by an alternative technique a systematic phenotypic expression analysis of surface antigens via flow cytometry was performed. This screening expanded the previous data set, resulting in 229 surface proteins, which were expressed on naive unstimulated and activated CD4+ T cells. Furthermore, we generated a surface expression atlas based on transcriptome data, experimental annotation, and predicted subcellular localization, and correlated the proteomics result with this transcriptional data set. This extensive surface atlas provides an overall naive CD4+ T cell surface resource and will enable future studies aiming at a deeper understanding of mechanisms of T-cell biology allowing the identification of novel immune targets usable for the development of therapeutic treatments. PMID:25991687

  15. The azetidine derivative, KHG26792 protects against ATP-induced activation of NFAT and MAPK pathways through P2X7 receptor in microglia.

    PubMed

    Kim, Eun-A; Cho, Chang Hun; Kim, Jiae; Hahn, Hoh-Gyu; Choi, Soo Young; Yang, Seung-Ju; Cho, Sung-Woo

    2015-12-01

    Azetidine derivatives are of interest for drug development because they may be useful therapeutic agents. However, their mechanisms of action remain to be completely elucidated. Here, we have investigated the effects of 3-(naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride (KHG26792) on ATP-induced activation of NFAT and MAPK through P2X7 receptor in the BV-2 mouse microglial cell line. KHG26792 decreased ATP-induced TNF-α release from BV-2 microglia by suppressing, at least partly, P2X7 receptor stimulation. KHG26792 also inhibited the ATP-induced increase in IL-6, PGE2, NO, ROS, CXCL2, and CCL3. ATP induced NFAT activation through P2X7 receptor, with KHG26792 reducing the ATP-induced NFAT activation. KHG26792 inhibited an ATP-induced increase in iNOS protein and ERK phosphorylation. KHG26792 prevented an ATP-induced increase in MMP-9 activity through the P2X7 receptor as a result of degradation of TIMP-1 by cathepsin B. Our data provide mechanistic insights into the role of KHG26792 in the inhibition of TNF-α produced via P2X7 receptor-mediated activation of NFAT and MAPK pathways in ATP-treated BV-2 cells. This study highlights the potential use of KHG26792 as a therapeutic agent for the many diseases of the CNS related to activated microglia.

  16. Blockade of Astrocytic Calcineurin/NFAT Signaling Helps to Normalize Hippocampal Synaptic Function and Plasticity in a Rat Model of Traumatic Brain Injury

    PubMed Central

    Furman, Jennifer L.; Sompol, Pradoldej; Kraner, Susan D.; Pleiss, Melanie M.; Putman, Esther J.; Dunkerson, Jacob; Mohmmad Abdul, Hafiz; Roberts, Kelly N.; Scheff, Stephen W.

    2016-01-01

    Increasing evidence suggests that the calcineurin (CN)-dependent transcription factor NFAT (Nuclear Factor of Activated T cells) mediates deleterious effects of astrocytes in progressive neurodegenerative conditions. However, the impact of astrocytic CN/NFAT signaling on neural function/recovery after acute injury has not been investigated extensively. Using a controlled cortical impact (CCI) procedure in rats, we show that traumatic brain injury is associated with an increase in the activities of NFATs 1 and 4 in the hippocampus at 7 d after injury. NFAT4, but not NFAT1, exhibited extensive labeling in astrocytes and was found throughout the axon/dendrite layers of CA1 and the dentate gyrus. Blockade of the astrocytic CN/NFAT pathway in rats using adeno-associated virus (AAV) vectors expressing the astrocyte-specific promoter Gfa2 and the NFAT-inhibitory peptide VIVIT prevented the injury-related loss of basal CA1 synaptic strength and key synaptic proteins and reduced the susceptibility to induction of long-term depression. In conjunction with these seemingly beneficial effects, VIVIT treatment elicited a marked increase in the expression of the prosynaptogenic factor SPARCL1 (hevin), especially in hippocampal tissue ipsilateral to the CCI injury. However, in contrast to previous work on Alzheimer's mouse models, AAV-Gfa2-VIVIT had no effects on the levels of GFAP and Iba1, suggesting that synaptic benefits of VIVIT were not attributable to a reduction in glial activation per se. Together, the results implicate the astrocytic CN/NFAT4 pathway as a key mechanism for disrupting synaptic remodeling and homeostasis in the hippocampus after acute injury. SIGNIFICANCE STATEMENT Similar to microglia, astrocytes become strongly “activated” with neural damage and exhibit numerous morphologic/biochemical changes, including an increase in the expression/activity of the protein phosphatase calcineurin. Using adeno-associated virus (AAV) to inhibit the calcineurin

  17. GAD65-reactive T cells are activated in patients with autoimmune type 1a diabetes

    PubMed Central

    Viglietta, Vissia; Kent, Sally C.; Orban, Tihamer; Hafler, David A.

    2002-01-01

    Insulin-dependent type 1 diabetes is an autoimmune disease mediated by T lymphocytes recognizing pancreatic islet cell antigens. Glutamic acid decarboxylase 65 (GAD65) appears to be an important autoantigen in the disease. However, T cells from both patients with type 1 diabetes and healthy subjects vigorously proliferate in response to GAD65 stimulation ex vivo, leading us to postulate that the critical event in the onset of human diabetes is the activation of autoreactive T cells. Thus, we investigated whether GAD65-reactive T cells in patients with diabetes functioned as previously activated memory T cells, no longer requiring a second, costimulatory signal for clonal expansion. We found that in patients with new-onset type 1 diabetes, GAD65-reactive T cells were strikingly less dependent on CD28 and B7-1 costimulation to enter into cell cycle and proliferate than were equivalent cells derived from healthy controls. We hypothesize that these autoreactive T cells have been activated in vivo and have differentiated into memory cells, suggesting a pathogenic role in type 1 diabetes. In addition, we observed different effects with selective blockade of either B7-1 or B7-2 molecules; B7-1 appears to deliver a negative signal by engaging CTLA-4, while B7-2 engagement of CD28 upregulates T cell proliferation and cytokine secretion. PMID:11927616

  18. Activated T cells sustain myeloid-derived suppressor cell-mediated immune suppression

    PubMed Central

    Damuzzo, Vera; Francescato, Samuela; Pozzuoli, Assunta; Berizzi, Antonio; Mocellin, Simone; Rossi, Carlo Riccardo; Bronte, Vincenzo; Mandruzzato, Susanna

    2016-01-01

    The expansion of myeloid derived suppressor cells (MDSCs), a suppressive population able to hamper the immune response against cancer, correlates with tumor progression and overall survival in several cancer types. We have previously shown that MDSCs can be induced in vitro from precursors present in the bone marrow and observed that these cells are able to actively proliferate in the presence of activated T cells, whose activation level is critical to drive the suppressive activity of MDSCs. Here we investigated at molecular level the mechanisms involved in the interplay between MDSCs and activated T cells. We found that activated T cells secrete IL-10 following interaction with MDSCs which, in turn, activates STAT3 phosphorylation on MDSCs then leading to B7-H1 expression. We also demonstrated that B7-H1+ MDSCs are responsible for immune suppression through a mechanism involving ARG-1 and IDO expression. Finally, we show that the expression of ligands B7-H1 and MHC class II both on in vitro-induced MDSCs and on MDSCs in the tumor microenvironment of cancer patients is paralleled by an increased expression of their respective receptors PD-1 and LAG-3 on T cells, two inhibitory molecules associated with T cell dysfunction. These findings highlight key molecules and interactions responsible for the extensive cross-talk between MDSCs and activated T cells that are at the basis of immune suppression. PMID:26700461

  19. Activated T cells sustain myeloid-derived suppressor cell-mediated immune suppression.

    PubMed

    Pinton, Laura; Solito, Samantha; Damuzzo, Vera; Francescato, Samuela; Pozzuoli, Assunta; Berizzi, Antonio; Mocellin, Simone; Rossi, Carlo Riccardo; Bronte, Vincenzo; Mandruzzato, Susanna

    2016-01-12

    The expansion of myeloid derived suppressor cells (MDSCs), a suppressive population able to hamper the immune response against cancer, correlates with tumor progression and overall survival in several cancer types. We have previously shown that MDSCs can be induced in vitro from precursors present in the bone marrow and observed that these cells are able to actively proliferate in the presence of activated T cells, whose activation level is critical to drive the suppressive activity of MDSCs. Here we investigated at molecular level the mechanisms involved in the interplay between MDSCs and activated T cells. We found that activated T cells secrete IL-10 following interaction with MDSCs which, in turn, activates STAT3 phosphorylation on MDSCs then leading to B7-H1 expression. We also demonstrated that B7-H1+ MDSCs are responsible for immune suppression through a mechanism involving ARG-1 and IDO expression. Finally, we show that the expression of ligands B7-H1 and MHC class II both on in vitro-induced MDSCs and on MDSCs in the tumor microenvironment of cancer patients is paralleled by an increased expression of their respective receptors PD-1 and LAG-3 on T cells, two inhibitory molecules associated with T cell dysfunction. These findings highlight key molecules and interactions responsible for the extensive cross-talk between MDSCs and activated T cells that are at the basis of immune suppression.

  20. NFAT transcription factor regulation by urocortin II in cardiac myocytes and heart failure.

    PubMed

    Walther, Stefanie; Awad, Sawsan; Lonchyna, Vassyl A; Blatter, Lothar A

    2014-03-01

    Urocortin II (UcnII), a cardioactive peptide with beneficial effects in normal and failing hearts, is also arrhythmogenic and prohypertrophic. We demonstrated that cardiac effects are mediated by a phosphatidylinositol-3 kinase (PI3K)/Akt kinase (Akt)/endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathways. Nuclear factor of activated T-cells (NFAT) transcription factors play a key role in the regulation of gene expression in cardiac development, maintenance of an adult differentiated cardiac phenotype, and remodeling processes in cardiac hypertrophy and heart failure (HF). We tested the hypothesis that UcnII differentially regulates NFAT activity in cardiac myocytes from both normal and failing hearts through the PI3K/Akt/eNOS/NO pathway. Isoforms NFATc1 and NFATc3 revealed different basal subcellular distribution in normal and HF rabbit ventricular myocytes with a nuclear NFATc1 and a cytosolic localization of NFATc3. However, in HF, the nuclear localization of NFATc1 was less pronounced, whereas the nuclear occupancy of NFATc3 was increased. In normal myocytes, UcnII induced nuclear export of NFATc1 and attenuated NFAT-dependent transcriptional activity but did not affect the distribution of NFATc3. In HF UcnII facilitated nuclear export of both isoforms and reduced transcriptional activity. NFAT regulation was mediated by a PI3K/Akt/eNOS/NO signaling cascade that converged on the activation of several kinases, including glycogen synthase kinase-3β (GSK3β), c-Jun NH2-terminal kinase (JNK), p38 mitogen-activated kinase (p38), and PKG, resulting in phosphorylation, deactivation, and nuclear export of NFAT. In conclusion, while NFATc1 and NFATc3 reveal distinct subcellular distribution patterns, both are regulated by the UcnII-PI3K/Akt/eNOS/NO pathway that converges on the activation of NFAT kinases and NFAT inactivation. The data reconcile cardioprotective and prohypertrophic UcnII effects mediated by different NFAT isoforms.

  1. Unexpected T cell regulatory activity of anti-histone H1 autoantibody: Its mode of action in regulatory T cell-dependent and -independent manners

    SciTech Connect

    Takaoka, Yuki; Kawamoto, Seiji; Katayama, Akiko; Nakano, Toshiaki; Yamanaka, Yasushi; Takahashi, Miki; Shimada, Yayoi; Chiang, Kuei-Chen; Ohmori, Naoya; Aki, Tsunehiro; Goto, Takeshi; Sato, Shuji; Goto, Shigeru; Chen, Chao-Long; Ono, Kazuhisa

    2013-02-08

    Highlights: ► Anti-histone H1 autoantibody (anti-H1) acts on T cells to inhibit their activation. ► Anti-H1 suppresses T cell activation in Treg cell-dependent and -independent manners. ► Suboptimal dose of anti-H1 enhances suppressor function of Treg cells. ► High dose of anti-H1 directly inhibits T cell receptor signaling. -- Abstract: Induction of anti-nuclear antibodies against DNA or histones is a hallmark of autoimmune disorders, but their actual contribution to disease predisposition remains to be clarified. We have previously reported that autoantibodies against histone H1 work as a critical graft survival factor in a rat model of tolerogeneic liver transplantation. Here we show that an immunosuppressive anti-histone H1 monoclonal antibody (anti-H1 mAb) acts directly on T cells to inhibit their activation in response to T cell receptor (TCR) ligation. Intriguingly, the T cell activation inhibitory activity of anti-H1 mAb under suboptimal dosages required regulatory T (Treg) cells, while high dose stimulation with anti-H1 mAb triggered a Treg cell-independent, direct negative regulation of T cell activation upon TCR cross-linking. In the Treg cell-dependent mode of immunosuppressive action, anti-H1 mAb did not induce the expansion of CD4{sup +}Foxp3{sup +} Treg cells, but rather potentiated their regulatory capacity. These results reveal a previously unappreciated T cell regulatory role of anti-H1 autoantibody, whose overproduction is generally thought to be pathogenic in the autoimmune settings.

  2. Metabolic control of T-cell activation and death in SLE

    PubMed Central

    Fernandez, David; Perl, Andras

    2009-01-01

    Systemic lupus erythematosus (SLE) is characterized by abnormal T-cell activation and death, processes which are crucially dependent on the controlled production of reactive oxygen intermediates (ROI) and of ATP in mitochondria. The mitochondrial transmembrane potential (Δψm) has conclusively emerged as a critical checkpoint of ATP synthesis and cell death. Lupus T cells exhibit persistent elevation of Δψm or mitochondrial hyperpolarization (MHP) as well as depletion of ATP and glutathione which decrease activation-induced apoptosis and instead predispose T cells for necrosis, thus stimulating inflammation in SLE. NO-induced mitochondrial biogenesis in normal T cells accelerates the rapid phase and reduces the plateau of Ca2+ influx upon CD3/CD28 co-stimulation, thus mimicking the Ca2+ signaling profile of lupus T cells. Treatment of SLE patients with rapamycin improves disease activity, normalizes CD3/CD28-induced Ca2+ fluxing but fails to affect MHP, suggesting that altered Ca2+ fluxing is downstream or independent of mitochondrial dysfunction. Understanding the molecular basis and consequences of MHP is essential for controlling T-cell activation and death signaling in SLE. Lupus T cells exhibit mitochondrial dysfunctionMitochondrial hyperpolarization (MHP) and ATP depletion predispose lupus T cells to death by necrosis which is pro-inflammatoryMHP is caused by depletion of glutathione and exposure to nitric oxide (NO)NO-induced mitochondrial biogenesis regenerates the Ca2+ signaling profile of lupus T cellsRapamycin treatment normalizes Ca2+ fluxing but not MHP, suggesting that the mammalian target of rapamycin, acts as a sensor and effector of MHP in SLE PMID:18722557

  3. Interleukin-7 and Toll-Like Receptor 7 Induce Synergistic B Cell and T Cell Activation

    PubMed Central

    Bikker, Angela; Kruize, Aike A.; van der Wurff-Jacobs, Kim M. G.; Peters, Rogier P.; Kleinjan, Marije; Redegeld, Frank; de Jager, Wilco; Lafeber, Floris P. J. G.; van Roon, Joël A. G.

    2014-01-01

    Objectives To investigate the potential synergy of IL-7-driven T cell-dependent and TLR7-mediated B cell activation and to assess the additive effects of monocyte/macrophages in this respect. Methods Isolated CD19 B cells and CD4 T cells from healthy donors were co-cultured with TLR7 agonist (TLR7A, Gardiquimod), IL-7, or their combination with or without CD14 monocytes/macrophages (T/B/mono; 1 : 1 : 0,1). Proliferation was measured using 3H-thymidine incorporation and Ki67 expression. Activation marker (CD19, HLA-DR, CD25) expression was measured by FACS analysis. Immunoglobulins were measured by ELISA and release of cytokines was measured by Luminex assay. Results TLR7-induced B cell activation was not associated with T cell activation. IL-7-induced T cell activation alone and together with TLR7A synergistically increased numbers of both proliferating (Ki67+) B cells and T cells, which was further increased in the presence of monocytes/macrophages. This was associated by up regulation of activation markers on B cells and T cells. Additive or synergistic induction of production of immunoglobulins by TLR7 and IL-7 was associated by synergistic induction of T cell cytokines (IFNγ, IL-17A, IL-22), which was only evident in the presence of monocytes/macrophages. Conclusions IL-7-induced CD4 T cell activation and TLR7-induced B cell activation synergistically induce T helper cell cytokine and B cell immunoglobulin production, which is critically dependent on monocytes/macrophages. Our results indicate that previously described increased expression of IL-7 and TLR7 together with increased numbers of macrophages at sites of inflammation in autoimmune diseases like RA and pSS significantly contributes to enhanced lymphocyte activation. PMID:24740301

  4. CCAAT Enhancer Binding Protein and Nuclear Factor of Activated T Cells Regulate HIV-1 LTR via a Novel Conserved Downstream Site in Cells of the Monocyte-Macrophage Lineage

    PubMed Central

    Dahiya, Satinder; Liu, Yujie; Nonnemacher, Michael R.; Dampier, Will; Wigdahl, Brian

    2014-01-01

    Transcriptional control of the human immunodeficiency virus type 1 (HIV-1) promoter, the long terminal repeat (LTR), is achieved by interactions with cis-acting elements present both upstream and downstream of the start site. In silico transcription factor binding analysis of the HIV-1 subtype B LTR sequences revealed a potential downstream CCAAT enhancer binding protein (C/EBP) binding site. This binding site (+158 to+172), designated DS3, was found to be conserved in 67% of 3,858 unique subtype B LTR sequences analyzed in terms of nucleotide sequence as well as physical location in the LTR. DS3 was found to be well represented in other subtypes as well. Interestingly, DS3 overlaps with a previously identified region that bind members of the nuclear factor of activated T cells (NFAT) family of proteins. NFATc2 exhibited a higher relative affinity for DS3 as compared with members of the C/EBP family (C/EBP α and β). DS3 was able to compete efficiently with the low-affinity upstream C/EBP binding site I with respect to C/EBP binding, suggesting utilization of both NFAT and C/EBP. Moreover, cyclosporine A treatment, which has been shown to prevent dephosphorylation and nuclear translocation of NFAT isoforms, resulted in enhanced C/EBPα binding. The interactions at DS3 were also validated in an integrated HIV-1 LTR in chronically infected U1 cells. A binding knockout of DS3 demonstrated reduced HIV-1 LTR-directed transcription under both basal and interleukin-6-stimulated conditions only in cells of the monocyte-macrophage lineage cells and not in cells of T-cell origin. Thus, the events at DS3 positively regulate the HIV-1 promoter in cells of the monocyte-macrophage lineage. PMID:24551078

  5. Chronic ethanol consumption by mice results in activated splenic T cells.

    PubMed

    Song, Kejing; Coleman, Ruth A; Zhu, Xiaoyan; Alber, Carol; Ballas, Zuhair K; Waldschmidt, Thomas J; Cook, Robert T

    2002-12-01

    Previous studies have shown that T cells from human alcoholics overexpress activation or memory markers such as human leukocyte antigen-DR, CD45RO, CD57, and CD11b and may have reduced levels of CD62L. In those studies, we demonstrated that the increased CD57(+) T cell population rapidly produces interferon-gamma (IFN-gamma) and tumor necrosis factor alpha, independent of a second signal requirement, consistent with an increased effector T cell population. In contrast to the length of alcohol abuse by human alcoholics, most work with mice has involved 2-week ethanol exposures or less, which result in decreased IFN-gamma responses. In the present work, we have evaluated C57Bl/6 or BALB/c mice, which were administered 20% w/v ethanol in water for 3-13 weeks. In these mice, rapid cytoplasmic IFN-gamma expression by T cells after stimulation through the T cell receptor was significantly increased versus normals. Studies of surface-activation markers showed that T cells from chronically ethanol-fed mice had reduced CD62L expression and an increased percentage of CD44(hi) T cells. The CD44(hi) subset was largely second signal-independent for secreted IFN-gamma and interleukin (IL)-4 production at early times after stimulation. The enriched T cells of chronic ethanol mice secreted more IFN-gamma and IL-4 than controls and equivalent IL-2 at early times after stimulation (6-24 h). The overall results support the concept that in humans and mice, chronic alcohol exposure of sufficient duration results in T cell activation or sensitization in vivo and an increased percentage of the effector/memory subset.

  6. Azithromycin suppresses CD4+ T-cell activation by direct modulation of mTOR activity

    PubMed Central

    Ratzinger, F.; Haslacher, H.; Poeppl, W.; Hoermann, G.; Kovarik, J. J.; Jutz, S.; Steinberger, P.; Burgmann, H.; Pickl, W. F.; Schmetterer, K. G.

    2014-01-01

    Advanced macrolides, such as azithromycin (AZM) or clarithromycin (CLM), are antibiotics with immunomodulatory properties. Here we have sought to evaluate their in vitro influence on the activation of CD4+ T-cells. Isolated CD4+ T-cells were stimulated with agonistic anti-CD3/anti-CD28 monoclonal antibodies in the presence of 0.6 mg/L, 2.5 mg/L, 10 mg/L or 40 mg/L AZM or CLM. Cell proliferation, cytokine level in supernatants and cell viability was assessed. Intracellular signaling pathways were evaluated using reporter cell lines, FACS analysis, immunoblotting and in vitro kinase assays. AZM inhibited cell proliferation rate and cytokine secretion of CD4+ T-cells in a dose-dependent manner. Similarly, high concentrations of CLM (40 mg/L) also suppressed these T-cell functions. Analysis of molecular signaling pathways revealed that exposure to AZM reduced the phosphorylation of the S6 ribosomal protein, a downstream target of mTOR. This effect was also observed at 40 mg/L CLM. In vitro kinase studies using recombinant mTOR showed that AZM inhibited mTOR activity. In contrast to rapamycin, this inhibition was independent of FKBP12. We show for the first time that AZM and to a lesser extent CLM act as immunosuppressive agents on CD4+ T-cells by inhibiting mTOR activity. Our results might have implications for the clinical use of macrolides. PMID:25500904

  7. Janus particles as artificial antigen-presenting cells for T cell activation.

    PubMed

    Chen, Bo; Jia, Yilong; Gao, Yuan; Sanchez, Lucero; Anthony, Stephen M; Yu, Yan

    2014-01-01

    Here we show that the multifunctionality of Janus particles can be exploited for in vitro T cell activation. We engineer bifunctional Janus particles on which the spatial distribution of two ligands, anti-CD3 and fibronectin, mimics the "bull's eye" protein pattern formed in the membrane junction between a T cell and an antigen-presenting cell. Different levels of T cell activation can be achieved by simply switching the spatial distribution of the two ligands on the surfaces of the "bull's eye" particles. We find that the ligand pattern also affects clustering of intracellular proteins. This study demonstrates that anisotropic particles, such as Janus particles, can be developed as artificial antigen-presenting cells for modulating T cell activation. PMID:25343426

  8. Loss of interferon-induced Stat1 phosphorylation in activated T cells.

    PubMed

    Van De Wiele, C Justin; Marino, Julie H; Whetsell, Michael E; Vo, Stephen S; Masengale, Rhonda M; Teague, T Kent

    2004-03-01

    Modulation of cytokine responsiveness following T cell activation represents an important mechanism that shapes the fate of T cells after encounters with antigens. We activated T cells in mice with superantigen and assessed their ability to phosphorylate Stat1 in response to interferon-gamma (IFN-gamma) and IFN-alpha. After 4 h of activation in vivo, T cells became deficient in their ability to phosphorylate Stat1 in response to either cytokine. The loss of IFN sensitivity was accompanied by increased mRNA transcription for multiple suppressors of cytokine signaling (SOCS) genes (SOCS1, SOCS3, and SOCS7). The transcript levels of these SOCS were elevated only during the early hours after activation and were at or below normal levels by 60 h. Likewise, the activation-induced inhibition of IFN-alpha signaling was transient, and sensitivity was restored by 3 days postactivation. The loss of sensitivity to IFN-gamma persisted, however, and was still evident at 3 days. These data suggest that SOCS-independent mechanisms specific for inhibition of IFN-gamma signaling may be present at later stages of the T cell response. The loss of Stat1 signaling may be a factor in differentiation of T cells during and after activation, and it could also represent a protective mechanism against the toxic effects of IFN-gamma during immune responses.

  9. Epigenetic mechanisms, T-cell activation, and CCR5 genetics interact to regulate T-cell expression of CCR5, the major HIV-1 coreceptor

    PubMed Central

    Gornalusse, German G.; Mummidi, Srinivas; Gaitan, Alvaro A.; Jimenez, Fabio; Ramsuran, Veron; Picton, Anabela; Rogers, Kristen; Manoharan, Muthu Saravanan; Avadhanam, Nymisha; Murthy, Krishna K.; Martinez, Hernan; Molano Murillo, Angela; Chykarenko, Zoya A.; Hutt, Richard; Daskalakis, Demetre; Shostakovich-Koretskaya, Ludmila; Abdool Karim, Salim; Martin, Jeffrey N.; Deeks, Steven G.; Hecht, Frederick; Sinclair, Elizabeth; Clark, Robert A.; Okulicz, Jason; Valentine, Fred T.; Martinson, Neil; Tiemessen, Caroline Tanya; Ndung’u, Thumbi; Hunt, Peter W.; He, Weijing; Ahuja, Sunil K.

    2015-01-01

    T-cell expression levels of CC chemokine receptor 5 (CCR5) are a critical determinant of HIV/AIDS susceptibility, and manifest wide variations (i) between T-cell subsets and among individuals and (ii) in T-cell activation-induced increases in expression levels. We demonstrate that a unifying mechanism for this variation is differences in constitutive and T-cell activation-induced DNA methylation status of CCR5 cis-regulatory regions (cis-regions). Commencing at an evolutionarily conserved CpG (CpG −41), CCR5 cis-regions manifest lower vs. higher methylation in T cells with higher vs. lower CCR5 levels (memory vs. naïve T cells) and in memory T cells with higher vs. lower CCR5 levels. HIV-related and in vitro induced T-cell activation is associated with demethylation of these cis-regions. CCR5 haplotypes associated with increased vs. decreased gene/surface expression levels and HIV/AIDS susceptibility magnify vs. dampen T-cell activation-associated demethylation. Methylation status of CCR5 intron 2 explains a larger proportion of the variation in CCR5 levels than genotype or T-cell activation. The ancestral, protective CCR5-HHA haplotype bears a polymorphism at CpG −41 that is (i) specific to southern Africa, (ii) abrogates binding of the transcription factor CREB1 to this cis-region, and (iii) exhibits a trend for overrepresentation in persons with reduced susceptibility to HIV and disease progression. Genotypes lacking the CCR5-Δ32 mutation but with hypermethylated cis-regions have CCR5 levels similar to genotypes heterozygous for CCR5-Δ32. In HIV-infected individuals, CCR5 cis-regions remain demethylated, despite restoration of CD4+ counts (≥800 cells per mm3) with antiretroviral therapy. Thus, methylation content of CCR5 cis-regions is a central epigenetic determinant of T-cell CCR5 levels, and possibly HIV-related outcomes. PMID:26307764

  10. Epigenetic mechanisms, T-cell activation, and CCR5 genetics interact to regulate T-cell expression of CCR5, the major HIV-1 coreceptor.

    PubMed

    Gornalusse, German G; Mummidi, Srinivas; Gaitan, Alvaro A; Jimenez, Fabio; Ramsuran, Veron; Picton, Anabela; Rogers, Kristen; Manoharan, Muthu Saravanan; Avadhanam, Nymisha; Murthy, Krishna K; Martinez, Hernan; Molano Murillo, Angela; Chykarenko, Zoya A; Hutt, Richard; Daskalakis, Demetre; Shostakovich-Koretskaya, Ludmila; Abdool Karim, Salim; Martin, Jeffrey N; Deeks, Steven G; Hecht, Frederick; Sinclair, Elizabeth; Clark, Robert A; Okulicz, Jason; Valentine, Fred T; Martinson, Neil; Tiemessen, Caroline Tanya; Ndung'u, Thumbi; Hunt, Peter W; He, Weijing; Ahuja, Sunil K

    2015-08-25

    T-cell expression levels of CC chemokine receptor 5 (CCR5) are a critical determinant of HIV/AIDS susceptibility, and manifest wide variations (i) between T-cell subsets and among individuals and (ii) in T-cell activation-induced increases in expression levels. We demonstrate that a unifying mechanism for this variation is differences in constitutive and T-cell activation-induced DNA methylation status of CCR5 cis-regulatory regions (cis-regions). Commencing at an evolutionarily conserved CpG (CpG -41), CCR5 cis-regions manifest lower vs. higher methylation in T cells with higher vs. lower CCR5 levels (memory vs. naïve T cells) and in memory T cells with higher vs. lower CCR5 levels. HIV-related and in vitro induced T-cell activation is associated with demethylation of these cis-regions. CCR5 haplotypes associated with increased vs. decreased gene/surface expression levels and HIV/AIDS susceptibility magnify vs. dampen T-cell activation-associated demethylation. Methylation status of CCR5 intron 2 explains a larger proportion of the variation in CCR5 levels than genotype or T-cell activation. The ancestral, protective CCR5-HHA haplotype bears a polymorphism at CpG -41 that is (i) specific to southern Africa, (ii) abrogates binding of the transcription factor CREB1 to this cis-region, and (iii) exhibits a trend for overrepresentation in persons with reduced susceptibility to HIV and disease progression. Genotypes lacking the CCR5-Δ32 mutation but with hypermethylated cis-regions have CCR5 levels similar to genotypes heterozygous for CCR5-Δ32. In HIV-infected individuals, CCR5 cis-regions remain demethylated, despite restoration of CD4+ counts (≥800 cells per mm(3)) with antiretroviral therapy. Thus, methylation content of CCR5 cis-regions is a central epigenetic determinant of T-cell CCR5 levels, and possibly HIV-related outcomes.

  11. Phagocytosis-dependent activation of a TLR9–BTK–calcineurin–NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus

    PubMed Central

    Herbst, Susanne; Shah, Anand; Mazon Moya, Maria; Marzola, Vanessa; Jensen, Barbara; Reed, Anna; Birrell, Mark A; Saijo, Shinobu; Mostowy, Serge; Shaunak, Sunil; Armstrong-James, Darius

    2015-01-01

    Transplant recipients on calcineurin inhibitors are at high risk of invasive fungal infection. Understanding how calcineurin inhibitors impair fungal immunity is a key priority for defining risk of infection. Here, we show that the calcineurin inhibitor tacrolimus impairs clearance of the major mould pathogen Aspergillus fumigatus from the airway, by inhibiting macrophage inflammatory responses. This leads to defective early neutrophil recruitment and fungal clearance. We confirm these findings in zebrafish, showing an evolutionarily conserved role for calcineurin signalling in neutrophil recruitment during inflammation. We find that calcineurin–NFAT activation is phagocytosis dependent and collaborates with NF-κB for TNF-α production. For yeast zymosan particles, activation of macrophage calcineurin–NFAT occurs via the phagocytic Dectin-1–spleen tyrosine kinase pathway, but for A. fumigatus, activation occurs via a phagosomal TLR9-dependent and Bruton's tyrosine kinase-dependent signalling pathway that is independent of MyD88. We confirm the collaboration between NFAT and NF-κB for TNF-α production in primary alveolar macrophages. These observations identify inhibition of a newly discovered macrophage TLR9–BTK–calcineurin–NFAT signalling pathway as a key immune defect that leads to organ transplant-related invasive aspergillosis. PMID:25637383

  12. Angiotensin II activates the calcineurin/NFAT signaling pathway and induces cyclooxygenase-2 expression in rat endometrial stromal cells.

