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Sample records for activated t-cell nfat

  1. Nuclear Factor of Activated T-cells (NFAT) plays a role in SV40 infection

    PubMed Central

    Manley, Kate; O’Hara, Bethany A; Atwood, Walter J

    2008-01-01

    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 κB sites located within the 72bp 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. PMID:18031784

  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. Hyperactivation of nuclear factor of activated T cells 1 (NFAT1) in T cells attenuates severity of murine autoimmune encephalomyelitis

    PubMed Central

    Ghosh, Srimoyee; Koralov, Sergei B.; Stevanovic, Irena; Sundrud, Mark S.; Sasaki, Yoshiteru; Rajewsky, Klaus; Rao, Anjana; Müller, Martin R.

    2010-01-01

    Nuclear factor of activated T cells (NFAT) proteins are a group of Ca2+-regulated transcription factors residing in the cytoplasm of resting cells. Dephosphorylation by calcineurin results in nuclear translocation of NFAT and subsequent expression of target genes; rephosphorylation by kinases, including casein kinase 1 (CK1), restores NFAT to its latent state in the cytoplasm. We engineered a hyperactivable version of NFAT1 with increased affinity for calcineurin and decreased affinity for casein kinase 1. Mice expressing hyperactivable NFAT1 in their T-cell compartment exhibited a dramatically increased frequency of both IL-17– and IL-10–producing cells after differentiation under Th17 conditions—this was associated with direct binding of NFAT1 to distal regulatory regions of Il-17 and Il-10 gene loci in Th17 cells. Despite higher IL-17 production in culture, the mice were significantly less prone to myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis than controls, correlating with increased production of the immunomodulatory cytokine IL-10 and enhanced accumulation of regulatory T cells within the CNS. Thus, NFAT hyperactivation paradoxically leads to decreased susceptibility to experimental autoimmune encephalomyelitis, supporting previous observations linking defects in Ca2+/NFAT signaling to lymphoproliferation and autoimmune disease. PMID:20696888

  4. 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. PMID:25583478

  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. The transcription factor NFAT promotes exhaustion of activated CD8⁺ T cells.

    PubMed

    Martinez, Gustavo J; Pereira, Renata M; Äijö, Tarmo; Kim, Edward Y; Marangoni, Francesco; Pipkin, Matthew E; Togher, Susan; Heissmeyer, Vigo; Zhang, Yi Chen; Crotty, Shane; Lamperti, Edward D; Ansel, K Mark; Mempel, Thorsten R; Lähdesmäki, Harri; Hogan, Patrick G; Rao, Anjana

    2015-02-17

    During persistent antigen stimulation, CD8(+) T cells show a gradual decrease in effector function, referred to as exhaustion, which impairs responses in the setting of tumors and infections. Here we demonstrate that the transcription factor NFAT controls the program of T cell exhaustion. When expressed in cells, an engineered form of NFAT1 unable to interact with AP-1 transcription factors diminished T cell receptor (TCR) signaling, increased the expression of inhibitory cell surface receptors, and interfered with the ability of CD8(+) T cells to protect against Listeria infection and attenuate tumor growth in vivo. We defined the genomic regions occupied by endogenous and engineered NFAT1 in primary CD8(+) T cells and showed that genes directly induced by the engineered NFAT1 overlapped with genes expressed in exhausted CD8(+) T cells in vivo. Our data show that NFAT promotes T cell anergy and exhaustion by binding at sites that do not require cooperation with AP-1. PMID:25680272

  7. 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

  8. Lithium Regulates Keratinocyte Proliferation Via Glycogen Synthase Kinase 3 and NFAT2 (Nuclear Factor of Activated T Cells 2)

    PubMed Central

    Hampton, Philip J; Jans, Ralph; Flockhart, Ross J; Parker, Graeme; Reynolds, Nick J

    2012-01-01

    Certain environmental factors including drugs exacerbate or precipitate psoriasis. Lithium is the commonest cause of drug-induced psoriasis but underlying mechanisms are currently unknown. Lithium inhibits glycogen synthase kinase 3 (GSK-3). As lithium does not exacerbate other T-cell-mediated chronic inflammatory diseases, we investigated whether lithium may be acting directly on epidermal keratinocytes by inhibiting GSK-3. We report that lithium-induced keratinocyte proliferation at therapeutically relevant doses (1–2 mM) and increased the proportion of cells in S phase of the cell cycle. Inhibition of GSK-3 in keratinocytes by retroviral transduction of GSK-binding protein (an endogenous inhibitory protein) or through a highly selective pharmacological inhibitor also resulted in increased keratinocyte proliferation. Nuclear factor of activated T cells (NFAT) is an important substrate for GSK-3 and for cyclosporin, an effective treatment for psoriasis that inhibits NFAT activation in keratinocytes as well as in lymphocytes. Both lithium and genetic/pharmacological inhibition of GSK-3 resulted in increased nuclear localization of NFAT2 (NFATc1) and increased NFAT transcriptional activation. Finally, retroviral transduction of NFAT2 increased keratinocyte proliferation whereas siRNA-mediated knockdown of NFAT2 reduced keratinocyte proliferation and decreased epidermal thickness in an organotypic skin equivalent model. Taken together, these data identify GSK-3 and NFAT2 as key regulators of keratinocyte proliferation and as potential molecular targets relevant to lithium-provoked psoriasis. J. Cell. Physiol. 227: 1529–1537, 2012. © 2011 Wiley Periodicals, Inc. PMID:21678407

  9. Inhibition of Nuclear Factor of Activated T-Cells (NFAT) Suppresses Accelerated Atherosclerosis in Diabetic Mice

    PubMed Central

    Zetterqvist, Anna V.; Berglund, Lisa M.; Blanco, Fabiana; Garcia-Vaz, Eliana; Wigren, Maria; Dunér, Pontus; Andersson, Anna-Maria Dutius; To, Fong; Spegel, Peter; Nilsson, Jan; Bengtsson, Eva; Gomez, Maria F.

    2013-01-01

    Objective of the Study Diabetic patients have a much more widespread and aggressive form of atherosclerosis and therefore, higher risk for myocardial infarction, peripheral vascular disease and stroke, but the molecular mechanisms leading to accelerated damage are still unclear. Recently, we showed that hyperglycemia activates the transcription factor NFAT in the arterial wall, inducing the expression of the pro-atherosclerotic protein osteopontin. Here we investigate whether NFAT activation may be a link between diabetes and atherogenesis. Methodology and Principal Findings Streptozotocin (STZ)-induced diabetes in apolipoprotein E−/− mice resulted in 2.2 fold increased aortic atherosclerosis and enhanced pro-inflammatory burden, as evidenced by elevated blood monocytes, endothelial activation- and inflammatory markers in aorta, and pro-inflammatory cytokines in plasma. In vivo treatment with the NFAT blocker A-285222 for 4 weeks completely inhibited the diabetes-induced aggravation of atherosclerosis, having no effect in non-diabetic mice. STZ-treated mice exhibited hyperglycemia and higher plasma cholesterol and triglycerides, but these were unaffected by A-285222. NFAT-dependent transcriptional activity was examined in aorta, spleen, thymus, brain, heart, liver and kidney, but only augmented in the aorta of diabetic mice. A-285222 completely blocked this diabetes-driven NFAT activation, but had no impact on the other organs or on splenocyte proliferation or cytokine secretion, ruling out systemic immunosuppression as the mechanism behind reduced atherosclerosis. Instead, NFAT inhibition effectively reduced IL-6, osteopontin, monocyte chemotactic protein 1, intercellular adhesion molecule 1, CD68 and tissue factor expression in the arterial wall and lowered plasma IL-6 in diabetic mice. Conclusions Targeting NFAT signaling may be a novel and attractive approach for the treatment of diabetic macrovascular complications. PMID:23755169

  10. The Profile of Immune Modulation by Cannabidiol (CBD) Involves Deregulation of Nuclear Factor of Activated T Cells (NFAT)

    PubMed Central

    Kaplan, Barbara L. F.; Springs, Alison E. B.; Kaminski, Norbert E.

    2009-01-01

    Cannabidiol (CBD) is a cannabinoid compound derived from Cannabis Sativa that does not possess high affinity for either the CB1 or CB2 cannabinoid receptors. Similar to other cannabinoids, we demonstrated previously that CBD suppressed interleukin-2 (IL-2) production from phorbol ester plus calcium ionophore (PMA/Io)-activated murine splenocytes. Thus, the focus of the present studies was to further characterize the effect of CBD on immune function. CBD also suppressed IL-2 and interferon-γ (IFN-γ) mRNA expression, proliferation, and cell surface expression of the IL-2 receptor alpha chain, CD25. While all of these observations support the fact that CBD suppresses T cell function, we now demonstrate that CBD suppressed IL-2 and IFN-γ production in purified splenic T cells. CBD also suppressed activator protein-1 (AP-1) and nuclear factor of activated T cells (NFAT) transcriptional activity, which are critical regulators of IL-2 and IFN-γ. Furthermore, CBD suppressed the T cell-dependent anti-sheep red blood cell immunoglobulin M antibody forming cell (anti-sRBC IgM AFC) response. Finally, using splenocytes derived from CB1-/-/CB2-/- mice, it was determined that suppression of IL-2 and IFN-γ and suppression of the in vitro anti-sRBC IgM AFC response occurred independently of both CB1 and CB2. However, the magnitude of the immune response to sRBC was significantly depressed in CB1-/-/CB2-/- mice. Taken together, these data suggest that CBD suppresses T cell function and that CB1 and/or CB2 play a critical role in the magnitude of the in vitro anti-sRBC IgM AFC response. PMID:18656454

  11. Physical interactions between Ets and NF-kappaB/NFAT proteins play an important role in their cooperative activation of the human immunodeficiency virus enhancer in T cells.

    PubMed Central

    Bassuk, A G; Anandappa, R T; Leiden, J M

    1997-01-01

    The transcriptional regulatory elements of many inducible T-cell genes contain adjacent or overlapping binding sites for the Ets and NF-kappaB/NFAT families of transcription factors. Similar arrays of functionally important NF-kappaB/NFAT and Ets binding sites are present in the transcriptional enhancers of human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2), suggesting that this pattern of nuclear protein binding sites reflects an evolutionarily conserved mechanism for regulating inducible T-cell gene expression that has been co-opted during HIV evolution. Despite these findings, the molecular mechanisms by which Ets and NF-kappaB/NFAT proteins cooperatively regulate inducible T-cell gene expression remained unknown. In the studies described in this report, we demonstrated a physical interaction between multiple Ets and NF-kappaB/NFAT proteins both in vitro and in activated normal human T cells. This interaction is mediated by the Ets domain of Ets proteins and the C-terminal region of the Rel homology domains of NF-kappaB/NFAT proteins. In addition, the Ets-NF-kappaB/NFAT interaction requires the presence of DNA binding sites for both proteins, as it is abolished by the DNA intercalating agents propidium iodide and ethidium bromide and enhanced by the presence of synthetic oligonucleotides containing binding sites for Ets and NF-kappaB proteins. A dominant-negative mutant of NF-kappaB p50 that binds DNA but fails to interact with Ets proteins inhibits the synergistic activation of the HIV-1 and HIV-2 enhancers by NF-kappaB (p50 + p65) and Ets-1, suggesting that physical interaction between Ets and NF-kappaB proteins is required for the transcriptional activity of the HIV-1 and HIV-2 enhancers. Taken together, these findings suggest that evolutionarily conserved physical interactions between Ets and NF-kappaB/NFAT proteins are important in regulating the inducible expression of T-cell genes and viruses. These interactions represent a potential target

  12. 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. PMID:26324768

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. microRNA 184 regulates expression of NFAT1 in umbilical cord blood CD4+ T cells

    PubMed Central

    Weitzel, R. Patrick; Lesniewski, Mathew L.; Haviernik, Peter; Kadereit, Suzanne; Leahy, Patrick; Greco, Nicholas J.

    2009-01-01

    The reduced expression of nuclear factor of activated T cells-1 (NFAT1) protein in umbilical cord blood (UCB)–derived CD4+ T cells and the corresponding reduction in inflammatory cytokine secretion after stimulation in part underlies their phenotypic differences from adult blood (AB) CD4+ T cells. This muted response may contribute to the lower incidence and severity of high-grade acute graft-versus-host disease (aGVHD) exhibited by UCB grafts. Here we provide evidence that a specific microRNA, miR-184, inhibits NFAT1 protein expression elicited by UCB CD4+ T cells. Endogenous expression of miR-184 in UCB is 58.4-fold higher compared with AB CD4+ T cells, and miR-184 blocks production of NFAT1 protein through its complementary target sequence on the NFATc2 mRNA without transcript degradation. Furthermore, its negative effects on NFAT1 protein and downstream interleukin-2 (IL-2) transcription are reversed through antisense blocking in UCB and can be replicated via exogenous transfection of precursor miR-184 into AB CD4+ T cells. Our findings reveal a previously uncharacterized role for miR-184 in UCB CD4+ T cells and a novel function for microRNA in the early adaptive immune response. PMID:19286996

  19. Oxytocin-Stimulated NFAT Transcriptional Activation in Human Myometrial Cells

    PubMed Central

    McArdle, Craig A.; López Bernal, Andrés

    2012-01-01

    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 Ca2+ 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. PMID:22902539

  20. Cutting Edge: NFAT Transcription Factors Promote the Generation of Follicular Helper T Cells in Response to Acute Viral Infection.

    PubMed

    Martinez, Gustavo J; Hu, Joyce K; Pereira, Renata M; Crampton, Jordan S; Togher, Susan; Bild, Nicholas; Crotty, Shane; Rao, Anjana

    2016-03-01

    Follicular CD4(+) Th (Tfh) cells provide B cell help in germinal center reactions that support class switching, somatic hypermutation, and the generation of high-affinity Abs. In this article, we show that deficiency in NFAT1 and NFAT2 in CD4(+) T cells leads to impaired germinal center reactions upon viral infection because of reduced Tfh cell differentiation and defective expression of proteins involved in T/B interactions and B cell help, including ICOS, PD-1, and SLAM family receptors. Genome-wide chromatin immunoprecipitation data suggest that NFAT proteins likely directly participate in regulation of genes important for Tfh cell differentiation and function. NFAT proteins are important TCR and Ca(2+)-dependent regulators of T cell biology, and in this article we demonstrate a major positive role of NFAT family members in Tfh differentiation. PMID:26851216

  1. Co-operative interactions between NFAT (nuclear factor of activated T cells) c1 and the zinc finger transcription factors Sp1/Sp3 and Egr-1 regulate MT1-MMP (membrane type 1 matrix metalloproteinase) transcription by glomerular mesangial cells.

    PubMed Central

    Alfonso-Jaume, Maria Alejandra; Mahimkar, Rajeev; Lovett, David H

    2004-01-01

    The transition of normally quiescent glomerular MCs (mesangial cells) to a highly proliferative phenotype with characteristics of myofibroblasts is a process commonly observed in inflammatory diseases affecting the renal glomerulus, the ultimate result of which is glomerulosclerosis. Generation of proteolytically active MMP (matrix metalloproteinase)-2 by the membrane-associated membrane type 1 (MT1)-MMP is responsible for the transition of mesangial cells to the myofibroblast phenotype [Turck, Pollock, Lee, Marti and Lovett (1996) J. Biol. Chem. 271, 15074-15083]. In the present study, we show that the expression of MT1-MMP within the context of MCs is mediated by three discrete cis -acting elements: a proximal non-canonical Sp1 site that preferentially binds Sp1; an overlapping Sp1/Egr-1-binding site that preferentially binds Egr-1; and a more distal binding site for the NFAT (nuclear factor of activated T cells) that binds the NFAT c1 isoform present in MC nuclear extracts. Transfection with an NFAT c1 expression plasmid, or activation of calcineurin with a calcium ionophore, yielded major increases in NFAT c1 nuclear DNA-binding activity, MT1-MMP transcription and protein synthesis, which were additive with the lower levels of transactivation provided by the proximal Sp1 and the overlapping Sp1/Egr-1 sites. Specific binding of NFAT c1 to the MT1-MMP promoter was confirmed by chromatin immunoprecipitation studies, while MT1-MMP expression was suppressed by treatment with the calcineurin inhibitor, cyclosporin A. These studies are the first demonstration that a specific NFAT isoform enhances transcription of an MMP (MT1-MMP) that plays a major role in the proteolytic events that are a dominant feature of acute glomerular inflammation. Suppression of MT1-MMP by commonly used calcineurin inhibitors may play a role in the development of renal fibrosis following renal transplantation. PMID:14979875

  2. 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

  3. 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

  4. Disease relevance of T11TS-induced T-cell signal transduction through the CD2-mediated calcineurin-NFAT pathway: Perspectives in glioma immunotherapy.

    PubMed

    Chaudhuri, Suhnrita; Bhattacharya, Debanjan; Singh, Manoj Kumar; Moitra, Saibal; Ronsard, Larance; Ghosh, Tushar Kanti; Chaudhuri, Swapna

    2015-10-01

    Malignant glioma is the most lethal of a wide array of CNS neoplasms. Its onset and progression are markedly associated with profound immunosupression and paralysis of T-cell survival and proliferation. Myriad immunotherapeutic strategies are presently used to target such T-cell anomalies in glioma. Our recent work has highlighted use of the novel glycopeptide, the CD2 ligand, T11 target structure (T11TS) as an immunotherapeutic agent against experimentally induced glioma in rats. We have shown that T11TS causes multi-target modulation of key components of the T-cell - antigen presenting cell (APC) immunological synapse. This consequently triggers T-cell activation so as to reverse glioma-induced changes to physiological levels. T11TS administration also causes CD2 upregulation. Earlier we also found T11TS to cause enhanced proliferation of both CD4+ and CD8+ T-cells in glioma conditions. These findings led us to believe that downstream CD2-stimulated "alternative pathway" of calcineurin-NFAT could be a possible target for modulation by T11TS. In the present paper we thus show that immunotherapy with T11TS induces a multi-targeted approach towards activation of this "alternative pathway" of T-cell signaling providing an immunotherapeutic advantage against glioma. We show here that T11TS immunotherapy causes positive modulations of the CD2 pathway-associated proteins, viz., p59fyn, protein kinase C-θ (PKC-θ), calcineurin and nuclear factor for activation of T-cells (NFAT) and hint that this may accord greater survival and proliferation advantage to T-cells of the glioma-bearing animals for augmented defence against glioma. These findings help open a molecular immunotherapeutic door - one which is directed towards clinical studies for glioma-immunotherapy using T11TS. PMID:26105805

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. AKAP-Anchored PKA Maintains Neuronal L-type Calcium Channel Activity and NFAT Transcriptional Signaling

    PubMed Central

    Murphy, Jonathan G.; Sanderson, Jennifer L.; Gorski, Jessica A.; Scott, John D.; Catterall, William A.; Sather, William A.; Dell’Acqua, Mark L.

    2014-01-01

    Summary In neurons, Ca2+ influx through L-type voltage-gated Ca2+ channels (LTCC) couples electrical activity to changes in transcription. LTCC activity is elevated by the cAMP-dependent protein kinase (PKA) and depressed by the Ca2+-dependent phosphatase calcineurin (CaN), with both enzymes localized to the channel by A-kinase anchoring protein (AKAP) 79/150. AKAP79/150 anchoring of CaN also promotes LTCC activation of transcription through dephosphorylation of the nuclear factor of activated T-cells (NFAT). We report here that genetic disruption of PKA anchoring to AKAP79/150 also interferes with LTCC activation of CaN-NFAT signaling in neurons. Disruption of AKAP-PKA anchoring promoted redistribution of the kinase out of dendritic spines, profound decreases in LTCC phosphorylation and Ca2+ influx, and impaired NFAT movement to the nucleus and activation of transcription. Our findings support a model wherein basal activity of AKAP79/150-anchored PKA opposes CaN to preserve LTCC phosphorylation, thereby sustaining LTCC activation of CaN-NFAT signaling to the neuronal nucleus. PMID:24835999

  10. 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

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

    PubMed Central

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

    2015-01-01

    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. PMID:25719243

  12. CD95 co-stimulation blocks activation of naive T cells by inhibiting T cell receptor signaling

    PubMed Central

    Lindquist, Jonathan A.; Arhel, Nathalie; Felder, Edward; Karl, Sabine; Haas, Tobias L.; Fulda, Simone; Walczak, Henning; Kirchhoff, Frank; Debatin, Klaus-Michael

    2009-01-01

    CD95 is a multifunctional receptor that induces cell death or proliferation depending on the signal, cell type, and cellular context. Here, we describe a thus far unknown function of CD95 as a silencer of T cell activation. Naive human T cells triggered by antigen-presenting cells expressing a membrane-bound form of CD95 ligand (CD95L) or stimulated by anti-CD3 and -CD28 antibodies in the presence of recombinant CD95L had reduced activation and proliferation, whereas preactivated, CD95-sensitive T cells underwent apoptosis. Triggering of CD95 during T cell priming interfered with proximal T cell receptor signaling by inhibiting the recruitment of ζ-chain–associated protein of 70 kD, phospholipase-γ, and protein kinase C-θ into lipid rafts, thereby preventing their mutual tyrosine protein phosphorylation. Subsequently, Ca2+ mobilization and nuclear translocation of transcription factors NFAT, AP1, and NF-κB were strongly reduced, leading to impaired cytokine secretion. CD95-mediated inhibition of proliferation in naive T cells could not be reverted by the addition of exogenous interleukin-2 and T cells primed by CD95 co-stimulation remained partially unresponsive upon secondary T cell stimulation. HIV infection induced CD95L expression in primary human antigeen-presenting cells, and thereby suppressed T cell activation, suggesting that CD95/CD95L-mediated silencing of T cell activation represents a novel mechanism of immune evasion. PMID:19487421

  13. Activation and Cellular Localization of the Cyclosporine A-sensitive Transcription Factor NF-AT in Skeletal Muscle Cells

    PubMed Central

    Abbott, Karen L.; Friday, Bret B.; Thaloor, Deepa; Murphy, T.J.; Pavlath, Grace K.

    1998-01-01

    The widely used immunosuppressant cyclosporine A (CSA) blocks nuclear translocation of the transcription factor, NF-AT (nuclear factor of activated T cells), preventing its activity. mRNA for several NF-AT isoforms has been shown to exist in cells outside of the immune system, suggesting a possible mechanism for side effects associated with CSA treatment. In this study, we demonstrate that CSA inhibits biochemical and morphological differentiation of skeletal muscle cells while having a minimal effect on proliferation. Furthermore, in vivo treatment with CSA inhibits muscle regeneration after induced trauma in mice. These results suggest a role for NF-AT–mediated transcription outside of the immune system. In subsequent experiments, we examined the activation and cellular localization of NF-AT in skeletal muscle cells in vitro. Known pharmacological inducers of NF-AT in lymphoid cells also stimulate transcription from an NF-AT–responsive reporter gene in muscle cells. Three isoforms of NF-AT (NF-ATp, c, and 4/x/c3) are present in the cytoplasm of muscle cells at all stages of myogenesis tested. However, each isoform undergoes calcium-induced nuclear translocation from the cytoplasm at specific stages of muscle differentiation, suggesting specificity among NF-AT isoforms in gene regulation. Strikingly, one isoform (NF-ATc) can preferentially translocate to a subset of nuclei within a single multinucleated myotube. These results demonstrate that skeletal muscle cells express functionally active NF-AT proteins and that the nuclear translocation of individual NF-AT isoforms, which is essential for the ability to coordinate gene expression, is influenced markedly by the differentiation state of the muscle cell. PMID:9763451

  14. TonEBP/NFAT5 regulates ACTBL2 expression in biomechanically activated vascular smooth muscle cells

    PubMed Central

    Hödebeck, Maren; Scherer, Clemens; Wagner, Andreas H.; Hecker, Markus; Korff, Thomas

    2014-01-01

    Cytoskeletal reorganization and migration are critical responses which enable vascular smooth muscle cells (VSMCs) cells to evade, compensate, or adapt to alterations in biomechanical stress. An increase in wall stress or biomechanical stretch as it is elicited by arterial hypertension promotes their reorganization in the vessel wall which may lead to arterial stiffening and contractile dysfunction. This adaptive remodeling process is dependent on and driven by subtle phenotype changes including those controlling the cytoskeletal architecture and motility of VSMCs. Recently, it has been reported that the transcription factor nuclear factor of activated T-cells 5 (TonEBP/NFAT5) controls critical aspects of the VSMC phenotype and is activated by biomechanical stretch. We therefore hypothesized that NFAT5 controls the expression of gene products orchestrating cytoskeletal reorganization in stretch-stimulated VSMCs. Automated immunofluorescence and Western blot analyses revealed that biomechanical stretch enhances the expression and nuclear translocation of NFAT5 in VSMCs. Subsequent in silico analyses suggested that this transcription factor binds to the promotor region of ACTBL2 encoding kappa-actin which was shown to be abundantly expressed in VSMCs upon exposure to biomechanical stretch. Furthermore, ACTBL2 expression was inhibited in these cells upon siRNA-mediated knockdown of NFAT5. Kappa-actin appeared to be aligned with stress fibers under static culture conditions, dispersed in lamellipodia and supported VSMC migration as its knockdown diminishes lateral migration of these cells. In summary, our findings delineated biomechanical stretch as a determinant of NFAT5 expression and nuclear translocation controlling the expression of the cytoskeletal protein ACTBL2. This response may orchestrate the migratory activity of VSMCs and thus promote maladaptive rearrangement of the arterial vessel wall during hypertension. PMID:25520667

  15. 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

  16. Brx Mediates the Response of Lymphocytes to Osmotic Stress Through the Activation of NFAT5

    PubMed Central

    Kino, Tomoshige; Takatori, Hiroaki; Manoli, Irini; Wang, Yonghong; Tiulpakov, Anatoly; Blackman, Marc R.; Su, Yan A.; Chrousos, George P.; DeCherney, Alan H.; Segars, James H.

    2010-01-01

    Extracellular hyperosmolarity, or osmotic stress, generally caused by differences in salt and macromolecule concentrations across the plasma membrane, occurs in lymphoid organs and at inflammatory sites. The response of immune cells to osmotic stress is regulated by nuclear factor of activated T cells 5 (NFAT5), a transcription factor that induces the expression of hyperosmolarity-responsive genes and stimulates cytokine production. We report that the guanine nucleotide exchange factor (GEF) Brx [also known as protein kinase A–anchoring protein 13 (AKAP13)] is essential for the expression of nfat5 in response to osmotic stress, thus transmitting the extracellular hyperosmolarity signal and enabling differentiation of splenic B cells and production of immunoglobulin. This process required the activity of p38 mitogen-activated protein kinase (MAPK) and NFAT5 and involved a physical interaction between Brx and c-Jun N-terminal kinase (JNK)–interacting protein 4 (JIP4), a scaffold molecule specific to activation of the p38 MAPK cascade. Our results indicate that Brx integrates the responses of immune cells to osmotic stress and inflammation by elevating intracellular osmolarity and stimulating the production of cytokines. PMID:19211510

  17. Interaction of calcineurin with a domain of the transcription factor NFAT1 that controls nuclear import.

    PubMed Central

    Luo, C; Shaw, K T; Raghavan, A; Aramburu, J; Garcia-Cozar, F; Perrino, B A; Hogan, P G; Rao, A

    1996-01-01

    The nuclear import of the nuclear factor of activated T cells (NFAT)-family transcription factors is initiated by the protein phosphatase calcineurin. Here we identify a regulatory region of NFAT1, N terminal to the DNA-binding domain, that controls nuclear import of NFAT1. The regulatory region of NFAT1 binds directly to calcineurin, is a substrate for calcineurin in vitro, and shows regulated subcellular localization identical to that of full-length NFAT1. The corresponding region of NFATc likewise binds calcineurin, suggesting that the efficient activation of NFAT1 and NFATc by calcineurin reflects a specific targeting of the phosphatase to these proteins. The presence in other NFAT-family transcription factors of several sequence motifs from the regulatory region of NFAT1, including its probable nuclear localization sequence, indicates that a conserved protein domain may control nuclear import of all NFAT proteins. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8799126

  18. NFAT1 Directly Regulates IL8 and MMP3 to Promote Melanoma Tumor Growth and Metastasis.

    PubMed

    Shoshan, Einav; Braeuer, Russell R; Kamiya, Takafumi; Mobley, Aaron K; Huang, Li; Vasquez, Mayra E; Velazquez-Torres, Guermarie; Chakravarti, Nitin; Ivan, Cristina; Prieto, Victor; Villares, Gabriel J; Bar-Eli, Menashe

    2016-06-01

    Nuclear factor of activated T cell (NFAT1, NFATC2) is a transcription factor that binds and positively regulates IL2 expression during T-cell activation. NFAT1 has important roles in both innate and adaptive immune responses, but its involvement in cancer is not completely understood. We previously demonstrated that NFAT1 contributes to melanoma growth and metastasis by regulating the autotaxin gene (Enpp2). Here, we report a strong correlation between NFAT1 expression and metastatic potential in melanoma cell lines and tumor specimens. To elucidate the mechanisms underlying NFAT1 overexpression during melanoma progression, we conducted a microarray on a highly metastatic melanoma cell line in which NFAT1 expression was stably silenced. We identified and validated two downstream targets of NFAT1, IL8, and MMP3. Accordingly, NFAT1 depletion in metastatic melanoma cell lines was associated with reduced IL8 and MMP3 expression, whereas NFAT1 overexpression in a weakly metastatic cell line induced expression of these targets. Restoration of NFAT1 expression recovered IL8 and MMP3 expression levels back to baseline, indicating that both are direct targets of NFAT1. Moreover, in vivo studies demonstrated that NFAT1 and MMP3 promoted melanoma tumor growth and lung metastasis. Collectively, our findings assign a new role for NFAT1 in melanoma progression, underscoring the multifaceted functions that immunomodulatory factors may acquire in an unpredictable tumor microenvironment. Cancer Res; 76(11); 3145-55. ©2016 AACR. PMID:27013197

  19. 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

  20. 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

  1. 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

  2. Immunosuppressive drugs prevent a rapid dephosphorylation of transcription factor NFAT1 in stimulated immune cells.

    PubMed Central

    Shaw, K T; Ho, A M; Raghavan, A; Kim, J; Jain, J; Park, J; Sharma, S; Rao, A; Hogan, P G

    1995-01-01

    The immunosuppressive drugs cyclosporin A and FK506 interfere with the inducible transcription of cytokine genes in T cells and in other immune cells, in part by preventing the activation of NF-AT (nuclear factor of activated T cells). We show that transcription factor NFAT1 in T cells is rapidly dephosphorylated on stimulation, that dephosphorylation occurs before translocation of NFAT1 into the cell nucleus, and that dephosphorylation increases the affinity of NFAT1 for its specific sites in DNA. Cyclosporin A prevents the dephosphorylation and the nuclear translocation of NFAT1 in T cells, B cells, macrophages, and mast cells, delineating at least one mechanism that contributes to the profound immunosuppressive effects of this compound. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7479966

  3. 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

  4. 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. PMID:26374065

  5. TRAIL-receptor costimulation inhibits proximal TCR signaling and suppresses human T cell activation and proliferation.

    PubMed

    Lehnert, Corinna; Weiswange, Maxi; Jeremias, Irmela; Bayer, Carina; Grunert, Michaela; Debatin, Klaus-Michael; Strauss, Gudrun

    2014-10-15

    The TRAIL-receptor/TRAIL system originally described to induce apoptosis preferentially in malignant cells is also known to be involved in T cell homeostasis and the response to viral infections and autoimmune diseases. Whereas the expression of TRAIL on activated NK and T cells increases their cytotoxicity, induction of TRAIL on APCs can turn them into apoptosis inducers but might also change their immunostimulatory capacity. Therefore, we analyzed how TRAIL-receptor (TRAIL-R) costimulation is modulating TCR-mediated activation of human T cells. T cells triggered by rTRAIL in combination with anti-CD3 and -CD28 Abs exhibited a strong decrease in the expression of activation markers and Th1 and Th2 cytokines compared with CD3/CD28-activated T cells. Most importantly, proliferation of TRAIL-R costimulated T cells was strongly impaired, but no apoptosis was induced. Addition of exogenous IL-2 could not rescue T cells silenced by TRAIL-R costimulation, and TRAIL-mediated inhibition of T cell proliferation only prevented TCR-triggered proliferation but was ineffective if T cells were activated downstream of the TCR. Inhibition of T cell proliferation was associated with abrogation of proximal TCR signaling by inhibiting recruitment of TCR-associated signaling molecules to lipid rafts, followed by abrogation of protein tyrosine phosphorylation of ZAP70, phospholipase C-γ1, and protein kinase C-θ, and impaired nuclear translocation of NFAT, AP-1, and NF-κB. Most importantly, TRAIL-R costimulation efficiently inhibited alloantigen-induced T cell proliferation and CD3/28-induced activation and proliferation of autoreactive T cells derived from patients with Omenn syndrome, indicating that coactivation of TRAIL-R and TCR represents a mechanism to downmodulate T cell immune responses. PMID:25217163

  6. Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling

    PubMed Central

    Sena, Laura A.; Li, Sha; Jairaman, Amit; Prakriya, Murali; Ezponda, Teresa; Hildeman, David A.; Wang, Chyung-Ru; Schumacker, Paul T.; Licht, Jonathan D.; Perlman, Harris; Bryce, Paul J.; Chandel, Navdeep S.

    2013-01-01

    SUMMARY It is widely appreciated that T cells increase glycolytic flux during activation, however the role of mitochondrial flux is unclear. Here we have shown that mitochondrial metabolism, in the absence of glucose metabolism, was sufficient to support interleukin-2 (IL-2) induction. Furthermore, we used mice with reduced mitochondrial reactive oxygen species (mROS) production in T cells (T-Uqcrfs−/− mice) to show that mitochondria are required for T cell activation to produce mROS for activation of nuclear factor of activated T cells (NFAT) and subsequent IL-2 induction. These mice could not induce antigen-specific expansion of T cells in vivo, however Uqcrfs1−/− T cells retained the ability to proliferate in vivo under lymphopenic conditions. This suggests that Uqcrfs1−/− T cells were not lacking bioenergetically, but rather lacked specific ROS-dependent signaling events needed for antigen-specific expansion. Thus, mitochondrial metabolism is a critical component of T cell activation through production of complex III ROS. PMID:23415911

  7. 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.

  8. 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

  9. Arterial Wall Stress Controls NFAT5 Activity in Vascular Smooth Muscle Cells

    PubMed Central

    Scherer, Clemens; Pfisterer, Larissa; Wagner, Andreas H.; Hödebeck, Maren; Cattaruzza, Marco; Hecker, Markus; Korff, Thomas

    2014-01-01

    Background Nuclear factor of activated T‐cells 5 (NFAT5) has recently been described to control the phenotype of vascular smooth muscle cells (VSMCs). Although an increase in wall stress or stretch (eg, elicited by hypertension) is a prototypic determinant of VSMC activation, the impact of this biomechanical force on the activity of NFAT5 is unknown. This study intended to reveal the function of NFAT5 and to explore potential signal transduction pathways leading to its activation in stretch‐stimulated VSMCs. Methods and Results Human arterial VSMCs were exposed to biomechanical stretch and subjected to immunofluorescence and protein‐biochemical analyses. Stretch promoted the translocation of NFAT5 to the nucleus within 24 hours. While the protein abundance of NFAT5 was regulated through activation of c‐Jun N‐terminal kinase under these conditions, its translocation required prior activation of palmitoyltransferases. DNA microarray and ChiP analyses identified the matrix molecule tenascin‐C as a prominent transcriptional target of NFAT5 under these conditions that stimulates migration of VSMCs. Analyses of isolated mouse femoral arteries exposed to hypertensive perfusion conditions verified that NFAT5 translocation to the nucleus is followed by an increase in tenascin‐C abundance in the vessel wall. Conclusions Collectively, our data suggest that biomechanical stretch is sufficient to activate NFAT5 both in native and cultured VSMCs where it regulates the expression of tenascin‐C. This may contribute to an improved migratory activity of VSMCs and thus promote maladaptive vascular remodeling processes such as hypertension‐induced arterial stiffening. PMID:24614757

  10. Photochemical approaches to T-cell activation

    PubMed Central

    Huse, Morgan

    2010-01-01

    Despite decades of intensive research, T-cell activation has remained mysterious because of both the dizzying diversity of antigen recognition and the speed and comprehensiveness of the T-cell-receptor signalling network. Further progress will require new approaches and reagents that provide added levels of control. Photochemistry allows specific biochemical processes to be controlled with light and is well suited to mechanistic studies in complex cellular environments. In recent years, several laboratories have adopted approaches based on photoreactive peptide-major histocompatibility complex reagents in order to study T-cell activation and function with high precision. Here, I review these efforts and outline future directions for this exciting area of research. PMID:20406301

  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. Fus1/Tusc2 Is a Novel Regulator of Mitochondrial Calcium Handling, Ca2+-Coupled Mitochondrial Processes, and Ca2+-Dependent NFAT and NF-κB Pathways in CD4+ T Cells

    PubMed Central

    Uzhachenko, Roman; Ivanov, Sergey V.; Yarbrough, Wendell G.; Shanker, Anil; Medzhitov, Ruslan

    2014-01-01

    Abstract Aims: Fus1 has been established as mitochondrial tumor suppressor, immunomodulator, and antioxidant protein, but molecular mechanism of these activities remained to be identified. Based on putative calcium-binding and myristoyl-binding domains that we identified in Fus1, we explored our hypothesis that Fus1 regulates mitochondrial calcium handling and calcium-coupled processes. Results: Fus1 loss resulted in reduced rate of mitochondrial calcium uptake in calcium-loaded epithelial cells, splenocytes, and activated CD4+ T cells. The reduced rate of mitochondrial calcium uptake in Fus1-deficient cells correlated with cytosolic calcium increase and dysregulation of calcium-coupled mitochondrial parameters, such as reactive oxygen species production, ΔμH+, mitochondrial permeability transition pore opening, and GSH content. Inhibition of calcium efflux via mitochondria, Na+/Ca2+ exchanger significantly improved the mitochondrial calcium uptake in Fus1−/− cells. Ex vivo analysis of activated CD4+ T cells showed Fus1-dependent changes in calcium-regulated processes, such as surface expression of CD4 and PD1/PD-L1, proliferation, and Th polarization. Fus1−/− T cells showed increased basal expression of calcium-dependent NF-κB and NFAT targets but were unable to fully activate these pathways after stimulation. Innovation: Our results establish Fus1 as one of the few identified regulators of mitochondrial calcium handling. Our data support the idea that alterations in mitochondrial calcium dynamics could lead to the disruption of metabolic coupling in mitochondria that, in turn, may result in multiple cellular and systemic abnormalities. Conclusion: Our findings suggest that Fus1 achieves its protective role in inflammation, autoimmunity, and cancer via the regulation of mitochondrial calcium and calcium-coupled parameters. Antioxid. Redox Signal. 20, 1533–1547. PMID:24328503

  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. 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

  15. 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

  16. 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

  17. NFAT5 regulates the canonical Wnt pathway and is required for cardiomyogenic differentiation

    SciTech Connect

    Adachi, Atsuo; Takahashi, Tomosaburo; Ogata, Takehiro; Imoto-Tsubakimoto, Hiroko; Nakanishi, Naohiko; Ueyama, Tomomi; Matsubara, Hiroaki

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer NFAT5 protein expression is downregulated during cardiomyogenesis. Black-Right-Pointing-Pointer Inhibition of NFAT5 function suppresses canonical Wnt signaling. Black-Right-Pointing-Pointer Inhibition of NFAT5 function attenuates mesodermal induction. Black-Right-Pointing-Pointer NFAT5 function is required for cardiomyogenesis. -- Abstract: While nuclear factor of activated T cells 5 (NFAT5), a transcription factor implicated in osmotic stress response, is suggested to be involved in other processes such as migration and proliferation, its role in cardiomyogenesis is largely unknown. Here, we examined the role of NFAT5 in cardiac differentiation of P19CL6 cells, and observed that it was abundantly expressed in undifferentiated P19CL6 cells, and its protein expression was significantly downregulated by enhanced proteasomal degradation during DMSO-induced cardiomyogenesis. Expression of a dominant negative mutant of NFAT5 markedly attenuated cardiomyogenesis, which was associated with the inhibition of mesodermal differentiation. TOPflash reporter assay revealed that the transcriptional activity of canonical Wnt signaling was activated prior to mesodermal differentiation, and this activation was markedly attenuated by NFAT5 inhibition. Pharmacological activation of canonical Wnt signaling by [2 Prime Z, 3 Prime E]-6-bromoindirubin-3 Prime -oxime (BIO) restored Brachyury expression in NFAT5DN-expressing cells. Inhibition of NFAT5 markedly attenuated Wnt3 and Wnt3a induction. Expression of Dkk1 and Cerberus1, which are secreted Wnt antagonists, was also inhibited by NFAT5 inhibition. Thus, endogenous NFAT5 regulates the coordinated expression of Wnt ligands and antagonists, which are essential for cardiomyogenesis through the canonical Wnt pathway. These results demonstrated a novel role of NFAT5 in cardiac differentiation of stem cells.

  18. 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

  19. 4-1BB ligand signaling to T cells limits T cell activation.

    PubMed

    Eun, So-Young; Lee, Seung-Woo; Xu, Yanfei; Croft, Michael

    2015-01-01

    4-1BB ligand (4-1BBL) and its receptor, 4-1BB, are both induced on T cells after activation, but little is known about the role of 4-1BBL. In this study we show that 4-1BBL can transmit signals that limit T cell effector activity under tolerogenic conditions. Cross-linking 4-1BBL inhibited IL-2 production in vitro, primarily with suboptimal TCR stimulation. Furthermore, naive 4-1BBL-deficient OT-II transgenic T cells displayed a greater conversion to effector T cells in vivo when responding to soluble OVA peptide in wild-type hosts, whereas development of Foxp3(+) regulatory T cells was not altered. A greater number of effector T cells also differentiated from naive wild-type OT-II T cells when transferred into 4-1BB-deficient hosts, suggesting that APC-derived 4-1BB is likely to trigger 4-1BBL. Indeed, effector T cells that could not express 4-1BBL accumulated in larger numbers in vitro when stimulated with 4-1BB-expressing mesenteric lymph node dendritic cells. 4-1BBL was expressed on T cells when Ag presentation was limiting, and 4-1BBL was aberrantly expressed at very high levels on T cells that could not express 4-1BB. Trans-ligation, Ab capture, and endocytosis experiments additionally showed that T cell-intrinsic 4-1BB regulated internalization of membrane 4-1BBL, implying that the strong induction of 4-1BB on T cells may counteract the suppressive function of 4-1BBL by limiting its availability. These data suggest that 4-1BBL expressed on T cells can restrain effector T cell development, creating a more favorable regulatory T cell to effector cell balance under tolerogenic conditions, and this may be particularly active in mucosal barrier tissues where 4-1BB-expressing regulatory dendritic cells present Ag. PMID:25404362

  20. 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.

  1. Early kinetic window of target T cell susceptibility to CD25+ regulatory T cell activity.

    PubMed

    Sojka, Dorothy K; Hughson, Angela; Sukiennicki, Teresa L; Fowell, Deborah J

    2005-12-01

    Peripheral tolerance is maintained in part by thymically derived CD25+CD4+ T cells (regulatory T cells (Tregs)). Their mechanism of action has not been well characterized. Therefore, to get a better understanding of Treg action, we investigated the kinetics of murine Treg activity in vitro. Tregs were suppressive within a surprisingly narrow kinetic window: necessary and sufficient only in the first 6-10 h of culture. Visualization of this time frame, using a sensitive single-cell assay for IL-2, revealed the early elaboration of target cell IL-2 producers in the first 6 h despite the presence of CD25+CD4+ Tregs. However, after 6 h, a rapid rise in the number of IL-2 producers in the absence of Tregs was dramatically abrogated by the presence of Tregs. Importantly, the timing of suppression was dictated by the kinetics of target T cell activation suggesting that early target T cell signals may alter susceptibility to suppression. Modulating target T cell activation signals with provision of CD28, IL-2, or high Ag dose all abrogated suppression of proliferation late in culture. However, only CD28 signals enabled target T cells to resist the early Treg-induced down-regulation of IL-2. Therefore the quality of early target T cell activation signals, in particular engagement of CD28, represents an important control point in the balance between vulnerability and resistance to Treg suppression. PMID:16301632

  2. 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. PMID:27080132

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. Helenalin suppresses essential immune functions of activated CD4+ T cells by multiple mechanisms.

