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Sample records for cd40 ligand-treated human

  1. CD40L-Tri, a novel formulation of recombinant human CD40L that effectively activates B cells.

    PubMed

    Naito, Masayasu; Hainz, Ursula; Burkhardt, Ute E; Fu, Buyin; Ahove, Deborah; Stevenson, Kristen E; Rajasagi, Mohini; Zhu, Baogong; Alonso, Anselmo; Witten, Elizabeth; Matsuoka, Ken-Ichi; Neuberg, Donna; Duke-Cohan, Jonathan S; Wu, Catherine J; Freeman, Gordon J

    2013-02-01

    CD40L has a well-established role in enhancing the immunostimulatory capacity of normal and malignant B cells, but a formulation suitable for clinical use has not been widely available. Like other TNF family members, in vivo and in vitro activity of CD40L requires a homotrimeric configuration, and growing evidence suggests that bioactivity depends on higher-order clustering of CD40. We generated a novel formulation of human recombinant CD40L (CD40L-Tri) in which the CD40L extracellular domain and a trimerization motif are connected by a long flexible peptide linker. We demonstrate that CD40L-Tri significantly expands normal CD19+ B cells by over 20- to 30-fold over 14 days and induces B cells to become highly immunostimulatory antigen-presenting cells (APCs). Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers. In addition, CD40L-Tri could induce malignant B cells to become effective APCs, such that tumor-directed immune responses could be probed. Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells. The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker.

  2. Mechanisms of Nifedipine-Downregulated CD40L/sCD40L Signaling in Collagen Stimulated Human Platelets

    PubMed Central

    Hsu, Wen-Lin; Chou, Tz-Chong

    2015-01-01

    The platelet-derived soluble CD40L (sCD40L) release plays a critical role in the development of atherosclerosis. Nifedipine, a dihydropyridine-based L-type calcium channel blocker (CCB), has been reported to have an anti-atherosclerotic effect beyond its blood pressure-lowering effect, but the molecular mechanisms remain unclear. The present study was designed to investigate whether nifedipine affects sCD40L release from collagen-stimulated human platelets and to determine the potential role of peroxisome proliferator-activated receptor-β/-γ (PPAR-β/-γ). We found that treatment with nifedipine significantly inhibited the platelet surface CD40L expression and sCD40L release in response to collagen, while the inhibition was markedly reversed by blocking PPAR-β/-γ activity with specific antagonist such as GSK0660 and GW9662. Meanwhile, nifedipine also enhanced nitric oxide (NO) and cyclic GMP formation in a PPAR-β/-γ-dependent manner. When the NO/cyclic GMP pathway was suppressed, nifedipine-mediated inhibition of sCD40L release was abolished significantly. Collagen-induced phosphorylation of p38MAPK, ERK1/2 and HSP27, matrix metalloproteinase-2 (MMP-2) expression/activity and reactive oxygen species (ROS) formation were significantly inhibited by nifedipine, whereas these alterations were all attenuated by co-treatment with PPAR-β/-γ antagonists. Collectively, these results demonstrate that PPAR-β/-γ-dependent pathways contribute to nifedipine-mediated downregulation of CD40L/sCD40L signaling in activated platelets through regulation of NO/ p38MAPK/ERK1/2/HSP27/MMP-2 signalings and provide a novel mechanism regarding the anti-atherosclerotic effect of nifedipine. PMID:25970603

  3. Mechanisms of Nifedipine-Downregulated CD40L/sCD40L Signaling in Collagen Stimulated Human Platelets.

    PubMed

    Chen, Tso-Hsiao; Shih, Ching-Yu; Hsu, Wen-Lin; Chou, Tz-Chong

    2015-01-01

    The platelet-derived soluble CD40L (sCD40L) release plays a critical role in the development of atherosclerosis. Nifedipine, a dihydropyridine-based L-type calcium channel blocker (CCB), has been reported to have an anti-atherosclerotic effect beyond its blood pressure-lowering effect, but the molecular mechanisms remain unclear. The present study was designed to investigate whether nifedipine affects sCD40L release from collagen-stimulated human platelets and to determine the potential role of peroxisome proliferator-activated receptor-β/-γ (PPAR-β/-γ). We found that treatment with nifedipine significantly inhibited the platelet surface CD40L expression and sCD40L release in response to collagen, while the inhibition was markedly reversed by blocking PPAR-β/-γ activity with specific antagonist such as GSK0660 and GW9662. Meanwhile, nifedipine also enhanced nitric oxide (NO) and cyclic GMP formation in a PPAR-β/-γ-dependent manner. When the NO/cyclic GMP pathway was suppressed, nifedipine-mediated inhibition of sCD40L release was abolished significantly. Collagen-induced phosphorylation of p38MAPK, ERK1/2 and HSP27, matrix metalloproteinase-2 (MMP-2) expression/activity and reactive oxygen species (ROS) formation were significantly inhibited by nifedipine, whereas these alterations were all attenuated by co-treatment with PPAR-β/-γ antagonists. Collectively, these results demonstrate that PPAR-β/-γ-dependent pathways contribute to nifedipine-mediated downregulation of CD40L/sCD40L signaling in activated platelets through regulation of NO/ p38MAPK/ERK1/2/HSP27/MMP-2 signalings and provide a novel mechanism regarding the anti-atherosclerotic effect of nifedipine.

  4. [Liposome-mediated human CD40 gene transfection and human umbilical vein endothelial ECV-304 cells].

    PubMed

    Wang, Wei-rong; Lin, Rong; Yang, Yu-cong; Gan, Wei-jie; Liu, Jun-tian; Lü, She-min

    2005-12-01

    To construct an eukaryotic expression vector containing human CD40 gene for its efficient, continuous and stable expression in human umbilical vein endothelial ECV-304 cells. The recombinant plasmid pUCD40 was digested with endonucleases to obtain human CD40 gene fragment, which was cloned into pCDNA3.1 vector to construct recombinant eukaryotic expression vector pCDNA3.1(+)/CD40. The recombinant vector was identified by enzyme digestion before introduced into ECV-304 cells via liposome, with the positive cell clones selected with G418. The stable transfection and expression of CD40 in ECV-304 cells were identified by reverse transcription (RT)-PCR, Western blotting and flow cytometry, respectively. Enzyme digestion analysis showed that target gene had been cloned into the recombinant vector. The transfected ECV-304 cells successfully expressed human CD40 as determined by RT-PCR and Western-blotting, and 95% of the cells were CD40-positive as shown by flow cytometry. The recombinant eukaryotic expression vector pCDNA3.1(+)/CD40 has been successfully constructed, which is capable of stable transfection and expression of CD40 in ECV-304 cells to facilitate further investigation of the roles of CD40 molecule in antiatherosclerotic drug development.

  5. CD40 expressed on thymic epithelial cells provides costimulation for proliferation but not for apoptosis of human thymocytes.

    PubMed

    Ruggiero, G; Martinez Cáceres, E; Voordouw, A; Noteboom, E; Graf, D; Kroczek, R A; Spits, H

    1996-05-15

    Human thymic epithelial cells express CD40, so we examined the possible role of CD40 in activation of thymocytes. We observed that both CD4+CD8- and CD4-CD8+ thymocytes proliferate after stimulation by anti-CD3 mAb in the presence of cultured thymic epithelial cells. Costimulation of CD4+ thymocytes by thymic epithelial cells is partly inhibited by an anti-CD40 mAb, but this mAb has no effect on costimulation of CD8+ thymocytes. The selective costimulatory ability of CD40 for CD4+ thymocytes was confirmed in experiments in which thymocytes were stimulated with anti-CD3 in the presence of murine P815 cells transfected with CD40 cDNA. The level of costimulation induced by P815-CD40 was comparable with that induced by P815 cells expressing CD80 (B7.1). Treatment of thymocytes with the Ca2+ ionophore ionomycin and the phorbol ester PMA or with anti-CD3 mAb resulted in up-regulation of the CD40 ligand, suggesting that this molecule is involved in CD40-mediated costimulation of human thymocytes. Costimulation of thymocytes by CD80 strongly increased anti-CD3-induced death of fetal thymocytes. In contrast, costimulation by CD40 did not increase anti-CD3-mediated apoptosis of these thymocytes. To confirm that CD40 does not affect anti-CD3-induced cell death, we established a variant of the Jurkat T leukemic cell line that constitutively expresses CD40L and analyzed the sensitivity of this cell line for activation-induced apoptosis. In contrast to CD80, CD40 failed to increase anti-CD3-mediated apoptosis in CD40L+ Jurkat cells, whereas both CD40 and CD80 strongly increased IL-2 production induced by anti-CD3. These findings suggest that costimulation by CD40 is involved in clonal expansion of CD4+ thymocytes but not in activation-induced cell death.

  6. Human Genetics in Rheumatoid Arthritis Guides a High-Throughput Drug Screen of the CD40 Signaling Pathway

    PubMed Central

    Li, Gang; Diogo, Dorothée; Wu, Di; Spoonamore, Jim; Dancik, Vlado; Franke, Lude; Kurreeman, Fina; Rossin, Elizabeth J.; Duclos, Grant; Hartland, Cathy; Zhou, Xuezhong; Li, Kejie; Liu, Jun; De Jager, Philip L.; Siminovitch, Katherine A.; Zhernakova, Alexandra; Raychaudhuri, Soumya; Bowes, John; Eyre, Steve; Padyukov, Leonid; Gregersen, Peter K.; Worthington, Jane; Gupta, Namrata; Clemons, Paul A.; Stahl, Eli; Tolliday, Nicola; Plenge, Robert M.

    2013-01-01

    Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10−9). Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10−9), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA–approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in

  7. Enhanced activation of human dendritic cells by inducible CD40 and Toll-like receptor-4 ligation.

    PubMed

    Lapteva, Natalia; Seethammagari, Mamatha R; Hanks, Brent A; Jiang, Jianghong; Levitt, Jonathan M; Slawin, Kevin M; Spencer, David M

    2007-11-01

    Despite the potency of dendritic cells (DC) as antigen-presenting cells for priming adaptive immunity, DC-based cancer vaccines have been largely insufficient to effectively reduce tumor burden or prevent tumor progression in most patients. To enhance DC-based vaccines, we used the combination of a synthetic ligand-inducible CD40 receptor (iCD40) along with Toll-like receptor-4 (TLR-4) ligation in human monocyte-derived DCs. The iCD40 receptor permits targeted, reversible activation of CD40 in vivo, potentially bypassing the essential role of CD4(+) T cells for activation of DCs. As a rigorous preclinical study of this approach, we evaluated key parameters of DC activation and function. Whereas neither iCD40 nor TLR-4 signaling alone led to high levels of interleukin (IL)-12p70 and IL-6, using iCD40 in combination with lipopolysaccharide (LPS) or monophosphoryl lipid A led to strongly synergistic production of both. Furthermore, this approach led to high expression of DC maturation markers, epitope-specific CTL and T helper 1 responses, as well as DC migration in vitro and in vivo. Moreover, use of iCD40-modified and LPS-stimulated DCs led to targeted expansion of autologous T cells against tumor-associated antigens, including prostate-specific membrane antigen, and elimination of preestablished tumors, supporting this technology as a potent strategy for DC-based cancer immunotherapy.