    PubMed

    Abraham, Florencia; Sacerdoti, Flavia; De León, Romina; Gentile, Teresa; Canellada, Andrea

    2012-01-01

    Cyclooxygenase (COX)-2, the inducible isoform of cyclooxygenase, plays a role in the process of uterine decidualization and blastocyst attachment. On the other hand, overexpression of COX-2 is involved in the proliferation of the endometrial tissue during endometriosis. Deregulation of the renin-angiotensin-system plays a role in the pathophysiology of endometriosis and pre-eclampsia. Angiotensin II increases intracellular Ca(2+) concentration by targeting phospholypase C-gamma in endometrial stromal cells (ESC). A key element of the cellular response to Ca(2+) signals is the activity of the Ca(2+)- and calmodulin-dependent phosphatase calcineurin. Our first aim was to study whether angiotensin II stimulated Cox-2 gene expression in rat ESC and to analyze whether calcineurin activity was involved. In cells isolated from non-pregnant uteri, COX-2 expression--both mRNA and protein--was induced by co-stimulation with phorbol ester and calcium ionophore (PIo), as well as by angiotensin II. Pretreatment with the calcineurin inhibitor cyclosporin A inhibited this induction. We further analyzed the role of the calcineurin/NFAT signaling pathway in the induction of Cox-2 gene expression in non-pregnant rat ESC. Cyclosporin A abolished NFATc1 dephosphorylation and translocation to the nucleus. Cyclosporin A also inhibited the transcriptional activity driven by the Cox-2 promoter. Exogenous expression of the peptide VIVIT -specific inhibitor of calcineurin/NFAT binding- blocked the activation of Cox-2 promoter and the up-regulation of COX-2 protein in these cells. Finally we analyzed Cox-2 gene expression in ESC of early-pregnant rats. COX-2 expression--both mRNA and protein--was induced by stimulation with PIo as well as by angiotensin II. This induction appears to be calcineurin independent, since it was not abrogated by cyclosporin A. In conclusion, angiotensin II induced Cox-2 gene expression by activating the calcineurin/NFAT signaling pathway in endometrial stromal

  13. Angiotensin II Activates the Calcineurin/NFAT Signaling Pathway and Induces Cyclooxygenase-2 Expression in Rat Endometrial Stromal Cells

    PubMed Central

    Abraham, Florencia; Sacerdoti, Flavia; De León, Romina; Gentile, Teresa; Canellada, Andrea

    2012-01-01

    Cyclooxygenase (COX)-2, the inducible isoform of cyclooxygenase, plays a role in the process of uterine decidualization and blastocyst attachment. On the other hand, overexpression of COX-2 is involved in the proliferation of the endometrial tissue during endometriosis. Deregulation of the renin-angiotensin-system plays a role in the pathophysiology of endometriosis and pre-eclampsia. Angiotensin II increases intracellular Ca2+ concentration by targeting phospholypase C-gamma in endometrial stromal cells (ESC). A key element of the cellular response to Ca2+ signals is the activity of the Ca2+- and calmodulin-dependent phosphatase calcineurin. Our first aim was to study whether angiotensin II stimulated Cox-2 gene expression in rat ESC and to analyze whether calcineurin activity was involved. In cells isolated from non-pregnant uteri, COX-2 expression -both mRNA and protein- was induced by co-stimulation with phorbol ester and calcium ionophore (PIo), as well as by angiotensin II. Pretreatment with the calcineurin inhibitor cyclosporin A inhibited this induction. We further analyzed the role of the calcineurin/NFAT signaling pathway in the induction of Cox-2 gene expression in non-pregnant rat ESC. Cyclosporin A abolished NFATc1 dephosphorylation and translocation to the nucleus. Cyclosporin A also inhibited the transcriptional activity driven by the Cox-2 promoter. Exogenous expression of the peptide VIVIT -specific inhibitor of calcineurin/NFAT binding- blocked the activation of Cox-2 promoter and the up-regulation of COX-2 protein in these cells. Finally we analyzed Cox-2 gene expression in ESC of early-pregnant rats. COX-2 expression -both mRNA and protein- was induced by stimulation with PIo as well as by angiotensin II. This induction appears to be calcineurin independent, since it was not abrogated by cyclosporin A. In conclusion, angiotensin II induced Cox-2 gene expression by activating the calcineurin/NFAT signaling pathway in endometrial stromal cells of

  14. Phosphoantigen Burst upon Plasmodium falciparum Schizont Rupture Can Distantly Activate Vγ9Vδ2 T Cells.

    PubMed

    Guenot, Marianne; Loizon, Séverine; Howard, Jennifer; Costa, Giulia; Baker, David A; Mohabeer, Shaneel Y; Troye-Blomberg, Marita; Moreau, Jean-François; Déchanet-Merville, Julie; Mercereau-Puijalon, Odile; Mamani-Matsuda, Maria; Behr, Charlotte

    2015-10-01

    Malaria induces potent activation and expansion of the Vγ9Vδ2 subpopulation of γδT cells, which inhibit the Plasmodium falciparum blood cycle through soluble cytotoxic mediators, abrogating merozoite invasion capacity. Intraerythrocytic stages efficiently trigger Vγ9Vδ2 T-cell activation and degranulation through poorly understood mechanisms. P. falciparum blood-stage extracts are known to contain phosphoantigens able to stimulate Vγ9Vδ2 T cells, but how these are presented by intact infected red blood cells (iRBCs) remains elusive. Here we show that, unlike activation by phosphoantigen-expressing cells, Vγ9Vδ2 T-cell activation by intact iRBCs is independent of butyrophilin expression by the iRBC, and contact with an intact iRBC is not required. Moreover, blood-stage culture supernatants proved to be as potent activators of Vγ9Vδ2 T cells as iRBCs. Bioactivity in the microenvironment is attributable to phosphoantigens, as it is dependent on the parasite DOXP pathway, on Vγ9Vδ2 TCR signaling, and on butyrophilin expression by Vγ9Vδ2 T cells. Kinetic studies showed that the phosphoantigens were released at the end of the intraerythrocytic cycle at the time of parasite egress. We document exquisite sensitivity of Vγ9Vδ2 T cells, which respond to a few thousand parasites. These data unravel a novel framework, whereby release of phosphoantigens into the extracellular milieu by sequestered parasites likely promotes activation of distant Vγ9Vδ2 T cells that in turn exert remote antiparasitic functions.

  15. Reduced Frequency of Memory T Cells and Increased Th17 Responses in Patients with Active Tuberculosis

    PubMed Central

    Marín, Nancy D.; París, Sara C.; Rojas, Mauricio

    2012-01-01

    Phenotypic and functional alterations in Mycobacterium tuberculosis T cell subsets have been reported in patients with active tuberculosis. A better understanding of these alterations will increase the knowledge about immunopathogenesis and also may contribute to the development of new diagnostics and prophylactic strategies. Here, the ex vivo phenotype of CD4+ and CD8+ T cells and the frequency and phenotype of gamma interferon (IFN-γ)- and interleukin 17 (IL-17)-producing cells elicited in short-term and long-term cultures following CFP-10 and purified protein derivative (PPD) stimulation were determined in noninfected persons (non-TBi), latently infected persons (LTBi), and patients with active tuberculosis (ATB). Phenotypic characterization of T cells was done based on the expression of CD45RO and CD27. Results show that ATB had a reduced frequency of circulating CD4+ CD45RO+ CD27+ T cells and an increased frequency of CD4+ CD45RO− CD27+ T cells. ATB also had a higher frequency of circulating IL-17-producing CD4+ T cells than did LTBi after PPD stimulation, whereas LTBi had more IFN-γ-producing CD4+ T cells than did non-TBi. The phenotype of IFN-γ-producing cells at 24 h differs from the phenotype of IL-17-producing cells with no differences between LTBi and ATB. At 144 h, IFN-γ- and IL-17-producing cells were mainly CD45RO+ CD27+ T cells and they were more frequent in ATB. These results suggest that M. tuberculosis infection induces alterations in T cells which interfere with an adequate specific immune response. PMID:22914361

  16. Reduced frequency of memory T cells and increased Th17 responses in patients with active tuberculosis.

    PubMed

    Marín, Nancy D; París, Sara C; Rojas, Mauricio; García, Luis F

    2012-10-01

    Phenotypic and functional alterations in Mycobacterium tuberculosis T cell subsets have been reported in patients with active tuberculosis. A better understanding of these alterations will increase the knowledge about immunopathogenesis and also may contribute to the development of new diagnostics and prophylactic strategies. Here, the ex vivo phenotype of CD4(+) and CD8(+) T cells and the frequency and phenotype of gamma interferon (IFN-γ)- and interleukin 17 (IL-17)-producing cells elicited in short-term and long-term cultures following CFP-10 and purified protein derivative (PPD) stimulation were determined in noninfected persons (non-TBi), latently infected persons (LTBi), and patients with active tuberculosis (ATB). Phenotypic characterization of T cells was done based on the expression of CD45RO and CD27. Results show that ATB had a reduced frequency of circulating CD4(+) CD45RO(+) CD27(+) T cells and an increased frequency of CD4(+) CD45RO(-) CD27(+) T cells. ATB also had a higher frequency of circulating IL-17-producing CD4(+) T cells than did LTBi after PPD stimulation, whereas LTBi had more IFN-γ-producing CD4(+) T cells than did non-TBi. The phenotype of IFN-γ-producing cells at 24 h differs from the phenotype of IL-17-producing cells with no differences between LTBi and ATB. At 144 h, IFN-γ- and IL-17-producing cells were mainly CD45RO(+) CD27(+) T cells and they were more frequent in ATB. These results suggest that M. tuberculosis infection induces alterations in T cells which interfere with an adequate specific immune response.

  17. Ectonucleotidase activity and immunosuppression in astrocyte-CD4 T cell bidirectional signaling

    PubMed Central

    Filipello, Fabia; Romagnani, Andrea; Mazzitelli, Sonia; Matteoli, Michela; Verderio, Claudia; Grassi, Fabio

    2016-01-01

    Astrocytes play a crucial role in neuroinflammation as part of the glia limitans, which regulates infiltration of the brain parenchyma by leukocytes. The signaling pathways and molecular events, which result from the interaction of activated T cells with astrocytes are poorly defined. Here we show that astrocytes promote the expression and enzymatic activity of CD39 and CD73 ectonucleotidases in recently activated CD4 cells by a contact dependent mechanism that is independent of T cell receptor interaction with class II major histocompatibility complex (MHC). Transforming growth factor-β (TGF-β) is robustly upregulated and sufficient to promote ectonucleotidases expression. T cell adhesion to astrocyte results in differentiation to an immunosuppressive phenotype defined by expression of the transcription factor Rorγt, which characterizes the CD4 T helper 17 subset. CD39 activity in T cells in turn inhibits spontaneous calcium oscillations in astrocytes that correlated with enhanced and reduced transcription of CCL2 chemokine and Sonic hedgehog (Shh), respectively. We hypothesize this TCR-independent interaction promote an immunosuppressive program in T cells to control possible brain injury by deregulated T cell activation during neuroinflammation. On the other hand, the increased secretion of CCL2 with concomitant reduction of Shh might promote leukocytes extravasation into the brain parenchyma. PMID:26784253

  18. The glycosyl phosphatidylinositol anchor is critical for Ly-6A/E- mediated T cell activation

    PubMed Central

    1991-01-01

    Ly-6E, a glycosyl phosphatidylinositol (GPI)-anchored murine alloantigen that can activate T cells upon antibody cross-linking, has been converted into an integral membrane protein by gene fusion. This fusion product, designated Ly-6EDb, was characterized in transiently transfected COS cells and demonstrated to be an integral cell surface membrane protein. Furthermore, the fusion antigen can be expressed on the surface of the BW5147 class "E" mutant cell line, which only expresses integral membrane proteins but not GPI-anchored proteins. The capability of this fusion antigen to activate T cells was examined by gene transfer studies in D10G4.1, a type 2 T cell helper clones. When transfected into D10 cells, the GPI-anchored Ly-6E antigen, as well as the endogenous GPI-anchored Ly-6A antigen, can initiate T cell activation upon antibody cross-linking. In contrast, the transmembrane anchored Ly-6EDb antigen was unable to mediate T cell activation. Our results demonstrate that the GPI-anchor is critical to Ly-6A/E-mediated T cell activation. PMID:1825084

  19. CD4 T cell activation by B cells in human Leishmania (Viannia) infection

    PubMed Central

    2014-01-01

    Background An effective adaptive immune response requires activation of specific CD4 T cells. The capacity of B cells to activate CD4 T cells in human cutaneous leishmaniasis caused by Leishmania (Viannia) has not been evaluated. Methods CD4 T cell activation by B cells of cutaneous leishmaniasis patients was evaluated by culture of PBMCs or purified B cells and CD4 T cells with Leishmania panamensis antigens. CD4 T cell and B cell activation markers were evaluated by flow cytometry and 13 cytokines were measured in supernatants with a bead-based capture assay. The effect of Leishmania antigens on BCR-mediated endocytosis of ovalbumin was evaluated in the Ramos human B cell line by targeting the antigen with anti-IgM-biotin and anti-biotin-ovalbumin-FITC. Results Culture of PBMCs from cutaneous leishmaniasis patients with Leishmania antigens resulted in upregulation of the activation markers CD25 and CD69 as well as increased frequency of CD25hiCD127- cells among CD4 T cells. Concomitantly, B cells upregulated the costimulatory molecule CD86. These changes were not observed in PBMCs from healthy subjects, indicating participation of Leishmania-specific lymphocytes expanded in vivo. Purified B cells from these patients, when interacting with purified CD4 T cells and Leishmania antigens, were capable of inducing significant increases in CD25 and CD69 expression and CD25hiCD127- frequency in CD4 T cells. These changes were associated with upregulation of CD86 in B cells. Comparison of changes in CD4 T cell activation parameters between PBMC and B cell/CD4 T cell cultures showed no statistically significant differences; further, significant secretion of IFN-γ, TNF-α, IL-6 and IL-13 was induced in both types of cultures. Additionally, culture with Leishmania antigens enhanced BCR-mediated endocytosis of ovalbumin in Ramos human B cells. Conclusions The capacity of B cells specific for Leishmania antigens in peripheral blood of cutaneous leishmaniasis patients to

  20. Longitudinal characterization of dysfunctional T cell-activation during human acute Ebola infection

    PubMed Central

    Agrati, C; Castilletti, C; Casetti, R; Sacchi, A; Falasca, L; Turchi, F; Tumino, N; Bordoni, V; Cimini, E; Viola, D; Lalle, E; Bordi, L; Lanini, S; Martini, F; Nicastri, E; Petrosillo, N; Puro, V; Piacentini, M; Di Caro, A; Kobinger, G P; Zumla, A; Ippolito, G; Capobianchi, M R

    2016-01-01

    Data on immune responses during human Ebola virus disease (EVD) are scanty, due to limitations imposed by biosafety requirements and logistics. A sustained activation of T-cells was recently described but functional studies during the acute phase of human EVD are still missing. Aim of this work was to evaluate the kinetics and functionality of T-cell subsets, as well as the expression of activation, autophagy, apoptosis and exhaustion markers during the acute phase of EVD until recovery. Two EVD patients admitted to the Italian National Institute for Infectious Diseases, Lazzaro Spallanzani, were sampled sequentially from soon after symptom onset until recovery and analyzed by flow cytometry and ELISpot assay. An early and sustained decrease of CD4 T-cells was seen in both patients, with an inversion of the CD4/CD8 ratio that was reverted during the recovery period. In parallel with the CD4 T-cell depletion, a massive T-cell activation occurred and was associated with autophagic/apoptotic phenotype, enhanced expression of the exhaustion marker PD-1 and impaired IFN-gamma production. The immunological impairment was accompanied by EBV reactivation. The association of an early and sustained dysfunctional T-cell activation in parallel to an overall CD4 T-cell decline may represent a previously unknown critical point of Ebola virus (EBOV)-induced immune subversion. The recent observation of late occurrence of EBOV-associated neurological disease highlights the importance to monitor the immuno-competence recovery at discharge as a tool to evaluate the risk of late sequelae associated with resumption of EBOV replication. Further studies are required to define the molecular mechanisms of EVD-driven activation/exhaustion and depletion of T-cells. PMID:27031961

  1. Longitudinal characterization of dysfunctional T cell-activation during human acute Ebola infection.

    PubMed

    Agrati, C; Castilletti, C; Casetti, R; Sacchi, A; Falasca, L; Turchi, F; Tumino, N; Bordoni, V; Cimini, E; Viola, D; Lalle, E; Bordi, L; Lanini, S; Martini, F; Nicastri, E; Petrosillo, N; Puro, V; Piacentini, M; Di Caro, A; Kobinger, G P; Zumla, A; Ippolito, G; Capobianchi, M R

    2016-01-01

    Data on immune responses during human Ebola virus disease (EVD) are scanty, due to limitations imposed by biosafety requirements and logistics. A sustained activation of T-cells was recently described but functional studies during the acute phase of human EVD are still missing. Aim of this work was to evaluate the kinetics and functionality of T-cell subsets, as well as the expression of activation, autophagy, apoptosis and exhaustion markers during the acute phase of EVD until recovery. Two EVD patients admitted to the Italian National Institute for Infectious Diseases, Lazzaro Spallanzani, were sampled sequentially from soon after symptom onset until recovery and analyzed by flow cytometry and ELISpot assay. An early and sustained decrease of CD4 T-cells was seen in both patients, with an inversion of the CD4/CD8 ratio that was reverted during the recovery period. In parallel with the CD4 T-cell depletion, a massive T-cell activation occurred and was associated with autophagic/apoptotic phenotype, enhanced expression of the exhaustion marker PD-1 and impaired IFN-gamma production. The immunological impairment was accompanied by EBV reactivation. The association of an early and sustained dysfunctional T-cell activation in parallel to an overall CD4 T-cell decline may represent a previously unknown critical point of Ebola virus (EBOV)-induced immune subversion. The recent observation of late occurrence of EBOV-associated neurological disease highlights the importance to monitor the immuno-competence recovery at discharge as a tool to evaluate the risk of late sequelae associated with resumption of EBOV replication. Further studies are required to define the molecular mechanisms of EVD-driven activation/exhaustion and depletion of T-cells.

  2. Longitudinal characterization of dysfunctional T cell-activation during human acute Ebola infection.

    PubMed

    Agrati, C; Castilletti, C; Casetti, R; Sacchi, A; Falasca, L; Turchi, F; Tumino, N; Bordoni, V; Cimini, E; Viola, D; Lalle, E; Bordi, L; Lanini, S; Martini, F; Nicastri, E; Petrosillo, N; Puro, V; Piacentini, M; Di Caro, A; Kobinger, G P; Zumla, A; Ippolito, G; Capobianchi, M R

    2016-01-01

    Data on immune responses during human Ebola virus disease (EVD) are scanty, due to limitations imposed by biosafety requirements and logistics. A sustained activation of T-cells was recently described but functional studies during the acute phase of human EVD are still missing. Aim of this work was to evaluate the kinetics and functionality of T-cell subsets, as well as the expression of activation, autophagy, apoptosis and exhaustion markers during the acute phase of EVD until recovery. Two EVD patients admitted to the Italian National Institute for Infectious Diseases, Lazzaro Spallanzani, were sampled sequentially from soon after symptom onset until recovery and analyzed by flow cytometry and ELISpot assay. An early and sustained decrease of CD4 T-cells was seen in both patients, with an inversion of the CD4/CD8 ratio that was reverted during the recovery period. In parallel with the CD4 T-cell depletion, a massive T-cell activation occurred and was associated with autophagic/apoptotic phenotype, enhanced expression of the exhaustion marker PD-1 and impaired IFN-gamma production. The immunological impairment was accompanied by EBV reactivation. The association of an early and sustained dysfunctional T-cell activation in parallel to an overall CD4 T-cell decline may represent a previously unknown critical point of Ebola virus (EBOV)-induced immune subversion. The recent observation of late occurrence of EBOV-associated neurological disease highlights the importance to monitor the immuno-competence recovery at discharge as a tool to evaluate the risk of late sequelae associated with resumption of EBOV replication. Further studies are required to define the molecular mechanisms of EVD-driven activation/exhaustion and depletion of T-cells. PMID:27031961

  3. FK506 ameliorates podocyte injury in type 2 diabetic nephropathy by down-regulating TRPC6 and NFAT expression.

    PubMed

    Ma, Ruixia; Liu, Liqiu; Jiang, Wei; Yu, Yanjuan; Song, Haifeng

    2015-01-01

    Diabetic nephropathy (DN) is the leading cause of end-stage renal failure, and podocyte injury plays a major role in the development of DN. In this study, we investigated whether tacrolimus (FK506), an immunosuppressor, can attenuate podocyte injury in a type 2 diabetic mellitus (T2DM) rat model with DN. Transmission electron microcopy was used to morphologically evaluate renal injury. The urinary albumin (UAL), creatinine clearance rate (Ccr) and major biochemical parameters, including glucose, insulin, serum creatinine (Scr), urea nitrogen, total cholesterol (CHO) and triglyceride (TG), were examined 12 weeks after the administration of FK506. The expressions of the canonical transient receptor potential 6 (TRPC6), nuclear factor of activated T-cells (NFAT) and nephrin were detected by Western blotting and qPCR. In the rat model of DN, the expressions of TRPC6 and NFAT were significantly elevated compared with the normal rat group; however, the treatment with FK506 normalized the increased expression of TRPC6 and NFAT and attenuated podocyte ultrastructure injury. UAL, Ccr and the biochemical parameters were also improved by the use of FK506. In cell experiments, FK506 improved the decreased expression of nephrin and suppressed the elevated expression of both TRPC6 and NFAT caused by high glucose in accordance with TRPC6 blocker U73122. Our results demonstrated that FK506 could ameliorate podocyte injury in T2DM, which may be related to suppressed expressions of TRPC6 and NFAT.

  4. Distinct fates of monocytes and T cells directly activated by Pseudomonas aeruginosa exoenzyme S.

    PubMed

    Epelman, Slava; Neely, Graham G; Ma, Ling Ling; Gjomarkaj, Mark; Pace, Elisabetta; Melis, Mario; Woods, Donald E; Mody, Christopher H

    2002-03-01

    Gram-negative infections can cause overwhelming inflammatory responses. Although factors other than LPS are clearly involved, these factors and their mechanisms of action have been poorly defined. During studies of LPS-independent inflammatory responses of the gram-negative pathogen Pseudomonas aeruginosa, an important virulence factor (exoenzyme S) was shown to be a potent mitogen for T cells. The current work demonstrates that exoenzyme S selectively induced transcription and secretion of biologically active cytokines and chemokines (chemotactic for neutrophils and T cells) from monocytes. Exoenzyme S stimulated highly purified monocytes independent of T cells. In addition, exoenzyme S stimulated T cells directly; neither T-cell activation (CD69) nor apoptosis (hypodiploidy) required the presence of monocytes. However, T-cell activation was enhanced via a noncontact-dependent mechanism as a result of the secretion of TNF-alpha and IL-6. This study identifies a unique property of a gram-negative-derived microbial product capable of activating multiple cell types and suggests a mechanism by which exoenzyme S contributes to the immunopathogenesis of cystic fibrosis and sepsis in patients infected with P. aeruginosa. PMID:11867683

  5. Friend of GATA-1 Represses GATA-3–dependent Activity in CD4+ T Cells

    PubMed Central

    Zhou, Meixia; Ouyang, Wenjun; Gong, Qian; Katz, Samuel G.; White, J. Michael; Orkin, Stuart H.; Murphy, Kenneth M.

    2001-01-01

    The development of naive CD4+ T cells into a T helper (Th) 2 subset capable of producing interleukin (IL)-4, IL-5, and IL-13 involves a signal transducer and activator of transcription (Stat)6-dependent induction of GATA-3 expression, followed by Stat6-independent GATA-3 autoactivation. The friend of GATA (FOG)-1 protein regulates GATA transcription factor activity in several stages of hematopoietic development including erythrocyte and megakaryocyte differentiation, but whether FOG-1 regulates GATA-3 in T cells is uncertain. We show that FOG-1 can repress GATA-3–dependent activation of the IL-5 promoter in T cells. Also, FOG-1 overexpression during primary activation of naive T cells inhibited Th2 development in CD4+ T cells. FOG-1 fully repressed GATA-3–dependent Th2 development and GATA-3 autoactivation, but not Stat6-dependent induction of GATA-3. FOG-1 overexpression repressed development of Th2 cells from naive T cells, but did not reverse the phenotype of fully committed Th2 cells. Thus, FOG-1 may be one factor capable of regulating the Th2 development. PMID:11714753

  6. Necroptosis of Dendritic Cells Promotes Activation of γδ T Cells.

    PubMed

    Collins, Cheryl C; Bashant, Kathleen; Erikson, Cuixia; Thwe, Phyu Myat; Fortner, Karen A; Wang, Hong; Morita, Craig T; Budd, Ralph C

    2016-01-01

    γδ T cells function at the interface between innate and adaptive immunity and have well-demonstrated roles in response to infection, autoimmunity and tumors. A common characteristic of these seemingly disparate conditions may be cellular stress or death. However, the conditions under which ligands for γδ T cells are induced or exposed remain largely undefined. We observed that induction of necroptosis of murine or human dendritic cells (DC) by inhibition of caspase activity paradoxically augments their ability to activate γδ T cells. Furthermore, upregulation of the stabilizer of caspase-8 activity, c-FLIP, by IL-4, not only greatly reduced the susceptibility of DC to necroptosis, but also considerably decreased their ability to activate γδ T cells. Collectively, these findings suggest that the induction of necroptosis in DC upregulates or exposes the expression of γδ T cell ligands, and they support the view that γδ T cells function in the immune surveillance of cell stress. PMID:27431410

  7. Tissue signatures influence the activation of intrahepatic CD8+ T cells against malaria sporozoites

    PubMed Central

    Morrot, Alexandre; Rodrigues, Maurício M.

    2014-01-01

    Plasmodium sporozoites and liver stages express antigens that are targeted to the MHC-Class I antigen-processing pathway. After the introduction of Plasmodium sporozoites by Anopheles mosquitoes, bone marrow-derived dendritic cells in skin-draining lymph nodes are the first cells to cross-present parasite antigens and elicit specific CD8+ T cells. One of these antigens is the immunodominant circumsporozoite protein (CSP). The CD8+ T cell-mediated protective immune response against CSP is dependent on the interleukin loop involving IL-4 receptor expression on CD8+ cells and IL-4 secretion by CD4+ T cell helpers. In a few days, these CD8+ T cells re-circulate to secondary lymphoid organs and the liver. In the liver, the hepatic sinusoids are enriched with cells, such as dendritic, sinusoidal endothelial and Kupffer cells, that are able to cross-present MHC class I antigens to intrahepatic CD8+ T cells. Specific CD8+ T cells actively find infected hepatocytes and target intra-cellular parasites through mechanisms that are both interferon-γ-dependent and -independent. Immunity is mediated by CD8+ T effector or effector-memory cells and, when present in high numbers, these cells can provide sterilizing immunity. Human vaccination trials with recombinant formulations or attenuated sporozoites have yet to achieve the high numbers of specific effector T cells that are required for sterilizing immunity. In spite of the limited number of specific CD8+ T cells, attenuated sporozoites provided multiple times by the endovenous route provided a high degree of protective immunity. These observations highlight that CD8+ T cells may be useful for improving antibody-mediated protective immunity to pre-erythrocytic stages of malaria parasites. PMID:25202304

  8. Cish actively silences TCR signaling in CD8+ T cells to maintain tumor tolerance

    PubMed Central

    Guittard, Geoffrey C.; Franco, Zulmarie; Crompton, Joseph G.; Eil, Robert L.; Patel, Shashank J.; Ji, Yun; Van Panhuys, Nicholas; Klebanoff, Christopher A.; Sukumar, Madhusudhanan; Clever, David; Chichura, Anna; Roychoudhuri, Rahul; Varma, Rajat; Wang, Ena; Gattinoni, Luca; Marincola, Francesco M.; Balagopalan, Lakshmi; Samelson, Lawrence E.

    2015-01-01

    Improving the functional avidity of effector T cells is critical in overcoming inhibitory factors within the tumor microenvironment and eliciting tumor regression. We have found that Cish, a member of the suppressor of cytokine signaling (SOCS) family, is induced by TCR stimulation in CD8+ T cells and inhibits their functional avidity against tumors. Genetic deletion of Cish in CD8+ T cells enhances their expansion, functional avidity, and cytokine polyfunctionality, resulting in pronounced and durable regression of established tumors. Although Cish is commonly thought to block STAT5 activation, we found that the primary molecular basis of Cish suppression is through inhibition of TCR signaling. Cish physically interacts with the TCR intermediate PLC-γ1, targeting it for proteasomal degradation after TCR stimulation. These findings establish a novel targetable interaction that regulates the functional avidity of tumor-specific CD8+ T cells and can be manipulated to improve adoptive cancer immunotherapy. PMID:26527801

  9. T-cell activation or tolerization: the Yin and Yang of bacterial superantigens

    PubMed Central

    Sähr, Aline; Förmer, Sandra; Hildebrand, Dagmar; Heeg, Klaus

    2015-01-01

    Bacterial superantigens (SAg) are exotoxins from pathogens which interact with innate and adaptive immune cells. The paradox that SAgs cause activation and inactivation/anergy of T-cells was soon recognized. The structural and molecular events following SAg binding to antigen presenting cells (APCs) followed by crosslinking of T-cell receptors were characterized in detail. Activation, cytokine burst and T-cell anergy have been described in vitro and in vivo. Later it became clear that SAg-induced T-cell anergy is in part caused by SAg-dependent activation of T-regulatory cells (Tregs). Although the main focus of analyses was laid on T-cells, it was also shown that SAg binding to MHC class II molecules on APCs induces a signal, which leads to activation and secretion of pro-inflammatory cytokines. Accordingly APCs are mandatory for T-cell activation. So far it is not known, whether APCs play a role during SAg-triggered activation of Tregs. We therefore tested whether in SAg (Streptococcal pyrogenic exotoxin A) -treated APCs an anti-inflammatory program is triggered in addition. We show here that not only the anti-inflammatory cytokine IL-10 and the co-inhibitory surface molecule PD-L1 (CD274) but also inhibitory effector systems like indoleamine 2,3-dioxygenase (IDO) or intracellular negative feedback loops (suppressor of cytokine signaling molecules, SOCS) are induced by SAgs. Moreover, cyclosporine A completely prevented induction of this program. We therefore propose that APCs triggered by SAgs play a key role in T-cell activation as well as inactivation and induction of Treg cells. PMID:26539181

  10. Astragaloside II triggers T cell activation through regulation of CD45 protein tyrosine phosphatase activity

    PubMed Central

    Wan, Chun-ping; Gao, Li-xin; Hou, Li-fei; Yang, Xiao-qian; He, Pei-lan; Yang, Yi-fu; Tang, Wei; Yue, Jian-min; Li, Jia; Zuo, Jian-ping

    2013-01-01

    Aim: To investigate the immunomodulating activity of astragalosides, the active compounds from a traditional tonic herb Astragalus membranaceus Bge, and to explore the molecular mechanisms underlying the actions, focusing on CD45 protein tyrosine phosphatase (CD45 PTPase), which plays a critical role in T lymphocyte activation. Methods: Primary splenocytes and T cells were prepared from mice. CD45 PTPase activity was assessed using a colorimetric assay. Cell proliferation was measured using a [3H]-thymidine incorporation assay. Cytokine proteins and mRNAs were examined with ELISA and RT-PCR, respectively. Activation markers, including CD25 and CD69, were analyzed using flow cytometry. Activation of LCK (Tyr505) was detected using Western blot analysis. Mice were injected with the immunosuppressant cyclophosphamide (CTX, 80 mg/kg), and administered astragaloside II (50 mg/kg). Results: Astragaloside I, II, III, and IV concentration-dependently increased the CD45-mediated of pNPP/OMFP hydrolysis with the EC50 values ranged from 3.33 to 10.42 μg/mL. Astragaloside II (10 and 30 nmol/L) significantly enhanced the proliferation of primary splenocytes induced by ConA, alloantigen or anti-CD3. Astragaloside II (30 nmol/L) significantly increased IL-2 and IFN-γ secretion, upregulated the mRNA levels of IFN-γ and T-bet in primary splenocytes, and promoted CD25 and CD69 expression on primary CD4+ T cells upon TCR stimulation. Furthermore, astragaloside II (100 nmol/L) promoted CD45-mediated dephosphorylation of LCK (Tyr505) in primary T cells, which could be blocked by a specific CD45 PTPase inhibitor. In CTX-induced immunosuppressed mice, oral administration of astragaloside II restored the proliferation of splenic T cells and the production of IFN-γ and IL-2. However, astragaloside II had no apparent effects on B cell proliferation. Conclusion: Astragaloside II enhances T cell activation by regulating the activity of CD45 PTPase, which may explain why Astragalus

  11. Frequency of IFNγ-producing T cells correlates with seroreactivity and activated T cells during canine Trypanosoma cruzi infection.