    PubMed

    Berges, Carsten; Fuchs, Dominik; Opelz, Gerhard; Daniel, Volker; Naujokat, Cord

    2009-09-01

    Helenalin is a naturally occuring sesquiterpene lactone extracted from Arnica montana and Arnica chamissonis ssp. foliosa. Helenalin and its derivatives are known for anti-cancer and anti-inflammatory effects via inhibiting NF-kappaB and telomerase activity and impairing protein and DNA synthesis, suggesting that helenalin is a potential candidate for the treatment of deregulated and unwanted T cell-mediated immune responses. Here we show that helenalin induces apoptosis in activated CD4+ T cells by triggering the mitochondrial pathway of apoptosis. Induction of apoptosis is accompanied by rapid stabilization of p53, nuclear localization of p53 and AIF, and an increase in ROS production that results in loss of mitochondrial membrane potential (DeltaPsim). Activated CD4+ T cells which survive exposure to helenalin undergo inhibition of proliferation by induction of G2/M cell cycle arrest. Cell cycle arrest is accompanied by the accumulation of cell cycle regulator proteins p21(WAF/CIP1), p2(KIP1) and cyclin D2, whereas abundance of cyclin A and B(1) is decreased. Cell surface expression of the activation-associated receptors CD25, CD27, CD28, CD120b as well as production of IL-2 are impaired. Transcriptional activation of genes encoding for CD25, IL-2 and IFN-gamma is mediated by transcription factors of the NFAT family, and we demonstrate that helenalin suppresses nuclear translocation of NFATc2 in activated CD4+ T cells. Thus, helenalin can be defined as a new immunosuppressive compound suited for the treatment of deregulated and unwanted T cell-mediated immune responses. PMID:19656571

  9. NGF Upregulates the Plasminogen Activation Inhibitor-1 in Neurons via the Calcineurin/NFAT Pathway and the Down Syndrome-Related Proteins DYRK1A and RCAN1 Attenuate This Effect

    PubMed Central

    Stefos, Georgios C.; Soppa, Ulf; Dierssen, Mara; Becker, Walter

    2013-01-01

    Background Plasminogen activator inhibitor 1 (PAI-1) is a key regulator of the plasminogen activation system. Although several lines of evidence support a significant role of PAI-1 in the brain, the regulation of its expression in neurons is poorly understood. In the present study we tested the hypothesis that NGF induces the upregulation of PAI-1 via the calcineurin/nuclear factor of activated T cells (NFAT) pathway and analysed whether the overexpression of the Down syndrome-related proteins DYRK1A and RCAN1 modulated the effect of NGF on PAI-1 expression. Results NGF upregulated PAI-1 mRNA levels in primary mouse hippocampal neurons cultured for 3 days in vitro and in the rat pheochromocytoma cell line PC12. Reporter gene assays revealed that NGF activated the calcineurin/NFAT pathway in PC12 cells. Induction of PAI-1 by NGF was sensitive to the calcineurin inhibitor FK506 and the specific inhibition of NFAT activation by the cell permeable VIVIT peptide. Activation of calcineurin/NFAT signalling through other stimuli resulted in a much weaker induction of PAI-1 expression, suggesting that other NGF-induced pathways are involved in PAI-1 upregulation. Overexpression of either DYRK1A or RCAN1 negatively regulated NFAT-dependent transcriptional activity and reduced the upregulation of PAI-1 levels by NGF. Conclusion The present results show that the calcineurin/NFAT pathway mediates the upregulation of PAI-1 by NGF. The negative effect of DYRK1A and RCAN1 overexpression on NGF signal transduction in neural cells may contribute to the altered neurodevelopment and brain function in Down syndrome. PMID:23825664

  10. Osmotic induction of placental growth factor in retinal pigment epithelial cells in vitro: contribution of NFAT5 activity.

    PubMed

    Hollborn, Margrit; Reichmuth, Konrad; Prager, Philipp; Wiedemann, Peter; Bringmann, Andreas; Kohen, Leon

    2016-08-01

    One risk factor of neovascular age-related macular degeneration is systemic hypertension; hypertension is mainly caused by extracellular hyperosmolarity after consumption of dietary salt. In retinal pigment epithelial (RPE) cells, high extracellular osmolarity induces vascular endothelial growth factor (VEGF)-A (Hollborn et al. in Mol Vis 21:360-377, 2015). The aim of the present study was to determine whether extracellular hyperosmolarity and chemical hypoxia trigger the expression of further VEGF family members including placental growth factor (PlGF) in human RPE cells. Hyperosmotic media were made up by addition of 100 mM NaCl or sucrose. Chemical hypoxia was induced by CoCl2. Gene expression was quantified by real-time RT-PCR, and secretion of PlGF-2 was investigated with ELISA. Nuclear factor of activated T cell 5 (NFAT5) was depleted using siRNA. Extracellular hyperosmolarity triggered expression of VEGF-A, VEGF-D, and PlGF genes, and secretion of PlGF-2. Hypoosmolarity decreased PlGF gene expression. Hypoxia induced expression of VEGF-A, VEGF-B, VEGF-D, and PlGF genes. Extracellular hyperosmolarity and hypoxia produced additive PlGF gene expression. Both hyperosmolarity and hypoxia induced expression of KDR and FLT-4 receptor genes, while hyperosmolarity caused neuropilin-2 and hypoxia neuropilin-1 gene expression. The hyperosmotic, but not the hypoxic, PlGF gene expression was in part mediated by NFAT5. The expression of PlGF in RPE cells depends on the extracellular osmolarity. The data suggest that high consumption of dietary salt may exacerbate the angiogenic response of RPE cells in the hypoxic retina via transcriptional activation of various VEGF family member genes. PMID:27230578

  11. 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

  12. NFAT Gene Family in Inflammation and Cancer

    PubMed Central

    Pan, M.-G.; Xiong, Y.; Chen, F.

    2013-01-01

    Calcineurin-NFAT signaling is critical for numerous aspects of vertebrate function during and after embryonic development. Initially discovered in T cells, the NFAT gene family, consisting of five members, regulates immune system, inflammatory response, angiogenesis, cardiac valve formation, myocardial development, axonal guidance, skeletal muscle development, bone homeostasis, development and metastasis of cancer, and many other biological processes. In this review we will focus on the NFAT literature relevant to the two closely related pathological systems: inflammation and cancer. PMID:22950383

  13. T Cell-Extrinsic CD18 Attenuates Antigen-Dependent CD4+ T cell Activation In Vivo1

    PubMed Central

    Wu, Xingxin; Lahiri, Amit; Sarin, Ritu; Abraham, Clara

    2015-01-01

    The β2 integrins (CD11/CD18) are heterodimeric leukocyte adhesion molecules expressed on hematopoietic cells. The role of T cell-intrinsic CD18 in trafficking of naïve T cells to secondary lymphoid organs, and in antigen-dependent T cell activation in vitro and in vivo has been well-defined. However, the T cell-extrinsic role for CD18, including on antigen presenting cells (APC), in contributing to T cell activation in vivo is less well understood. We examined the role for T cell-extrinsic CD18 in the activation of WT CD4+ T cells in vivo through the adoptive transfer of DO11.10 Ag-specific CD4+ T cells into CD18−/− mice. We found that T cell-extrinsic CD18 was required for attenuating OVA-induced T cell proliferation in peripheral lymph nodes (PLN). The increased proliferation of WT DO11.10 CD4+ T cells in CD18−/− PLN was associated with a higher percentage of APC, and these APC demonstrated an increased activation profile and increased Ag-uptake, in particular in F4/80+ APC. Depletion of F4/80+ cells both reduced and equalized antigen-dependent T cell proliferation in CD18−/− relative to littermate control PLN, demonstrating that these cells play a critical role in the enhanced T cell proliferation in CD18−/− mice. Consistently, CD11b blockade, which is expressed on F4/80+ macrophages, enhanced the proliferation of DO11.10+ T cells in CD18+/− PLN. Thus, in contrast to the T cell-intrinsic essential role for CD18 in T cell activation, T cell-extrinsic expression of CD18 attenuates antigen-dependent CD4+ T cell activation in PLN in vivo. PMID:25801431

  14. 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.

  15. Ezrin tunes T-cell activation by controlling Dlg1 and microtubule positioning at the immunological synapse

    PubMed Central

    Lasserre, Rémi; Charrin, Stéphanie; Cuche, Céline; Danckaert, Anne; Thoulouze, Maria-Isabel; de Chaumont, Fabrice; Duong, Tarn; Perrault, Nathalie; Varin-Blank, Nadine; Olivo-Marin, Jean-Christophe; Etienne-Manneville, Sandrine; Arpin, Monique; Di Bartolo, Vincenzo; Alcover, Andrés

    2010-01-01

    T-cell receptor (TCR) signalling is triggered and tuned at immunological synapses by the generation of signalling complexes that associate into dynamic microclusters. Microcluster movement is necessary to tune TCR signalling, but the molecular mechanism involved remains poorly known. We show here that the membrane-microfilament linker ezrin has an important function in microcluster dynamics and in TCR signalling through its ability to set the microtubule network organization at the immunological synapse. Importantly, ezrin and microtubules are important to down-regulate signalling events leading to Erk1/2 activation. In addition, ezrin is required for appropriate NF-AT activation through p38 MAP kinase. Our data strongly support the notion that ezrin regulates immune synapse architecture and T-cell activation through its interaction with the scaffold protein Dlg1. These results uncover a crucial function for ezrin, Dlg1 and microtubules in the organization of the immune synapse and TCR signal down-regulation. Moreover, they underscore the importance of ezrin and Dlg1 in the regulation of NF-AT activation through p38. PMID:20551903

  16. Studies of T-cell activation in chronic inflammation

    PubMed Central

    2002-01-01

    Chapter summary The strong association between specific alleles encoded within the MHC class II region and the development of rheumatoid arthritis (RA) has provided the best evidence to date that CD4+ T cells play a role in the pathogenesis of this chronic inflammatory disease. However, the unusual phenotype of synovial T cells, including their profound proliferative hyporesponsiveness to TCR ligation, has challenged the notion that T-cell effector responses are driven by cognate cartilage antigens in inflamed synovial joints. The hierarchy of T-cell dysfunction from peripheral blood to inflamed joint suggests that these defects are acquired through prolonged exposure to proinflammatory cytokines such as tumour necrosis factor (TNF)-α. Indeed, there are now compelling data to suggest that chronic cytokine activation may contribute substantially to the phenotype and effector function of synovial T cells. Studies reveal that chronic exposure of T cells to TNF uncouples TCR signal transduction pathways by impairing the assembly and stability of the TCR/CD3 complex at the cell surface. Despite this membrane-proximal effect, TNF selectively uncouples downstream signalling pathways, as is shown by the dramatic suppression of calcium signalling responses, while Ras/ERK activation is spared. On the basis of these data, it is proposed that T-cell survival and effector responses are driven by antigen-independent, cytokine-dependent mechanisms, and that therapeutic strategies that seek to restore T-cell homeostasis rather than further depress T-cell function should be explored in the future. PMID:12110140

  17. Improved anti-leukemia activities of adoptively transferred T cells expressing bispecific T-cell engager in mice.

    PubMed

    Liu, X; Barrett, D M; Jiang, S; Fang, C; Kalos, M; Grupp, S A; June, C H; Zhao, Y

    2016-01-01

    Despite the impressive clinical efficacy of T cells engineered to express chimeric antigen receptors (CAR-Ts), the current applications of CAR-T cell therapy are limited by major treatment-related toxicity. Thus, safer yet effective alternative approaches must be developed. In this study, we compared CD19 bispecific T-cell engager (BiTE)-transferred T cells that had been transfected by RNA electroporation with CD19 CAR RNA-transferred T cells both in vitro and in an aggressive Nalm6 leukemia mouse model. BiTEs were secreted from the transferred T cells and enabled both the transferred and bystander T cells to specifically recognize CD19(+) cell lines, with increased tumor killing ability, prolonged functional persistence, increased cytokine production and potent proliferation compared with the CAR-T cells. More interestingly, in comparison with CD3/CD28 bead-stimulated T cells, T cells that were expanded by a rapid T-cell expansion protocol (REP) showed enhanced anti-tumor activities for both CAR and BiTE RNA-electroporated T cells both in vitro and in a Nalm6 mouse model (P<0.01). Furthermore, the REP T cells with BiTE RNAs showed greater efficacy in the Nalm6 leukemia model compared with REP T cells with CAR RNA (P<0.05) and resulted in complete leukemia remission. PMID:27258611

  18. 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. PMID:26903518

  19. 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.

  20. The Amiloride Derivative Phenamil Attenuates Pulmonary Vascular Remodeling by Activating NFAT and the Bone Morphogenetic Protein Signaling Pathway ▿

    PubMed Central

    Chan, Mun Chun; Weisman, Alexandra S.; Kang, Hara; Nguyen, Peter H.; Hickman, Tyler; Mecker, Samantha V.; Hill, Nicholas S.; Lagna, Giorgio; Hata, Akiko

    2011-01-01

    Pulmonary artery hypertension (PAH) is characterized by elevated pulmonary artery resistance and increased medial thickness due to deregulation of vascular remodeling. Inactivating mutations of the BMPRII gene, which encodes a receptor for bone morphogenetic proteins (BMPs), are identified in ∼60% of familial PAH (FPAH) and ∼30% of idiopathic PAH (IPAH) patients. It has been hypothesized that constitutive reduction in BMP signal by BMPRII mutations may cause abnormal vascular remodeling by promoting dedifferentiation of vascular smooth muscle cells (vSMCs). Here, we demonstrate that infusion of the amiloride analog phenamil during chronic-hypoxia treatment in rat attenuates development of PAH and vascular remodeling. Phenamil induces Tribbles homolog 3 (Trb3), a positive modulator of the BMP pathway that acts by stabilizing the Smad family signal transducers. Through induction of Trb3, phenamil promotes the differentiated, contractile vSMC phenotype characterized by elevated expression of contractile genes and reduced cell growth and migration. Phenamil activates the Trb3 gene transcription via activation of the calcium-calcineurin-nuclear factor of activated T cell (NFAT) pathway. These results indicate that constitutive elevation of Trb3 by phenamil is a potential therapy for IPAH and FPAH. PMID:21135135

  1. 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.

  2. 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

  3. Elevated Intracellular Calcium Increases Ferritin H Expression Through an NFAT-Independent Posttranscriptional Mechanism Involving mRNA Stabilization

    PubMed Central

    MacKenzie, Elizabeth L.; Tsuji, Yoshiaki

    2009-01-01

    An increase in intracellular Ca2+ is one of the initiating events in T cell activation. A calcium-mediated signaling cascade in T cells involves activation of calcineurin and the dephosphorylation and translocation of Nuclear Factor of Activated T-cells (NFAT), resulting in the transcriptional activation of target genes such as IL-2. In the present study, we found that increased intracellular calcium leads to induction of the antioxidant protein ferritin H. We previously reported that the ferritin H gene is transcriptionally activated under oxidative stress conditions through an antioxidant responsive element (ARE). The facts that the ferritin H ARE contains a composite AP1 site, and that NFAT collaborates with AP1 transcription factors, led us to test whether calcium-activated NFAT is involved in the ferritin H induction through the ARE. Treatment of Jurkat T cells with the calcium ionophore, ionomycin, increased ferritin H mRNA and protein expression. Though NFAT translocated to the nucleus and bound a consensus NFAT sequence located in the IL-2 promoter following ionomycin treatment, it did not activate ferritin H transcription despite the presence of a putative NFAT binding sequence in the ferritin H ARE. In addition, the calcineurin inhibitor cyclosporin A treatment blocked ionomycin-mediated NFAT nuclear translocation but failed to abrogate the increase in ferritin H mRNA. Analysis of mRNA stability following actinomycin D treatment revealed that ionomycin prolongs ferritin H mRNA half-life. Taken together, these results suggest that ionomycin-mediated induction of ferritin H may occur in an NFAT-independent manner but through posttranscriptional stabilization of the ferritin H mRNA. PMID:18076382

  4. 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.

  5. 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

  6. T cells conditioned with MDSC show an increased anti-tumor activity after adoptive T cell based immunotherapy

    PubMed Central

    Raber, Patrick L.; Sierra, Rosa A.; Thevenot, Paul T.; Shuzhong, Zhang; Wyczechowska, Dorota D.; Kumai, Takumi; Celis, Esteban; Rodriguez, Paulo C.

    2016-01-01

    The success of adoptive T cell-based immunotherapy (ACT) in cancer is limited in part by the accumulation of myeloid-derived suppressor cells (MDSC), which block several T cell functions, including T cell proliferation and the expression of various cytotoxic mediators. Paradoxically, the inhibition of CD8+ T cell differentiation into cytotoxic populations increased their efficacy after ACT into tumor-bearing hosts. Therefore, we aimed to test the impact of conditioning CD8+ T cells with MDSC on their differentiation potential and ACT efficacy. Our results indicate that MDSC impaired the progression of CD8+ T cells into effector populations, without altering their activation status, production of IL-2, or signaling through the T cell receptor. In addition, culture of CD8+ T cells with MDSC resulted in an increased ACT anti-tumor efficacy, which correlated with a higher frequency of the transferred T cells and elevated IFNγ production. Interestingly, activated CD62L+ CD8+ Tcells were responsible for the enhanced anti-tumor activity showed by MDSC-exposed T cells. Additional results showed a decreased protein synthesis rate and lower activity of the mammalian/mechanistic target of rapamycin (mTOR) in T cells conditioned with MDSC. Silencing of the negative mTOR regulator tuberous sclerosis complex-2 in T cells co-cultured with MDSC restored mTOR activity, but resulted in T cell apoptosis. These results indicate that conditioning of T cells with MDSC induces stress survival pathways mediated by a blunted mTOR signaling, which regulated T cell differentiation and ACT efficacy. Continuation of this research will enable the development of better strategies to increase ACT responses in cancer. PMID:27007050

  7. 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.

  8. Master switches of T-cell activation and differentiation.

    PubMed

    Beier, K C; Kallinich, T; Hamelmann, E

    2007-04-01

    T-cells play a central role in allergic airway diseases such as bronchial asthma. The imbalance between allergen-specific pro-inflammatory and pro-allergic T-cell responses on one hand and regulatory or suppressive T-cell responses on the other may best explain the development of unwanted immune responses against environmental allergens, which lead to immunoglobulin E production and airway inflammation. A key role in the fine tuning of any T-cell response is provided by the engagement of so-called co-stimulatory molecules that are required for the full activation of T-cells and the recognition of antigens via the antigen-specific T-cell receptor. Many of these co-stimulatory molecules have been identified only recently, leading to a fundamental change in the overall understanding of T-cell regulation. Due to their pivotal impact on T-cell differentiation and control, co-stimulatory molecules are promising targets for therapeutic intervention in T-cell-regulated or -mediated immune disorders, including allergic diseases and asthma. In the present article, an attempt is made to summarise the current knowledge on the basic concept of co-stimulation, the presently known co-stimulatory molecules and their various functions on T-cell activation or suppression. The mini-series will be completed by two more articles describing the recent experimental studies and preliminary clinical findings regarding the role of co-stimulatory molecules in allergic disorders and bronchial asthma, and a discussion regarding the feasibility of co-stimulatory molecules as potential targets for the treatment of allergic airway disease. Although it is too early for any clinical implication or utilisation at this moment, the authors are convinced that a better understanding of co-stimulation in the context of allergic asthma will finally provide novel and promising approaches for treatment and prevention. PMID:17400879

  9. 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. PMID:27144475

  10. Hydrogen Sulfide Is an Endogenous Potentiator of T Cell Activation*

    PubMed Central

    Miller, Thomas W.; Wang, Evelyn A.; Gould, Serge; Stein, Erica V.; Kaur, Sukhbir; Lim, Langston; Amarnath, Shoba; Fowler, Daniel H.; Roberts, David D.

    2012-01-01

    H2S is an endogenous signaling molecule that may act via protein sulfhydrylation to regulate various physiological functions. H2S is also a byproduct of dietary sulfate metabolism by gut bacteria. Inflammatory bowel diseases such as ulcerative colitis are associated with an increase in the colonization of the intestine by sulfate reducing bacteria along with an increase in H2S production. Consistent with its increased production, H2S is implicated as a mediator of ulcerative colitis both in its genesis or maintenance. As T cells are well established mediators of inflammatory bowel disease, we investigated the effect of H2S exposure on T cell activation. Using primary mouse T lymphocytes (CD3+), OT-II CD4+ T cells, and the human Jurkat T cell line, we show that physiological levels of H2S potentiate TCR-induced activation. Nanomolar levels of H2S (50–500 nm) enhance T cell activation assessed by CD69 expression, interleukin-2 expression, and CD25 levels. Exposure of T cells to H2S dose-dependently enhances TCR-stimulated proliferation with a maximum at 300 nm (30% increase, p < 0.01). Furthermore, activation increases the capacity of T cells to make H2S via increased expression of cystathionine γ-lyase and cystathionine β-synthase. Disrupting this response by silencing these H2S producing enzymes impairs T cell activation, and proliferation and can be rescued by the addition of 300 nm H2S. Thus, H2S represents a novel autocrine immunomodulatory molecule in T cells. PMID:22167178

  11. 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

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

    PubMed

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

    2016-06-30

    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 CIITA(Tg)Plzf(lu/lu) and BALB/c.CD1d(-/-) mice as well as in an IL-4-deficient background, such as in CIITA(Tg)IL-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 CIITA(Tg)PIV(-/-) 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

  13. Functional Implications of Plasma Membrane Condensation for T Cell Activation

    PubMed Central

    Quinn, Carmel M.; Engelhardt, Karin; Williamson, David; Grewal, Thomas; Jessup, Wendy; Harder, Thomas; Gaus, Katharina

    2008-01-01

    The T lymphocyte plasma membrane condenses at the site of activation but the functional significance of this receptor-mediated membrane reorganization is not yet known. Here we demonstrate that membrane condensation at the T cell activation sites can be inhibited by incorporation of the oxysterol 7-ketocholesterol (7KC), which is known to prevent the formation of raft-like liquid-ordered domains in model membranes. We enriched T cells with 7KC, or cholesterol as control, to assess the importance of membrane condensation for T cell activation. Upon 7KC treatment, T cell antigen receptor (TCR) triggered calcium fluxes and early tyrosine phosphorylation events appear unaltered. However, signaling complexes form less efficiently on the cell surface, fewer phosphorylated signaling proteins are retained in the plasma membrane and actin restructuring at activation sites is impaired in 7KC-enriched cells resulting in compromised downstream activation responses. Our data emphasizes lipids as an important medium for the organization at T cell activation sites and strongly indicates that membrane condensation is an important element of the T cell activation process. PMID:18509459

  14. 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.

  15. Canine CD4+CD8+ double positive T cells in peripheral blood have features of activated T cells.

    PubMed

    Bismarck, Doris; Schütze, Nicole; Moore, Peter; Büttner, Mathias; Alber, Gottfried; Buttlar, Heiner v

    2012-10-15

    In dogs a CD4(+)CD8(+) double positive T cell subpopulation exists that has not been phenotypically defined yet. We demonstrate that canine CD4(+)CD8(+) T cells are mature CD1a(-) and TCRαβ(+) T cells. To analyse the activation potential of CD4(+)CD8(+) T cells, PBMC from dogs vaccinated against canine distemper virus (CDV) were re-stimulated with CDV. Upon antigen-specific stimulation, the CD4(+)CD8(+) T cell fraction increases and consists nearly exclusively of proliferated cells. Similarly, other features of activated effector/memory T cells such as up-regulation of CD25 and MHC-II as well as down-regulation of CD62L (L-selectin) were observed in CD4(+)CD8(+) T cells after stimulation. Canine CD4(+)CD8(+) T cells are less abundant, but more heterogeneous than porcine ones, comprising a small proportion expressing the β chain of CD8 in addition to the CD8α chain, like human CD4(+)CD8(+) T cells. In summary, this analysis provides the basis for functional characterisation of the in vivo relevance of CD4(+)CD8(+) T cells in T-cell mediated immunity. PMID:22789871

  16. Role of NFAT5 in Inflammatory Disorders Associated with Osmotic Stress

    PubMed Central

    Neuhofer, Wolfgang

    2010-01-01

    Nuclear factor of activated T cells 5 (NFAT5) is the most recently described member of the Rel family of transcription factors, including NF-κB and NFAT1-4, which play central roles in inducible gene expression during the immune response. NFAT5 was initially described to drive osmoprotective gene expression in renal medullary cells, which are routinely faced by high extracellular osmolalities. Recent data however indicate profound biological importance of the mammalian osmotic stress response in view of NFAT5 dependent gene regulation in non-renal tissues. In mononuclear cells and epithelial cells, NFAT5 stimulates the expression of various pro-inflammatory cytokines during elevated ambient tonicity. Accordingly, compared to plasma, the interstitial tonicity of lymphoid organs like spleen and thymus and that of liver is substantially hypertonic under physiological conditions. In addition, anisotonic disorders (hypernatremia, diabetes mellitus, dehydration) entail systemic hyperosmolality, and, in inflammatory disorders, the skin, intestine, and cornea are sites of local hyperosmolality. This article summarizes the current knowledge regarding systemic and local osmotic stress in anisotonic and inflammatory disorders in view of NFAT5 activation and regulation, and NFAT5 dependent cytokine production. PMID:21629436

  17. Ionizing Radiation Impairs T Cell Activation by Affecting Metabolic Reprogramming

    PubMed Central

    Li, Heng-Hong; Wang, Yi-wen; Chen, Renxiang; Zhou, Bin; Ashwell, Jonathan D.; Fornace, Albert J.

    2015-01-01

    Ionizing radiation has a variety of acute and long-lasting adverse effects on the immune system. Whereas measureable effects of radiation on immune cell cytotoxicity and population change have been well studied in human and animal models, little is known about the functional alterations of the surviving immune cells after ionizing radiation. The objective of this study was to delineate the effects of radiation on T cell function by studying the alterations of T cell receptor activation and metabolic changes in activated T cells isolated from previously irradiated animals. Using a global metabolomics profiling approach, for the first time we demonstrate that ionizing radiation impairs metabolic reprogramming of T cell activation, which leads to substantial decreases in the efficiency of key metabolic processes required for activation, such as glucose uptake, glycolysis, and energy metabolism. In-depth understanding of how radiation impacts T cell function highlighting modulation of metabolism during activation is not only a novel approach to investigate the pivotal processes in the shift of T cell homeostasis after radiation, it also may lead to new targets for therapeutic manipulation in the combination of radiotherapy and immune therapy. Given that appreciable effects were observed with as low as 10 cGy, our results also have implications for low dose environmental exposures. PMID:26078715

  18. 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

  19. 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

  20. 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 ...

  1. Mitochondrial ROS fire up T cell activation.

    PubMed

    Murphy, Michael P; Siegel, Richard M

    2013-02-21

    Metabolic reprogramming has emerged as an important feature of immune cell activation. Two new studies, including Sena et al. (2013) in this issue of Immunity, identify mitochondrial reactive oxygen species (ROS) arising from metabolic reprogramming as signaling molecules in T cell activation. PMID:23438817

  2. Effect of substrate mechanical properties on T cell activation

    NASA Astrophysics Data System (ADS)

    Hui, King; Upadhyaya, Arpita

    2013-03-01

    T cell activation is a key process in cell-mediated immunity, and engagement of T cell receptors by peptides on antigen presenting cells leads to activation of signaling cascades as well as cytoskeletal reorganization and large scale membrane deformations. While significant advances have been made in understanding the biochemical signaling pathways, the effects imposed by the physical environment and the role of mechanical forces on cell activation are not well understood. In this study, we have used anti-CD3 coated elastic polyacrylamide gels as stimulatory substrates to enable the spreading of Jurkat T cells and the measurement of cellular traction forces. We have investigated the effect of substrate stiffness on the dynamics of T cell spreading and cellular force generation. We found that T cells display more active and sustained edge dynamics on softer gels and that they exert increased traction stresses with increasing gel stiffness. A dynamic actin cytoskeleton was required to maintain the forces generated during activation, as inferred from small molecule inhibition experiments. Our results indicate an important role for physical properties of the antigen presenting cell as well as cytoskeleton-driven forces in signaling activation.

  3. 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.

  4. Phytocomponent 4-hydroxy-3-methoxycinnamaldehyde ablates T-cell activation by targeting protein kinase C-θ and its downstream pathways.

    PubMed

    Akber, Uroos; Na, Bo-Ra; Ko, Yoo-Seung; Ko, You-Seung; Lee, Hyun-Su; Kim, Hye-Ran; Kwon, Min-Sung; Park, Zee-Yong; Choi, Eun-Ju; Han, Weon-Cheol; Lee, Seung-Ho; Oh, Hyun-Mee; Jun, Chang-Duk

    2015-03-01

    Autoreactive T-cell responses have a crucial role in the pathology and clinical course of autoimmune diseases. Therefore, controlling the activation of these cells is an important strategy for developing therapies and therapeutics. Here, we identified that 4-hydroxy-3-methoxycinnamaldehyde (4H3MC) has a therapeutic potential for T-cell activation by modulating protein kinase C-θ (PKCθ) and its downstream pathways. Pre- and post-treatment with 4H3MC prevented IL-2 release from human transformed and untransformed T cells at the micromolar concentrations without any cytotoxic effects, in fact more efficiently than its structural analogue 4-hydroxycinnamic acid-a previously reported T-cell inhibitor. In silico analysis showed that 4H3MC is a potential inhibitor of PKC isotypes, including PKCθ-a crucial PKC isotype in T cells. Consistently, 4H3MC significantly blocked PKC activity in vitro and also inhibited the phosphorylation of PKCθ in T cells. 4H3MC had no effect on TCR-mediated membrane-proximal-signalling events such as phosphorylation of Zap70. Instead, it attenuated the phosphorylation of mitogen-activated protein kinases (ERK and p38) and promoter activities of NF-κB, AP-1 and NFAT. Taken together, our results provide the evidences that 4H3MC may have curative potential as a novel immune modulator in a broad range of immunopathological disorders by modulating PKCθ activity. PMID:25637768

  5. Low SAMHD1 expression following T-cell activation and proliferation renders CD4+ T cells susceptible to HIV-1

    PubMed Central

    Ruffin, Nicolas; Brezar, Vedran; Ayinde, Diana; Lefebvre, Cécile; Wiesch, Julian Schulze Zur; van Lunzen, Jan; Bockhorn, Maximilian; Schwartz, Olivier; Hocini, Hakim; Lelievre, Jean-Daniel; Banchereau, Jacques; Levy, Yves; Seddiki, Nabila

    2015-01-01

    Objectives: HIV-1 replication depends on the state of cell activation and division. It is established that SAMHD1 restricts HIV-1 infection of resting CD4+ T cells. The modulation of SAMHD1 expression during T-cell activation and proliferation, however, remains unclear, as well as a role for SAMHD1 during HIV-1 pathogenesis. Methods: SAMHD1 expression was assessed in CD4+ T cells after their activation and in-vitro HIV-1 infection. We performed phenotype analyzes using flow cytometry on CD4+ T cells from peripheral blood and lymph nodes from cohorts of HIV-1-infected individuals under antiretroviral treatment or not, and controls. Results: We show that SAMHD1 expression decreased during CD4+ T-cell proliferation in association with an increased susceptibility to in-vitro HIV-1 infection. Additionally, circulating memory CD4+ T cells are enriched in cells with low levels of SAMHD1. These SAMHD1low cells are highly differentiated, exhibit a large proportion of Ki67+ cycling cells and are enriched in T-helper 17 cells. Importantly, memory SAMHD1low cells were depleted from peripheral blood of HIV-infected individuals. We also found that follicular helper T cells present in secondary lymphoid organs lacked the expression of SAMHD1, which was accompanied by a higher susceptibility to HIV-1 infection in vitro. Conclusion: We demonstrate that SAMHD1 expression is decreased during CD4+ T-cell activation and proliferation. Also, CD4+ T-cell subsets known to be more susceptible to HIV-1 infection, for example, T-helper 17 and follicular helper T cells, display lower levels of SAMHD1. These results pin point a role for SAMHD1 expression in HIV-1 infection and the concomitant depletion of CD4+ T cells. PMID:25715102

  6. NFAT5 Contributes to Osmolality-Induced MCP-1 Expression in Mesothelial Cells

    PubMed Central

    Küper, Christoph; Beck, Franz-X.; Neuhofer, Wolfgang

    2012-01-01

    Increased expression of the C-C chemokine monocyte chemoattractant protein-1 (MCP-1) in mesothelial cells in response to high glucose concentrations and/or high osmolality plays a crucial role in the development of peritoneal fibrosis during continuous ambulatory peritoneal dialysis (CAPD). Recent studies suggest that in kidney cells osmolality-induced MCP-1 upregulation is mediated by the osmosensitive transcription factor, nuclear factor of activated T cells 5 (NFAT5). The present study addressed the question of whether activation of NFAT5 by hyperosmolality, as present in PD fluids, contributes to MCP-1 expression in the mesothelial cell line Met5A. Hyperosmolality, induced by addition of glucose, NaCl, or mannitol to the growth medium, increased NFAT5 activity and stimulated MCP-1 expression in Met5A cells. siRNA-mediated knockdown of NFAT5 attenuated osmolality-induced MCP-1 upregulation substantially. Hyperosmolality also induced activation of nuclear factor-κB (NF-κB). Accordingly, pharmacological inhibition of NF-κB significantly decreased osmolality-induced MCP-1 expression. Taken together, these results indicate that high osmolalities activate the transcription factor NFAT5 in mesothelial cells. NFAT5 in turn upregulates MCP-1, likely in combination with NF-κB, and thus may participate in the development of peritoneal fibrosis during CAPD. PMID:22619484

  7. 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

  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. Oxidized phospholipids stimulate tissue factor expression in human endothelial cells via activation of ERK/EGR-1 and Ca(++)/NFAT.

    PubMed

    Bochkov, Valery N; Mechtcheriakova, Diana; Lucerna, Marcus; Huber, Joakim; Malli, Roland; Graier, Wolfgang F; Hofer, Erhard; Binder, Bernd R; Leitinger, Norbert

    2002-01-01

    Activation of endothelial cells by lipid oxidation products is a key event in the initiation and progression of the atherosclerotic lesion. Minimally modified low-density lipoprotein (MM-LDL) induces the expression of certain inflammatory molecules such as tissue factor (TF) in endothelial cells. This study examined intracellular signaling pathways leading to TF up-regulation by oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a biologically active component of MM-LDL. OxPAPC induced TF activity and protein expression in human umbilical vein endothelial cells (HUVECs). However, OxPAPC neither induced phosphorylation or degradation of I kappa B alpha nor DNA binding of nuclear factor-kappa B (NF-kappa B). Furthermore, OxPAPC-induced TF expression was not inhibited by overexpression of I kappa B alpha. These results strongly indicate that OxPAPC-induced TF expression is independent of the classical NF-kappa B pathway. However, OxPAPC stimulated phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and expression of early growth response factor 1 (EGR-1). Inhibitors of mitogen-activated kinase/ERK (MEK) or protein kinase C (PKC) blocked elevation of both EGR-1 and TF. Furthermore, overexpression of NAB2, a corepressor of EGR-1, inhibited effects of OxPAPC. In addition, OxPAPC induced rapid and reversible elevation of free cytosolic Ca(++) levels and nuclear factor of activated T cells (NFAT)/DNA binding. Induction of TF expression by OxPAPC was partially inhibited by cyclosporin A, known to block calcineurin, a Ca(++)-dependent phosphatase upstream of NFAT. Treatment of OxPAPC with phospholipase A(2) destroyed its biologic activity and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine was identified as one biologically active component of OxPAPC that induces TF expression. Together, the results demonstrate that OxPAPC induces TF expression in HUVECs through activation of PKC/ERK/EGR-1 and Ca(++)/calcineurin/NFAT pathways

  10. 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

  11. 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

  12. Ligand Mobility Modulates Immunological Synapse Formation and T Cell Activation

    PubMed Central

    Hsu, Chih-Jung; Hsieh, Wan-Ting; Waldman, Abraham; Clarke, Fiona; Huseby, Eric S.; Burkhardt, Janis K.; Baumgart, Tobias

    2012-01-01

    T cell receptor (TCR) engagement induces clustering and recruitment to the plasma membrane of many signaling molecules, including the protein tyrosine kinase zeta-chain associated protein of 70 kDa (ZAP70) and the adaptor SH2 domain-containing leukocyte protein of 76 kDa (SLP76). This molecular rearrangement results in formation of the immunological synapse (IS), a dynamic protein array that modulates T cell activation. The current study investigates the effects of apparent long-range ligand mobility on T cell signaling activity and IS formation. We formed stimulatory lipid bilayers on glass surfaces from binary lipid mixtures with varied composition, and characterized these surfaces with respect to diffusion coefficient and fluid connectivity. Stimulatory ligands coupled to these surfaces with similar density and orientation showed differences in their ability to activate T cells. On less mobile membranes, central supramolecular activation cluster (cSMAC) formation was delayed and the overall accumulation of CD3ζ at the IS was reduced. Analysis of signaling microcluster (MC) dynamics showed that ZAP70 MCs exhibited faster track velocity and longer trajectories as a function of increased ligand mobility, whereas movement of SLP76 MCs was relatively insensitive to this parameter. Actin retrograde flow was observed on all surfaces, but cell spreading and subsequent cytoskeletal contraction were more pronounced on mobile membranes. Finally, increased tyrosine phosphorylation and persistent elevation of intracellular Ca2+ were observed in cells stimulated on fluid membranes. These results point to ligand mobility as an important parameter in modulating T cell responses. PMID:22384241

  13. Vav-2 controls NFAT-dependent transcription inB- but not T-lymphocytes

    PubMed Central

    Doody, Gina M.; Billadeau, Daniel D.; Clayton, Elizabeth; Hutchings, Amanda; Berland, Robert; McAdam, Simon; Leibson, Paul J.; Turner, Martin

    2000-01-01

    We show here that Vav-2 is tyrosine phosphorylated following antigen receptor engagement in both B- and T-cells, but potentiates nuclear factor of activated T cells (NFAT)-dependent transcription only in B cells. Vav-2 function requires the N-terminus, as well as functional Dbl homology and SH2 domains. More over, the enhancement of NFAT-dependent transcription by Vav-2 can be inhibited by a number of dominant-negative GTPases. The ability of Vav-2 to potentiate NFAT-dependent transcription correlates with its ability to promote a sustained calcium flux. Thus, Vav-2 augments the calcium signal in B cells but not T cells, and a truncated form of Vav-2 can neither activate NFAT nor augment calcium signaling. The CD19 co-receptor physically interacts with Vav-2 and synergistically enhances Vav-2 phosphorylation induced by the B-cell receptor (BCR). In addition, we found that Vav-2 augments CD19-stimulated NFAT- dependent transcription, as well as transcription from the CD5 enhancer. These data suggest a role for Vav-2 in transducing BCR signals to the transcription factor NFAT and implicate Vav-2 in the integration of BCR and CD19 signaling. PMID:11080163

  14. Functional Anatomy of T Cell Activation and Synapse Formation

    PubMed Central

    Fooksman, David R.; Vardhana, Santosh; Vasiliver-Shamis, Gaia; Liese, Jan; Blair, David; Waite, Janelle; Sacristán, Catarina; Victora, Gabriel; Zanin-Zhorov, Alexandra; Dustin, Michael L.

    2010-01-01

    T cell activation and function require a structured engagement of antigen-presenting cells. These cell contacts are characterized by two distinct dynamics in vivo: transient contacts resulting from promigratory junctions called immunological kinapses or prolonged contacts from stable junctions called immunological synapses. Kinapses operate in the steady state to allow referencing to self-peptide-MHC (pMHC) and searching for pathogen-derived pMHC. Synapses are induced by T cell receptor (TCR) interactions with agonist pMHC under specific conditions and correlate with robust immune responses that generate effector and memory T cells. High-resolution imaging has revealed that the synapse is highly coordinated, integrating cell adhesion, TCR recognition of pMHC complexes, and an array of activating and inhibitory ligands to promote or prevent T cell signaling. In this review, we examine the molecular components, geometry, and timing underlying kinapses and synapses. We integrate recent molecular and physiological data to provide a synthesis and suggest ways forward. PMID:19968559

  15. Key gravity-sensitive signaling pathways drive T cell activation.

    PubMed

    Boonyaratanakornkit, J B; Cogoli, A; Li, C-F; Schopper, T; Pippia, P; Galleri, G; Meloni, M A; Hughes-Fulford, M

    2005-12-01

    Returning astronauts have experienced altered immune function and increased vulnerability to infection during spaceflights dating back to Apollo and Skylab. Lack of immune response in microgravity occurs at the cellular level. We analyzed differential gene expression to find gravity-dependent genes and pathways. We found inhibited induction of 91 genes in the simulated freefall environment of the random positioning machine. Altered induction of 10 genes regulated by key signaling pathways was verified using real-time RT-PCR. We discovered that impaired induction of early genes regulated primarily by transcription factors NF-kappaB, CREB, ELK, AP-1, and STAT after crosslinking the T-cell receptor contributes to T-cell dysfunction in altered gravity environments. We have previously shown that PKA and PKC are key early regulators in T-cell activation. Since the majority of the genes were regulated by NF-kappaB, CREB, and AP-1, we studied the pathways that regulated these transcription factors. We found that the PKA pathway was down-regulated in vg. In contrast, PI3-K, PKC, and its upstream regulator pLAT were not significantly down-regulated by vectorless gravity. Since NF-kappaB, AP-1, and CREB are all regulated by PKA and are transcription factors predicted by microarray analysis to be involved in the altered gene expression in vectorless gravity, the data suggest that PKA is a key player in the loss of T-cell activation in altered gravity. PMID:16210397

  16. 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...

  17. Early T cell activation: integrating biochemical, structural, and biophysical cues.

    PubMed

    Malissen, Bernard; Bongrand, Pierre

    2015-01-01

    T cells carry out the formidable task of identifying small numbers of foreign antigenic peptides rapidly and specifically against a very noisy environmental background of endogenous self-peptides. Early steps in T cell activation have thus fascinated biologists and are among the best-studied models of cell stimulation. This remarkable process, critical in adaptive immune responses, approaches and even seems to exceed the limitations set by the physical laws ruling molecular behavior. Despite the enormous amount of information concerning the nature of molecules involved in the T cell antigen receptor (TCR) signal transduction network, and the description of the nanoscale organization and real-time analysis of T cell responses, the general principles of information gathering and processing remain incompletely understood. Here we review currently accepted key data on TCR function, discuss the limitations of current research strategies, and suggest a novel model of TCR triggering and a few promising ways of going further into the integration of available data. PMID:25861978

  18. 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

  19. T-Cell Immunophenotyping Distinguishes Active From Latent Tuberculosis

    PubMed Central

    Pollock, Katrina M.; Whitworth, Hilary S.; Montamat-Sicotte, Damien J.; Grass, Lisa; Cooke, Graham S.; Kapembwa, Moses S.; Kon, Onn M.; Sampson, Robert D.; Taylor, Graham P.; Lalvani, Ajit

    2013-01-01

    Background. Changes in the phenotype and function of Mycobacterium tuberculosis (M. tuberculosis)-specific CD4+ and CD8+ T-cell subsets in response to stage of infection may allow discrimination between active tuberculosis and latent tuberculosis infection. Methods. A prospective comparison of M. tuberculosis-specific cellular immunity in subjects with active tuberculosis and latent tuberculosis infection, with and without human immunodeficiency virus (HIV) coinfection. Polychromatic flow cytometry was used to measure CD4+ and CD8+ T-cell subset phenotype and secretion of interferon γ (IFN-γ), interleukin 2 (IL-2), and tumor necrosis factor α (TNF-α). Results. Frequencies of CD4+ and CD8+ cells secreting IFN-γ-only, TNF-α-only and dual IFN-γ/TNF-α were greater in active tuberculosis vs latent tuberculosis infection. All M. tuberculosis-specific CD4+ subsets, with the exception of IL-2-only cells, switched from central to effector memory phenotype in active tuberculosis vs latent tuberculosis infection, accompanied by a reduction in IL-7 receptor α (CD127) expression. The frequency of PPD-specific CD4+ TNF-α-only-secreting T cells with an effector phenotype accurately distinguished active tuberculosis from latent tuberculosis infection with an area under the curve of 0.99, substantially more discriminatory than measurement of function alone. Conclusions. Combined measurement of T-cell phenotype and function defines a highly discriminatory biomarker of tuberculosis disease activity. Unlocking the diagnostic and monitoring potential of this combined approach now requires validation in large-scale prospective studies. PMID:23966657

  20. 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.