  8. Interaction with damaged vessel wall in vivo in humans induces platelets to express CD40L resulting in endothelial activation with no effect of aspirin intake.

    PubMed

    Giannini, Silvia; Falcinelli, Emanuela; Bury, Loredana; Guglielmini, Giuseppe; Rossi, Roberta; Momi, Stefania; Gresele, Paolo

    2011-06-01

    Activated platelets express CD40L on their plasma membrane and release the soluble fragment sCD40L. The interaction between platelet surface CD40L and endothelial cell CD40 leads to the activation of endothelium contributing to atherothrombosis. Few studies have directly demonstrated an increased expression of platelet CD40L in conditions of in vivo platelet activation in humans, and no data are available on its relevance for endothelial activation. We aimed to assess whether platelets activated in vivo at a localized site of vascular injury in humans express CD40L and release sCD40L, whether the level of platelet CD40L expression attained in vivo is sufficient to induce endothelial activation, and whether platelet CD40L expression is inhibited by aspirin intake. We used the skin-bleeding-time test as a model to study the interaction between platelets and a damaged vessel wall by measuring CD40L in the blood emerging from a skin wound in vivo in healthy volunteers. In some experiments, shed blood was analyzed before and 1 h after the intake of 500 mg of aspirin. Platelets from the bleeding-time blood express CD40L and release soluble sCD40L, in a time-dependent way. In vivo platelet CD40L expression was mild but sufficient to induce VCAM-1 expression and IL-8 secretion in coincubation experiments with cultured human endothelial cells. Moreover, platelets recovered from the bleeding-time blood activated endothelial cells; an anti-CD40L antibody blocked this effect. On the contrary, the amount of sCD40L released by activated platelets at a localized site of vascular injury did not reach the concentrations required to induce endothelial cell activation. Soluble monocyte chemoattractant protein-1, a marker of endothelium activation, was increased in shed blood and correlated with platelet CD40L expression. Aspirin intake did not inhibit CD40L expression by platelets in vivo. We concluded that CD40L expressed by platelets in vivo in humans upon contact with a damaged

  9. Introduction of a CD40L genomic fragment via a human artificial chromosome vector permits cell-type-specific gene expression and induces immunoglobulin secretion.

    PubMed

    Yamada, Hidetoshi; Li, Yanze C; Nishikawa, Mitsuo; Oshimura, Mitsuo; Inoue, Toshiaki

    2008-01-01

    Gene therapy using cDNA driven by an exogenous promoter is not suited for genetic disorders that require intrinsic expression of a transgene, such as hyperimmunoglobulin (Ig)M syndrome (HIGM), which is caused by mutations in the CD40L gene. The human artificial chromosome (HAC) vector has the potential to solve this problem, because it can be used to transfer large genomic fragments containing their own regulatory elements. In this study, we examined whether introduction of a genomic fragment of CD40L via the HAC vector permits intrinsic expression of the transgene and has an effect on immunoglobulin secretion. We constructed an HAC vector carrying the mouse CD40L genomic fragment (mCD40L-HAC) in Chinese hamster ovary (CHO) cells and transferred the mCD40L-HAC vector into a human CD4-positive active T-cell line (Jurkat) and a human myeloid cell line (U937) via microcell-mediated chromosome transfer (MMCT). The mCD40L-HAC vector permits mCD40L expression in human active T cells but not in human myeloid cells. The mCD40L-HAC also functions to stimulate mouse B cells derived from CD40L(-/-) mice, inducing secretion of IgG. This study may be an initial step toward the therapeutic application of HAC vectors for intrinsic expression of genes, a potential new direction for genome-based gene therapy.

  10. Involvement of mitogen-activated protein kinases and NF{kappa}B in LPS-induced CD40 expression on human monocytic cells

    SciTech Connect

    Wu Weidong | Alexis, Neil E. |; Chen Xian |; Bromberg, Philip A. |; Peden, David B. ||

    2008-04-15

    CD40 is a costimulatory molecule linking innate and adaptive immune responses to bacterial stimuli, as well as a critical regulator of functions of other costimulatory molecules. The mechanisms regulating lipopolysaccharide (LPS)-induced CD40 expression have not been adequately characterized in human monocytic cells. In this study we used a human monocytic cell line, THP-1, to investigate the possible mechanisms of CD40 expression following LPS exposure. Exposure to LPS resulted in a dose- and time-dependent increase in CD40 expression. Further studies using immunoblotting and pharmacological inhibitors revealed that mitogen-activated protein kinases (MAPKs) and NF{kappa}B were activated by LPS exposure and involved in LPS-induced CD40 expression. Activation of MAPKs was not responsible for LPS-induced NF{kappa}B activation. TLR4 was expressed on THP-1 cells and pretreatment of cells with a Toll-like receptor 4 (TLR4) neutralizing antibody (HTA125) significantly blunted LPS-induced MAPK and NF{kappa}B activation and ensuing CD40 expression. Additional studies with murine macrophages expressing wild type and mutated TLR4 showed that TLR4 was implicated in LPS-induced ERK and NF{kappa}B activation, and CD40 expression. Moreover, blockage of MAPK and NF{kappa}B activation inhibited LPS-induced TLR4 expression. In summary, LPS-induced CD40 expression in monocytic cells involves MAPKs and NF{kappa}B.

  11. Tuning of CD40-CD154 interactions in human B-lymphocyte activation: a broad array of in vitro models for a complex in vivo situation.

    PubMed

    Néron, Sonia; Nadeau, Philippe J; Darveau, André; Leblanc, Jean-François

    2011-02-01

    Naive and memory B-lymphocyte populations can be activated through the binding of CD154 to CD40, a receptor that is constitutively expressed on the surface of these cells. Models based on the in vitro stimulation of human B lymphocytes through CD40 have greatly contributed to our understanding of the human immune response in healthy individuals and patients suffering from immune disorders. The nature of the engineered CD40 ligands is as diverse as the in vitro models used in studies of CD40-activated B lymphocytes. Monoclonal anti-CD40 antibodies, recombinant CD154 proteins, soluble CD154(+) membranes as well as CD154(+) cell lines have turned out to be very useful tools, and are still in use today. As for any receptor-ligand interaction, parameters such as duration and strength of contact, timing, affinity, and receptor density are major determinants of CD40 binding by CD154 or anti-CD40. Furthermore, variation in the intensity of CD40 stimulation has been shown to influence proliferation, differentiation and immunoglobulin secretion of human hybridomas, B-cell lines, tonsil and blood B lymphocytes. The objective of this review is to present an overview of the great diversity of CD40 agonists used in in vitro models of B-lymphocyte activation, with a particular emphasis on variations in the resulting strength of CD40 signaling generated by these models. A better understanding of these models could open up new avenues for the rational use of human B lymphocytes as antigen-presenting cells in cellular therapies.

  12. Human CD40L-expressing type 3 innate lymphoid cells induce IL-10-producing immature transitional regulatory B cells.

    PubMed

    Komlósi, Zsolt István; Kovács, Nóra; van de Veen, Willem; Kirsch, Anna; Fahrner, Heinz Benedikt; Wawrzyniak, Marcin; Rebane, Ana; Stanic, Barbara; Palomares, Oscar; Rückert, Beate; Menz, Günter; Akdis, Mübeccel; Losonczy, György; Akdis, Cezmi A

    2017-09-19

    Type 3 innate lymphoid cells (ILC3s) are involved in the maintenance of mucosal homeostasis; however, their role in immunoregulation has been unknown. Immature transitional regulatory B (itBreg) cells are innate-like B cells with immunosuppressive properties, and the in vivo mechanisms by which they are induced have not been fully clarified. We aimed to investigate the ILC3-B cell interaction that probably takes place in human tonsils. ILC3s were isolated from peripheral blood and palatine tonsils, expanded and cocultured with naïve B cells. Tonsillar ILC3s and Breg cells were visualized with immunofluorescence histology. The frequencies of ILC3s were measured in tonsil tissue of allergic and non-allergic patients; and in peripheral blood of allergic asthmatics and healthy controls. A mutually beneficial relationship was revealed between ILC3s and B cells: ILC3s induced IL-15 production in B cells via BAFF-receptor, while IL-15, a potent growth factor for ILC3s, induced the expression of CD40L on circulating and tonsillar ILC3s. IL-15-activated CD40L(+)ILC3s helped B cell survival, proliferation and the differentiation of IL-10-secreting, functional itBreg cells in a CD40L- and BAFF-receptor-dependent manner. ILC3s and Breg cells were in close connection with each other in palatine tonsils. The frequency of ILC3s was reduced in tonsil tissue of allergic patients and in peripheral blood of allergic asthmatics. Human CD40L(+)ILC3s provide innate B cell help, and are involved in an innate immunoregulatory mechanism by the induction of itBreg cell differentiation, which takes place in palatine tonsils in vivo. This mechanism may contribute to the maintenance of the immune tolerance and become insufficient in allergic diseases. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  13. The antileukemia activity of a human anti-CD40 antagonist antibody, HCD122, on human chronic lymphocytic leukemia cells

    PubMed Central

    Klabunde, Sha; Lin, Karen; Georgakis, Georgios V.; Cherukuri, Anu; Holash, Jocelyn; Goldbeck, Cheryl; Xu, Xiaomei; Kadel, Edward E.; Lee, Sang Hoon; Aukerman, Sharon Lea; Jallal, Bahija; Aziz, Natasha; Weng, Wen-Kai; Wierda, William; O'Brien, Susan; Younes, Anas

    2008-01-01

    B-cell chronic lymphocytic leukemia (B-CLL) is a lymphoproliferative disorder characterized by the surface expression of CD20, CD5 antigens, as well as the receptor CD40. Activation of CD40 by its ligand (CD40L) induces proliferation and rescues the cells from spontaneous and chemotherapy-induced apoptosis. CD40 activation also induces secretion of cytokines, such as IL-6, IL-10, TNF-α, IL-8, and GM-CSF, which are involved in tumor cell survival, migration, and interaction with cells in the tumor microenvironment. Here we demonstrate that in primary B-CLL tumor cells, the novel antagonist anti-CD40 monoclonal antibody, HCD122, inhibits CD40L-induced activation of signaling pathways, proliferation and survival, and secretion of cytokines. Furthermore, HCD122 is also a potent mediator of antibody-dependent cellular cytotoxicity (ADCC), lysing B-CLL cells more efficiently than rituximab in vitro, despite a significantly higher number of cell surface CD20 binding sites compared with CD40. Unlike rituximab, however, HCD122 (formerly CHIR-12.12) does not internalize upon binding to the cells. Our data suggest that HCD122 may inhibit B-CLL growth by blocking CD40 signaling and by ADCC-mediated cell lysis. PMID:18497318

  14. The antileukemia activity of a human anti-CD40 antagonist antibody, HCD122, on human chronic lymphocytic leukemia cells.