    PubMed

    Hartley, Ashley N; Cooley, Gretchen; Gwyn, Sarah; Orozco, Marcela M; Tarleton, Rick L

    2014-01-01

    Vaccines to prevent Trypanosoma cruzi infection in humans or animals are not available, and in many settings, dogs are an important source of domestic infection for the insect vector. Identification of infected canines is crucial for evaluating peridomestic transmission dynamics and parasite control strategies. As immune control of T. cruzi infection is dependent on humoral and cell-mediated immune responses, we aimed to define a serodiagnostic assay and T cell phenotypic markers for identifying infected dogs and studying the canine T. cruzi-specific immune response. Plasma samples and peripheral blood mononuclear cells (PBMCs) were obtained from forty-two dogs living in a T. cruzi-endemic region. Twenty dogs were known to be seropositive and nine seronegative by conventional serologic tests two years prior to our study. To determine canine seroreactivity, we tested sera or plasma samples in a multiplex bead array against eleven recombinant T. cruzi proteins. Ninety-four percent (17/18) of dogs positive by multiplex serology were initially positive by conventional serology. The frequency of IFNγ-producing cells in PBMCs responding to T. cruzi correlated to serological status, identifying 95% of multiplex seropositive dogs. Intracellular staining identified CD4+ and CD8+ T cell populations as the sources of T. cruzi lysate-induced IFNγ. Low expression of CCR7 and CD62L on CD4+ and CD8+ T cells suggested a predominance of effector/effector memory T cells in seropositive canines. These results are the first, to our knowledge, to correlate T. cruzi-specific antibody responses with T cell responses in naturally infected dogs and validate these methods for identifying dogs exposed to T. cruzi. PMID:24456537

  12. Allopurinol reduces antigen-specific and polyclonal activation of human T cells

    PubMed Central

    Pérez-Mazliah, Damián; Albareda, María C.; Alvarez, María G.; Lococo, Bruno; Bertocchi, Graciela L.; Petti, Marcos; Viotti, Rodolfo J.; Laucella, Susana A.

    2012-01-01

    Allopurinol is the most popular commercially available xanthine oxidase inhibitor and it is widely used for treatment of symptomatic hyperuricaemia, or gout. Although, several anti-inflammatory actions of allopurinol have been demonstrated in vivo and in vitro, there have been few studies on the action of allopurinol on T cells. In the current study, we have assessed the effect of allopurinol on antigen-specific and mitogen-driven activation and cytokine production in human T cells. Allopurinol markedly decreased the frequency of IFN-γ and IL-2-producing T cells, either after polyclonal or antigen-specific stimulation with Herpes Simplex virus 1, Influenza (Flu) virus, tetanus toxoid and Trypanosoma cruzi-derived antigens. Allopurinol attenuated CD69 upregulation after CD3 and CD28 engagement and significantly reduced the levels of spontaneous and mitogen-induced intracellular reactive oxygen species in T cells. The diminished T cell activation and cytokine production in the presence of allopurinol support a direct action of allopurinol on human T cells, offering a potential pharmacological tool for the management of cell-mediated inflammatory diseases. PMID:23049532

  13. LYSOPHOSPHATIDIC ACID INHIBITS CD8 T CELL ACTIVATION AND CONTROL OF TUMOR PROGRESSION

    PubMed Central

    Oda, Shannon K.; Strauch, Pamela; Fujiwara, Yuko; Al-Shami, Amin; Oravecz, Tamas; Tigyi, Gabor; Pelanda, Roberta; Torres, Raul M.

    2013-01-01

    CD8 T lymphocytes are able to eliminate nascent tumor cells through a process referred to as immune surveillance. However, multiple inhibitory mechanisms within the tumor microenvironment have been described that impede tumor rejection by CD8 T cells, including increased signaling by inhibitory receptors. Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that has been shown repeatedly to promote diverse cellular processes benefiting tumorigenesis. Accordingly, the increased expression of LPA and LPA receptors is a common feature of diverse tumor cell lineages and can result in elevated systemic LPA levels. LPA is recognized by at least 6 distinct G-protein-coupled receptors and several of which are expressed by T cells, although the precise role of LPA signaling in CD8 T cell activation and function has not been defined. Here, we demonstrate that LPA signaling via the LPA5 receptor expressed by CD8 T cells suppresses antigen receptor signaling, cell activation and proliferation in vitro and in vivo. Importantly, in a mouse melanoma model tumor-specific CD8 T cells that are LPA5-deficient are able to control tumor growth significantly better than wild-type tumor-specific CD8 T cells. Together, these data suggest that the production of LPA by tumors serves not only in an autocrine manner to promote tumorigenesis but also as a mechanism to suppress adaptive immunity and highlights a potential novel target for cancer treatment. PMID:24455753

  14. B7-H4 Treatment of T Cells Inhibits ERK, JNK, p38, and AKT Activation

    PubMed Central

    Wang, Xiaojie; Hao, Jianqiang; Metzger, Daniel L.; Ao, Ziliang; Chen, Lieping; Ou, Dawei; Verchere, C. Bruce; Mui, Alice; Warnock, Garth L.

    2012-01-01

    B7-H4 is a newly identified B7 homolog that plays an important role in maintaining T-cell homeostasis by inhibiting T-cell proliferation and lymphokine-secretion. In this study, we investigated the signal transduction pathways inhibited by B7-H4 engagement in mouse T cells. We found that treatment of CD3+ T cells with a B7-H4.Ig fusion protein inhibits anti-CD3 elicited T-cell receptor (TCR)/CD28 signaling events, including phosphorylation of the MAP kinases, ERK, p38, and JNK. B7-H4.Ig treatment also inhibited the phosphorylation of AKT kinase and impaired its kinase activity as assessed by the phosphorylation of its endogenous substrate GSK-3. Expression of IL-2 is also reduced by B7-H4. In contrast, the phosphorylation state of the TCR proximal tyrosine kinases ZAP70 and lymphocyte-specific protein tyrosine kinase (LCK) are not affected by B7-H4 ligation. These results indicate that B7-H4 inhibits T-cell proliferation and IL-2 production through interfering with activation of ERK, JNK, and AKT, but not of ZAP70 or LCK. PMID:22238573

  15. Lysophosphatidic acid inhibits CD8 T cell activation and control of tumor progression.

    PubMed

    Oda, Shannon K; Strauch, Pamela; Fujiwara, Yuko; Al-Shami, Amin; Oravecz, Tamas; Tigyi, Gabor; Pelanda, Roberta; Torres, Raul M

    2013-10-01

    CD8 T lymphocytes are able to eliminate nascent tumor cells through a process referred to as immune surveillance. However, multiple inhibitory mechanisms within the tumor microenvironment have been described that impede tumor rejection by CD8 T cells, including increased signaling by inhibitory receptors. Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that has been shown repeatedly to promote diverse cellular processes benefiting tumorigenesis. Accordingly, the increased expression of LPA and LPA receptors is a common feature of diverse tumor cell lineages and can result in elevated systemic LPA levels. LPA is recognized by at least 6 distinct G-protein-coupled receptors and several of which are expressed by T cells, although the precise role of LPA signaling in CD8 T cell activation and function has not been defined. Here, we demonstrate that LPA signaling via the LPA5 receptor expressed by CD8 T cells suppresses antigen receptor signaling, cell activation and proliferation in vitro and in vivo. Importantly, in a mouse melanoma model tumor-specific CD8 T cells that are LPA5-deficient are able to control tumor growth significantly better than wild-type tumor-specific CD8 T cells. Together, these data suggest that the production of LPA by tumors serves not only in an autocrine manner to promote tumorigenesis but also as a mechanism to suppress adaptive immunity and highlights a potential novel target for cancer treatment. PMID:24455753

  16. Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies.

    PubMed

    Rodgers, David T; Mazagova, Magdalena; Hampton, Eric N; Cao, Yu; Ramadoss, Nitya S; Hardy, Ian R; Schulman, Andrew; Du, Juanjuan; Wang, Feng; Singer, Oded; Ma, Jennifer; Nunez, Vanessa; Shen, Jiayin; Woods, Ashley K; Wright, Timothy M; Schultz, Peter G; Kim, Chan Hyuk; Young, Travis S

    2016-01-26

    Chimeric antigen receptor T (CAR-T) cell therapy has produced impressive results in clinical trials for B-cell malignancies. However, safety concerns related to the inability to control CAR-T cells once infused into the patient remain a significant challenge. Here we report the engineering of recombinant antibody-based bifunctional switches that consist of a tumor antigen-specific Fab molecule engrafted with a peptide neo-epitope, which is bound exclusively by a peptide-specific switchable CAR-T cell (sCAR-T). The switch redirects the activity of the bio-orthogonal sCAR-T cells through the selective formation of immunological synapses, in which the sCAR-T cell, switch, and target cell interact in a structurally defined and temporally controlled manner. Optimized switches specific for CD19 controlled the activity, tissue-homing, cytokine release, and phenotype of sCAR-T cells in a dose-titratable manner in a Nalm-6 xenograft rodent model of B-cell leukemia. The sCAR-T-cell dosing regimen could be tuned to provide efficacy comparable to the corresponding conventional CART-19, but with lower cytokine levels, thereby offering a method of mitigating cytokine release syndrome in clinical translation. Furthermore, we demonstrate that this methodology is readily adaptable to targeting CD20 on cancer cells using the same sCAR-T cell, suggesting that this approach may be broadly applicable to heterogeneous and resistant tumor populations, as well as other liquid and solid tumor antigens. PMID:26759369

  17. Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies

    PubMed Central

    Rodgers, David T.; Mazagova, Magdalena; Hampton, Eric N.; Cao, Yu; Ramadoss, Nitya S.; Hardy, Ian R.; Schulman, Andrew; Du, Juanjuan; Wang, Feng; Singer, Oded; Ma, Jennifer; Nunez, Vanessa; Shen, Jiayin; Woods, Ashley K.; Wright, Timothy M.; Schultz, Peter G.; Kim, Chan Hyuk; Young, Travis S.

    2016-01-01

    Chimeric antigen receptor T (CAR-T) cell therapy has produced impressive results in clinical trials for B-cell malignancies. However, safety concerns related to the inability to control CAR-T cells once infused into the patient remain a significant challenge. Here we report the engineering of recombinant antibody-based bifunctional switches that consist of a tumor antigen-specific Fab molecule engrafted with a peptide neo-epitope, which is bound exclusively by a peptide-specific switchable CAR-T cell (sCAR-T). The switch redirects the activity of the bio-orthogonal sCAR-T cells through the selective formation of immunological synapses, in which the sCAR-T cell, switch, and target cell interact in a structurally defined and temporally controlled manner. Optimized switches specific for CD19 controlled the activity, tissue-homing, cytokine release, and phenotype of sCAR-T cells in a dose-titratable manner in a Nalm-6 xenograft rodent model of B-cell leukemia. The sCAR–T-cell dosing regimen could be tuned to provide efficacy comparable to the corresponding conventional CART-19, but with lower cytokine levels, thereby offering a method of mitigating cytokine release syndrome in clinical translation. Furthermore, we demonstrate that this methodology is readily adaptable to targeting CD20 on cancer cells using the same sCAR-T cell, suggesting that this approach may be broadly applicable to heterogeneous and resistant tumor populations, as well as other liquid and solid tumor antigens. PMID:26759369

  18. Intracellular complement activation sustains T cell homeostasis and mediates effector differentiation.

    PubMed

    Liszewski, M Kathryn; Kolev, Martin; Le Friec, Gaelle; Leung, Marilyn; Bertram, Paula G; Fara, Antonella F; Subias, Marta; Pickering, Matthew C; Drouet, Christian; Meri, Seppo; Arstila, T Petteri; Pekkarinen, Pirkka T; Ma, Margaret; Cope, Andrew; Reinheckel, Thomas; Rodriguez de Cordoba, Santiago; Afzali, Behdad; Atkinson, John P; Kemper, Claudia

    2013-12-12

    Complement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generated C3a. While "tonic" intracellular C3a generation was required for homeostatic T cell survival, shuttling of this intracellular C3-activation-system to the cell surface upon T cell stimulation induced autocrine proinflammatory cytokine production. Furthermore, T cells from patients with autoimmune arthritis demonstrated hyperactive intracellular complement activation and interferon-γ production and CTSL inhibition corrected this deregulated phenotype. Importantly, intracellular C3a was observed in all examined cell populations, suggesting that intracellular complement activation might be of broad physiological significance. PMID:24315997

  19. Recurrent activating mutations of CD28 in peripheral T-cell lymphomas.

    PubMed

    Rohr, J; Guo, S; Huo, J; Bouska, A; Lachel, C; Li, Y; Simone, P D; Zhang, W; Gong, Q; Wang, C; Cannon, A; Heavican, T; Mottok, A; Hung, S; Rosenwald, A; Gascoyne, R; Fu, K; Greiner, T C; Weisenburger, D D; Vose, J M; Staudt, L M; Xiao, W; Borgstahl, G E O; Davis, S; Steidl, C; McKeithan, T; Iqbal, J; Chan, W C

    2016-05-01

    Peripheral T-cell lymphomas (PTCLs) comprise a heterogeneous group of mature T-cell neoplasms with a poor prognosis. Recently, mutations in TET2 and other epigenetic modifiers as well as RHOA have been identified in these diseases, particularly in angioimmunoblastic T-cell lymphoma (AITL). CD28 is the major co-stimulatory receptor in T cells which, upon binding ligand, induces sustained T-cell proliferation and cytokine production when combined with T-cell receptor stimulation. We have identified recurrent mutations in CD28 in PTCLs. Two residues-D124 and T195-were recurrently mutated in 11.3% of cases of AITL and in one case of PTCL, not otherwise specified (PTCL-NOS). Surface plasmon resonance analysis of mutations at these residues with predicted differential partner interactions showed increased affinity for ligand CD86 (residue D124) and increased affinity for intracellular adaptor proteins GRB2 and GADS/GRAP2 (residue T195). Molecular modeling studies on each of these mutations suggested how these mutants result in increased affinities. We found increased transcription of the CD28-responsive genes CD226 and TNFA in cells expressing the T195P mutant in response to CD3 and CD86 co-stimulation and increased downstream activation of NF-κB by both D124V and T195P mutants, suggesting a potential therapeutic target in CD28-mutated PTCLs. PMID:26719098

  20. Metabolic reprogramming of alloantigen-activated T cells after hematopoietic cell transplantation

    PubMed Central

    Nguyen, Hung D.; Chatterjee, Shilpak; Haarberg, Kelley M.K.; Wu, Yongxia; Bastian, David; Heinrichs, Jessica; Fu, Jianing; Daenthanasanmak, Anusara; Schutt, Steven; Shrestha, Sharad; Liu, Chen; Wang, Honglin; Chi, Hongbo; Mehrotra, Shikhar

    2016-01-01

    Alloreactive donor T cells are the driving force in the induction of graft-versus-host disease (GVHD), yet little is known about T cell metabolism in response to alloantigens after hematopoietic cell transplantation (HCT). Here, we have demonstrated that donor T cells undergo metabolic reprograming after allogeneic HCT. Specifically, we employed a murine allogeneic BM transplant model and determined that T cells switch from fatty acid β-oxidation (FAO) and pyruvate oxidation via the tricarboxylic (TCA) cycle to aerobic glycolysis, thereby increasing dependence upon glutaminolysis and the pentose phosphate pathway. Glycolysis was required for optimal function of alloantigen-activated T cells and induction of GVHD, as inhibition of glycolysis by targeting mTORC1 or 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) ameliorated GVHD mortality and morbidity. Together, our results indicate that donor T cells use glycolysis as the predominant metabolic process after allogeneic HCT and suggest that glycolysis has potential as a therapeutic target for the control of GVHD. PMID:26950421

  1. Structure-activity relationship between carboxylic acids and T cell cycle blockade.

    PubMed

    Gilbert, Kathleen M; DeLoose, Annick; Valentine, Jimmie L; Fifer, E Kim

    2006-04-01

    This study was designed to examine the potential structure-activity relationship between carboxylic acids, histone acetylation and T cell cycle blockade. Toward this goal a series of structural homologues of the short-chain carboxylic acid n-butyrate were studied for their ability to block the IL-2-stimulated proliferation of cloned CD4+ T cells. The carboxylic acids were also tested for their ability to inhibit histone deacetylation. In addition, Western blotting was used to examine the relative capacity of the carboxlic acids to upregulate the cyclin kinase-dependent inhibitor p21cip1 in T cells. As shown earlier n-butyrate effectively inhibited histone deacetylation. The increased acetylation induced by n-butyrate was associated with the upregulation of the cyclin-dependent kinase inhibitor p21cip1 and the cell cycle blockade of CD4+ T cells. Of the other carboxylic acids studied, the short chain acids, C3-C5, without branching were the best inhibitors of histone deacetylase. This inhibition correlated with increased expression of the cell cycle blocker p21cip1, and the associated suppression of CD4+ T cell proliferation. The branched-chain carboxylic acids tested were ineffective in all the assays. These results underline the relationship between the ability of a carboxylic acid to inhibit histone deacetylation, and their ability to block T cell proliferation, and suggests that branching inhibits these effects.

  2. Bromelain treatment reduces CD25 expression on activated CD4+ T cells in vitro.

    PubMed

    Secor, Eric R; Singh, Anurag; Guernsey, Linda A; McNamara, Jeff T; Zhan, Lijun; Maulik, Nilanjana; Thrall, Roger S

    2009-03-01

    Bromelain (Br), an extract from pineapple stem with cysteine protease activity, exerts anti-inflammatory effects in a number of inflammatory models. We have previously shown that Br treatment decreased activated CD4(+) T cells and has a therapeutic role in an ovalbumin-induced murine model of allergic airway disease. The current study was designed to determine the effect of Br on CD4(+) T cell activation, specifically the expression of CD25 in vitro. CD25 is up regulated upon T cell activation, found as a soluble fraction (sCD25) and is a therapeutic target in inflammation, autoimmunity and allergy. Br treatment of anti-CD3 stimulated CD4(+) T cells reduced CD25 expression in a dose and time dependent manner. This reduction of CD25 was dependent on the proteolytic action of Br as the addition of E64 (a cysteine protease inhibitor) abrogated this response. The concentration of sCD25 was increased in supernatants of Br treated activated CD4(+) T cells as compared to control cells, suggesting that Br proteolytically cleaved cell-surface CD25. This novel mechanism of action identifies how Br may exert its therapeutic benefits in inflammatory conditions.

  3. Micro-adhesion rings surrounding TCR microclusters are essential for T cell activation.

    PubMed

    Hashimoto-Tane, Akiko; Sakuma, Machie; Ike, Hiroshi; Yokosuka, Tadashi; Kimura, Yayoi; Ohara, Osamu; Saito, Takashi

    2016-07-25

    The immunological synapse (IS) formed at the interface between T cells and antigen-presenting cells represents a hallmark of initiation of acquired immunity. T cell activation is initiated at T cell receptor (TCR) microclusters (MCs), in which TCRs and signaling molecules assemble at the interface before IS formation. We found that each TCR-MC was transiently bordered by a ring structure made of integrin and focal adhesion molecules in the early phase of activation, which is similar in structure to the IS in microscale. The micro-adhesion ring is composed of LFA-1, focal adhesion molecules paxillin and Pyk2, and myosin II (MyoII) and is supported by F-actin core and MyoII activity through LFA-1 outside-in signals. The formation of the micro-adhesion ring was transient but especially sustained upon weak TCR stimulation to recruit linker for activation of T cells (LAT) and SLP76. Perturbation of the micro-adhesion ring induced impairment of TCR-MC development and resulted in impaired cellular signaling and cell functions. Thus, the synapse-like structure composed of the core TCR-MC and surrounding micro-adhesion ring is a critical structure for initial T cell activation through integrin outside-in signals.

  4. CIP2A Promotes T-Cell Activation and Immune Response to Listeria monocytogenes Infection.

    PubMed

    Côme, Christophe; Cvrljevic, Anna; Khan, Mohd Moin; Treise, Irina; Adler, Thure; Aguilar-Pimentel, Juan Antonio; Au-Yeung, Byron; Sittig, Eleonora; Laajala, Teemu Daniel; Chen, Yiling; Oeder, Sebastian; Calzada-Wack, Julia; Horsch, Marion; Aittokallio, Tero; Busch, Dirk H; Ollert, Markus W; Neff, Frauke; Beckers, Johannes; Gailus-Durner, Valerie; Fuchs, Helmut; Hrabě de Angelis, Martin; Chen, Zhi; Lahesmaa, Riitta; Westermarck, Jukka

    2016-01-01

    The oncoprotein Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is overexpressed in most malignancies and is an obvious candidate target protein for future cancer therapies. However, the physiological importance of CIP2A-mediated PP2A inhibition is largely unknown. As PP2A regulates immune responses, we investigated the role of CIP2A in normal immune system development and during immune response in vivo. We show that CIP2A-deficient mice (CIP2AHOZ) present a normal immune system development and function in unchallenged conditions. However when challenged with Listeria monocytogenes, CIP2AHOZ mice display an impaired adaptive immune response that is combined with decreased frequency of both CD4+ T-cells and CD8+ effector T-cells. Importantly, the cell autonomous effect of CIP2A deficiency for T-cell activation was confirmed. Induction of CIP2A expression during T-cell activation was dependent on Zap70 activity. Thus, we reveal CIP2A as a hitherto unrecognized mediator of T-cell activation during adaptive immune response. These results also reveal CIP2AHOZ as a possible novel mouse model for studying the role of PP2A activity in immune regulation. On the other hand, the results also indicate that CIP2A targeting cancer therapies would not cause serious immunological side-effects.

  5. CIP2A Promotes T-Cell Activation and Immune Response to Listeria monocytogenes Infection

    PubMed Central

    Cvrljevic, Anna; Khan, Mohd Moin; Treise, Irina; Adler, Thure; Aguilar-Pimentel, Juan Antonio; Au-Yeung, Byron; Sittig, Eleonora; Laajala, Teemu Daniel; Chen, Yiling; Oeder, Sebastian; Calzada-Wack, Julia; Horsch, Marion; Aittokallio, Tero; Busch, Dirk H.; Ollert, Markus W.; Neff, Frauke; Beckers, Johannes; Gailus-Durner, Valerie; Fuchs, Helmut; de Angelis, Martin Hrabě; Chen, Zhi; Lahesmaa, Riitta; Westermarck, Jukka

    2016-01-01

    The oncoprotein Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is overexpressed in most malignancies and is an obvious candidate target protein for future cancer therapies. However, the physiological importance of CIP2A-mediated PP2A inhibition is largely unknown. As PP2A regulates immune responses, we investigated the role of CIP2A in normal immune system development and during immune response in vivo. We show that CIP2A-deficient mice (CIP2AHOZ) present a normal immune system development and function in unchallenged conditions. However when challenged with Listeria monocytogenes, CIP2AHOZ mice display an impaired adaptive immune response that is combined with decreased frequency of both CD4+ T-cells and CD8+ effector T-cells. Importantly, the cell autonomous effect of CIP2A deficiency for T-cell activation was confirmed. Induction of CIP2A expression during T-cell activation was dependent on Zap70 activity. Thus, we reveal CIP2A as a hitherto unrecognized mediator of T-cell activation during adaptive immune response. These results also reveal CIP2AHOZ as a possible novel mouse model for studying the role of PP2A activity in immune regulation. On the other hand, the results also indicate that CIP2A targeting cancer therapies would not cause serious immunological side-effects. PMID:27100879

  6. T cell Receptor Signal Transduction in T lymphocytes

    PubMed Central

    Gorentla, Balachandra K; Zhong, Xiao-Ping

    2012-01-01

    The T cell receptor (TCR) recognizes self or foreign antigens presented by major histocompatibility complex (MHC) molecules. Engagement of the TCR triggers the formation of multi-molecular signalosomes that lead to the generation of second messengers and subsequent activation of multiple distal signaling cascades, such as the Ca+2-calcineurin-NFAT, RasGRP1-Ras-Erk1/2, PKCθ-IKK-NFκB, and TSC1/2-mTOR pathways. These signaling cascades control many aspects of T cell biology. Mechanisms have been evolved to fine-tune TCR signaling to maintain T cell homeostasis and self-tolerance, and to properly mount effective responses to microbial infection. Defects or deregulation of TCR signaling has been implicated in the pathogenesis of multiple human diseases. PMID:23946894

  7. Mechanisms of T Cell Activation and Pathways of Hypertension

    PubMed Central

    Abais-Battad, Justine M.; Rudemiller, Nathan P.; Mattson, David L.

    2015-01-01

    Summary Significant advancements have been made in the search for antigens and pathways responsible for activation of the adaptive immune response, furthering our understanding of the factors contributing to hypertension and potentially leading to the development of new and more effective therapies. PMID:26125645

  8. Complexity of the primary genetic response to mitogenic activation of human T cells

    SciTech Connect

    Zipfel, P.F.; Siebenlist, U. ); Irving, S.G.; Kelly, K. )

    1989-03-01

    The authors describe the isolation and characterization of more than 60 novel cDNA clones that constitute part of the immediate genetic response to resting human peripheral blood T cells after mitogen activation. This primary response was highly complex, both in the absolute number of inducible genes and in the diversity of regulation. Although most of the genes expressed in activated T cells were shared with the activation response of normal human fibroblasts, a significant number were more restricted in tissue specificity and thus likely encode or effect the differentiated functions of activated T cells. The activatable genes could be further differentiated on the basis of kinetics of induction, response to cycloheximide, and sensitivity to the immunosuppressive drug cylcosporin A. It is of note that cyclosporin A inhibited the expression of more than 10 inducible genes, which suggests that this drug has a broad genetic mechanism of action.

  9. Otud7b facilitates T cell activation and inflammatory responses by regulating Zap70 ubiquitination.

    PubMed

    Hu, Hongbo; Wang, Hui; Xiao, Yichuan; Jin, Jin; Chang, Jae-Hoon; Zou, Qiang; Xie, Xiaoping; Cheng, Xuhong; Sun, Shao-Cong

    2016-03-01

    Signal transduction from the T cell receptor (TCR) is crucial for T cell-mediated immune responses and, when deregulated, also contributes to the development of autoimmunity. How TCR signaling is regulated is incompletely understood. In this study, we demonstrate a ubiquitin-dependent mechanism in which the deubiquitinase Otud7b has a crucial role in facilitating TCR signaling. Upon TCR ligation, Otud7b is rapidly recruited to the tyrosine kinase Zap70, a central mediator of TCR-proximal signaling. Otud7b deficiency attenuates the activation of Zap70 and its downstream pathways and impairs T cell activation and differentiation, rendering mice refractory to T cell-mediated autoimmune and inflammatory responses. Otud7b facilitated Zap70 activation by deubiquitinating Zap70, thus preventing the association of Zap70 with the negative-regulatory phosphatases Sts1 and Sts2. These findings establish Otud7b as a positive regulator of TCR-proximal signaling and T cell activation, highlighting the importance of deubiquitination in regulating Zap70 function. PMID:26903241

  10. βig-h3 Represses T-Cell Activation in Type 1 Diabetes.

    PubMed

    Patry, Maeva; Teinturier, Romain; Goehrig, Delphine; Zetu, Cornelia; Ripoche, Doriane; Kim, In-San; Bertolino, Philippe; Hennino, Ana

    2015-12-01

    βig-h3/TGF-βi is a secreted protein capable of binding to both extracellular matrix and cells. Human genetic studies recently revealed that in the tgfbi gene encoding for βig-h3, three single nucleotide polymorphisms were significantly associated with type 1 diabetes (T1D) risk. Pancreatic islets express βig-h3 in physiological conditions, but this expression is reduced in β-cell insult in T1D. Since the integrity of islets is destroyed by autoimmune T lymphocytes, we thought to investigate the impact of βig-h3 on T-cell activation. We show here that βig-h3 inhibits T-cell activation markers as well as cytotoxic molecule production as granzyme B and IFN-γ. Furthermore, βig-h3 inhibits early T-cell receptor signaling by repressing the activation of the early kinase protein Lck. Moreover, βig-h3-treated T cells are unable to induce T1D upon transfer in Rag2 knockout mice. Our study demonstrates for the first time that T-cell activation is modulated by βig-h3, an islet extracellular protein, in order to efficiently avoid autoimmune response.

  11. Mechanism for macrophage activation against Corynebacterium parvum--participation of T cells and its lymphokines.

    PubMed

    Mori, H; Mihara, M; Uesugi, Y; Nagai, H; Koda, A

    1994-01-01

    It is well known that Corynebacterium parvum activates macrophages to produce tumor necrosis factor (TNF). It is suspected that the activation of macrophages by C. parvum requires T-cell participation. The purpose of this study was to confirm that T cells participate in the activation of macrophages by C. parvum. TNF production in vitro from the spleen cells of BALB/c(-)+/+ mice was abrogated completely by the pre-treatment of spleen cells with anti-Ia antiserum and complement, indicating that Ia+ cells are the source of TNF. TNF production was not elicited at all in BALB/c-nu/nu mice. However, there was an increase in the number of Ia+ cells as well as an increase in the weight of spleen and liver. Supernatant from a culture of spleen cells stimulated with phytohemagglutinin-P (a PHA-induced lymphokine) made it possible for BALB/c-nu/nu mice to produce TNF, associated with an induction of Lyt-1+ cells and Lyt-2+ cells. However, treatment with the lymphokine did not augment the increases of Ia+ cells or liver and spleen weights. These results suggest that increasing the number of Ia+ cells is not sufficient to bring about TNF production; Ia+ cells must also be stimulated by T cells or T-cell lymphokines in order to produce TNF. These results suggest that T cells play an essential role in the activation of Ia+ cells against C. parvum. PMID:7723692

  12. Ipilimumab augments antitumor activity of bispecific antibody-armed T cells

    PubMed Central

    2014-01-01

    Background Ipilimumab is an antagonistic monoclonal antibody against cytotoxic T-lymphocyte antigen-4 (CTLA-4) that enhances antitumor immunity by inhibiting immunosuppressive activity of regulatory T cells (Treg). In this study, we investigated whether inhibiting Treg activity with ipilimumab during ex vivo T cell expansion could augment anti-CD3-driven T cell proliferation and enhance bispecific antibody (BiAb)-redirected antitumor cytotoxicity of activated T cells (ATC). Methods PBMC from healthy individuals were stimulated with anti-CD3 monoclonal antibody with or without ipilimumab and expanded for 10-14 days. ATC were harvested and armed with anti-CD3 x anti-EGFR BiAb (EGFRBi) or anti-CD3 x anti-CD20 BiAb (CD20Bi) to test for redirected cytotoxicity against COLO356/FG pancreatic cancer cell line or Burkitt’s lymphoma cell line (Daudi). Results In PBMC from healthy individuals, the addition of ipilimumab at the initiation of culture significantly enhanced T cell proliferation (p = 0.0029). ATC grown in the presence of ipilimumab showed significantly increased mean tumor-specific cytotoxicity at effector:target (E:T) ratio of 25:1 directed at COLO356/FG and Daudi by 37.71% (p < 0.0004) and 27.5% (p < 0.0004), respectively, and increased the secretion of chemokines (CCL2, CCL3, CCL4,CCL5, CXCL9, and granulocyte-macrophage colony stimulating factor(GM-CSF)) and cytokines (IFN-γ, IL-2R, IL-12, and IL-13), while reducing IL-10 secretion. Conclusions Expansion of ATC in the presence of ipilimumab significantly improves not only the T cell proliferation but it also enhances cytokine secretion and the specific cytotoxicity of T cells armed with bispecific antibodies. PMID:25008236

  13. A Cross-Talk Between NFAT and NF-κB Pathways is Crucial for Nickel-Induced COX-2 Expression in Beas-2B Cells

    PubMed Central

    Cai, T.; Li, X.; Ding, J.; Luo, W.; Li, J.; Huang, C.