  1. 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

  2. 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

  3. NFAT1 and NFAT3 Cooperate with HDAC4 during Regulation of Alternative Splicing of PMCA Isoforms in PC12 Cells

    PubMed Central

    Kosiorek, Michalina; Podszywalow-Bartnicka, Paulina; Zylinska, Ludmila; Pikula, Slawomir

    2014-01-01

    Background The bulk of human genes undergo alternative splicing (AS) upon response to physiological stimuli. AS is a great source of protein diversity and biological processes and is associated with the development of many diseases. Pheochromocytoma is a neuroendocrine tumor, characterized by an excessive Ca2+-dependent secretion of catecholamines. This underlines the importance of balanced control of calcium transport via regulation of gene expression pattern, including different calcium transport systems, such as plasma membrane Ca2+-ATPases (PMCAs), abundantly expressed in pheochromocytoma chromaffin cells (PC12 cells). PMCAs are encoded by four genes (Atp2b1, Atp2b2, Atp2b3, Atp2b4), whose transcript products undergo alternative splicing giving almost 30 variants. Results In this scientific report, we propose a novel mechanism of regulation of PMCA alternative splicing in PC12 cells through cooperation of the nuclear factor of activated T-cells (NFAT) and histone deacetylases (HDACs). Luciferase assays showed increased activity of NFAT in PC12 cells, which was associated with altered expression of PMCA. RT-PCR experiments suggested that inhibition of the transcriptional activity of NFAT might result in the rearrangement of PMCA splicing variants in PC12 cells. NFAT inhibition led to dominant expression of 2x/c, 3x/a and 4x/a PMCA variants, while in untreated cells the 2w,z/b, 3z,x/b,c,e,f, and 4x/b variants were found as well. Furthermore, chromatin immunoprecipitation experiments showed that NFAT1-HDAC4 or NFAT3-HDAC4 complexes might be involved in regulation of PMCA2x splicing variant generation. Conclusions We suggest that the influence of NFAT/HDAC on PMCA isoform composition might be important for altered dopamine secretion by PC12 cells. PMID:24905014

  4. The Effects of TLR Activation on T-Cell Development and Differentiation

    PubMed Central

    Jin, Bo; Sun, Tao; Yu, Xiao-Hong; Yang, Ying-Xiang; Yeo, Anthony E. T.

    2012-01-01

    Invading pathogens have unique molecular signatures that are recognized by Toll-like receptors (TLRs) resulting in either activation of antigen-presenting cells (APCs) and/or costimulation of T cells inducing both innate and adaptive immunity. TLRs are also involved in T-cell development and can reprogram Treg cells to become helper cells. T cells consist of various subsets, that is, Th1, Th2, Th17, T follicular helper (Tfh), cytotoxic T lymphocytes (CTLs), regulatory T cells (Treg) and these originate from thymic progenitor thymocytes. T-cell receptor (TCR) activation in distinct T-cell subsets with different TLRs results in differing outcomes, for example, activation of TLR4 expressed in T cells promotes suppressive function of regulatory T cells (Treg), while activation of TLR6 expressed in T cells abrogates Treg function. The current state of knowledge of regarding TLR-mediated T-cell development and differentiation is reviewed. PMID:22737174

  5. 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

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

    PubMed Central

    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

  7. 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

  8. Chemically programmed bispecific antibodies that recruit and activate T cells.

    PubMed

    Cui, Huiting; Thomas, Joshua D; Burke, Terrence R; Rader, Christoph

    2012-08-17

    Bispecific antibodies (biAbs) that mediate cytotoxicity by recruiting and activating endogenous immune cells are an emerging class of next-generation antibody therapeutics. Of particular interest are biAbs of relatively small size (∼50 kDa) that can redirect cytotoxic T cells through simultaneous binding of tumor cells. Here we describe a conceptually unique class of biAbs in which the tumor cell specificity of a humanized antibody fragment that recognizes CD3 on T cells is chemically programmed through a C-terminal selenocysteine (Sec) residue. We demonstrate that through chemically programmed specificity for integrin α(4)β(1) or folate receptor 1 (FOLR1), and common specificity for CD3, these hybrid molecules exert potent and specific in vitro and ex vivo cytotoxicity toward tumor cell lines and primary tumor cells in the presence of primary T cells. Importantly, the generic nature of chemical programming allows one to apply our approach to virtually any specificity, promising a broad utility of chemically programmed biAbs in cancer therapy. PMID:22761439

  9. Stochasticity and spatial heterogeneity in T-cell activation.

    PubMed

    Burroughs, Nigel J; van der Merwe, P Anton

    2007-04-01

    Stochastic and spatial aspects are becoming increasingly recognized as an important factor in T-cell activation. Activation occurs in an intrinsically noisy environment, requiring only a handful of agonist peptide-major histocompatibility complex molecules, thus making consideration of signal to noise of prime importance in understanding sensitivity and specificity. Furthermore, it is widely established that surface-bound ligands are more effective at activation than soluble forms, while surface patternation has highlighted the role of spatial relocation in activation. Here we consider the results of a number of models of T-cell activation, from a realistic model of kinetic segregation-induced T-cell receptor (TCR) triggering through to simple queuing theory models. These studies highlight the constraints on cell activation by a surface receptor that recruits kinases. Our analysis shows that TCR triggering based on trapping of bound TCRs in regions of close proximity that exclude large ectodomain-containing molecules, such as the phosphatases CD45 and CD148, can effectively reproduce known signaling characteristics and is a viable 'signal transduction' mechanism distinct from oligomerization and conformation-based mechanisms. A queuing theory analysis shows the interrelation between sensitivity and specificity, emphasizing that these are properties of individual cell functions and need not be, nor are likely to be, uniform across different functions. In fact, threshold-based mechanisms of detection are shown to be poor at ligand discrimination because, although they can be highly specific, that specificity is limited to a small range of peptide densities. Time integration mechanisms however are able to control noise effectively, while kinetic proofreading mechanisms endow them with good specificity properties. Thus, threshold mechanisms are likely to be important for rapidly detecting minimal signaling requirements, thus achieving efficient scanning of antigen

  10. Suplatast tosilate alleviates nasal symptoms through the suppression of nuclear factor of activated T-cells-mediated IL-9 gene expression in toluene-2,4-diisocyanate-sensitized rats.

    PubMed

    Mizuguchi, Hiroyuki; Orimoto, Naoki; Kadota, Takuya; Kominami, Takahiro; Das, Asish K; Sawada, Akiho; Tamada, Misaki; Miyagi, Kohei; Adachi, Tsubasa; Matsumoto, Mayumi; Kosaka, Tomoya; Kitamura, Yoshiaki; Takeda, Noriaki; Fukui, Hiroyuki

    2016-03-01

    Histamine H1 receptor (H1R) gene is upregulated in patients with pollinosis; its expression level is highly correlated with the nasal symptom severity. Antihistamines are widely used as allergy treatments because they inhibit histamine signaling by blocking H1R or suppressing H1R signaling as inverse agonists. However, long-term treatment with antihistamines does not completely resolve toluene-2,4-diisocyanate (TDI)-induced nasal symptoms, although it can decrease H1R gene expression to the basal level, suggesting additional signaling is responsible for the pathogenesis of the allergic symptoms. Here, we show that treatment with suplatast tosilate in combination with antihistamines markedly alleviates nasal symptoms in TDI-sensitized rats. Suplatast suppressed TDI-induced upregulation of IL-9 gene expression. Suplatast also suppressed ionomycin/phorbol-12-myristate-13-acetate-induced upregulation of IL-2 gene expression in Jurkat cells, in which calcineurin (CN)/nuclear factor of activated T-cells (NFAT) signaling is known to be involved. Immunoblot analysis demonstrated that suplatast inhibited binding of NFAT to DNA. Furthermore, suplatast suppressed ionomycin-induced IL-9 mRNA upregulation in RBL-2H3 cells, in which CN/NFAT signaling is also involved. These data suggest that suplatast suppressed NFAT-mediated IL-9 gene expression in TDI-sensitized rats and this might be the underlying mechanism of the therapeutic effects of combined therapy of suplatast with antihistamine. PMID:26874672

  11. Mast cells enhance T cell activation: Importance of mast cell-derived TNF

    NASA Astrophysics Data System (ADS)

    Nakae, Susumu; Suto, Hajime; Kakurai, Maki; Sedgwick, Jonathon D.; Tsai, Mindy; Galli, Stephen J.

    2005-05-01

    Mast cells are not only important effector cells in immediate hypersensitivity reactions and immune responses to pathogens but also can contribute to T cell-mediated disorders. However, the mechanisms by which mast cells might influence T cells in such settings are not fully understood. We find that mast cells can enhance proliferation and cytokine production in multiple T cell subsets. Mast cell-dependent enhancement of T cell activation can be promoted by FcRI-dependent mast cell activation, TNF production by both mast cells and T cells, and mast cell-T cell contact. However, at high concentrations of cells, mast cells can promote T cell activation independent of IgE or TNF. Finally, mast cells also can promote T cell activation by means of soluble factors. These findings identify multiple mechanisms by which mast cells can influence T cell proliferation and cytokine production. allergy | asthma | autoimmunity | cytokines | immune response

  12. 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

  13. Nod2 Activates NF-kB in CD4+ T Cells but Its Expression Is Dispensable for T Cell-Induced Colitis

    PubMed Central

    Zanello, Galliano; Goethel, Ashleigh; Forster, Katharina; Geddes, Kaoru; Philpott, Dana J.; Croitoru, Kenneth

    2013-01-01

    Although the etiology of Crohn's disease (CD) remains elusive this disease is characterized by T cell activation that leads to chronic inflammation and mucosal damage. A potential role for maladaptation between the intestinal microbiota and the mucosal immune response is suggested by the fact that mutations in the pattern recognition receptor Nod2 are associated with higher risks for developing CD. Although Nod2 deletion in CD4+ T cells has been shown to impair the induction of colitis in the murine T cell transfer model, the analysis of T cell intrinsic Nod2 function in T cell differentiation and T cell-mediated immunity is inconsistent between several studies. In addition, the role of T cell intrinsic Nod2 in regulatory T cell (Treg) development and function during colitis remain to be analyzed. In this study, we show that Nod2 expression is higher in activated/memory CD4+ T cells and its expression was inducible after T cell receptor (TCR) ligation. Nod2 stimulation with muramyl dipeptide (MDP) led to a nuclear accumulation of c-Rel NF-kB subunit. Although functionally active in CD4+ T cells, the deletion of Nod2 did not impair the induction and the prevention of colitis in the T cell transfer model. Moreover, Nod2 deletion did not affect the development of Foxp3+ Treg cells in the spleen of recipient mice and Nod2 deficient CD4 T cells expressing the OVA specific transgenic TCR were able to differentiate in Foxp3+ Treg cells after OVA feeding. In vitro, CD25+ Nod2 deficient T cells suppressed T cell proliferation as well as wild type counter parts and T cell stimulation with MDP did not affect the proliferation and the cytokine secretion of T cells. In conclusion, our data indicate that Nod2 is functional in murine CD4+ T cells but its expression is dispensable for the T cell regulation of colitis. PMID:24324812

  14. 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

  15. CD4+ and CD8+ T Cell Activation Are Associated with HIV DNA in Resting CD4+ T Cells

    PubMed Central

    Cockerham, Leslie R.; Siliciano, Janet D.; Sinclair, Elizabeth; O'Doherty, Una; Palmer, Sarah; Yukl, Steven A.; Strain, Matt C.; Chomont, Nicolas; Hecht, Frederick M.; Siliciano, Robert F.; Richman, Douglas D.; Deeks, Steven G.

    2014-01-01

    The association between the host immune environment and the size of the HIV reservoir during effective antiretroviral therapy is not clear. Progress has also been limited by the lack of a well-accepted assay for quantifying HIV during therapy. We examined the association between multiple measurements of HIV and T cell activation (as defined by markers including CD38, HLA-DR, CCR5 and PD-1) in 30 antiretroviral-treated HIV-infected adults. We found a consistent association between the frequency of CD4+ and CD8+ T cells expressing HLA-DR and the frequency of resting CD4+ T cells containing HIV DNA. This study highlights the need to further examine this relationship and to better characterize the biology of markers commonly used in HIV studies. These results may also have implications for reactivation strategies. PMID:25340755

  16. Soluble peptide-MHC monomers cause activation of CD8+ T cells through transfer of the peptide to T cell MHC molecules

    NASA Astrophysics Data System (ADS)

    Ge, Qing; Stone, Jennifer D.; Thompson, M. Todd; Cochran, Jennifer R.; Rushe, Mia; Eisen, Herman N.; Chen, Jianzhu; Stern, Lawrence J.

    2002-10-01

    T cell receptor (TCR)-mediated activation of CD4+ T cells is known to require multivalent engagement of the TCR by, for example, oligomeric peptide-MHC complexes. In contrast, for CD8+ T cells, there is evidence for TCR-mediated activation by univalent engagement of the TCR. We have here compared oligomeric and monomeric Ld and Kb peptide-MHC complexes and free peptide as stimulators of CD8+ T cells expressing the 2C TCR. We found that the monomers are indeed effective in activating naïve and effector CD8+ T cells, but through an unexpected mechanism that involves transfer of peptide from soluble monomers to T cell endogenous MHC (Kb) molecules. The result is that T cells, acting as antigen-presenting cells, are able to activate other naïve T cells.

  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. TCR-induced sumoylation of the kinase PKC-θ controls T cell synapse organization and T cell activation.

    PubMed

    Wang, Xu-Dong; Gong, Yu; Chen, Zhi-Long; Gong, Bei-Ni; Xie, Ji-Ji; Zhong, Chuan-Qi; Wang, Qi-Long; Diao, Liang-Hui; Xu, Anlong; Han, Jiahuai; Altman, Amnon; Li, Yingqiu

    2015-11-01

    Sumoylation regulates many cellular processes, but its role in signaling via the T cell antigen receptor (TCR) remains unknown. We found that the kinase PKC-θ was sumoylated upon costimulation with antigen or via the TCR plus the coreceptor CD28, with Lys325 and Lys506 being the main sumoylation sites. We identified the SUMO E3 ligase PIASxβ as a ligase for PKC-θ. Analysis of primary mouse and human T cells revealed that sumoylation of PKC-θ was essential for T cell activation. Desumoylation did not affect the catalytic activity of PKC-θ but inhibited the association of CD28 with PKC-θ and filamin A and impaired the assembly of a mature immunological synapse and central co-accumulation of PKC-θ and CD28. Our findings demonstrate that sumoylation controls TCR-proximal signaling and that sumoylation of PKC-θ is essential for the formation of a mature immunological synapse and T cell activation. PMID:26390157

  19. NFATc1 deficiency in T cells protects mice from experimental autoimmune encephalomyelitis.

    PubMed

    Reppert, Sarah; Zinser, Elisabeth; Holzinger, Corinna; Sandrock, Lena; Koch, Sonja; Finotto, Susetta

    2015-05-01

    NFATc1 is a member of the nuclear factor of activated T cells (NFAT) family of transcription factors. NFAT is activated upon T-cell receptor activation followed by intracytoplasmatic calcium influx where calmodulin, a calcium sensor protein, activates the phosphatase calcineurin that dephosphorylates NFAT proteins and results in NFAT nuclear import. Here, we show the analysis of conditional NFATc1-deficient mice bearing a deletion of NFATc1 in CD4(+) and CD8(+) T cells. NFATc1-deficient CD4(+) T cells polarized under Th17 conditions express reduced levels of the Th17-associated transcription factor RORγT (where ROR is RAR-related orphan receptor) as well as the Th17-associated cytokines IL-17A, IL-17F, IL-21, and IL-10. In the murine model of experimental EAE, we found a strong reduction of the disease outcome in conditional NFATc1-deficient mice, as compared with control littermates. This was accompanied by a diminished inflammation in the brain and spinal cord and reduced IL-17A and IFN-γ expression by antigen-specific spleen, spinal cord, and brain cells. Altogether, these results reveal an important role of NFATc1 in inducing Th17-cell responses and IFN-γ, both being relevant for the EAE development. PMID:25689841

  20. Profiles of activation, differentiation-markers, or β-integrins on T cells contribute to predict T cells' antileukemic responses after stimulation with leukemia-derived dendritic cells.

    PubMed

    Vogt, Valentin; Schick, Julia; Ansprenger, Christian; Braeu, Marion; Kroell, Tanja; Kraemer, Doris; Köhne, Claus-Henning; Hausmann, Andreas; Buhmann, Raymund; Tischer, Johanna; Schmetzer, Helga

    2014-01-01

    Stem cell transplantations and donor lymphocyte infusions are promising immunotherapies to cure acute myeloid leukemia (AML). Leukemia-derived dendritic cells are known to improve antileukemic functionality of T cells. We evaluated the composition and development of distinct T-cell subtypes in AML patients (n=12) compared with healthy probands (n=5) before and during stimulation with leukemia-derived dendritic cells-containing DC (DC) or blast-containing mononuclear cells (MNC) in 0-7 days mixed lymphocyte cultures (MLC) by flow cytometry. AML patients' T-cell subgroups were correlated with antileukemic functionality before and after DC/MNC stimulation by functional fluorolysis assays. (1) Unstimulated T cells from AML patients presented with significantly lower proportions of activated, Tcm, CD137, and β-integrin T cells, and significantly higher proportions of Tnaive and Teff compared with healthy probands. (2) After 7 days of DC or MNC stimulation, T-cell profiles were characterized by (significantly) increased proportions of activated T cells with effector function and significantly decreased proportions of β-integrin T cells. (3) Antileukemic cytotoxicity was achieved in 40% of T cells after MNC stimulation compared with 64% after DC stimulation. Antileukemic activity after DC stimulation but not after MNC stimulation correlated with higher proportions of Tcm and Tnaive before stimulation, as well as with significantly higher proportions of activated and β-integrin T cells. Furthermore, cutoff values for defined T-cell activation/differentiation markers and β-integrin T cells could be defined, allowing a prediction of antileukemic reactivity. We could demonstrate the potential of the composition of unstimulated/DC-stimulated T cells for the lysis of AML blasts. Especially, AML patients with high numbers of Tnaive and Tcm could benefit from DC stimulation; proportions of activated and β-integrin T cells correlated with increased antileukemic functionality

  1. 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

  2. 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

  3. 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

  4. 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. PMID:27035718

  5. 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

  6. Expression of inhibitory receptors on intratumoral T cells modulates the activity of a T cell-bispecific antibody targeting folate receptor

    PubMed Central

    Schreiner, Jens; Thommen, Daniela S.; Herzig, Petra; Bacac, Marina; Klein, Christian; Roller, Andreas; Belousov, Anton; Levitsky, Victor; Savic, Spasenija; Moersig, Wolfgang; Uhlenbrock, Franziska; Heinzelmann-Schwarz, Viola A.; Umana, Pablo; Pisa, Pavel; Lardinois, Didier; Müller, Philipp; Karanikas, Vaios; Zippelius, Alfred

    2016-01-01

    ABSTRACT T-cell bispecific antibodies (TCBs) are a novel therapeutic tool designed to selectively recruit T-cells to tumor cells and simultaneously activate them. However, it is currently unknown whether the dysfunctional state of T-cells, embedded into the tumor microenvironment, imprints on the therapeutic activity of TCBs. We performed a comprehensive analysis of activation and effector functions of tumor-infiltrating T-cells (TILs) in different tumor types, upon stimulation by a TCB targeting folate receptor 1 and CD3 (FolR1-TCB). We observed a considerable heterogeneity in T-cell activation, cytokine production and tumor cell killing upon exposure to FolR1-TCB among different FolR1-expressing tumors. Of note, tumors presenting with a high frequency of PD-1hi TILs displayed significantly impaired tumor cell killing and T-cell function. Further characterization of additional T-cell inhibitory receptors revealed that PD-1hi TILs defined a T-cell subset with particularly high levels of multiple inhibitory receptors compared with PD-1int and PD-1neg T-cells. PD-1 blockade could restore cytokine secretion but not cytotoxicity of TILs in a subset of patients with scarce PD-1hi expressing cells; in contrast, patients with abundance of PD-1hi expressing T-cells did not benefit from PD-1 blockade. Our data highlight that FolR1-TCB is a promising novel immunotherapeutic treatment option which is capable of activating intratumoral T-cells in different carcinomas. However, its therapeutic efficacy may be substantially hampered by a pre-existing dysfunctional state of T-cells, reflected by abundance of intratumoral PD-1hi T-cells. These findings present a rationale for combinatorial approaches of TCBs with other therapeutic strategies targeting T-cell dysfunction. PMID:27057429

  7. 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

  8. 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

  9. Tim-1-Mediated T Cell Activation Requires Recruitment and Activation of PI 3-Kinase

    PubMed Central

    de Souza, Anjali J.; Oak, Jean S.; Jordanhazy, Ryan; DeKruyff, Rosemarie H.; Fruman, David A.; Kane, Lawrence P.

    2009-01-01

    Ligation of the transmembrane protein Tim-1 can co-stimulate T cell activation. Agonistic antibodies to Tim-1 are also capable of inducing T cell activation without additional stimuli. However, little is known about the biochemical mechanisms underlying T cell stimulation or co-stimulation through Tim-1. We show that a tyrosine in Tim-1 becomes phosphorylated in an lck-dependent manner, whereupon it can directly recruit p85 adaptor subunits of PI 3-kinase. This results in PI3K activation, which is required for Tim-1 function. We also provide genetic evidence that p85 expression is required for optimal Tim-1 function. Thus, we describe a pathway from Tim-1 tyrosine phosphorylation to the PI3K signaling pathway, which appears to be a major effector of Tim-1-mediated T cell activation. PMID:18453570

  10. 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. PMID:26522449

  11. Distinct Mechanisms Regulate Lck Spatial Organization in Activated T Cells

    PubMed Central

    Kapoor-Kaushik, Natasha; Hinde, Elizabeth; Compeer, Ewoud B.; Yamamoto, Yui; Kraus, Felix; Yang, Zhengmin; Lou, Jieqiong; Pageon, Sophie V.; Tabarin, Thibault; Gaus, Katharina; Rossy, Jérémie

    2016-01-01

    Phosphorylation of the T cell receptor (TCR) by the kinase Lck is the first detectable signaling event upon antigen engagement. The distribution of Lck within the plasma membrane, its conformational state, kinase activity, and protein–protein interactions all contribute to determine how efficiently Lck phosphorylates the engaged TCR. Here, we used cross-correlation raster image correlation spectroscopy and photoactivated localization microscopy to identify two mechanisms of Lck clustering: an intrinsic mechanism of Lck clustering induced by locking Lck in its open conformation and an extrinsic mechanism of clustering controlled by the phosphorylation of tyrosine 192, which regulates the affinity of Lck SH2 domain. Both mechanisms of clustering were differently affected by the absence of the kinase Zap70 or the adaptor Lat. We further observed that the adaptor TSAd bound to and promoted the diffusion of Lck when it is phosphorylated on tyrosine 192. Our data suggest that while Lck open conformation drives aggregation and clustering, the spatial organization of Lck is further controlled by signaling events downstream of TCR phosphorylation. PMID:27014263

  12. T cell stimulator cells, an efficient and versatile cellular system to assess the role of costimulatory ligands in the activation of human T cells

    PubMed Central

    Leitner, Judith; Kuschei, Werner; Grabmeier-Pfistershammer, Katharina; Woitek, Ramona; Kriehuber, Ernst; Majdic, Otto; Zlabinger, Gerhard; Pickl, Winfried F.; Steinberger, Peter

    2010-01-01

    It is well established that full activation of T cells requires the interaction of the TCR complex with the peptide–MHC complex (Signal 1) and additional signals (Signal 2). These second signals are generated by the interaction of costimulatory ligands expressed on antigen presenting cells with activating receptors on T cells. In addition, T cell responses are negatively regulated by inhibitory costimulatory pathways. Since professional antigen presenting cells (APC) harbour a plethora of stimulating and inhibitory surface molecules, the contribution of individual costimulatory molecules is difficult to assess on these cells. We have developed a system of stimulator cells that can give signal 1 to human T cells via a membrane bound anti-CD3 antibody fragment. By expressing human costimulatory ligands on these cells, their role in T cell activation processes can readily be analyzed. We demonstrate that T cell stimulator cells are excellent tools to study various aspects of human T cell costimulation, including the effects of immunomodulatory drugs or how costimulatory signals contribute to the in vitro expansion of T cells. T cell stimulator cells are especially suited for the functional evaluation of ligands that are implicated in costimulatory processes. In this study we have evaluated the role of the CD2 family member CD150 (SLAM) and the TNF family member TL1A (TNFSF15) in the activation of human T cells. Whereas our results do not point to a significant role of CD150 in T cell activation we found TL1A to potently costimulate human T cells. Taken together our results demonstrate that T cell stimulator cells are excellent tools to study various aspects of costimulatory processes. PMID:20858499

  13. Activated rat T cells synthesize and express functional major histocompatibility class II antigens.

    PubMed Central

    Broeren, C P; Wauben, M H; Lucassen, M A; Van Meurs, M; Van Kooten, P J; Boog, C J; Claassen, E; Van Eden, W

    1995-01-01

    In the present report, we studied the presence and functional significance of major histocompatibility complex (MHC) class II antigen on rat T cells. Most rat T-cell lines cultured in vitro were found to be MHC class II+. Also, these T-cell lines were shown to synthesize MHC class II molecules. Immunohistochemical and flow cytometric double stainings for T-cell receptor (TCR) and MHC class II showed that in vivo as well a large proportion of T cells was MHC class II+. The immunohistochemical staining of spleen sections enabled us to characterize the MHC class II+ and MHC class II- T cells. It was shown that resting T cells in vivo were MHC class II-. In contrast, activated T cells, as determined by their localization in the marginal zone of the spleen, proved to be MHC class II+. Finally, T-cell clones were found to be able to present peptidic antigens, but could only poorly present more complex exogenous antigens, probably due to inefficient uptake of such antigens. These features would endow activated rat T cells with the capacity to present cell-specific self-proteins, such as TCR, to regulatory CD4+ MHC class II-restricted T cells, as was described by our group elsewhere. Images Figure 2 Figure 5 PMID:7750994

  14. 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

  15. Efficient T-cell priming and activation requires signaling through prostaglandin E2 (EP) receptors.

    PubMed

    Sreeramkumar, Vinatha; Hons, Miroslav; Punzón, Carmen; Stein, Jens V; Sancho, David; Fresno, Manuel; Cuesta, Natalia

    2016-01-01

    Understanding the regulation of T-cell responses during inflammation and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. In this regard, prostaglandin E2 (PGE2) is mostly considered a myeloid-derived immunosuppressive molecule. We describe for the first time that T cells secrete PGE2 during T-cell receptor stimulation. In addition, we show that autocrine PGE2 signaling through EP receptors is essential for optimal CD4(+) T-cell activation in vitro and in vivo, and for T helper 1 (Th1) and regulatory T cell differentiation. PGE2 was found to provide additive co-stimulatory signaling through AKT activation. Intravital multiphoton microscopy showed that triggering EP receptors in T cells is also essential for the stability of T cell-dendritic cell (DC) interactions and Th-cell accumulation in draining lymph nodes (LNs) during inflammation. We further demonstrated that blocking EP receptors in T cells during the initial phase of collagen-induced arthritis in mice resulted in a reduction of clinical arthritis. This could be attributable to defective T-cell activation, accompanied by a decline in activated and interferon-γ-producing CD4(+) Th1 cells in draining LNs. In conclusion, we prove that T lymphocytes secret picomolar concentrations of PGE2, which in turn provide additive co-stimulatory signaling, enabling T cells to attain a favorable activation threshold. PGE2 signaling in T cells is also required for maintaining long and stable interactions with DCs within LNs. Blockade of EP receptors in vivo impairs T-cell activation and development of T cell-mediated inflammatory responses. This may have implications in various pathophysiological settings. PMID:26051593

  16. 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

  17. Leucine Metabolism in T Cell Activation: mTOR Signaling and Beyond.

    PubMed

    Ananieva, Elitsa A; Powell, Jonathan D; Hutson, Susan M

    2016-07-01

    In connection with the increasing interest in metabolic regulation of the immune response, this review discusses current advances in understanding the role of leucine and leucine metabolism in T lymphocyte (T cell) activation. T cell activation during the development of an immune response depends on metabolic reprogramming to ensure that sufficient nutrients and energy are taken up by the highly proliferating T cells. Leucine has been described as an important essential amino acid and a nutrient signal that activates complex 1 of the mammalian target of rapamycin (mTORC1), which is a critical regulator of T cell proliferation, differentiation, and function. The role of leucine in these processes is further discussed in relation to amino acid transporters, leucine-degrading enzymes, and other metabolites of leucine metabolism. A new model of T cell regulation by leucine is proposed and outlines a chain of events that leads to the activation of mTORC1 in T cells. PMID:27422517

  18. Role of Accessory Proteins of HTLV-1 in Viral Replication, T Cell Activation, and Cellular Gene Expression

    PubMed Central

    Michael, Bindhu; Nair, Amithraj; Lairmore, Michael D.

    2010-01-01

    Human T-cell lymphotropic virus type 1 (HTLV-1), causes adult T cell leukemia/lymphoma (ATLL), and initiates a variety of immune mediated disorders. The viral genome encodes common structural and enzymatic proteins characteristic of all retroviruses and utilizes alternative splicing and alternate codon usage to make several regulatory and accessory proteins encoded in the pX region (pX ORF I to IV). Recent studies indicate that the accessory proteins p12I, p27I, p13II, and p30II, encoded by pX ORF I and II, contribute to viral replication and the ability of the virus to maintain typical in vivo expression levels. Proviral clones that are mutated in either pX ORF I or II, while fully competent in cell culture, are severely limited in their replicative capacity in a rabbit model. These HTLV-1 accessory proteins are critical for establishment of viral infectivity, enhance T- lymphocyte activation and potentially alter gene transcription and mitochondrial function. HTLV-1 pX ORF I expression is critical to the viral infectivity in resting primary lymphocytes suggesting a role for the calcineurin-binding protein p12I in lymphocyte activation. The endoplasmic reticulum and cis-Golgi localizing p12I activates NFAT, a key T cell transcription factor, through calcium-mediated signaling pathways and may lower the threshold of lymphocyte activation via the JAK/STAT pathway. In contrast p30II localizes to the nucleus and represses viral promoter activity, but may regulate cellular gene expression through p300/CBP or related co-activators of transcription. The mitochondrial localizing p13II induces morphologic changes in the organelle and may influence energy metabolism infected cells. Future studies of the molecular details HTLV-1 “accessory” proteins interactions will provide important new directions for investigations of HTLV-1 and related viruses associated with lymphoproliferative diseases. Thus, the accessory proteins of HTLV-1, once thought to be dispensable for

  19. 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

  20. CD40 Activation Rescues Antiviral CD8+ T Cells from PD-1-Mediated Exhaustion

    PubMed Central

    Isogawa, Masanori; Chung, Josan; Murata, Yasuhiro; Kakimi, Kazuhiro; Chisari, Francis V.

    2013-01-01

    The intrahepatic immune environment is normally biased towards tolerance. Nonetheless, effective antiviral immune responses can be induced against hepatotropic pathogens. To examine the immunological basis of this paradox we studied the ability of hepatocellularly expressed hepatitis B virus (HBV) to activate immunologically naïve HBV-specific CD8+ T cell receptor (TCR) transgenic T cells after adoptive transfer to HBV transgenic mice. Intrahepatic priming triggered vigorous in situ T cell proliferation but failed to induce interferon gamma production or cytolytic effector function. In contrast, the same T cells differentiated into cytolytic effector T cells in HBV transgenic mice if Programmed Death 1 (PD-1) expression was genetically ablated, suggesting that intrahepatic antigen presentation per se triggers negative regulatory signals that prevent the functional differentiation of naïve CD8+ T cells. Surprisingly, coadministration of an agonistic anti-CD40 antibody (αCD40) inhibited PD-1 induction and restored T cell effector function, thereby inhibiting viral gene expression and causing a necroinflammatory liver disease. Importantly, the depletion of myeloid dendritic cells (mDCs) strongly diminished the αCD40 mediated functional differentiation of HBV-specific CD8+ T cells, suggesting that activation of mDCs was responsible for the functional differentiation of HBV-specific CD8+ T cells in αCD40 treated animals. These results demonstrate that antigen-specific, PD-1-mediated CD8+ T cell exhaustion can be rescued by CD40-mediated mDC-activation. PMID:23853599

  1. 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

  2. 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

  3. Transcription regulates HIF-1α expression in CD4(+) T cells.

    PubMed

    Bollinger, Thomas; Bollinger, Annalena; Gies, Sydney; Feldhoff, Lea; Solbach, Werner; Rupp, Jan

    2016-01-01

    The transcription factor hypoxia inducible factor-1α (HIF-1α) mediates the metabolic adaptation of cells to hypoxia and T-helper cell fate. However, HIF-1α regulation in CD4(+) T cells (T cells) remains elusive. Here we observed that depletion of oxygen (O2⩽2%) alone was not sufficient to induce HIF-1α expression in T cells. However, when hypoxic T cells were stimulated, HIF-1α was expressed and this was dependent on nuclear factor-κB- and nuclear factor of activated T cell (NFAT)-mediated transcriptional upregulation of Hif-1α mRNA. HIF-1α upregulation could be blocked by drugs inhibiting NF-κB, NFAT or mammalian target of rapamycin precluding CD4(+) T-cell stimulation or translation in T cells, as well as by blocking transcription. CD3, CD28, phorbol-12-myristat-13-acetat (PMA) or ionomycin-stimulated T cells did not express HIF-1α under normoxic conditions. In conclusion, regulation of HIF-1α expression in CD4(+) T cells in hypoxia gravely relies on its transcriptional upregulation and subsequent enhanced protein stabilization. PMID:26150319

  4. 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

  5. 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

  6. Resistance of CD45RA- T cells to apoptosis and functional impairment, and activation of tumor-antigen specific T cells during radiation therapy of prostate cancer.

    PubMed

    Tabi, Zsuzsanna; Spary, Lisa K; Coleman, Sharon; Clayton, Aled; Mason, Malcolm D; Staffurth, John

    2010-07-15

    The effect of radiation therapy (RT) to the pelvis on circulating T cells was studied in prostate cancer (PCa) patients to provide a baseline for a more informed design of combination radioimmunotherapy. Peripheral blood samples taken from 12 PCa patients with locally advanced tumor before, during, and after hypofractionated RT were analyzed for T cell phenotype and function. There was significantly more loss of naive and early memory compared with more differentiated T cells during RT. The proportions of annexin-V(+) and Fas-expressing T cells were elevated in patients during RT and in PBMC irradiated in vitro (< or = 5.0 Gy), with preferential increases in CD45RA(+) T cells. The baseline level of apoptosis of CD45RA(-) T cells increased > 2-fold in the presence of an IkappaB-kinase inhibitor, indicating a protective effect via this pathway. T cell proliferation was impaired during RT with IL-2-dependent recovery post-RT. Recall T cell responses to common viral Ags, measured by IFN-gamma production, were little affected by RT. In vitro irradiation of healthy donor PBMCs resulted in a significantly increased frequency of responding T cells, due at least partly to the preferential elimination of CD45RA(+) T cells. Most importantly, antitumor CD4(+) and CD8(+) T cell responses were detectable after, but not before or during RT. The results indicate that generating tumor-specific T cell responses before RT and boosting their activity post-RT are ways likely to amplify the frequency and function of antitumor T cells, with implications for scheduling immunotherapy in PCa. PMID:20548027

  7. mTOR and metabolic pathways in T cell quiescence and functional activation

    PubMed Central

    Yang, Kai; Chi, Hongbo

    2013-01-01

    The mechanistic target of rapamycin (mTOR), an evolutionally conserved serine and threonine kinase, plays a critical role in the promotion of cell growth and proliferation via integration of cellular and environmental cues. In adaptive immunity, the mTOR pathway orchestrates multiple physiological processes including the development and homeostasis of T cells under steady state, and their subsequent activation and differentiation upon antigen recognition. Associated with such fate decisions is the dynamic reprogramming of T cell metabolic pathways, as naïve, activated and memory cells are defined by distinct bioenergetic and biosynthetic activities. Emerging evidence indicates that mTOR signaling intersects with T cell metabolism at two major levels to constitute a critical control mechanism of T cell fate decisions. First, as a central environmental sensor, mTOR links immune signaling and the availability of nutrients, especially amino acids. Second, mTOR activates specific metabolic pathways in T cells such as aerobic glycolysis (also known as the “Warburg effect”) in a process dependent upon the induction of transcription factors MYC and HIF1α. Understanding how mTOR interplays with T cell metabolism to dictate T cell fates and functions will provide fundamental insights into the mechanism of immune responses and the development of novel therapeutics against immune-mediated diseases. In this review, we summarize the current advances on mTOR signaling and T cell metabolism in the control of development, homeostasis, activation and differentiation of T cells. PMID:23375549

  8. Activation of T cell death-associated gene 8 regulates the cytokine production of T cells and macrophages in vitro.

    PubMed

    Onozawa, Yoshiko; Fujita, Yoshifumi; Kuwabara, Harumi; Nagasaki, Miyuki; Komai, Tomoaki; Oda, Tomiichiro

    2012-05-15

    An orphan G-protein-coupled receptor, T cell death-associated gene 8 (TDAG8) which has been reported to be a proton sensor, inhibits the production of pro-inflammatory cytokines induced by extracellular acidification. Recently, we have found that TDAG8 knockout mice showed significant exacerbation in various immune-mediated inflammation disease models. To elucidate the role of TDAG8, we screened an in-house library to find compounds which have a profile as a TDAG8 agonist using a cyclic adenosine 5'-monophosphate assay. Among the screening hits, we focused on (3-[(2,4-dichlorobenzyl)thio]-1,6-dimethyl-5,6-dihydro-1H-pyridazino[4,5-e][1,3,4]thiadiazin-5-one) (named BTB09089). BTB09089 did not act on other proton sensing G-protein-coupled receptors such as G-protein-coupled receptor 4 (GPR4) nor ovarian cancer G-protein-coupled receptor 1 (OGR1). Moreover, BTB09089 increased cAMP level in the splenocytes from wild-type littermates but not from TDAG8-deficient mice. Thus, BTB09089 was found to be a TDAG8 specific agonist. We then investigated the effects of BTB09089 on T cells and macrophages in vitro. In splenocytes, BTB09089 suppressed the production of IL-2 stimulated with anti-CD3 and anti-CD28 antibodies. In peritoneal exuded macrophages induced by thioglycollate, BTB09089 suppressed the production of TNF-α and IL-6 while it increased that of IL-10 when stimulated with lipopolysaccharide. These effects were observed in cells from wild type mice, but not those from TDAG8 knockout mice. These results indicate that activation of TDAG8 attenuates immune-mediated inflammation by regulating the cytokine production of T cells and macrophages. PMID:22445881

  9. THE INITIAL PHASE OF AN IMMUNE RESPONSE FUNCTIONS TO ACTIVATE REGULATORY T CELLS

    PubMed Central

    O’Gorman, William E.; Dooms, Hans; Thorne, Steve H.; Kuswanto, Wilson F.; Simonds, Erin F.; Krutzik, Peter O.; Nolan, Garry P.; Abbas, Abul K.

    2009-01-01

    An early reaction of CD4+ T lymphocytes to antigen is the production of cytokines, notably IL-2. In order to detect cytokine dependent responses, naive antigen-specific T cells were stimulated in vivo and the presence of phosphorylated STAT5 molecules was used to identify the cell populations responding to IL-2. Within hours of T-cell priming, IL-2-dependent STAT5 phosphorylation occurred primarily in Foxp3+ regulatory T cells. In contrast, the antigen-specific T cells received STAT5 signals only after repeated antigen exposure or memory differentiation. Regulatory T cells receiving IL-2 signals proliferated and developed enhanced suppressive activity. These results indicate that one of the earliest events in a T cell response is the activation of endogenous regulatory cells, potentially to prevent autoimmunity. PMID:19542444

  10. Enhanced effector responses in activated CD8+ T cells deficient in diacylglycerol kinases.

    PubMed

    Riese, Matthew J; Wang, Liang-Chuan S; Moon, Edmund K; Joshi, Rohan P; Ranganathan, Anjana; June, Carl H; Koretzky, Gary A; Albelda, Steven M

    2013-06-15

    Recent clinical trials have shown promise in the use of chimeric antigen receptor (CAR)-transduced T cells; however, augmentation of their activity may broaden their clinical use and improve their efficacy. We hypothesized that because CAR action requires proteins essential for T-cell receptor (TCR) signal transduction, deletion of negative regulators of these signaling pathways would enhance CAR signaling and effector T-cell function. We tested CAR activity and function in T cells that lacked one or both isoforms of diacylglycerol kinase (dgk) expressed highly in T cells, dgkα and dgkζ, enzymes that metabolize the second messenger diacylglycerol (DAG) and limit Ras/ERK activation. We found that primary murine T cells transduced with CARs specific for the human tumor antigen mesothelin showed greatly enhanced cytokine production and cytotoxicity when cocultured with a murine mesothelioma line that stably expresses mesothelin. In addition, we found that dgk-deficient CAR-transduced T cells were more effective in limiting the growth of implanted tumors, both concurrent with and after establishment of tumor. Consistent with our studies in mice, pharmacologic inhibition of dgks also augments function of primary human T cells transduced with CARs. These results suggest that deletion of negative regulators of TCR signaling enhances the activity and function of CAR-expressing T cells and identify dgks as potential targets for improving the clinical potential of CARs. PMID:23576561

  11. 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

  12. Mapping the polarity and stimulus density requirements for T-cell activation

    NASA Astrophysics Data System (ADS)

    Wei, Xunbin; Krasieva, Tatiana B.; Zhang, Zhanxiang; Negulescu, Paul A.; Sun, Chung-Ho; Berns, Michael W.; Cahalan, Michael D.; Tromberg, Bruce J.

    1998-08-01

    T-cell contact with antigen-presenting cells (APC) initiates an activation cascade which includes an increase in T-cell intracellular calcium [(Ca2+)i] and leads to T-cell proliferation and differentiation. Although T-cell/APC physical contact is required for an immune response, little is known about the patterns of cellular interaction and their relation to activation. We have combined fluorescence spectroscopy and imaging with optical manipulation to investigate the contact requirements for T-cell activation, using optical tweezers to control the orientation of T- cell/APC pairs and fluorescence microscopy to measure the subsequent (Ca2+)i response, detected as an emission shift from the combination of fura-red and oregon- green, two cytoplasmic (Ca2+) indicators. APCs or beads coated with antibodies to the T-cell receptor (TCR) are trapped with a near-infrared titanium-sapphire laser and placed at different locations along the T-cell, which has a polarized appearance defined by the shape and direction of crawling (2-5 micrometers /min). T cells contacted with antigen- presenting cells or antibody-coated beads entered a dynamic and reproducible program in the first 10 - 20 mins, including (Ca2+)i increase, changes in shape and motility, engulfment, and stable contact. T cells presented with antigen at the leading edge had a higher probability of responding (85%) and a shorter latency of response (50 secs) than those contacting APCs or beads with their trailing end (APCs: 30%, 150 secs; beads: 6%, 300 secs). Alterations in antibody density, quantified by FACS analysis, and bead size were used to determine the spatial requirements for T cell activation and the minimum number of receptors which must be engaged in order to transmit a positive signal. Preliminary data show that T cell responses [response percentage, latency and (Ca2+)i pattern] depend on both antibody density and bead size.