    PubMed

    Luqman, Mohammad; Klabunde, Sha; Lin, Karen; Georgakis, Georgios V; Cherukuri, Anu; Holash, Jocelyn; Goldbeck, Cheryl; Xu, Xiaomei; Kadel, Edward E; Lee, Sang Hoon; Aukerman, Sharon Lea; Jallal, Bahija; Aziz, Natasha; Weng, Wen-Kai; Wierda, William; O'Brien, Susan; Younes, Anas

    2008-08-01

    B-cell chronic lymphocytic leukemia (B-CLL) is a lymphoproliferative disorder characterized by the surface expression of CD20, CD5 antigens, as well as the receptor CD40. Activation of CD40 by its ligand (CD40L) induces proliferation and rescues the cells from spontaneous and chemotherapy-induced apoptosis. CD40 activation also induces secretion of cytokines, such as IL-6, IL-10, TNF-alpha, IL-8, and GM-CSF, which are involved in tumor cell survival, migration, and interaction with cells in the tumor microenvironment. Here we demonstrate that in primary B-CLL tumor cells, the novel antagonist anti-CD40 monoclonal antibody, HCD122, inhibits CD40L-induced activation of signaling pathways, proliferation and survival, and secretion of cytokines. Furthermore, HCD122 is also a potent mediator of antibody-dependent cellular cytotoxicity (ADCC), lysing B-CLL cells more efficiently than rituximab in vitro, despite a significantly higher number of cell surface CD20 binding sites compared with CD40. Unlike rituximab, however, HCD122 (formerly CHIR-12.12) does not internalize upon binding to the cells. Our data suggest that HCD122 may inhibit B-CLL growth by blocking CD40 signaling and by ADCC-mediated cell lysis.

  15. The CD40/CD40 ligand interactions exert pleiotropic effects on bone marrow granulopoiesis.

    PubMed

    Mavroudi, Irene; Papadaki, Vassiliki; Pyrovolaki, Katerina; Katonis, Pavlos; Eliopoulos, Aristides G; Papadaki, Helen A

    2011-05-01

    CD40 is a member of the TNFR family and upon interaction with its cognate ligand (CD40L), induces diverse biologic responses related to cell survival/growth. As altered CD40/CD40L interactions have been associated with neutropenia, we investigated the role of CD40/CD40L on human granulopoiesis using immunomagnetically sorted CD34(+), CD34(-)/CD33(+), and CD34(-)/CD33(-)/CD15(+) BM cells, which represent sequential stages of the granulocytic development, the KG-1 cells that constantly express CD34 and CD33, and LTBMCs that mimic the BM microenvironment. CD40 and CD40L were minimally expressed on CD34(+), CD34(-)/CD33(+), and CD34(-)/CD33(-)/CD15(+) cells, but CD40 was substantially induced in the presence of TNF-α. Cross-linking of CD40 in the above cell populations resulted in induction of apoptosis that was enhanced further in the presence of FasL. CD40 activation in primary as wells as in KG-1 cells resulted in Fas up-regulation, providing a mechanism for the CD40-mediated apoptosis. Addition of CD40L in clonogenic assays resulted in a significant decrease in the colony-forming capacity of BMMCs from patients with chronic neutropenia, presumably expressing high levels of CD40 in the progenitor cells, and this effect was reversed upon CD40 blockade. CD40 was constitutively expressed on LTBMC stromal cells and upon activation, resulted in an increase in G-CSF and GM-CSF production. These data show that CD40/CD40L interactions may promote granulopoiesis under steady-state conditions by inducing the stromal release of granulopoiesis-supporting cytokines, whereas under inflammatory conditions, they may affect the granulocytic progenitor/precursor cell survival by accelerating the Fas-mediated apoptosis.

  16. Ligation of CD40 in Human Müller Cells Induces P2X7 Receptor–Dependent Death of Retinal Endothelial Cells

    PubMed Central

    Portillo, Jose-Andres C.; Lopez Corcino, Yalitza; Dubyak, George R.; Kern, Timothy S.; Matsuyama, Shigemi; Subauste, Carlos S.

    2016-01-01

    Purpose Cluster of differentiation 40 (CD40) is required for retinal capillary degeneration in diabetic mice, a process mediated by the retinal endothelial cells (REC) death. However, CD40 activates prosurvival signals in endothelial cells. The purpose of this study was to identify a mechanism by which CD40 triggers programmed cell death (PCD) of RECs and address this paradox. Methods Human RECs and Müller cells were incubated with CD154 and L-N6-(1-Iminoethyl)lysine (L-Nil, nitric oxide synthase 2 inhibitor), α-lipoic acid (inhibitor of oxidative stress), anti-Fas ligand antibody, or A-438079 (P2X7 adenosine triphosphate [ATP] receptor inhibitor). Programmed cell death was analyzed by fluorescence-activated cell sorting (FACS) or Hoechst/propidium iodide staining. Release of ATP was measured using a luciferase-based assay. Mice were made diabetic with streptozotocin. Expression of P2X7 was assessed by FACS, quantitative PCR, or immunohistochemistry. Results Ligation of CD40 in primary RECs did not induce PCD. In contrast, in the presence of primary CD40+ Müller cells, CD40 stimulation caused PCD of RECs that was not impaired by L-Nil, α-lipoic acid, or anti-Fas ligand antibody. We found CD40 did not trigger TNF-α or IL-1β secretion. Primary Müller cells released extracellular ATP in response to CD40 ligation. Inhibition of P2X7 (A-438079) impaired PCD of RECs; CD40 upregulated P2X7 in RECs, making them susceptible to ATP/P2X7–mediated PCD. Diabetic mice upregulated P2X7 in the retina and RECs in a CD40-dependent manner. Conclusions Cluster of differentiation 40 induces PCD of RECs through a dual mechanism: ATP release by Müller cells and P2X7 upregulation in RECs. These findings are likely of in vivo relevance since CD40 upregulates P2X7 in RECs in diabetic mice and CD40 is known to be required for retinal capillary degeneration. PMID:27893093

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

    PubMed

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

    2013-11-15

    We have previously reported that Δ(9)-tetrahydrocannabinol (Δ(9)-THC), the main psychoactive cannabinoid in marijuana, suppresses CD40 ligand (CD40L) expression by activated mouse CD4(+) T cells. CD40L is involved in pathogenesis of many autoimmune and inflammatory diseases. In the present study, we investigated the molecular mechanism of Δ(9)-THC-mediated suppression of CD40L expression using peripheral blood human T cells. Pretreatment with Δ(9)-THC attenuated CD40L expression in human CD4(+) 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(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(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(9)-THC suppresses human T cell function.

  18. Increased secretion of hyperimmune antibodies following lipopolysaccharide stimulation of CD40-activated human B cells in vitro.

    PubMed

    Dumont, Nellie; Aubin, Eric; Proulx, Dominic P; Lemieux, Réal; Bazin, Renée

    2009-04-01

    Human B cells can be cultured ex vivo for a few weeks, following stimulation of the CD40 cell surface molecule in the presence of recombinant cytokines such as interleukin-4 (IL-4). However, attempts to produce polyclonal antigen-specific human antibodies by in vitro culture of human B cells obtained from immunized donors have not been successful. It has been shown in mice that lipopolysaccharide (LPS) is a potent mitogen for B cells and plays an important role in the generation of antigen-specific antibody responses. Although it has long been believed that LPS has no direct effect on human B cells, recent data indicating that IL-4-activated human B cells are induced to express Toll-like receptor-4, the main LPS receptor, prompted us to study the effects of LPS on the proliferation and antibody secretion of human B cells. Our results showed that LPS caused a reduction in the expansion of CD40-activated human B cells, accompanied by an increase in antigen-specific antibody secretion. This result suggested that some, but not all, B cells were able to differentiate into antibody-secreting cells in response to LPS. This increased differentiation could be explained by the observation that LPS-stimulated human B cells were induced to secrete higher amounts of IL-6, a pleiotropic cytokine well-known for its B-cell differentiation activity. In vivo, the effect of LPS on cytokine secretion by B cells may not only enhance B-cell differentiation but also help to sustain a local ongoing immune response to invading Gram-negative bacteria, until all pathogens have been cleared from the organism.

  19. NF-κB Is Involved in Regulation of CD40 Ligand Expression on Mycobacterium bovis Bacillus Calmette-Guérin-Activated Human T Cells

    PubMed Central

    Méndez-Samperio, Patricia; Ayala, Hilda; Vázquez, Abraham

    2003-01-01

    Interaction between CD40L (CD154) on activated T cells and its receptor CD40 on antigen-presenting cells has been reported to be important in the resolution of infection by mycobacteria. However, the mechanism(s) by which Mycobacterium bovis bacillus Calmette-Guérin (BCG) up-regulates membrane expression of CD40L molecules is poorly understood. This study was done to investigate the role of the nuclear factor κB (NF-κB) signaling pathway in the regulation of CD40L expression in human CD4+ T cells stimulated with BCG. Specific pharmacologic inhibition of the NF-κB pathway revealed that this signaling cascade was required in the regulation of CD40L expression on the surface of BCG-activated CD4+ T cells. These results were further supported by the fact that treatment of BCG-activated CD4+ T cells with these pharmacological inhibitors significantly down-regulated CD40L mRNA. In this study, inhibitor κBα (IκBα) and IκBβ protein production was not affected by the chemical protease inhibitors and, more importantly, BCG led to the rapid but transient induction of NF-κB activity. Our results also indicated that CD40L expression on BCG-activated CD4+ T cells resulted from transcriptional up-regulation of the CD40L gene by a mechanism which is independent of de novo protein synthesis. Interestingly, BCG-induced activation of NF-κB and the increased CD40L cell surface expression were blocked by the protein kinase C (PKC) inhibitors 1-[5-isoquinolinesulfonyl]-2-methylpiperazine and salicylate, both of which block phosphorylation of IκB. Moreover, rottlerin a Ca2+-independent PKC isoform inhibitor, significantly down-regulated CD40L mRNA in BCG-activated CD4+ T cells. These data strongly suggest that CD40L expression by BCG-activated CD4+ T cells is regulated via the PKC pathway and by NF-κB DNA binding activity. PMID:12738634

  20. IL-12-mediated STAT4 signaling and TCR signal strength cooperate in the induction of CD40L in human and mouse CD8+ T cells.