    2013-01-01

    Cyclooxygenase-2 (COX-2) is a critical enzyme implicated in chronic inflammation-associated cancer development. Our studies have shown that the exposure of Beas-2B cells, a human bronchial epithelial cell line, to lung carcinogenic nickel compounds results in increased COX-2 expression. However, the signaling pathways leading to nickel-induced COX-2 expression are not well understood. In the current study, we found that the exposure of Beas-2B cells to nickel compounds resulted in the activation of both nuclear factor of activated T cell (NFAT) and nuclear factor-κB (NF-κB). The expression of COX-2 induced upon nickel exposure was inhibited by either a NFAT pharmacological inhibitor or the knockdown of NFAT3 by specific siRNA. We further found that the activation of NFAT and NF-κB was dependent on each other. Since our previous studies have shown that NF-κB activation is critical for nickel-induced COX-2 expression in Beas-2B cells exposed to nickel compounds under same experimental condition, we anticipate that there might be a cross-talk between the activation of NFAT and NF-κB for the COX-2 induction due to nickel exposure in Beas-2B cells. Furthermore, we showed that the scavenging of reactive oxygen species (ROS) by introduction of mitochondrial catalase inhibited the activation of both NFAT and NF-κB, and the induction of COX-2 due to nickel exposure. Taken together, our results defining the evidence showing a key role of the cross-talk between NFAT and NF-κB pathways in regulating nickel-induced COX-2 expression, further provide insight into the understanding of the molecular mechanisms linking nickel exposure to its lung carcinogenic effects. PMID:21486220

  14. T cell activation regulates CD6 alternative splicing by transcription dynamics and SRSF1.

    PubMed

    da Glória, Vânia G; Martins de Araújo, Mafalda; Mafalda Santos, Ana; Leal, Rafaela; de Almeida, Sérgio F; Carmo, Alexandre M; Moreira, Alexandra

    2014-07-01

    The T cell-surface glycoprotein CD6 is a modulator of cellular responses and has been implicated in several autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and psoriasis. During Ag presentation, CD6 is targeted to the immunological synapse in a ligand binding-dependent manner, in which CD6 domain 3 directly contacts CD166, expressed on the APC. T cell activation results in the induction of CD6Δd3, an alternatively spliced isoform that lacks the ligand-binding domain and thus no longer localizes at the immunological synapse. In this study, we investigated the molecular mechanisms regulating the expression of CD6Δd3 upon human primary T cell activation. Using chromatin immunoprecipitation, we observed an increase in RNA polymerase II occupancy along the CD6 gene and augmented CD6 transcription. We showed that activation leads to transcription-related chromatin modifications, revealed by higher CD6 acetylation levels. Modulation of chromatin conformation using a histone deacetylase inhibitor that increases transcription rate causes an increase of exon 5 skipping. We further showed that the splicing factor SRSF1 binds to a regulatory element in CD6 intron 4, activating exon 5 splicing and promoting exon 5 inclusion. Concomitant with T cell activation-induced exon 5 skipping, we observed a downregulation of SRSF1. Using RNA immunoprecipitation, we showed that in activated T cells, SRSF1 recruitment to the CD6 transcript is impaired by increased chromatin acetylation levels. We propose that upon T cell activation, SRSF1 becomes limiting, and its function in CD6 exon 5 splicing is countered by an increase in CD6 transcription, dependent on chromatin acetylation.

  15. Wasp recruitment to the T cell:APC contact site occurs independently of Cdc42 activation.

    PubMed

    Cannon, J L; Labno, C M; Bosco, G; Seth, A; McGavin, M H; Siminovitch, K A; Rosen, M K; Burkhardt, J K

    2001-08-01

    Cdc42 and WASP are critical regulators of actin polymerization whose function during T cell signaling is poorly understood. Using a novel reagent that specifically detects Cdc42-GTP in fixed cells, we found that activated Cdc42 localizes to the T cell:APC contact site in an antigen-dependent manner. TCR signaling alone was sufficient to induce localization of Cdc42-GTP, and functional Lck and Zap-70 kinases were required. WASP also localized to the T cell:APC contact site in an antigen-dependent manner. Surprisingly, WASP localization was independent of the Cdc42 binding domain but required the proline-rich domain. Our results indicate that localized WASP activation requires the integration of multiple signals: WASP is recruited via interaction with SH3 domain-containing proteins and is activated by Cdc42-GTP concentrated at the same site. PMID:11520460

  16. Early Gag Immunodominance of the HIV-Specific T-Cell Response during Acute/Early Infection Is Associated with Higher CD8+ T-Cell Antiviral Activity and Correlates with Preservation of the CD4+ T-Cell Compartment

    PubMed Central

    Ghiglione, Yanina; Falivene, Juliana; Socias, María Eugenia; Laufer, Natalia; Coloccini, Romina Soledad; Rodriguez, Ana María; Ruiz, María Julia; Pando, María Ángeles; Giavedoni, Luis David; Cahn, Pedro; Sued, Omar; Salomon, Horacio; Gherardi, María Magdalena

    2013-01-01

    The important role of the CD8+ T-cell response on HIV control is well established. Moreover, the acute phase of infection represents a proper scenario to delineate the antiviral cellular functions that best correlate with control. Here, multiple functional aspects (specificity, ex vivo viral inhibitory activity [VIA] and polyfunctionality) of the HIV-specific CD8+ T-cell subset arising early after infection, and their association with disease progression markers, were examined. Blood samples from 44 subjects recruited within 6 months from infection (primary HIV infection [PHI] group), 16 chronically infected subjects, 11 elite controllers (EC), and 10 healthy donors were obtained. Results indicated that, although Nef dominated the anti-HIV response during acute/early infection, a higher proportion of early anti-Gag T cells correlated with delayed progression. Polyfunctional HIV-specific CD8+ T cells were detected at early time points but did not associate with virus control. Conversely, higher CD4+ T-cell set points were observed in PHI subjects with higher HIV-specific CD8+ T-cell VIA at baseline. Importantly, VIA levels correlated with the magnitude of the anti-Gag cellular response. The advantage of Gag-specific cells may result from their enhanced ability to mediate lysis of infected cells (evidenced by a higher capacity to degranulate and to mediate VIA) and to simultaneously produce IFN-γ. Finally, Gag immunodominance was associated with elevated plasma levels of interleukin 2 (IL-2) and macrophage inflammatory protein 1β (MIP-1β). All together, this study underscores the importance of CD8+ T-cell specificity in the improved control of disease progression, which was related to the capacity of Gag-specific cells to mediate both lytic and nonlytic antiviral mechanisms at early time points postinfection. PMID:23616666

  17. PD-L1/PD-1 Co-Stimulation, a Brake for T cell Activation and a T cell Differentiation Signal.

    PubMed

    Liechtenstein, Therese; Dufait, Ines; Bricogne, Christopher; Lanna, Alessio; Pen, Joeri; Breckpot, Karine; Escors, David

    2012-10-30

    For T cell activation, three signals have to be provided from the antigen presenting cell; Signal 1 (antigen recognition), signal 2 (co-stimulation) and signal 3 (cytokine priming). Blocking negative co-stimulation during antigen presentation to T cells is becoming a promising therapeutic strategy to enhance cancer immunotherapy. Here we will focus on interference with PD-1/PD-L1 negative co-stimulation during antigen presentation to T cells as a therapeutic approach. We will discuss the potential mechanisms and the therapeutic consequences by which interference/inhibition with this interaction results in anti-tumour immunity. Particularly, we will comment on whether blocking negative co-stimulation provides differentiation signals to T cells undergoing antigen presentation. A major dogma in immunology states that T cell differentiation signals are given by cytokines and chemokines (signal 3) rather than co-stimulation (signal 2). We will discuss whether this is the case when blocking PD-L1/PD-1 negative co-stimulation.

  18. Amplification of the polyclonal activation of human T cells. I. Null-cell products promote the polyclonal proliferation of T cells.

    PubMed Central

    Caraux, J; Klein, B; Thierry, C; Serrou, B

    1982-01-01

    Synergy can be observed in the proliferative response to mitogens of cultures containing human T and Null cells when compared with those containing only highly purified cells of those two types. This synergy was analysed (i) by evaluation of the proliferative response at each step of the purification process leading to separation of T and Null cells; (ii) by back-mixing T and Nul cells at different rations; and (iii) by evaluation of the proliferative response of free suspension cultures of T cells overlaying a semi-solid layer containing Null cells, or of free suspension cultures of Null cells over a semi-solid culture layer of T cells. The following conclusions were reached: (i) purified Null cells are unresponsive to mitogen when cultured alone or in the presence of diffusible T-cell products; (ii) the T cells are less responsive when cultured alone than in the presence of Null cells or diffusible Null cell products. Thus the synergistic effect observed between T and Null cells is not due to the promotion of Null-cell proliferation by T -cell products but can be accounted for by diffusible Null-cell products enhancing the process of T lymphocyte activation by mitogens. PMID:6977479

  19. Expression of CD11c Is Associated with Unconventional Activated T Cell Subsets with High Migratory Potential.

    PubMed

    Qualai, Jamal; Li, Lin-Xi; Cantero, Jon; Tarrats, Antoni; Fernández, Marco Antonio; Sumoy, Lauro; Rodolosse, Annie; McSorley, Stephen J; Genescà, Meritxell

    2016-01-01

    CD11c is an α integrin classically employed to define myeloid dendritic cells. Although there is little information about CD11c expression on human T cells, mouse models have shown an association of CD11c expression with functionally relevant T cell subsets. In the context of genital tract infection, we have previously observed increased expression of CD11c in circulating T cells from mice and women. Microarray analyses of activated effector T cells expressing CD11c derived from naïve mice demonstrated enrichment for natural killer (NK) associated genes. Here we find that murine CD11c+ T cells analyzed by flow cytometry display markers associated with non-conventional T cell subsets, including γδ T cells and invariant natural killer T (iNKT) cells. However, in women, only γδ T cells and CD8+ T cells were enriched within the CD11c fraction of blood and cervical tissue. These CD11c+ cells were highly activated and had greater interferon (IFN)-γ secretory capacity than CD11c- T cells. Furthermore, circulating CD11c+ T cells were associated with the expression of multiple adhesion molecules in women, suggesting that these cells have high tissue homing potential. These data suggest that CD11c expression distinguishes a population of circulating T cells during bacterial infection with innate capacity and mucosal homing potential. PMID:27119555

  20. Expression of CD11c Is Associated with Unconventional Activated T Cell Subsets with High Migratory Potential

    PubMed Central

    Cantero, Jon; Tarrats, Antoni; Fernández, Marco Antonio; Sumoy, Lauro; Rodolosse, Annie; McSorley, Stephen J.

    2016-01-01

    CD11c is an α integrin classically employed to define myeloid dendritic cells. Although there is little information about CD11c expression on human T cells, mouse models have shown an association of CD11c expression with functionally relevant T cell subsets. In the context of genital tract infection, we have previously observed increased expression of CD11c in circulating T cells from mice and women. Microarray analyses of activated effector T cells expressing CD11c derived from naïve mice demonstrated enrichment for natural killer (NK) associated genes. Here we find that murine CD11c+ T cells analyzed by flow cytometry display markers associated with non-conventional T cell subsets, including γδ T cells and invariant natural killer T (iNKT) cells. However, in women, only γδ T cells and CD8+ T cells were enriched within the CD11c fraction of blood and cervical tissue. These CD11c+ cells were highly activated and had greater interferon (IFN)-γ secretory capacity than CD11c- T cells. Furthermore, circulating CD11c+ T cells were associated with the expression of multiple adhesion molecules in women, suggesting that these cells have high tissue homing potential. These data suggest that CD11c expression distinguishes a population of circulating T cells during bacterial infection with innate capacity and mucosal homing potential. PMID:27119555

  1. Immune complexes that contain HIV antigens activate peripheral blood T cells.

    PubMed

    Korolevskaya, L B; Shmagel, K V; Saidakova, E V; Shmagel, N G; Chereshnev, V A

    2016-07-01

    Uninfected donor T cells were treated in vitro by model immune complexes that contained either HIV or hepatitis C virus (HCV) antigens. Unlike HCV antigen-containing complexes, the immune complexes that contained HIV antigens have been shown to activate peripheral blood T cells of uninfected donors under in vitro conditions. Both the antiviral antibodies and HIV antigen were involved in the activation process. The unique properties of the immune complexes formed by HIV antigens and antiviral antibodies are believed to result from the virus-specific antibody properties and molecular conformation of the antigen-antibody complex. PMID:27595830

  2. Antiproliferative Activity of T. welwitschii Extract on Jurkat T Cells In Vitro

    PubMed Central

    Moyo, Batanai; Mukanganyama, Stanley

    2015-01-01

    Triumfetta welwitschii is a plant used traditionally for the treatment of fever and diarrhoea. Previous work has shown that T. welwitschii has antibacterial activity. The purpose of this study was to investigate T. welwitschii extract for anticancer activity against Jurkat T cells. The Jurkat T cell line is used to study acute T cell leukaemia. An antiproliferation assay, determination of induction of apoptosis, the determination of the effect of the combination of the extract and GSH, and effects of the extract on DNA leakage were conducted. T. welwitschii was found to decrease cell viability in a dose- and time-dependent manner. T. welwitschii caused apoptosis in the Jurkat T cells as shown by DNA fragmentation. When T. welwitschii was combined with reduced GSH, it was found that the growth of the Jurkat T cells was significantly reduced compared to untreated cells after 72 h of treatment. This was unexpected, as cancer cells have elevated levels of GSH compared to normal cells. The results of this study show that T. welwitschii is a potential source of compounds that may serve as leads for anticancer compounds. PMID:26557698

  3. Mucosal Regulatory T Cells and T Helper 17 Cells in HIV-Associated Immune Activation

    PubMed Central

    Pandiyan, Pushpa; Younes, Souheil-Antoine; Ribeiro, Susan Pereira; Talla, Aarthi; McDonald, David; Bhaskaran, Natarajan; Levine, Alan D.; Weinberg, Aaron; Sekaly, Rafick P.

    2016-01-01

    Residual mucosal inflammation along with chronic systemic immune activation is an important feature in individuals infected with human immunodeficiency virus (HIV), and has been linked to a wide range of co-morbidities, including malignancy, opportunistic infections, immunopathology, and cardiovascular complications. Although combined antiretroviral therapy (cART) can reduce plasma viral loads to undetectable levels, reservoirs of virus persist, and increased mortality is associated with immune dysbiosis in mucosal lymphoid tissues. Immune-based therapies are pursued with the goal of improving CD4+ T-cell restoration, as well as reducing chronic immune activation in cART-treated patients. However, the majority of research on immune activation has been derived from analysis of circulating T cells. How immune cell alterations in mucosal tissues contribute to HIV immune dysregulation and the associated risk of non-infectious chronic complications is less studied. Given the significant differences between mucosal T cells and circulating T cells, and the immediate interactions of mucosal T cells with the microbiome, more attention should be devoted to mucosal immune cells and their contribution to systemic immune activation in HIV-infected individuals. Here, we will focus on mucosal immune cells with a specific emphasis on CD4+ T lymphocytes, such as T helper 17 cells and CD4+Foxp3+ regulatory T cells (Tregs), which play crucial roles in maintaining mucosal barrier integrity and preventing inflammation, respectively. We hypothesize that pro-inflammatory milieu in cART-treated patients with immune activation significantly contributes to enhanced loss of Th17 cells and increased frequency of dysregulated Tregs in the mucosa, which in turn may exacerbate immune dysfunction in HIV-infected patients. We also present initial evidence to support this hypothesis. A better comprehension of how pro-inflammatory milieu impacts these two types of cells in the mucosa will shed light

  4. Identification of lineariifolianoid A as a novel dual NFAT1 and MDM2 inhibitor for human cancer therapy

    PubMed Central

    Qin, Jiang-Jiang; Sarkar, Sushanta; Voruganti, Sukesh; Agarwal, Rajesh; Wang, Wei; Zhang, Ruiwen

    2016-01-01

    Abstract There is an increasing interest in development of novel anticancer agents that target oncogenes. We have recently discovered that nuclear factor of activated T cells 1 (NFAT1) is a novel regulator of the Mouse Double Minute 2 (MDM2) oncogene and the NFAT1-MDM2 pathway has been implicated in human cancer development and progression, justifying that targeting the NFAT1-MDM2 pathway could be a novel strategy for discovery and development of novel cancer therapeutics. The present study was designed to examine the anticancer activity and underlying mechanisms of action of lineariifolianoid A (LinA), a novel natural product inhibitor of the NFAT1-MDM2 pathway. The cytotoxicity of LinA was first tested in various human cancer cell lines in comparison with normal cell lines. The results showed that the breast cancer cells were highly sensitive to LinA treatment. We next demonstrated the effects of LinA on cell proliferation, colony formation, cell cycle progression, and apoptosis in breast cancer MCF7 and MDA-MB-231 cells, in dose-dependent and p53-independent manners. LinA also inhibited the migration and invasion of these cancer cells. Our mechanistic studies further indicated that its anticancer activities were attributed to its inhibitory effects on the NFAT1-MDM2 pathway and modulatory effects on the expression of key proteins involved in cell cycle progression, apoptosis, and DNA damage. In summary, LinA is a novel NFAT1-MDM2 inhibitor and may be developed as a preventive and therapeutic agent against human cancer. PMID:27533941

  5. Asynchronous combinatorial action of four regulatory factors activates Bcl11b for T cell commitment.

    PubMed

    Kueh, Hao Yuan; Yui, Mary A; Ng, Kenneth K H; Pease, Shirley S; Zhang, Jingli A; Damle, Sagar S; Freedman, George; Siu, Sharmayne; Bernstein, Irwin D; Elowitz, Michael B; Rothenberg, Ellen V

    2016-08-01

    During T cell development, multipotent progenitors relinquish competence for other fates and commit to the T cell lineage by turning on Bcl11b, which encodes a transcription factor. To clarify lineage commitment mechanisms, we followed developing T cells at the single-cell level using Bcl11b knock-in fluorescent reporter mice. Notch signaling and Notch-activated transcription factors collaborate to activate Bcl11b expression irrespectively of Notch-dependent proliferation. These inputs work via three distinct, asynchronous mechanisms: an early locus 'poising' function dependent on TCF-1 and GATA-3, a stochastic-permissivity function dependent on Notch signaling, and a separate amplitude-control function dependent on Runx1, a factor already present in multipotent progenitors. Despite their necessity for Bcl11b expression, these inputs act in a stage-specific manner, providing a multitiered mechanism for developmental gene regulation. PMID:27376470

  6. Gene encoding T-cell-activating protein TAP maps to the Ly-6 locus.

    PubMed Central

    Reiser, H; Yeh, E T; Gramm, C F; Benacerraf, B; Rock, K L

    1986-01-01

    Recently we described two murine T-cell membrane proteins, TAP (T-cell-activating protein) and TAPa (TAP-associated protein). Previous experiments suggested that TAP is involved in physiologic T-cell activation. The subject of this report is a genetic analysis of these molecules. TAP and TAPa map to the Ly-6 locus. The relationship of these molecules to other antigens encoded in this locus is examined. Based on tissue distribution, molecular structure, and functional properties, TAP is distinct from any previously described Ly-6 antigen, whereas TAPa is probably identical to the 34-11-3 antigen. TAP and TAPa are coexpressed on all cell types examined so far. Moreover, comparative studies demonstrate a complex developmentally regulated pattern in the expression of molecules encoded in this locus. Images PMID:3010324

  7. Application of intact cell-based NFAT-β-lactamase reporter assay for Pasteurella multocida toxin-mediated activation of calcium signaling pathway

    PubMed Central

    Luo, Shuhong; Ho, Mengfei; Wilson, Brenda A.

    2009-01-01

    Pasteurella multocida toxin (PMT) stimulates and subsequently uncouples phospholipase C β1 (PLCβ1) signal transduction through its selective action on the alpha subunit of the Gq protein. Here, we describe the application of an NFAT-β-lactamase reporter assay as a functional readout for PMT-induced activation of the Gq-protein-coupled PLCβ1-IP3-Ca2+ signaling pathway. Use of the NFAT-β-lactamase reporter assay with a cell-permeable fluorogenic substrate provides high sensitivity due to the absence of endogenous β-lactamase activity in mammalian cells. This assay system was optimized for cell density, dose and time exposure of PMT stimulation. It is suited for quantitative characterization of PMT activity in mammalian cells and for use as a high-throughput screening method for PMT deletion and point mutants suitable for vaccine development. This method has application for diagnostic screening of clinical isolates of toxinogenic P. multocida. PMID:18190943

  8. Activated platelet–T-cell conjugates in peripheral blood of patients with HIV infection: coupling coagulation/inflammation and T cells

    PubMed Central

    Green, Samantha A.; Smith, Mindy; Hasley, Rebecca B.; Stephany, David; Harned, Adam; Nagashima, Kunio; Abdullah, Shahed; Pittaluga, Stefania; Imamichi, Tomozumi; Qin, Jing; Rupert, Adam; Ober, Alex; Lane, H. Clifford; Catalfamo, Marta

    2015-01-01

    Background: Despite successfully suppressed viremia by treatment, patients with high levels of biomarkers of coagulation/inflammation are at an increased risk of developing non-AIDS defining serious illnesses such as cardiovascular diseases. Thus, there is a relationship between persistent immune activation and coagulation/inflammation, although the mechanisms are poorly understood. Platelets play an important role in this process. Although interactions between platelets and elements of the innate immune system, such as monocytes, are well described, little is known about the interaction between platelets and the adaptive immune system. Design: We investigated the interaction of a component of the coagulation system, platelets, and the adaptive immune system T cells. Methods: Healthy controls and combination antiretroviral therapy (cART)-treated HIV-infected patients with viral loads of less than 40 copies/ml for more than 15 months were analysed for platelet–T-cell conjugate formation. Results: Platelets can form conjugates with T cells and were preferentially seen in CD4+ and CD8+ T-cell subsets with more differentiated phenotypes [memory, memory/effector and terminal effector memory (TEM)]. Compared with healthy controls, these conjugates in patients with HIV infection were more frequent, more often composed of activated platelets (CD42b+CD62P+), and were significantly associated with the D-dimer serum levels. Conclusion: These data support a model in which platelet–T-cell conjugates may play a critical role in the fast recruitment of antigen-experienced T cells to the place of injury. This mechanism can contribute in maintaining a state of coagulation/inflammation observed in these patients contributing to the pathology of the disease. PMID:26002800

  9. Interleukin-13-induced MUC5AC expression is regulated by a PI3K–NFAT3 pathway in mouse tracheal epithelial cells

    SciTech Connect

    Yan, Fugui; Li, Wen; Zhou, Hongbin; Wu, Yinfang; Ying, Songmin; Chen, Zhihua; Shen, Huahao

    2014-03-28

    Highlights: • IL-13 specifically induced NFAT3 activation in mouse tracheal epithelial cells. • CsA and LY294002 significantly blocked IL-13-induced MUC5AC production. • The PI3K–NFAT3 pathway is positively involved in IL-13-induced MUC5AC production. - Abstract: Interleukin-13 (IL-13) plays a critical role in asthma mucus overproduction, while the mechanisms underlying this process are not fully elucidated. Previous studies showed that nuclear factor of activated T cells (NFAT) is involved in the pathogenesis of asthma, but whether it can directly regulate IL-13-induced mucus (particularly MUC5AC) production is still not clear. Here we showed that IL-13 specifically induced NFAT3 activation through promoting its dephosphorylation in air–liquid interface (ALI) cultures of mouse tracheal epithelial cells (mTECs). Furthermore, both Cyclosporin A (CsA, a specific NFAT inhibitor) and LY294002 (a Phosphoinositide 3-kinase (PI3K) inhibitor) significantly blocked IL-13-induced MUC5AC mRNA and protein production through the inhibition of NFAT3 activity. We also confirmed that CsA could not influence the forkhead Box A2 (Foxa2) and mouse calcium dependent chloride channel 3 (mClca3) expression in IL-13-induced MUC5AC production, which both are known to be important in IL-13-stimulated mucus expression. Our study is the first to demonstrate that the PI3K–NFAT3 pathway is positively involved in IL-13-induced mucus production, and provided novel insights into the molecular mechanism of asthma mucus hypersecretion.

  10. Exhaustion of Activated CD8 T Cells Predicts Disease Progression in Primary HIV-1 Infection

    PubMed Central

    Hickling, Stephen; Hurst, Jacob; Meyerowitz, Jodi; Willberg, Christian B.; Robinson, Nicola; Brown, Helen; Kinloch, Sabine; Babiker, Abdel; Nwokolo, Nneka; Fox, Julie; Fidler, Sarah; Phillips, Rodney; Frater, John

    2016-01-01

    The rate at which HIV-1 infected individuals progress to AIDS is highly variable and impacted by T cell immunity. CD8 T cell inhibitory molecules are up-regulated in HIV-1 infection and associate with immune dysfunction. We evaluated participants (n = 122) recruited to the SPARTAC randomised clinical trial to determine whether CD8 T cell exhaustion markers PD-1, Lag-3 and Tim-3 were associated with immune activation and disease progression. Expression of PD-1, Tim-3, Lag-3 and CD38 on CD8 T cells from the closest pre-therapy time-point to seroconversion was measured by flow cytometry, and correlated with surrogate markers of HIV-1 disease (HIV-1 plasma viral load (pVL) and CD4 T cell count) and the trial endpoint (time to CD4 count <350 cells/μl or initiation of antiretroviral therapy). To explore the functional significance of these markers, co-expression of Eomes, T-bet and CD39 was assessed. Expression of PD-1 on CD8 and CD38 CD8 T cells correlated with pVL and CD4 count at baseline, and predicted time to the trial endpoint. Lag-3 expression was associated with pVL but not CD4 count. For all exhaustion markers, expression of CD38 on CD8 T cells increased the strength of associations. In Cox models, progression to the trial endpoint was most marked for PD-1/CD38 co-expressing cells, with evidence for a stronger effect within 12 weeks from confirmed diagnosis of PHI. The effect of PD-1 and Lag-3 expression on CD8 T cells retained statistical significance in Cox proportional hazards models including antiretroviral therapy and CD4 count, but not pVL as co-variants. Expression of ‘exhaustion’ or ‘immune checkpoint’ markers in early HIV-1 infection is associated with clinical progression and is impacted by immune activation and the duration of infection. New markers to identify exhausted T cells and novel interventions to reverse exhaustion may inform the development of novel immunotherapeutic approaches. PMID:27415828

  11. Exhaustion of Activated CD8 T Cells Predicts Disease Progression in Primary HIV-1 Infection.

    PubMed

    Hoffmann, Matthias; Pantazis, Nikos; Martin, Genevieve E; Hickling, Stephen; Hurst, Jacob; Meyerowitz, Jodi; Willberg, Christian B; Robinson, Nicola; Brown, Helen; Fisher, Martin; Kinloch, Sabine; Babiker, Abdel; Weber, Jonathan; Nwokolo, Nneka; Fox, Julie; Fidler, Sarah; Phillips, Rodney; Frater, John

    2016-07-01

    The rate at which HIV-1 infected individuals progress to AIDS is highly variable and impacted by T cell immunity. CD8 T cell inhibitory molecules are up-regulated in HIV-1 infection and associate with immune dysfunction. We evaluated participants (n = 122) recruited to the SPARTAC randomised clinical trial to determine whether CD8 T cell exhaustion markers PD-1, Lag-3 and Tim-3 were associated with immune activation and disease progression. Expression of PD-1, Tim-3, Lag-3 and CD38 on CD8 T cells from the closest pre-therapy time-point to seroconversion was measured by flow cytometry, and correlated with surrogate markers of HIV-1 disease (HIV-1 plasma viral load (pVL) and CD4 T cell count) and the trial endpoint (time to CD4 count <350 cells/μl or initiation of antiretroviral therapy). To explore the functional significance of these markers, co-expression of Eomes, T-bet and CD39 was assessed. Expression of PD-1 on CD8 and CD38 CD8 T cells correlated with pVL and CD4 count at baseline, and predicted time to the trial endpoint. Lag-3 expression was associated with pVL but not CD4 count. For all exhaustion markers, expression of CD38 on CD8 T cells increased the strength of associations. In Cox models, progression to the trial endpoint was most marked for PD-1/CD38 co-expressing cells, with evidence for a stronger effect within 12 weeks from confirmed diagnosis of PHI. The effect of PD-1 and Lag-3 expression on CD8 T cells retained statistical significance in Cox proportional hazards models including antiretroviral therapy and CD4 count, but not pVL as co-variants. Expression of 'exhaustion' or 'immune checkpoint' markers in early HIV-1 infection is associated with clinical progression and is impacted by immune activation and the duration of infection. New markers to identify exhausted T cells and novel interventions to reverse exhaustion may inform the development of novel immunotherapeutic approaches. PMID:27415828

  12. Circulating gamma delta T cells are activated and depleted during progression of high-grade gliomas: Implications for gamma delta T cell therapy of GBM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glioblastoma multiforme (GBM) remains frustratingly impervious to any existing therapy. We have previously shown that GBM is sensitive to recognition and lysis by ex vivo activated gamma delta T cells, a minor subset of lymphocytes that innately recognize autologous stress-associated target antigens...

  13. Mechanism of Activation-Induced Downregulation of Mitofusin 2 in Human Peripheral Blood T Cells.

    PubMed

    Dasgupta, Asish; Chen, Kuang-Hueih; Munk, Rachel B; Sasaki, Carl Y; Curtis, Jessica; Longo, Dan L; Ghosh, Paritosh

    2015-12-15

    Mitofusin 2 (Mfn2), a mitochondrial protein, was shown to have antiproliferative properties when overexpressed. In this article, we show that activation of resting human peripheral blood T cells caused downregulation of Mfn2 levels. This downregulation of Mfn2 was blocked by different inhibitors (mTOR inhibitor rapamycin, PI3K inhibitor LY294002, and Akt inhibitor A443654), producing cells that were arrested in the G0/G1 stage of the cell cycle. Furthermore, the activation-induced downregulation of Mfn2 preceded the entry of the cells into the cell cycle, suggesting that Mfn2 downregulation is a prerequisite for activated T cell entry into the cell cycle. Accordingly, small interfering RNA-mediated knockdown of Mfn2 resulted in increased T cell proliferation. Overexpression of constitutively active AKT resulted in the downregulation of Mfn2, which can be blocked by a proteasome inhibitor. Akt-mediated downregulation of Mfn2 was via the mTORC1 pathway because this downregulation was blocked by rapamycin, and overexpression of wild-type, but not kinase-dead mTOR, caused Mfn2 downregulation. Our data suggested that activation-induced reactive oxygen species production plays an important role in the downregulation of Mfn2. Collectively, our data suggest that the PI3K-AKT-mTOR pathway plays an important role in activation-induced downregulation of Mfn2 and subsequent proliferation of resting human T cells. PMID:26566676

  14. Murine T cell activation is regulated by surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide)

    SciTech Connect

    Warford, Jordan; Doucette, Carolyn D.; Hoskin, David W.; Easton, Alexander S.