  13. The actin-bundling protein L-plastin supports T-cell motility and activation

    PubMed Central

    Morley, Sharon Celeste

    2013-01-01

    Summary Tight regulation of actin dynamics is essential for T-cell trafficking and activation. Recent studies in human and murine T cells reveal that T-cell motility and full T-cell activation require the hematopoietic-specific, actin-bundling protein L-plastin. T cells lacking L-plastin do not form fully mature synapses and thus demonstrate reduced cytokine production and proliferation. Reduction or loss of L-plastin expression also reduces the velocity of T cells and impairs thymic egress and intranodal motility. While dispensable for proximal T-cell receptor and chemokine receptor signaling, L-plastin is critical to the later stages of synapse maturation and cellular polarization. Serine phosphorylation, calcium, and calmodulin binding regulate the bundling activity and localization of LPL following T-cell receptor and chemokine receptor engagement. However, the interaction between these regulatory domains and resulting changes in local control of actin cytoskeletal structures has not been fully elucidated. Circumstantial evidence suggests a function for L-plastin in either the formation or maintenance of integrin-associated adhesion structures. As L-plastin may be a target of the commonly used immunosuppressive agent dexamethasone, full elucidation of the regulation and function of L-plastin in T-cell biology may illuminate new pathways for clinically useful immunotherapeutics. PMID:24117812

  14. Interleukin-7 is required for CD4(+) T cell activation and autoimmune neuroinflammation.

    PubMed

    Lawson, Brian R; Gonzalez-Quintial, Rosana; Eleftheriadis, Theodoros; Farrar, Michael A; Miller, Stephen D; Sauer, Karsten; McGavern, Dorian B; Kono, Dwight H; Baccala, Roberto; Theofilopoulos, Argyrios N

    2015-12-01

    IL-7 is known to be vital for T cell homeostasis but has previously been presumed to be dispensable for TCR-induced activation. Here, we show that IL-7 is critical for the initial activation of CD4(+) T cells in that it provides some of the necessary early signaling components, such as activated STAT5 and Akt. Accordingly, short-term in vivo IL-7Rα blockade inhibited the activation and expansion of autoantigen-specific CD4(+) T cells and, when used to treat experimental autoimmune encephalomyelitis (EAE), prevented and ameliorated disease. Our studies demonstrate that IL-7 signaling is a prerequisite for optimal CD4(+) T cell activation and that IL-7R antagonism may be effective in treating CD4(+) T cell-mediated neuroinflammation and other autoimmune inflammatory conditions. PMID:26319414

  15. Aggressive Peripheral CD70-positive T-cell Lymphoma Associated with Severe Chronic Active EBV Infection

    PubMed Central

    Shaffer, Donald R.; Sheehan, Andrea M.; Yi, Zhongzhen; Rodgers, Cheryl C; Bollard, Catherine M; Brenner, Malcolm K; Rooney, Cliona M; Heslop, Helen E; Gottschalk, Stephen

    2011-01-01

    Severe chronic active Epstein-Barr virus infection (CAEBV) in T or NK cells is a rare complication of latent EBV infection. CAEBV associated T-cell lymphoproliferative disease (LPD) consists of polyclonal lesions as well as aggressive lymphomas. Here we report such a patient. In addition, we show that this primary CAEBV associated T-cell lymphoma expresses CD70 and is sensitive to killing by CD70-specific T cells, identifying CD70 as a potential immunotherapeutic target for CAEBV-associated T-cell lymphoma. PMID:21994111

  16. The tetraspanin CD9 is preferentially expressed on the human CD4+CD45RA+ naive T cell population and is involved in T cell activation

    PubMed Central

    KOBAYASHI, H; HOSONO, O; IWATA, S; KAWASAKI, H; KUWANA, M; TANAKA, H; DANG, N H; MORIMOTO, C

    2004-01-01

    Human CD4+ T cells can be divided into reciprocal memory and naive T cell subsets based on their expression of CD45 isoforms and CD29/integrin beta1 subunit. To identify unique cell surface molecules on human T cells, we developed a new monoclonal antibody termed anti5H9. Binding of anti5H9 triggers a co-stimulatory response in human peripheral blood T cells. Retrovirus-mediated expression cloning has revealed that the antigen recognized by anti5H9 is identical to the tetraspanin CD9. We now show that human CD9 is preferentially expressed on the CD4+CD45RA+ naive T cell subset, and that CD9+CD45RA+ T cells respond preferentially to the recombinant beta2-glycoprotein I, compared to CD9–CD45RA+ T cells. Furthermore, anti5H9 inhibits both the recombinant beta2-glycoprotein I- and the recall antigen tetanus toxoid-specific T cell proliferation. These results suggest that the tetraspanin CD9 plays an important role in T cell activation. PMID:15196249

  17. Increased numbers and functional activity of CD56+ T cells in healthy cytomegalovirus positive subjects

    PubMed Central

    Almehmadi, Mazen; Flanagan, Brian F; Khan, Naeem; Alomar, Suliman; Christmas, Stephen E

    2014-01-01

    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. PMID:24433347

  18. An essential regulatory role for macrophage migration inhibitory factor in T-cell activation.

    PubMed Central

    Bacher, M; Metz, C N; Calandra, T; Mayer, K; Chesney, J; Lohoff, M; Gemsa, D; Donnelly, T; Bucala, R

    1996-01-01

    The protein known as macrophage migration inhibitory factor (MIF) was one of the first cytokines to be discovered and was described 30 years ago to be a T-cell-derived factor that inhibited the random migration of macrophages in vitro. A much broader role for MIF has emerged recently as a result of studies that have demonstrated it to be released from the anterior pituitary gland in vivo. MIF also is the first protein that has been identified to be secreted from monocytes/macrophages upon glucocorticoid stimulation. Once released, MIF acts to "override" or counter-regulate the suppressive effects of glucocorticoids on macrophage cytokine production. We report herein that MIF plays an important regulatory role in the activation of T cells induced by mitogenic or antigenic stimuli. Activated T cells produce MIF and neutralizing anti-MIF antibodies inhibit T-cell proliferation and interleukin 2 production in vitro, and suppress antigen-driven T-cell activation and antibody production in vivo. T cells also release MIF in response to glucocorticoid stimulation and MIF acts to override glucocorticoid inhibition of T-cell proliferation and interleukin 2 and interferon gamma production. These studies indicate that MIF acts in concert with glucocorticoids to control T-cell activation and assign a previously unsuspected but critical role for MIF in antigen-specific immune responses. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8755565

  19. Regulatory T cells actively infiltrate metastatic brain tumors.

    PubMed

    Sugihara, Adam Quasar; Rolle, Cleo E; Lesniak, Maciej S

    2009-06-01

    Regulatory T cells (CD4+CD25+FoxP3+, Treg) have been shown to play a major role in suppression of the immune response to malignant gliomas. In this study, we investigated the kinetics of Treg infiltration in metastatic brain tumor models, including melanoma, breast and colon cancers. Our data indicate that both CD4+ and Treg infiltration are significantly increased throughout the time of metastatic tumor progression. These findings were recapitulated in human CNS tumor samples of metastatic melanoma and non-small cell lung carcinoma. Collectively, these data support investigating immunotherapeutic strategies targeting Treg in metastatic CNS tumors. PMID:19424570

  20. Five Layers of Receptor Signaling in γδ T-Cell Differentiation and Activation

    PubMed Central

    Ribeiro, Sérgio T.; Ribot, Julie C.; Silva-Santos, Bruno

    2015-01-01

    The contributions of γδ T-cells to immunity to infection or tumors critically depend on their activation and differentiation into effectors capable of secreting cytokines and killing infected or transformed cells. These processes are molecularly controlled by surface receptors that capture key extracellular cues and convey downstream intracellular signals that regulate γδ T-cell physiology. The understanding of how environmental signals are integrated by γδ T-cells is critical for their manipulation in clinical settings. Here, we discuss how different classes of surface receptors impact on human and murine γδ T-cell differentiation, activation, and expansion. In particular, we review the role of five receptor types: the T-cell receptor (TCR), costimulatory receptors, cytokine receptors, NK receptors, and inhibitory receptors. Some of the key players are the costimulatory receptors CD27 and CD28, which differentially impact on pro-inflammatory subsets of γδ T-cells; the cytokine receptors IL-2R, IL-7R, and IL-15R, which drive functional differentiation and expansion of γδ T-cells; the NK receptor NKG2D and its contribution to γδ T-cell cytotoxicity; and the inhibitory receptors PD-1 and BTLA that control γδ T-cell homeostasis. We discuss these and other receptors in the context of a five-step model of receptor signaling in γδ T-cell differentiation and activation, and discuss its implications for the manipulation of γδ T-cells in immunotherapy. PMID:25674089

  1. 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

  2. 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

  3. Role of Increased Guanosine Triphosphate Cyclohydrolase-1 Expression and Tetrahydrobiopterin Levels upon T Cell Activation

    PubMed Central

    Chen, Wei; Li, Li; Brod, Torben; Saeed, Omar; Thabet, Salim; Jansen, Thomas; Dikalov, Sergey; Weyand, Cornelia; Goronzy, Jorg; Harrison, David G.

    2011-01-01

    Tetrahydrobiopterin (BH4) is an essential co-factor for the nitric-oxide (NO) synthases, and in its absence these enzymes produce superoxide (O2˙̄) rather than NO. The rate-limiting enzyme for BH4 production is guanosine triphosphate cyclohydrolase-1 (GTPCH-1). Because endogenously produced NO affects T cell function, we sought to determine whether antigen stimulation affected T cell GTPCH-1 expression and ultimately BH4 levels. Resting T cells had minimal expression of inducible NOS (NOS2), endothelial NOS (NOS3), and GTPCH-1 protein and nearly undetectable levels of BH4. Anti-CD3 stimulation of T cells robustly stimulated the coordinated expression of NOS2, NOS3, and GTPCH-1 and markedly increased both GTPCH-1 activity and T cell BH4 levels. The newly expressed GTPCH-1 was phosphorylated on serine 72 and pharmacological inhibition of casein kinase II reduced GTPCH-1 phosphorylation and blunted the increase in T cell BH4. Inhibition of GTPCH-1 with diaminohydroxypyrimidine (1 mmol/liter) prevented T cell BH4 accumulation, reduced NO production, and increased T cell O2˙̄ production, due to both NOS2 and NOS3 uncoupling. GTPCH-1 inhibition also promoted TH2 polarization in memory CD4 cells. Ovalbumin immunization of mice transgenic for an ovalbumin receptor (OT-II mice) confirmed a marked increase in T cell BH4 in vivo. These studies identify a previously unidentified consequence of T cell activation, promoting BH4 levels, NO production, and modulating T cell cytokine production. PMID:21343293

  4. Surfactant protein A integrates activation signal strength to differentially modulate T cell proliferation.

    PubMed

    Mukherjee, Sambuddho; Giamberardino, Charles; Thomas, Joseph; Evans, Kathy; Goto, Hisatsugu; Ledford, Julie G; Hsia, Bethany; Pastva, Amy M; Wright, Jo Rae

    2012-02-01

    Pulmonary surfactant lipoproteins lower the surface tension at the alveolar-airway interface of the lung and participate in host defense. Previous studies reported that surfactant protein A (SP-A) inhibits lymphocyte proliferation. We hypothesized that SP-A-mediated modulation of T cell activation depends upon the strength, duration, and type of lymphocyte activating signals. Modulation of T cell signal strength imparted by different activating agents ex vivo and in vivo in different mouse models and in vitro with human T cells shows a strong correlation between strength of signal (SoS) and functional effects of SP-A interactions. T cell proliferation is enhanced in the presence of SP-A at low SoS imparted by exogenous mitogens, specific Abs, APCs, or in homeostatic proliferation. Proliferation is inhibited at higher SoS imparted by different doses of the same T cell mitogens or indirect stimuli such as LPS. Importantly, reconstitution with exogenous SP-A into the lungs of SP-A(-/-) mice stimulated with a strong signal also resulted in suppression of T cell proliferation while elevating baseline proliferation in unstimulated T cells. These signal strength and SP-A-dependent effects are mediated by changes in intracellular Ca(2+) levels over time, involving extrinsic Ca(2+)-activated channels late during activation. These effects are intrinsic to the global T cell population and are manifested in vivo in naive as well as memory phenotype T cells. Thus, SP-A appears to integrate signal thresholds to control T cell proliferation. PMID:22219327

  5. Activated T cell exosomes promote tumor invasion via Fas signaling pathway.

    PubMed

    Cai, Zhijian; Yang, Fei; Yu, Lei; Yu, Zhou; Jiang, Lingling; Wang, Qingqing; Yang, Yunshan; Wang, Lie; Cao, Xuetao; Wang, Jianli

    2012-06-15

    Activated T cells release bioactive Fas ligand (FasL) in exosomes, which subsequently induce self-apoptosis of T cells. However, their potential effects on cell apoptosis in tumors are still unknown. In this study, we purified exosomes expressing FasL from activated CD8(+) T cell from OT-I mice and found that activated T cell exosomes had little effect on apoptosis and proliferation of tumor cells but promoted the invasion of B16 and 3LL cancer cells in vitro via the Fas/FasL pathway. Activated T cell exosomes increased the amount of cellular FLICE inhibitory proteins and subsequently activated the ERK and NF-κB pathways, which subsequently increased MMP9 expression in the B16 murine melanoma cells. In a tumor-invasive model in vivo, we observed that the activated T cell exosomes promoted the migration of B16 tumor cells to lung. Interestingly, pretreatment with FasL mAb significantly reduced the migration of B16 tumor cells to lung. Furthermore, CD8 and FasL double-positive exosomes from tumor mice, but not normal mice, also increased the expression of MMP9 and promoted the invasive ability of B16 murine melanoma and 3LL lung cancer cells. In conclusion, our results indicate that activated T cell exosomes promote melanoma and lung cancer cell metastasis by increasing the expression of MMP9 via Fas signaling, revealing a new mechanism of tumor immune escape. PMID:22573809

  6. 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

  7. T-cell death following immune activation is mediated by mitochondria-localized SARM.

    PubMed

    Panneerselvam, P; Singh, L P; Selvarajan, V; Chng, W J; Ng, S B; Tan, N S; Ho, B; Chen, J; Ding, J L

    2013-03-01

    Following acute-phase infection, activated T cells are terminated to achieve immune homeostasis, failure of which results in lymphoproliferative and autoimmune diseases. We report that sterile α- and heat armadillo-motif-containing protein (SARM), the most conserved Toll-like receptors adaptor, is proapoptotic during T-cell immune response. SARM expression is significantly reduced in natural killer (NK)/T lymphoma patients compared with healthy individuals, suggesting that decreased SARM supports NK/T-cell proliferation. T cells knocked down of SARM survived and proliferated more significantly compared with wild-type T cells following influenza infection in vivo. During activation of cytotoxic T cells, the SARM level fell before rising, correlating inversely with cell proliferation and subsequent T-cell clearance. SARM knockdown rescued T cells from both activation- and neglect-induced cell deaths. The mitochondria-localized SARM triggers intrinsic apoptosis by generating reactive oxygen species and depolarizing the mitochondrial potential. The proapoptotic function is attributable to the C-terminal sterile alpha motif and Toll/interleukin-1 receptor domains. Mechanistically, SARM mediates intrinsic apoptosis via B cell lymphoma-2 (Bcl-2) family members. SARM suppresses B cell lymphoma-extra large (Bcl-xL) and downregulates extracellular signal-regulated kinase phosphorylation, which are cell survival effectors. Overexpression of Bcl-xL and double knockout of Bcl-2 associated X protein and Bcl-2 homologous antagonist killer substantially reduced SARM-induced apoptosis. Collectively, we have shown how T-cell death following infection is mediated by SARM-induced intrinsic apoptosis, which is crucial for T-cell homeostasis. PMID:23175186

  8. Increased Mucosal CD4+ T Cell Activation in Rhesus Macaques following Vaccination with an Adenoviral Vector

    PubMed Central

    Bukh, Irene; Calcedo, Roberto; Roy, Soumitra; Carnathan, Diane G.; Grant, Rebecca; Qin, Qiuyue; Boyd, Surina; Ratcliffe, Sarah J.; Veeder, Christin L.; Bellamy, Scarlett L.; Betts, Michael R.

    2014-01-01

    ABSTRACT The possibility that vaccination with adenovirus (AdV) vectors increased mucosal T cell activation remains a central hypothesis to explain the potential enhancement of HIV acquisition within the Step trial. Modeling this within rhesus macaques is complicated because human adenoviruses, including human adenovirus type 5 (HAdV-5), are not endogenous to macaques. Here, we tested whether vaccination with a rhesus macaque-derived adenoviral vector (simian adenovirus 7 [SAdV-7]) enhances mucosal T cell activation within rhesus macaques. Following intramuscular SAdV-7 vaccination, we observed a pronounced increase in SAdV-7-specific CD4+ T cell responses in peripheral blood and, more dramatically, in rectal mucosa tissue. Vaccination also induced a significant increase in the frequency of activated memory CD4+ T cells in SAdV-7- and HAdV-5-vaccinated animals in the rectal mucosa but not in peripheral blood. These fluctuations within the rectal mucosa were also associated with a pronounced decrease in the relative frequency of naive resting CD4+ T cells. Together, these results indicate that peripheral vaccination with an AdV vector can increase the activation of mucosal CD4+ T cells, potentially providing an experimental model to further evaluate the role of host-vector interactions in increased HIV acquisition after AdV vector vaccination. IMPORTANCE The possibility that vaccination with a human adenovirus 5 vector increased mucosal T cell activation remains a central hypothesis to explain the potential enhancement of human immunodeficiency virus (HIV) acquisition within the Step trial. In this study, we tested whether vaccination with a rhesus macaque-derived adenoviral vector in rhesus macaques enhances mucosal CD4+ T cell activation, the main cell target of simian immunodeficiency virus (SIV)/HIV. The results showed that vaccination with an adenoviral vector indeed increases activation of mucosal CD4+ T cells and potentially increases susceptibility to SIV

  9. 5-Azacytidine Promotes an Inhibitory T-Cell Phenotype and Impairs Immune Mediated Antileukemic Activity

    PubMed Central

    Stübig, Thomas; Luetkens, Tim; Hildebrandt, York; Atanackovic, Djordje; Binder, Thomas M. C.; Fehse, Boris; Kröger, Nicolaus

    2014-01-01

    Demethylating agent, 5-Azacytidine (5-Aza), has been shown to be active in treatment of myeloid malignancies. 5-Aza enhances anticancer immunity, by increasing expression of tumor-associated antigens. However, the impact of 5-Aza immune responses remains poorly understood. Here, T-cell mediated tumor immunity effects of 5-Aza, are investigated in vitro and in vivo. T-cells from healthy donors were treated with 5-Aza and analyzed by qRT-PCR and flow cytometry for changes in gene expression and phenotype. Functionality was assessed by a tumor lysis assay. Peripheral blood from patients treated with 5-Aza after alloSCT was monitored for changes in T-cell subpopulations. 5-Aza treatment resulted in a decrease in CD8+ T-cells, whereas CD4+ T-cells increased. Furthermore, numbers of IFN-γ+ T-helper 1 cells (Th1) were reduced, while Treg-cells showed substantial increase. Additionally, CD8+ T-cells exhibited limited killing capacity against leukemic target cells. In vivo data confirm the increase of Treg compartment, while CD8+ T-effector cell numbers were reduced. 5-Aza treatment results in a shift from cytotoxic to regulatory T-cells with a functional phenotype and a major reduction in proinflammatory Th1-cells, indicating a strong inhibition of tumor-specific T-cell immunity by 5-Aza. PMID:24757283

  10. 5-azacytidine promotes an inhibitory T-cell phenotype and impairs immune mediated antileukemic activity.

    PubMed

    Stübig, Thomas; Badbaran, Anita; Luetkens, Tim; Hildebrandt, York; Atanackovic, Djordje; Binder, Thomas M C; Fehse, Boris; Kröger, Nicolaus

    2014-01-01

    Demethylating agent, 5-Azacytidine (5-Aza), has been shown to be active in treatment of myeloid malignancies. 5-Aza enhances anticancer immunity, by increasing expression of tumor-associated antigens. However, the impact of 5-Aza immune responses remains poorly understood. Here, T-cell mediated tumor immunity effects of 5-Aza, are investigated in vitro and in vivo. T-cells from healthy donors were treated with 5-Aza and analyzed by qRT-PCR and flow cytometry for changes in gene expression and phenotype. Functionality was assessed by a tumor lysis assay. Peripheral blood from patients treated with 5-Aza after alloSCT was monitored for changes in T-cell subpopulations. 5-Aza treatment resulted in a decrease in CD8+ T-cells, whereas CD4+ T-cells increased. Furthermore, numbers of IFN-γ + T-helper 1 cells (Th1) were reduced, while Treg-cells showed substantial increase. Additionally, CD8+ T-cells exhibited limited killing capacity against leukemic target cells. In vivo data confirm the increase of Treg compartment, while CD8+ T-effector cell numbers were reduced. 5-Aza treatment results in a shift from cytotoxic to regulatory T-cells with a functional phenotype and a major reduction in proinflammatory Th1-cells, indicating a strong inhibition of tumor-specific T-cell immunity by 5-Aza. PMID:24757283

  11. CD47-dependent immunomodulatory and angiogenic activities of extracellular vesicles produced by T cells.

    PubMed

    Kaur, Sukhbir; Singh, Satya P; Elkahloun, Abdel G; Wu, Weiwei; Abu-Asab, Mones S; Roberts, David D

    2014-07-01

    Intercellular communication is critical for integrating complex signals in multicellular eukaryotes. Vascular endothelial cells and T lymphocytes closely interact during the recirculation and trans-endothelial migration of T cells. In addition to direct cell-cell contact, we show that T cell derived extracellular vesicles can interact with endothelial cells and modulate their cellular functions. Thrombospondin-1 and its receptor CD47 are expressed on exosomes/ectosomes derived from T cells, and these extracellular vesicles are internalized and modulate signaling in both T cells and endothelial cells. Extracellular vesicles released from cells expressing or lacking CD47 differentially regulate activation of T cells induced by engaging the T cell receptor. Similarly, T cell-derived extracellular vesicles modulate endothelial cell responses to vascular endothelial growth factor and tube formation in a CD47-dependent manner. Uptake of T cell derived extracellular vesicles by recipient endothelial cells globally alters gene expression in a CD47-dependent manner. CD47 also regulates the mRNA content of extracellular vesicles in a manner consistent with some of the resulting alterations in target endothelial cell gene expression. Therefore, the thrombospondin-1 receptor CD47 directly or indirectly regulates intercellular communication mediated by the transfer of extracellular vesicles between vascular cells. PMID:24887393

  12. 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

  13. 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

  14. 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

  15. 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. PMID:26700461

  16. Ly-6A is required for T cell receptor expression and protein tyrosine kinase fyn activity.

    PubMed Central

    Lee, S K; Su, B; Maher, S E; Bothwell, A L

    1994-01-01

    To characterize the function of the Ly-6A antigen in T cell activation, antisense Ly-6 RNA was expressed in a stably transfected antigen-specific T cell clone. Reduced Ly-6A expression results in inhibition of responses to antigen, anti-TCR (anti-T cell receptor) crosslinking and concanavalin A plus recombinant interleukin 1 and causes impairment of in vitro fyn tyrosine kinase activity. More substantial reduction of Ly-6A results in reduction of TCR expression. Analysis of mRNA species indicates that the reduction is specific for the TCR beta chain. These data demonstrate that Ly-6A may regulate TCR expression and may be involved in early events of T cell activation via regulation of fyn tyrosine kinase activity. Images PMID:8187770

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. Human endogenous retrovirus envelope proteins target dendritic cells to suppress T-cell activation.

    PubMed

    Hummel, Jonas; Kämmerer, Ulrike; Müller, Nora; Avota, Elita; Schneider-Schaulies, Sibylle

    2015-06-01

    Though mostly defective, human endogenous retroviruses (HERV) can retain open reading frames, which are especially expressed in the placenta. There, the envelope (env) proteins of HERV-W (Syncytin-1), HERV-FRD (Syncytin-2), and HERV-K (HML-2) were implicated in tolerance against the semi-allogenic fetus. Here, we show that the known HERV env-binding receptors ASCT-1 and -2 and MFSD2 are expressed by DCs and T-cells. When used as effectors in coculture systems, CHO cells transfected to express Syncytin-1, -2, or HML-2 did not affect T-cell expansion or overall LPS-driven phenotypic DC maturation, however, promoted release of IL-12 and TNF-α rather than IL-10. In contrast, HERV env expressing choriocarcinoma cell lines suppressed T-cell proliferation and LPS-induced TNF-α and IL-12 release, however, promoted IL-10 accumulation, indicating that these effects might not rely on HERV env interactions. However, DCs conditioned by choriocarcinoma, but also transgenic CHO cells failed to promote allogenic T-cell expansion. This was associated with a loss of DC/T-cell conjugate frequencies, impaired Ca(2+) mobilization, and aberrant patterning of f-actin and tyrosine phosphorylated proteins in T-cells. Altogether, these findings suggest that HERV env proteins target T-cell activation indirectly by modulating the stimulatory activity of DCs. PMID:25752285

  2. Tolerance induction in memory CD4 T cells requires two rounds of antigen-specific activation.

    PubMed

    David, Alexandria; Crawford, Frances; Garside, Paul; Kappler, John W; Marrack, Philippa; MacLeod, Megan

    2014-05-27

    A major goal for immunotherapy is to tolerize the immune cells that coordinate tissue damage in autoimmune and alloantigen responses. CD4 T cells play a central role in many of these conditions and improved antigen-specific regulation or removal of these cells could revolutionize current treatments. A confounding factor is that little is known about whether and how tolerance is induced in memory CD4 T cells. We used MHC class II tetramers to track and analyze a population of endogenous antigen-specific memory CD4 T cells exposed to soluble peptide in the absence of adjuvant. We found that such memory T cells proliferated and reentered the memory pool apparently unperturbed by the incomplete activation signals provided by the peptide. Upon further restimulation in vivo, CD4 memory T cells that had been previously exposed to peptide proliferated, provided help to primary responding B cells, and migrated to inflamed sites. However, these reactivated memory cells failed to survive. The reduction in T-cell number was marked by low expression of the antiapoptotic molecule B cell lymphoma 2 (Bcl2) and increased expression of activated caspase molecules. Consequently, these cells failed to sustain a delayed-type hypersensitivity response. Moreover, following two separate exposures to soluble antigen, no T-cell recall response and no helper activity for B cells could be detected. These results suggest that the induction of tolerance in memory CD4 T cells is possible but that deletion and permanent removal of the antigen-specific T cells requires reactivation following exposure to the tolerogenic antigen. PMID:24821788

  3. 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

  4. Persistent HIV Type 1 Seronegative Status Is Associated With Lower CD8+ T-Cell Activation.

    PubMed

    Kuebler, Peter J; Mehrotra, Megha L; Shaw, Brian I; Leadabrand, Kaitlyn S; Milush, Jeffrey M; York, Vanessa A; Defechereux, Patricia; Grant, Robert M; Kallás, Esper G; Nixon, Douglas F

    2016-02-15

    We leveraged data from the Preexposure Prophylaxis Initiative (iPrEx), a global trial of preexposure chemoprophylaxis against human immunodeficiency virus type 1 (HIV-1) infection, to compare T-cell activation between those who remained negative for HIV-1 and those who became infected during the trial. The frequency of CD38(+)HLA-DR(+) CD8(+) T cells was greater in those who seroconverted, relative to the frequency in those who remained uninfected (1.30% vs 0.82%, respectively; P = .005). This translated to an odds ratio of 4.26 (95% confidence interval, 1.54-11.78) for the association between CD8(+) T-cell activation and infection with HIV-1. T-cell activation may be a biomarker for elevated HIV-1 infection risk. PMID:26310308

  5. CD137 Agonist Therapy Can Reprogram Regulatory T Cells into Cytotoxic CD4+ T Cells with Antitumor Activity.

    PubMed

    Akhmetzyanova, Ilseyar; Zelinskyy, Gennadiy; Littwitz-Salomon, Elisabeth; Malyshkina, Anna; Dietze, Kirsten K; Streeck, Hendrik; Brandau, Sven; Dittmer, Ulf

    2016-01-01

    Recent successes in immune therapeutic strategies aimed to improve control over tumor growth have sparked hope that long-lived control of cancer through stimulation of the immune system can be possible. However, the underlying immunological mechanisms that are induced by immunotherapeutic strategies are not well understood. In this study, we used the highly immunogenic Friend virus-induced FBL-3 tumor as a model to study the mechanisms of immunological tumor control by CD4(+) T cells in the course of CD137 (4-1BB) agonist immunotherapy in the absence of a CD8 T cell response. We demonstrate that treatment with a CD137 agonist resulted in complete FBL-3 tumor regression in CD8(+) T cell-deficient mice. CD137 signaling enhanced the production of proinflammatory cytokines and cytotoxic molecules in tumor-specific CD4(+) T cells. Interestingly, a subset of CD4(+)Foxp3(+) regulatory T cells was reprogrammed to eliminate immunogenic virus-induced tumor cells in response to CD137 agonist treatment. These cells expressed markers characteristic for Th cells (CD154) and produced the cytokine TNF-α or the T-box transcriptional factor Eomesodermin and granzyme B without loss of Foxp3 expression. Foxp3 Eomes double-positive CD4(+) T cells were capable of eliminating immunogenic virus-induced tumor cells in vivo. Thus, our data show that tumor-induced Foxp3(+)CD4(+) T cells can be reprogrammed into cytotoxic effector cells upon therapeutic costimulatory signaling and restore antitumor immunity. PMID:26608920

  6. 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

  7. 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. PMID:26307764

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

    PubMed Central

    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; Behr, Charlotte

    2015-01-01

    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. PMID:26169273

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

    PubMed

    Filipello, Fabia; Pozzi, Davide; Proietti, Michele; Romagnani, Andrea; Mazzitelli, Sonia; Matteoli, Michela; Verderio, Claudia; Grassi, Fabio

    2016-02-01

    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

  10. 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

  11. 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

  12. 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

  13. NFAT restricts osteochondroma formation from entheseal progenitors

    PubMed Central

    Ge, Xianpeng; Tsang, Kelly; He, Lizhi; Garcia, Roberto A.; Ermann, Joerg; Mizoguchi, Fumitaka; Zhang, Minjie; Zhou, Bin; Zhou, Bin; 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

  14. 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

  15. Interleukin 17-Producing γδ T Cells Increased in Patients with Active Pulmonary Tuberculosis

    PubMed Central

    Peng, Meiyu; Wang, Zhaohua; Yao, Chunyan; Jiang, Lina; Jin, Qili; Wang, Jing; Li, Baiqing

    2008-01-01

    Although it has been known that γδ T cells may play an important role in the immune response to infection of Mycobacterium tuberculosis (M. tb), the mechanisms by which the γδ T cells participate in the innate and/or acquired immunity to tuberculosis (TB) have not been full elucidated. In the present study, 27 patients with active pulmonary TB and 16 healthy donors (HD) were performed. We found that proportion of IL-17-producing cells among lymphocyte was similar between TB patients and HD, whereas the proportions of γδ T cells in IL-17-producing cells (59.2%) and IL-17-producing cells in γδ T cells (19.4%) in peripheral blood were markedly increased in TB patients when compared to those in HD (43.9% and 7.7%, respectively). In addition, the proportions of IFN-γ-producing γδ T cells in TB patients were obviously lower than that in HD. Upon re-stimulated with M. tb heat-treated antigen (M. tb-HAg) in vitro, fewer IL-17-producing γδ T cells were generated from HD and TB patients, whereas IFN-γ-producing γδ T cells were increased in TB patients compared to that in HD. Our findings in TB patients and healthy human were consistent with other murine investigation that the IL-17-producing γδ T cells were main source of IL-17 in mouse model of BCG infection, suggesting that γδ T cells might be involved in the formation of tubercular granuloma in pulmonary TB patients, but need further identification. PMID:18582402

  16. SF2 and SRp55 regulation of CD45 exon 4 skipping during T cell activation.

    PubMed

    Lemaire, R; Winne, A; Sarkissian, M; Lafyatis, R

    1999-03-01

    CD45 is an alternatively spliced membrane phosphatase required for T cell activation. Exons 4, 5 and 6 can be included or skipped from spliced mRNA resulting in several protein isoforms that include or exclude epitopes referred to as RA, RB or RC, respectively. T cells reciprocally express CD45RA or CD45RO (lacking all three exons), corresponding to naive versus memory T cells. Overexpression of the alternative splicing regulators, SF2 or SWAP, induces skipping of CD45 exon 4 in transfected COS cells. We show here that the arginine/serine-rich domain of SWAP and the RNA recognition motifs of SF2 are required for skipping of CD45 exon 4. Unlike SWAP, SF2 specifically regulated alternative splicing of CD45 exon 4, having no effect on a non-regulated exon of CD45 (exon 9). Like SF2 and SWAP, the SR proteins SC35, SRp40 and SRp75, but not SRp55 also induced CD45 exon 4 skipping. In contrast, antisense inhibition of SRp55 induced exon 4 skipping. SF2 and SRp55 proteins were not detectable or expressed at a very low level in freshly isolated CD45RA+ and CD45RO+ T cells. Activation of CD45RA+ T cells shifted CD45 expression from CD45RA to CD45RO, and induced a large increase in expression of both SF2 and SRp55. Thus, SF2 at least in part determines splicing of CD45 exon 4 during T cell activation. SRp55, SR protein phosphorylation, or other splicing factors likely regulate CD45 splicing in CD45RO+ memory T cells. The different SR proteins expressed by memory and activated T cells emphasize the different phenotypes of these cell types that both express CD45RO. PMID:10092085

  17. A77 1726, the active metabolite of leflunomide, attenuates lupus nephritis by promoting the development of regulatory T cells and inhibiting IL-17-producing double negative T cells.

    PubMed

    Qiao, Guilin; Yang, Lifen; Li, Zhenping; Williams, James W; Zhang, Jian

    2015-04-01

    Lupus nephritis (LN) is a challenging problem that affects 50% of patients with systemic lupus erythematosus (SLE) without effective therapy. Here, we report that A77 1726, the active metabolite of leflunomide, effectively inhibits development of LN and attenuates the generalized autoimmune features. A77 1726 suppresses the expansion of double negative (DN) T cells, and inhibits T and B cell activation. Intriguingly, A77 1726 treatment significantly increases CD4(+)Foxp3(+) regulatory T cells but suppresses potential "pathogenic" IL-17-producing DN T cells in lymph nodes. In vitro experiment shows that A77 1726 potentiates the conversion of naive CD4(+)CD25(-) T cells into CD4(+)CD25(+)Foxp3(+) inducible regulatory T cells (iTregs) by inhibiting Akt. Taken together, our data indicate that the therapeutic effects of A77 1726 in murine LN are mediated, at least in part, by augmenting iTregs which suppress pathogenic IL-17-producing DN T cells through an Akt-dependent mechanism. PMID:25638413

  18. Global intron retention mediated gene regulation during CD4+ T cell activation.

    PubMed

    Ni, Ting; Yang, Wenjing; Han, Miao; Zhang, Yubo; Shen, Ting; Nie, Hongbo; Zhou, Zhihui; Dai, Yalei; Yang, Yanqin; Liu, Poching; Cui, Kairong; Zeng, Zhouhao; Tian, Yi; Zhou, Bin; Wei, Gang; Zhao, Keji; Peng, Weiqun; Zhu, Jun

    2016-08-19

    T cell activation is a well-established model for studying cellular responses to exogenous stimulation. Using strand-specific RNA-seq, we observed that intron retention is prevalent in polyadenylated transcripts in resting CD4(+) T cells and is significantly reduced upon T cell activation. Several lines of evidence suggest that intron-retained transcripts are less stable than fully spliced transcripts. Strikingly, the decrease in intron retention (IR) levels correlate with the increase in steady-state mRNA levels. Further, the majority of the genes upregulated in activated T cells are accompanied by a significant reduction in IR. Of these 1583 genes, 185 genes are predominantly regulated at the IR level, and highly enriched in the proteasome pathway, which is essential for proper T cell proliferation and cytokine release. These observations were corroborated in both human and mouse CD4(+) T cells. Our study revealed a novel post-transcriptional regulatory mechanism that may potentially contribute to coordinated and/or quick cellular responses to extracellular stimuli such as an acute infection. PMID:27369383

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

    PubMed Central

    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

    2016-01-01

    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

  20. 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

  1. Quantifying activity: a new assay reveals T-cell signalling in tiny skin biopsy samples.

    PubMed

    Dornmair, Klaus

    2014-06-01

    Tissue-invasive T cells are observed in many inflammatory dermatological diseases, but in most cases, it is not known how they were attracted, what they might recognize, and to which extent they are activated. Answering these questions is surely essential for understanding pathogeneses of the diseases. In a recent issue of Experimental Dermatology, Smith et al. showed that early signalling events in skin-resident T cells may be investigated by multiplex immunoprecipitation flow cytometry, even if only few T cells are available from skin biopsy samples. This new technology will most likely contribute to elucidating the role of skin-invasive T cells and to understanding the pathology of dermatological diseases. PMID:24665970

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

    PubMed

    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

  3. T-cell accumulation and regulated on activation, normal T cell expressed and secreted upregulation in adipose tissue in obesity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Obesity is associated with chronic inflammation, which includes increased macrophage accumulation in adipose tissue (AT) and upregulation of chemokines and cytokines. T cells also play important roles in chronic inflammatory diseases such as atherosclerosis but have not been well studied in obesity....

  4. Angiopoietin 2 stimulates TIE2-expressing monocytes to suppress T cell activation and to promote regulatory T cell expansion.

    PubMed

    Coffelt, Seth B; Chen, Yung-Yi; Muthana, Munitta; Welford, Abigail F; Tal, Andrea O; Scholz, Alexander; Plate, Karl H; Reiss, Yvonne; Murdoch, Craig; De Palma, Michele; Lewis, Claire E

    2011-04-01

    Angiopoietin 2 (ANGPT2) is a proangiogenic cytokine whose expression is often upregulated by endothelial cells in tumors. Expression of its receptor, TIE2, defines a highly proangiogenic subpopulation of myeloid cells in circulation and tumors called TIE2-expressing monocytes/macrophages (TEMs). Genetic depletion of TEMs markedly reduces tumor angiogenesis in various tumor models, emphasizing their essential role in driving tumor progression. Previously, we demonstrated that ANGPT2 augments the expression of various proangiogenic genes, the potent immunosuppressive cytokine, IL-10, and a chemokine for regulatory T cells (Tregs), CCL17 by TEMs in vitro. We now show that TEMs also express higher levels of IL-10 than TIE2(-) macrophages in tumors and that ANGPT2-stimulated release of IL-10 by TEMs suppresses T cell proliferation, increases the ratio of CD4(+) T cells to CD8(+) T cells, and promotes the expansion of CD4(+)CD25(high)FOXP3(+) Tregs. Furthermore, syngeneic murine tumors expressing high levels of ANGPT2 contained not only high numbers of TEMs but also increased numbers of Tregs, whereas genetic depletion of tumor TEMs resulted in a marked reduction in the frequency of Tregs in tumors. Taken together, our data suggest that ANGPT2-stimulated TEMs represent a novel, potent immunosuppressive force in tumors. PMID:21368233

  5. 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

  6. Protective Role of β-arrestin2 in Colitis Through Modulation of T-cell Activation.

    PubMed

    Sharma, Deepika; Malik, Ankit; Steury, Michael D; Lucas, Peter C; Parameswaran, Narayanan

    2015-12-01

    β-arrestin2 (β-arr2), identified as a scaffolding protein in G-protein-coupled receptor desensitization, is a negative regulator of inflammation in polymicrobial sepsis. In this study, we wanted to investigate the role of β-arr2 in intestinal inflammation, a site of persistent microbial stimulation. In the absence of β-arr2, mice exhibited greater extent of mucosal inflammation determined by cellular infiltration and expression of inflammatory mediators even under homeostatic conditions. Furthermore, β-arr2-deficient mice were more susceptible to dextran sulfate sodium-induced colitis as demonstrated by greater body weight loss, higher disease activity index, and shortened colon as compared with wild-type mice. We also show that T cells from β-arr2 knockout mice exhibit altered activation status under both basal and colitic conditions, implicating their involvement in disease induction. Further assessment of the role of β-arr2 in intrinsic T-cell differentiation confirmed its importance in T-cell polarization. Using the T-cell transfer model of colitis, we demonstrate that T-cell-specific β-arr2 is important in limiting colitic inflammation; however, it plays a paradoxical role in concurrent systemic wasting disease. Together, our study highlights a critical negative regulatory role of β-arr2 in intestinal inflammation and demonstrates a distinct role of T-cell-specific β-arr2 in systemic wasting disease. PMID:26296063

  7. Short telomeres and high telomerase activity in T-cell prolymphocytic leukemia.

    PubMed

    Röth, A; Dürig, J; Himmelreich, H; Bug, S; Siebert, R; Dührsen, U; Lansdorp, P M; Baerlocher, G M

    2007-12-01

    To test the role of telomere biology in T-cell prolymphocytic leukemia (T-PLL), a rare aggressive disease characterized by the expansion of a T-cell clone derived from immuno-competent post-thymic T-lymphocytes, we analyzed telomere length and telomerase activity in subsets of peripheral blood leukocytes from 11 newly diagnosed or relapsed patients with sporadic T-PLL. Telomere length values of the leukemic T cells (mean+/-s.d.: 1.53+/-0.65 kb) were all below the 1st percentile of telomere length values observed in T cells from healthy age-matched controls whereas telomere length of normal T- and B cells fell between the 1st and 99th percentile of the normal distribution. Leukemic T cells exhibited high levels of telomerase and were sensitive to the telomerase inhibitor BIBR1532 at doses that showed no effect on normal, unstimulated T cells. Targeting the short telomeres and telomerase activity in T-PLL seems an attractive strategy for the future treatment of this devastating disease. PMID:17898784

  8. 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

  9. The Dendritic Cell Synapse: A Life Dedicated to T Cell Activation.

    PubMed

    Benvenuti, Federica

    2016-01-01

    T-cell activation within immunological synapses is a complex process whereby different types of signals are transmitted from antigen-presenting cells to T cells. The molecular strategies developed by T cells to interpret and integrate these signals have been systematically dissected in recent years and are now in large part understood. On the other side of the immune synapse, dendritic cells (DCs) participate actively in synapse formation and maintenance by remodeling of membrane receptors and intracellular content. However, the details of such changes have been only partially characterized. The DCs actin cytoskeleton has been one of the first systems to be identified as playing an important role in T-cell priming and some of the underlying mechanisms have been elucidated. Similarly, the DCs microtubule cytoskeleton undergoes major spatial changes during synapse formation that favor polarization of the DCs subcellular space toward the interacting T cell. Recently, we have begun to investigate the trafficking machinery that controls polarized delivery of endosomal vesicles at the DC-T immune synapse with the aim of understanding the functional relevance of polarized secretion of soluble factors during T-cell priming. Here, we will review the current knowledge of events occurring in DCs during synapse formation and discuss the open questions that still remain unanswered. PMID:27014259

  10. 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

  11. Developmental Heterogeneity in DNA Packaging Patterns Influences T-Cell Activation and Transmigration

    PubMed Central

    Garg, Megha; R., Indulaxmi; Perumalsamy, Lakshmi R.; Sarin, Apurva; Shivashankar, G. V.

    2012-01-01

    Cellular differentiation programs are accompanied by large-scale changes in nuclear organization and gene expression. In this context, accompanying transitions in chromatin assembly that facilitates changes in gene expression and cell behavior in a developmental system are poorly understood. Here, we address this gap and map structural changes in chromatin organization during murine T-cell development, to describe an unusual heterogeneity in chromatin organization and associated functional correlates in T-cell lineage. Confocal imaging of DNA assembly in cells isolated from bone marrow, thymus and spleen reveal the emergence of heterogeneous patterns in DNA organization in mature T-cells following their exit from the thymus. The central DNA pattern dominated in immature precursor cells in the thymus whereas both central and peripheral DNA patterns were observed in naïve and memory cells in circulation. Naïve T-cells with central DNA patterns exhibited higher mechanical pliability in response to compressive loads in vitro and transmigration assays in vivo, and demonstrated accelerated expression of activation-induced marker CD69. T-cell activation was characterized by marked redistribution of DNA assembly to a central DNA pattern and increased nuclear size. Notably, heterogeneity in DNA patterns recovered in cells induced into quiescence in culture, suggesting an internal regulatory mechanism for chromatin reorganization. Taken together, our results uncover an important component of plasticity in nuclear organization, reflected in chromatin assembly, during T-cell development, differentiation and transmigration. PMID:22957031

  12. 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

  13. The Dendritic Cell Synapse: A Life Dedicated to T Cell Activation

    PubMed Central

    Benvenuti, Federica

    2016-01-01

    T-cell activation within immunological synapses is a complex process whereby different types of signals are transmitted from antigen-presenting cells to T cells. The molecular strategies developed by T cells to interpret and integrate these signals have been systematically dissected in recent years and are now in large part understood. On the other side of the immune synapse, dendritic cells (DCs) participate actively in synapse formation and maintenance by remodeling of membrane receptors and intracellular content. However, the details of such changes have been only partially characterized. The DCs actin cytoskeleton has been one of the first systems to be identified as playing an important role in T-cell priming and some of the underlying mechanisms have been elucidated. Similarly, the DCs microtubule cytoskeleton undergoes major spatial changes during synapse formation that favor polarization of the DCs subcellular space toward the interacting T cell. Recently, we have begun to investigate the trafficking machinery that controls polarized delivery of endosomal vesicles at the DC–T immune synapse with the aim of understanding the functional relevance of polarized secretion of soluble factors during T-cell priming. Here, we will review the current knowledge of events occurring in DCs during synapse formation and discuss the open questions that still remain unanswered. PMID:27014259

  14. Prolonged Activation of Virus-Specific CD8+T Cells after Acute B19 Infection

    PubMed Central

    2005-01-01

    Background Human parvovirus B19 (B19) is a ubiquitous and clinically significant pathogen, causing erythema infectiosum, arthropathy, transient aplastic crisis, and intrauterine fetal death. The phenotype of CD8+ T cells in acute B19 infection has not been studied previously. Methods and Findings The number and phenotype of B19-specific CD8+ T cell responses during and after acute adult infection was studied using HLA–peptide multimeric complexes. Surprisingly, these responses increased in magnitude over the first year post-infection despite resolution of clinical symptoms and control of viraemia, with T cell populations specific for individual epitopes comprising up to 4% of CD8+ T cells. B19-specific T cells developed and maintained an activated CD38+ phenotype, with strong expression of perforin and CD57 and downregulation of CD28 and CD27. These cells possessed strong effector function and intact proliferative capacity. Individuals tested many years after infection exhibited lower frequencies of B19-specific cytotoxic T lymphocytes, typically 0.05%–0.5% of CD8+ T cells, which were perforin, CD38, and CCR7 low. Conclusion This is the first example to our knowledge of an “acute” human viral infection inducing a persistent activated CD8+ T cell response. The likely explanation—analogous to that for cytomegalovirus infection—is that this persistent response is due to low-level antigen exposure. CD8+ T cells may contribute to the long-term control of this significant pathogen and should be considered during vaccine development. PMID:16253012

  15. 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

  16. Vaginal Immunization to Elicit Primary T-Cell Activation and Dissemination

    PubMed Central

    Pettini, Elena; Prota, Gennaro; Ciabattini, Annalisa; Boianelli, Alessandro; Fiorino, Fabio; Pozzi, Gianni; Vicino, Antonio; Medaglini, Donata

    2013-01-01

    Primary T-cell activation at mucosal sites is of utmost importance for the development of vaccination strategies. T-cell priming after vaginal immunization, with ovalbumin and CpG oligodeoxynucleotide adjuvant as model vaccine formulation, was studied in vivo in hormone-synchronized mice and compared to the one induced by the nasal route. Twenty-four hours after both vaginal or nasal immunization, antigen-loaded dendritic cells were detected within the respective draining lymph nodes. Vaginal immunization elicited a strong recruitment of antigen-specific CD4+ T cells into draining lymph nodes that was more rapid than the one observed following nasal immunization. T-cell clonal expansion was first detected in iliac lymph nodes, draining the genital tract, and proliferated T cells disseminated towards distal lymph nodes and spleen similarly to what observed following nasal immunization. T cells were indeed activated by the antigen encounter and acquired homing molecules essential to disseminate towards distal lymphoid organs as confirmed by the modulation of CD45RB, CD69, CD44 and CD62L marker expression. A multi-type Galton Watson branching process, previously used for in vitro analysis of T-cell proliferation, was applied to model in vivo CFSE proliferation data in draining lymph nodes 57 hours following immunization, in order to calculate the probabilistic decision of a cell to enter in division, rest in quiescence or migrate/die. The modelling analysis indicated that the probability of a cell to proliferate was higher following vaginal than nasal immunization. All together these data show that vaginal immunization, despite the absence of an organized mucosal associated inductive site in the genital tract, is very efficient in priming antigen-specific CD4+ T cells and inducing their dissemination from draining lymph nodes towards distal lymphoid organs. PMID:24349003

  17. 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

  18. 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

  19. Activating and propagating polyclonal gamma delta T cells with broad specificity for malignancies

    PubMed Central

    Deniger, Drew C.; Maiti, Sourindra N.; Mi, Tiejuan; Switzer, Kirsten C.; Ramachandran, Vijaya; Hurton, Lenka V.; Ang, Sonny; Olivares, Simon; Rabinovich, Brian A.; Huls, Helen; Lee, Dean A.; Bast, Robert C.; Champlin, Richard E.; Cooper, Laurence J.N.