    PubMed

    Stark, Regina; Hartung, Anett; Zehn, Dietmar; Frentsch, Marco; Thiel, Andreas

    2013-06-01

    CD40L is one of the key molecules bridging the activation of specific T cells and the maturation of professional and nonprofessional antigen-presenting cells including B cells. CD4(+) T cells have been regarded as the major T-cell subset that expresses CD40L upon cognate activation; however, we demonstrate here that a putative CD8(+) helper T-cell subset expressing CD40L is induced in human and murine CD8(+) T cells in vitro and in mice immunized with antigen-pulsed dendritic cells. IL-12 and STAT4-mediated signaling was the major instructive cytokine signal boosting the ability of CD8(+) T cells to express CD40L both in vitro and in vivo. Additionally, TCR signaling strength modulated CD40L expression in CD8(+) T cells after primary differentiation in vitro as well as in vivo. The induction of CD40L in CD8(+) T cells regulated by IL-12 and TCR signaling may enable CD8(+) T cells to respond autonomously of CD4(+) T cells. Thus, we propose that under proinflammatory conditions, a self-sustaining positive feedback loop could facilitate the efficient priming of T cells stimulated by high affinity peptide displaying APCs.

  1. Antibodies to CD40 prevent Epstein-Barr virus-mediated human B-cell lymphomagenesis in severe combined immune deficient mice given human peripheral blood lymphocytes.

    PubMed

    Murphy, W J; Funakoshi, S; Beckwith, M; Rushing, S E; Conley, D K; Armitage, R J; Fanslow, W C; Rager, H C; Taub, D D; Ruscetti, F W

    1995-09-01

    CD40 is expressed on both normal and neoplastic B lymphocytes. Signal transduction through CD40 in vitro has been shown to exert stimulatory effects on normal B cells and inhibitory effects on Epstein-Barr virus (EBV)-induced B-cell lymphoma lines and some other cell lines derived from patients with aggressive histology lymphoma. The transfer of normal human peripheral blood lymphocytes (huPBL) from EBV-seropositive donors into severe combined immune deficient (SCID) mice has been previously shown to result in the generation of human B-cell lymphomas. These tumors are similar to the highly aggressive EBV-induced lymphomas that can arise clinically after transplantation or in the setting of immunodeficiency. Treatment of huPBL-SCID chimeric mice with anti-CD40 or anti-CD20 monoclonal antibodies (MoAb) significantly delayed the development of EBV-induced B-cell lymphoma. However, the effects of the two MoAb were mechanistically distinct. Anti-CD40 treatment prevented lymphoma generation, while still allowing for functional human B-cell engraftment in the huPBL-SCID mice compared with mice receiving no treatment, all of which succumbed to lymphoma. By contrast, treatment with anti-CD20 significantly inhibited total human B-cell engraftment in the SCID recipients, which accounted for the absence of lymphomas. In vitro assays examining the transformation of human B cells by EBV also indicated that anti-CD40 could directly inhibit EBV-transformation, whereas anti-CD20 antibodies had no effect. Thus, anti-CD40 exerts selective effects to allow for the engraftment of normal human B cells and prevent the emergence of EBV lymphomas. Stimulation of CD40 by antibodies or its physiologic ligand may, therefore, be of significant clinical use in the prevention of EBV-induced B lymphomas that may arise when EBV-seropositive individuals receive immunosuppressive regimens after transplantation or in immune deficiency states, such as acquired immune deficiency syndrome.

  2. Glucocorticoids upregulate CD40 ligand expression and induce CD40L-dependent immunoglobulin isotype switching

    PubMed Central

    Jabara, Haifa H.; Brodeur, Scott R.; Geha, Raif S.

    2001-01-01

    IL-4 and CD40 ligation are essential for IgE synthesis by B cells. We have shown previously that hydrocortisone (HC) induces IgE synthesis in IL-4–stimulated human B cells. In this study we demonstrate that HC fails to induce IgE synthesis in B cells from CD40 ligand–deficient (CD40L-deficient) patients. Disruption of CD40L-CD40 interactions by soluble CD40-Ig fusion protein or anti-CD40L mAb blocked the capacity of HC to induce IgE synthesis in normal B cells. HC upregulated CD40L mRNA expression in PBMCs and surface expression of CD40L in PBMCs as well as in purified populations of T and B cells. Upregulation of CD40L mRNA in PBMCs occurred 3 hours after stimulation with HC and was inhibited by actinomycin D. Upregulation of CD40L mRNA and induction of IgE synthesis by HC were inhibited by the steroid hormone receptor antagonist RU-486. These results indicate that ligand-mediated activation of the glucocorticoid receptor upregulates CD40L expression in human lymphocytes. PMID:11160161

  3. Stimulation of CD40 in human bladder carcinoma cells inhibits anti-Fas/APO-1 (CD95)-induced apoptosis.

    PubMed

    Jakobson, E; Jönsson, G; Björck, P; Paulie, S

    1998-09-11

    CD40 and the CD95 (Fas/APO-1 antigen) are both members of the tumor necrosis factor receptor family. Whereas CD40 mediates a strong growth stimulatory signal in B cells, engagement of the CD95 receptor leads to growth inhibition and induction of apoptosis. As it has been reported that CD40 activation may rescue B cells from undergoing apoptosis, we were interested to see whether it had a similar effect in other cells expressing the CD40 receptor. We used epithelial tumor cells from the urinary bladder, a cell type that frequently expresses CD40 but for which no clear function of the molecule has been assigned. We found that the ligation of CD95 with the antibody anti-APO-1 induced apoptosis in most of the cell lines tested. Stimulation of CD40 with antibodies or a soluble construct of the CD40 ligand was shown to protect cells from apoptosis, as demonstrated by their ability to suppress the growth inhibition exerted by the anti-APO-1 antibody. Our results show that CD40 stimulation make cells less vulnerable to apoptosis induced via CD95 and suggest that CD40 expression on epithelial tumors may be associated with cell survival.

  4. A novel fully human anti-CD40 monoclonal antibody, 4D11, for kidney transplantation in cynomolgus monkeys.

    PubMed

    Imai, Atsushi; Suzuki, Tomomi; Sugitani, Atsushi; Itoh, Tomoo; Ueki, Shinya; Aoyagi, Takeshi; Yamashita, Kenichiro; Taniguchi, Masahiko; Takahashi, Nobuaki; Miura, Toru; Shimamura, Tsuyoshi; Furukawa, Hiroyuki; Todo, Satoru

    2007-10-27

    CD40-CD154 pathway blockade by anti-CD154 monoclonal antibodies (mAbs) significantly prolongs allograft survival in nonhuman primates. However, thromboembolic complications have prevented clinical application. Thus, blockade of the counter molecule by a novel fully human anti-CD40 mAb, 4D11, is an attractive alternative. Kidney transplantations were performed between outbred cynomolgus monkeys (stimulation index >3 in a mixed lymphocyte reaction). The animals were divided into five groups: nontreatment control (Group 1, n=3), 10-week treatment with either 10 mg/kg (Group 2, n=3), 20 mg/kg (Group 3, n=3), or 40 mg/kg (Group 4, n=1), and 4-week treatment (Group 5, n=1 each) with 10 mg/kg, 20 mg/kg, or 40 mg/kg followed by monthly administration. Graft survival, biochemistry, complete blood counts, lymphocyte phenotypes, blood drug levels, antidonor and antidrug antibodies, and renal histology were examined. Survival (days) was as follows: Group 1 (5, 6, 7), Group 2 (150, 108, 108), Group 3 (84, 108, 379), Group 4 (147), and Group 5 (147, 102, 112). Two animals in Group 3 with normal graft function were killed upon development of hydronephrosis and cerebral infarction. B lymphocytes fell to one-third of the preoperative value at 4 weeks after transplantation in all animals. Antidonor antibodies developed in most of the animals after stopping drug treatment or at the time of death. No animals except for one formed anti-4D11 antibody. 4D11 appears to be a promising agent for antirejection treatment in clinical organ transplantation.

  5. Modulation of neuronal differentiation by CD40 isoforms

    SciTech Connect

    Hou Huayu; Obregon, Demian; Lou, Deyan; Ehrhart, Jared; Fernandez, Frank; Silver, Archie; Tan Jun

    2008-05-02

    Neuron differentiation is a complex process involving various cell-cell interactions, and multiple signaling pathways. We showed previously that CD40 is expressed and functional on mouse and human neurons. In neurons, ligation of CD40 protects against serum withdrawal-induced injury and plays a role in survival and differentiation. CD40 deficient mice display neuron dysfunction, aberrant neuron morphologic changes, and associated gross brain abnormalities. Previous studies by Tone and colleagues suggested that five isoforms of CD40 exist with two predominant isoforms expressed in humans: signal-transducible CD40 type I and a C-terminal truncated, non-signal-transducible CD40 type II. We hypothesized that differential expression of CD40 isoform type I and type II in neurons may modulate neuron differentiation. Results show that adult wild-type, and CD40{sup -/-} deficient mice predominantly express CD40 type I and II isoforms. Whereas adult wild-type mice express mostly CD40 type I in cerebral tissues at relatively high levels, in age and gender-matched CD40{sup -/-} mice CD40 type I expression was almost completely absent; suggesting a predominance of the non-signal-transducible CD40 type II isoform. Younger, 1 day old wild-type mice displayed less CD40 type I, and more CD40 type II, as well as, greater expression of soluble CD40 (CD40L/CD40 signal inhibitor), compared with 1 month old mice. Neuron-like N2a cells express CD40 type I and type II isoforms while in an undifferentiated state, however once induced to differentiate, CD40 type I predominates. Further, differentiated N2a cells treated with CD40 ligand express high levels of neuron specific nuclear protein (NeuN); an effect reduced by anti-CD40 type I siRNA, but not by control (non-targeting) siRNA. Altogether these data suggest that CD40 isoforms may act in a temporal fashion to modulate neuron differentiation during brain development. Thus, modulation of neuronal CD40 isoforms and CD40 signaling may

  6. Human epidermal Langerhans cells differ from monocyte-derived Langerhans cells in CD80 expression and in secretion of IL-12 after CD40 cross-linking.

    PubMed

    Peiser, Matthias; Wanner, Reinhard; Kolde, Gerhard

    2004-09-01

    Langerhans cells (LCs) represent an immature population of myeloid dendritic cells (DCs). As a result of their unique Birbeck granules (BGs), langerin expression, and heterogeneous maturation process, they differ from other immature DCs. Monocyte-derived LCs (MoLCs) mimic epidermal LCs. MoLCs with characteristic BGs are generated by culturing blood-derived monocytes with granulocyte macrophage-colony stimulating factor, interleukin (IL)-4, and transforming growth factor-beta1. Here, we compare maturation-induced antigen expression and cytokine release of LCs with MoLCs. To achieve comparable cell populations, LCs and MoLCs were isolated by CD1c cell sorting, resulting in high purity. In unstimulated cells, CD40 was expressed at equal levels. After stimulation with CD40 ligand (CD40L), LCs and MoLCs acquired CD83 and increased CD86. High CD80 expression was exclusively detected in CD1c-sorted MoLCs. Human leukocyte antigen-DR and CD54 expression was found in all cell populations, however, at different intensities. CD40 triggering increased the potency of LCs and MoLCs to stimulate CD4+ T cell proliferation. Activated MoLCs released IL-12p70 and simultaneously, anti-inflammatory IL-10. The application of the Toll-like receptor ligands peptidoglycan, flagellin, and in particular, lipopolysaccharide (LPS) increased the corelease of these cytokines. LCs secreted IL-10 at a comparable level with MoLCs but failed to produce high amounts of IL-12p70 after application of danger signals. These data indicate that MoLCs as well as LCs display no maturation arrest concerning CD83 and CD86 expression. In difference to MoLCs, LCs resisted activation by CD40L and LPS in terms of IL-12 production. This shows that natural and generated LCs share similar features but differ in relevant functions.