    2014-01-10

    Highlights: •Surfen is the first inhibitor of glycosaminoglycan function to be studied in murine T cells. •Surfen reduces T cell proliferation stimulated in vitro and in vivo. •Surfen reduces CD25 expression in T cells activated in vivo but not in vitro. •Surfen increases T cell proliferation when T cell receptor activation is bypassed. •Surfen’s effects are blocked by co-administration of heparin sulfate. -- Abstract: Surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide) binds to glycosaminoglycans (GAGs) and has been shown to influence their function, and the function of proteoglycans (complexes of GAGs linked to a core protein). T cells synthesize, secrete and express GAGs and proteoglycans which are involved in several aspects of T cell function. However, there are as yet no studies on the effect of GAG-binding agents such as surfen on T cell function. In this study, surfen was found to influence murine T cell activation. Doses between 2.5 and 20 μM produced a graduated reduction in the proliferation of T cells activated with anti-CD3/CD28 antibody-coated T cell expander beads. Surfen (20 mg/kg) was also administered to mice treated with anti-CD3 antibody to activate T cells in vivo. Lymphocytes from surfen-treated mice also showed reduced proliferation and lymph node cell counts were reduced. Surfen reduced labeling with a cell viability marker (7-ADD) but to a much lower extent than its effect on proliferation. Surfen also reduced CD25 (the α-subunit of the interleukin (IL)-2 receptor) expression with no effect on CD69 expression in T cells treated in vivo but not in vitro. When receptor activation was bypassed by treating T cells in vitro with phorbyl myristate acetate (10 ng/ml) and ionomycin (100 ng/ml), surfen treatment either increased proliferation (10 μM) or had no effect (2.5, 5 and 20 μM). In vitro treatment of T cells with surfen had no effect on IL-2 or interferon-γ synthesis and did not alter proliferation of the IL-2 dependent cell

  15. Increased per cell IFN-γ productivity indicates recent in vivo activation of T cells

    PubMed Central

    Schlingmann, Tobias R.; Shive, Carey L.; Targoni, Oleg S.; Tary-Lehmann, Magdalena; Lehmann, Paul V.

    2009-01-01

    Immunization with vaccinia virus causes long-term immunity. Efforts have been made to characterize the T cells responsible for this protection. Recently, T cell subsets were described that not only co-express multiple cytokines, but also show increased per cell cytokine productivity. These highly productive cells are often considered to be the most protective. We used ELISPOT assays to measure per cell IFN-γ productivity of vaccinia specific T cells in childhood immunized adults immediately before and at different time points after vaccinia re-vaccination. Apart from an increase in frequency, we found a marked increase of IFN-γ productivity following vaccinia re-vaccination. However, these changes were short-lived as both parameters quickly returned to baseline values within 22 days after re-vaccination. Therefore, increased per cell IFN-γ productivity seems to be a sign of recent in vivo T cell activation rather than a stable marker of a distinct T cell subset responsible for long-term immune protection. PMID:19427634

  16. Microbial antigen mimics activate diabetogenic CD8 T cells in NOD mice.

    PubMed

    Tai, Ningwen; Peng, Jian; Liu, Fuqiang; Gulden, Elke; Hu, Youjia; Zhang, Xiaojun; Chen, Li; Wong, F Susan; Wen, Li

    2016-09-19

    Both animal model and human studies indicate that commensal bacteria may modify type 1 diabetes (T1D) development. However, the underlying mechanisms by which gut microbes could trigger or protect from diabetes are not fully understood, especially the interaction of commensal bacteria with pathogenic CD8 T cells. In this study, using islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-reactive CD8 T cell receptor NY8.3 transgenic nonobese diabetic mice, we demonstrated that MyD88 strongly modulates CD8(+) T cell-mediated T1D development via the gut microbiota. Some microbial protein peptides share significant homology with IGRP. Both the microbial peptide mimic of Fusobacteria and the bacteria directly activate IGRP-specific NY8.3 T cells and promote diabetes development. Thus, we provide evidence of molecular mimicry between microbial antigens and an islet autoantigen and a novel mechanism by which the diabetogenicity of CD8(+) T cells can be regulated by innate immunity and the gut microbiota. PMID:27621416

  17. Multiple Receptor-Ligand Interactions Direct Tissue-Resident γδ T Cell Activation

    PubMed Central

    Witherden, Deborah. A.; Ramirez, Kevin; Havran, Wendy L.

    2014-01-01

    γδ T cells represent a major T cell population in epithelial tissues, such as skin, intestine, and lung, where they function in maintenance of the epithelium and provide a crucial first line defense against environmental and pathogenic insults. Despite their importance, the molecular mechanisms directing their activation and function have remained elusive. Epithelial-resident γδ T cells function through constant communication with neighboring cells, either via direct cell-to-cell contact or cell-to-matrix interactions. These intimate relationships allow γδ T cells to facilitate the maintenance of epithelial homeostasis, tissue repair following injury, inflammation, and protection from malignancy. Recent studies have identified a number of molecules involved in these complex interactions, under both homeostatic conditions, as well as following perturbation of these barrier tissues. These interactions are crucial to the timely production of cytokines, chemokines, growth factors, and extracellular matrix proteins for restoration of homeostasis. In this review, we discuss recent advances in understanding the mechanisms directing epithelial-T cell crosstalk and the distinct roles played by individual receptor-ligand pairs of cell surface molecules in this process. PMID:25505467

  18. Analysis of T Cell Responses during Active Varicella-Zoster Virus Reactivation in Human Ganglia

    PubMed Central

    Steain, Megan; Sutherland, Jeremy P.; Rodriguez, Michael; Cunningham, Anthony L.; Slobedman, Barry

    2014-01-01

    ABSTRACT Varicella-zoster virus (VZV) is responsible for both varicella (chickenpox) and herpes zoster (shingles). During varicella, the virus establishes latency within the sensory ganglia and can reactivate to cause herpes zoster, but the immune responses that occur in ganglia during herpes zoster have not previously been defined. We examined ganglia obtained from individuals who, at the time of death, had active herpes zoster. Ganglia innervating the site of the cutaneous herpes zoster rash showed evidence of necrosis, secondary to vasculitis, or localized hemorrhage. Despite this, there was limited evidence of VZV antigen expression, although a large inflammatory infiltrate was observed. Characterization of the infiltrating T cells showed a large number of infiltrating CD4+ T cells and cytolytic CD8+ T cells. Many of the infiltrating T cells were closely associated with neurons within the reactivated ganglia, yet there was little evidence of T cell-induced neuronal apoptosis. Notably, an upregulation in the expression of major histocompatibility complex class I (MHC-I) and MHC-II molecules was observed on satellite glial cells, implying these cells play an active role in directing the immune response during herpes zoster. This is the first detailed characterization of the interaction between T cells and neuronal cells within ganglia obtained from patients suffering herpes zoster at the time of death and provides evidence that CD4+ and cytolytic CD8+ T cell responses play an important role in controlling VZV replication in ganglia during active herpes zoster. IMPORTANCE VZV is responsible for both varicella (chickenpox) and herpes zoster (shingles). During varicella, the virus establishes a life-long dormant infection within the sensory ganglia and can reawaken to cause herpes zoster, but the immune responses that occur in ganglia during herpes zoster have not previously been defined. We examined ganglia obtained from individuals who, at the time of death, had

  19. Non-Canonical Notch Signaling Drives Activation and Differentiation of Peripheral CD4+ T Cells

    PubMed Central

    Dongre, Anushka; Surampudi, Lalitha; Lawlor, Rebecca G.; Fauq, Abdul H.; Miele, Lucio; Golde, Todd E.; Minter, Lisa M.; Osborne, Barbara A.

    2014-01-01

    Cleavage of the Notch receptor via a γ-secretase, results in the release of the active intra-cellular domain of Notch that migrates to the nucleus and interacts with RBP-Jκ, resulting in the activation of downstream target genes. This canonical Notch signaling pathway has been documented to influence T cell development and function. However, the mechanistic details underlying this process remain obscure. In addition to RBP-Jκ, the intra-cellular domain of Notch also interacts with other proteins in the cytoplasm and nucleus, giving rise to the possibility of an alternate, RBP-Jκ independent Notch pathway. However, the contribution of such RBP-Jκ independent, “non-canonical” Notch signaling in regulating peripheral T cell responses is unknown. In this report, we specifically demonstrate the requirement of Notch1 for regulating signal strength and signaling events distal to the T cell receptor in peripheral CD4+ T cells. By using mice with a conditional deletion in Notch1 or RBP-Jκ, we show that Notch1 regulates activation and proliferation of CD4+ T cells independently of RBP-Jκ. Furthermore, differentiation to TH1 and iTreg lineages although Notch dependent, is RBP-Jκ independent. Our striking observations demonstrate that many of the cell-intrinsic functions of Notch occur independently of RBP-Jκ. Such non-canonical regulation of these processes likely occurs through NF-κ B. This reveals a previously unknown, novel role of non-canonical Notch signaling in regulating peripheral T cell responses. PMID:24611064

  20. Regulation of gene expression by NFAT transcription factors in hibernating ground squirrels is dependent on the cellular environment.

    PubMed

    Zhang, Yichi; Storey, Kenneth B

    2016-09-01

    Calcineurin is a calmodulin-stimulated phosphatase that regulates the nuclear translocation of nuclear factor of activated T cell (NFAT) c1-4 through dephosphorylation. We believe that this mechanism plays various roles in the remodeling and maintenance of Ictidomys tridecemlineatus skeletal muscle. During hibernation, bouts of torpor and arousal take place, and squirrels do not lose muscle mass despite being inactive. Protein expression of Ca(2+) signaling proteins were studied using immunoblotting. A DNA-protein interaction ELISA technique was created to test the binding of NFATs in the nucleus to DNA probes containing the NFAT response element under environmental conditions reflective of those during hibernation. Calcineurin protein levels increased by 3.08-fold during torpor (compared to euthermic control), whereas calpain1 levels also rose by 3.66-fold during torpor. Calmodulin levels were elevated upon entering torpor. NFATc4 binding to DNA showed a 1.4-fold increase during torpor, and we found that this binding was further enhanced when 600 nM of Ca(2+) was supplemented. We also found that decreasing the temperature of ELISAs resulted in progressive decreases in the binding of NFATs c1, c3, and c4 to DNA. In summary, calmodulin and calpain1 appear to activate calcineurin and NFATc4 during torpor. NFAT binding to target promoters is affected by intranuclear [Ca(2+)] and environmental temperatures. Therefore, Ca(2+) signaling and temperature changes play key roles in regulation of the NFAT-calcineurin pathway in skeletal muscle of hibernating 13-lined ground squirrels over the torpor-arousal cycle, and they may contribute to the avoidance of disuse-induced muscle atrophy that occurs naturally in these animals.

  1. Regulation of gene expression by NFAT transcription factors in hibernating ground squirrels is dependent on the cellular environment.

    PubMed

    Zhang, Yichi; Storey, Kenneth B

    2016-09-01

    Calcineurin is a calmodulin-stimulated phosphatase that regulates the nuclear translocation of nuclear factor of activated T cell (NFAT) c1-4 through dephosphorylation. We believe that this mechanism plays various roles in the remodeling and maintenance of Ictidomys tridecemlineatus skeletal muscle. During hibernation, bouts of torpor and arousal take place, and squirrels do not lose muscle mass despite being inactive. Protein expression of Ca(2+) signaling proteins were studied using immunoblotting. A DNA-protein interaction ELISA technique was created to test the binding of NFATs in the nucleus to DNA probes containing the NFAT response element under environmental conditions reflective of those during hibernation. Calcineurin protein levels increased by 3.08-fold during torpor (compared to euthermic control), whereas calpain1 levels also rose by 3.66-fold during torpor. Calmodulin levels were elevated upon entering torpor. NFATc4 binding to DNA showed a 1.4-fold increase during torpor, and we found that this binding was further enhanced when 600 nM of Ca(2+) was supplemented. We also found that decreasing the temperature of ELISAs resulted in progressive decreases in the binding of NFATs c1, c3, and c4 to DNA. In summary, calmodulin and calpain1 appear to activate calcineurin and NFATc4 during torpor. NFAT binding to target promoters is affected by intranuclear [Ca(2+)] and environmental temperatures. Therefore, Ca(2+) signaling and temperature changes play key roles in regulation of the NFAT-calcineurin pathway in skeletal muscle of hibernating 13-lined ground squirrels over the torpor-arousal cycle, and they may contribute to the avoidance of disuse-induced muscle atrophy that occurs naturally in these animals. PMID:27344571

  2. Regulatory Activity of Polyunsaturated Fatty Acids in T-Cell Signaling

    PubMed Central

    Kim, Wooki; Khan, Naim A.; McMurray, David N.; Prior, Ian A.; Wang, Naisyin; Chapkin, Robert S.

    2010-01-01

    n-3 polyunsaturated fatty acids (PUFA) are considered to be authentic immunosuppressors and appear to exert beneficial effects with respect to certain immune-mediated diseases. In addition to promoting T-helper 1 (Th1) cell to T-helper 2 (Th2) cell effector T-cell differentiation, n-3 PUFA may also exert anti-inflammatory actions by inducing apoptosis in Th1 cells. With respect to mechanisms of action, effects range from the modulation of membrane receptors to gene transcription via perturbation of a number of second messenger cascades. In this review, the putative targets of anti-inflammatory n-3 PUFA, activated during early and late events of T-cell activation will be discussed. Studies have demonstrated that these fatty acids alter plasma membrane micro-organization (lipid rafts) at the immunological synapse, the site where T-cells and antigen presenting cells (APC) form a physical contact for antigen initiated T-cell signaling. In addition, the production of diacylglycerol and the activation of different isoforms of protein kinase C (PKC), mitogen activated protein kinase (MAPK), calcium signaling, and nuclear translocation/activation of transcriptional factors, can be modulated by n-3 PUFA. Advantages and limitations of diverse methodologies to study the membrane lipid raft hypothesis, as well as apparent contradictions regarding the effect of n-3 PUFA on lipid rafts will be critically presented. PMID:20176053

  3. The SWI/SNF chromatin-remodeling complex modulates peripheral T cell activation and proliferation by controlling AP-1 expression.

    PubMed

    Jeong, Seung Min; Lee, Changjin; Lee, Sung Kyu; Kim, Jieun; Seong, Rho Hyun

    2010-01-22

    The SWI/SNF chromatin-remodeling complex has been implicated in the activation and proliferation of T cells. After T cell receptor signaling, the SWI/SNF complex rapidly associates with chromatin and controls gene expression in T cells. However, the process by which the SWI/SNF complex regulates peripheral T cell activation has not been elucidated. In this study, we show that the SWI/SNF complex regulates cytokine production and proliferation of T cells. During T cell activation, the SWI/SNF complex is recruited to the promoter of the transcription factor AP-1, and it increases the expression of AP-1. Increased expression of the SWI/SNF complex resulted in enhanced AP-1 activity, cytokine production, and proliferation of peripheral T cells, whereas knockdown of the SWI/SNF complex expression impaired the AP-1 expression and reduced the activation and proliferation of T cells. Moreover, mice that constitutively expressed the SWI/SNF complex in T cells were much more susceptible to experimentally induced autoimmune encephalomyelitis than the normal mice were. These results suggest that the SWI/SNF complex plays a critical role during T cell activation and subsequent immune responses.

  4. Increased prevalence of late stage T cell activation antigen (VLA-1) in active juvenile chronic arthritis.

    PubMed Central

    Odum, N; Morling, N; Platz, P; Hofmann, B; Ryder, L P; Heilmann, C; Pedersen, F K; Nielsen, L P; Friis, J; Svejgaard, A

    1987-01-01

    The presence of activated T cells as judged from the reaction with monoclonal antibodies (MoAb) against (a) a late stage T cell activation antigen (VLA-1), (b) the interleukin 2 (IL2) receptor (CD25), and (c) four different HLA class II molecules (HLA-DR, DRw52, DQ, and DP) was studied in 15 patients with active juvenile chronic arthritis (JCA), 10 patients with JCA in remission, and 11 age matched, healthy controls. In addition, the distribution of T 'helper/inducer' (CD4+), T 'suppressor/inducer' (CD4+, Leu8+), T 'suppressor/cytotoxic' (CD8+), and 'natural killer' (NK) cells (CD16+) was studied. Twenty patients and six controls were investigated for the capability to stimulate alloreactivated primed lymphocytes. The prevalence of VLA-1 positive, large cells was significantly increased to 5% (median value) in active JCA as compared with JCA in remission (2%, p less than 0.05) and controls (1%, p less than 0.05), whereas no significant difference between JCA in remission and controls was observed. Except for two patients with active JCA, less than 1% IL2 receptor bearing cells were found in patients with JCA and controls. No significant difference in the prevalence and expression of the various HLA class II antigens was observed between the groups. Similarly, no significant differences in stimulatory capability in secondary mixed lymphocyte culture (MLC) were seen. The distribution of T helper/inducer (CD4+), T suppressor/cytotoxic (CD8+), and NK cells was similar in active JCA, JCA in remission, and controls. The prevalence of T suppressor/inducer (CD4+,Leu8+) cells was higher in remission JCA (17%) than in active JCA (11%) and controls (10%). This increase, however, did not reach statistical significance. In conclusion, late stage but not early stage T cell activation antigens were increased in patients with active JCA as compared with patients with JCA in remission and control, whereas some patients in remission had an increased prevalence of T suppressor

  5. Targeting human CD27 with an agonist antibody stimulates T-cell activation and antitumor immunity.

    PubMed

    Thomas, Lawrence J; He, Li-Zhen; Marsh, Henry; Keler, Tibor

    2014-01-01

    CD27 is an important co-stimulatory receptor of T cells that can potentially be exploited for immunotherapy. We developed a human IgG1 antibody that targets human CD27, and demonstrated its immunostimulatory and antineoplastic activity in various preclinical models. Currently, the antibody (1F5, CDX-1127) is being tested in patients affected by advanced malignancies. PMID:24605266

  6. Endogenous n-3 Polyunsaturated Fatty Acids Attenuate T Cell-Mediated Hepatitis via Autophagy Activation.

    PubMed

    Li, Yanli; Tang, Yuan; Wang, Shoujie; Zhou, Jing; Zhou, Jia; Lu, Xiao; Bai, Xiaochun; Wang, Xiang-Yang; Chen, Zhengliang; Zuo, Daming

    2016-01-01

    Omega-3 polyunsaturated fatty acids (n-3 PUFAs) exert anti-inflammatory effects in several liver disorders, including cirrhosis, acute liver failure, and fatty liver disease. To date, little is known about their role in immune-mediated liver diseases. In this study, we used fat-1 transgenic mice rich in endogenous n-3 PUFAs to examine the role of n-3 PUFAs in immune-mediated liver injury. Concanavalin A (Con A) was administered intravenously to wild-type (WT) and fat-1 transgenic mice to induce T cell-mediated hepatitis. Reduced liver damage was shown in Con A-administrated fat-1 transgenic mice, as evidenced by decreased mortality, attenuated hepatic necrosis, lessened serum alanine aminotransferase activity, and inhibited production of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-17A, and IFN-γ). In vivo and in vitro studies demonstrated that n-3 PUFAs significantly inhibited the activation of hepatic T cells and the differentiation of Th1 cells after Con A challenge. Further studies showed that n-3 PUFAs markedly increased autophagy level in Con A-treated fat-1 T cells compared with the WT counterparts. Blocking hepatic autophagy activity with chloroquine diminished the differences in T cell activation and liver injury between Con A-injected WT and fat-1 transgenic mice. We conclude that n-3 PUFAs limit Con A-induced hepatitis via an autophagy-dependent mechanism and could be exploited as a new therapeutic approach for autoimmune hepatitis.

  7. Endogenous n-3 Polyunsaturated Fatty Acids Attenuate T Cell-Mediated Hepatitis via Autophagy Activation

    PubMed Central

    Li, Yanli; Tang, Yuan; Wang, Shoujie; Zhou, Jing; Zhou, Jia; Lu, Xiao; Bai, Xiaochun; Wang, Xiang-Yang; Chen, Zhengliang; Zuo, Daming

    2016-01-01

    Omega-3 polyunsaturated fatty acids (n-3 PUFAs) exert anti-inflammatory effects in several liver disorders, including cirrhosis, acute liver failure, and fatty liver disease. To date, little is known about their role in immune-mediated liver diseases. In this study, we used fat-1 transgenic mice rich in endogenous n-3 PUFAs to examine the role of n-3 PUFAs in immune-mediated liver injury. Concanavalin A (Con A) was administered intravenously to wild-type (WT) and fat-1 transgenic mice to induce T cell-mediated hepatitis. Reduced liver damage was shown in Con A-administrated fat-1 transgenic mice, as evidenced by decreased mortality, attenuated hepatic necrosis, lessened serum alanine aminotransferase activity, and inhibited production of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-17A, and IFN-γ). In vivo and in vitro studies demonstrated that n-3 PUFAs significantly inhibited the activation of hepatic T cells and the differentiation of Th1 cells after Con A challenge. Further studies showed that n-3 PUFAs markedly increased autophagy level in Con A-treated fat-1 T cells compared with the WT counterparts. Blocking hepatic autophagy activity with chloroquine diminished the differences in T cell activation and liver injury between Con A-injected WT and fat-1 transgenic mice. We conclude that n-3 PUFAs limit Con A-induced hepatitis via an autophagy-dependent mechanism and could be exploited as a new therapeutic approach for autoimmune hepatitis.

  8. Dietary fish oil increases CD8+ T-cells and decreases autoreactive T-cell activity in autoimmune NZB/W F1 mice.

    PubMed

    Wu, W M; Chiang, B L; Chang, S C; Lin, B F

    2001-03-01

    To further elucidate the effect of different dietary fats on the pathogenesis of autoimmune diseases, five groups of New Zealand black/white (NZB/W) F1 mice were fed diets containing 200 g of different dietary fats including palm oil, lard-soybean oil (1:1, w/w), soybean oil, canola oil or fish oil. Serum levels of anti-DNA antibodies, proteinuria were followed every month and life span of the mice was determined. After 5 months of the respective diets, mice were killed at the age of 7 months and phenotypic analysis of splenic cells and peritoneal resident cells was performed. The pattern of production of cytokines in splenic T-cells was also investigated. The peritoneal resident cells were isolated for measurement of prostaglandin E2 (PGE2) levels. Significantly lower immunoglobulin G (IgG) anti-single-stranded DNA (ssDNA) and anti-double-stranded DNA (dsDNA) antibody levels were associated with less severe proteinuria and prolonged life span in mice fed dietary fish oil compared to mice fed other dietary oils. Phenotypic analysis of spleen cells showed increased CD8+ T-cells in the mice fed dietary fish oil compared to mice of the other dietary groups, and the percentage of natural killer (NK) cells in the mice fed dietary fish oil was also higher compared to the other dietary groups. The peritoneal resident cells produced lower PGE2 in mice fed fish oil compared to mice in the other dietary groups. To further investigate the effect of fish oil on autoreactive T-cells, splenic T-cells purified using a nylon wool column were stimulated with non-T-cells of young NZB/W F1 mice. Our data suggest that the anti-DNA antibody augmentation ability of T-cells in mice fed dietary fish oil was significantly decreased compared to mice in the other dietary groups. These data indicate that dietary fish oil might maintain the existence of CD8+ T-cells, decrease autoreactive T-cell activity and alleviate subsequent autoimmune processes in autoimmune prone NZB/W F1 mice. PMID

  9. Human T cell activation. III. Induction of an early activation antigen, EA 1 by TPA, mitogens and antigens

    SciTech Connect

    Hara, T.; Jung, L.K.L.; FU, S.M.

    1986-03-01

    With human T cells activated for 12 hours by 12-o-tetradecanoyl phorbol-13-acetate (TPA) as immunogen, an IgG/sub 2a/ monoclonal antibody, mAb Ea 1, has been generated to a 60KD phosphorylated protein with 32KD and 28KD subunits. The antigen, Ea 1, is readily detected on 60% of isolated thymocytes by indirect immunofluorescence. A low level of Ea 1 expression is detectable on 2-6% of blood lymphocytes. Isolated T cells have been induced to express Ea 1 by TPA, mitogens and anitgens. TPA activated T cells express Ea 1 as early as 1 hour after activation. By 4 hours, greater than 95% of the T cells stain with mAb Ea 1. About 50% of the PHA or Con A activated T cells express Ea 1 with a similar kinetics. Ea 1 expression proceeds that of IL-2 receptor in these activation processes. T cells activated by soluble antigens (tetanus toxoid and PPD) and alloantigens in MLR also express Ea 1 after a long incubation. About 20% of the T cells stain for Ea 1 at day 6. Ea 1 expression is not limited to activated T cells. B cells activated by TPA or anti-IgM Ab plus B cell growth factor express Ea 1. The kinetics of Ea 1 expression is slower and the staining is less intense. Repeated attempts to detect Ea 1 on resting and activated monocytes and granulocytes have not been successful. Ea 1 expression is due to de novo synthesis for its induction is blocked by cycloheximide and actinomycin D. Ea 1 is the earliest activation antigen detectable to-date.

  10. MiR-16 regulates mouse peritoneal macrophage polarization and affects T-cell activation.

    PubMed

    Jia, Xiaoqin; Li, Xiaomin; Shen, Yating; Miao, Junjun; Liu, Hao; Li, Guoli; Wang, Zhengbing

    2016-10-01

    MiR-16 is a tumour suppressor that is down-regulated in certain human cancers. However, little is known on its activity in other cell types. In this study, we examined the biological significance and underlying mechanisms of miR-16 on macrophage polarization and subsequent T-cell activation. Mouse peritoneal macrophages were isolated and induced to undergo either M1 polarization with 100 ng/ml of interferon-γ and 20 ng/ml of lipopolysaccharide, or M2 polarization with 20 ng/ml of interleukin (IL)-4. The identity of polarized macrophages was determined by profiling cell-surface markers by flow cytometry and cytokine production by ELISA. Macrophages were infected with lentivirus-expressing miR-16 to assess the effects of miR-16. Effects on macrophage-T cell interactions were analysed by co-culturing purified CD4(+) T cells with miR-16-expressing peritoneal macrophages, and measuring activation marker CD69 by flow cytometry and cytokine secretion by ELISA. Bioinformatics analysis was applied to search for potential miR-16 targets and understand its underlying mechanisms. MiR-16-induced M1 differentiation of mouse peritoneal macrophages from either the basal M0- or M2-polarized state is indicated by the significant up-regulation of M1 marker CD16/32, repression of M2 marker CD206 and Dectin-1, and increased secretion of M1 cytokine IL-12 and nitric oxide. Consistently, miR-16-expressing macrophages stimulate the activation of purified CD4(+) T cells. Mechanistically, miR-16 significantly down-regulates the expression of PD-L1, a critical immune suppressor that controls macrophage-T cell interaction and T-cell activation. MiR-16 plays an important role in shifting macrophage polarization from M2 to M1 status, and functionally activating CD4(+) T cells. This effect is potentially mediated through the down-regulation of immune suppressor PD-L1.

  11. Nuclear Factor of Activated T Cells Transcription Factor Nfatp Controls Superantigen-Induced Lethal Shock

    PubMed Central

    Tsytsykova, Alla V.; Goldfeld, Anne E.

    2000-01-01

    Tumor necrosis factor α (TNF-α) is the key mediator of superantigen-induced T cell lethal shock. Here, we show that nuclear factor of activated T cells transcription factor, NFATp, controls susceptibility to superantigen-induced lethal shock in mice through its activation of TNF-α gene transcription. In NFATp-deficient mice, T cell stimulation leads to delayed induction and attenuation of TNF-α mRNA levels, decreased TNF-α serum levels, and resistance to superantigen-induced lethal shock. By contrast, after lipopolysaccharide (LPS) challenge, serum levels of TNF-α and susceptibility to shock are unaffected. These results demonstrate that NFATp is an essential activator of immediate early TNF-α gene expression in T cells and they present in vivo evidence of the inducer- and cell type–specific regulation of TNF-α gene expression. Furthermore, they suggest NFATp as a potential selective target in the treatment of superantigen-induced lethal shock. PMID:10952728

  12. Positive and negative regulation of T-cell activation through kinases and phosphatases.

    PubMed Central

    Mustelin, Tomas; Taskén, Kjetil

    2003-01-01

    The sequence of events in T-cell antigen receptor (TCR) signalling leading to T-cell activation involves regulation of a number of protein tyrosine kinases (PTKs) and the phosphorylation status of many of their substrates. Proximal signalling pathways involve PTKs of the Src, Syk, Csk and Tec families, adapter proteins and effector enzymes in a highly organized tyrosine-phosphorylation cascade. In intact cells, tyrosine phosphorylation is rapidly reversible and generally of a very low stoichiometry even under induced conditions due to the fact that the enzymes removing phosphate from tyrosine-phosphorylated substrates, the protein tyrosine phosphatases (PTPases), have a capacity that is several orders of magnitude higher than that of the PTKs. It follows that a relatively minor change in the PTK/PTPase balance can have a major impact on net tyrosine phosphorylation and thereby on activation and proliferation of T-cells. This review focuses on the involvement of PTKs and PTPases in positive and negative regulation of T-cell activation, the emerging theme of reciprocal regulation of each type of enzyme by the other, as well as regulation of phosphotyrosine turnover by Ser/Thr phosphorylation and regulation of localization of signal components. PMID:12485116

  13. T-cell activation is an immune correlate of risk in BCG vaccinated infants

    PubMed Central

    Fletcher, Helen A.; Snowden, Margaret A.; Landry, Bernard; Rida, Wasima; Satti, Iman; Harris, Stephanie A.; Matsumiya, Magali; Tanner, Rachel; O'Shea, Matthew K.; Dheenadhayalan, Veerabadran; Bogardus, Leah; Stockdale, Lisa; Marsay, Leanne; Chomka, Agnieszka; Harrington-Kandt, Rachel; Manjaly-Thomas, Zita-Rose; Naranbhai, Vivek; Stylianou, Elena; Darboe, Fatoumatta; Penn-Nicholson, Adam; Nemes, Elisa; Hatherill, Mark; Hussey, Gregory; Mahomed, Hassan; Tameris, Michele; McClain, J Bruce; Evans, Thomas G.; Hanekom, Willem A.; Scriba, Thomas J.; McShane, Helen

    2016-01-01

    Vaccines to protect against tuberculosis (TB) are urgently needed. We performed a case–control analysis to identify immune correlates of TB disease risk in Bacille Calmette–Guerin (BCG) immunized infants from the MVA85A efficacy trial. Among 53 TB case infants and 205 matched controls, the frequency of activated HLA-DR+ CD4+ T cells associates with increased TB disease risk (OR=1.828, 95% CI=1.25–2.68, P=0.002, FDR=0.04, conditional logistic regression). In an independent study of Mycobacterium tuberculosis-infected adolescents, activated HLA-DR+ CD4+ T cells also associate with increased TB disease risk (OR=1.387, 95% CI=1.068–1.801, P=0.014, conditional logistic regression). In infants, BCG-specific T cells secreting IFN-γ associate with reduced risk of TB (OR=0.502, 95% CI=0.29–0.86, P=0.013, FDR=0.14). The causes and impact of T-cell activation on disease risk should be considered when designing and testing TB vaccine candidates for these populations. PMID:27068708

  14. Simvastatin requires activation in accessory cells to modulate T-cell responses in asthma and COPD.

    PubMed

    Knobloch, Jürgen; Yakin, Yakup; Körber, Sandra; Grensemann, Barbara; Bendella, Zeynep; Boyaci, Niyazi; Gallert, Willem-Jakob; Yanik, Sarah Derya; Jungck, David; Koch, Andrea

    2016-10-01

    T-cell-dependent airway and systemic inflammation triggers the progression of chronic obstructive pulmonary disease (COPD) and asthma. Retrospective studies suggest that simvastatin has anti-inflammatory effects in both diseases but it is unclear, which cell types are targeted. We hypothesized that simvastatin modulates T-cell activity. Circulating CD4+ and CD8+ T-cells, either pure, co-cultured with monocytes or alveolar macrophages (AM) or in peripheral blood mononuclear cells (PBMCs), were ex vivo activated towards Th1/Tc1 or Th2/Tc2 and incubated with simvastatin. Markers for Th1/Tc1 (IFNγ) and Th2/Tc2 (IL-5, IL-13) were measured by ELISA; with PBMCs this was done comparative between 11 healthy never-smokers, 11 current smokers without airflow limitation, 14 smokers with COPD and 11 never-smokers with atopic asthma. T-cell activation induced IFNγ, IL-5 and IL-13 in the presence and absence of accessory cells. Simvastatin did not modulate cytokine expression in pure T-cell fractions. β-hydroxy-simvastatin acid (activated simvastatin) suppressed IL-5 and IL-13 in pure Th2- and Tc2-cells. Simvastatin suppressed IL-5 and IL-13 in Th2-cells co-cultivated with monocytes or AM, which was partially reversed by the carboxylesterase inhibitor benzil. Simvastatin suppressed IL-5 production of Th2/Tc2-cells in PBMCs without differences between cohorts and IL-13 stronger in never-smokers and asthma compared to COPD. Simvastatin induced IFNγ in Th1/Tc1-cells in PBMCs of all cohorts except asthmatics. Simvastatin requires activation in accessory cells likely by carboxylesterase to suppress IL-5 and IL-13 in Th2/Tc2-cells. The effects on Il-13 are partially reduced in COPD. Asthma pathogenesis prevents simvastatin-induced IFNγ up-regulation. Simvastatin has anti-inflammatory effects that could be of interest for asthma therapy.