    2014-01-01

    Purpose To activate and propagate populations of γδT cells expressing polyclonal repertoire of γ and δ TCR chains for adoptive immunotherapy for cancer, which has yet to be achieved. Experimental Design Clinical-grade artificial antigen presenting cells (aAPC) derived from K562 tumor cells were used as irradiated feeders to activate and expand human γδT cells to clinical scale. These cells were tested for proliferation, TCR expression, memory phenotype, cytokine secretion, and tumor killing. Results γδT cell proliferation was dependent upon CD137L expression on aAPC and addition of exogenous IL-2 and IL-21. Propagated γδT cells were polyclonal as they expressed Vδ1, Vδ2, Vδ3, Vδ5, Vδ7, and Vδ8 with Vγ2, Vγ3, Vγ7, Vγ8, Vγ9, Vγ10, and Vγ11 TCR chains. Interferon-γ production by Vδ1, Vδ2, and Vδ1negVδ2neg subsets was inhibited by pan-TCRγδantibody when added to co-cultures of polyclonal γδT cells and tumor cell lines. Polyclonal γδT cells killed acute and chronic leukemia, colon, pancreatic, and ovarian cancer cell lines, but not healthy autologous or allogeneic normal B cells. Blocking antibodies demonstrated that polyclonal γδT cells mediated tumor cell lysis through combination of DNAM1, NKG2D, and TCRγδ. The adoptive transfer of activated and propagated γδT cells expressing polyclonal versus defined Vδ TCR chains imparted a hierarchy (polyclonal>Vδ1>Vδ1negVδ2neg>Vδ2) of survival of mice with ovarian cancer xenografts. Conclusions Polyclonal γδT cells can be activated and propagated with clinical-grade aAPC and demonstrate broad anti-tumor activities, which will facilitate the implementation of γδT cell cancer immunotherapies in humans. PMID:24833662

  20. 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

  1. In vivo activated cytotoxic T cells in the thyroid infiltrate of patients with Hashimoto's thyroiditis.

    PubMed Central

    Del Prete, G F; Vercelli, D; Tiri, A; Maggi, E; Mariotti, S; Pinchera, A; Ricci, M; Romagnani, S

    1986-01-01

    High proportions of T8+ cells with inverted T4/T8 ratio were found in freshly isolated thyroid lymphocytes from patients with Hashimoto's thyroiditis. In addition, about one third of thyroid infiltrating cells expressed the TAC antigen, whereas in patient peripheral blood (PB) or normal lymphocytes from PB or lymphoid organs the percentage of TAC-positive cells was consistently lower than 10%. Following negative selection with OKT4 or OKT8 monoclonal antibodies and complement, TAC+ T cells were enriched in the T8+ cell population. Thyroid infiltrating T cells from two patients underwent two different cloning procedures. In the first, single T cells were initially activated with phytohaemagglutinin (PHA) and interleukin 2 (IL-2), in the other with recombinant IL-2 (rIL-2) alone. The majority of T cell clones obtained by initial PHA-stimulation (55-65%) had the T8+ phenotype, but the frequency of T8+ clones obtained by stimulating T cells with rIL-2 alone was even higher (78 & 71%, respectively). The majority of T8+ clones elicited by PHA (35/37 & 36/38) and all the T8+ clones (36/36 & 22/22) obtained from thyroid infiltrates with initial stimulation by rIL-2 displayed cytolytic activity. Most of cytolytic T8+ clones obtained from thyroid infiltrates with both cloning procedures, displayed NK activity against human K562 and MOLT-4 target cells, but not against a NK-resistant target, such as Raji cells. These data suggest that in Hashimoto's disease a considerable proportion of thyroid infiltrating T cells are in vivo activated T8+ cytolytic T cells with NK activity, which may be of importance in determining or maintaining the tissue damage of the target gland. PMID:3024884

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

    PubMed

    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; Yu, Xue-Zhong

    2016-04-01

    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

  3. 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

  4. 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

  5. Differential effects of mercury, lead, and cadmium on IL-2 production by Jurkat T cells.

    PubMed

    Colombo, Myrian; Hamelin, Claudine; Kouassi, Edouard; Fournier, Michel; Bernier, Jacques

    2004-06-01

    Mercury, lead, and cadmium are widespread and highly toxic pollutants. The aim of this study was to determine the effects of sublethal doses of CH(3)HgCl, CdCl(2), and PbCl(2) on IL-2 production by T lymphocytes. Jurkat T cells were stimulated by triggering CD3 and CD28 molecules before, in conjunction with, or following heavy metal exposure. Heavy metals, individually or mixed together at equimolar concentrations, were used. Results demonstrated that low, noncytotoxic doses of metals induce tyrosine phosphorylation. Mercury and lead (1 microM) inhibit IL-2 production regardless of the state of T cell activation. Cadmium stimulated IL-2 production only in preactivated T cells. Surprisingly, a mixture of these three metals had no effect. We subsequently determined the effects of heavy metals on NFAT (nuclear factors of activated T cells) activity. When cells were stimulated by potent stimulation involving the CD3 and CD28 molecules, an increased NFAT activation was noted when the cells were exposed to mercury and to the metal mixture. Activation with PMA/calcium ionophores indicated that the target of heavy metals is located downstream from PKC and calcium mobilization. These results suggest that the state and mode of T cell activation are important parameters to consider in heavy metal toxicity. PMID:15183152

  6. Nuclear Envelope Lamin-A Couples Actin Dynamics with Immunological Synapse Architecture and T Cell Activation

    PubMed Central

    González-Granado, José María; Trigueros-Motos, Laia; Cibrián, Danay; Morlino, Giulia; Blanco-Berrocal, Marta; Osorio, Fernando Garcia; Freije, José María Pérez; López-Otín, Carlos; Sánchez-Madrid, Francisco; Andrés, Vicente

    2014-01-01

    In many cell types, nuclear A-type lamins have been implicated in structural and functional activities, including higher-order genome organization, DNA replication and repair, gene transcription, and signal transduction. However, their role in specialized immune cells remains largely unexplored. Here, we showed that the abundance of A-type lamins is almost negligible in resting naïve T lymphocytes, but that it is substantially increased upon activation of the T cell receptor (TCR), and is an early event that accelerates formation of the immunological synapse between T cells and antigen-presenting cells. We found that lamin-A enhanced the polymerization of F-actin in T cells, a critical step for immunological synapse formation, by physically connecting the nucleus to the plasma membrane through the linker of nucleoskeleton and cytoskeleton (LINC) complex. We also showed that lamin-A played a key role in other membrane, cytoplasmic, and nuclear events related to TCR activation, including receptor-clustering, downstream signaling, and target gene expression. Notably, the presence of lamin-A was associated with enhanced extracellular signal–regulated kinase 1/2 signaling, and pharmacological inhibition of this pathway reduced the extent of lamin-A–dependent T cell activation. Moreover, mice deficient in lamin-A exhibited impaired T cell responses in vivo. These findings underscore the importance of A-type lamins for TCR activation, and identify lamin-A as a previously unappreciated regulator of the immune response. PMID:24757177

  7. 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. PMID:27354546

  8. Polymerization-dependent activation of porcine γδ T-cells by proanthocyanidins.

    PubMed

    Williams, Andrew R; Fryganas, Christos; Reichwald, Kirsten; Skov, Søren; Mueller-Harvey, Irene; Thamsborg, Stig M

    2016-04-01

    Plant-derived proanthocyanidins (PAC) have been promoted as a natural method of improving health and immune function in livestock. It has previously been shown that PAC are effective agonists for activating ruminant γδ T-cells in vitro, however effects on other livestock species are not yet clear. Moreover, the fine structural characteristics of the PAC which contribute to this stimulatory effect have not been elucidated. Here, we demonstrate activation of porcine γδ T-cells by PAC via up-regulation of CD25 (IL-2Rα) and show that 1) activation is dependent on degree of polymerization (DP), with PAC fractions containing polymers with mean DP >6 significantly more effective than fractions with mean DP <6, whilst flavan-3-ol monomers (the constituent monomeric units of PAC) did not induce CD25 expression and 2) both procyanidin and prodelphinidin-type PAC are effective agonists. Furthermore, we show that this effect of PAC is restricted to the γδ T-cell population within porcine peripheral mononuclear cells as significant CD25 up-regulation was not observed in non γδ T-cells, and no activation (via CD80/86 up-regulation) was evident in monocytes. Our results show that dietary PAC may contribute to enhancement of innate immunity in swine via activation of γδ T-cells. PMID:27033935

  9. 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. PMID:27100879

  10. Human Dendritic Cells Activated by TSLP and CD40L Induce Proallergic Cytotoxic T Cells

    PubMed Central

    Gilliet, Michel; Soumelis, Vassili; Watanabe, Norihiko; Hanabuchi, Shino; Antonenko, Svetlana; de Waal-Malefyt, Rene; Liu, Yong-Jun

    2003-01-01

    Human thymic stromal lymphopoietin (TSLP) is a novel epithelial cell–derived cytokine, which induces dendritic cell (DC)-mediated CD4+ T cell responses with a proallergic phenotype. Although the participation of CD8+ T cells in allergic inflammation is well documented, their functional properties as well as the pathways leading to their generation remain poorly understood. Here, we show that TSLP-activated CD11c+ DCs potently activate and expand naive CD8+ T cells, and induce their differentiation into interleukin (IL)-5 and IL-13–producing effectors exhibiting poor cytolytic activity. Additional CD40L triggering of TSLP-activated DCs induced CD8+ T cells with potent cytolytic activity, producing large amounts of interferon (IFN)-γ, while retaining their capacity to produce IL-5 and IL-13. These data further support the role of TSLP as initial trigger of allergic T cell responses and suggest that CD40L-expressing cells may act in combination with TSLP to amplify and sustain pro-allergic responses and cause tissue damage by promoting the generation of IFN-γ–producing cytotoxic effectors. PMID:12707303

  11. 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

  12. IL-1β-dependent activation of dendritic epidermal T cells in contact hypersensitivity1

    PubMed Central

    Nielsen, Morten M.; Lovato, Paola; MacLeod, Amanda S.; Witherden, Deborah A.; Skov, Lone; Dyring-Andersen, Beatrice; Dabelsteen, Sally; Woetmann, Anders; Ødum, Niels; Havran, Wendy L.; Geisler, Carsten; Bonefeld, Charlotte M.

    2014-01-01

    Substances that penetrate the skin surface can act as allergens and induce a T cell-mediated inflammatory skin disease called contact hypersensitivity (CHS). IL-17 is a key cytokine in CHS and was originally thought to be produced solely by CD4+ T cells. However, it is now known that several cell types including γδ T cells can produce IL-17. Here, we determine the role of γδ T cells, especially the dendritic epidermal T cells (DETC), in CHS. By use of a well-established model for CHS where dinitroflourobenzen (DNFB) is used as allergen, we found that γδ T cells are important players in CHS. Thus, an increased number of IL-17 producing DETC appear in the skin following exposure to DNFB in WT mice, and DNFB-induced ear-swelling is reduced by approximately 50% in TCRδ−/− mice compared to WT mice. In accordance, DNFB-induced ear-swelling was reduced by approximately 50% in IL-17−/− mice. We show that DNFB triggers DETC activation and IL-1β production in the skin, and that keratinocytes produce IL-1β when stimulated with DNFB. We find that DETC activated in vitro by incubation with anti-CD3 and IL-1β produce IL-17. Importantly, we demonstrate that the IL-1 receptor antagonist anakinra significantly reduces CHS responses as measured by decreased ear-swelling, inhibition of local DETC activation and a reduction in the number of IL-17+ γδ T cells and DETC in the draining lymph nodes. Taken together, we show that DETC become activated and produce IL-17 in an IL-1β-dependent manner during CHS suggesting a key role for DETC in CHS. PMID:24600030

  13. 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.

  14. Complexity of the primary genetic response to mitogenic activation of human T cells.

    PubMed Central

    Zipfel, P F; Irving, S G; Kelly, K; Siebenlist, U

    1989-01-01

    We 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 cyclosporin 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. Images PMID:2498643

  15. Role of acid sphingomyelinase bioactivity in human CD4+ T-cell activation and immune responses

    PubMed Central

    Bai, A; Kokkotou, E; Zheng, Y; Robson, S C

    2015-01-01

    Acid sphingomyelinase (ASM), a lipid hydrolase enzyme, has the potential to modulate various cellular activation responses via the generation of ceramide and by interaction with cellular receptors. We have hypothesized that ASM modulates CD4+ T-cell receptor activation and impacts immune responses. We first observed interactions of ASM with the intracellular domains of both CD3 and CD28. ASM further mediates T-cell proliferation after anti-CD3/CD28 antibody stimulation and alters CD4+ T-cell activation signals by generating ceramide. We noted that various pharmacological inhibitors of ASM or knockdown of ASM using small hairpin RNA inhibit CD3/CD28-mediated CD4+ T-cell proliferation and activation. Furthermore, such blockade of ASM bioactivity by biochemical inhibitors and/or molecular-targeted knockdown of ASM broadly abrogate T-helper cell responses. In conclusion, we detail immune, pivotal roles of ASM in adaptive immune T-cell responses, and propose that these pathways might provide novel targets for the therapy of autoimmune and inflammatory diseases. PMID:26203857

  16. 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

  17. Effects of dietary n-3 fatty acids on T cell activation and T cell receptor-mediated signaling in a murine model.

    PubMed

    McMurray, D N; Jolly, C A; Chapkin, R S

    2000-09-01

    A short-term feeding paradigm in mice, with diets enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), was used to study the modulation of T cell activation via the T cell receptor (TcR) and the downstream pathways of intracellular signaling. Diets enriched in EPA and DHA suppressed antigen-specific delayed hypersensitivity reactions and mitogen-induced proliferation of T cells. Cocultures of accessory cells and T cells from mice given different diets revealed that purified fatty acid ethyl esters acted directly on the T cell, rather than through the accessory cell. The loss of proliferative capacity was accompanied by reductions in interleukin (IL)-2 secretion and IL-2 receptor alpha chain mRNA transcription, suggesting that dietary EPA and DHA act, in part, by interrupting the autocrine IL-2 activation pathway. Dietary EPA and DHA blunted the production of intracellular second messengers, including diacylglycerol and ceramide, following mitogen stimulation in vitro. Dietary effects appear to vary with the agonist employed (i.e., anti-CD3 [TcR], anti-CD28, exogenous IL-2, or phorbol myristate acetate and ionomycin). PMID:10944491

  18. CD4+ T Cell Depletion, Immune Activation and Increased Production of Regulatory T Cells in the Thymus of HIV-Infected Individuals

    PubMed Central

    Bandera, Alessandra; Ferrario, Giulio; Saresella, Marina; Marventano, Ivana; Soria, Alessandro; Zanini, Fabio; Sabbatini, Francesca; Airoldi, Monica; Marchetti, Giulia; Franzetti, Fabio; Trabattoni, Daria; Clerici, Mario; Gori, Andrea

    2010-01-01

    Mechanisms by which HIV affects the thymus are multiple and only partially known, and the role of thymic dysfunction in HIV/AIDS immunopathogenesis remains poorly understood. To evaluate the effects of HIV infection on intra-thymic precursors of T cells in HIV-infected adults, we conducted a detailed immunophenotypic study of thymic tissue isolated from 7 HIV-infected and 10 HIV-negative adults who were to undergo heart surgery. We found that thymuses of HIV-infected individuals were characterized by a relative depletion of CD4+ single positive T cells and a corresponding enrichment of CD8+ single positive T cells. In addition, thymocytes derived from HIV-infected subjects showed increased levels of activated and proliferating cells. Our analysis also revealed a decreased expression of interleukin-7 receptor in early thymocytes from HIV-infected individuals, along with an increase in this same expression in mature double- and single-positive cells. Frequency of regulatory T cells (CD25+FoxP3+) was significantly increased in HIV-infected thymuses, particularly in priorly-committed CD4 single positive cells. Our data suggest that HIV infection is associated with a complex set of changes in the immunophenotype of thymocytes, including a reduction of intrathymic CD4+ T cell precursors, increased expression of activation markers, changes in the expression pattern of IL-7R and enrichment of T regulatory cells generation. PMID:20520721

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

    PubMed

    Bertin, Samuel; Aoki-Nonaka, Yukari; de Jong, Petrus Rudolf; Nohara, Lilian L; Xu, Hongjian; 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

    2014-11-01

    TRPV1 is a Ca(2+)-permeable channel studied mostly as a pain receptor in sensory neurons. However, its role in other cell types is poorly understood. Here we found that TRPV1 was functionally expressed in CD4(+) T cells, where it acted as a non-store-operated Ca(2+) channel and contributed to T cell antigen receptor (TCR)-induced Ca(2+) influx, TCR signaling and T cell activation. In models of T cell-mediated colitis, TRPV1 promoted colitogenic T cell responses and intestinal inflammation. Furthermore, genetic and pharmacological inhibition of TRPV1 in human CD4(+) T cells recapitulated the phenotype of mouse Trpv1(-/-) CD4(+) T cells. Our findings suggest that inhibition of TRPV1 could represent a new therapeutic strategy for restraining proinflammatory T cell responses. PMID:25282159

  20. Antigen Presentation and T-Cell Activation Are Critical for RBP4-Induced Insulin Resistance.

    PubMed

    Moraes-Vieira, Pedro M; Castoldi, Angela; Aryal, Pratik; Wellenstein, Kerry; Peroni, Odile D; Kahn, Barbara B

    2016-05-01

    Adipose tissue (AT) inflammation contributes to impaired insulin action, which is a major cause of type 2 diabetes. RBP4 is an adipocyte- and liver-derived protein with an important role in insulin resistance, metabolic syndrome, and AT inflammation. RBP4 elevation causes AT inflammation by activating innate immunity, which elicits an adaptive immune response. RBP4-overexpressing mice (RBP4-Ox) are insulin resistant and glucose intolerant and have increased AT macrophages and T-helper 1 cells. We show that high-fat diet-fed RBP4(-/-) mice have reduced AT inflammation and improved insulin sensitivity versus wild type. We also elucidate the mechanism for RBP4-induced macrophage antigen presentation and subsequent T-cell activation. In RBP4-Ox, AT macrophages display enhanced c-Jun N-terminal kinase, extracellular signal-related kinase, and p38 phosphorylation. Inhibition of these pathways and of NF-κB reduces activation of macrophages and CD4 T cells. MyD88 is an adaptor protein involved in proinflammatory signaling. In macrophages from MyD88(-/-) mice, RBP4 fails to stimulate secretion of tumor necrosis factor, IL-12, and IL-6 and CD4 T-cell activation. In vivo blockade of antigen presentation by treating RBP4-Ox mice with CTLA4-Ig, which blocks costimulation of T cells, is sufficient to reduce AT inflammation and improve insulin resistance. Thus, MyD88 and downstream mitogen-activated protein kinase and NF-κB pathways are necessary for RBP4-induced macrophage antigen presentation and subsequent T-cell activation. Also, blocking antigen presentation with CTLA4-Ig improves RBP4-induced insulin resistance and macrophage-induced T-cell activation. PMID:26936962

  1. Increased frequency of activated T-cells in the Helicobacter pylori-infected antrum and duodenum.

    PubMed

    Strömberg, E; Lundgren, A; Edebo, A; Lundin, S; Svennerholm, A-M; Lindholm, C

    2003-05-25

    Helicobacter pylori colonize the human stomach and duodenum. The infection has been shown to induce a strong T-cell response in the stomach, whereas the response within the duodenum has been poorly characterized. Furthermore, it remains to be elucidated whether the T-cell response may contribute to ulcer formation in the host. In this study, the frequency of different T-cell subsets, their degree of activation and expression of co-stimulatory receptors in biopsies from the duodenum as well as the antrum were studied by immunohistochemistry and flow cytometry. It was also evaluated whether there are differences in the T-cell responses between duodenal ulcer patients and asymptomatic carriers that might explain why only 10-15% of the infected subjects develop duodenal ulcers. The frequencies of CD4+, CD8+ and CD45RO+, i.e. memory T-cells, were significantly increased in the antrum, and the number of CD25+ cells was considerably higher in both the antrum and duodenum of duodenal ulcer patients and asymptomatic carriers as compared to uninfected individuals. Interestingly, the levels of immunosuppressive CTLA-4+ cells were significantly higher in the duodenum of duodenal ulcer patients, as compared to the asymptomatic carriers. H. pylori cause activation of T-cells in the duodenum as well as in the stomach. Our observation of higher levels of CTLA-4+ cells in the duodenum of duodenal ulcer patients than in the asymptomatic carriers suggests that a suppressive T-cell response may be related to the development of duodenal ulcers. PMID:12738386

  2. TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation

    PubMed Central

    Lillemeier, Björn F; Mörtelmaier, Manuel A; Forstner, Martin B; Huppa, Johannes B; Groves, Jay T; Davis, Mark M

    2010-01-01

    The organization and dynamics of receptors and other molecules in the plasma membrane are not well understood. Here we analyzed the spatio-temporal dynamics of T cell antigen receptor (TCR) complexes and linker for activation of T cells (Lat), a key adaptor molecule in the TCR signaling pathway, in T cell membranes using high-speed photoactivated localization microscopy, dual-color fluorescence cross-correlation spectroscopy and transmission electron microscopy. In quiescent T cells, both molecules existed in separate membrane domains (protein islands), and these domains concatenated after T cell activation. These concatemers were identical to signaling microclusters, a prominent hallmark of T cell activation. This separation versus physical juxtapositioning of receptor domains and domains containing downstream signaling molecules in quiescent versus activated T cells may be a general feature of plasma membrane–associated signal transduction. PMID:20010844

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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...

  12. 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

  13. 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

  14. Clonal expansions of activated gamma/delta T cells in recent-onset multiple sclerosis.

    PubMed Central

    Shimonkevitz, R; Colburn, C; Burnham, J A; Murray, R S; Kotzin, B L

    1993-01-01

    Multiple sclerosis (MS) is a chronic disease characterized by focal demyelination of the white matter of the brain and spinal cord. Central nervous system damage appears to be mediated by infiltrating T lymphocytes and macrophages, and a central role for autoreactive CD4+ T cells has been proposed. However, the initial immune events that lead to the chronic process of MS remain unidentified. We now present evidence that a subset of T lymphocytes bearing gamma/delta T-cell antigen receptors has been activated in patients with recent-onset disease. Cells recovered from the cerebrospinal fluid of subjects with MS were cultured for short periods of time in medium supplemented with T-cell growth factors. Expansions of V delta 1 and V delta 2 T-cell receptor-bearing lymphocytes were found only in cell populations obtained from subjects with recent-onset disease. Similar populations were not expanded in subjects with chronic MS or other neurological diseases. Junctional region sequencing showed the expanded gamma/delta T cells to be oligoclonal in nature, suggestive of specific stimulation by antigen. These results reveal a fundamental difference in the immunopathogenesis of acute vs. chronic disease and provide additional insight into the autoimmune nature of MS. PMID:8430106

  15. Cytolytic activity in T cell clones derived from human synovial rheumatoid membrane: inhibition by synovial fluid.

    PubMed Central

    Miltenburg, A M; Van Laar, J M; De Kuiper, P; Daha, M R; Breedveld, F C

    1990-01-01

    A panel of T cell clones was derived from the synovial membrane of a patient with rheumatoid arthritis (RA). We investigated whether T cell clones with cytolytic properties were present and whether T cell cytotoxicity was influenced by the presence of synovial fluid. These issues were studied using anti-CD3 and lectin-induced cytotoxicity assays. The majority of the T cell clones derived from the synovial membrane showed cytotoxic properties although non-cytotoxic clones were also found. Three clones (N11, N6 and N15) showed strong cytotoxicity (more than 40% lysis at an effector-to-target cell ratio of 10:1) whereas three clones (N16, N4 and N14) were non-cytotoxic (less than 20% lysis at an effector-to-target cell ratio of 10:1). The induction of cytotoxicity in the anti-CD3-driven system was shown to be dependent on the dose of anti-CD3 present. When synovial fluid was added to these assays a strong inhibition of cytotoxicity was found. This inhibition of cytotoxicity was found with synovial fluid samples of RA patients, as well as with non-RA synovial fluids. Both anti-CD3 and lectin-dependent cytotoxicity assays were strongly inhibited. In conclusion, T cell clones with cytotoxic activity can be isolated from rheumatoid synovial membrane. In the presence of synovial fluid these cytotoxic cells are inhibited to exert their cytotoxic function. PMID:2148285

  16. Active targeting of chemotherapy to disseminated tumors using nanoparticle-carrying T cells

    PubMed Central

    Huang, Bonnie; Abraham, Wuhbet D.; Zheng, Yiran; Bustamante López, Sandra C.; Luo, Samantha S.; Irvine, Darrell J.

    2015-01-01

    Tumor cells disseminate into compartments that are poorly accessible from circulation, which necessitates high doses of systemic chemotherapy. However, the effectiveness of many drugs, such as the potent topoisomerase I poison SN-38, are hampered by poor pharmacokinetics. To deliver SN-38 to lymphoma tumors in vivo, we took advantage of the fact that healthy lymphocytes can be programmed to phenocopy the biodistribution of the tumor cells. In a murine model of disseminated lymphoma, we expanded autologous polyclonal T cells ex vivo under conditions that retained homing receptors mirroring lymphoma cells, and functionalized these T cells to carry SN-38–loaded nanocapsules on their surfaces. Nanocapsule-functionalized T cells were resistant to SN-38, but mediated efficient killing of lymphoma cells in vitro. Upon adoptive transfer into tumor-bearing mice, these T cells served as active vectors to deliver the chemotherapeutic into tumor-bearing lymphoid organs. Cell-mediated delivery concentrated SN-38 in lymph nodes at levels 90-fold greater than free drug systemically administered at 10-fold higher doses. The live T cell delivery approach reduced tumor burden significantly after two weeks of treatment and enhanced survival under conditions where free SN-38 and SN-38-loaded nanocapsules alone were ineffective. These results suggest that tissue-homing lymphocytes can serve as specific targeting agents to deliver nanoparticles into sites difficult to access from the circulation, and thus improve the therapeutic index of chemotherapeutic drugs with unfavorable pharmacokinetics. PMID:26062846

  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. 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, because they also regulate other cellular activities and even opposite activities in some cases. The second one is why these many kinases, some of which might have redundant functions, are needed to regulate NFAT5 activity. This review reiterates the concept of signaling through cooperation. Cells need these kinases working in a coordinated way to provide the signaling specificity that is lacking in the individual one. Redundancy in regulation of NFAT5 is a critical strategy for cells to

  19. T-cell activation is required for efficient replication of human herpesvirus 6.

    PubMed Central

    Frenkel, N; Schirmer, E C; Katsafanas, G; June, C H

    1990-01-01

    We have investigated whether T-cell activation is required for the replication of the T-lymphotropic human herpesvirus 6. The virus did not replicate in quiescent peripheral blood lymphocytes but replicated efficiently following exposure of the cells to the polyclonal mitogen phytohemagglutinin (PHA). When purified T cells were treated with PHA in the absence of accessory cells, no virus replication was observed unless exogenous interleukin-2 (IL-2) was added to the medium, promoting cell division. Incubation of peripheral blood lymphocytes in the absence of PHA but in the presence of IL-2 resulted in delayed cell blastogenesis and virus replication. Cell blastogenesis and virus replication did not occur in the purified T-cell cultures incubated with IL-2 alone. Taken together, the results show that human herpesvirus 6 replication requires full progression of the cell cycle. This finding might have implications for the pathogenicity of the virus in the human host. Images PMID:2166835

  20. Nuclear envelope lamin-A as a coordinator of T cell activation

    PubMed Central

    Rocha-Perugini, Vera; González-Granado, José M

    2014-01-01

    Nuclear lamins A/C control several critical cellular functions, e.g., chromatin organization, gene transcription, DNA replication, DNA damage responses, cell cycle progression, cell differentiation, and cell polarization during migration. However, few studies have addressed the role of lamins A/C in the control of the functions of immune cells. Recently, we have demonstrated that lamins A/C are induced in T cells upon antigen recognition. Lamins A/C enhance T cell responses by coupling the plasma membrane to the nucleus via the linker of nucleoskeleton and cytoskeleton (LINC) complex and the actin cytoskeleton. Here, we discuss the possible physiological relevance and functional context of lamin A/C in T cell activation and propose a model in which lamins A/C are key modulators of immune cell functions. PMID:25482193

  1. CD8+ T Cell-Independent Immune-Mediated Mechanisms of Anti-Tumor Activity

    PubMed Central

    Pluhar, G. Elizabeth; Pennell, Christopher A.; Olin, Michael R.

    2016-01-01

    Despite the growing number of preclinical and clinical trials focused on immunotherapy for the treatment of malignant gliomas, the prognosis for this disease remains grim. Cancer immunotherapy seeks to recruit an effective immune response to eliminate tumor cells. To date, cancer vaccines have shown only limited effectiveness because of our incomplete understanding of the necessary effector cells and mechanisms that yield efficient tumor clearance. CD8+ T cell cytotoxic activity has long been proposed as the primary effector function necessary for tumor regression. However, there is increasing evidence that indicates that components of the immune system other than CD8+ T cells play important roles in tumor eradication and control. The following review should provide an understanding of the mechanisms involved in an effective antitumor response to guide future therapeutic designs. The information provided suggests an alternate means of effective tumor clearance in malignant glioma to the canonical CD8+ cytotoxic T cell mechanism. PMID:26351148

  2. Effect of antiviral treatment of chronic hepatitis C on the frequency of regulatory T cells, T-cell activation, and serum levels of TGF-beta.

    PubMed

    Chalupa, Pavel; Davidová, Alžběta; Beran, Ondřej; Arientová, Simona; Boštík, Pavel; Kapla, Jaroslav; Kondělková, Kateřina; Plíšek, Stanislav; Holub, Michal

    2016-08-01

    The aim was to analyze T-regulatory cells (Tregs), activated CD8(+) T cells, and transforming growth factor-beta (TGF)-β in hepatitis C patients. We enrolled 31 patients with chronic genotype 1 hepatitis C virus (HCV) infection, 30 seropositive persons with spontaneous HCV elimination, and 23 healthy volunteers. The patients were examined at the beginning of the interferon-alpha (IFN-α)-based therapy (baseline) and at weeks 4 (W4) and 12 (W12) of the therapy. The percentage of Tregs and the expression of activation markers CD38 and HLA-DR on CD8(+) T cells were analyzed in the peripheral blood by flow cytometry. Serum levels of TGF-β were measured in a multiplex assay using flow cytometry. The percentage of Tregs in patients was higher than in controls and seropositive persons. Similarly, the percentage of CD8(+) T cells expressing CD38 and HLA-DR was higher in patients compared with controls and seropositive persons. Chronic HCV infection is associated with elevated circulating Tregs and activated CD8(+) T cells. During IFN-α-based therapy these cells gradually increase, whereas TGF-β serum levels decrease. PMID:27307383

  3. Narcolepsy: autoimmunity, effector T cell activation due to infection, or T cell independent, major histocompatibility complex class II induced neuronal loss?

    PubMed

    Fontana, Adriano; Gast, Heidemarie; Reith, Walter; Recher, Mike; Birchler, Thomas; Bassetti, Claudio L

    2010-05-01

    Human narcolepsy with cataplexy is a neurological disorder, which develops due to a deficiency in hypocretin producing neurons in the hypothalamus. There is a strong association with human leucocyte antigens HLA-DR2 and HLA-DQB1*0602. The disease typically starts in adolescence. Recent developments in narcolepsy research support the hypothesis of narcolepsy being an immune-mediated disease. Narcolepsy is associated with polymorphisms of the genes encoding T cell receptor alpha chain, tumour necrosis factor alpha and tumour necrosis factor receptor II. Moreover the rate of streptococcal infection is increased at onset of narcolepsy. The hallmarks of anti-self reactions in the tissue--namely upregulation of major histocompatibility antigens and lymphocyte infiltrates--are missing in the hypothalamus. These findings are questionable because they were obtained by analyses performed many years after onset of disease. In some patients with narcolepsy autoantibodies to Tribbles homolog 2, which is expressed by hypocretin neurons, have been detected recently. Immune-mediated destruction of hypocretin producing neurons may be mediated by microglia/macrophages that become activated either by autoantigen specific CD4(+) T cells or superantigen stimulated CD8(+) T cells, or independent of T cells by activation of DQB1*0602 signalling. Activation of microglia and macrophages may lead to the release of neurotoxic molecules such as quinolinic acid, which has been shown to cause selective destruction of hypocretin neurons in the hypothalamus. PMID:20403960

  4. Calcineurin/NFAT Signaling Represses Genes Vamp1 and Vamp2 via PMCA-Dependent Mechanism during Dopamine Secretion by Pheochromocytoma Cells

    PubMed Central

    Kosiorek, Michalina; Zylinska, Ludmila; Zablocki, Krzysztof; Pikula, Slawomir

    2014-01-01

    Background Plasma membrane Ca2+-ATPases (PMCA) extrude Ca2+ ions out of the cell and contribute to generation of calcium oscillations. Calcium signaling is crucial for transcriptional regulation of dopamine secretion by neuroendocrine PC12 cells. Low resting [Ca2+]c in PC12 cells is maintained mainly by two Ca2+-ATPases, PMCA2 and PMCA3. Recently, we found that Ca2+ dependent phosphatase calcineurin was excessively activated under conditions of experimental downregulation of PMCA2 or PMCA3. Thus, the aim of this study was to explain if, via modulation of the Ca2+/calcineurin-dependent nuclear factor of activated T cells (NFAT) pathway, PMCA2 and PMCA3 affect intracellular signaling in pheochromocytoma/neuronal cells/PC12 cells. Secondly, we tested whether this might influence dopamine secretion by PC12 cells. Results PMCA2- and PMCA3-deficient cells displayed profound decrease in dopamine secretion accompanied by a permanent increase in [Ca2+]c. Reduction in secretion might result from changes in NFAT signaling, following altered PMCA pattern. Consequently, activation of NFAT1 and NFAT3 transcription factors was observed in PMCA2- or PMCA3-deficient cells. Furthermore, chromatin immunoprecipitation assay indicated that NFATs could be involved in repression of Vamp genes encoding vesicle associated membrane proteins (VAMP). Conclusions PMCA2 and PMCA3 are crucial for dopamine secretion in PC12 cells. Reduction in PMCA2 or PMCA3 led to calcium-dependent activation of calcineurin/NFAT signaling and, in consequence, to repression of the Vamp gene and deterioration of the SNARE complex formation in PC12 cells. PMID:24667359

  5. CTLA4-Ig immunosuppressive activity at the level of dendritic cell/T cell crosstalk

    PubMed Central

    Mayer, Edda; Hölzl, Markus; Ahmadi, Sarah; Dillinger, Barbara; Pilat, Nina; Fuchs, Dietmar; Wekerle, Thomas; Heitger, Andreas

    2013-01-01

    Immunosuppressive cytotoxic T lymphocyte associated antigen-4 immunoglobulin fusion proteins (CTLA4-Ig) block the CD28:CD80/86 costimulatory pathway. On a cellular level, CTLA4-Ig is understood to dampen T cell responses. As a mechanism, CTLA4-Ig has been reported to affect dendritic cell (DC) function via inducing the immunosuppressive indoleamine 2,3 dioxygenase (IDO) pathway and promoting a DC regulatory phenotype. We here probed cellular mechanisms of CTLA4-Ig immunoregulation in an allogeneic setting using C57BL/6 splenic or bone marrow derived DCs (BMDCs) as stimulators of allogeneic Balb/c derived T cells. To address whether CTLA4-Ig immunosuppression affected DCs, we pre-exposed C57BL/6 splenic or BMDCs to CTLA4-Ig and removed unbound CTLA4-Ig before co-culture with allogeneic T cells. CTLA4-Ig disappeared rapidly (within 4 h) from the cell membrane by combined internalization and dissociation. These CTLA4-Ig pre-exposed DCs were fully capable of stimulating allogeneic T cell proliferation, suggesting that CTLA4-Ig does not impair the DC stimulatory capacity. Only the presence of CTLA4-Ig during DC/T cell co-culture resulted in the expected inhibition of proliferation. C57BL/6 splenic or BMDCs exposed to CTLA4-Ig did not display IDO activity. We conclude that CTLA4-Ig immunosuppressive activity does not depend on a DC regulatory phenotype but on its presence during DC/T cell interaction. PMID:23434857

  6. Systematic identification of regulatory proteins critical for T-cell activation

    PubMed Central

    Chu, Peter; Pardo, Jorge; Zhao, Haoran; Li, Connie C; Pali, Erlina; Shen, Mary M; Qu, Kunbin; Yu, Simon X; Huang, Betty CB; Yu, Peiwen; Masuda, Esteban S; Molineaux, Susan M; Kolbinger, Frank; Aversa, Gregorio; de Vries, Jan; Payan, Donald G; Liao, X Charlene

    2003-01-01

    Background The activation of T cells, mediated by the T-cell receptor (TCR), activates a battery of specific membrane-associated, cytosolic and nuclear proteins. Identifying the signaling proteins downstream of TCR activation will help us to understand the regulation of immune responses and will contribute to developing therapeutic agents that target immune regulation. Results In an effort to identify novel signaling molecules specific for T-cell activation we undertook a large-scale dominant effector genetic screen using retroviral technology. We cloned and characterized 33 distinct genes from over 2,800 clones obtained in a screen of 7 × 108 Jurkat T cells on the basis of a reduction in TCR-activation-induced CD69 expression after expressing retrovirally derived cDNA libraries. We identified known signaling molecules such as Lck, ZAP70, Syk, PLCγ1 and SHP-1 (PTP1C) as truncation mutants with dominant-negative or constitutively active functions. We also discovered molecules not previously known to have functions in this pathway, including a novel protein with a RING domain (found in a class of ubiquitin ligases; we call this protein TRAC-1), transmembrane molecules (EDG1, IL-10Rα and integrin α2), cytoplasmic enzymes and adaptors (PAK2, A-Raf-1, TCPTP, Grb7, SH2-B and GG2-1), and cytoskeletal molecules (moesin and vimentin). Furthermore, using truncated Lck, PLCγ1, EDG1 and PAK2 mutants as examples, we showed that these dominant immune-regulatory molecules interfere with IL-2 production in human primary lymphocytes. Conclusions This study identified important signal regulators in T-cell activation. It also demonstrated a highly efficient strategy for discovering many components of signal transduction pathways and validating them in physiological settings. PMID:12974981

  7. A virtual lymph node model to dissect the requirements for T-cell activation by synapses and kinapses.

    PubMed

    Moreau, Hélène D; Bogle, Gib; Bousso, Philippe

    2016-08-01

    The initiation of T-cell responses in lymph nodes requires T cells to integrate signals delivered by dendritic cells (DCs) during long-lasting contacts (synapses) or more transient interactions (kinapses). However, it remains extremely challenging to understand how a specific sequence of contacts established by T cells ultimately dictates T-cell fate. Here, we have coupled a computational model of T-cell migration and interactions with DCs with a real-time, flow cytometry-like representation of T-cell activation. In this model, low-affinity peptides trigger T-cell proliferation through kinapses but we show that this process is only effective under conditions of high DC densities and prolonged antigen availability. By contrast, high-affinity peptides favor synapse formation and a vigorous proliferation under a wide range of antigen presentation conditions. In line with the predictions, decreasing the DC density in vivo selectively abolished proliferation induced by the low-affinity peptide. Finally, our results suggest that T cells possess a biochemical memory of previous stimulations of at least 1-2 days. We propose that the stability of T-cell-DC interactions, apart from their signaling potency, profoundly influences the robustness of T-cell activation. By offering the ability to control parameters that are difficult to manipulate experimentally, the virtual lymph node model provides new possibilities to tackle the fundamental mechanisms that regulate T-cell responses elicited by infections or vaccines. PMID:27089942

  8. Etoposide selectively ablates activated T cells to control the immunoregulatory disorder hemophagocytic lymphohistiocytosis

    PubMed Central

    Johnson, Theodore S.; Terrell, Catherine E.; Millen, Scott H.; Katz, Jonathan D.; Hildeman, David A.; Jordan, Michael B.

    2014-01-01

    Hemophagocytic lymphohistiocytosis (HLH) is an inborn disorder of immune regulation caused by mutations affecting perforin-dependent cytotoxicity. Defects of this pathway impair negative feedback between cytotoxic lymphocytes and APCs, leading to prolonged and pathologic activation of T cells. Etoposide, a widely used chemotherapeutic drug which inhibits topoisomerase II, is the mainstay of treatment for HLH, though its therapeutic mechanism remains unknown. We utilized a murine model of HLH, involving lymphocytic choriomeningitis virus infection of perforin deficient mice to study the activity and mechanism of etoposide for treating HLH and found that it substantially alleviated all symptoms of murine HLH and allowed prolonged survival. This therapeutic effect was relatively unique among chemotherapeutic agents tested, suggesting distinctive effects on the immune response. We found that the therapeutic mechanism of etoposide in this model system involved potent deletion of activated T cells and efficient suppression of inflammatory cytokine production. This effect was remarkably selective; etoposide did not exert a direct anti-inflammatory effect on macrophages or dendritic cells and it did not cause deletion of quiescent naive or memory T cells. Finally, etoposide’s immunomodulatory effects were similar in wild type and perforin deficient animals. Thus, etoposide treats HLH by selectively eliminating pathologic, activated T cells and may have utility as a novel immune modulator in a broad array of immunopathologic disorders. PMID:24259502

  9. Etoposide selectively ablates activated T cells to control the immunoregulatory disorder hemophagocytic lymphohistiocytosis.