  7. Human Immunodeficiency Virus (HIV) Type 1 Vpu Induces the Expression of CD40 in Endothelial Cells and Regulates HIV-Induced Adhesion of B-Lymphoma Cells

    PubMed Central

    Henderson, Winnie W.; Ruhl, Rebecca; Lewis, Paul; Bentley, Matthew; Nelson, Jay A.; Moses, Ashlee V.

    2004-01-01

    AIDS-related B-cell non-Hodgkin's lymphoma (AIDS-NHL) is a significant cause of morbidity and mortality among individuals infected with human immunodeficiency virus type 1 (HIV-1). AIDS-NHL is clinically and histologically heterogeneous, but common features include an aggressive clinical course and frequent extranodal presentation. HIV-1 infection of nonimmune cells that interact with malignant B cells at extranodal sites may influence both the development and the clinical presentation of disease. Our previous studies have shown that coculture of B-lymphoma (BL) cells with HIV-1-infected endothelial cells (EC) leads to contact activation of EC and firm BL-cell adhesion. The key event promoting EC-BL-cell adhesion was HIV-1 upregulation of endothelial CD40, which allowed induction of vascular cell adhesion molecule 1 (VCAM-1) in a CD40-dependent manner. The present study was designed to identify the HIV-1 protein(s) that influence EC-BL-cell adhesion. When HIV-1 proteins were individually expressed in EC by using recombinant adenoviruses, cultured BL cells adhered exclusively to Vpu-transduced EC. As with HIV-infected EC, adhesive properties were linked to the capacity of Vpu to upregulate CD40, which in turn allowed efficient expression of VCAM-1. When EC were infected with an HIV-1 pseudotype lacking the Vpu gene, CD40 upregulation and BL-cell adhesive properties were lost, indicating an essential role for Vpu in EC-BL-cell interactions. Thus, these data reveal a novel function for HIV-1 Vpu and further suggest a role for Vpu in the development of AIDS-NHL at EC-rich extranodal sites. PMID:15078922

  8. Internalization of IgG-Coated Targets Results in Activation and Secretion of Soluble CD40 Ligand and RANTES by Human Platelets ▿

    PubMed Central

    Antczak, Adam J.; Vieth, Joshua A.; Singh, Navinderjit; Worth, Randall G.

    2011-01-01

    Platelets are crucial elements for maintenance of hemostasis. Other functions attributable to platelets are now being appreciated, such as their role in inflammatory reactions and host defense. Platelets have been reported to bind immunological stimuli like IgG complexes, and for nearly 50 years it has been speculated that platelets may participate in immunological reactions. Platelets have been reported to bind and internalize various substances, similar to other leukocytes, such as macrophages and dendritic cells. In the present study, we tested the hypothesis that human platelets can bind and internalize IgG-coated particles, similar to leukocytes. To this end, we observed that interaction with IgG-coated beads resulted in platelet activation (as measured by CD62P expression), internalization of targets, and significant soluble CD40 ligand (sCD40L) and RANTES (regulated upon activation, normal T cell expresses and secreted) secretion. Blocking FcγRIIA with monoclonal antibody (MAb) IV.3 or inhibiting actin remodeling with cytochalasin D inhibited platelet activation, internalization, and cytokine production. These data suggest that platelets are capable of mediating internalization of IgG-coated particles, resulting in platelet activation and release of both sCD40L and RANTES. PMID:21177916

  9. The CD40-CD40L system in cardiovascular disease.

    PubMed

    Pamukcu, Burak; Lip, Gregory Y H; Snezhitskiy, Viktor; Shantsila, Eduard

    2011-08-01

    The CD40-CD40L system is a pathway which is associated with both prothrombotic and proinflammatory effects. CD40 and its ligand were first discovered on the surface of activated T cells, but its presence on B cells, antigen-presenting cells, mast cells, and finally platelets, is evident. The soluble form of CD40L (sCD40L) is derived mainly from activated platelets and contributes to the pathophysiology of atherosclerosis and atherothrombosis. Indeed, sCD40L has autocrine, paracrine, and endocrine activities, and it enhances platelet activation, aggregation, and platelet-leucocyte conjugation that may lead to atherothrombosis. It has even been suggested that sCD40L may play a pathogenic role in triggering acute coronary syndromes. Conversely, blockade of this pathway with anti-CD40L antibodies may prevent or delay the progression of atherosclerosis. Concentrations of sCD40L also predict risk of future cardiovascular disease in healthy women and clinical outcomes in patients with acute coronary syndromes. However, there are controversial and uncertain points over the application of this biomarker to clinical cardiology. In this review, we provide an overview of potential implications of CD40-CD40L signalling and sCD40L as a biomarker in patients with atherosclerotic vascular diseases.

  10. 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.

  11. Organization of the human CD40L gene: Implications for molecular defects in X chromosome-linked hyper-IgM syndrome and prenatal diagnosis

    SciTech Connect

    Villa, A.; Macchi, P.P.; Strina, D.; Frattini, A.; Lucchini, F.; Patrosso, C.M.; Vezzoni, P.; Notarangelo, L.D.; Giliani, S.; Mantuano, E.

    1994-03-15

    Recently, CD40L has been identified as the gene responsible for X chromosome-linked hyper-IgM syndrome (HIGM1). CD40L on activated T cells from HIGM1 patients fails to bind B-cell CD40 molecules, and subsequent analysis of CD40L transcripts by reverse transcription PCR demonstrated coding region mutations in these patients. This approach, however, is of limited use for prenatal diagnosis of HIGM1 in the early-gestation fetus. In this report, the authors have defined the genomic structure of the CD40L gene, which is composed of five exons and four intervening introns. With this information, the authors have defined at the genomic level the CD40L coding region. These different deletions arose from three distinct mechanisms, including (i) a splice donor mutation with exon skipping, (ii) a splice acceptor mutation with utilization of a cryptic splice site, and (iii) a deletion/insertion event with the creation of a new splice acceptor site. In addition, they have performed prenatal evaluation of an 11-week-old fetus at risk for HIGM1. CD40L genomic clones provide a starting point for further studies of the genetic elements that control CD40L expression. Knowledge of the CD40L gene structure will prove useful for the identification of additional mutations in HIGM1 and for performing genetic counseling about this disease. 54 refs., 4 figs., 1 tab.

  12. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated disruption of the CD40 ligand-induced activation of primary human B cells.

    PubMed

    Lu, Haitian; Crawford, Robert B; Kaplan, Barbara L F; Kaminski, Norbert E

    2011-09-15

    Suppression of the primary antibody response is particularly sensitive to suppression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice; however, surprisingly little is known concerning the effects of TCDD on humoral immunity or B cell function in humans. Results from a limited number of previous studies, primarily employing in vitro activation models, suggested that human B cell effector function is suppressed by TCDD. The present study sought to extend these findings by investigating, in primary human B cells, the effects of TCDD on several critical stages leading to antibody secretion including activation and plasmacytic differentiation using an in vitro CD40 ligand activation model. These studies revealed important differences in the response of human and mouse B cells to TCDD, the most striking being altered expression of plasmacytic differentiation regulators, B lymphocyte-induced maturation protein 1 and paired box protein 5, in mouse but not human B cells. The activation of human B cells was profoundly impaired by TCDD, as evidenced by decreased expression of activation markers CD80, CD86, and CD69. The impaired activation correlated with decreased cell viability, which prevented the progression of human B cells toward plasmacytic differentiation. TCDD treatment also attenuated the early activation of mitogen-activated protein kinases (MAPK) and Akt signaling in human B cells. Collectively, the present study provided experimental evidence for novel mechanisms by which TCDD impairs the effector function of primary human B cells.

  13. CD40L confers helper functions to human intra-melanoma class-I-restricted CD4+CD8+ double positive T cells

    PubMed Central

    Parrot, Tiphaine; Oger, Romain; Benlalam, Houssem; Raingeard de la Blétière, Diane; Jouand, Nicolas; Coutolleau, Anne; Preisser, Laurence; Khammari, Amir; Dréno, Brigitte; Guardiola, Philippe; Delneste, Yves; Labarrière, Nathalie; Gervois, Nadine

    2016-01-01

    ABSTRACT Although CD4+CD8+ double positive (DP) T cells represent a small fraction of peripheral T lymphocytes in healthy human donors, their frequency is often increased under pathological conditions (in blood and targeted tissues). In solid cancers such as melanoma, we previously demonstrated an enrichment of tumor reactive CD4lowCD8highαβ DP T cells among tumor-infiltrating lymphocytes of unknown function. Similarly to their single positive (SP) CD8+ counterparts, intra-melanoma DP T cells recognized melanoma cell lines in an HLA-class-I restricted context. However, they presented a poor cytotoxic activity but a strong production of diverse Th1 and Th2 cytokines. The aim of this study was to clearly define the role of intra-melanoma CD4lowCD8highαβ DP T cells in the antitumor immune response. Based on a comparative transcriptome analysis between intra-melanoma SP CD4+, SP CD8+ and DP autologous melanoma-infiltrating T-cell compartments, we evidenced an overexpression of the CD40L co-stimulatory molecule on activated DP T cells. We showed that, like SP CD4+ T cells, and through CD40L involvement, DP T cells are able to induce both proliferation and differentiation of B lymphocytes and maturation of functional DCs able to efficiently prime cytotoxic melanoma-specific CD8 T-cell responses. Taken together, these results highlight the helper potential of atypical DP T cells and their role in potentiating antitumor response. PMID:28123891

  14. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated disruption of the CD40 ligand-induced activation of primary human B cells

    SciTech Connect

    Lu Haitian Crawford, Robert B. Kaplan, Barbara L.F. Kaminski, Norbert E.