  15. Virulent Salmonella enterica serovar typhimurium evades adaptive immunity by preventing dendritic cells from activating T cells.

    PubMed

    Tobar, Jaime A; Carreño, Leandro J; Bueno, Susan M; González, Pablo A; Mora, Jorge E; Quezada, Sergio A; Kalergis, Alexis M

    2006-11-01

    Dendritic cells (DCs) constitute the link between innate and adaptive immunity by directly recognizing pathogen-associated molecular patterns (PAMPs) in bacteria and by presenting bacterial antigens to T cells. Recognition of PAMPs renders DCs as professional antigen-presenting cells able to prime naïve T cells and initiate adaptive immunity against bacteria. Therefore, interfering with DC function would promote bacterial survival and dissemination. Understanding the molecular mechanisms that have evolved in virulent bacteria to evade activation of adaptive immunity requires the characterization of virulence factors that interfere with DC function. Salmonella enterica serovar Typhimurium, the causative agent of typhoid-like disease in the mouse, can prevent antigen presentation to T cells by avoiding lysosomal degradation in DCs. Here, we show that this feature of virulent Salmonella applies in vivo to prevent activation of adaptive immunity. In addition, this attribute of virulent Salmonella requires functional expression of a type three secretion system (TTSS) and effector proteins encoded within the Salmonella pathogenicity island 2 (SPI-2). In contrast to wild-type virulent Salmonella, mutant strains carrying specific deletions of SPI-2 genes encoding TTSS components or effectors proteins are targeted to lysosomes and are no longer able to prevent DCs from activating T cells in vitro or in vivo. SPI-2 mutant strains are attenuated in vivo, showing reduced tissue colonization and enhanced T-cell activation, which confers protection against a challenge with wild-type virulent Salmonella. Our data suggest that impairment of DC function by the activity of SPI-2 gene products is crucial for Salmonella pathogenesis.

  16. A Functionalized Sphingolipid Analogue for Studying Redistribution during Activation in Living T Cells.

    PubMed

    Collenburg, Lena; Walter, Tim; Burgert, Anne; Müller, Nora; Seibel, Jürgen; Japtok, Lukasz; Kleuser, Burkhard; Sauer, Markus; Schneider-Schaulies, Sibylle

    2016-05-01

    Sphingolipids are major components of the plasma membrane. In particular, ceramide serves as an essential building hub for complex sphingolipids, but also as an organizer of membrane domains segregating receptors and signalosomes. Sphingomyelin breakdown as a result of sphingomyelinase activation after ligation of a variety of receptors is the predominant source of ceramides released at the plasma membrane. This especially applies to T lymphocytes where formation of ceramide-enriched membrane microdomains modulates TCR signaling. Because ceramide release and redistribution occur very rapidly in response to receptor ligation, novel tools to further study these processes in living T cells are urgently needed. To meet this demand, we synthesized nontoxic, azido-functionalized ceramides allowing for bio-orthogonal click-reactions to fluorescently label incorporated ceramides, and thus investigate formation of ceramide-enriched domains. Azido-functionalized C6-ceramides were incorporated into and localized within plasma membrane microdomains and proximal vesicles in T cells. They segregated into clusters after TCR, and especially CD28 ligation, indicating efficient sorting into plasma membrane domains associated with T cell activation; this was abolished upon sphingomyelinase inhibition. Importantly, T cell activation was not abrogated upon incorporation of the compound, which was efficiently excluded from the immune synapse center as has previously been seen in Ab-based studies using fixed cells. Therefore, the functionalized ceramides are novel, highly potent tools to study the subcellular redistribution of ceramides in the course of T cell activation. Moreover, they will certainly also be generally applicable to studies addressing rapid stimulation-mediated ceramide release in living cells. PMID:27036914

  17. T Cell Dynamic Activation and Functional Analysis in Nanoliter Droplet Microarray

    PubMed Central

    Sarkar, Saheli; Motwani, Vinny; Sabhachandani, Pooja; Cohen, Noa; Konry, Tania

    2015-01-01

    Objective Characterization of the heterogeneity in immune reactions requires assessing dynamic single cell responses as well as interactions between the various immune cell subsets. Maturation and activation of effector cells is regulated by cell contact-dependent and soluble factor-mediated paracrine signalling. Currently there are few methods available that allow dynamic investigation of both processes simultaneously without physically constraining non-adherent cells and eliminating crosstalk from neighboring cell pairs. We describe here a microfluidic droplet microarray platform that permits rapid functional analysis of single cell responses and co-encapsulation of heterotypic cell pairs, thereby allowing us to evaluate the dynamic activation state of primary T cells. Methods The microfluidic droplet platform enables generation and docking of monodisperse nanoliter volume (0.523 nl) droplets, with the capacity of monitoring a thousand droplets per experiment. Single human T cells were encapsulated in droplets and stimulated on-chip with the calcium ionophore ionomycin. T cells were also co-encapsulated with dendritic cells activated by ovalbumin peptide, followed by dynamic calcium signal monitoring. Results Ionomycin-stimulated cells depicted fluctuation in calcium signalling compared to control. Both cell populations demonstrated marked heterogeneity in responses. Calcium signalling was observed in T cells immediately following contact with DCs, suggesting an early activation signal. T cells further showed non-contact mediated increase in calcium level, although this response was delayed compared to contact-mediated signals. Conclusions Our results suggest that this nanoliter droplet array-based microfluidic platform is a promising technique for assessment of heterogeneity in various types of cellular responses, detection of early/delayed signalling events and live cell phenotyping of immune cells. PMID:26613065

  18. Nanovesicle-targeted Kv1.3 knockdown in memory T cells suppresses CD40L expression and memory phenotype.

    PubMed

    Chimote, Ameet A; Hajdu, Peter; Kottyan, Leah C; Harley, John B; Yun, Yeoheung; Conforti, Laura

    2016-05-01

    Ca(2+) signaling controls activation and effector functions of T lymphocytes. Ca(2+) levels also regulate NFAT activation and CD40 ligand (CD40L) expression in T cells. CD40L in activated memory T cells binds to its cognate receptor, CD40, on other cell types resulting in the production of antibodies and pro-inflammatory mediators. The CD40L/CD40 interaction is implicated in the pathogenesis of autoimmune disorders and CD40L is widely recognized as a therapeutic target. Ca(2+) signaling in T cells is regulated by Kv1.3 channels. We have developed lipid nanoparticles that deliver Kv1.3 siRNAs (Kv1.3-NPs) selectively to CD45RO(+) memory T cells and reduce the activation-induced Ca(2+) influx. Herein we report that Kv1.3-NPs reduced NFAT activation and CD40L expression exclusively in CD45RO(+) T cells. Furthermore, Kv1.3-NPs suppressed cytokine release and induced a phenotype switch of T cells from predominantly memory to naïve. These findings indicate that Kv1.3-NPs operate as targeted immune suppressive agents with promising therapeutic potentials.

  19. In Vitro Inhibition of NFAT5-Mediated Induction of CCL2 in Hyperosmotic Conditions by Cyclosporine and Dexamethasone on Human HeLa-Modified Conjunctiva-Derived Cells

    PubMed Central

    Baudouin, Christophe; Gard, Carole; Brignole-Baudouin, Françoise

    2016-01-01

    Purpose To investigate the pro-inflammatory intracellular mechanisms induced by an in vitro model of dry eye disease (DED) on a Hela-modified conjunctiva-derived cells in hyperosmolarity (HO) stress conditions. This study focused on CCL2 induction and explored the implications of the nuclear factor of activated T-cells 5 (NFAT5) as well as mitogen-activated protein kinases (MAPK) and nuclear factor kappa B (NFĸB). This work was completed by an analysis of the effects of cyclosporine A (CsA), dexamethasone (Dex) and doxycycline (Dox) on HO-induced CCL2 and NFAT5 induction. Methods A human HeLa-modified conjunctiva-derived cell line was cultured in NaCl-hyperosmolar medium for various exposure times. Cellular viability, CCL2 secretion, NFAT5 and CCL2 gene expression, and intracytoplasmic NFAT5 were assessed using the Cell Titer Blue® assay, enzyme-linked immunosorbent assay (ELISA), RT-qPCR and immunostaining, respectively. In selected experiments, inhibitors of MAPKs or NFκB, therapeutic agents or NFAT5 siRNAs were added before the hyperosmolar stimulations. Results HO induced CCL2 secretion and expression as well as NFAT5 gene expression and translocation. Adding NFAT5-siRNA before hyperosmolar stimulation led to a complete inhibition of CCL2 induction and to a decrease in cellular viability. p38 MAPK (p38), c-Jun NH2-terminal kinase (JNK) and NFĸB inhibitors, CsA and Dex induced a partial inhibition of HO-induced CCL2, while Dox and extracellular signal-regulated kinase (ERK) inhibitor did not. Dex also induced a partial inhibition of HO-induced NFAT5 gene expression but not CsA or Dox. Conclusions These in vitro results suggest a potential role of CCL2 in DED and highlight the crucial role of NFAT5 in the pro-inflammatory effect of HO on HeLa-modified conjunctiva-derived cells, a rarely studied cellular type. This inflammatory pathway involving NFAT5 and CCL2 could offer a promising target for developing new therapies to treat DED, warranting further

  20. Whole Blood Activation Results in Altered T Cell and Monocyte Cytokine Production Profiles by Flow Cytometry

    NASA Technical Reports Server (NTRS)

    Crucian, Brian E.; Sams, Clarence F.

    2001-01-01

    An excellent monitor of the immune balance of peripheral circulating cells is to determine their cytokine production patterns in response to stimuli. Using flow cytometry, a positive identification of cytokine producing cells in a mixed culture may be achieved. Recently, the ability to assess cytokine production following a whole-blood activation culture has been described. In this study, whole blood activation was compared to traditional PBMC activation and the individual cytokine secretion patterns for both T cells, T cell subsets and monocytes was determined by flow cytometry. RESULTS: For T cell cytokine assessment (IFNg/IL-10 and IL-21/L-4) following PMA +ionomycin activation: (1) a Significantly greater percentages of T cells producing IFNgamma and IL-2 were observed following whole-blood culture and (2) altered T cell cytokine production kinetics were observed by varying whole blood culture times. Four-color analysiS was used to allow assessment of cytokine production by specific T cell subsets. It was found that IFNgamma production was significantly elevated in the CD3+/CD8+ T cell population as compared to the CD3+/CD8- population following five hours of whole blood activation. Conversely, IL-2 and IL-10 production were Significantly elevated in the CD3+/CD8- T cell population as compared to the CD3+/CD8+ population. Monocyte cytokine production was assessed in both culture systems following LPS activation for 24 hours. A three-color flow cytometric was used to assess two cytokines (IL-1a/IL-12 and TNFa/IL-10) in conjunction with CD14. Nearly all monocytes were stimulated to produce IL-1a, IL-12 and TNFa. equally well in both culture systems, however monocyte production of IL-10 was significantly elevated in whole blood culture as compared to PBMC culture. IL-12 producing monocytes appeared to be a distinct subpopulation of the IL-1a producing set, whereas IL-10 and TNFa producing monocytes were largely mutually exclusive. IL-10 and TNFa producing

  1. Inhibition of Th2 cytokine production in T cells by monascin via PPAR-γ activation.

    PubMed

    Hsu, Wei-Hsuan; Lee, Bao-Hong; Hsu, Ya-Wen; Pan, Tzu-Ming

    2013-08-28

    Yellow pigment monascin (MS) is a secondary metabolite isolated from Monascus -fermented products and has numerous physiological activities. However, the potential use of MS for immunomodulation remains unclear. We showed that MS and the synthetic peroxisome proliferator-activated receptor (PPAR)-γ ligand rosiglitazone (RG) significantly inhibited the production of Th2 cytokines, including IL-4, IL-5, and IL-13, in PMA/ionomycin-activated mouse EL-4 T cells. Moreover, we showed that this was due to cellular PPAR-γ translocation. These results indicate that MS and RG promote PPAR-γ-DNA interactions and suggest that the regulatory effects of MS and RG on Th2 cytokine production could be abolished with PPAR-γ antagonist treatment. MS and RG also suppressed Th2 transcription factor translocation (e.g., GATA-3 and nuclear factor of activated T cells) by preventing the phosphorylation of protein kinase C and signal transducer and activator of transcription 6. PMID:23848565

  2. Aurora A drives early signalling and vesicle dynamics during T-cell activation

    PubMed Central

    Blas-Rus, Noelia; Bustos-Morán, Eugenio; Pérez de Castro, Ignacio; de Cárcer, Guillermo; Borroto, Aldo; Camafeita, Emilio; Jorge, Inmaculada; Vázquez, Jesús; Alarcón, Balbino; Malumbres, Marcos; Martín-Cófreces, Noa B.; Sánchez-Madrid, Francisco

    2016-01-01

    Aurora A is a serine/threonine kinase that contributes to the progression of mitosis by inducing microtubule nucleation. Here we have identified an unexpected role for Aurora A kinase in antigen-driven T-cell activation. We find that Aurora A is phosphorylated at the immunological synapse (IS) during TCR-driven cell contact. Inhibition of Aurora A with pharmacological agents or genetic deletion in human or mouse T cells severely disrupts the dynamics of microtubules and CD3ζ-bearing vesicles at the IS. The absence of Aurora A activity also impairs the activation of early signalling molecules downstream of the TCR and the expression of IL-2, CD25 and CD69. Aurora A inhibition causes delocalized clustering of Lck at the IS and decreases phosphorylation levels of tyrosine kinase Lck, thus indicating Aurora A is required for maintaining Lck active. These findings implicate Aurora A in the propagation of the TCR activation signal. PMID:27091106

  3. Calmodulin-dependent phosphatase, kinases, and transcriptional corepressors involved in T-cell activation

    PubMed Central

    Liu, Jun O.

    2009-01-01

    Summary The second messenger calcium plays an essential role in mediating the TCR signaling pathway leading to cytokine production and T cell clonal expansion. The immunosuppressive drugs cyclosproin A (CsA) and FK506 have served both as therapeutic agents and as molecular probes for unraveling the protein phosphatase calcineurin as a rate-limiting enzyme involved in the transmission of calcium signal from the cytosol into the nucleus to reprogram gene expression. The use of mouse knockout models has helped to verify and further elucidate the functions of different isoforms of calcineurin in both helper T cell activation and thymocyte development. In addition to calcineurin, three other classes of calmodulin-binding proteins have also been shown to play important roles in calcium signaling in T cells. Thus, Cabin1 and class II HDACs have been found to constitute a novel calcium-signaling module in conjunction with the transcription factor myocyte enhance factor family and the transcriptional coactivator p300 to suppress and activate cytokine gene transcription in a calcium-dependent manner. The calmodulin-dependent protein kinases (CaMK) II and IV were also shown to play negative and positive regulatory functions, respectively, in TCR-mediated cytokine production. The crosstalks among these and other signal transducers in T cells form an extensive non-linear signaling network that dictates the final outcome of the TCR signaling pathway. PMID:19290928

  4. Inhibition of caspase-8 activity reduces IFN-gamma expression by T cells from Leishmania major infection.

    PubMed

    Pereira, Wânia F; Guillermo, Landi V C; Ribeiro-Gomes, Flávia L; Lopes, Marcela F

    2008-03-01

    Following infection with Leishmania major, T cell activation and apoptosis can be detected in draining lymph nodes of C57BL/6-infected mice. We investigated the mechanisms involved in apoptosis and cytokine expression following T cell activation. After two weeks of infection, apoptotic T cells were not detected in draining lymph nodes but activation with anti-CD3 induced apoptosis in both CD4 and CD8 T cells. Treatment with anti-Fas Ligand, caspase-8 or caspase- 9 inhibitors did not block activation-induced T-cell death. We also investigated whether the blockade of caspase-8 activity would affect the expression of type-1 or type-2 cytokines. At early stages of infection, both CD4 and CD8 T cells expressed IFN-gamma upon activation. Treatment with the caspase-8 inhibitor zIETD-fmk (benzyl-oxycarbonyl-Ile- Glu(OMe)-Thr-Asp(OMe)-fluoromethyl ketone) reduced the proportion of CD8 T cells and IFN-gamma expression in both CD4 and CD8 T cells. We conclude that a non apoptotic role of caspase-8 activity may be required for T cell-mediated type-1 responses during L. major infection.

  5. NFAT restricts osteochondroma formation from entheseal progenitors

    PubMed Central

    Tsang, Kelly; He, Lizhi; Garcia, Roberto A.; Ermann, Joerg; Mizoguchi, Fumitaka; Zhang, Minjie; Aliprantis, Antonios O.

    2016-01-01

    Osteochondromas are common benign osteocartilaginous tumors in children and adolescents characterized by cartilage-capped bony projections on the surface of bones. These tumors often cause pain, deformity, fracture, and musculoskeletal dysfunction, and they occasionally undergo malignant transformation. The pathogenesis of osteochondromas remains poorly understood. Here, we demonstrate that nuclear factor of activated T cells c1 and c2 (NFATc1 and NFATc2) suppress osteochondromagenesis through individual and combinatorial mechanisms. In mice, conditional deletion of NFATc1 in mesenchymal limb progenitors, Scleraxis-expressing (Scx-expressing) tendoligamentous cells, or postnatally in Aggrecan-expressing cells resulted in osteochondroma formation at entheses, the insertion sites of ligaments and tendons onto bone. Combinatorial deletion of NFATc1 and NFATc2 gave rise to larger and more numerous osteochondromas in inverse proportion to gene dosage. A population of entheseal NFATc1- and Aggrecan-expressing cells was identified as the osteochondroma precursor, previously believed to be growth plate derived or perichondrium derived. Mechanistically, we show that NFATc1 restricts the proliferation and chondrogenesis of osteochondroma precursors. In contrast, NFATc2 preferentially inhibits chondrocyte hypertrophy and osteogenesis. Together, our findings identify and characterize a mechanism of osteochondroma formation and suggest that regulating NFAT activity is a new therapeutic approach for skeletal diseases characterized by defective or exaggerated osteochondral growth. PMID:27158674

  6. Reconstitution of CD4 T Cells in Bronchoalveolar Lavage Fluid after Initiation of Highly Active Antiretroviral Therapy▿

    PubMed Central

    Knox, Kenneth S.; Vinton, Carol; Hage, Chadi A.; Kohli, Lisa M.; Twigg, Homer L.; Klatt, Nichole R.; Zwickl, Beth; Waltz, Jeffrey; Goldman, Mitchell; Douek, Daniel C.; Brenchley, Jason M.

    2010-01-01

    The massive depletion of gastrointestinal-tract CD4 T cells is a hallmark of the acute phase of HIV infection. In contrast, the depletion of the lower-respiratory-tract mucosal CD4 T cells as measured in bronchoalveolar lavage (BAL) fluid is more moderate and similar to the depletion of CD4 T cells observed in peripheral blood (PB). To understand better the dynamics of disease pathogenesis and the potential for the reconstitution of CD4 T cells in the lung and PB following the administration of effective antiretroviral therapy, we studied cell-associated viral loads, CD4 T-cell frequencies, and phenotypic and functional profiles of antigen-specific CD4 T cells from BAL fluid and blood before and after the initiation of highly active antiretroviral therapy (HAART). The major findings to emerge were the following: (i) BAL CD4 T cells are not massively depleted or preferentially infected by HIV compared to levels for PB; (ii) BAL CD4 T cells reconstitute after the initiation of HAART, and their infection frequencies decrease; (iii) BAL CD4 T-cell reconstitution appears to occur via the local proliferation of resident BAL CD4 T cells rather than redistribution; and (iv) BAL CD4 T cells are more polyfunctional than CD4 T cells in blood, and their functional profile is relatively unchanged after the initiation of HAART. Taken together, these data suggest mechanisms for mucosal CD4 T-cell depletion and interventions that might aid in the reconstitution of mucosal CD4 T cells. PMID:20610726

  7. Regulation of human tonsillar T-cell proliferation by the active metabolite of vitamin D3.

    PubMed Central

    Nunn, J D; Katz, D R; Barker, S; Fraher, L J; Hewison, M; Hendy, G N; O'Riordan, J L

    1986-01-01

    We have examined the effects of 1,25(OH)2D3 on T-cell populations isolated by buoyant density and E rosetting from human tonsils. Cell proliferation was assessed by measuring the incorporation of 125iododeoxyuridine; interleukin-2 (IL-2) production was measured using an IL-2-dependent cell line, and the number of 1,25(OH)2D3 receptors was measured by whole-cell nuclear association assay. At a concentration of 10(-7) M, 1,25(OH)2D3 inhibited mitogen-induced T-cell proliferation in all E+ T-cell populations. This effect was more pronounced in the cells from the intermediate and high density layers and was reflected both in cell proliferative responses and in relative IL-2 synthesis. By adding the 1,25(OH)2D3 during the course of the mitogen assay, we demonstrated that activation of the T cell precedes the 1,25(OH)2D3-mediated inhibition. Cells that had been preincubated with mitogen in the presence of the 1,25(OH)2D3 were refractory to further stimulation by mitogens. Receptors for 1,25(OH)2D3 could not be detected in unstimulated T cells. However, activation led to the expression of high-affinity receptors for 1,25(OH)2D3. Co-incubation of the cells with mitogen and 1,25(OH)2D3 increased the number of receptors compared with mitogen alone. The effects provide further evidence for the hypothesis that 1,25(OH)2D3 is an important potential modulator of the immune system through its action on T cells. Taking our observations in conjunction with the known capacity of monocytes to hydroxylate the precursor metabolite (and thus synthesize the active form of cholecalciferol), the results support the suggestion that 1,25(OH)2D3 plays a role as a local mediator of mononuclear phagocyte-T cell interaction in human lymphomedullary tissues. PMID:3026959

  8. Antiviral activity of human Vδ2 T-cells against WNV includes both cytolytic and non-cytolytic mechanisms.

    PubMed

    Agrati, Chiara; Castilletti, Concetta; Cimini, Eleonora; Romanelli, Antonella; Lapa, Daniele; Quartu, Serena; Martini, Federico; Capobianchi, Maria Rosaria

    2016-04-01

    West Nile virus (WNV) causes a severe central nervous system infection in humans, primarily in the elderly and immunocompromised subjects. Human γδ T-cells play a critical role in the immune response against viruses, and studies of WNV meningoencephalitis in laboratory mice described a role of γδ T-cells in the protective immune response. Aim of this study was to analyze the cytolytic and non-cytolytic antiviral activity of human Vδ2 T-cells against WNV replication. The anti-WNV activity of soluble factor released by zoledronic acid (ZA)-activated Vδ2 T-cell lines and the cytotoxic capability of Vδ2 T-cell lines against WNV-infected cells were tested in vitro. The activation of Vδ2 T-cell lines was able to inhibit WNV replication through the release of soluble factors. IFN-γ is massively released by activated Vδ2 T-cell lines and is involved in the anti-WNV activity. Moreover, the Vδ2 T-cell lines can efficiently kill WNV-infected cells possibly through perforin-mediated mechanism. Altogether, our results provide insight into the effector functions of human Vδ2 T-cells against WNV. The possibility to target these cells by ZA, a commercially available drug used in humans, could potentially offer a new immunotherapeutic strategy for WNV infection. PMID:27196553

  9. Dissection of macrophage tumoricidal and protozoacidal activities using T-cell hybridomas and recombinant lymphokines.

    PubMed Central

    Futch, W S; Schook, L B

    1985-01-01

    Macrophage (M phi) phenotype and function can be modulated by various T-cell lymphokines (LK). The alteration of M phi phenotype is a result of LK concentration, duration of exposure, and the level of M phi activation when obtained from in vivo sources through elicitation by either sterile irritants or cellular immune mechanisms. To dissect M phi activation into discrete signals, we constructed T-cell hybridomas by fusing hypoxanthine-aminopterin-thymidine-sensitive BW5147 cells with nylon wool-purified, concanavalin A-stimulated T cells. The resulting T-cell hybrids were screened for their ability to (i) protect M phi from the cytopathic effect of Naegleria lysates, (ii) induce class II major histocompatibility complex gene product (Ia antigen) expression, (iii) increase tumoricidal and cytostatic activity, and (iv) alter ectoenzyme profiles on either resident or thioglycolate-elicited M phi. Two hybridomas (T-3 and T-9) were selected for further evaluation because of their activity patterns. Supernatants from T-3 and T-9 were compared with cloned gamma-interferon (IFN-gamma) for alterations of biological activities. Both T-3 and T-9 were able to protect resident-M phi cells from Naegleria lysate but had no protective effect on thioglycolate-induced M phi. T-9 supernatant had patterns of activity similar to IFN-gamma, whereas T-3 patterns were different. The addition of anti- IFN-gamma removed T-9 cytostatic activity while not affecting T-3-induced activity. The LK inducing protection from the cytopathic effect of Naegleria lysate is not IFN-gamma but another molecular moiety. We conclude that the activation of M phi for the destruction of tumor cells and amoebae may occur via different mechanisms. PMID:2998999

  10. CaMKII Negatively Regulates Calcineurin-NFAT Signaling in Cardiac Myocytes

    PubMed Central

    MacDonnell, Scott M.; Weisser-Thomas, Jutta; Kubo, Hajime; Hanscome, Marie; Liu, Qinghang; Jaleel, Naser; Berretta, Remus; Chen, Xiongwen; Brown, Joan H.; Sabri, Abdel-Karim; Molkentin, Jeffery D.; Houser, Steven R.

    2009-01-01

    Rationale Pathologic cardiac myocyte hypertrophy is thought to be induced by the persistent increases in intracellular Ca2+ needed to maintain cardiac function when systolic wall stress is increased. Hypertrophic Ca2+ binds to calmodulin (CaM) and activates the phosphatase calcineurin (Cn) and CaM kinase (CaMKII). Cn dephosphorylates cytoplasmic nuclear factor of activated T-cells (NFAT), inducing its translocation to the nucleus where it activates anti-apoptotic and hypertrophic target genes. Cytoplasmic CaMKII regulates Ca2+ handling proteins but whether or not it is directly involved in hypertrophic and survival signaling is not known. Objective This study explored the hypothesis that cytoplasmic CaMKII reduces NFAT nuclear translocation by inhibiting the phosphatase activity of Cn. Methods and Results GFP-tagged NFATc3 was used to determine the cellular location of NFAT in cultured neonatal rat ventricular myocytes (NRVM) and adult feline ventricular myocytes. Constitutively active (CaMKII-CA) or dominant negative (CaMKII-DN) mutants of cytoplasmic targeted CaMKIIδc were used to activate and inhibit cytoplasmic CaMKII activity. In NRVM CaMKII-DN (48.5±3%, P<0.01 vs control) increased while CaMKII-CA decreased (5.9±1%, P<0.01 vs control) NFAT nuclear translocation (Control: 12.3±1%). Cn inhibitors were used to show that these effects were caused by modulation of Cn activity. Increasing Ca2+ increased Cn-dependent NFAT translocation (to 71.7±7%, p<0.01) and CaMKII-CA reduced this effect (to 17.6±4%). CaMKII-CA increased TUNEL and caspase-3 activity (P<0.05). CaMKII directly phosphorylated Cn at Ser197 in CaMKII-CA infected NRVM and in hypertrophied feline hearts. Conclusion These data show that activation of cytoplasmic CaMKII inhibits NFAT nuclear translocation by phosphorylation and subsequent inhibition of Cn. PMID:19608982

  11. Hyaluronan synthesis is necessary for autoreactive T-cell trafficking, activation, and Th1 polarization

    PubMed Central

    Kuipers, Hedwich F.; Rieck, Mary; Gurevich, Irina; Nagy, Nadine; Negrin, Robert S.; Wight, Thomas N.; Steinman, Lawrence; Bollyky, Paul L.

    2016-01-01

    The extracellular matrix polysaccharide hyaluronan (HA) accumulates at sites of autoimmune inflammation, including white matter lesions in multiple sclerosis (MS), but its functional importance in pathogenesis is unclear. We have evaluated the impact of 4-methylumbelliferone (4-MU), an oral inhibitor of HA synthesis, on disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Treatment with 4-MU decreases the incidence of EAE, delays its onset, and reduces the severity of established disease. 4-MU inhibits the activation of autoreactive T cells and prevents their polarization toward a Th1 phenotype. Instead, 4-MU promotes polarization toward a Th2 phenotpye and induction of Foxp3+ regulatory T cells. Further, 4-MU hastens trafficking of T cells through secondary lymphoid organs, impairs the infiltration of T cells into the CNS parenchyma, and limits astrogliosis. Together, these data suggest that HA synthesis is necessary for disease progression in EAE and that treatment with 4-MU may be a potential therapeutic strategy in CNS autoimmunity. Considering that 4-MU is already a therapeutic, called hymecromone, that is approved to treat biliary spasm in humans, we propose that it could be repurposed to treat MS. PMID:26787861

  12. Phosphatidylinositol 4-Phosphate 5-Kinases in the Regulation of T Cell Activation

    PubMed Central

    Porciello, Nicla; Kunkl, Martina; Viola, Antonella; Tuosto, Loretta

    2016-01-01

    Phosphatidylinositol 4,5-biphosphate kinases (PIP5Ks) are critical regulators of T cell activation being the main enzymes involved in the synthesis of phosphatidylinositol 4,5-biphosphate (PIP2). PIP2 is indeed a pivotal regulator of the actin cytoskeleton, thus controlling T cell polarization and migration, stable adhesion to antigen-presenting cells, spatial organization of the immunological synapse, and co-stimulation. Moreover, PIP2 also serves as a precursor for the second messengers inositol triphosphate, diacylglycerol, and phosphatidylinositol 3,4,5-triphosphate, which are essential for the activation of signaling pathways regulating cytokine production, cell cycle progression, survival, metabolism, and differentiation. Here, we discuss the impact of PIP5Ks on several T lymphocyte functions with a specific focus on the role of CD28 co-stimulation in PIP5K compartimentalization and activation. PMID:27242793

  13. Phosphatidylinositol 4-Phosphate 5-Kinases in the Regulation of T Cell Activation.

    PubMed

    Porciello, Nicla; Kunkl, Martina; Viola, Antonella; Tuosto, Loretta

    2016-01-01

    Phosphatidylinositol 4,5-biphosphate kinases (PIP5Ks) are critical regulators of T cell activation being the main enzymes involved in the synthesis of phosphatidylinositol 4,5-biphosphate (PIP2). PIP2 is indeed a pivotal regulator of the actin cytoskeleton, thus controlling T cell polarization and migration, stable adhesion to antigen-presenting cells, spatial organization of the immunological synapse, and co-stimulation. Moreover, PIP2 also serves as a precursor for the second messengers inositol triphosphate, diacylglycerol, and phosphatidylinositol 3,4,5-triphosphate, which are essential for the activation of signaling pathways regulating cytokine production, cell cycle progression, survival, metabolism, and differentiation. Here, we discuss the impact of PIP5Ks on several T lymphocyte functions with a specific focus on the role of CD28 co-stimulation in PIP5K compartimentalization and activation.