    PubMed

    Johnson, Theodore S; Terrell, Catherine E; Millen, Scott H; Katz, Jonathan D; Hildeman, David A; Jordan, Michael B

    2014-01-01

    Hemophagocytic lymphohistiocytosis (HLH) is an inborn disorder of immune regulation caused by mutations affecting perforin-dependent cytotoxicity. Defects in this pathway impair negative feedback between cytotoxic lymphocytes and APCs, leading to prolonged and pathologic activation of T cells. Etoposide, a widely used chemotherapeutic drug that inhibits topoisomerase II, is the mainstay of treatment for HLH, although its therapeutic mechanism remains unknown. We used a murine model of HLH, involving lymphocytic choriomeningitis virus infection of perforin-deficient mice, to study the activity and mechanism of etoposide for treating HLH and found that it substantially alleviated all symptoms of murine HLH and allowed prolonged survival. This therapeutic effect was relatively unique among chemotherapeutic agents tested, suggesting distinctive effects on the immune response. We found that the therapeutic mechanism of etoposide in this model system involved potent deletion of activated T cells and efficient suppression of inflammatory cytokine production. This effect was remarkably selective; etoposide did not exert a direct anti-inflammatory effect on macrophages or dendritic cells, and it did not cause deletion of quiescent naive or memory T cells. Finally, etoposide's immunomodulatory effects were similar in wild-type and perforin-deficient animals. Thus, etoposide treats HLH by selectively eliminating pathologic, activated T cells and may have usefulness as a novel immune modulator in a broad array of immunopathologic disorders. PMID:24259502

  10. Thrombospondin-1 is a CD47-dependent endogenous inhibitor of hydrogen sulfide signaling in T cell activation

    PubMed Central

    Miller, Thomas W.; Kaur, Sukhbir; Ivins-O’Keefe, Kelly; Roberts, David D.

    2013-01-01

    Thrombospondin-1 is a potent suppressor of T cell activation via its receptor CD47. However, the precise mechanism for this inhibition remains unclear. Because H2S is an endogenous potentiator of T cell activation and is necessary for full T cell activation, we hypothesized that thrombospondin-1 signaling through CD47 inhibits T cell activation by antagonizing H2S signaling. Primary T cells from thrombospondin-1 null mice were more sensitive to H2S-dependent activation assessed by proliferation and induction of interleukin-2 and CD69 mRNAs. Exogenous thrombospondin-1 inhibited H2S responses in wild type and thrombospondin-1 null T cells but enhanced the same responses in CD47 null T cells. Fibronectin, which shares integrin and glycosaminoglycan binding properties with thrombospondin-1 but not CD47 binding, did not inhibit H2S signaling. A CD47-binding peptide derived from thrombospondin-1 inhibited H2S-induced activation, whereas two other functional sequences from thrombospondin-1 enhanced H2S signaling. Therefore, engaging CD47 is necessary and sufficient for thrombospondin-1 to inhibit H2S-dependent T cell activation. H2S stimulated T cell activation by potentiating MEK-dependent ERK phosphorylation, and thrombospondin-1 inhibited this signaling in a CD47-dependent manner. Thrombospondin-1 also limited activation-dependent T cell expression of the H2S biosynthetic enzymes cystathionine β-synthase and cystathionine γ-lyase, thereby limiting the autocrine role of H2S in T cell activation. Thus, thrombospondin-1 signaling through CD47 is the first identified endogenous inhibitor of H2S signaling and constitutes a novel mechanism that negatively regulates T cell activation. PMID:23499828

  11. 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

  12. T-cell activation is an immune correlate of risk in BCG vaccinated infants.

    PubMed

    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; Hatheril, 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

  13. Immunosuppressive activity of pogostone on T cells: Blocking proliferation via S phase arrest.

    PubMed

    Su, Ji-Yan; Luo, Xia; Zhang, Xiao-Jun; Deng, Xiang-Liang; Su, Zi-Ren; Zhou, Lian; Li, Shan-Shan; Dai, Zhenhua; Xu, Yang; Lai, Xiao-Ping

    2015-06-01

    Pogostone (PO) is one of the major chemical constituents of the essential oil of Pogostemon cablin (Blanco) Benth. In the present study, the effect of PO on T cell responsiveness was investigated to explore its potential in immunosuppression by a Concanavalin A (ConA)-stimulation model using splenocytes isolated from C57BL/6 mice. Cytotoxicity by PO on normal splenocytes was evaluated by MTS assays. Characteristics of apoptosis, proliferation, and cell cycle were analyzed by flow cytometry. Related expressions of cyclins and cyclin-dependent kinases (CDKs) were also determined by flow cytometry. Inflammatory cytokine profiling was performed emplying cytometric beads assays (CBA). Moreover, the T cell-mediated delayed Type hepersensity (DTH) model was applied to evaluate the immunosuppressive activity of PO. Neither viability reduction in normal splenocytes nor apoptosis in ConA-stimulated splenocytes was observed under PO treatments. Meanwhile, PO remarkably reduced the total population of ConA-stimulated T cell, blocked T cell proliferation induced by Con A, and inhibited the production of IFN-γ and IL-10. This blockade of stimulated T cell proliferation by PO was likely attributed to down-regulation of cyclin E, cyclin B and CDK1 and the subsequent S-phase arrest. Additionally, PO could inhibit the DTH reaction by alleviating ear swelling and inflammatory infiltrations in the DNCB-challenged ear. Taken together, PO exhibited an immunosuppressive property by directly blocking T cell proliferation as well as altering inflammatory cytokine profile, suggesting that PO may have clinical implications for treating autoimmune diseases and other immune-based disorders. PMID:25912345

  14. 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

  15. Interferon-alpha stimulates production of interleukin-10 in activated CD4+ T cells and monocytes.

    PubMed

    Aman, M J; Tretter, T; Eisenbeis, I; Bug, G; Decker, T; Aulitzky, W E; Tilg, H; Huber, C; Peschel, C

    1996-06-01

    In the present study, we investigated the effect of interferon-alpha (IFN-alpha) on the expression of interleukin-10 (IL-10) mRNA and protein synthesis in human monocytes and CD4+ T cells. In mononuclear cells, IFN-alpha induced expression of IL-10 mRNA and further enhanced lipopolysaccharide (LPS)-stimulated IL-10 expression. In purified monocytes, a strong expression of IL-10 mRNA induced by LPS was not further enhanced by IFN-alpha. In highly purified CD4+ T cells, IFN-alpha upregulated IL-10 mRNA upon activation with phytohemagglutinin and phorbol myristate acetate. In purified monocytes, an effect of IFN-alpha on IL-10 protein synthesis was dependent on costimulation with LPS. Maximal stimulation of IL-10 protein by IFN-alpha was seen after prolonged incubation periods of 48 to 96 hours, whereas IFN-gamma reduced IL-10 production in the early incubation period. Similar effects of IFN-alpha were observed in CD4+ T cells activated with CD3 and CD28 monoclonal antibodies. Addition of IFN-alpha caused an increase of IL-10 in culture supernatants of activated T-helper cells of more than 100% after 96 hours of incubation. In contrast, other cytokines, including IFN-gamma and IL-4, had no influence on IL-10 secretion stimulated by CD3 and CD28 in CD4+ T cells. In serum samples of IFN-alpha-treated individuals, we failed to detect an influence of cytokine treatment on IL-10 serum levels, confirming the requirement of additional activating signals for IFN-alpha-mediated effects on IL-10 synthesis. In conclusion, IFN-alpha enhances the late induction of IL-10, which physiologically occurs upon stimulation of monocytes and T cells. Biologically, this effect might enhance the negative-feedback mechanism ascribed to IL-10, which limits inflammatory reactions. PMID:8639843

  16. 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

  17. 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

  18. 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

  19. Aurora A drives early signalling and vesicle dynamics during T-cell activation.

    PubMed

    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

  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. Murine allogeneic CD19 CAR T cells harbor potent antileukemic activity but have the potential to mediate lethal GVHD.

    PubMed

    Jacoby, Elad; Yang, Yinmeng; Qin, Haiying; Chien, Christopher D; Kochenderfer, James N; Fry, Terry J

    2016-03-10

    Acute lymphoblastic leukemia (ALL) persisting or relapsing following bone marrow transplantation (BMT) has a dismal prognosis. Success with chimeric antigen receptor (CAR) T cells offers an opportunity to treat these patients with leukemia-redirected donor-derived T cells, which may be more functional than T cells derived from patients with leukemia but have the potential to mediate graft-versus-host disease (GVHD). We, together with others, have previously demonstrated tumor-specific T-cell dysfunction in the allogeneic environment. Here, we studied CAR T-cell function following BMT using an immunocompetent murine model of minor mismatched allogeneic transplantation followed by donor-derived CD19-CAR T cells. Allogeneic donor-derived CD19-CAR T cells eliminated residual ALL with equal potency to those administered after syngeneic BMT. Surprisingly, allogeneic CAR T cells mediated lethal acute GVHD with early mortality, which is atypical for this minor mismatch model. We demonstrated that both allogeneic and syngeneic CAR T cells show initial expansion as effector T cells, with a higher peak but rapid deletion of allogeneic CAR T cells. Interestingly, CAR-mediated acute GVHD was only seen in the presence of leukemia, suggesting CAR-target interactions induced GVHD. Indeed, serum interleukin (IL)-6 was elevated only in the presence of both leukemia and CAR T cells, and IL-6 neutralization ameliorated the severity of GVHD in a delayed donor lymphocyte infusion model. Finally, allogeneic CD4(+) CAR T cells were responsible for GVHD, which correlated with their ability to produce IL-6 upon CAR stimulation. Altogether, we demonstrate that donor-derived allogeneic CAR T cells are active but have the capacity to drive GVHD. PMID:26660684

  2. 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

  3. 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

  4. Immunomodulatory effect of procainamide in man. Inhibition of human suppressor T-cell activity in vitro.

    PubMed Central

    Ochi, T; Goldings, E A; Lipsky, P E; Ziff, M

    1983-01-01

    Procainamide (PA) induces the production of a number of autoantibodies in a high proportion of treated individuals and in some a syndrome closely resembling systemic lupus erythematosus. The mechanism underlying this action of PA is unclear. To examine the possibility that PA might induce autoantibody formation by altering normal immunoregulatory mechanisms, the action of this drug on an in vitro model of antibody formation in man was examined. PA was found to augment the generation of immunoglobulin-secreting cells (ISC) from human peripheral blood mononuclear cells (PBM) in response to pokeweed mitogen but had no effect on pokeweed mitogen-induced tritiated thymidine incorporation. When purified populations of B and T cells were used, PA enhanced the generation of ISC in B-cell cultures supported by untreated T cells but not by T cells treated with mitomycin C. These results indicate that PA augmented B-cell responses by inhibiting suppressor T-cell activity and not by augmenting helper T-cell or B-cell function. N-Acetyl-procainamide had no effect on the generation of ISC in this system. The effect of PA on concanavalin A (Con A)-induced suppressor cell activity was also examined to determine whether PA altered the generation or expression of suppressor T-cell function. PBM were cultured with 30 microgram/ml of Con A for 48 h to generate suppressor cells. When these were co-cultured with fresh PBM, the number of ISC generated was decreased by 58.1 +/- 3.4% (mean +/- SEM, n = 6). Cells that had been similarly incubated without Con A were not inhibitory. The addition of PA to the Con A-stimulated cultures inhibited the generation of suppressor cells as indicated by the fact that the response of fresh cells co-cultured with the Con A-stimulated cells was diminished by only 27.2 +/- 4.3%. In this system too, N-acetyl-procaimamide had no effect. By contrast, adding PA only to the co-culture of Con A-stimulated cells with fresh PBM had a less marked effect on

  5. Regulatory T cells and chronic immune activation in human immunodeficiency virus 1 (HIV-1)-infected children

    PubMed Central

    Freguja, R; Gianesin, K; Mosconi, I; Zanchetta, M; Carmona, F; Rampon, O; Giaquinto, C; De Rossi, A

    2011-01-01

    The function of CD4+ T cells with regulatory activity (Tregs) is the down-regulation of immune responses. This suppressive activity may limit the magnitude of effector responses, resulting in failure to control human immunodeficiency virus 1 (HIV-1) infection, but may also suppress chronic immune activation, a characteristic feature of HIV-1 disease. We evaluated the correlation between viral load, immune activation and Tregs in HIV-1-infected children. Eighty-nine HIV-1-infected children (aged 6–14 years) were included in the study and analysed for HIV-1 plasmaviraemia, HIV-1 DNA load, CD4 and CD8 cell subsets. Treg cells [CD4+ CD25highCD127lowforkhead box P3 (FoxP3high)] and CD8-activated T cells (CD8+CD38+) were determined by flow cytometry. Results showed that the number of activated CD8+CD38+ T cells increased in relation to HIV-1 RNA plasmaviraemia (r = 0·403, P < 0·0001). The proportion of Tregs also correlated positively with HIV-1 plasmaviraemia (r = 0·323, P = 0·002), but correlated inversely with CD4+ cells (r = −0·312, P = 0·004), thus suggesting a selective expansion along with increased viraemia and CD4+ depletion. Interestingly, a positive correlation was found between the levels of Tregs and CD8+CD38+ T cells (r = 0·305, P = 0·005), and the percentage of Tregs tended to correlate with HIV-1 DNA load (r = 0·224, P = 0·062). Overall, these findings suggest that immune activation contributes to the expansion of Treg cells. In turn, the suppressive activity of Tregs may impair effector responses against HIV-1, but appears to be ineffective in limiting immune activation. PMID:21438872

  6. A virtual lymph node model to dissect the requirements for T-cell activation by synapses and kinapses

    PubMed Central

    Moreau, Hélène D; Bogle, Gib; Bousso, Philippe

    2016-01-01

    The initiation of T-cell responses in lymph nodes requires T cells to integrate signals delivered by dendritic cells (DCs) during long-lasting contacts (synapses) or more transient interactions (kinapses). However, it remains extremely challenging to understand how a specific sequence of contacts established by T cells ultimately dictates T-cell fate. Here, we have coupled a computational model of T-cell migration and interactions with DCs with a real-time, flow cytometry-like representation of T-cell activation. In this model, low-affinity peptides trigger T-cell proliferation through kinapses but we show that this process is only effective under conditions of high DC densities and prolonged antigen availability. By contrast, high-affinity peptides favor synapse formation and a vigorous proliferation under a wide range of antigen presentation conditions. In line with the predictions, decreasing the DC density in vivo selectively abolished proliferation induced by the low-affinity peptide. Finally, our results suggest that T cells possess a biochemical memory of previous stimulations of at least 1–2 days. We propose that the stability of T-cell–DC interactions, apart from their signaling potency, profoundly influences the robustness of T-cell activation. By offering the ability to control parameters that are difficult to manipulate experimentally, the virtual lymph node model provides new possibilities to tackle the fundamental mechanisms that regulate T-cell responses elicited by infections or vaccines. PMID:27089942

  7. GATA3 induces human T-cell commitment by restraining Notch activity and repressing NK-cell fate.

    PubMed

    Van de Walle, Inge; Dolens, Anne-Catherine; Durinck, Kaat; De Mulder, Katrien; Van Loocke, Wouter; Damle, Sagar; Waegemans, Els; De Medts, Jelle; Velghe, Imke; De Smedt, Magda; Vandekerckhove, Bart; Kerre, Tessa; Plum, Jean; Leclercq, Georges; Rothenberg, Ellen V; Van Vlierberghe, Pieter; Speleman, Frank; Taghon, Tom

    2016-01-01

    The gradual reprogramming of haematopoietic precursors into the T-cell fate is characterized by at least two sequential developmental stages. Following Notch1-dependent T-cell lineage specification during which the first T-cell lineage genes are expressed and myeloid and dendritic cell potential is lost, T-cell specific transcription factors subsequently induce T-cell commitment by repressing residual natural killer (NK)-cell potential. How these processes are regulated in human is poorly understood, especially since efficient T-cell lineage commitment requires a reduction in Notch signalling activity following T-cell specification. Here, we show that GATA3, in contrast to TCF1, controls human T-cell lineage commitment through direct regulation of three distinct processes: repression of NK-cell fate, upregulation of T-cell lineage genes to promote further differentiation and restraint of Notch activity. Repression of the Notch1 target gene DTX1 hereby is essential to prevent NK-cell differentiation. Thus, GATA3-mediated positive and negative feedback mechanisms control human T-cell lineage commitment. PMID:27048872

  8. GATA3 induces human T-cell commitment by restraining Notch activity and repressing NK-cell fate

    PubMed Central

    Van de Walle, Inge; Dolens, Anne-Catherine; Durinck, Kaat; De Mulder, Katrien; Van Loocke, Wouter; Damle, Sagar; Waegemans, Els; De Medts, Jelle; Velghe, Imke; De Smedt, Magda; Vandekerckhove, Bart; Kerre, Tessa; Plum, Jean; Leclercq, Georges; Rothenberg, Ellen V.; Van Vlierberghe, Pieter; Speleman, Frank; Taghon, Tom

    2016-01-01

    The gradual reprogramming of haematopoietic precursors into the T-cell fate is characterized by at least two sequential developmental stages. Following Notch1-dependent T-cell lineage specification during which the first T-cell lineage genes are expressed and myeloid and dendritic cell potential is lost, T-cell specific transcription factors subsequently induce T-cell commitment by repressing residual natural killer (NK)-cell potential. How these processes are regulated in human is poorly understood, especially since efficient T-cell lineage commitment requires a reduction in Notch signalling activity following T-cell specification. Here, we show that GATA3, in contrast to TCF1, controls human T-cell lineage commitment through direct regulation of three distinct processes: repression of NK-cell fate, upregulation of T-cell lineage genes to promote further differentiation and restraint of Notch activity. Repression of the Notch1 target gene DTX1 hereby is essential to prevent NK-cell differentiation. Thus, GATA3-mediated positive and negative feedback mechanisms control human T-cell lineage commitment. PMID:27048872

  9. Gamma delta T cells are activated by polysaccharide K (PSK) and contribute to the anti-tumor effect of PSK

    PubMed Central

    Inatsuka, Carol; Yang, Yi; Gad, Ekram; Rastetter, Lauren; Disis, Mary L.

    2013-01-01

    Polysaccharide K (PSK) is a widely used mushroom extract that has shown anti-tumor and immunomodulatory effects in both preclinical and clinical studies. Therefore, it is important to understand the mechanism of actions of PSK. We recently reported that PSK can activate toll-like receptor 2 and enhances the function of NK cells. The current study was undertaken to study the effect of PSK on gamma delta (γδ) T cells, another important arm of the innate immunity. In vitro experiments using mouse splenocytes showed that γδ T cells produce IFN-γ after treatment with PSK and have up-regulated expression of CD25, CD69, and CD107a. To investigate whether the effect of PSK on γδ T cells is direct or indirect, purified γδ T cells were cultured either alone or together with bone marrow-derived DC in a co-culture or trans-well system and then stimulated with PSK. Results showed that direct cell-to-cell contact between γδ T cells and DC is required for optimal activation of γδ T cells. There was also reciprocal activation of DC by PSK-activated γδ T cells, as demonstrated by higher expression of costimulatory molecules and enhanced production of IL-12 by DC in the presence of γδ T cells. PSK can also co-stimulate γδ T cells with anti-TCR and anti-CD3 stimulation, in the absence of DC. Finally, in vivo treatment with PSK activates γδ T cells among the tumor infiltrating lymphocytes, and depleting γδ T cells during PSK treatment attenuated the anti-tumor effect of PSK. All together, these results demonstrated that γδ T cells are activated by PSK and contribute to the anti-tumor effect of PSK. PMID:23685781

  10. HIV integration site distributions in resting and activated CD4+ T cells infected in culture

    PubMed Central

    Brady, Troy; Agosto, Luis M.; Malani, Nirav; Berry, Charles C.; O'Doherty, Una; Bushman, Frederic

    2010-01-01

    Objective The goal of this study was to investigate whether the location of HIV integration differs in resting versus activated T cells, a feature that could contribute to the formation of latent viral reservoirs via effects on integration targeting. Design Primary resting or activated CD4+ T cells were infected with purified X4-tropic HIV in the presence and absence of nucleoside triphosphates and genomic locations of integrated provirus determined. Methods We sequenced and analyzed a total of 2661 HIV integration sites using linker-mediated PCR and 454 sequencing. Integration site data sets were then compared to each other and to computationally generated random distributions. Results HIV integration was favored in active transcription units in both cell types, but integration sites from activated cells were found more often in genomic regions that were dense in genes, dense in CpG islands, and enriched in G/C bases. Integration sites from activated cells were also more strongly correlated with histone methylation patterns associated with active genes. Conclusion These data indicate that integration site distributions show modest but significant differences between resting and activated CD4+ T cells, and that integration in resting cells occurs more often in regions that may be suboptimal for proviral gene expression. PMID:19550285

  11. Bacterial capsular polysaccharide prevents the onset of asthma through T-cell activation.

    PubMed

    Johnson, Jenny L; Jones, Mark B; Cobb, Brian A

    2015-04-01

    Over the last four decades, increases in the incidence of immune-mediated diseases in the Western world have been linked to changes in microbial exposure. It is becoming increasingly clear that the normal microbiota in the gut can profoundly alter susceptibility to a wide range of diseases, such as asthma, in which immune homeostasis is disrupted, yet the mechanisms governing this microbial influence remains poorly defined. In this study, we show that gastrointestinal exposure to PSA, a capsular polysaccharide derived from the commensal bacterium Bacteroides fragilis, significantly limits susceptibility to the induction of experimental asthma. We report that direct treatment of mice with PSA generates protection from asthma, and this effect can be given to a naïve recipient by adoptive transfer of CD4(+) T cells from PSA-exposed mice. Remarkably, we found that these PSA-induced T cells are not canonical FoxP3(+) regulatory T cells, but that they potently inhibit both Th1 and Th2 models of asthma in an IL-10-dependent fashion. These findings reveal that bacterial polysaccharides link the microbiota with the peripheral immune system by activating CD4(+)Foxp3(-) T cells upon exposure in the gut, and they facilitate resistance to unnecessary inflammatory responses via the production of IL-10. PMID:25347992

  12. 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

  13. Piperine impairs the migration and T cell-activating function of dendritic cells.

    PubMed

    Rodgers, Gemma; Doucette, Carolyn D; Soutar, David A; Liwski, Robert S; Hoskin, David W

    2016-02-01

    Piperine, a major alkaloid found in the fruits of black and long pepper plants, has anti-inflammatory properties; however, piperine's effect on dendritic cell (DC) migration and T cell-activating function has not been investigated. Bone marrow-derived mouse DCs that were matured in the presence of 100 μM piperine showed reduced in vitro migration in response to CCL21, as well as reduced in vivo migration to lymph nodes. In addition, piperine-treated DCs had reduced CCR7 expression and elevated CCR5 expression, as well as reduced expression of CD40 and class II major histocompatibility complex molecules and decreased nuclear accumulation of RelB. DC production of interleukin (IL)-6, tumor necrosis factor α, and monocyte chemoattractant protein-1 in response to lipopolysaccharide stimulation was also reduced following piperine treatment. Exposure to piperine during maturation therefore caused DCs to retain an immature phenotype, which was associated with a reduced capacity to promote T cell activation since co-culture of ovalbumin (OVA323-339)-specific T cells with OVA323-339-pulsed DCs that were previously matured in the presence of piperine showed reduced interferon-γ and IL-2 expression. OVA323-339-specific T cell proliferation was also reduced in vivo in the presence of piperine-treated DCs. Inhibition of DC migration and function by piperine may therefore be a useful strategy to down-regulate potentially harmful DC-driven T cell responses to self-antigens and transplantation antigens. PMID:26640239

  14. Organ transplantation: modulation of T-cell activation pathways initiated by cell surface receptors to suppress graft rejection.

    PubMed

    Weatherly, Kathleen; Braun, Michel Y

    2011-01-01

    T-cell activation depends upon two types of signals: a T-cell-receptor-mediated antigen-specific signal and several non-antigen-specific ones provided by the engagement of costimulatory and/or inhibitory T-cell surface molecules. In clinical transplantation, T-cell costimulatory/inhibitory molecules are involved in determining cytokine production, vascular endothelial cell damage, and induction of transplant rejection. Several of the latest new immunotherapeutic strategies being currently developed to control graft rejection aim at inhibiting alloreactive T-cell function by regulating activating and costimulatory/inhibitory signals to T cells. This article describes the recent development and potential application of these therapies in experimental and pre-clinical transplantation. PMID:20941624

  15. STAT3 Impairs STAT5 Activation in the Development of IL-9-Secreting T Cells.

    PubMed

    Olson, Matthew R; Verdan, Felipe Fortino; Hufford, Matthew M; Dent, Alexander L; Kaplan, Mark H

    2016-04-15

    Th cell subsets develop in response to multiple activating signals, including the cytokine environment. IL-9-secreting T cells develop in response to the combination of IL-4 and TGF-β, although they clearly require other cytokine signals, leading to the activation of transcription factors including STAT5. In Th17 cells, there is a molecular antagonism of STAT5 with STAT3 signaling, although whether this paradigm exists in other Th subsets is not clear. In this paper, we demonstrate that STAT3 attenuates the ability of STAT5 to promote the development of IL-9-secreting T cells. We demonstrate that production of IL-9 is increased in the absence of STAT3 and cytokines that result in a sustained activation of STAT3, including IL-6, have the greatest potency in repressing IL-9 production in a STAT3-dependent manner. Increased IL-9 production in the absence of STAT3 correlates with increased endogenous IL-2 production and STAT5 activation, and blocking IL-2 responses eliminates the difference in IL-9 production between wild-type and STAT3-deficient T cells. Moreover, transduction of developing Th9 cells with a constitutively active STAT5 eliminates the ability of IL-6 to reduce IL-9 production. Thus, STAT3 functions as a negative regulator of IL-9 production through attenuation of STAT5 activation and function. PMID:26976954

  16. Replication of HIV-1 deleted Nef mutants in chronically immune activated human T cells.

    PubMed

    Shapira-Nahor, Orit; Maayan, Shlomo; Peden, Keith W C; Rabinowitz, Ruth; Schlesinger, Michael; Alian, Akram; Panet, Amos

    2002-11-10

    Lymphocytes (PBMC) obtained from blood of HIV-sera negative Ethiopian immigrants (ETH) were highly susceptible to HIV-1 infection in vitro with no need for stimulation by mitogens. As the HIV nef gene product has been shown to enhance viral replication in stimulated primary lymphocytes, we investigated in this work the role of Nef in viral replication in the ETH cells. Lymphocytes obtained from ETH individuals supported high replication of wild-type HIV-1 and low but significant replication level of the two deleted Nef mutants (encode truncated Nef proteins consisting only of either the first 35 or the first 86 amino acids of Nef). In contrast, no replication was observed in nonactivated cells obtained from non-ETH individuals. After activation of the PBMC from ETH individuals with PHA, replication of both wild-type strains and the two deleted Nef mutant viruses further increased. The CD4(+) T cells of ETH individuals exhibited elevated levels of the surface activation markers CD45RO and HLA-DR, compared with T cells derived from non-ETH group. Likewise, expression of the chemokine receptors CCR5 and CXCR4 on these cells was higher in the ETH group than in the non-ETH group. Replication of HIV-1 wild-type and the isogenic-deleted Nef mutants was significantly correlated with the proportion of ETH cells expressing CD45RO and the chemokine receptors. This study suggests that HIV-1 may respond differently to several activation states characteristic of T cells. One activation state, defined by chronically activated lymphocytes from ETH individuals, is permissive to the wild-type HIV-1 and, to a lesser degree, to the Nef mutants. Further activation of these cells by exogenous stimuli enhances replication of the virus. Our results support the notion that Nef enhances the basal level of T cell activation and consequently, viral replication. PMID:12482665

  17. Stable inhibitory activity of regulatory T cells requires the transcription factor Helios.

    PubMed

    Kim, Hye-Jung; Barnitz, R Anthony; Kreslavsky, Taras; Brown, Flavian D; Moffett, Howell; Lemieux, Madeleine E; Kaygusuz, Yasemin; Meissner, Torsten; Holderried, Tobias A W; Chan, Susan; Kastner, Philippe; Haining, W Nicholas; Cantor, Harvey

    2015-10-16

    The maintenance of immune homeostasis requires regulatory T cells (T(regs)). Given their intrinsic self-reactivity, T(regs) must stably maintain a suppressive phenotype to avoid autoimmunity. We report that impaired expression of the transcription factor (TF) Helios by FoxP3(+) CD4 and Qa-1-restricted CD8 T(regs) results in defective regulatory activity and autoimmunity in mice. Helios-deficient T(regs) develop an unstable phenotype during inflammatory responses characterized by reduced FoxP3 expression and increased effector cytokine expression secondary to diminished activation of the STAT5 pathway. CD8 T(regs) also require Helios-dependent STAT5 activation for survival and to prevent terminal T cell differentiation. The definition of Helios as a key transcription factor that stabilizes T(regs) in the face of inflammatory responses provides a genetic explanation for a core property of T(regs). PMID:26472910

  18. 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

  19. 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

  20. The adenovirus e3 promoter is sensitive to activation signals in human T cells.

    PubMed

    Mahr, Jeffrey A; Boss, Jeremy M; Gooding, Linda R

    2003-01-01

    The group C adenoviruses typically cause acute respiratory disease in young children. In addition, a persistent phase of infection has been observed in which virus may be shed for years without producing overt pathology. Our laboratory recently reported that group C adenovirus DNA can be found in tonsil and adenoid T lymphocytes from the majority of pediatric donors (C. T. Garnett, D. Erdman, W. Xu, and L. R. Gooding, J. Virol. 76:10608-10616, 2002). This finding suggests that immune evasion strategies of human adenoviruses may be directed, in part, toward protection of persistently or latently infected T lymphocytes. Many of the adenoviral gene products implicated in prevention of immune destruction of virus-infected cells are encoded within the E3 transcription unit. In this study, the E3 promoter was evaluated for sensitivity to T-cell activation signals by using a promoter reporter plasmid. Indeed, this promoter is extremely sensitive to T-cell activation, with phorbol myristate acetate (PMA) plus ionomycin increasing E3-directed transcription 100-fold. By comparison, in the same cells E1A expression leads to a 5.5-fold increase in transcription from the E3 promoter. In contrast to induction by E1A, activation by PMA plus ionomycin requires the two E3 NF-kappaB binding sites. Interestingly, expression of E1A inhibits induction of the E3 promoter in response to T-cell activation while increasing E3 promoter activity in unactivated cells. Collectively, these data suggest that the E3 promoter may have evolved the capacity to respond to T-cell activation in the absence of E1A expression and may act to upregulate antiapoptotic gene expression in order to promote survival of persistently infected T lymphocytes. PMID:12502827

  1. Constitutive activation of Pim1 kinase is a therapeutic target for adult T-cell leukemia.

    PubMed

    Bellon, Marcia; Lu, Ling; Nicot, Christophe

    2016-05-19

    Human T-cell leukemia virus type 1 (HTLV-1)-associated adult T-cell leukemia and T-cell lymphoma (ATL) are aggressive diseases with poor prognoses, limited therapeutic options, and no curative treatment. In this study, we used a mouse model of ATL and restored expression of the microRNA, miR-124a, to identify in vivo downstream effectors responsible for its tumor-suppressive functions in ATL cells. Our results revealed that STAT3, a direct target of miR-124a, is constitutively activated in HTLV-I-transformed cells and ATL cells, and activating STAT3 mutations were detected in 25.5% of primary ATL patients. Interestingly, we found that the STAT3 downstream kinase effector, Pim1, is constitutively activated in ATL cells. The dependence of ATL cells to Pim1 activity was demonstrated using 2 Pim1 small inhibitors, SMI-4a and AZD1208. These studies indicated that HTLV-I-transformed and ATL cells, but not normal peripheral blood mononuclear cells, are highly sensitive to AZD1208, and the inhibition of Pim1 signaling triggers an apoptotic signal in leukemic cells. Finally, preclinical testing of AZD1208 in a mouse model of ATL resulted in significant prevention of tumor growth in vivo. In conclusion, our studies suggest that constitutive activation of the STAT3-Pim1 pathway represents a novel therapeutic target for the treatment of ATL. PMID:26813676

  2. 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. PMID:25433234

  3. Quantitative Phosphoproteomic Analysis Reveals a Role for Serine and Threonine Kinases in the Cytoskeletal Reorganization in Early T Cell Receptor Activation in Human Primary T Cells*

    PubMed Central

    Ruperez, Patricia; Gago-Martinez, Ana; Burlingame, A. L.; Oses-Prieto, Juan A.

    2012-01-01

    Protein phosphorylation-dephosphorylation events play a primary role in regulation of almost all aspects of cell function including signal transduction, cell cycle, or apoptosis. Thus far, T cell phosphoproteomics have focused on analysis of phosphotyrosine residues, and little is known about the role of serine/threonine phosphorylation in early activation of the T cell receptor (TCR). Therefore, we performed a quantitative mass spectrometry-based analysis of the global phosphoproteome of human primary T cells in response to 5 min of TCR activation with anti-CD3 antibody. Combining immunoprecipitation with an antiphosphotyrosine antibody, titanium dioxide phosphopeptide enrichment, isobaric tag for the relative and absolute quantitation methodology, and strong cation exchange separation, we were able to identify 2814 phosphopeptides. These unique sites were employed to investigate the site-specific phosphorylation dynamics. Five hundred and seventeen phosphorylation sites showed TCR-responsive changes. We found that upon 5 min of stimulation of the TCR, specific serine and threonine kinase motifs are overrepresented in the set of responsive phosphorylation sites. These phosphorylation events targeted proteins with many different activities and are present in different subcellular locations. Many of these proteins are involved in intracellular signaling cascades related mainly to cytoskeletal reorganization and regulation of small GTPase-mediated signal transduction, probably involved in the formation of the immune synapse. PMID:22499768

  4. Regulatory T cells and antigen-specific tolerance.

    PubMed

    Kretschmer, Karsten; Apostolou, Irina; Verginis, Panos; von Boehmer, Harald

    2008-01-01

    Foxp3-expressing regulatory T cells (Tregs) have an essential function of preventing autoimmune disease in man and mouse. Foxp3 binds to forkhead motifs of about 1,100 genes and the strength of binding increases upon PMA/ionomycin stimulation. In Foxp3-expressing T-cell hybridomas, Foxp3 promoter binding does not lead to activation or suppression of genes which becomes only visible after T-cell activation. These findings are in line with observations by others that Foxp3 exerts important functions through association with Tcell receptor (TCR)-dependent transcription factors in a DNAbinding complex. Tregs can be generated when developing T cells encounter TCR agonist ligands in the thymus. This pro - cess requires costimulatory signals. In contrast, extra thymic conversion of naive T cells into Tregs is inhibited by costimulation. In fact, DC-derived retinoic acid (RA) helps the conversion process by counteracting the negative impact of costimulation. Since AP-1 is produced after costimulation and appears to interfere with Foxp3-NFAT transcription complexes, it is of interest to note that RA interferes with AP-1-dependent transcription. Thus, RA may interfere with the negative impact of costimulation on Treg conversion by interfering with the generation and/or function of AP-1. PMID:18802332

  5. 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

  6. FOXP3+Helios+ Regulatory T Cells, Immune Activation, and Advancing Disease in HIV-Infected Children.

    PubMed

    Khaitan, Alka; Kravietz, Adam; Mwamzuka, Mussa; Marshed, Fatma; Ilmet, Tiina; Said, Swalehe; Ahmed, Aabid; Borkowsky, William; Unutmaz, Derya

    2016-08-15

    Regulatory T cells (Tregs) are functionally suppressive CD4 T cells, critical for establishing peripheral tolerance and controlling inflammatory responses. Previous reports of Tregs during chronic HIV disease have conflicting results with higher or lower levels compared with controls. Identifying true Tregs with suppressive activity proves challenging during HIV infection, as traditional Treg markers, CD25 and FOXP3, may transiently upregulate expression as a result of immune activation (IA). Helios is an Ikaros family transcription factor that marks natural Tregs with suppressive activity and does not upregulate expression after activation. Coexpression of FOXP3 and Helios has been suggested as a highly specific marker of "bona fide" Tregs. We evaluated Treg subsets by FOXP3 coexpressed with either CD25 or Helios and their association with HIV disease progression in perinatally infected HIV-positive children. Identifying Tregs by FOXP3 coexpression with Helios rather than CD25 revealed markedly higher Treg frequencies, particularly in HIV+ children. Regardless of antiretroviral therapy, HIV-infected children had a selective expansion of memory FOXP3+Helios+ Tregs. The rise in memory Tregs correlated with declining HIV clinical status, indicated by falling CD4 percentages and CD4:CD8 ratios and increasing HIV plasma viremia and IA. In addition, untreated HIV+ children exhibited an imbalance between the levels of Tregs and activated T cells. Finally, memory Tregs expressed IA markers CD38 and Ki67 and exhaustion marker, PD-1, that tightly correlated with a similar phenotype in memory CD4 T cells. Overall, HIV-infected children had significant disruptions of memory Tregs that associated with advancing HIV disease. PMID:27003495

  7. 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

  8. In vivo activity of a nonspecific T cell-replacing factor.

    PubMed

    Kindred, B; Bösing-Schneider, R; Corley, R B

    1979-01-01

    Nonspecific T cell-replacing factors prepared as supernatants from mixed lymphocyte cultures or concanavalin A-stimulated spleen cells are active in vivo iv injected into nude mice at least 3 days before antigen. The supernatants appear to act by enhancing the week IgM responses that occur in untreated nudes. Secondary responses and IgG antibody were not found. PMID:153929

  9. Biomarkers on patient T cells diagnose active tuberculosis and monitor treatment response

    PubMed Central

    Adekambi, Toidi; Ibegbu, Chris C.; Cagle, Stephanie; Kalokhe, Ameeta S.; Wang, Yun F.; Hu, Yijuan; Day, Cheryl L.; Ray, Susan M.; Rengarajan, Jyothi

    2015-01-01

    BACKGROUND. The identification and treatment of individuals with tuberculosis (TB) is a global public health priority. Accurate diagnosis of pulmonary active TB (ATB) disease remains challenging and relies on extensive medical evaluation and detection of Mycobacterium tuberculosis (Mtb) in the patient’s sputum. Further, the response to treatment is monitored by sputum culture conversion, which takes several weeks for results. Here, we sought to identify blood-based host biomarkers associated with ATB and hypothesized that immune activation markers on Mtb-specific CD4+ T cells would be associated with Mtb load in vivo and could thus provide a gauge of Mtb infection. METHODS. Using polychromatic flow cytometry, we evaluated the expression of immune activation markers on Mtb-specific CD4+ T cells from individuals with asymptomatic latent Mtb infection (LTBI) and ATB as well as from ATB patients undergoing anti-TB treatment. RESULTS. Frequencies of Mtb-specific IFN-γ+CD4+ T cells that expressed immune activation markers CD38 and HLA-DR as well as intracellular proliferation marker Ki-67 were substantially higher in subjects with ATB compared with those with LTBI. These markers accurately classified ATB and LTBI status, with cutoff values of 18%, 60%, and 5% for CD38+IFN-γ+, HLA-DR+IFN-γ+, and Ki-67+IFN-γ+, respectively, with 100% specificity and greater than 96% sensitivity. These markers also distinguished individuals with untreated ATB from those who had successfully completed anti-TB treatment and correlated with decreasing mycobacterial loads during treatment. CONCLUSION. We have identified host blood-based biomarkers on Mtb-specific CD4+ T cells that discriminate between ATB and LTBI and provide a set of tools for monitoring treatment response and cure. TRIAL REGISTRATION. Registration is not required for observational studies. FUNDING. This study was funded by Emory University, the NIH, and the Yerkes National Primate Center. PMID:25822019

  10. Quantal concept of T-cell activation: adhesion domains as immunological synapses

    NASA Astrophysics Data System (ADS)

    Sackmann, Erich

    2011-06-01

    Adhesion micro-domains (ADs) formed during encounters of lymphocytes with antigen-presenting cells (APC) mediate the genetic expression of quanta of cytokines interleukin-2 (IL-2). The IL-2-induced activation of IL-2 receptors promotes the stepwise progression of the T-cells through the cell cycle, hence their name, immunological synapses. The ADs form short-lived reaction centres controlling the recruitment of activators of the biochemical pathway (the kinases Lck and ZAP) while preventing the access of inhibitors (phosphatase CD45) through steric repulsion forces. CD45 acts as the generator of adhesion domains and, through its role as a spacer protein, also as the promoter of the reaction. In a second phase of T-cell-APC encounters, long-lived global reaction spaces (called supramolecular activation complexes (SMAC)) form by talin-mediated binding of the T-cell integrin (LFA-1) to the counter-receptor ICAM-1, resulting in the formation of ring-like tight adhesion zones (peripheral SMAC). The ADs move to the centre of the intercellular adhesion zone forming the central SMAC, which serve in the recycling of the AD. We propose that cell stimulation is triggered by integrating the effect evoked by the short-lived adhesion domains. Similar global reaction platforms are formed by killer cells to destruct APC. We present a testable mechanical model showing that global reaction spaces (SMAC or dome-like contacts between cytotoxic cells and APC) form by self-organization through delayed activation of the integrin-binding affinity and stabilization of the adhesion zones by F-actin recruitment. The mechanical stability and the polarization of the adhering T-cells are mediated by microtubule-actin cross-talk.

  11. Vγ9Vδ2 T cells and zoledronate mediate antitumor activity in an orthotopic mouse model of human chondrosarcoma.

    PubMed

    Sun, L; Li, Y; Jiang, Z; Zhang, J; Li, H; Li, B; Ye, Z

    2016-06-01

    Chondrosarcoma (CS) is a cartilaginous malignant neoplasm characterized by resistance to conventional adjuvant therapy. The prognosis of unresectable or metastatic CS is poor. Therefore, it is imperative to explore novel therapeutic approaches to improve the treatment efficacy for those CS patients. Emerging data has implicated the synergistic antitumor activity of zoledronate (ZOL) and Vγ9Vδ2 T cells. However, whether ZOL-stimulated Vγ9Vδ2 T cells could infiltrate bone sarcoma and inhibit tumor growth has not been thoroughly answered yet. In this study, Vγ9Vδ2 T cells from healthy donors and CS patients were expanded in the presence of ZOL (1 μM) and IL-2 (400 IU/ml). The antitumor activity of Vγ9Vδ2 T cells to ZOL-pretreated human CS was examined both in vitro and in vivo. ZOL pretreatment substantially enhanced the cytotoxicity of Vγ9Vδ2 T cells to SW1353 and primary CS cells. ZOL potentiated the migration and cytotoxicity of Vγ9Vδ2 T cells to SW1353 in dose- and time-dependent manner. Moreover, weekly intravenous ZOL followed by Vγ9Vδ2 T cells inhibited subcutaneous xenograft growth. Thus, Vγ9Vδ2 T cells were able to infiltrate bone tumor and significantly suppressed the development of orthotopic SW1353 xenografts. Altogether, the study raises the possibility of combining ZOL with Vγ9Vδ2 T cells for CS treatment. PMID:26676633

  12. F-actin polymerization and retrograde flow drive sustained PLCγ1 signaling during T cell activation

    PubMed Central

    Babich, Alexander; Li, Shuixing; O'Connor, Roddy S.; Milone, Michael C.; Freedman, Bruce D.

    2012-01-01

    Activation of T cells by antigen-presenting cells involves assembly of signaling molecules into dynamic microclusters (MCs) within a specialized membrane domain termed the immunological synapse (IS). Actin and myosin IIA localize to the IS, and depletion of F-actin abrogates MC movement and T cell activation. However, the mechanisms that coordinate actomyosin dynamics and T cell receptor signaling are poorly understood. Using pharmacological inhibitors that perturb individual aspects of actomyosin dynamics without disassembling the network, we demonstrate that F-actin polymerization is the primary driver of actin retrograde flow, whereas myosin IIA promotes long-term integrity of the IS. Disruption of F-actin retrograde flow, but not myosin IIA contraction, arrested MC centralization and inhibited sustained Ca2+ signaling at the level of endoplasmic reticulum store release. Furthermore, perturbation of retrograde flow inhibited PLCγ1 phosphorylation within MCs but left Zap70 activity intact. These studies highlight the importance of ongoing actin polymerization as a central driver of actomyosin retrograde flow, MC centralization, and sustained Ca2+ signaling. PMID:22665519

  13. 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

  14. A CD28 superagonistic antibody elicits 2 functionally distinct waves of T cell activation in rats

    PubMed Central

    Müller, Nora; van den Brandt, Jens; Odoardi, Francesca; Tischner, Denise; Herath, Judith; Flügel, Alexander; Reichardt, Holger M.