    2011-09-15

    Suppression of the primary antibody response is particularly sensitive to suppression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice; however, surprisingly little is known concerning the effects of TCDD on humoral immunity or B cell function in humans. Results from a limited number of previous studies, primarily employing in vitro activation models, suggested that human B cell effector function is suppressed by TCDD. The present study sought to extend these findings by investigating, in primary human B cells, the effects of TCDD on several critical stages leading to antibody secretion including activation and plasmacytic differentiation using an in vitro CD40 ligand activation model. These studies revealed important differences in the response of human and mouse B cells to TCDD, the most striking being altered expression of plasmacytic differentiation regulators, B lymphocyte-induced maturation protein 1 and paired box protein 5, in mouse but not human B cells. The activation of human B cells was profoundly impaired by TCDD, as evidenced by decreased expression of activation markers CD80, CD86, and CD69. The impaired activation correlated with decreased cell viability, which prevented the progression of human B cells toward plasmacytic differentiation. TCDD treatment also attenuated the early activation of mitogen-activated protein kinases (MAPK) and Akt signaling in human B cells. Collectively, the present study provided experimental evidence for novel mechanisms by which TCDD impairs the effector function of primary human B cells. - Highlights: > In this study primary human and mouse B cell toxicity to TCDD was compared. > TCDD altered the expression of Blimp-1 and Pax5 in mouse but not human B cells. > TCDD markedly suppressed human B cell activation as characterized by CD80, CD86 and CD69 expression. > TCDD inhibited ERK, p38, and Akt phosphorylation in human B cells.

  15. Involvement of nuclear factor {kappa}B in platelet CD40 signaling

    SciTech Connect

    Hachem, Ahmed; Yacoub, Daniel; Zaid, Younes; Mourad, Walid; Merhi, Yahye

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer sCD40L induces TRAF2 association to CD40 and NF-{kappa}B activation in platelets. Black-Right-Pointing-Pointer I{kappa}B{alpha} phosphorylation downstream of CD40L/CD40 signaling is independent of p38 MAPK phosphorylation. Black-Right-Pointing-Pointer I{kappa}B{alpha} is required for sCD40L-induced platelet activation and potentiation of aggregation. -- Abstract: CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-{kappa}B). Given that platelets contain NF-{kappa}B, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of I{kappa}B{alpha}, which are abolished by CD40L blockade. Inhibition of I{kappa}B{alpha} phosphorylation reverses sCD40L-induced I{kappa}B{alpha} phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on I{kappa}B{alpha} phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of I{kappa}B{alpha} phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-{kappa}B activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against thrombo

  16. CD40: a mediator of pro- and anti-inflammatory signals in renal tubular epithelial cells.

    PubMed

    Laxmanan, Sreenivas; Datta, Dipak; Geehan, Christopher; Briscoe, David M; Pal, Soumitro

    2005-09-01

    Infiltration of immune cells into the renal interstitium is characteristic of chronic inflammatory kidney diseases. CD4+ T cells and platelets express CD40 ligand (CD40L) and are reported to mediate proinflammatory events in renal proximal tubular epithelial cells (RPTEC) via interaction with CD40. In other cell types, CD40 signals can also induce protective genes. Here, human RPTEC were treated with sCD40L to ligate CD40, and a significant increase in the generation of proinflammatory reactive oxygen species was found; however, CD40-activated cells did not undergo apoptosis. This suggests that CD40 signals may simultaneously induce antiapoptotic genes for cytoprotection of RPTEC. Heme oxygenase-1 (HO-1) expressed in RPTEC serves as a protective gene, but it is not known whether it is regulated by CD40. Next, RPTEC were transiently transfected with a full-length HO-1 promoter-luciferase construct and were treated with sCD40L. CD40 ligation was found to significantly increase HO-1 promoter activity. By electrophoretic mobility shift assay, it was confirmed that CD40 signaling induced the transcriptional activation of HO-1 through the binding of NF-kappaB to its promoter. By Western blot analysis, a marked increase in HO-1 protein expression following CD40 ligation was also found. These observations are of clinical significance because it was found that CD40 and HO-1 are induced in expression in vivo in inflamed rejecting kidney biopsies and co-expressed in renal tubules. Therefore, ligation of CD40 in RPTEC promotes both inflammatory and anti-inflammatory processes. Regulating the balance between these two events may be of importance in the prevention of tubular injury associated with renal disease.

  17. Cloning and high level expression of the biologically active extracellular domain of Macaca mulatta CD40 in Pichia pastoris.

    PubMed

    Zhu, Shengyun; Wan, Lin; Yang, Hao; Cheng, Jingqiu; Lu, Xiaofeng

    2016-03-01

    The CD40-mediated immune response contributes to a wide variety of chronic inflammatory diseases. CD40 antagonists have potential as novel therapies for immune disorders. However, the CD40 pathway has not been well characterized in the rhesus monkey Macaca mulatta, which is a valuable animal model for human immune disease. An 834 bp transcript was cloned from peripheral blood mononuclear cells (PBMCs) of rhesus monkey using specific primers designed according to the predicted sequence of M. mulatta CD40 (mmCD40) in GenBank. Sequence analysis demonstrated that mmCD40 is highly homologous to human CD40 (hCD40), with an amino acid sequence identity of 94%. Genes encoding the extracellular domain of mmCD40 and the Fc fragment of the hIgG1 were inserted into a pPIC9K plasmid to produce mmCD40Ig by Pichia pastoris. Approximately 15-20 mg of the mmCD40Ig protein with ∼90% purity could be recovered from 1 L of culture. The purified mmCD40Ig protein can form dimers and can specifically bind CD40L-positive cells. Additionally, the mmCD40Ig protein can bind hCD40L protein in phosphate buffered saline and form a stable combination in a size-exclusion chromatography assay using a Superdex 200 column. Moreover, mmCD40Ig is as efficient as M. mulatta CTLA4Ig (mmCTLA4Ig) to suppress Con A-stimulated lymphocyte proliferation. Additionally, mmCD40Ig only showed mild immunosuppressive activity in a one-way mixed lymphocyte reaction (MLR) system. These results suggest that mmCD40Ig secreted by P. pastoris was productive and functional, and it could be used as a tool for pathogenesis and therapies for chronic inflammatory diseases in a M. mulatta model.

  18. The role of CD40 and CD40L in bone mineral density and in osteoporosis risk: A genetic and functional study.

    PubMed

    Panach, Layla; Pineda, Begoña; Mifsut, Damián; Tarín, Juan J; Cano, Antonio; García-Pérez, Miguel Ángel

    2016-02-01

    Compelling data are revealing that the CD40/CD40L system is involved in bone metabolism. Furthermore, we have previously demonstrated that polymorphisms in both genes are associated with bone phenotypes. The aim of this study is to further characterize this association and to identify the causal functional mechanism. We conducted an association study of BMD with 15 SNPs in CD40/CD40L genes in a population of 779 women. In addition, we assessed the functionality of this association through the study of the allele-dependent expression of CD40 and CD40L in peripheral blood leukocytes (PBLs) and in human osteoblasts (OBs) obtained from bone explants by qPCR and by sequencing. When an allelic imbalance (AI) was detected, studies on allele-dependent in vitro transcription rate and on CpG methylation in the gene promoter were also performed. Our results confirm the genetic association between SNP rs116535 (T>C) of CD40L gene with LS-BMD. Regarding CD40 gene, two SNPs showed nominal P-values<0.05 for FN- and LS-BMD (Z-scores), although the association was not significant after correcting for multiple testing. Homozygous TT women for SNP rs1883832 (C>T) of CD40 gene showed a trend to have lower levels of OPG (Q-value=0.059), especially when women of BMD-quartile ends were selected (P<0.05). Regarding functionality, we detected an AI for rs1883832 with the C allele the most expressed in OBs and in PBLs. Since the rs116535 of CD40L gene did not show AI, it was not further analyzed. Finally, we described a differential methylation of CpGs in the CD40 promoter among women of high in comparison to low BMD. Our results suggest that the CD40/CD40L system plays a role in regulating BMD. Effectively, our data suggest that a decreased production of OPG could be the cause of the lower BMD observed in TT women for rs1883832 of the CD40 gene and that the degree of methylation of CpGs in the CD40 promoter could contribute to the acquisition of BMD. One possibility that deserves further

  19. CD40/CD40L contributes to hypercholesterolemia-induced microvascular inflammation

    PubMed Central

    Stokes, Karen Y.; Calahan, LeShanna; Hamric, Candiss M.; Russell, Janice M.; Granger, D. Neil

    2009-01-01

    Hypercholesterolemia is associated with phenotypic changes in endothelial cell function that lead to a proinflammatory and prothrombogenic state in different segments of the microvasculature. CD40 ligand (CD40L) and its receptor CD40 are ubiquitously expressed and mediate inflammatory responses and platelet activation. The objective of this study was to determine whether CD40/CD40L, in particular T-cell CD40L, contributes to microvascular dysfunction induced by hypercholesterolemia. Intravital microscopy was used to quantify blood cell adhesion in cremasteric postcapillary venules, endothelium-dependent vasodilation responses in arterioles, and microvascular oxidative stress in wild-type (WT) C57BL/6, CD40-deficient (−/−), CD40L−/−, or severe combined immune deficient (SCID) mice placed on a normal (ND) or high-cholesterol (HC) diet for 2 wk. WT-HC mice exhibited an exaggerated leukocyte and platelet recruitment in venules and impaired vasodilation responses in arterioles compared with ND counterparts. A deficiency of CD40, CD40L, or lymphocytes attenuated these responses to HC. The HC phenotype was rescued in CD40L−/− and SCID mice by a transfer of WT T cells. Bone marrow chimeras revealed roles for both vascular- and blood cell-derived CD40 and CD40L in the HC-induced vascular responses. Hypercholesterolemia induced an oxidative stress in both arterioles and venules of WT mice, which was abrogated by either CD40 or CD40L deficiency. The transfer of WT T cells into CD40L−/− mice restored the oxidative stress. These results implicate CD40/CD40L interactions between circulating cells and the vascular wall in both the arteriolar and venular dysfunction elicited by hypercholesterolemia and identify T-cell-associated CD40L as a key mediator of these responses. PMID:19112095

  20. TWEAK inhibits TRAF2-mediated CD40 signaling by destabilization of CD40 signaling complexes.

    PubMed

    Salzmann, Steffen; Lang, Isabell; Rosenthal, Alevtina; Schäfer, Viktoria; Weisenberger, Daniela; Carmona Arana, José Antonio; Trebing, Johannes; Siegmund, Daniela; Neumann, Manfred; Wajant, Harald

    2013-09-01

    We found recently that TNF-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible-14 (Fn14) by virtue of their strong capability to reduce the freely available cytoplasmic pool of TNFR-associated factor (TRAF)2 and cellular inhibitors of apoptosis (cIAPs) antagonize the functions of these molecules in TNFR1 signaling, resulting in sensitization for apoptosis and inhibition of classical NF-κB signaling. In this study, we demonstrate that priming of cells with TWEAK also interferes with activation of the classical NF-κB pathway by CD40. Likewise, there was strong inhibition of CD40 ligand (CD40L)-induced activation of MAPKs in TWEAK-primed cells. FACS analysis and CD40L binding studies revealed unchanged CD40 expression and normal CD40L-CD40 interaction in TWEAK-primed cells. CD40L immunoprecipitates, however, showed severely reduced amounts of CD40 and CD40-associated proteins, indicating impaired formation or reduced stability of CD40L-CD40 signaling complexes. The previously described inhibitory effect of TWEAK on TNFR1 signaling has been traced back to reduced activity of the TNFR1-associated TRAF2-cIAP1/2 ubiquitinase complex and did not affect the stability of the immunoprecipitable TNFR1 receptor complex. Thus, the inhibitory effect of TWEAK on CD40 signaling must be based at least partly on other mechanisms. In line with this, signaling by the CD40-related TRAF2-interacting receptor TNFR2 was also attenuated but still immunoprecipitable in TWEAK-primed cells. Collectively, we show that Fn14 activation by soluble TWEAK impairs CD40L-CD40 signaling complex formation and inhibits CD40 signaling and thus identify the Fn14-TWEAK system as a potential novel regulator of CD40-related cellular functions.