  14. Phosphatidylinositol 4-Phosphate 5-Kinases in the Regulation of T Cell Activation.

    PubMed

    Porciello, Nicla; Kunkl, Martina; Viola, Antonella; Tuosto, Loretta

    2016-01-01

    Phosphatidylinositol 4,5-biphosphate kinases (PIP5Ks) are critical regulators of T cell activation being the main enzymes involved in the synthesis of phosphatidylinositol 4,5-biphosphate (PIP2). PIP2 is indeed a pivotal regulator of the actin cytoskeleton, thus controlling T cell polarization and migration, stable adhesion to antigen-presenting cells, spatial organization of the immunological synapse, and co-stimulation. Moreover, PIP2 also serves as a precursor for the second messengers inositol triphosphate, diacylglycerol, and phosphatidylinositol 3,4,5-triphosphate, which are essential for the activation of signaling pathways regulating cytokine production, cell cycle progression, survival, metabolism, and differentiation. Here, we discuss the impact of PIP5Ks on several T lymphocyte functions with a specific focus on the role of CD28 co-stimulation in PIP5K compartimentalization and activation. PMID:27242793

  15. Visualizing TCR-induced POLKADOTS formation and NF-κB activation in the D10 T-cell clone and mouse primary effector T cells.

    PubMed

    Paul, Suman; Schaefer, Brian C

    2015-01-01

    T cells are an immune cell lineage that play a central role in protection against pathogen infection. Antigen, in the form of pathogen-derived peptides, stimulates the T-cell receptor (TCR), leading to activation of the transcription factor, nuclear factor kappa B (NF-κB). The subsequent NF-κB-dependent gene expression program drives expansion and effector differentiation of antigen-specific T cells, leading to the adaptive anti-pathogen immune response. The cell surface TCR transmits activating signals to cytosolic NF-κB by a complex signaling cascade, in which the adapter protein Bcl10 plays a key role. We have previously demonstrated that TCR engagement leads to the formation of cytosolic Bcl10 clusters, called POLKADOTS, that provide a platform for the assembly of the terminal signaling complex that ultimately mediates NF-κB activation. In this chapter, we describe the methods utilized to visualize the formation of TCR-induced POLKADOTS and to study the temporal association between POLKADOTS formation and nuclear translocation of the NF-κB subunit, RelA/p65.

  16. In vitro exposure to the herbicide atrazine inhibits T cell activation, proliferation, and cytokine production and significantly increases the frequency of Foxp3+ regulatory T cells.

    PubMed

    Thueson, Lindsay E; Emmons, Tiffany R; Browning, Dianna L; Kreitinger, Joanna M; Shepherd, David M; Wetzel, Scott A

    2015-02-01

    The herbicide atrazine (2-chloro-4-[ethylamino]-6-[isopropylamino]-s-triazine) is the most common water contaminant in the United States. Atrazine is a phosphodiesterase inhibitor and is classified as an estrogen disrupting compound because it elevates estrogen levels via induction of the enzyme aromatase. Previous studies have shown that atrazine exposure alters the function of innate immune cells such as NK cells, DC, mast cells, and macrophages. In this study we have examined the impact of in vitro atrazine exposure on the activation, proliferation, and effector cytokine production by primary murine CD4(+) T lymphocytes. We found that atrazine exposure significantly inhibited CD4(+) T cell proliferation and accumulation as well as the expression of the activation markers CD25 and CD69 in a dose-dependent manner. Interestingly, the effects were more pronounced in cells from male animals. These effects were partially mimicked by pharmacological reagents that elevate intracellular cAMP levels and addition of exogenous rmIL-2 further inhibited proliferation and CD25 expression. Consistent with these findings, atrazine exposure during T cell activation resulted in a 2- to 5-fold increase in the frequency of Foxp3(+) CD4(+) T cells.

  17. Streptococcus induces circulating CLA(+) memory T-cell-dependent epidermal cell activation in psoriasis.

    PubMed

    Ferran, Marta; Galván, Ana B; Rincón, Catalina; Romeu, Ester R; Sacrista, Marc; Barboza, Erika; Giménez-Arnau, Ana; Celada, Antonio; Pujol, Ramon M; Santamaria-Babí, Luis F

    2013-04-01

    Streptococcal throat infection is associated with a specific variant of psoriasis and with HLA-Cw6 expression. In this study, activation of circulating psoriatic cutaneous lymphocyte-associated antigen (CLA)(+) memory T cells cultured together with epidermal cells occurred only when streptococcal throat extracts were added. This triggered the production of Th1, Th17, and Th22 cytokines, as well as epidermal cell mediators (CXCL8, CXCL9, CXCL10, and CXCL11). Streptococcal extracts (SEs) did not induce any activation with either CLA(-) cells or memory T cells cultured together with epidermal cells from healthy subjects. Intradermal injection of activated culture supernatants into mouse skin induced epidermal hyperplasia. SEs also induced activation when we used epidermal cells from nonlesional skin of psoriatic patients with CLA(+) memory T cells. Significant correlations were found between SE induced upregulation of mRNA expression for ifn-γ, il-17, il-22, ip-10, and serum level of antistreptolysin O in psoriatic patients. This study demonstrates the direct involvement of streptococcal infection in pathological mechanisms of psoriasis, such as IL-17 production and epidermal cell activation.

  18. Constitutive Lck Activity Drives Sensitivity Differences between CD8+ Memory T Cell Subsets.

    PubMed

    Moogk, Duane; Zhong, Shi; Yu, Zhiya; Liadi, Ivan; Rittase, William; Fang, Victoria; Dougherty, Janna; Perez-Garcia, Arianne; Osman, Iman; Zhu, Cheng; Varadarajan, Navin; Restifo, Nicholas P; Frey, Alan B; Krogsgaard, Michelle

    2016-07-15

    CD8(+) T cells develop increased sensitivity following Ag experience, and differences in sensitivity exist between T cell memory subsets. How differential TCR signaling between memory subsets contributes to sensitivity differences is unclear. We show in mouse effector memory T cells (TEM) that >50% of lymphocyte-specific protein tyrosine kinase (Lck) exists in a constitutively active conformation, compared with <20% in central memory T cells (TCM). Immediately proximal to Lck signaling, we observed enhanced Zap-70 phosphorylation in TEM following TCR ligation compared with TCM Furthermore, we observed superior cytotoxic effector function in TEM compared with TCM, and we provide evidence that this results from a lower probability of TCM reaching threshold signaling owing to the decreased magnitude of TCR-proximal signaling. We provide evidence that the differences in Lck constitutive activity between CD8(+) TCM and TEM are due to differential regulation by SH2 domain-containing phosphatase-1 (Shp-1) and C-terminal Src kinase, and we use modeling of early TCR signaling to reveal the significance of these differences. We show that inhibition of Shp-1 results in increased constitutive Lck activity in TCM to levels similar to TEM, as well as increased cytotoxic effector function in TCM Collectively, this work demonstrates a role for constitutive Lck activity in controlling Ag sensitivity, and it suggests that differential activities of TCR-proximal signaling components may contribute to establishing the divergent effector properties of TCM and TEM. This work also identifies Shp-1 as a potential target to improve the cytotoxic effector functions of TCM for adoptive cell therapy applications. PMID:27271569

  19. TAP, a novel T cell-activating protein involved in the stimulation of MHC-restricted T lymphocytes

    PubMed Central

    1986-01-01

    Five mAbs have been generated and used to characterize TAP (T cell activating protein) a novel, functional murine T cell membrane antigen. The TAP molecule is a 12-kD protein that is synthesized by T cells. By antibody crossblocking, it appears to be closely associated with a 16- kD protein on the T cell membrane also identified with a novel mAb. These molecules are clearly distinct from the major well-characterized murine T cell antigens previously described. Antibody binding to TAP can result in the activation of MHC-restricted, antigen-specific inducer T cell hybridomas that is equivalent in magnitude to maximal antigen or lectin stimulation. This is a direct effect of soluble antibody and does not require accessory cells or other factors. The activating anti-TAP mAbs are also mitogenic for normal heterogeneous T lymphocytes in the presence of accessory cells or IL-1. In addition, these antibodies are observed to modulate specific immune stimulation. Thus, the activating anti-TAP mAbs synergise with antigen-specific stimulation of T cells, while a nonactivating anti-TAP mAb inhibits antigen driven activation. These observations suggest that the TAP molecule may participate in physiologic T cell activation. The possible relationship of TAP to known physiologic triggering structures, the T3- T cell receptor complex, is considered. TAP is expressed on 70% of peripheral T cells and therefore defines a major T cell subset, making it perhaps the first example of a murine subset-specific activating protein. PMID:2418146

  20. Increased Caspase Activity Primes Human Lyme Arthritis Synovial γδ T cells for Proliferation and Death

    PubMed Central

    Thai, Phan T.; Collins, Cheryl C.; Fortner, Karen A.; Koenig, Andreas; Hayes, Sandra M.; Budd, Ralph C.

    2011-01-01

    γδ T cells function between the innate and adaptive immune responses, promoting antigen-presenting cell function, and manifesting cytolytic activity. Their numbers often increase during infections, such as HIV, and at sites of chronic inflammation. However, the turnover dynamics of human γδ T cells are poorly understood. Here we find that despite more rapid proliferation in vitro by human Lyme arthritis synovial γδ T cells of the Vδ1 subset, they have reduced surviving cell numbers compared to αβ T cells due to increased cell death by the γδ T cells. Because caspases are involved in cell proliferation and death, and signaling is more efficient through TCR-γδ than TCR-αβ, we examined the levels of active caspases during cell cycling and following TCR restimulation. We observed higher overall caspase activity in Borrelia-reactive γδ T cells than comparable αβ T cells. This was paralleled by greater spontaneous cell death and TCR restimulation-induced cell death of the γδ T cells, which was caspase dependent. Our current findings thus are consistent with a model where human γδ T cells evolved to function quickly and transiently, in an innate fashion. PMID:21983117

  1. Bystander stimulation of activated CD4+ T cells of unrelated specificity following a booster vaccination with tetanus toxoid.

    PubMed

    Di Genova, Gianfranco; Savelyeva, Natalia; Suchacki, Amy; Thirdborough, Stephen M; Stevenson, Freda K

    2010-04-01

    Antigen-specific CD4(+) T cells are central to natural and vaccine-induced immunity. An ongoing antigen-specific T-cell response can, however, influence surrounding T cells with unrelated antigen specificities. We previously observed this bystander effect in healthy human subjects following recall vaccination with tetanus toxoid (TT). Since this interplay could be important for maintenance of memory, we have moved to a mouse model for further analysis. We investigated whether boosting memory CD4(+) T cells against TT in vivo would influence injected CD4(+) TCR transgenic T cells (OT-II) specific for an unrelated OVA peptide. If OT-II cells were pre-activated with OVA peptide in vitro, these cells showed a bystander proliferative response during the ongoing parallel TT-specific response. Bystander proliferation was dependent on boosting of the TT-specific memory response in the recipients, with no effect in naive mice. Bystander stimulation was also proportional to the strength of the TT-specific memory T-cell response. T cells activated in vitro displayed functional receptors for IL-2 and IL-7, suggesting these as potential mediators. This crosstalk between a stimulated CD4(+) memory T-cell response and CD4(+) T cells activated by an unrelated antigen could be important in human subjects continually buffeted by environmental antigens.

  2. An inducible transcription factor activates expression of human immunodeficiency virus in T cells

    NASA Astrophysics Data System (ADS)

    Nabel, Gary; Baltimore, David

    1987-04-01

    Human immunodeficiency virus (HIV) production from latently infected T lymphocytes can be induced with compounds that activate the cells to secrete lymphokines1,2. The elements in the HIV genome which control activation are not known but expression might be regulated through a variety of DNA elements. The cis-acting control elements of the viral genome are enhancer and promoter regions. The virus also encodes trans-acting factors specified by the tat-III (refs 3-6) and art genes7. We have examined whether products specific to activated T cells might stimulate viral transcription by binding to regions on viral DNA. Activation of T cells, which increases HIV expression up to 50-fold, correlated with induction of a DNA binding protein indistinguishable from a recognized transcription factor, called NF-κB (ref. 8), with binding sites in the viral enhancer. Mutation of these binding sites abolished inducibility. That NF-κB acts in synergy with the viral tat-III gene product to enhance HIV expression in T cells may have implications for the pathogenesis of AIDS (acquired immune deficiency syndrome).

  3. Microglia Induce Neurotoxic IL-17+ γδ T Cells Dependent on TLR2, TLR4, and TLR9 Activation

    PubMed Central

    Derkow, Katja; Krüger, Christina; Dembny, Paul; Lehnardt, Seija

    2015-01-01

    Background Interleukin-17 (IL-17) acts as a key regulator in central nervous system (CNS) inflammation. γδ T cells are an important innate source of IL-17. Both IL-17+ γδ T cells and microglia, the major resident immune cells of the brain, are involved in various CNS disorders such as multiple sclerosis and stroke. Also, activation of Toll-like receptor (TLR) signaling pathways contributes to CNS damage. However, the mechanisms underlying the regulation and interaction of these cellular and molecular components remain unclear. Objective In this study, we investigated the crosstalk between γδ T cells and microglia activated by TLRs in the context of neuronal damage. To this end, co-cultures of IL-17+ γδ T cells, neurons, and microglia were analyzed by immunocytochemistry, flow cytometry, ELISA and multiplex immunoassays. Results We report here that IL-17+ γδ T cells but not naïve γδ T cells induce a dose- and time-dependent decrease of neuronal viability in vitro. While direct stimulation of γδ T cells with various TLR ligands did not result in up-regulation of CD69, CD25, or in IL-17 secretion, supernatants of microglia stimulated by ligands specific for TLR2, TLR4, TLR7, or TLR9 induced activation of γδ T cells through IL-1β and IL-23, as indicated by up-regulation of CD69 and CD25 and by secretion of vast amounts of IL-17. This effect was dependent on the TLR adaptor myeloid differentiation primary response gene 88 (MyD88) expressed by both γδ T cells and microglia, but did not require the expression of TLRs by γδ T cells. Similarly to cytokine-primed IL-17+ γδ T cells, IL-17+ γδ T cells induced by supernatants derived from TLR-activated microglia also caused neurotoxicity in vitro. While these neurotoxic effects required stimulation of TLR2, TLR4, or TLR9 in microglia, neuronal injury mediated by bone marrow-derived macrophages did not require TLR signaling. Neurotoxicity mediated by IL-17+ γδ T cells required a direct cell

  4. Asymmetric inheritance of mTORC1 kinase activity during division dictates CD8 T cell differentiation

    PubMed Central

    Pollizzi, Kristen N.; Sun, Im-Hong; Patel, Chirag H.; Lo, Ying-Chun; Oh, Min-Hee; Waickman, Adam T.; Tam, Ada J.; Blosser, Richard L.; Wen, Jiayu; Delgoffe, Greg M.; Powell, Jonathan D.

    2016-01-01

    The asymmetric partitioning of fate determining proteins has been shown to contribute to the generation of effector and memory CD8+ T cell precursors. Here, we demonstrate the asymmetric partitioning of mTORC1 activity upon activation of naïve CD8+ T cells. This results in the generation of one daughter T cell with increased mTORC1 activity, increased glycolytic activity and increased expression of effector molecules. The other daughter T cell inherits relatively low levels of mTORC1 activity, possesses increased lipid metabolism, expresses increased anti-apoptotic molecules and subsequently displays enhanced long-term survival. Mechanistically, we demonstrate a link between TCR-induced asymmetric expression of amino acid transporters and RagC-mediated translocation of mTOR to the lysosomes. Overall, our data provide important insight into how mTORC1-mediated metabolic reprogramming affects the fate decisions of T cells. PMID:27064374

  5. Affinity and dose of TCR engagement yield proportional enhancer and gene activity in CD4+ T cells

    PubMed Central

    Allison, Karmel A; Sajti, Eniko; Collier, Jana G; Gosselin, David; Troutman, Ty Dale; Stone, Erica L; Hedrick, Stephen M; Glass, Christopher K

    2016-01-01

    Affinity and dose of T cell receptor (TCR) interaction with antigens govern the magnitude of CD4+ T cell responses, but questions remain regarding the quantitative translation of TCR engagement into downstream signals. We find that while the response of mouse CD4+ T cells to antigenic stimulation is bimodal, activated cells exhibit analog responses proportional to signal strength. Gene expression output reflects TCR signal strength, providing a signature of T cell activation. Expression changes rely on a pre-established enhancer landscape and quantitative acetylation at AP-1 binding sites. Finally, we show that graded expression of activation genes depends on ERK pathway activation, suggesting that an ERK-AP-1 axis plays an important role in translating TCR signal strength into proportional activation of enhancers and genes essential for T cell function. DOI: http://dx.doi.org/10.7554/eLife.10134.001 PMID:27376549

  6. T cells respond to heat shock protein 60 via TLR2: activation of adhesion and inhibition of chemokine receptors.

    PubMed

    Zanin-Zhorov, Alexandra; Nussbaum, Gabriel; Franitza, Susanne; Cohen, Irun R; Lider, Ofer

    2003-08-01

    Soluble 60 kDa heat shock protein (HSP60) activates macrophages via TLR4. We now report that soluble HSP60 activates T cells via the innate receptor TLR2. HSP60 activated T cell adhesion to fibronectin to a degree similar to other activators: IL-2, SDF-1alpha, and RANTES. T cell type and state of activation was important; nonactivated CD45RA+ and IL-2-activated CD45RO+ T cells responded optimally (1 h) at low concentrations (0.1-1 ng/ml), but nonactivated CD45RO+ T cells required higher concentrations (approximately 1 microg/ml) of HSP60. T cell HSP60 signaling was inhibited specifically by monoclonal antibodies (mAb) to TLR2 but not by a mAb to TLR4. Indeed, T cells from mice with mutated TLR4 could still respond to HSP60, whereas Chinese hamster T cells with mutated TLR2 did not respond. The human T cell response to soluble HSP60 depended on phosphatidylinositol 3-kinase and protein kinase C signaling and involved the phosphorylation of Pyk-2. Soluble HSP60 also inhibited actin polymerization and T cell chemotaxis through extracellular matrix-like gels toward the chemokines SDF-1alpha (CXCL12) or ELC (CCL19). Exposure to HSP60 for longer times (18 h) down-regulated chemokine receptor expression: CXCR4 and CCR7. These results suggest that soluble HSP60, through TLR2-dependent interactions, can regulate T cell behavior in inflammation. PMID:12824285

  7. CD27-CD70 interactions regulate B-cell activation by T cells.

    PubMed Central

    Kobata, T; Jacquot, S; Kozlowski, S; Agematsu, K; Schlossman, S F; Morimoto, C

    1995-01-01

    CD27, a member of the tumor necrosis factor (TNF) receptor family, binds to its ligand CD70, a member of the TNF family, and subsequently induces T-cell costimulation and B-cell activation. CD27 is expressed on resting T and B cells, whereas CD70 is expressed on activated T and B cells. Utilizing transfected murine pre-B-cell lines expressing human CD27 or CD70, we have examined the effect of such transfectant cells on human B-cell IgG production and B-cell proliferation. We show that the addition of CD27-transfected cells to a T-cell-dependent, pokeweed mitogen-driven B-cell IgG synthesis system resulted in marked inhibition of IgG production, whereas the addition of CD70-transfected cells enhanced IgG production. The inhibition and enhancement of pokeweed mitogen-driven IgG production by CD27 and CD70 transfectants were abrogated by pretreatment with anti-CD27 and anti-CD70 monoclonal antibodies, respectively. In contrast, little or no inhibition of IgG production and B-cell proliferation was noted with CD27-transfected cells or either anti-CD27 or CD70 monoclonal antibody in a T-cell-independent Staphylococcus aureus/interleukin 2-driven B-cell activation system. In this same system CD70-transfected cells enhanced B-cell IgG production and B-cell proliferation, and this enhancement could be gradually abrogated by addition of increasing numbers of CD27-transfected cells. These results clearly demonstrate that interactions among subsets of T cells expressing CD27 and CD70 play a key role in regulating B-cell activation and immunoglobulin synthesis. PMID:7479974

  8. Endogenous n-3 Polyunsaturated Fatty Acids Attenuate T Cell-Mediated Hepatitis via Autophagy Activation.

    PubMed

    Li, Yanli; Tang, Yuan; Wang, Shoujie; Zhou, Jing; Zhou, Jia; Lu, Xiao; Bai, Xiaochun; Wang, Xiang-Yang; Chen, Zhengliang; Zuo, Daming

    2016-01-01

    Omega-3 polyunsaturated fatty acids (n-3 PUFAs) exert anti-inflammatory effects in several liver disorders, including cirrhosis, acute liver failure, and fatty liver disease. To date, little is known about their role in immune-mediated liver diseases. In this study, we used fat-1 transgenic mice rich in endogenous n-3 PUFAs to examine the role of n-3 PUFAs in immune-mediated liver injury. Concanavalin A (Con A) was administered intravenously to wild-type (WT) and fat-1 transgenic mice to induce T cell-mediated hepatitis. Reduced liver damage was shown in Con A-administrated fat-1 transgenic mice, as evidenced by decreased mortality, attenuated hepatic necrosis, lessened serum alanine aminotransferase activity, and inhibited production of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-17A, and IFN-γ). In vivo and in vitro studies demonstrated that n-3 PUFAs significantly inhibited the activation of hepatic T cells and the differentiation of Th1 cells after Con A challenge. Further studies showed that n-3 PUFAs markedly increased autophagy level in Con A-treated fat-1 T cells compared with the WT counterparts. Blocking hepatic autophagy activity with chloroquine diminished the differences in T cell activation and liver injury between Con A-injected WT and fat-1 transgenic mice. We conclude that n-3 PUFAs limit Con A-induced hepatitis via an autophagy-dependent mechanism and could be exploited as a new therapeutic approach for autoimmune hepatitis. PMID:27679638

  9. Endogenous n-3 Polyunsaturated Fatty Acids Attenuate T Cell-Mediated Hepatitis via Autophagy Activation

    PubMed Central

    Li, Yanli; Tang, Yuan; Wang, Shoujie; Zhou, Jing; Zhou, Jia; Lu, Xiao; Bai, Xiaochun; Wang, Xiang-Yang; Chen, Zhengliang; Zuo, Daming

    2016-01-01

    Omega-3 polyunsaturated fatty acids (n-3 PUFAs) exert anti-inflammatory effects in several liver disorders, including cirrhosis, acute liver failure, and fatty liver disease. To date, little is known about their role in immune-mediated liver diseases. In this study, we used fat-1 transgenic mice rich in endogenous n-3 PUFAs to examine the role of n-3 PUFAs in immune-mediated liver injury. Concanavalin A (Con A) was administered intravenously to wild-type (WT) and fat-1 transgenic mice to induce T cell-mediated hepatitis. Reduced liver damage was shown in Con A-administrated fat-1 transgenic mice, as evidenced by decreased mortality, attenuated hepatic necrosis, lessened serum alanine aminotransferase activity, and inhibited production of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-17A, and IFN-γ). In vivo and in vitro studies demonstrated that n-3 PUFAs significantly inhibited the activation of hepatic T cells and the differentiation of Th1 cells after Con A challenge. Further studies showed that n-3 PUFAs markedly increased autophagy level in Con A-treated fat-1 T cells compared with the WT counterparts. Blocking hepatic autophagy activity with chloroquine diminished the differences in T cell activation and liver injury between Con A-injected WT and fat-1 transgenic mice. We conclude that n-3 PUFAs limit Con A-induced hepatitis via an autophagy-dependent mechanism and could be exploited as a new therapeutic approach for autoimmune hepatitis. PMID:27679638

  10. Modulation of both activator protein-1 and nuclear factor-kappa B signal transduction of human T cells by amiodarone

    PubMed Central

    Cheng, Shu-Meng; Lin, Wei-Hsiang; Lin, Chin-Sheng; Ho, Ling-Jun; Tsai, Tsung-Neng; Wu, Chun-Hsien; Lai, Jenn-Haung

    2015-01-01

    Amiodarone, a common and effective antiarrhythmic drug, has been reported to have anti-inflammatory effects such as reducing the activation and movement of neutrophils. However, its effects on human T cells remain unclear. The aim of this study was to elucidate the effects and possible underlying mechanisms of amiodarone on human T cells. We isolated human primary T cells from the peripheral blood of healthy volunteers and performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, electrophoretic mobility shift assay, luciferase assay, and Western blotting to evaluate the modulatory effects of amiodarone on human T cells. We found that amiodarone dose dependently inhibited the production of cytokines, including interleukin-2 (IL-2), IL-4, tumor necrosis factor-alpha, and interferon-gamma in activated human T cells. By flow cytometry, we demonstrated that amiodarone suppressed the expression of IL-2 receptor-alpha (CD25) and CD69, the cell surface markers of activated T cells. Moreover, molecular investigations revealed that amiodarone down-regulated activator protein-1 (AP-1) and nuclear factor kappa-B (NF-κB) DNA-binding activities in activated human T cells and also inhibited DNA binding and transcriptional activities of both AP-1 and NF-κB in Jurkat cells. Finally, by Western blotting, we showed that amiodarone reduced the activation of c-Jun NH2-terminal protein kinase and P38 mitogen-activated protein kinase, and suppressed stimuli-induced I-kappa B-alpha degradation in activated human T cells. Through regulation of AP-1 and NF-κB signaling, amiodarone inhibits cytokine production and T cell activation. These results show the pleiotropic effects of amiodarone on human T cells and suggest its therapeutic potential in inflammation-related cardiovascular disorders. PMID:25073960

  11. Bone Morphogenetic Protein Signaling Regulates Development and Activation of CD4(+) T Cells.

    PubMed

    Kuczma, Michal; Kraj, Piotr

    2015-01-01

    Bone morphogenetic proteins (BMPs) are growth factors belonging to the TGF-β (transforming growth factor β) superfamily. BMPs were found to regulate multiple cell processes such as proliferation, survival, differentiation, and apoptosis. They were originally described to play a pivotal role in inducing bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites but were found to play a significant role in embryogenesis and development of multiple tissues and organs. Activities of BMPs are regulated by a number of secreted proteins, which modulate their availability to bind cellular receptors. The functions of individual BMPs are highly redundant due to binding the same receptors and inducing overlapping signal transduction pathways. Recently, BMPs were found to regulate cells of the innate and adaptive immune system. BMPs are involved in thymic development of T cells at the early, double negative, as well as later, double positive, stages of thymopoesis. They specifically modulate thymic development of regulatory T cells (T(reg)). In the periphery, BMPs affect T cell activation, promoting generation of T(reg) cells. We found that mice deficient for one of the receptors activated by BMPs demonstrated slower growth of transplantable melanoma tumors.

  12. The dendritic cell cytoskeleton promotes T cell adhesion and activation by constraining ICAM-1 mobility

    PubMed Central

    Comrie, William A.; Li, Shuixing; Boyle, Sarah

    2015-01-01

    Integrity of the dendritic cell (DC) actin cytoskeleton is essential for T cell priming, but the underlying mechanisms are poorly understood. We show that the DC F-actin network regulates the lateral mobility of intracellular cell adhesion molecule 1 (ICAM-1), but not MHCII. ICAM-1 mobility and clustering are regulated by maturation-induced changes in the expression and activation of moesin and α-actinin-1, which associate with actin filaments and the ICAM-1 cytoplasmic domain. Constrained ICAM-1 mobility is important for DC function, as DCs expressing a high-mobility ICAM-1 mutant lacking the cytoplasmic domain exhibit diminished antigen-dependent conjugate formation and T cell priming. These defects are associated with inefficient induction of leukocyte functional antigen 1 (LFA-1) affinity maturation, which is consistent with a model in which constrained ICAM-1 mobility opposes forces on LFA-1 exerted by the T cell cytoskeleton, whereas ICAM-1 clustering enhances valency and further promotes ligand-dependent LFA-1 activation. Our results reveal an important new mechanism through which the DC cytoskeleton regulates receptor activation at the immunological synapse. PMID:25666808

  13. Symptom Severity Predicts Degree of T Cell Activation In Adult Women Following Childhood Maltreatment

    PubMed Central

    Lemieux, Andrine; Coe, Christopher L.; Carnes, Molly

    2008-01-01

    Although depression is often associated with a reduction in cellular immune responses, other types of emotional disturbance and psychopathology can activate certain aspects of immunity. Activation markers on T cells, in particular, have been found to be elevated in post-traumatic stress states. However, little is known about the relationship between the severity of PTSD symptoms and the degree of change in T cell phenotypes, or about the potential role of neuroendocrine factors in mediating the association. Twenty-four women with a history of sexual trauma during childhood, including 11 who met diagnostic criteria for PTSD, were compared to 12 age-matched, healthy women without a history of maltreatment. The women provided fasted blood samples for enumeration of cell subsets by immunofluorescence and 24-hour urine samples for analysis of catecholamine and cortisol levels. The percent of T cells expressing CD45RA, an early activation marker, was higher in the PTSD diagnosed women, and the levels correlated positively with intrusive symptoms and negatively with avoidant symptoms. These alterations in cell surface markers did not appear to be mediated by norepinephrine (NE) or cortisol, making them a distinctive and independent biomarker of arousal and disturbance in PTSD. PMID:18396007

  14. Spaceflight alters expression of microRNA during T-cell activation.

    PubMed

    Hughes-Fulford, Millie; Chang, Tammy T; Martinez, Emily M; Li, Chai-Fei

    2015-12-01

    Altered immune function has been demonstrated in astronauts during spaceflights dating back to Apollo and Skylab; this could be a major barrier to long-term space exploration. We tested the hypothesis that spaceflight causes changes in microRNA (miRNA) expression. Human leukocytes were stimulated with mitogens on board the International Space Station using an onboard normal gravity control. Bioinformatics showed that miR-21 was significantly up-regulated 2-fold during early T-cell activation in normal gravity, and gene expression was suppressed under microgravity. This was confirmed using quantitative real-time PCR (n = 4). This is the first report that spaceflight regulates miRNA expression. Global microarray analysis showed significant (P < 0.05) suppression of 85 genes under microgravity conditions compared to normal gravity samples. EGR3, FASLG, BTG2, SPRY2, and TAGAP are biologically confirmed targets and are co-up-regulated with miR-21. These genes share common promoter regions with pre-mir-21; as the miR-21 matures and accumulates, it most likely will inhibit translation of its target genes and limit the immune response. These data suggest that gravity regulates T-cell activation not only by transcription promotion but also by blocking translation via noncoding RNA mechanisms. Moreover, this study suggests that T-cell activation itself may induce a sequence of gene expressions that is self-limited by miR-21. PMID:26276131

  15. Spaceflight alters expression of microRNA during T-cell activation.

    PubMed

    Hughes-Fulford, Millie; Chang, Tammy T; Martinez, Emily M; Li, Chai-Fei

    2015-12-01

    Altered immune function has been demonstrated in astronauts during spaceflights dating back to Apollo and Skylab; this could be a major barrier to long-term space exploration. We tested the hypothesis that spaceflight causes changes in microRNA (miRNA) expression. Human leukocytes were stimulated with mitogens on board the International Space Station using an onboard normal gravity control. Bioinformatics showed that miR-21 was significantly up-regulated 2-fold during early T-cell activation in normal gravity, and gene expression was suppressed under microgravity. This was confirmed using quantitative real-time PCR (n = 4). This is the first report that spaceflight regulates miRNA expression. Global microarray analysis showed significant (P < 0.05) suppression of 85 genes under microgravity conditions compared to normal gravity samples. EGR3, FASLG, BTG2, SPRY2, and TAGAP are biologically confirmed targets and are co-up-regulated with miR-21. These genes share common promoter regions with pre-mir-21; as the miR-21 matures and accumulates, it most likely will inhibit translation of its target genes and limit the immune response. These data suggest that gravity regulates T-cell activation not only by transcription promotion but also by blocking translation via noncoding RNA mechanisms. Moreover, this study suggests that T-cell activation itself may induce a sequence of gene expressions that is self-limited by miR-21.