    2008-01-01

    Administration of the CD28 superagonistic antibody JJ316 is an efficient means to treat autoimmune diseases in rats, but the humanized antibody TGN1412 caused devastating side effects in healthy volunteers during a clinical trial. Here we show that JJ316 treatment of rats induced a dramatic redistribution of T lymphocytes from the periphery to the secondary lymphoid organs, resulting in severe T lymphopenia. Live imaging of secondary lymphoid organs revealed that JJ316 administration almost instantaneously (<2 minutes) arrested T cells in situ. This reduction in T cell motility was accompanied by profound cytoskeletal rearrangements and increased cell size. In addition, surface expression of lymphocyte function–associated antigen-1 was enhanced, endothelial differentiation sphingolipid G protein–coupled receptor 1 and L selectin levels were downregulated, and the cells lost their responsiveness to sphingosine 1–phosphate–directed migration. These proadhesive alterations were accompanied by signs of strong activation, including upregulation of CD25, CD69, CD134, and proinflammatory mediators. However, this did not lead to a cytokine storm similar to the clinical trial. While most of the early changes disappeared within 48 hours, we observed that CD4+CD25+FoxP3+ regulatory T cells experienced a second phase of activation, which resulted in massive cell enlargement, extensive polarization, and increased motility. These data suggest that CD28 superagonists elicit 2 qualitatively distinct waves of activation. PMID:18357346

  15. 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

  16. S152 (CD27). A modulating disulfide-linked T cell activation antigen.

    PubMed

    Bigler, R D; Bushkin, Y; Chiorazzi, N

    1988-07-01

    A large subset of normal resting peripheral blood T cells express a protein at low density defined by the murine mAb S152. Reactive T cells are present in both the CD4+ and CD8+ subpopulations. This determinant, however, is not expressed on growth factor-independent T cell lines. After activation of mononuclear cells by either Con A or PHA, greater than 80% of the cells stained at a mean fluorescence intensity that was more than six times that seen on resting cells. When PWM- or mixed lymphocyte reaction-activated cultures were studied, 7 to 22% of S152+ cells stained at high intensity whereas most cells stained at the baseline low intensity. The increased fraction of S152+ cells staining at high intensity after activation paralleled both the increased percentage of anti-Tac+ and 5E9+ cells and cellular proliferation measured by thymidine incorporation. Modulation of the S152 Ag was induced when either Con A- or PHA-activated mononuclear cells were placed into secondary cultures containing S152 mAb. Expression of the S152 Ag began to decrease after 2 h and reached a minimum after 6 h. Resting T cells, however, did not appear to modulate when cultured with S152 mAb. Immunoprecipitation and gel electrophoresis analysis revealed the S152 molecule to have a mobility of 120 kDa before reduction. After reduction the molecule was shown to be composed of two 55-kDa molecules with an isoelectric point of 5 to 6 indicating that the S152 Ag is a disulfide-linked homodimer. These studies confirm that the S152 mAb reacts with the newly defined CD27 molecule. The presence of the S152 Ag on resting and activated cells, its parallel increase with the Tac and 5E9 Ag, and its ability to modulate on activated cells suggest that this molecule may play a functional role during T cell activation. PMID:2837508

  17. Systematic study of constitutive cyclooxygenase-2 expression: Role of NF-κB and NFAT transcriptional pathways

    PubMed Central

    Kirkby, Nicholas S.; Chan, Melissa V.; Zaiss, Anne K.; Garcia-Vaz, Eliana; Jiao, Jing; Berglund, Lisa M.; Verdu, Elena F.; Ahmetaj-Shala, Blerina; Wallace, John L.; Herschman, Harvey R.; Gomez, Maria F.; Mitchell, Jane A.

    2016-01-01

    Cyclooxygenase-2 (COX-2) is an inducible enzyme that drives inflammation and is the therapeutic target for widely used nonsteroidal antiinflammatory drugs (NSAIDs). However, COX-2 is also constitutively expressed, in the absence of overt inflammation, with a specific tissue distribution that includes the kidney, gastrointestinal tract, brain, and thymus. Constitutive COX-2 expression is therapeutically important because NSAIDs cause cardiovascular and renal side effects in otherwise healthy individuals. These side effects are now of major concern globally. However, the pathways driving constitutive COX-2 expression remain poorly understood. Here we show that in the kidney and other sites, constitutive COX-2 expression is a sterile response, independent of commensal microorganisms and not associated with activity of the inflammatory transcription factor NF-κB. Instead, COX-2 expression in the kidney but not other regions colocalized with nuclear factor of activated T cells (NFAT) transcription factor activity and was sensitive to inhibition of calcineurin-dependent NFAT activation. However, calcineurin/NFAT regulation did not contribute to constitutive expression elsewhere or to inflammatory COX-2 induction at any site. These data address the mechanisms driving constitutive COX-2 and suggest that by targeting transcription it may be possible to develop antiinflammatory therapies that spare the constitutive expression necessary for normal homeostatic functions, including those important to the cardiovascular-renal system. PMID:26712011

  18. The neuropeptide catestatin promotes vascular smooth muscle cell proliferation through the Ca{sup 2+}-calcineurin-NFAT signaling pathway

    SciTech Connect

    Guo, Xiaoxia; Zhou, Chunyan; Sun, Ningling

    2011-04-22

    Highlights: {yields} Catestatin stimulates proliferation of vascular smooth muscle cells in a dose-dependent manner. {yields} Catestatin provokes sustained increase in intracellular Ca{sup 2+}. {yields} Catestatin produces increased activation of calcineurin and promotes NFATc1 translocation into the nucleus. -- Abstract: The Chromogranin A-derived neuropeptide catestatin is an endogenous nicotinic cholinergic antagonist that acts as a pleiotropic hormone. Since catestatin shares several functions with other members derived from the chromogranin/secretogranin protein family and other neuropeptides which exert proliferative effects on vascular smooth muscle cells (VSMCs), we therefore hypothesized that catestatin would regulate VSMC proliferation. The present study demonstrates that catestatin caused a dose-dependent induction of proliferation in rat aortic smooth muscle cells and furthermore evoked a sustained increase in intracellular calcium. This subsequently leaded to enhanced activation of the Ca{sup 2+}/calmodulin-dependent phosphatase, calcineurin and resulted in an activation of the Ca{sup 2+}-dependent transcription factor, nuclear factor of activated T cells (NFAT), initiating transcription of proliferative genes. In addition, cyclosporin A (CsA), a potent inhibitor of calcineurin, abrogated catestatin-mediated effect on VSMCs, indicating that the calcineurin-NFAT signaling is strongly required for catestatin-induced growth of VSMCs. The present study establishes catestatin as a novel proliferative cytokine on vascular smooth muscle cells and this effect is mediated by the Ca{sup 2+}-calcineurin-NFAT signaling pathway.

  19. Lentivirally Engineered DC activate AFP-specific T cells which Inhibit Hepatocellular Carcinoma Growth in vitro and in vivo

    PubMed Central

    Liu, Yang; Butterfield, Lisa H.; Fu, Xiaohui; Song, Zhenshun; Zhang, Xiaoping; Lu, Chongde; Ding, Guanghui; Wu, Mengchao

    2012-01-01

    Alpha-fetoprotein (AFP), a tumor-associated antigen for hepatocellular carcinoma (HCC), is an established biomarker for HCC. In this study, we created a lentivirus expressing the AFP antigen and investigated the antitumor activity of AFP-specific CD8+ T cells, with and without CD4+ T cells, which were activated by either AFP peptide-pulsed or Lenti-AFP-engineered DC in vitro and in vivo. AFP-specific T cells could efficiently kill HepG2 HCC cells, and produced IL-2, IFN-γ, TNF-α, perforin and granzyme B, with minimal production of IL-10 (a negative regulator of T cell activation). Both strategies activated AFP-specific T cells, but the lentiviral strategy was superior by several measures. Data also support an impact of CD4+ T cells in supporting anti-tumor activity. In vivo studies in a xenograft HCC tumor model also showed that AFP-specific T cells could markedly suppress HCC tumor formation and morbidity in tumor-bearing nude mice, as well as regulate serum levels of related cytokines and antitumor molecules. In parallel with human in vitro T cell cultures, the in vivo model demonstrated superior anti-tumor effects and Th1-skewing with Lenti-AFP-DC. This study supports the superiority of a full-length antigen lentivirus-based DC vaccine strategy over peptides, and provides new insight into the design of DC-based vaccines. PMID:21491085

  20. Vitamin E reverses impaired linker for activation of T cells activation in T cells from aged C57BL/6 mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Supplemental vitamin E restores age-related defects in IL-2 production, T cell proliferation, and immune synapse formation. Here, we evaluated the effect of vitamin E on TCR-proximal signaling events. In aged murine CD4+ T cells stimulated via CD3 and CD28, tyrosine 191 of the adaptor protein LAT wa...

  1. Inhibition of antigen receptor-dependent Ca(2+) signals and NF-AT activation by P2X7 receptors in human B lymphocytes.

    PubMed

    Pippel, Anja; Beßler, Björn; Klapperstück, Manuela; Markwardt, Fritz

    2015-04-01

    One of the first intracellular signals after antigen binding by the antigen receptor of B lymphocytes is the increased intracellular Ca(2+) concentration ([Ca(2+)]i), which is followed by several intracellular signaling events like the nuclear translocation of the transcription factor NF-AT controlling the fate of B lymphocytes after their activation. Extracellular ATP, which is released from cells under several pathological conditions, is considered a danger-associated signal serving as an immunomodulator. We investigated the interaction of antigen receptor (BCR) and P2X7 receptor (P2X7R) activation on [Ca(2+)]i signaling and on nuclear translocation of the transcription factor NF-AT in human B lymphocytes. Although the P2X7R is an ATP-gated Ca(2+)-permeable ion channel, P2X7R activation inhibits the BCR-mediated [Ca(2+)]i responses. This effect is mimicked by cell membrane depolarization induced by an increase in the extracellular K(+) concentration or by application of the Na(+) ionophore gramicidin, but is abolished by stabilization of the membrane potential using the K(+) ionophore valinomycin, by extracellular Mg(2+), which is known to inhibit P2X7R-dependent effects, or by replacing Na(+) by the less P2X7R-permeable Tris(+) ion. Furthermore, P2X7R activation by ATP inhibits the BCR-dependent translocation of the transcription factor NF-ATc1 to the nucleus. We therefore conclude that extracellular ATP via the P2X7R mediates inhibitory effects on B cell activation. This may be of relevance for understanding of the activation of the BCR under pathological conditions and for the development of therapeutic strategies targeting human B lymphocytes or P2X7 receptors. PMID:25678443

  2. Calcineurin-NFAT Signaling Controls Somatic Cell Reprogramming in a Stage-Dependent Manner.

    PubMed

    Sun, Ming; Liao, Bing; Tao, Yu; Chen, Hao; Xiao, Feng; Gu, Junjie; Gao, Shaorong; Jin, Ying

    2016-05-01

    Calcineurin-NFAT signaling is critical for early lineage specification of mouse embryonic stem cells and early embryos. However, its roles in somatic cell reprogramming remain unknown. Here, we report that calcineurin-NFAT signaling has a dynamic activity and plays diverse roles at different stages of reprogramming. At the early stage, calcineurin-NFAT signaling is transiently activated and its activation is required for successful reprogramming. However, at the late stage of reprogramming, activation of calcineurin-NFAT signaling becomes a barrier for reprogramming and its inactivation is critical for successful induction of pluripotency. Mechanistically, calcineurin-NFAT signaling contributes to the reprogramming through regulating multiple early events during reprogramming, including mesenchymal to epithelial transition (MET), cell adhesion and emergence of SSEA1(+) intermediate cells. Collectively, this study reveals for the first time the important roles of calcineurin-NFAT signaling during somatic cell reprogramming and provides new insights into the molecular regulation of reprogramming. PMID:26448199

  3. 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

  4. Activated CD4+ T cells preferentially take up lipid microspheres, but resting cells do not.

    PubMed Central

    Suzuki, K

    1995-01-01

    Lipid microspheres (LM) used as drug carriers increase the effectiveness and reduce the toxicity of incorporated drugs. The present study is designed to determine whether or not activated T lymphocytes, which were the cells chosen first from the 'inflammatory cells', can take up LM in vitro. LM were labelled with a fluorescent probe, DiI (DiI-LM), to examine the kinetics. Flow cytometric analysis demonstrated that in freshly isolated peripheral blood mononuclear cells (PBMC), monocytes principally took up DiI-LM, while lymphocytes and granulocytes did not. When PBMC were stimulated with immobilized anti-CD3 MoAb and IL-2, cells expressing CD3, CD4, CD8 and CD16 incorporated DiI-LM. Purified CD4+ T cells, obtained by positive panning selection, were stimulated with this system. They were CD25, CD71, LFA-1-positive, and also showed an ability to take up DiI-LM, which resting cells did not. The findings were confirmed by flow cytometry and quantitative analysis of DiI. Confocal micrographs showed fluorescent granules from the probe in the cytoplasm of stimulated CD4+ T cells after incubation with DiI-LM. These results suggest that immunomodulatory agents incorporated into LM might selectively regulate the function of CD4+ or CD8+ T cells when these are activated. Images Fig. 4 PMID:7882572

  5. Exchange Protein Directly Activated by cAMP Modulates Regulatory T-Cell-Mediated Immunosuppression

    PubMed Central

    Almahariq, Muayad; Mei, Fang C.; Wang, Hui; Cao, Anthony T.; Yao, Suxia; Soong, Lynn; Sun, Jiaren; Cong, Yingzi; Chen, Ju; Cheng, Xiaodong

    2016-01-01

    The cyclic adenosine monophosphate (cAMP) signaling pathway plays an essential role in immune functions. In this study we examined the role of the cAMP/EPAC1 (exchange protein directly activated by cAMP) axis in regulatory T-cell (Treg)-mediated immune suppression using genetic and pharmacologic approaches. Genetic deletion of EPAC1 in Treg and effector T-cells (Teff) synergistically attenuated Treg-mediated suppression of Teff. Mechanistically, EPAC1 inhibition enhanced activation of the transcription factor STAT3 and up-regulated SMAD7 expression while down-regulating expression of SMAD4. Consequently, CD4+T-cells were desensitized to TGF-β1, a cytokine employed by Treg cells to exert a broad inhibitory function within the immune system. Furthermore, deletion of EPAC1 led to production of significant levels of OVA-IgG antibodies in a low dose oral tolerance mouse mode. These in vivo observations are consistent with the finding that EPAC1 plays an important role in Treg-mediated suppression. More importantly, pharmacological inhibition of EPAC1 using an EPAC specific inhibitor recapitulates the EPAC1 deletion phenotype both in vivo and in vitro. Our results show that EPAC1 boosts Treg-mediated suppression, and identify EPAC1 as a target with broad therapeutic potential since Treg cells are involved in numerous pathologies including autoimmunity, infections, and a wide range of cancers. PMID:25339598

  6. B-cell antigens within normal and activated human T cells

    PubMed Central

    Sandilands, G P; Perry, M; Wootton, M; Hair, J; More, I A R

    1999-01-01

    In this study we compared cell surface staining for human peripheral blood lymphocyte (PBL) CD antigens by flow cytometry, with staining obtained following permeabilization of PBL using the Cytoperm method (Serotec). Six CD antigens (CD20, CD21, CD22, CD32, CD35 and major histocompatibility complex class II antigen) normally found on the surface of B cells, were also found to be expressed within T cells. We also showed, by immunoelectron microscopy, that these inappropriately expressed (‘occult’) CD antigens are located within cytoplasmic vesicles or within the rough endoplasmic reticulum. Following in vitro activation of T cells a distinct increase in expression of all of these cytoplasmic antigens was observed but staining at the cell surface was, by comparison, weak. We therefore propose that up-regulation of various B-cell CD antigens occurs within the cytoplasm of T cells following activation and that these antigens may be synthesized and released into the fluid-phase as soluble immunoregulatory molecules. PMID:10233724

  7. B-cell antigens within normal and activated human T cells.

    PubMed

    Sandilands, G P; Perry, M; Wootton, M; Hair, J; More, I A

    1999-03-01

    In this study we compared cell surface staining for human peripheral blood lymphocyte (PBL) CD antigens by flow cytometry, with staining obtained following permeabilization of PBL using the Cytoperm method (Serotec). Six CD antigens (CD20, CD21, CD22, CD32, CD35 and major histocompatibility complex class II antigen) normally found on the surface of B cells, were also found to be expressed within T cells. We also showed, by immunoelectron microscopy, that these inappropriately expressed ('occult') CD antigens are located within cytoplasmic vesicles or within the rough endoplasmic reticulum. Following in vitro activation of T cells a distinct increase in expression of all of these cytoplasmic antigens was observed but staining at the cell surface was, by comparison, weak. We therefore propose that up-regulation of various B-cell CD antigens occurs within the cytoplasm of T cells following activation and that these antigens may be synthesized and released into the fluid-phase as soluble immunoregulatory molecules. PMID:10233724

  8. Temporal protein expression pattern in intracellular signalling cascade during T-cell activation: a computational study.

    PubMed

    Ganguli, Piyali; Chowdhury, Saikat; Bhowmick, Rupa; Sarkar, Ram Rup

    2015-10-01

    Various T-cell co-receptor molecules and calcium channel CRAC play a pivotal role in the maintenance of cell's functional responses by regulating the production of effector molecules (mostly cytokines) that aids in immune clearance and also maintaining the cell in a functionally active state. Any defect in these co-receptor signalling pathways may lead to an altered expression pattern of the effector molecules. To study the propagation of such defects with time and their effect on the intracellular protein expression patterns, a comprehensive and largest pathway map of T-cell activation network is reconstructed manually. The entire pathway reactions are then translated using logical equations and simulated using the published time series microarray expression data as inputs. After validating the model, the effect of in silico knock down of co-receptor molecules on the expression patterns of their downstream proteins is studied and simultaneously the changes in the phenotypic behaviours of the T-cell population are predicted, which shows significant variations among the proteins expression and the signalling routes through which the response is propagated in the cytoplasm. This integrative computational approach serves as a valuable technique to study the changes in protein expression patterns and helps to predict variations in the cellular behaviour. PMID:26564978

  9. TGF-β and IL-21 cooperatively stimulate activated CD8(+) T cells to differentiate into Tc17 cells.

    PubMed

    Chen, Hsin-Wei; Tsai, Jy-Ping; Yao, Tsung-You; Hsieh, Chia-Ling; Chen, I-Hua; Liu, Shin-Jen

    2016-06-01

    TGF-β together with IL-21 or IL-6 can drive the differentiation of naïve CD8(+) T cells into IL-17-producing CD8(+) T cells. These IL-17-producing CD8(+) T cells are termed Tc17 cells. Tc17 cells preserve plasticity under various conditions in vitro and in vivo. IFN-γ-producing CD8(+) T cells are termed Tc1 cells. However, Tc1 cells are considered relatively stable. In the present study, we show that the combination of TGF-β plus IL-21, but not IL-6, converts Tc1 cells into Tc17 cells; this conversion is associated with elevated RORα, RORγt, and Batf mRNA levels. These results indicate that Tc1 cells are skewed to the Tc17 cell phenotype under TGF-β plus IL-21-polarizing conditions. Furthermore, IL-6R is expressed on naïve, but not activated, CD8(+) T cells. In contrast, IL-21R is expressed on both naïve and activated CD8(+) T cells. Thus, differential expression profiles of IL-6R and IL-21R on naïve and activated CD8(+) T cells may be one mechanism by which TGF-β plus IL-21, but not IL-6, can drive activated CD8(+) T cells to differentiate into IL-17-producing cells. Taken together, these results provide a novel viewpoint for the plasticity of Tc1 cells. PMID:27085379

  10. Modest Interference with Actin Dynamics in Primary T Cell Activation by Antigen Presenting Cells Preferentially Affects Lamellal Signaling

    PubMed Central

    Roybal, Kole T.; Mace, Emily M.; Clark, Danielle J.; Leard, Alan D.; Herman, Andrew; Verkade, Paul; Orange, Jordan S.; Wülfing, Christoph

    2015-01-01

    Dynamic subcellular distributions of signaling system components are critical regulators of cellular signal transduction through their control of molecular interactions. Understanding how signaling activity depends on such distributions and the cellular structures driving them is required for comprehensive insight into signal transduction. In the activation of primary murine T cells by antigen presenting cells (APC) signaling intermediates associate with various subcellular structures, prominently a transient, wide, and actin-associated lamellum extending from an interdigitated T cell:APC interface several micrometers into the T cell. While actin dynamics are well established as general regulators of cellular organization, their role in controlling signaling organization in primary T cell:APC couples and the specific cellular structures driving it is unresolved. Using modest interference with actin dynamics with a low concentration of Jasplakinolide as corroborated by costimulation blockade we show that T cell actin preferentially controls lamellal signaling localization and activity leading downstream to calcium signaling. Lamellal localization repeatedly related to efficient T cell function. This suggests that the transient lamellal actin matrix regulates T cell signaling associations that facilitate T cell activation. PMID:26237588

  11. 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

  12. Messenger RNA encoding constitutively active Toll-like receptor 4 enhances effector functions of human T cells.

    PubMed

    Pato, A; Eisenberg, G; Machlenkin, A; Margalit, A; Cafri, G; Frankenburg, S; Merims, S; Peretz, T; Lotem, M; Gross, G

    2015-11-01

    Adoptive T cell therapy of cancer employs a large number of ex-vivo-propagated T cells which recognize their targets either by virtue of their endogenous T cell receptor (TCR) or via genetic reprogramming. However, both cell-extrinsic and intrinsic mechanisms often diminish the in-vivo potency of these therapeutic T cells, limiting their clinical efficacy and broader use. Direct activation of human T cells by Toll-like receptor (TLR) ligands induces T cell survival and proliferation, boosts the production of proinflammatory cytokines and augments resistance to regulatory T cell (Treg) suppression. Removal of the TLR ligand-binding region results in constitutive signalling triggered by the remaining cytosolic Toll/interleukin-1 receptor (TIR) domain. The use of such TIR domains therefore offers an ideal means for equipping anti-tumour T cells with the arsenal of functional attributes required for improving current clinical protocols. Here we show that constitutively active (ca)TLR-4 can be expressed efficiently in human T cells using mRNA electroporation. The mere expression of caTLR-4 mRNA in polyclonal CD8 and CD4 T cells induced the production of interferon (IFN)-γ, triggered the surface expression of CD25, CD69 and 4-1BB and up-regulated a panel of cytokines and chemokines. In tumour-infiltrating lymphocytes prepared from melanoma patients, caTLR-4 induced robust IFN-γ secretion in all samples tested. Furthermore, caTLR-4 enhanced the anti-melanoma cytolytic activity of tumour-infiltrating lymphocytes and augmented the secretion of IFN-γ, tumour necrosis factor (TNF)-α and granulocyte-macrophage colony-stimulating factor (GM-CSF) for at least 4 days post-transfection. Our results demonstrate that caTLR-4 is capable of exerting multiple T cell-enhancing effects and can potentially be used as a genetic adjuvant in adoptive cell therapy. PMID:26212048

  13. 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

  14. A tumor-derived protein which provides T-cell costimulation through accessory cell activation.

    PubMed

    Powell, T J; Schreck, R; McCall, M; Hui, T; Rice, A; App, H; Azam, M; Ullrich, A; Shawver, L K

    1995-05-01

    A recently described tumor-derived glycoprotein, designated 90K, has been shown to have positive effects on the generation of cytotoxic effector cells (NK/LAK) from human PBMC. To determine the mechanism of these effects, we have examined the effects of 90K on cytokine production by human PBMC. A culture of normal PBMC with 90K alone did not result in IL-2 secretion; however, in coculture with suboptimal doses of ConA, 90K increased IL-2 secretion by PBMC. Coculture of PBMC with 90K and ConA also resulted in increased production of the cytokines IL-1, IL-6, GM-CSF, and TNF alpha. T cells depleted of accessory cells failed to respond to ConA alone, 90K alone, or the combination of ConA and 90K, suggesting that this protein does not have a direct effect on T cells. However, 90K alone was sufficient to induce cytokine production by unfractionated PBMC (IL-1, IL-6, GM-CSF, and TNF alpha) or by CD14-enriched PBMC (IL-1 and IL-6). In addition, expression of ICAM-1 was increased on a human monocytic cell line cultured with purified 90K in the absence of any other stimulus. This 90K-induced upregulation of ICAM-1 expression was accompanied by an increased accessory function of the monocytes, demonstrated by their ability to support ConA-induced activation of peripheral blood T cells. Based on the current data, we propose a model in which 90K activates accessory cells, resulting in the secretion of cytokines and the expression of adhesion molecules, which in turn act as costimulatory signals for T-cell activation. Activated T cells then produce cytokines such as IL-2, which lead to a more vigorous cell-mediated immune response to tumor cells and virus-infected cells. Thus, 90K shows promise as an immunotherapeutic reagent for diseases such as cancer and viral infection. PMID:7582257

  15. 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

  16. Lower Pre-Treatment T Cell Activation in Early- and Late-Onset Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome

    PubMed Central

    Goovaerts, Odin; Jennes, Wim; Massinga-Loembé, Marguerite; Ondoa, Pascale; Ceulemans, Ann; Vereecken, Chris; Worodria, William; Mayanja-Kizza, Harriet; Colebunders, Robert; Kestens, Luc

    2015-01-01

    Background Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an inflammatory complication in HIV-TB co-infected patients receiving antiretroviral therapy (ART). The role of disturbed T cell reconstitution in TB-IRIS is not well understood. We investigated T cell activation and maturation profiles in patients who developed TB-IRIS at different intervals during ART. Methods Twenty-two HIV-TB patients who developed early-onset TB-IRIS and 10 who developed late-onset TB-IRIS were matched for age, sex and CD4 count to equal numbers of HIV-TB patients who did not develop TB-IRIS. Flow cytometry analysis was performed on fresh blood, drawn before and after ART initiation and during TB-IRIS events. T cell activation and maturation was measured on CD4+ and CD8+ T cells using CD45RO, CD38, HLA-DR, CCR7 and CD27 antibodies. Results CD8+ T cell activation before ART was decreased in both early-onset (77% vs. 82%, p = 0.014) and late-onset (71% vs. 83%, p = 0.012) TB-IRIS patients compared to non-IRIS controls. After ART initiation, the observed differences in T cell activation disappeared. During late-onset, but not early-onset TB-IRIS, we observed a skewing from memory to terminal effector CD4+ and CD8+ T cell populations (p≤0.028). Conclusion Our data provide evidence of reduced CD8+ T cell activation before ART as a common predisposing factor of early- and late-onset TB-IRIS. The occurrence of TB-IRIS itself was not marked by an over-activated CD8+ T cell compartment. Late- but not early-onset TB-IRIS was characterized by a more terminally differentiated T cell phenotype. PMID:26208109

  17. Staphylococcal enterotoxin A (SEA) stimulates STAT3 activation and IL-17 expression in cutaneous T-cell lymphoma.

    PubMed

    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; Odum, Niels

    2016-03-10

    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

  18. 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

  19. TAGLN2 regulates T cell activation by stabilizing the actin cytoskeleton at the immunological synapse

    PubMed Central

    Na, Bo-Ra; Kim, Hye-Ran; Piragyte, Indre; Oh, Hyun-Mee; Kwon, Min-Sung; Akber, Uroos; Lee, Hyun-Su; Park, Do-Sim; Song, Woo Keun; Park, Zee-Yong; Im, Sin-Hyeog; Rho, Mun-Chual; Hyun, Young-Min; Kim, Minsoo

    2015-01-01

    The formation of an immunological synapse (IS) requires tight regulation of actin dynamics by many actin polymerizing/depolymerizing proteins. However, the significance of actin stabilization at the IS remains largely unknown. In this paper, we identify a novel function of TAGLN2—an actin-binding protein predominantly expressed in T cells—in stabilizing cortical F-actin, thereby maintaining F-actin contents at the IS and acquiring LFA-1 (leukocyte function-associated antigen-1) activation after T cell receptor stimulation. TAGLN2 blocks actin depolymerization and competes with cofilin both in vitro and in vivo. Knockout of TAGLN2 (TAGLN2−/−) reduced F-actin content and destabilized F-actin ring formation, resulting in decreased cell adhesion and spreading. TAGLN2−/− T cells displayed weakened cytokine production and cytotoxic effector function. These findings reveal a novel function of TAGLN2 in enhancing T cell responses by controlling actin stability at the IS. PMID:25869671

  20. Leukemia-associated activating mutation of Flt3 expands dendritic cells and alters T cell responses.

    PubMed

    Lau, Colleen M; Nish, Simone A; Yogev, Nir; Waisman, Ari; Reiner, Steven L; Reizis, Boris

    2016-03-01

    A common genetic alteration in acute myeloid leukemia is the internal tandem duplication (ITD) in FLT3, the receptor for cytokine FLT3 ligand (FLT3L). Constitutively active FLT3-ITD promotes the expansion of transformed progenitors, but also has pleiotropic effects on hematopoiesis. We analyzed the effect of FLT3-ITD on dendritic cells (DCs), which express FLT3 and can be expanded by FLT3L administration. Pre-leukemic mice with the Flt3(ITD) knock-in allele manifested an expansion of classical DCs (cDCs) and plasmacytoid DCs. The expansion originated in DC progenitors, was cell intrinsic, and was further enhanced in Flt3(ITD/ITD) mice. The mutation caused the down-regulation of Flt3 on the surface of DCs and reduced their responsiveness to Flt3L. Both canonical Batf3-dependent CD8(+) cDCs and noncanonical CD8(+) cDCs were expanded and showed specific alterations in their expression profiles. Flt3(ITD) mice showed enhanced capacity to support T cell proliferation, including a cell-extrinsic expansion of regulatory T (T reg) cells. Accordingly, these mice restricted alloreactive T cell responses during graft-versus-host reaction, but failed to control autoimmunity without T reg cells. Thus, the FLT3-ITD mutation directly affects DC development, indirectly modulating T cell homeostasis and supporting T reg cell expansion. We hypothesize that this effect of FLT3-ITD might subvert immunosurveillance and promote leukemogenesis in a cell-extrinsic manner. PMID:26903243

  1. Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation

    PubMed Central

    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

  2. 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

  3. Rac1 Protein Regulates Glycogen Phosphorylase Activation and Controls Interleukin (IL)-2-dependent T Cell Proliferation*

    PubMed Central

    Arrizabalaga, Onetsine; Lacerda, Hadriano M.; Zubiaga, Ana M.; Zugaza, José L.

    2012-01-01

    Small GTPases of the Rho family have been implicated in important cellular processes such as cell migration and adhesion, protein secretion, and/or gene transcription. In the lymphoid system, these GTPases participate in the signaling cascades that are activated after engagement of antigen receptors. However, little is known about the role that Rho GTPases play in IL-2-mediated responses. Here, we show that IL-2 induces Rac1 activation in Kit 225 T cells. We identified by mass spectrometry the muscle isoform of glycogen phosphorylase (PYGM) as a novel Rac1 effector molecule in IL-2-stimulated cells. The interaction between the active form of Rac1 (Rac1-GTP) and PYGM was established directly through a domain comprising amino acids 191–270 of PYGM that exhibits significant homology with the Rac binding domain of PAK1. The integrity of this region was crucial for PYGM activation. Importantly, IL-2-dependent cellular proliferation was inhibited upon blocking both the activation of Rac1 and the activity of PYGM. These results reveal a new role for Rac1 in cell signaling, showing that this GTPase triggers T cell proliferation upon IL-2 stimulation by associating with PYGM and modulating its enzymatic activity. PMID:22337875

  4. Intersection of population variation and autoimmunity genetics in human T cell activation.

    PubMed

    Ye, Chun Jimmie; Feng, Ting; Kwon, Ho-Keun; Raj, Towfique; Wilson, Michael T; Asinovski, Natasha; McCabe, Cristin; Lee, Michelle H; Frohlich, Irene; Paik, Hyun-il; Zaitlen, Noah; Hacohen, Nir; Stranger, Barbara; De Jager, Philip; Mathis, Diane; Regev, Aviv; Benoist, Christophe

    2014-09-12

    T lymphocyte activation by antigen conditions adaptive immune responses and immunopathologies, but we know little about its variation in humans and its genetic or environmental roots. We analyzed gene expression in CD4(+) T cells during unbiased activation or in T helper 17 (T(H)17) conditions from 348 healthy participants representing European, Asian, and African ancestries. We observed interindividual variability, most marked for cytokine transcripts, with clear biases on the basis of ancestry, and following patterns more complex than simple T(H)1/2/17 partitions. We identified 39 genetic loci specifically associated in cis with activated gene expression. We further fine-mapped and validated a single-base variant that modulates YY1 binding and the activity of an enhancer element controlling the autoimmune-associated IL2RA gene, affecting its activity in activated but not regulatory T cells. Thus, interindividual variability affects the fundamental immunologic process of T helper activation, with important connections to autoimmune disease. PMID:25214635

  5. B cells pulsed with Helicobacter pylori antigen efficiently activate memory CD8+ T cells from H. pylori-infected individuals.

    PubMed

    Azem, Josef; Svennerholm, Ann-Mari; Lundin, B Samuel

    2006-01-01

    Helicobacter pylori infection causes chronic gastritis that may progress to peptic ulcers or gastric adenocarcinoma and thereby cause major world-wide health problems. Previous studies have shown that CD4+ T cells are important in the immune response to H. pylori in humans, but the role of CD8+ T cells is less clear. In order to study the CD8+ T cell response to H. pylori in greater detail, we have evaluated efficient conditions for activation of CD8+ T cells in vitro. We show that H. pylori-reactive CD8+ T cells can be activated most efficiently by B cells or dendritic cells pulsed with H. pylori antigens. We further show that the majority of CD8+ T cells in H. pylori-infected gastric mucosa are memory cells, and that memory CD8+ T cells sorted from peripheral blood of H. pylori-infected individuals respond 15-fold more to H. pylori urease compared to memory cells from uninfected subjects. We conclude that CD8+ T cells do participate in the immune response to H. pylori, and this may have implications for the development of more severe disease outcomes in H. pylori-infected subjects. PMID:16324887

  6. Enhanced Anti-HIV Functional Activity Associated with Gag-Specific CD8 T-Cell Responses▿ †

    PubMed Central

    Julg, B.; Williams, K. L.; Reddy, S.; Bishop, K.; Qi, Y.; Carrington, M.; Goulder, P. J.; Ndung'u, T.; Walker, B. D.

    2010-01-01

    Effective HIV-specific T-cell immunity requires the ability to inhibit virus replication in the infected host, but the functional characteristics of cells able to mediate this effect are not well defined. Since Gag-specific CD8 T cells have repeatedly been associated with lower viremia, we examined the influence of Gag specificity on the ability of unstimulated CD8 T cells from chronically infected persons to inhibit virus replication in autologous CD4 T cells. Persons with broad (≥6; n = 13) or narrow (≤1; n = 13) Gag-specific responses, as assessed by gamma interferon enzyme-linked immunospot assay, were selected from 288 highly active antiretroviral therapy (HAART)-naive HIV-1 clade C-infected South Africans, matching groups for total magnitude of HIV-specific CD8 T-cell responses and CD4 T-cell counts. CD8 T cells from high Gag responders suppressed in vitro replication of a heterologous HIV strain in autologous CD4 cells more potently than did those from low Gag responders (P < 0.003) and were associated with lower viral loads in vivo (P < 0.002). As previously shown in subjects with low viremia, CD8 T cells from high Gag responders exhibited a more polyfunctional cytokine profile and a stronger ability to proliferate in response to HIV stimulation than did low Gag responders, which mainly exhibited monofunctional CD8 T-cell responses. Furthermore, increased polyfunctionality was significantly correlated with greater inhibition of viral replication in vitro. These data indicate that enhanced suppression of HIV replication is associated with broader targeting of Gag. We conclude that it is not the overall magnitude but rather the breadth, magnitude, and functional capacity of CD8 T-cell responses to certain conserved proteins, like Gag, which predict effective antiviral HIV-specific CD8 T-cell function. PMID:20335261

  7. IL-33 Receptor-Expressing Regulatory T Cells Are Highly Activated, Th2 Biased and Suppress CD4 T Cell Proliferation through IL-10 and TGFβ Release

    PubMed Central

    Datsi, Angeliki; Hegazy, Ahmed N.; Varga, Domonkos V.; Holecska, Vivien; Saito, Hirohisa; Nakae, Susumu; Löhning, Max

    2016-01-01

    Immunomodulatory Foxp3+ regulatory T cells (Tregs) form a heterogeneous population consisting of subsets with different activation states, migratory properties and suppressive functions. Recently, expression of the IL-33 receptor ST2 was shown on Tregs in inflammatory settings. Here we report that ST2 expression identifies highly activated Tregs in mice even under homeostatic conditions. ST2+ Tregs preferentially accumulate at non-lymphoid sites, likely mediated by their high expression of several chemokine receptors facilitating tissue homing. ST2+ Tregs exhibit a Th2-biased character, expressing GATA-3 and producing the Th2 cytokines IL-5 and IL-13 –especially in response to IL-33. Yet, IL-33 is dispensable for the generation and maintenance of these cells in vivo. Furthermore, ST2+ Tregs are superior to ST2− Tregs in suppressing CD4+ T cell proliferation in vitro independent of IL-33. This higher suppressive capacity is partially mediated by enhanced production and activation of the anti-inflammatory cytokines IL-10 and TGFβ. Thus, ST2 expression identifies a highly activated, strongly suppressive Treg subset preferentially located in non-lymphoid tissues. Here ST2+ Tregs may be well positioned to immediately react to IL-33 alarm signals. Their specific properties may render ST2+ Tregs useful targets for immunomodulatory therapies. PMID:27548066

  8. Identification of a novel gene expressed in activated natural killer cells and T cells

    SciTech Connect

    Dahl, C.A.; Schall, R.P.; He, H.; Cairns, J.S. )

    1992-01-15

    The authors have isolated a cDNA clone from a human activated NK cell-derived cDNA library that identifies a transcript [NK4] that is selectively expressed in lymphocytes. The expression of this transcript is increased after activation of T cells by mitogens or activation of NK cells by IL-2 (lymphokine-activated killer cells). The transcript levels demonstrated by Northern blot analysis increase by 12 h after activation, remain high for at least 48 h, and require protein synthesis for expression. Southern blot analysis of B lymphoblastoid lines derived from 18 unrelated individuals reveal variable banding patterns suggestive of polymorphism within the NK4 gene. No homology was found between the sequence of the coding region of this transcript and any sequences in the GenBank data base. Sequence homology to the U1 small nuclear RNA was found within the 3[prime] untranslated region immediately upstream of the site of polyadenylation, suggesting a possible role for U1 in the polyadenylation process. Sequence analysis indicates the transcript would encode a protein having a mass of 27 kDa. The presence of a signal sequence and lack of a transmembrane region suggests that the protein is secreted. In addition, the protein contains an RGD sequence that may be involved in cellular adhesion. This transcript appears to encode a novel product common to the activation pathways of both NK cells and T cells. 50 refs., 8 figs.

  9. Molecular Mechanisms of Treg-Mediated T Cell Suppression

    PubMed Central

    Schmidt, Angelika; Oberle, Nina; Krammer, Peter H.

    2012-01-01

    CD4+CD25highFoxp3+ regulatory T cells (Tregs) can suppress other immune cells and, thus, are critical mediators of peripheral self-tolerance. On the one hand, Tregs avert autoimmune disease and allergies. On the other hand, Tregs can prevent immune reactions against tumors and pathogens. Despite the importance of Tregs, the molecular mechanisms of suppression remain incompletely understood and controversial. Proliferation and cytokine production of CD4+CD25− conventional T cells (Tcons) can be inhibited directly by Tregs. In addition, Tregs can indirectly suppress Tcon activation via inhibition of the stimulatory capacity of antigen presenting cells. Direct suppression of Tcons by Tregs can involve immunosuppressive soluble factors or cell contact. Different mechanisms of suppression have been described, so far with no consensus on one universal mechanism. Controversies might be explained by the fact that different mechanisms may operate depending on the site of the immune reaction, on the type and activation state of the suppressed target cell as well as on the Treg activation status. Further, inhibition of T cell effector function can occur independently of suppression of proliferation. In this review, we summarize the described molecular mechanisms of suppression with a particular focus on suppression of Tcons and rapid suppression of T cell receptor-induced calcium (Ca2+), NFAT, and NF-κB signaling in Tcons by Tregs. PMID:22566933

  10. Naive human T cells are activated and proliferate in response to the heme oxygenase-1 inhibitor tin mesoporphyrin.

    PubMed

    Burt, Trevor D; Seu, Lillian; Mold, Jeffrey E; Kappas, Attallah; McCune, Joseph M

    2010-11-01

    Heme oxygenase-1 (HO-1) and its catabolic by-products have potent anti-inflammatory activity in many models of disease. It is not known, however, if HO-1 also plays a role in the homeostatic control of T cell activation and proliferation. We demonstrate here that the HO-1 inhibitor tin mesoporphyrin (SnMP) induces activation, proliferation, and maturation of naive CD4(+) and CD8(+) T cells via interactions with CD14(+) monocytes in vitro. This response is dependent upon interactions of T cells with MHC class I and II on the surface of CD14(+) monocytes. Furthermore, CD4(+)CD25(+)FoxP3(+) regulatory T cells were able to suppress this proliferation, even though their suppressive activity was itself impaired by SnMP. Given the magnitude of the Ag-independent T cell response induced by SnMP, we speculate that HO-1 plays an important role in dampening nonspecific T cell activation. Based on these findings, we propose a potential role for HO-1 in the control of naive T cell homeostatic proliferation. PMID:20921523

  11. The impact of Nucleofection® on the activation state of primary human CD4 T cells

    PubMed Central

    Zhang, Mingce; Ma, Zhengyu; Selliah, Nithianandan; Weiss, Greta; Genin, Anna; Finkel, Terri H.; Cron, Randy Q.