  1. Type II Toxoplasma gondii induction of CD40 on infected macrophages enhances interleukin-12 responses.

    PubMed

    Morgado, Pedro; Sudarshana, Dattanand M; Gov, Lanny; Harker, Katherine S; Lam, Tonika; Casali, Paolo; Boyle, Jon P; Lodoen, Melissa B

    2014-10-01

    Toxoplasma gondii is an obligate intracellular parasite that can cause severe neurological disease in infected humans. CD40 is a receptor on macrophages that plays a critical role in controlling T. gondii infection. We examined the regulation of CD40 on the surface of T. gondii-infected bone marrow-derived macrophages (BMdMs). T. gondii induced CD40 expression both at the transcript level and on the cell surface, and interestingly, the effect was parasite strain specific: CD40 levels were dramatically increased in type II T. gondii-infected BMdMs compared to type I- or type III-infected cells. Type II induction of CD40 was specific to cells harboring intracellular parasites and detectable as early as 6 h postinfection (hpi) at the transcript level. CD40 protein expression peaked at 18 hpi. Using forward genetics with progeny from a type II × type III cross, we found that CD40 induction mapped to a region of chromosome X that included the gene encoding the dense granule protein 15 (GRA15). Using type I parasites stably expressing the type II allele of GRA15 (GRA15II), we found that type I GRA15II parasites induced the expression of CD40 on infected cells in an NF-κB-dependent manner. In addition, stable expression of hemagglutinin-tagged GRA15II in THP-1 cells resulted in CD40 upregulation in the absence of infection. Since CD40 signaling contributes to interleukin-12 (IL-12) production, we examined IL-12 from infected macrophages and found that CD40L engagement of CD40 amplified the IL-12 response in type II-infected cells. These data indicate that GRA15II induction of CD40 promotes parasite immunity through the production of IL-12. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  2. AICAR reduces the collagen-stimulated secretion of PDGF-AB and release of soluble CD40 ligand from human platelets: Suppression of HSP27 phosphorylation via p44/p42 MAP kinase.

    PubMed

    Tsujimoto, Masanori; Tokuda, Haruhiko; Kuroyanagi, Gen; Yamamoto, Naohiro; Kainuma, Shingo; Matsushima-Nishiwaki, Rie; Onuma, Takashi; Iida, Yuko; Kojima, Akiko; Sawada, Shigenobu; Doi, Tomoaki; Enomoto, Yukiko; Tanabe, Kumiko; Akamatsu, Shigeru; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Kozawa, Osamu; Iwama, Toru

    2016-08-01

    We have previously reported that collagen-induced phosphorylation of heat shock protein (HSP) 27 via p44/p42 mitogen-activated protein (MAP) kinase in human platelets is sufficient to induce the secretion of platelet-derived growth factor (PDGF)-AB and the release of soluble cluster of differentiation 40 ligand (sCD40L). Adenosine monophosphate-activated protein kinase (AMPK), which is known to regulate energy homeostasis, has a crucial role as an energy sensor in various eukaryotic cells. The present study investigated the effects of 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl 5'-monophosphate (AICAR), which is an activator of AMPK, on the collagen-induced activation of human platelets. It was demonstrated that AICAR dose-dependently reduced collagen-stimulated platelet aggregation up to 1.0 µM. Analysis of the size of platelet aggregates demonstrated that AICAR decreased the ratio of large aggregates (50-70 µm), whereas the ratio of small aggregates (9-25 µm) was increased by AICAR administration. AICAR markedly attenuated the phosphorylation levels of p44/p42 MAP kinase and HSP27, which are induced by collagen. Furthermore, AICAR significantly decreased the secretion of PDGF-AB and the collagen-induced release of sCD40L. These results indicated that AICAR-activated AMPK may reduce the secretion of PDGF-AB and the collagen-induced release of sCD40L by inhibiting HSP27 phosphorylation via p44/p42 MAP kinase in human platelets.

  3. Differential modulation by delta9-tetrahydrocannabinol (∆9)-THC) of CD40 ligand (CD40L) expression in activated mouse splenic CD4+ T cells.

    PubMed

    Ngaotepprutaram, Thitirat; Kaplan, Barbara L F; Crawford, Robert B; Kaminski, Norbert E

    2012-12-01

    The anti-inflammatory activity of cannabinoids has been widely demonstrated in experimental animal models and in humans. CD40-CD40-ligand (L) interactions are among the most crucial initiators of inflammation. This study investigated the effects of ∆(9)-THC on CD40L expression in mouse splenic T cells after activation with various stimuli. Time course studies demonstrated that peak surface expression of CD40L by CD4(+) T cells after anti-CD3/CD28 or phorbol ester plus calcium ionophore (PMA/Io) occurred 8 h post activation. Peak CD40L mRNA levels were observed at 2 h post PMA/Io treatment and at 4 h post anti-CD3/CD28 treatment. Pretreatment with ∆(9)-THC significantly impaired the upregulation of CD40L induced by anti-CD3/CD28 at both the protein and mRNA level. By contrast, ∆(9)-THC did not affect PMA/Io-induced surface CD40L expression on CD4(+) T cells. Additionally, ∆(9)-THC also attenuated anti-CD3/CD28-induced CD40L expression on CD4(+) T cells derived from CB1(-/-)/CB2(-/-) mice. We investigated whether the mechanism by which ∆(9)-THC suppressed CD40L expression involved putative cannabinoid activation of the glucocorticoid receptor (GR). Although activation of GR resulted in suppression of CD40L induction by anti-CD3/CD28, no interaction between ∆(9)-THC and GR was observed by a glucocorticoid response element (GRE) luciferase reporter assay in HEK293T cells. Collectively, these results suggest that ∆(9)-THC targets proximal T cell receptor-associated signaling in a cannabinoid receptor- and glucocorticoid receptor-independent manner. These findings identify suppression of CD40L expression as a novel part of the mechanism by which ∆(9)-THC exerts anti-inflammatory activity.

  4. IL-4 plus CD40 monoclonal antibody induces human B cells gamma subclass-specific isotype switch: switching to gamma 1, gamma 3, and gamma 4, but not gamma 2.

    PubMed

    Fujieda, S; Zhang, K; Saxon, A

    1995-09-01

    Stimulation with IL-4 plus CD40 mAb is known to induce production of IgE and IgG4. In this study, we determined the IgG subclass specificity of IL-4 plus CD40 mAb stimulation for human purified B cells. We determined true in vitro switching by the generation of switch circular DNA (S gamma/S mu) representing primary S mu/S gamma events and production of gamma subclass-specific germ-line transcripts by a combination of reverse transcription-PCR and restriction endonuclease digestion. We simultaneously measured changes in the levels of IgG subclass proteins produced. Forty-two clones of circular switch DNA were identified and sequenced. The IgG subclass of S gamma fragment in the S gamma/S mu chimeric PCR products was determined by analyzing key S gamma nucleotides. The switch-deleted clones were found to consist of S gamma 1/S mu, S gamma 3/S mu, and S gamma 4/S mu chimeric switch sequences, showing that such switching had occurred. No S gamma 2/S mu chimeric switch sequences were found. While a consensus sequence was not identified at the S gamma/S mu breakpoints, four contiguous guanines (GGGG) were noticeably present in the S gamma region near the breakpoint. The induction of gamma 1, gamma 3, and gamma 4 switch circles in human purified B cells was accompanied by enhanced production of IgG1, IgG3, and IgG4 but not IgG2. Similarly, stimulation with IL-4 alone induced gamma 1, gamma 3, and gamma 4 but not gamma 2 germ-line transcripts. These results demonstrate that IL-4 plus CD40 mAb induces Ig isotype switch from mu to gamma 1, gamma 3, and gamma 4 but not to gamma 2.

  5. AdCD40L--crossing the valley of death?

    PubMed

    Ullenhag, Gustav; Loskog, Angelica S I

    2012-08-01

    CD40-mediated cancer therapy has been under development since it became clear that CD40 plays a profound role in the stimulation of adaptive immune responses. Further, CD40 signaling on tumor cells may lead to growth arrest or even apoptosis that improves therapy outcome. The therapeutic window is appealing since the immune system is selective and normal cells do not apoptose upon CD40 signaling. AdCD40L is an adenoviral-based immunostimulatory gene therapy under evaluation for its efficacy to treat cancer. Because of its nature, the adenoviral backbone will stimulate TLRs while CD40L potentiates the shifts toward Th1 type of immunity. AdCD40L has shown efficacy in various murine models, and safety studies have been performed on dog patients and in human clinical trials. AdCD40L has been used for both ex vivo gene modification of tumor cell vaccines as well as for direct intratumoral injections. Lately, an oncolytic vector has been used to further increase the eradication of solid tumors that as a consequence further boosts the release of tumor antigens and creates danger signaling in the tumor micro milieu. This review discusses the currently unfolding mechanisms of action of AdCD40L gene therapy and its possibilities to reach clinical care.