  16. Increased numbers and functional activity of CD56⁺ T cells in healthy cytomegalovirus positive subjects.

    PubMed

    Almehmadi, Mazen; Flanagan, Brian F; Khan, Naeem; Alomar, Suliman; Christmas, Stephen E

    2014-06-01

    Human T cells expressing CD56 are capable of tumour cell lysis following activation with interleukin-2 but their role in viral immunity has been less well studied. Proportions of CD56(+) T cells were found to be highly significantly increased in cytomegalovirus-seropositive (CMV(+) ) compared with seronegative (CMV(-) ) healthy subjects (9.1 ± 1.5% versus 3.7 ± 1.0%; P < 0.0001). Proportions of CD56(+) T cells expressing CD28, CD62L, CD127, CD161 and CCR7 were significantly lower in CMV(+) than CMV(-) subjects but those expressing CD4, CD8, CD45RO, CD57, CD58, CD94 and NKG2C were significantly increased (P < 0.05), some having the phenotype of T effector memory cells. Levels of pro-inflammatory cytokines and CD107a were significantly higher in CD56(+) T cells from CMV(+) than CMV(-) subjects following stimulation with CMV antigens. This also resulted in higher levels of proliferation in CD56(+) T cells from CMV(+) than CMV(-) subjects. Using Class I HLA pentamers, it was found that CD56(+) T cells from CMV(+) subjects contained similar proportions of antigen-specific CD8(+) T cells to CD56(-) T cells in donors of several different HLA types. These differences may reflect the expansion and enhanced functional activity of CMV-specific CD56(+) memory T cells. In view of the link between CD56 expression and T-cell cytotoxic function, this strongly implicates CD56(+) T cells as being an important component of the cytotoxic T-cell response to CMV in healthy carriers.

  17. CD26 surface molecule involvement in T cell activation and lymphokine synthesis in rheumatoid and other inflammatory synovitis.

    PubMed

    Gerli, R; Muscat, C; Bertotto, A; Bistoni, O; Agea, E; Tognellini, R; Fiorucci, G; Cesarotti, M; Bombardieri, S

    1996-07-01

    T cell surface expression and the functional role of CD26 antigen (Ag), a surface ectoenzyme involved in T cell activation and migration across the extracellular matrix, were analyzed in the peripheral blood (PB) and synovial fluid (SF) from patients with inflammatory arthritides. CD26 membrane expression on T cells was detected by cytofluorometry using two different monoclonal antibodies, anti-Ta1 and anti-1F7, while cell proliferation and both IL-2 and IFN-gamma production were evaluated in anti-CD3- or anti-CD2-stimulated cell cultures after Ag surface modulation with anti-1F7. The results showed that Ta1 and 1F7 Ag expression were increased on T cells from PB of patients with active, but not inactive, rheumatoid arthritis (RA). Most SF T cells from RA or other inflammatory arthritides displayed the memory marker CD45R0 and the Ta1 Ag, but lacked the 1F7 molecule. In addition, in vitro 1F7 modulation, which enhanced RA PB T cell proliferation and both IL-2 and IFN-gamma synthesis, did not synergize with anti-CD3 or anti-CD2 in inducing IL-2-dependent activation of SF T cells, but reduced IFN-gamma production. A spontaneous reappearance of 1F7 Ag on the SF T cell surface was seen after 2-5 days in culture. Phorbol myristate acetate, able to accelerate its reexpression, also restored a normal response of SF T cells to anti-1F7 comitogenic effects. These data confirm a role of the CD26 surface molecule in regulating T cell activation and lymphokine synthesis. This observation may have important implications in the regulation of T cell activity at the joint level during chronic inflammatory processes. PMID:8674237

  18. Artificial antigen presenting cell (aAPC) mediated activation and expansion of natural killer T cells.

    PubMed

    East, James E; Sun, Wenji; Webb, Tonya J

    2012-01-01

    Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells(1). Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules(2). NKT cells express an invariant TCRα chain rearrangement: Vα14Jα18 in mice and Vα24Jα18 in humans, which is associated with Vβ chains of limited diversity(3-6), and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d (7). The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with α-Galactosylceramide (α-GalCer) and a variety of cytokines(8). Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines(9,10). In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients(11). However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells(12, 13). Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is

  19. Deletion of mTORC1 Activity in CD4+ T Cells Is Associated with Lung Fibrosis and Increased γδ T Cells

    PubMed Central

    Vigeland, Christine L.; Collins, Samuel L.; Chan-Li, Yee; Hughes, Andrew H.; Oh, Min-Hee; Powell, Jonathan D.; Horton, Maureen R.

    2016-01-01

    Pulmonary fibrosis is a devastating, incurable disease in which chronic inflammation and dysregulated, excessive wound healing lead to progressive fibrosis, lung dysfunction, and ultimately death. Prior studies have implicated the cytokine IL-17A and Th17 cells in promoting the development of fibrosis. We hypothesized that loss of Th17 cells via CD4-specific deletion of mTORC1 activity would abrogate the development of bleomycin-induced pulmonary fibrosis. However, in actuality loss of Th17 cells led to increased mortality and fibrosis in response to bleomycin. We found that in the absence of Th17 cells, there was continued production of IL-17A by γδ T cells. These IL-17A+ γδ T cells were associated with increased lung neutrophils and M2 macrophages, accelerated development of fibrosis, and increased mortality. These data elucidate the critical role of IL-17A+ γδ T cells in promoting chronic inflammation and fibrosis, and reveal a novel therapeutic target for treatment of pulmonary fibrosis. PMID:27649073

  20. Deletion of mTORC1 Activity in CD4+ T Cells Is Associated with Lung Fibrosis and Increased γδ T Cells.

    PubMed

    Vigeland, Christine L; Collins, Samuel L; Chan-Li, Yee; Hughes, Andrew H; Oh, Min-Hee; Powell, Jonathan D; Horton, Maureen R

    2016-01-01

    Pulmonary fibrosis is a devastating, incurable disease in which chronic inflammation and dysregulated, excessive wound healing lead to progressive fibrosis, lung dysfunction, and ultimately death. Prior studies have implicated the cytokine IL-17A and Th17 cells in promoting the development of fibrosis. We hypothesized that loss of Th17 cells via CD4-specific deletion of mTORC1 activity would abrogate the development of bleomycin-induced pulmonary fibrosis. However, in actuality loss of Th17 cells led to increased mortality and fibrosis in response to bleomycin. We found that in the absence of Th17 cells, there was continued production of IL-17A by γδ T cells. These IL-17A+ γδ T cells were associated with increased lung neutrophils and M2 macrophages, accelerated development of fibrosis, and increased mortality. These data elucidate the critical role of IL-17A+ γδ T cells in promoting chronic inflammation and fibrosis, and reveal a novel therapeutic target for treatment of pulmonary fibrosis. PMID:27649073

  1. T cell activation responses are differentially regulated during clinorotation and in spaceflight

    NASA Technical Reports Server (NTRS)

    Hashemi, B. B.; Penkala, J. E.; Vens, C.; Huls, H.; Cubbage, M.; Sams, C. F.

    1999-01-01

    Studies of T lymphocyte activation with mitogenic lectins during spaceflight have shown a dramatic inhibition of activation as measured by DNA synthesis at 72 h, but the mechanism of this inhibition is unknown. We have investigated the progression of cellular events during the first 24 h of activation using both spaceflight microgravity culture and a ground-based model system that relies on the low shear culture environment of a rotating clinostat (clinorotation). Stimulation of human peripheral blood mononuclear cells (PBMCs) with soluble anti-CD3 (Leu4) in clinorotation and in microgravity culture shows a dramatic reduction in surface expression of the receptor for IL-2 (CD25) and CD69. An absence of bulk RNA synthesis in clinorotation indicates that stimulation with soluble Leu4 does not induce transition of T cells from G0 to the G1 stage of the cell cycle. However, internalization of the TCR by T cells and normal levels of IL-1 synthesis by monocytes indicate that intercellular interactions that are required for activation occur during clinorotation. Complementation of TCR-mediated signaling by phorbol ester restores the ability of PBMCs to express CD25 in clinorotation, indicating that a PKC-associated pathway may be compromised under these conditions. Bypassing the TCR by direct activation of intracellular pathways with a combination of phorbol ester and calcium ionophore in clinorotation resulted in full expression of CD25; however, only partial expression of CD25 occurred in microgravity culture. Though stimulation of purified T cells with Bead-Leu4 in microgravity culture resulted in the engagement and internalization of the TCR, the cells still failed to express CD25. When T cells were stimulated with Bead-Leu4 in microgravity culture, they were able to partially express CD69, a receptor that is constitutively stored in intracellular pools and can be expressed in the absence of new gene expression. Our results suggest that the inhibition of T cell

  2. T cell receptor junctional regions of V gamma 9+/V delta 2+ T cell clones in relation to non-MHC restricted cytotoxic activity.

    PubMed

    Flanagan, B F; Wheatcroft, N J; Thornton, S M; Christmas, S E

    1993-05-01

    Human gamma delta T cell clones having V gamma 9JP and V delta 2DJ1 T cell receptor (TCR) gene rearrangements were isolated form an individual donor and tested for non-MHC restricted cytotoxicity against the B lymphoblastoid cell line, BSM. Most clones were highly cytotoxic but 3/9 clones had very low activity, comparable to that of CD4+ alpha beta T cell clones. Although there was a tendency for clones with low cytotoxic function to produce high levels of interferon-gamma and tumor necrosis factor-alpha, this correlation was not complete. TCR gamma and delta junctional sequences were obtained and were found to be different for all clones. There were no consistent structural differences between gamma delta TCRs of cytotoxic and non-cytotoxic clones, but gamma or delta junctional regions of all three non-cytotoxic clones had unusual features. One clone had a particularly short gamma chain junctional sequence, one had a short delta chain junctional sequence and the third clone was the only one of the panel which failed to utilise the D delta 3 segment. If the gamma delta TCR is involved in target cell recognition in this model of non-MHC restricted killing, such variations in receptor structure may be sufficient to inhibit recognition and thereby reduce the cytotoxic capacity of a minority of V gamma 9+/V delta 2+ clones. Also, a panel of gamma delta T cell clones expressing V gamma 8/V delta 3 isolated from a different donor, were all highly cytotoxic against BSM, indicating that these target cells can be recognised by effector cells expressing a TCR other than the V gamma 9/V delta 2 receptor. The possible influence of other cell surface molecules on non-MHC restricted cytotoxic function is discussed.

  3. Dexamethasone promotes hypertrophy of H9C2 cardiomyocytes through calcineurin B pathway, independent of NFAT activation.

    PubMed

    Sangeetha, K N; Lakshmi, B S; Niranjali Devaraj, S

    2016-01-01

    Metabolic syndrome-induced cardiac hypertrophy is a global concern leading to an increase in the morbidity and mortality of patients, with the signalling mechanism associated with them still unclear. The present study attempts to understand the metabolic syndrome-associated cardiac hypertrophy through an in vitro model using external stimuli well known for inducing metabolic disorders, i.e. dexamethasone (DEX), a synthetic glucocorticoid. DEX (0.1 and 1 μM) promoted cardiac hypertrophy in H9C2 cells at 4 days of treatment as evidenced through increased cell size and protein content. A significant induction in foetal gene reprogramming was observed, confirming the establishment of hypertrophy. Moreover, the hypertrophic response at 4 days was perceived to be physiological at 0.1 μM and pathological at 1 μM based on α-MHC and IGF1R expression, but complete inhibition in the PKB/AKT expression confirmed it to be pathological hypertrophy at both the concentrations (0.1 and 1 μM). The present study reports for the first time the mechanistic insights into DEX-mediated hypertrophy. It is hypothesized to be orchestrated through the activation of AT1R that is involved in the alteration of the cardiac isoform of SERCA2 expression perturbing the calcium homeostasis. This leads to the activation of calcineurin B, independent of NFAT involvement, which in coordination with ROS induces the activation of JNK of the MAPK signalling. PMID:26511233

  4. Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse.

    PubMed

    Soares, Helena; Henriques, Ricardo; Sachse, Martin; Ventimiglia, Leandro; Alonso, Miguel A; Zimmer, Christophe; Thoulouze, Maria-Isabel; Alcover, Andrés

    2013-10-21

    How the vesicular traffic of signaling molecules contributes to T cell receptor (TCR) signal transduction at the immunological synapse remains poorly understood. In this study, we show that the protein tyrosine kinase Lck, the TCRζ subunit, and the adapter LAT traffic through distinct exocytic compartments, which are released at the immunological synapse in a differentially regulated manner. Lck vesicular release depends on MAL protein. Synaptic Lck, in turn, conditions the calcium- and synaptotagmin-7-dependent fusion of LAT and TCRζ containing vesicles. Fusion of vesicles containing TCRζ and LAT at the synaptic membrane determines not only the nanoscale organization of phosphorylated TCRζ, ZAP70, LAT, and SLP76 clusters but also the presence of phosphorylated LAT and SLP76 in interacting signaling nanoterritories. This mechanism is required for priming IL-2 and IFN-γ production and may contribute to fine-tuning T cell activation breadth in response to different stimulatory conditions.

  5. Premalignant Oral Lesion Cells Elicit Increased Cytokine Production and Activation of T-cells

    PubMed Central

    JOHNSON, SARA D.; LEVINGSTON, CORINNE; YOUNG, M. RITA I.

    2016-01-01

    Background Head and neck squamous cell carcinomas (HNSCC) are known to evade the host immune response. How premalignant oral lesions modulate the immune response, however, has yet to be elucidated. Materials and Methods A mouse model of oral carcinogenesis was used to determine how mediators from premalignant oral lesion cells vs. HNSCC cells impact on immune cytokine production and activation. Results Media conditioned by premalignant lesion cells elicited an increased production of T cell-associated cytokines and proinflammatory mediators from cervical lymph node cells compared to media conditioned by HNSCC cells or media alone. In the presence of premalignant lesion cell-conditioned media, CD4+ T cell expression of the IL-2 receptor CD25 and CD8+ T cell expression of the activation marker CD69 was greater, compared to what was induced in HNSCC cell-conditioned media or media alone. Conclusion Premalignant lesion cells promote a proinflammatory environment and induce immune changes before HNSCC tumors are established. PMID:27354582

  6. Tcf1 and Lef1 transcription factors establish CD8(+) T cell identity through intrinsic HDAC activity.

    PubMed

    Xing, Shaojun; Li, Fengyin; Zeng, Zhouhao; Zhao, Yunjie; Yu, Shuyang; Shan, Qiang; Li, Yalan; Phillips, Farrah C; Maina, Peterson K; Qi, Hank H; Liu, Chengyu; Zhu, Jun; Pope, R Marshall; Musselman, Catherine A; Zeng, Chen; Peng, Weiqun; Xue, Hai-Hui

    2016-06-01

    The CD4(+) and CD8(+) T cell dichotomy is essential for effective cellular immunity. How individual T cell identity is established remains poorly understood. Here we show that the high-mobility group (HMG) transcription factors Tcf1 and Lef1 are essential for repressing CD4(+) lineage-associated genes including Cd4, Foxp3 and Rorc in CD8(+) T cells. Tcf1- and Lef1-deficient CD8(+) T cells exhibit histone hyperacetylation, which can be ascribed to intrinsic histone deacetylase (HDAC) activity in Tcf1 and Lef1. Mutation of five conserved amino acids in the Tcf1 HDAC domain diminishes HDAC activity and the ability to suppress CD4(+) lineage genes in CD8(+) T cells. These findings reveal that sequence-specific transcription factors can utilize intrinsic HDAC activity to guard cell identity by repressing lineage-inappropriate genes. PMID:27111144

  7. Signal transducer and activator of transcription (STAT) signalling and T-cell lymphomas

    PubMed Central

    Mitchell, Tracey J; John, Susan

    2005-01-01

    Interaction of cytokines with their cognate receptors leads to the activation of latent transcription factors – the signal transducers and activators of transcription (STAT) proteins – whose biological activities ultimately regulate many critical aspects of cell growth, survival and differentiation. Dysregulation of the JAK-STAT pathway is frequently observed in many primary human tumours, reflecting the importance of this pathway in the maintenance of cellular integrity. Here we review the current progress in STAT structure and function, and the contribution of STAT signalling to the pathogenesis of T-cell lymphomas. PMID:15720432

  8. Magnetic-Activated Cell Sorting of TCR-Engineered T Cells, Using tCD34 as a Gene Marker, but Not Peptide–MHC Multimers, Results in Significant Numbers of Functional CD4+ and CD8+ T Cells

    PubMed Central

    Govers, Coen; Berrevoets, Cor; Treffers-Westerlaken, Elike; Broertjes, Marieke

    2012-01-01

    Abstract T cell-sorting technologies with peptide–MHC multimers or antibodies against gene markers enable enrichment of antigen-specific T cells and are expected to enhance the therapeutic efficacy of clinical T cell therapy. However, a direct comparison between sorting reagents for their ability to enrich T cells is lacking. Here, we compared the in vitro properties of primary human T cells gene-engineered with gp100280–288/HLA-A2-specific T cell receptor-αβ (TCRαβ) on magnetic-activated cell sorting (MACS) with various peptide–MHC multimers or an antibody against truncated CD34 (tCD34). With respect to peptide–MHC multimers, we observed that Streptamer®, when compared with pentamers and tetramers, improved T cell yield as well as level and stability of enrichment, of TCR-engineered T cells (>65% of peptide–MHC-binding T cells, stable for at least 6 weeks). In agreement with these findings, Streptamer, the only detachable reagent, revealed significant T cell expansion in the first week after MACS. Sorting TCR and tCD34 gene-engineered T cells with CD34 monoclonal antibody (mAb) resulted in the most significant T cell yield and enrichment of T cells (>95% of tCD34 T cells, stable for at least 6 weeks). Notably, T cells sorted with CD34 mAb, when compared with Streptamer, bound about 2- to 3-fold less peptide–MHC but showed superior antigen-specific upregulated expression of CD107a and production of interferon (IFN)-γ. Multiparametric flow cytometry revealed that CD4+ T cells, uniquely present in CD34 mAb-sorted T cells, contributed to enhanced IFN-γ production. Taken together, we postulate that CD34 mAb-based sorting of gene-marked T cells has benefits toward applications of T cell therapy, especially those that require CD4+ T cells. PMID:22871260

  9. Magnetic-activated cell sorting of TCR-engineered T cells, using tCD34 as a gene marker, but not peptide-MHC multimers, results in significant numbers of functional CD4+ and CD8+ T cells.

    PubMed

    Govers, Coen; Berrevoets, Cor; Treffers-Westerlaken, Elike; Broertjes, Marieke; Debets, Reno

    2012-06-01

    T cell-sorting technologies with peptide-MHC multimers or antibodies against gene markers enable enrichment of antigen-specific T cells and are expected to enhance the therapeutic efficacy of clinical T cell therapy. However, a direct comparison between sorting reagents for their ability to enrich T cells is lacking. Here, we compared the in vitro properties of primary human T cells gene-engineered with gp100(280-288)/HLA-A2-specific T cell receptor-αβ (TCRαβ) on magnetic-activated cell sorting (MACS) with various peptide-MHC multimers or an antibody against truncated CD34 (tCD34). With respect to peptide-MHC multimers, we observed that Streptamer(®), when compared with pentamers and tetramers, improved T cell yield as well as level and stability of enrichment, of TCR-engineered T cells (>65% of peptide-MHC-binding T cells, stable for at least 6 weeks). In agreement with these findings, Streptamer, the only detachable reagent, revealed significant T cell expansion in the first week after MACS. Sorting TCR and tCD34 gene-engineered T cells with CD34 monoclonal antibody (mAb) resulted in the most significant T cell yield and enrichment of T cells (>95% of tCD34 T cells, stable for at least 6 weeks). Notably, T cells sorted with CD34 mAb, when compared with Streptamer, bound about 2- to 3-fold less peptide-MHC but showed superior antigen-specific upregulated expression of CD107a and production of interferon (IFN)-γ. Multiparametric flow cytometry revealed that CD4(+) T cells, uniquely present in CD34 mAb-sorted T cells, contributed to enhanced IFN-γ production. Taken together, we postulate that CD34 mAb-based sorting of gene-marked T cells has benefits toward applications of T cell therapy, especially those that require CD4(+) T cells. PMID:22871260

  10. Staphylococcal enterotoxin A (SEA) stimulates STAT3 activation and IL-17 expression in cutaneous T-cell lymphoma

    PubMed Central

    Willerslev-Olsen, Andreas; Krejsgaard, Thorbjørn; Lindahl, Lise M.; Litvinov, Ivan V.; Fredholm, Simon; Petersen, David L.; Nastasi, Claudia; Gniadecki, Robert; Mongan, Nigel P.; Sasseville, Denis; Wasik, Mariusz A.; Bonefeld, Charlotte M.; Geisler, Carsten; Woetmann, Anders; Iversen, Lars; Kilian, Mogens; Koralov, Sergei B.

    2016-01-01

    Cutaneous T-cell lymphoma (CTCL) is characterized by proliferation of malignant T cells in a chronic inflammatory environment. With disease progression, bacteria colonize the compromised skin barrier and half of CTCL patients die of infection rather than from direct organ involvement by the malignancy. Clinical data indicate that bacteria play a direct role in disease progression, but little is known about the mechanisms involved. Here, we demonstrate that bacterial isolates containing staphylococcal enterotoxin A (SEA) from the affected skin of CTCL patients, as well as recombinant SEA, stimulate activation of signal transducer and activator of transcription 3 (STAT3) and upregulation of interleukin (IL)-17 in immortalized and primary patient–derived malignant and nonmalignant T cells. Importantly, SEA induces STAT3 activation and IL-17 expression in malignant T cells when cocultured with nonmalignant T cells, indicating an indirect mode of action. In accordance, malignant T cells expressing an SEA-nonresponsive T-cell receptor variable region β chain are nonresponsive to SEA in monoculture but display strong STAT3 activation and IL-17 expression in cocultures with SEA-responsive nonmalignant T cells. The response is induced via IL-2 receptor common γ chain cytokines and a Janus kinase 3 (JAK3)-dependent pathway in malignant T cells, and blocked by tofacitinib, a clinical-grade JAK3 inhibitor. In conclusion, we demonstrate that SEA induces cell cross talk–dependent activation of STAT3 and expression of IL-17 in malignant T cells, suggesting a mechanism whereby SEA-producing bacteria promote activation of an established oncogenic pathway previously implicated in carcinogenesis. PMID:26738536

  11. PKC-Theta is a Novel SC35 Splicing Factor Regulator in Response to T Cell Activation

    PubMed Central

    McCuaig, Robert Duncan; Dunn, Jennifer; Li, Jasmine; Masch, Antonia; Knaute, Tobias; Schutkowski, Mike; Zerweck, Johannes; Rao, Sudha

    2015-01-01

    Alternative splicing of nuclear pre-mRNA is essential for generating protein diversity and regulating gene expression. While many immunologically relevant genes undergo alternative splicing, the role of regulated splicing in T cell immune responses is largely unexplored, and the signaling pathways and splicing factors that regulate alternative splicing in T cells are poorly defined. Here, we show using a combination of Jurkat T cells, human primary T cells, and ex vivo naïve and effector virus-specific T cells isolated after influenza A virus infection that SC35 phosphorylation is induced in response to stimulatory signals. We show that SC35 colocalizes with RNA polymerase II in activated T cells and spatially overlaps with H3K27ac and H3K4me3, which mark transcriptionally active genes. Interestingly, SC35 remains coupled to the active histone marks in the absence of continuing stimulatory signals. We show for the first time that nuclear PKC-θ co-exists with SC35 in the context of the chromatin template and is a key regulator of SC35 in T cells, directly phosphorylating SC35 peptide residues at RNA recognition motif and RS domains. Collectively, our findings suggest that nuclear PKC-θ is a novel regulator of the key splicing factor SC35 in T cells. PMID:26594212

  12. Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation.

    PubMed

    Bruno, Stefania; Grange, Cristina; Tapparo, Marta; Pasquino, Chiara; Romagnoli, Renato; Dametto, Ennia; Amoroso, Antonio; Tetta, Ciro; Camussi, Giovanni

    2016-01-01

    Human liver stem cells (HLSCs) are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs), and dendritic cells (DCs) in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2) and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs), HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response. PMID:27127520

  13. DYRK1A Controls HIV-1 Replication at a Transcriptional Level in an NFAT Dependent Manner

    PubMed Central

    Booiman, Thijs; Loukachov, Vladimir V.; van Dort, Karel A.; van ’t Wout, Angélique B.; Kootstra, Neeltje A.

    2015-01-01

    Background Transcription of the HIV-1 provirus is regulated by both viral and host proteins and is very important in the context of viral latency. In latently infected cells, viral gene expression is inhibited as a result of the sequestration of host transcription factors and epigenetic modifications. Results In our present study we analyzed the effect of host factor dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) on HIV-1 replication. We show that DYRK1A controls HIV-1 replication by regulating provirus transcription. Downregulation or inhibition of DYRK1A increased LTR-driven transcription and viral replication in cell lines and primary PBMC. Furthermore, inhibition of DYRK1A resulted in reactivation of latent HIV-1 provirus to a similar extent as two commonly used broad-spectrum HDAC inhibitors. We observed that DYRK1A regulates HIV-1 transcription via the Nuclear Factor of Activated T-cells (NFAT) by promoting its translocation from the nucleus to the cytoplasm. Therefore, inhibition of DYRK1A results in increased nuclear levels of NFAT and increased NFAT binding to the viral LTR and thus increasing viral transcription. Conclusions Our data indicate that host factor DYRK1A plays a role in the regulation of viral transcription and latency. Therefore, DYRK1A might be an attractive candidate for therapeutic strategies targeting the viral reservoir. PMID:26641855

  14. Age-associated Failure to Adjust Type I Interferon Receptor Signaling Thresholds after T-cell Activation1

    PubMed Central

    Li, Guangjin; Ju, Jihang; Weyand, Cornelia M.; Goronzy, Jörg J.

    2015-01-01

    With increasing age, naïve CD4 T cells acquire intrinsic defects that compromise their ability to respond and differentiate. Type I IFNs, pervasive constituents of the environment in which adaptive immune responses occur, are known to regulate T cell differentiation and survival. Activated naïve CD4 T cells from older individuals have reduced responses to type I IFN, a defect that develops during activation and is not observed in quiescent naïve CD4 T cells. Naïve CD4 T cells from young adults upregulate the expression of STAT1 and STAT5 after activation, lowering their threshold to respond to type I IFN stimulation. The heightened STAT signaling is critical to maintain the expression of CD69 that regulates lymphocyte egress and the ability to produce IL-2 and to survive. Although activation of T cells from older adults also induces transcription of STAT1 and STAT5, failure to exclude SHP1 to the signaling complex blunts their type I IFN response. In summary, our data show that type I IFN signaling thresholds in naïve CD4 T cells after activation are dynamically regulated to respond environmental cues for clonal expansion and memory cell differentiation. Naïve CD4 T cells from older adults have a defect in this threshold calibration. Restoring their ability to respond to type I IFN emerges as a promising target to restore T cell responses and improve the induction of T cell memory. PMID:26091718

  15. Antigen-dependent and -independent contributions to primary memory CD8 T cell activation and protection following infection.

    PubMed

    Martin, Matthew D; Badovinac, Vladimir P

    2015-12-10

    Memory CD8 T-cell activation, including expression of IFN-γ and granzymeB, can be induced by antigen (Ag)-dependent signals through the T-cell-receptor, or by pathogen-derived inflammatory cytokines in an Ag-independent manner. Recent studies have come to conflicting results regarding the contributions of Ag and/or inflammation to memory CD8 T-cell activation. Additionally, research has indicated that inflammation-driven CD8 T-cell responses during un-related infections (bystander activation) have the potential to provide protection, but whether protection occurs in immuno-competent hosts is unclear. To investigate these questions, we examined activation of virus-specific memory CD8 T-cells following infection with L. monocytogenes either expressing or not cognate Ag. We show that Ag and inflammation act synergistically in vitro to induce memory activation. In vivo, we found that when memory CD8 T-cells significantly contribute to clearance of infection, early activation and continued responses by these cells are enhanced by cognate Ag recognition. Mechanistically, we show that bystander responses by memory are dependent upon the dose of infection and the amount of inflammation elicited following infection and are able to provide protection in IFN-γ deficient mice, but not in immuno-competent hosts. The data elucidate the requirements for memory CD8 T-cell activation and the protective role of bystander responses.

  16. Antigen-dependent and –independent contributions to primary memory CD8 T cell activation and protection following infection

    PubMed Central

    Martin, Matthew D.; Badovinac, Vladimir P.

    2015-01-01

    Memory CD8 T-cell activation, including expression of IFN-γ and granzymeB, can be induced by antigen (Ag)-dependent signals through the T-cell-receptor, or by pathogen-derived inflammatory cytokines in an Ag-independent manner. Recent studies have come to conflicting results regarding the contributions of Ag and/or inflammation to memory CD8 T-cell activation. Additionally, research has indicated that inflammation-driven CD8 T-cell responses during un-related infections (bystander activation) have the potential to provide protection, but whether protection occurs in immuno-competent hosts is unclear. To investigate these questions, we examined activation of virus-specific memory CD8 T-cells following infection with L. monocytogenes either expressing or not cognate Ag. We show that Ag and inflammation act synergistically in vitro to induce memory activation. In vivo, we found that when memory CD8 T-cells significantly contribute to clearance of infection, early activation and continued responses by these cells are enhanced by cognate Ag recognition. Mechanistically, we show that bystander responses by memory are dependent upon the dose of infection and the amount of inflammation elicited following infection and are able to provide protection in IFN-γ deficient mice, but not in immuno-competent hosts. The data elucidate the requirements for memory CD8 T-cell activation and the protective role of bystander responses. PMID:26658291

  17. How do kinases contribute to tonicity-dependent regulation of the transcription factor NFAT5?

    PubMed Central

    Zhou, Xiaoming

    2016-01-01

    NFAT5 plays a critical role in maintaining the renal functions. Its dis-regulation in the kidney leads to or is associated with certain renal diseases or disorders, most notably the urinary concentration defect. Hypertonicity, which the kidney medulla is normally exposed to, activates NFAT5 through phosphorylation of a signaling molecule or NFAT5 itself. Hypotonicity inhibits NFAT5 through a similar mechanism. More than a dozen of protein and lipid kinases have been identified to contribute to tonicity-dependent regulation of NFAT5. Hypertonicity activates NFAT5 by increasing its nuclear localization and transactivating activity in the early phase and protein abundance in the late phase. The known mechanism for inhibition of NFAT5 by hypotonicity is a decrease of nuclear NFAT5. The present article reviews the effect of each kinase on NFAT5 nuclear localization, transactivation and protein abundance, and the relationship among these kinases, if known. Cyclosporine A and tacrolimus suppress immune reactions by inhibiting the phosphatase calcineurin-dependent activation of NFAT1. It is hoped that this review would stimulate the interest to seek explanations from the NFAT5 regulatory pathways for certain clinical presentations and to explore novel therapeutic approaches based on the pathways. On the basic science front, this review raises two interesting questions. The first one is how these kinases can specifically signal to NFAT5 in the context of hypertonicity or hypotonicity,