    2014-01-01

    Gene transfer into primary human CD4 T lymphocytes is a critical tool in studying the mechanism of T cell-dependent immune responses and human immunodeficiency virus-1 (HIV-1) infection. Nucleofection® is an electroporation technique that allows efficient gene transfer into primary human CD4 T cells that are notoriously resistant to traditional electroporation. Despite its popularity in immunological research, careful characterization of its impact on the physiology of CD4 T cells has not been documented. Herein, using freshly-isolated primary human CD4 T cells, we examine the effects of Nucleofection® on CD4 T cell morphology, intracellular calcium levels, cell surface activation markers, and transcriptional activity. We find that immediately after Nucleofection®, CD4 T cells undergo dramatic morphological changes characterized by wrinkled and dilated plasma membranes before recovering 1 hour later. The intracellular calcium level also increases after Nucleofection®, peaking after 1 hour before recovering 8 hours post transfection. Moreover, Nucleofection® leads to increased expression of T cell activation markers, CD154 and CD69, for more than 24 hours, and enhances the activation effects of phytohemagglutinin (PHA) stimulation. In addition, transcriptional activity is increased in the first 24 hours after Nucleofection®, even in the absence of exogenous stimuli. Therefore, Nucleofection® significantly alters the activation state of primary human CD4 T cells. The effect of transferred gene products on CD4 T cell function by Nucleofection® should be assessed after sufficient resting time post transfection or analyzed in light of the activation caveats mentioned above. PMID:24910411

  12. The impact of Nucleofection® on the activation state of primary human CD4 T cells.

    PubMed

    Zhang, Mingce; Ma, Zhengyu; Selliah, Nithianandan; Weiss, Greta; Genin, Anna; Finkel, Terri H; Cron, Randy Q

    2014-06-01

    Gene transfer into primary human CD4 T lymphocytes is a critical tool in studying the mechanism of T cell-dependent immune responses and human immunodeficiency virus-1 (HIV-1) infection. Nucleofection® is an electroporation technique that allows efficient gene transfer into primary human CD4 T cells that are notoriously resistant to traditional electroporation. Despite its popularity in immunological research, careful characterization of its impact on the physiology of CD4 T cells has not been documented. Herein, using freshly-isolated primary human CD4 T cells, we examine the effects of Nucleofection® on CD4 T cell morphology, intracellular calcium levels, cell surface activation markers, and transcriptional activity. We find that immediately after Nucleofection®, CD4 T cells undergo dramatic morphological changes characterized by wrinkled and dilated plasma membranes before recovering 1h later. The intracellular calcium level also increases after Nucleofection®, peaking after 1h before recovering 8h post transfection. Moreover, Nucleofection® leads to increased expression of T cell activation markers, CD154 and CD69, for more than 24h, and enhances the activation effects of phytohemagglutinin (PHA) stimulation. In addition, transcriptional activity is increased in the first 24h after Nucleofection®, even in the absence of exogenous stimuli. Therefore, Nucleofection® significantly alters the activation state of primary human CD4 T cells. The effect of transferred gene products on CD4 T cell function by Nucleofection® should be assessed after sufficient resting time post transfection or analyzed in light of the activation caveats mentioned above. PMID:24910411

  13. Dominant-Activating, Germline Mutations in Phosphoinositide 3-Kinase p110δ Cause T Cell Senescence and Human Immunodeficiency

    PubMed Central

    Lucas, Carrie L.; Kuehn, Hye Sun; Zhao, Fang; Niemela, Julie E.; Deenick, Elissa K.; Palendira, Umaimainthan; Avery, Danielle T.; Moens, Leen; Cannons, Jennifer L.; Biancalana, Matthew; Stoddard, Jennifer; Ouyang, Weiming; Frucht, David L.; Rao, V. Koneti; Atkinson, T. Prescott; Agharahimi, Anahita; Hussey, Ashleigh A.; Folio, Les R.; Olivier, Kenneth N.; Fleisher, Thomas A.; Pittaluga, Stefania; Holland, Steven M.; Cohen, Jeffrey I.; Oliviera, Joao B.; Tangye, Stuart G.; Schwartzberg, Pamela L.; Lenardo, Michael J.; Uzel, Gulbu

    2014-01-01

    The p110δ subunit of phosphoinositide 3-kinase (PI(3)K) is selectively expressed in leukocytes and is critical for lymphocyte biology. Here we report three different germline, heterozygous, gain-of-function mutations in the PIK3CD gene encoding p110δ in fourteen patients from seven families. These patients presented with sinopulmonary infections, lymphadenopathy, nodular lymphoid hyperplasia and CMV and/or EBV viremia. Strikingly, naïve and central memory T cells were severely deficient, while senescent effector T cells were over-represented. In vitro, patient T cells exhibited increased phosphorylation of Akt and hyperactivation of mTOR, enhanced glucose uptake and terminal effector differentiation. Importantly, treatment with rapamycin to inhibit mTOR activity in vivo partially restored naïve T cells, largely rescued the in vitro T cell defects, and improved clinical course. PMID:24165795

  14. CD43 REGULATES THE THRESHOLD FOR T CELL ACTIVATION BY TARGETING CBL FUNCTIONS

    PubMed Central

    Pedraza-Alva, Gustavo; Lilia, B. Mérida; del Rio, Roxana; Nora, A. Fierro; Cruz-Muñoz, Mario E.; Olivares, Norma; Melchy, Erika; Igras, Vivian; Georg, A. Holländer; Steven, J. Burakoff; Rosenstein, Yvonne

    2013-01-01

    SUMMARY T cell (TC) activation requires the coordinated signaling of the T cell receptor (TCR) and co-receptor molecules, allowing TCs to respond to lower degrees of TCR occupancy. Co-receptor molecules set the threshold for TC activation by controlling different regulatory signaling loops. The Cbl family members prevent undesired activation of TCs by regulating TCR signals. In this report we show that TC pre-stimulation by the CD43 co-receptor molecule before TCR engagement inhibits TCR-dependent c-Cbl tyrosine phosphorylation, c-Cbl interaction with the adapter molecule Crk-L and promotes Cbl-b degradation in a PKCθ–dependent manner. Consequently, the prolonged tyrosine phosphorylation and delayed degradation of ZAP-70 and of the ζ chain lead to enhanced MAPK activation and robust TC response. These data indicates that CD43-mediated signals lower the threshold for TC activation by restricting the c-Cbl and Cbl-b inhibitory effects on TCR signaling. In addition to the strength and duration of intracellular signals, our data underscore temporality with which certain molecules are engaged as yet another mechanism to fine tune TC signal quality, and ultimately immune function. PMID:21905200

  15. MAP Kinase Inhibition Promotes T Cell and Anti-tumor Activity in Combination with PD-L1 Checkpoint Blockade.

    PubMed

    Ebert, Peter J R; Cheung, Jeanne; Yang, Yagai; McNamara, Erin; Hong, Rebecca; Moskalenko, Marina; Gould, Stephen E; Maecker, Heather; Irving, Bryan A; Kim, Jeong M; Belvin, Marcia; Mellman, Ira

    2016-03-15

    Targeted inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) can induce regression of tumors bearing activating mutations in the Ras pathway but rarely leads to tumor eradication. Although combining MEK inhibition with T-cell-directed immunotherapy might lead to more durable efficacy, T cell responses are themselves at least partially dependent on MEK activity. We show here that MEK inhibition did profoundly block naive CD8(+) T cell priming in tumor-bearing mice, but actually increased the number of effector-phenotype antigen-specific CD8(+) T cells within the tumor. MEK inhibition protected tumor-infiltrating CD8(+) T cells from death driven by chronic TCR stimulation while sparing cytotoxic activity. Combining MEK inhibition with anti-programmed death-ligand 1 (PD-L1) resulted in synergistic and durable tumor regression even where either agent alone was only modestly effective. Thus, despite the central importance of the MAP kinase pathway in some aspects of T cell function, MEK-targeted agents can be compatible with T-cell-dependent immunotherapy. PMID:26944201

  16. IL-12 and IL-4 activate a CD39-dependent intrinsic peripheral tolerance mechanism in CD8(+) T cells.

    PubMed

    Noble, Alistair; Mehta, Hema; Lovell, Andrew; Papaioannou, Eleftheria; Fairbanks, Lynette

    2016-06-01

    Immune responses to protein antigens involve CD4(+) and CD8(+) T cells, which follow distinct programs of differentiation. Naïve CD8 T cells rapidly develop cytotoxic T-cell (CTL) activity after T-cell receptor stimulation, and we have previously shown that this is accompanied by suppressive activity in the presence of specific cytokines, i.e. IL-12 and IL-4. Cytokine-induced CD8(+) regulatory T (Treg) cells are one of several Treg-cell phenotypes and are Foxp3(-) IL-10(+) with contact-dependent suppressive capacity. Here, we show they also express high level CD39, an ecto-nucleotidase that degrades extracellular ATP, and this contributes to their suppressive activity. CD39 expression was found to be upregulated on CD8(+) T cells during peripheral tolerance induction in vivo, accompanied by release of IL-12 and IL-10. CD39 was also upregulated during respiratory tolerance induction to inhaled allergen and on tumor-infiltrating CD8(+) T cells. Production of IL-10 and expression of CD39 by CD8(+) T cells was independently regulated, being respectively blocked by extracellular ATP and enhanced by an A2A adenosine receptor agonist. Our results suggest that any CTL can develop suppressive activity when exposed to specific cytokines in the absence of alarmins. Thus negative feedback controls CTL expansion under regulation from both nucleotide and cytokine environment within tissues. PMID:26990545

  17. Inhibiting NFAT1 for breast cancer therapy: New insights into the mechanism of action of MDM2 inhibitor JapA

    PubMed Central

    Voruganti, Sukesh; Wang, Hui; Zhang, Wei-Dong; Zhang, Ruiwen

    2015-01-01

    Transcription factor NFAT1 has been recently identified as a new regulator of the MDM2 oncogene. Targeting the NFAT1-MDM2 pathway represents a novel approach to cancer therapy. We have recently identified a natural product MDM2 inhibitor, termed JapA. As a specific and potent MDM2 inhibitor, JapA inhibits MDM2 at transcriptional and post-translational levels. However, the molecular mechanism remains to be fully elucidated for its inhibitory effects on MDM2 transcription. Herein, we reported that JapA inhibited NFAT1 and NFAT1-mediated MDM2 transcription, which contributed to the anticancer activity of JapA. Its effects on the expression and activity of NFAT1 were examined in various breast cancer cell lines in vitro and in MCF-7 and MDA-MB-231 xenograft tumors in vivo. The specificity of JapA in targeting NFAT1 and NFAT1-MDM2 pathway and the importance of NFAT1 inhibition in JapA's anticancer activity were demonstrated using NFAT1 overexpression and knockdown cell lines and the pharmacological activators and inhibitors of NFAT1 signaling. Our results indicated that JapA inhibited NFAT1 signaling in breast cancer cells in vitro and in vivo, which plays a pivotal role in its anticancer activity. JapA inhibited the nuclear localization of NFAT1, disrupted the NFAT1-MDM2 P2 promoter complex, and induced NFAT1 proteasomal degradation, resulting in the repression of MDM2 transcription. In conclusion, JapA is a novel NFAT1 inhibitor and the NFAT1 inhibition is responsible for the JapA-induced repression of MDM2 transcription, contributing to its anticancer activity. The results may pave an avenue for validating the NFAT1-MDM2 pathway as a novel molecular target for cancer therapy. PMID:26461225

  18. Long-range enhancer activity determines Myc sensitivity to Notch inhibitors in T cell leukemia

    PubMed Central

    Yashiro-Ohtani, Yumi; Wang, Hongfang; Zang, Chongzhi; Arnett, Kelly L.; Bailis, Will; Ho, Yugong; Knoechel, Birgit; Lanauze, Claudia; Louis, Lumena; Forsyth, Katherine S.; Chen, Sujun; Chung, Yoonjie; Schug, Jonathan; Blobel, Gerd A.; Liebhaber, Stephen A.; Bernstein, Bradley E.; Blacklow, Stephen C.; Liu, Xiaole Shirley; Aster, Jon C.; Pear, Warren S.

    2014-01-01

    Notch is needed for T-cell development and is a common oncogenic driver in T-cell acute lymphoblastic leukemia. The protooncogene c-Myc (Myc) is a critical target of Notch in normal and malignant pre-T cells, but how Notch regulates Myc is unknown. Here, we identify a distal enhancer located >1 Mb 3′ of human and murine Myc that binds Notch transcription complexes and physically interacts with the Myc proximal promoter. The Notch1 binding element in this region activates reporter genes in a Notch-dependent, cell-context–specific fashion that requires a conserved Notch complex binding site. Acute changes in Notch activation produce rapid changes in H3K27 acetylation across the entire enhancer (a region spanning >600 kb) that correlate with Myc expression. This broad Notch-influenced region comprises an enhancer region containing multiple domains, recognizable as discrete H3K27 acetylation peaks. Leukemia cells selected for resistance to Notch inhibitors express Myc despite epigenetic silencing of enhancer domains near the Notch transcription complex binding sites. Notch-independent expression of Myc in resistant cells is highly sensitive to inhibitors of bromodomain containing 4 (Brd4), a change in drug sensitivity that is accompanied by preferential association of the Myc promoter with more 3′ enhancer domains that are strongly dependent on Brd4 for function. These findings indicate that altered long-range enhancer activity can mediate resistance to targeted therapies and provide a mechanistic rationale for combined targeting of Notch and Brd4 in leukemia. PMID:25369933

  19. Pharmacological inhibition of interleukin-1 activity on T cells by hydrocortisone, cyclosporine, prostaglandins, and cyclic nucleotides.

    PubMed

    Tracey, D E; Hardee, M M; Richard, K A; Paslay, J W

    1988-01-01

    The effects of a panel of hormones and pharmacological agents on the activation of T cells by a combination of interleukin-1 and phytohemagglutinin (IL-1/PHA) was studied. Pharmacological effects on various stages of IL-1/PHA-induced interleukin-2 (IL-2) production by the cloned murine thymoma cell line LBRM-33-1A5.7 were dissected using a multi-step assay procedure. A 4-h lag phase in the kinetics of IL-2 production allowed the operational definition of an early, IL-1-dependent programming stage, followed by an IL-2-production stage of the assay. A cell-washing procedure between these stages was introduced in order to distinguish IL-1 receptor antagonists from functional IL-1/PHA antagonists. Hydrocortisone and cyclosporine were potent inhibitors (active in the nM range) of both stages of IL-2 production, suggesting that neither is an IL-1 receptor antagonist. The cyclic adenosine monophosphate (cAMP)-elevating agents prostaglandin E2, dibutyryl cAMP, and theophylline inhibited IL-2 production during the early, IL-1-dependent programming stage. By contrast, prostaglandin F2 alpha and dibutyryl cyclic guanosine monophosphate did not appreciably inhibit IL-1/PHA activity. These results are discussed in relationship to the effects of these test agents in thymocyte IL-1 assays or mitogenesis assays and the implications toward understanding the mechanisms underlying IL-1/PHA activation of T cells. PMID:3258857

  20. Investigation of biochemical property changes in activation-induced CD 8 + T cell apoptosis using Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Lee, Young Ju; Ahn, Hyung Joon; Lee, Gi-Ja; Jung, Gyeong Bok; Lee, Gihyun; Kim, Dohyun; Shin, Jae-Ho; Jin, Kyung-Hyun; Park, Hun-Kuk

    2015-07-01

    The study was to investigate the changes in biochemical properties of activated mature CD8+ T cells related to apoptosis at a molecular level. We confirmed the activation and apoptosis of CD8+ T cells by fluorescence-activated cell sorting and atomic force microscopy and then performed Raman spectral measurements on activated mature CD8+ T cells and cellular deoxyribose nucleic acid (DNA). In the activated mature CD8+ T cells, there were increases in protein spectra at 1002 and 1234 cm-1. In particular, to assess the apoptosis-related DNA spectral signatures, we investigated the spectra of the cellular DNA isolated from resting and activated mature CD8+ T cells. Raman spectra at 765 to 786 cm-1 and 1053 to 1087 cm-1 were decreased in activated mature DNA. In addition, we analyzed Raman spectrum using the multivariate statistical method including principal component analysis. Raman spectra of activated mature DNA are especially well-discriminated from those of resting DNA. Our findings regarding the biochemical and structural changes associated with apoptosis in activated mature T cells and cellular DNA according to Raman spectroscopy provide important insights into allospecific immune responses generated after organ transplantation, and may be useful for therapeutic manipulation of the immune response.

  1. Dissociation of peripheral T cell responses from thymocyte negative selection by weak agonists supports a spare receptor model of T cell activation

    PubMed Central

    McNeil, Lisa K.; Evavold, Brian D.

    2002-01-01

    We have focused on stability of the peptide-MHC complex as a determining factor of ligand potency for thymocytes and peripheral CD4+ T cell responses. MHC variant peptides that have low affinities and fast dissociation rates are different in that they stimulate proliferation and cytolysis of mature T cells (classifying the variant peptides as weak agonists) but do not induce thymocyte negative selection. The MHC variant weak agonists require significant receptor reserve, because decreasing the level of T cell receptor on mature T cells blocks the proliferative response. These results demonstrate that peripheral T cells are more sensitive to MHC variant ligands by virtue of increased T cell receptor expression; in addition, the data support a T cell model of the spare receptor theory. PMID:11904393

  2. Potent T Cell Activation with Dimeric Peptide–Major Histocompatibility Complex Class II Ligand: The Role of CD4 Coreceptor

    PubMed Central

    Hamad, Abdel Rahim A.; O'Herrin, Sean M.; Lebowitz, Michael S.; Srikrishnan, Ananth; Bieler, Joan; Schneck, Jonathan; Pardoll, Drew

    1998-01-01

    The interaction of the T cell receptor (TCR) with its cognate peptide–major histocompatibility complex (MHC) on the surface of antigen presenting cells (APCs) is a primary event during T cell activation. Here we used a dimeric IEk-MCC molecule to study its capacity to activate antigen-specific T cells and to directly analyze the role of CD4 in physically stabilizing the TCR–MHC interaction. Dimeric IEk-MCC stably binds to specific T cells. In addition, immobilized dimeric IEk-MCC can induce TCR downregulation and activate antigen-specific T cells more efficiently than anti-CD3. The potency of the dimeric IEk-MCC is significantly enhanced in the presence of CD4. However, CD4 does not play any significant role in stabilizing peptide-MHC–TCR interactions as it fails to enhance binding of IEk-MCC to specific T cells or influence peptide-MHC–TCR dissociation rate or TCR downregulation. Moreover, these results indicate that dimerization of peptide-MHC class II using an IgG molecular scaffold significantly increases its binding avidity leading to an enhancement of its stimulatory capacity while maintaining the physiological properties of cognate peptide–MHC complex. These peptide-MHC–IgG chimeras may, therefore, provide a novel approach to modulate antigen-specific T cell responses both in vitro and in vivo. PMID:9802975

  3. Human T lymphocytes express N-methyl-D-aspartate receptors functionally active in controlling T cell activation

    SciTech Connect

    Miglio, Gianluca; Varsaldi, Federica; Lombardi, Grazia . E-mail: lombardi@pharm.unipmn.it

    2005-12-30

    The aim of this study was to investigate the expression and the functional role of N-methyl-D-aspartate (NMDA) receptors in human T cells. RT-PCR analysis showed that human resting peripheral blood lymphocytes (PBL) and Jurkat T cells express genes encoding for both NR1 and NR2B subunits: phytohemagglutinin (PHA)-activated PBL also expresses both these genes and the NR2A and NR2D genes. Cytofluorimetric analysis showed that NR1 expression increases as a consequence of PHA (10 {mu}g/ml) treatment. D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [(+)-MK 801], competitive and non-competitive NMDA receptor antagonists, respectively, inhibited PHA-induced T cell proliferation, whereas they did not affect IL-2 (10 U/ml)-induced proliferation of PHA blasts. These effects were due to the prevention of T cell activation (inhibition of cell aggregate formation and CD25 expression), but not to cell cycle arrest or death. These results demonstrate that human T lymphocytes express NMDA receptors, which are functionally active in controlling cell activation.

  4. Differential modulation of CD8β by rat γδ and αβ T cells after activation

    PubMed Central

    Straube, Frank; Herrmann, Thomas

    2001-01-01

    Major histocompatibility complex (MHC) class I-restricted αβ T cells express the CD8αβ heterodimer, which acts as a MHC class I-specific co-receptor. Rats are so far the only species with frequent expression of the CD8αβ by MHC-unrestricted γδ T cells. This study compares CD8αβ expression by splenic rat αβ and γδ T cells and reveals a lineage-specific difference in the control of CD8β expression. After activation in vitro, many γδ T cells, but not αβ T cells, persistently down-modulate the expression of CD8β, but not CD8α, at the RNA level. Down-regulation occurred after stimulation with T-cell receptor (TCR)-specific monoclonal antibody (mAb) and interleukin-2 (IL-2) or CD28-mediated costimulation, and after activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Functional differences between modulating and non-modulating cells were not found with respect to interferon-γ (IFN-γ) production and cytolytic activity. The modulation could be indicative for a fundamental difference between αβ and γδ T cells and also limits the use of CD8β as a stable marker of γδ T-cell subsets. Possibly, CD8β modulation provides a mechanism to escape over-stimulation by (auto-)antigens by increasing the threshold of TCR-mediated activation in γδ T cells. PMID:11722639

  5. Dermal γδ T-Cells Can Be Activated by Mitochondrial Damage-Associated Molecular Patterns

    PubMed Central

    Schwacha, Martin G.; Rani, Meenakshi; Nicholson, Susannah E.; Lewis, Aaron M.; Holloway, Travis L.; Sordo, Salvador; Cap, Andrew P.

    2016-01-01

    Background Gamma delta T-cells have been shown to be important to the early immunoinflammatory response to injury, independent of infection. This unique T-cell population acts to regulate cell trafficking and the release of cytokines and growth factors. We propose this sterile inflammatory response is in part associated with damage associated molecular patterns (DAMPs) generated by major injury, such as burn, and mediated via toll-like receptors (TLRs). It is unknown whether DAMPs can activate resident γδ T-cells that reside in skin. Methods Gamma delta T-cells were isolated from the skin of male C57BL/6 mice by enzymatic digestion. Mitochondrial DAMPs (MTDs) were generated from mitochondria isolated from mouse livers by sonication and centrifugation. Dermal γδ T-cells were incubated with MTDs (0–500 μg/ml) for 24 hr and cells and supernatants were collected for analysis. Results MTDs activated dermal γδ T-cells, as evidenced by increased TLR2 and TLR4 expression following in vitro exposure. MTDs also induced the production of inflammatory cytokines (IL-1β, IL-6), and growth factors (PDGF and VEGF) by γδ T-cells. Conclusions These findings herein support the concept that MTDs released after tissue/cellular injury are capable of activating dermal γδ T-cells. We propose that the activation of this unique T-cell population is central in the initiation of sterile inflammation and also contributes to the subsequent healing processes. PMID:27403524

  6. A passive-flow microfluidic device for imaging latent HIV activation dynamics in single T cells

    PubMed Central

    Gearhart, Larisa M.; Miller-Jensen, Kathryn

    2015-01-01

    Quantifying cell-to-cell variability in drug response dynamics is important when evaluating therapeutic efficacy. For example, optimizing latency reversing agents (LRAs) for use in a clinical “activate-and-kill” strategy to purge the latent HIV reservoir in patients requires minimizing heterogeneous viral activation dynamics. To evaluate how heterogeneity in latent HIV activation varies across a range of LRAs, we tracked drug-induced response dynamics in single cells via live-cell imaging using a latent HIV–GFP reporter virus in a clonal Jurkat T cell line. To enable these studies in suspension cells, we designed a simple method to capture an array of single Jurkat T cells using a passive-flow microfluidic device. Our device, which does not require external pumps or tubing, can trap hundreds of cells within minutes with a high retention rate over 12 hours of imaging. Using this device, we quantified heterogeneity in viral activation stimulated by transcription factor (TF) activators and histone deacetylase (HDAC) inhibitors. Generally, TF activators resulted in both faster onset of viral activation and faster rates of production, while HDAC inhibitors resulted in more uniform onset times, but more heterogeneous rates of production. Finally, we demonstrated that while onset time of viral gene expression and rate of viral production together predict total HIV activation, rate and onset time were not correlated within the same individual cell, suggesting that these features are regulated independently. Overall, our results reveal drug-specific patterns of noisy HIV activation dynamics not previously identified in static single-cell assays, which may require consideration for the most effective activate-and-kill regime. PMID:26138068

  7. Grassypeptolides As Natural Inhibitors of Dipeptidyl Peptidase 8 and T-Cell Activation

    PubMed Central

    Kwan, Jason C.; Liu, Yanxia; Ratnayake, Ranjala; Hatano, Ryo; Kuribara, Akiko; Morimoto, Chiko; Ohnuma, Kei; Paul, Valerie J.; Ye, Tao

    2014-01-01

    Natural products made by marine cyanobacteria are often highly modified peptides and depsipeptides that have the potential to act as inhibitors for proteases. In the interest of finding novel protease inhibition activity and selectivity grassypeptolide A (1) was screened against a panel of proteases and found to selectively inhibit DPP8 over DPP4. Grassypeptolides were also found to inhibit IL-2 production and proliferation in activated T-cells, consistent with a putative role of DPP8 in the immune system. These effects were also observed in Jurkat cells, and DPP activity in Jurkat cell cytosol was shown to be inhibited by grassypeptolides. In silico docking suggests two possible binding modes of grassypeptolides – both at the active site of DPP8 and at one of the entrances to the internal cavity. Collectively these results suggest that grassypeptolides may be useful tool compounds in the study of DPP8 function. PMID:24591193

  8. NF-kappa B activity in T cells stably expressing the Tax protein of human T cell lymphotropic virus type I

    SciTech Connect

    Lacoste, J.; Cohen, L.; Hiscott, J. )

    1991-10-01

    The effect of constitutive Tax expression on the interaction of NF-{kappa} B with its recognition sequence and on NF-{kappa} B-dependent gene expression was examined in T lymphoid Jurkat cell lines (19D and 9J) stably transformed with a Tax expression vector. Tax expressing T cell lines contained a constitutive level of NF-{kappa} B binding activity, detectable by mobility shift assay and uv cross-linking using a palindromic NF-{kappa} B probe homologous to the interferon beta PRDII site. In Jurkat and NC2.10 induction with phorbol esters resulted in the appearance of new DNA binding proteins of 85, 75, and 54 kDa, whereas in Tax expressing cells the 85-kDa protein and a 92-kDa DNA binding protein were constitutively induced. Expression of Tax protein in 19D and 9J resulted in transcription of the endogenous NF-kappa B-dependent granulocyte-macrophage colony stimulating factor gene and increased basal level expression of transfected NF-kappa B-regulated promoters. Nonetheless transcription of both the endogenous and the transfected gene was inducible by PMA treatment. Tax expression in Jurkat T cells may alter the stoichiometry of NF-kappa B DNA binding proteins and thus change the expression of NF-kappa B-regulated promoters.

  9. In silico identified CCR4 antagonists target regulatory T cells and exert adjuvant activity in vaccination.

    PubMed

    Bayry, Jagadeesh; Tchilian, Elma Z; Davies, Matthew N; Forbes, Emily K; Draper, Simon J; Kaveri, Srini V; Hill, Adrian V S; Kazatchkine, Michel D; Beverley, Peter C L; Flower, Darren R; Tough, David F

    2008-07-22

    Adjuvants are substances that enhance immune responses and thus improve the efficacy of vaccination. Few adjuvants are available for use in humans, and the one that is most commonly used (alum) often induces suboptimal immunity for protection against many pathogens. There is thus an obvious need to develop new and improved adjuvants. We have therefore taken an approach to adjuvant discovery that uses in silico modeling and structure-based drug-design. As proof-of-principle we chose to target the interaction of the chemokines CCL22 and CCL17 with their receptor CCR4. CCR4 was posited as an adjuvant target based on its expression on CD4(+)CD25(+) regulatory T cells (Tregs), which negatively regulate immune responses induced by dendritic cells (DC), whereas CCL17 and CCL22 are chemotactic agents produced by DC, which are crucial in promoting contact between DC and CCR4(+) T cells. Molecules identified by virtual screening and molecular docking as CCR4 antagonists were able to block CCL22- and CCL17-mediated recruitment of human Tregs and Th2 cells. Furthermore, CCR4 antagonists enhanced DC-mediated human CD4(+) T cell proliferation in an in vitro immune response model and amplified cellular and humoral immune responses in vivo in experimental models when injected in combination with either Modified Vaccinia Ankara expressing Ag85A from Mycobacterium tuberculosis (MVA85A) or recombinant hepatitis B virus surface antigen (rHBsAg) vaccines. The significant adjuvant activity observed provides good evidence supporting our hypothesis that CCR4 is a viable target for rational adjuvant design. PMID:18621704

  10. Distinct activation thresholds of human conventional and innate-like memory T cells

    PubMed Central

    Slichter, Chloe K.; McDavid, Andrew; Miller, Hannah W.; Finak, Greg; Seymour, Brenda J.; McNevin, John P.; Diaz, Gabriela; Czartoski, Julie L.; McElrath, M. Juliana; Gottardo, Raphael; Prlic, Martin

    2016-01-01

    Conventional memory CD8+ T cells and mucosal-associated invariant T cells (MAIT cells) are found in blood, liver, and mucosal tissues and have similar effector potential following activation, specifically expression of IFN-γ and granzyme B. To better understand each subset’s unique contributions to immunity and pathology, we interrogated inflammation- and TCR-driven activation requirements using human memory CD8+ T and MAIT cells isolated from blood and mucosal tissue biopsies in ex vivo functional assays and single cell gene expression experiments. We found that MAIT cells had a robust IFN-γ and granzyme B response to inflammatory signals but limited responsiveness when stimulated directly via their TCR. Importantly, this is not due to an overall hyporesponsiveness to TCR signals. When delivered together, TCR and inflammatory signals synergize to elicit potent effector function in MAIT cells. This unique control of effector function allows MAIT cells to respond to the same TCR signal in a dichotomous and situation-specific manner. We propose that this could serve to prevent responses to antigen in noninflamed healthy mucosal tissue, while maintaining responsiveness and great sensitivity to inflammation-eliciting infections. We discuss the implications of these findings in context of inflammation-inducing damage to tissues such as BM transplant conditioning or HIV infection. PMID:27331143

  11. Fungal transformation and T-cell proliferation inhibitory activity of melengestrol acetate and its metabolite.

    PubMed

    Baydoun, Elias; Bano, Saira; Atia-tul-Wahab; Jabeen, Almas; Yousuf, Sammer; Mesaik, Ahmed; Smith, Colin; Choudhary, M Iqbal

    2014-08-01

    Biotransformation of melengestrol acetate (MGA, 17α-acetoxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione) (1) was investigated for the first time by using fungal cultures. Incubation of compound 1 with Cunninghamella blakesleeana yielded a new major metabolite, 17α-acetoxy-11β-hydroxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione (2). The metabolite 2 was purified by using HPLC, followed by characterization through (1)H- and (13)C-NMR and other spectroscopic techniques. Single crystal X-ray diffraction analysis was used to deduce the three dimensional structures of melengestrol acetate (1) and metabolite 2 for the first time. T-cell proliferation assay was employed to evaluate the immunosuppressant effect of compounds 1 and 2 with IC50=0.5±0.07 and 0.6±0.08μg/mL, respectively. The results indicated that these compounds possess sixfold potent T-cell proliferation inhibitory activity as compared to the standard prednisolone (IC50<3.1μg/mL). Both compounds were found to be non-toxic in a 3T3 (mouse fibroblast) cell-based cytotoxicity assay. This discovery of potent anti-inflammatory activity of compounds 1 and 2 can lead the way to develop new immunosuppressant compounds for clinical application. PMID:24793568

  12. T-cell activation: A queuing theory analysis at low agonist density.

    PubMed

    Wedagedera, J R; Burroughs, N J

    2006-09-01

    We analyze a simple linear triggering model of the T-cell receptor (TCR) within the framework of queuing theory, in which TCRs enter the queue upon full activation and exit by downregulation. We fit our model to four experimentally characterized threshold activation criteria and analyze their specificity and sensitivity: the initial calcium spike, cytotoxicity, immunological synapse formation, and cytokine secretion. Specificity characteristics improve as the time window for detection increases, saturating for time periods on the timescale of downregulation; thus, the calcium spike (30 s) has low specificity but a sensitivity to single-peptide MHC ligands, while the cytokine threshold (1 h) can distinguish ligands with a 30% variation in the complex lifetime. However, a robustness analysis shows that these properties are degraded when the queue parameters are subject to variation-for example, under stochasticity in the ligand number in the cell-cell interface and population variation in the cellular threshold. A time integration of the queue over a period of hours is shown to be able to control parameter noise efficiently for realistic parameter values when integrated over sufficiently long time periods (hours), the discrimination characteristics being determined by the TCR signal cascade kinetics (a kinetic proofreading scheme). Therefore, through a combination of thresholds and signal integration, a T cell can be responsive to low ligand density and specific to agonist quality. We suggest that multiple threshold mechanisms are employed to establish the conditions for efficient signal integration, i.e., coordinate the formation of a stable contact interface. PMID:16766611

  13. T-Cell Activation: A Queuing Theory Analysis at Low Agonist Density

    PubMed Central

    Wedagedera, J. R.; Burroughs, N. J.

    2006-01-01

    We analyze a simple linear triggering model of the T-cell receptor (TCR) within the framework of queuing theory, in which TCRs enter the queue upon full activation and exit by downregulation. We fit our model to four experimentally characterized threshold activation criteria and analyze their specificity and sensitivity: the initial calcium spike, cytotoxicity, immunological synapse formation, and cytokine secretion. Specificity characteristics improve as the time window for detection increases, saturating for time periods on the timescale of downregulation; thus, the calcium spike (30 s) has low specificity but a sensitivity to single-peptide MHC ligands, while the cytokine threshold (1 h) can distinguish ligands with a 30% variation in the complex lifetime. However, a robustness analysis shows that these properties are degraded when the queue parameters are subject to variation—for example, under stochasticity in the ligand number in the cell-cell interface and population variation in the cellular threshold. A time integration of the queue over a period of hours is shown to be able to control parameter noise efficiently for realistic parameter values when integrated over sufficiently long time periods (hours), the discrimination characteristics being determined by the TCR signal cascade kinetics (a kinetic proofreading scheme). Therefore, through a combination of thresholds and signal integration, a T cell can be responsive to low ligand density and specific to agonist quality. We suggest that multiple threshold mechanisms are employed to establish the conditions for efficient signal integration, i.e., coordinate the formation of a stable contact interface. PMID:16766611

  14. Activation-induced and damage-induced cell death in aging human T cells.

    PubMed

    Sikora, Ewa

    2015-11-01

    In multicellular organisms the proper system functionality is ensured by the balance between cell division, differentiation, senescence and death. This balance is changed during aging. Immunosenescence plays a crucial role in aging and leads to the shrinkage of T cell repertoire and the propensity to apoptosis. The elimination of expanded T cells at the end of immune response is crucial to maintain homeostasis and avoid any uncontrolled inflammation. Resting mature T lymphocytes, when activated via their antigen-specific receptor (TCR) and CD28 co-receptor, start to proliferate and then undergo the so called activation induced cell death (AICD), which mechanistically is triggered by the death receptor and leads to apoptosis. T lymphocytes, like other cells, are also exposed to damage, which can trigger the so called damage-induced cell death (DICD). It was hypothesized that oxidative stress and chronic antigenic load increasing with age reduced lymphocyte susceptibility to DICD and enhanced a proinflamatory status leading to increased AICD. However, data collected so far are inconsistent and does not support this assumption. Systematic and comprehensive studies are still needed for conclusive elucidation of the role of AICD and DICD in human immunosenescence, including the role of autophagy and necroptosis in the processes. PMID:25843236

  15. Dynamic Regulation of TCR–Microclusters and the Microsynapse for T Cell Activation

    PubMed Central

    Hashimoto-Tane, Akiko; Saito, Takashi

    2016-01-01

    The interaction between a T cell and an antigen-presenting cell is the initiating event in T cell-mediated adaptive immunity. The Immunological Synapse (IS) is formed at the interface between these two cell types, and is the site where antigen (Ag)-specific recognition and activation are induced through the T cell receptor (TCR). This occurs at the center of the IS, and cell adhesion is supported through integrins in the area surrounding the TCR. Recently, this model has been revised based on data indicating that the initial Ag-specific activation signal is triggered prior to IS formation at TCR–microclusters (MCs), sites where TCR, kinases and adaptors of TCR proximal downstream signaling molecules accumulate as an activation signaling cluster. TCR–MCs then move into the center of the cell–cell interface to generate the cSMAC. This translocation of TCR–MCs is mediated initially by the actin cytoskeleton and then by dynein-induced movement along microtubules. The translocation of TCR–MCs and cSMAC formation is induced upon strong TCR stimulation through the assembly of a TCR–dynein super complex with microtubules. The Ag-specific activation signal is induced at TCR–MCs, but the adhesion signal is now shown to be induced by generating a “microsynapse,” which is composed of a core of TCR–MCs and the surrounding adhesion ring of integrin and focal adhesion molecules. Since the microsynapse is critical for activation, particularly under weak TCR stimulation, this structure supports a weak TCR signal through a cell–cell adhesion signal. The microsynapse has a structure similar to the IS but on a micro-scale and regulates Ag-specific activation as well as cell–cell adhesion. We describe here the dynamic regulation of TCR–MCs, responsible for inducing Ag-specific activation signals, and the microsynapse, responsible for adhesion signals critical for cell–cell interactions, and their interrelationship. PMID:27446085

  16. A Modular Enhancer Is Differentially Regulated by GATA and NFAT Elements That Direct Different Tissue-Specific Patterns of Nucleosome Positioning and Inducible Chromatin Remodeling▿

    PubMed Central

    Bert, Andrew G.; Johnson, Brett V.; Baxter, Euan W.; Cockerill, Peter N.

    2007-01-01

    We investigated alternate mechanisms employed by enhancers to position and remodel nucleosomes and activate tissue-specific genes in divergent cell types. We demonstrated that the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene enhancer is modular and recruits different sets of transcription factors in T cells and myeloid cells. The enhancer recruited distinct inducible tissue-specific enhanceosome-like complexes and directed nucleosomes to different positions in these cell types. In undifferentiated T cells, the enhancer was activated by inducible binding of two NFAT/AP-1 complexes which disrupted two specifically positioned nucleosomes (N1 and N2). In myeloid cells, the enhancer was remodeled by GATA factors which constitutively displaced an upstream nucleosome (N0) and cooperated with inducible AP-1 elements to activate transcription. In mast cells, which express both GATA-2 and NFAT, these two pathways combined to activate the enhancer and generate high-level gene expression. At least 5 kb of the GM-CSF locus was organized as an array of nucleosomes with fixed positions, but the enhancer adopted different nucleosome positions in T cells and mast cells. Furthermore, nucleosomes located between the enhancer and promoter were mobilized upon activation in an enhancer-dependent manner. These studies reveal that distinct tissue-specific mechanisms can be used either alternately or in combination to activate the same enhancer. PMID:17283044

  17. Zizyphus lotus L. (Desf.) modulates antioxidant activity and human T-cell proliferation

    PubMed Central

    2010-01-01

    Background Zizyphus lotus L. (Desf.) also known as Jujube, is a deciduous shrub which belongs to Rhamnaceae family. This plant is used in Algerian traditional medicine for its anti-diabetic, sedative, analgesic, anti-inflammatory and hypoglycaemic activities. In the present study, we determined the concentrations of different vitamins (vitamin A, C and E) and fatty acids in root, stem, leaves, fruit pulp and seed of Zizyphus lotus L. (Desf.) and assessed the effects of their aqueous extracts on antioxidant status and human T-cell proliferation. Methods Aqueous filtrates from different parts, i.e, root, leaf, stem, fruit pulp and seed, of Zizyphus lotus L. (Desf.) were prepared. Vitamin C levels were determined by precipitating with 10% trichloroacetic acid and vitamin A and E were assessed by HPLC. Lipid composition of these extracts was determined by gas-liquid chromatography. Anti-oxidant capacity was evaluated by using anti-radical resistance kit [Kit Radicaux Libres (KRL@; Kirial International SA, Couternon, France)]. T-cell blastogenesis was assessed by the incorporation of 3H-thymidine. IL-2 gene expression was evaluated by RT-qPCR. Results Our results show that fruit pulp contained higher vitamin A and C contents than other parts of the plant. Furthermore, the fruit pulp was the richest source of linoleic acid (18:2n-6), a precursor of n-6 fatty acids. Fruit seeds possessed higher vitamin C levels than leaves, roots and stem. The leaves were the richest source of vitamin E and linolenic acid (18:3n-3), a precursor of n-3 fatty acids. The antioxidant capacity of the different extracts, measured by KRL@ test, was as follows: pulp < seedT-cell proliferation is concerned, we observed that the different extracts of Zizyphus lotus L. (Desf.) exerted immunosuppressive effects. Conclusion Seed extracts exerted the most potent immunosuppressive effects on T cell proliferation and IL-2 mRNA expression. The results of the present study are

  18. The Early Activation Marker CD69 Regulates the Expression of Chemokines and CD4 T Cell Accumulation in Intestine

    PubMed Central

    Radulovic, Katarina; Rossini, Valerio; Manta, Calin; Holzmann, Karlheinz; Kestler, Hans A.; Niess, Jan Hendrik

    2013-01-01

    Migration of naïve and activated lymphocytes is regulated by the expression of various molecules such as chemokine receptors and ligands. CD69, the early activation marker of C-type lectin domain family, is also shown to regulate the lymphocyte migration by affecting their egress from the thymus and secondary lymphoid organs. Here, we aimed to investigate the role of CD69 in accumulation of CD4 T cells in intestine using murine models of inflammatory bowel disease. We found that genetic deletion of CD69 in mice increases the expression of the chemokines CCL-1, CXCL-10 and CCL-19 in CD4+ T cells and/or CD4− cells. Efficient in vitro migration of CD69-deficient CD4 T cells toward the chemokine stimuli was the result of increased expression and/or affinity of chemokine receptors. In vivo CD69−/− CD4 T cells accumulate in the intestine in higher numbers than B6 CD4 T cells as observed in competitive homing assay, dextran sodium sulphate (DSS)-induced colitis and antigen-specific transfer colitis. In DSS colitis CD69−/− CD4 T cell accumulation in colonic lamina propria (cLP) was associated with increased expression of CCL-1, CXCL-10 and CCL-19 genes. Furthermore, treatment of DSS-administrated CD69−/− mice with the mixture of CCL-1, CXCL-10 and CCL-19 neutralizing Abs significantly decreased the histopathological signs of colitis. Transfer of OT-II×CD69−/− CD45RBhigh CD4 T cells into RAG−/− hosts induced CD4 T cell accumulation in cLP. This study showed CD69 as negative regulator of inflammatory responses in intestine as it decreases the expression of chemotactic receptors and ligands and reduces the accumulation of CD4 T cells in cLP during colitis. PMID:23776480

  19. Asymmetric inheritance of mTORC1 kinase activity during division dictates CD8(+) T cell differentiation.

    PubMed

    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

    2016-06-01

    The asymmetric partitioning of fate-determining proteins has been shown to contribute to the generation of CD8(+) effector and memory T cell precursors. Here we demonstrate the asymmetric partitioning of mTORC1 activity after the activation of naive CD8(+) T cells. This results in the generation of two daughter T cells, one of which shows increased mTORC1 activity, increased glycolytic activity and increased expression of effector molecules. The other daughter T cell has relatively low mTORC1 activity and increased lipid metabolism, expresses increased amounts of anti-apoptotic molecules and subsequently displays enhanced long-term survival. Mechanistically, we demonstrate a link between T cell antigen receptor (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

  20. HIV-induced T-cell activation/exhaustion in rectal mucosa is controlled only partially by antiretroviral treatment.

    PubMed

    Rueda, Cesar Mauricio; Velilla, Paula Andrea; Chougnet, Claire A; Montoya, Carlos Julio; Rugeles, Maria Teresa

    2012-01-01

    Peripheral blood T-cells from untreated HIV-1-infected patients exhibit reduced immune responses, usually associated with a hyperactivated/exhausted phenotype compared to HAART treated patients. However, it is not clear whether HAART ameliorates this altered phenotype of T-cells in the gastrointestinal-associated lymphoid tissue (GALT), the main site for viral replication. Here, we compared T-cells from peripheral blood and GALT of two groups of chronically HIV-1-infected patients: untreated patients with active viral replication, and patients on suppressive HAART. We characterized the T-cell phenotype by measuring PD-1, CTLA-4, HLA-DR, CD25, Foxp3 and granzyme A expression by flow cytometry; mRNA expression of T-bet, GATA-3, ROR-γt and Foxp3, and was also evaluated in peripheral blood mononuclear cells and rectal lymphoid cells. In HIV-1+ patients, the frequency of PD-1(+) and CTLA-4(+) T-cells (both CD4+ and CD8+ T cells) was higher in the GALT than in the blood. The expression of PD-1 by T-cells from GALT was higher in HIV-1-infected subjects with active viral replication compared to controls. Moreover, the expression per cell of PD-1 and CTLA-4 in CD4(+) T-cells from blood and GALT was positively correlated with viral load. HAART treatment decreased the expression of CTLA-4 in CD8(+) T cells from blood and GALT to levels similar as those observed in controls. Frequency of Granzyme A(+) CD8(+) T-cells in both tissues was low in the untreated group, compared to controls and HAART-treated patients. Finally, a switch towards Treg polarization was found in untreated patients, in both tissues. Together, these findings suggest that chronic HIV-1 infection results in an activated/exhausted T-cell phenotype, despite T-cell polarization towards a regulatory profile; these alterations are more pronounced in the GALT compared to peripheral blood, and are only partiality modulated by HAART. PMID:22276176

  1. Interluekin-12 enhances the function and anti-tumor activity in murine and human CD8+ T cells

    PubMed Central

    Rubinstein, Mark P.; Su, Ee Wern; Suriano, Samantha; Cloud, Colleen A.; Andrijauskaite, Kristina; Kesarwani, Pravin; Schwartz, Kristina M.; Williams, Katelyn; Johnson, C. Bryce; Li, Mingli; Scurti, Gina M.; Salem, Mohamed L.; Paulos, Chrystal M.; Garrett-Mayer, Elizabeth; Mehrotra, Shikhar; Cole, David J.

    2016-01-01

    Mouse CD8+ T cells conditioned with Interleukin (IL)-12 ex vivo mediate the potent regression of established melanoma when transferred into lymphodepleted mice. However, the quantitative and qualitative changes induced by IL-12 in the responding mouse CD8+ T cells have not been well defined. Moreover, the mechanisms by which IL-12-conditioning impacts human CD8+ T cells, and how such cells might be expanded prior to infusion into patients is not known. We found that ex vivo IL-12-conditioning of mouse CD8+ T cells led to a 10- to 100-fold increase in persistence and anti-tumor efficacy upon adoptive transfer into lymphodepleted mice. The enhancing effect of IL-12 was associated with maintenance of functional avidity. Importantly, in the context of ongoing ACT clinical trials, human CD8+ T cells genetically modified with a tyrosinase-specific T-cell receptor exhibited significantly enhanced functional activity when conditioned with IL-12 as indicated by heightened granzyme B expression and elevated peptide-specific CD107a degranulation. This effect was sustainable despite the 20 days of in vitro cellular expansion required to expand cells over 1,000-fold allowing adequate cell numbers for administration to cancer patients. Overall, these findings support the efficacy and feasibility of ex vivo IL-12-conditioning of TCR-modified human CD8+ T cells for adoptive transfer and cancer therapy. PMID:25676709

  2. Inhibition of NAMPT pathway by FK866 activates the function of p53 in HEK293T cells

    SciTech Connect

    Thakur, Basant Kumar; Dittrich, Tino; Chandra, Prakash; Becker, Annette; Lippka, Yannick; Selvakumar, Divakarvel; Klusmann, Jan-Henning; Reinhardt, Dirk; Welte, Karl

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer In 293T cells, p53 is considered to be inactive due to its interaction w