  6. Human B cells accumulate immunoglobulin V gene somatic mutations in a cell contact-dependent manner in cultures supported by activated T cells but not in cultures supported by CD40 ligand

    PubMed Central

    Huang, S-C; Glas, A M; Pinchuk, G V; Van Montfort, E H N; Rao, S P; Jiang, R; Milner, E C B

    1999-01-01

    The acquisition of somatic mutations in the rearranged immunoglobulin V regions in B cells occurs within the tightly regulated microenvironment of a germinal centre. The precise mechanism responsible for turning on the mutational process is unknown. To dissect the role of different components of the germinal centre in this mechanism, we have used in vitro cultures of normal human IgD+ peripheral blood B lymphocytes co-cultured with activated CD4+ T cells, or with resting CD4+ T cells, or with CD40 ligand and IL-4. We observed that if the cultures included activated CD4+ T cells, then up to 100% of VH transcripts on day 14 were somatically mutated. Transcripts were found to carry from one to 36 substitutions (median five). In contrast, in the absence of activated T cells, transcripts contained only background levels of somatic mutation irrespective of the presence of resting T cells or CD40 ligand and IL-4. Cell–cell contact was required for mutation because mutations were not detected when B cells were separated from activated T cells by a membrane. PMID:10361232

  7. CD40 Ligand and Appropriate Cytokines Induce Switching to IgG, IgA, and IgE and Coordinated Germinal Center and Plasmacytoid Phenotypic Differentiation in a Human Monoclonal IgM+IgD+ B Cell Line1

    PubMed Central

    Cerutti, Andrea; Zan, Hong; Schaffer, Andras; Bergsagel, Leif; Harindranath, Nagaradona; Max, Edward E.; Casali, Paolo

    2015-01-01

    B lymphocytes are induced to undergo Ig class switching and a complex phenotypic differentiation by the milieu of the germinal center. Partly as a result of the lack of a suitable in vitro B cell model, the relationship between these processes in the humans has never been formally established in vitro. We have identified a human monoclonal B cell line, CL-01, that expresses surface IgM and IgD and, upon induction with CD40 ligand, IL-4, and IL-10, switches to all seven downstream isotypes, showing typical DNA switch recombination preceded by germline transcription of targeted CH regions. In CL-01 cells, switch-inducing stimuli trigger concomitant changes in expression of surface IgD, CD23, CD38, and CD77 that parallel those reported in ex vivo isolated tonsillar centroblasts, centrocytes, and memory B cells. Eventually, in the presence of IL-6, CL-01 cells express CD56 and accumulate cytoplasmic IgG and IgA, both traits of plasmacytoid differentiation. Analysis of transcription and recombination of the Ig H locus in sorted CL-01 cells suggest that Ig class switching begins in centroblasts, it extends to all isotypes in centrocytes, and it is extinct in memory B cells. Thus, we have induced coordinated Ig class switching, progression through germinal center phenotypic stages, and differentiation to memory B cells and plasma cells at the level of a single B clonotype. Our data suggest that these processes are likely regulated by a common maturation program, the activation of which may require CD40 ligand, IL-4, IL-10, and IL-6 only. PMID:9498752

  8. Analysis of the association between CD40 and CD40 ligand polymorphisms and systemic sclerosis

    PubMed Central

    2012-01-01

    Introduction The aim of the present study was to investigate the possible role of CD40 and CD40 ligand (CD40LG) genes in the susceptibility and phenotype expression of systemic sclerosis (SSc). Methods In total, 2,670 SSc patients and 3,245 healthy individuals from four European populations (Spain, Germany, The Netherlands, and Italy) were included in the study. Five single-nucleotide polymorphisms (SNPs) of CD40 (rs1883832, rs4810485, rs1535045) and CD40LG (rs3092952, rs3092920) were genotyped by using a predesigned TaqMan allele-discrimination assay technology. Meta-analysis was assessed to determine whether an association exists between the genetic variants and SSc or its main clinical subtypes. Results No evidence of association between CD40 and CD40LG genes variants and susceptibility to SSc was observed. Similarly, no significant statistical differences were observed when SSc patients were stratified by the clinical subtypes, the serologic features, and pulmonary fibrosis. Conclusions Our results do not suggest an important role of CD40 and CD40LG gene polymorphisms in the susceptibility to or clinical expression of SSc. PMID:22731751

  9. CD40 Ligand Deficient C57BL/6 Mouse Is a Potential Surrogate Model of Human X-Linked Hyper IgM (X-HIGM) Syndrome for Characterizing Immune Responses against Pathogens

    PubMed Central

    Lopez-Saucedo, Catalina; Bernal-Reynaga, Rodolfo; Zayas-Jahuey, Jesus; Galindo-Gomez, Silvia; Shibayama, Mineko; Garcia-Galvez, Carlos; Estrada-Parra, Sergio; Estrada-Garcia, Teresa

    2015-01-01

    Individuals with X-HIGM syndrome fail to express functional CD40 ligand; consequently they cannot mount effective protective antibody responses against pathogenic bacteria. We evaluated, compared, and characterized the humoral immune response of wild type (WT) and C57-CD40L deficient (C57-CD40L−/−) mice infected with Citrobacter rodentium. Basal serum isotype levels were similar for IgM and IgG3 among mice, while total IgG and IgG2b concentrations were significantly lower in C57-CD40L−/− mice compared with WT. Essentially IgG1 and IgG2c levels were detectable only in WT mice. C57-CD40L−/− animals, orally inoculated with 2 × 109 CFU, presented several clinical manifestations since the second week of infection and eventually died. In contrast at this time point no clinical manifestations were observed among C57-CD40L−/− mice infected with 1 × 107 CFU. Infection was subclinical in WT mice inoculated with either bacterial dose. The serum samples from infected mice (1 × 107 CFU), collected at day 14 after infection, had similar C. rodentium-specific IgM titres. Although C57-CD40L−/− animals had lower IgG and IgG2b titres than WT mice, C57-CD40L−/− mice sera displayed complement-mediated bactericidal activity against C. rodentium. C. rodentium-infected C57-CD40L−/− mice are capable of producing antibodies that are protective. C57-CD40L−/− mouse is a useful surrogate model of X-HIGM syndrome for studying immune responses elicited against pathogens. PMID:26064940

  10. Soluble CD40 ligand accumulates in stored blood components, primes neutrophils through CD40, and is a potential cofactor in the development of transfusion-related acute lung injury

    PubMed Central

    Khan, Samina Yasmin; Kelher, Marguerite R.; Heal, Joanna M.; Blumberg, Neil; Boshkov, Lynn K.; Phipps, Richard; Gettings, Kelly F.; McLaughlin, Nathan J.; Silliman, Christopher C.

    2006-01-01

    Transfusion-related acute lung injury (TRALI) is a form of posttransfusion acute pulmonary insufficiency that has been linked to the infusion of biologic response modifiers (BRMs), including antileukocyte antibodies and lipids. Soluble CD40 ligand (sCD40L) is a platelet-derived proinflammatory mediator that accumulates during platelet storage. We hypothesized that human polymorpho-nuclear leukocytes (PMNs) express CD40, CD40 ligation rapidly primes PMNs, and sCD40L induces PMN-mediated cytotoxicity of human pulmonary microvascular endothelial cells (HMVECs). Levels of sCD40L were measured in blood components and in platelet concentrates (PCs) implicated in TRALI or control PCs that did not elicit a transfusion reaction. All blood components contained higher levels of sCD40L than fresh plasma, with apheresis PCs evidencing the highest concentration of sCD40L followed by PCs from whole blood, whole blood, and packed red blood cells (PRBCs). PCs implicated in TRALI reactions contained significantly higher sCD40L levels than control PCs. PMNs express functional CD40 on the plasma membrane, and recombinant sCD40L (10 ng/mL-1 μg/mL) rapidly (5 minutes) primed the PMN oxidase. Soluble CD40L promoted PMN-mediated cytotoxicity of HMVECs as the second event in a 2-event in vitro model of TRALI. We concluded that sCD40L, which accumulates during blood component storage, has the capacity to activate adherent PMNs, causing endothelial damage and possibly TRALI in predisposed patients. PMID:16772606

  11. The CD40/CD40 Ligand Interaction Is Required for Resistance to Toxoplasmic Encephalitis

    PubMed Central

    Reichmann, Gaby; Walker, William; Villegas, Eric N.; Craig, Linden; Cai, Guifang; Alexander, James; Hunter, Christopher A.

    2000-01-01

    Since the CD40/CD40 ligand (CD40L) interaction is involved in the regulation of macrophage production of interleukin 12 (IL-12) and T-cell production of gamma interferon (IFN-γ), effector cell functions associated with resistance to Toxoplasma gondii, the role of CD40L in immunity to this parasite was assessed. Infection of C57BL/6 mice with T. gondii results in an upregulation of CD40 expression on accessory cell populations at local sites of infection as well as in lymphoid tissues. Splenocytes from C57BL/6 mice infected with T. gondii for 5 days produced high levels of IL-12 and IFN-γ when stimulated with toxoplasma lysate antigen, and blocking CD40L did not significantly alter the production of IFN-γ or IL-12 by these cells. Similar results were observed with splenocytes and mononuclear cells isolated from the brains of chronically infected mice. Interestingly, although CD40L−/− mice infected with T. gondii produced less IL-12 than wild-type mice, they produced comparable levels of IFN-γ but succumbed to toxoplasmic encephalitis 4 to 5 weeks after infection. The inability of CD40L−/− mice to control parasite replication in the brain correlated with the ability of soluble CD40L, in combination with IFN-γ, to activate macrophages in vitro to control replication of T. gondii. Together, these results identify an important role for the CD40/CD40L interaction in resistance to T. gondii. However, this interaction may be more important in the control of parasite replication in the brain rather than the generation of protective T-cell responses during toxoplasmosis. PMID:10678943

  12. Enhancing Antitumor Efficacy of Chimeric Antigen Receptor T Cells Through Constitutive CD40L Expression

    PubMed Central

    Curran, Kevin J; Seinstra, Beatrijs A; Nikhamin, Yan; Yeh, Raymond; Usachenko, Yelena; van Leeuwen, Dayenne G; Purdon, Terence; Pegram, Hollie J; Brentjens, Renier J

    2015-01-01

    Adoptive cell therapy with genetically modified T cells expressing a chimeric antigen receptor (CAR) is a promising therapy for patients with B-cell acute lymphoblastic leukemia. However, CAR-modified T cells (CAR T cells) have mostly failed in patients with solid tumors or low-grade B-cell malignancies including chronic lymphocytic leukemia with bulky lymph node involvement. Herein, we enhance the antitumor efficacy of CAR T cells through the constitutive expression of CD40 ligand (CD40L, CD154). T cells genetically modified to constitutively express CD40L (CD40L-modified T cells) demonstrated increased proliferation and secretion of proinflammatory TH1 cytokines. Further, CD40L-modified T cells augmented the immunogenicity of CD40+ tumor cells by the upregulated surface expression of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58, and CD70), human leukocyte antigen (HLA) molecules (Class I and HLA-DR), and the Fas-death receptor (CD95). Additionally, CD40L-modified T cells induced maturation and secretion of the proinflammatory cytokine interleukin-12 by monocyte-derived dendritic cells. Finally, tumor-targeted CD19-specific CAR/CD40L T cells exhibited increased cytotoxicity against CD40+ tumors and extended the survival of tumor-bearing mice in a xenotransplant model of CD19+ systemic lymphoma. This preclinical data supports the clinical application of CAR T cells additionally modified to constitutively express CD40L with anticipated enhanced antitumor efficacy. PMID:25582824

  13. Targeted gene editing restores regulated CD40L function in X-linked hyper-IgM syndrome.

    PubMed

    Hubbard, Nicholas; Hagin, David; Sommer, Karen; Song, Yumei; Khan, Iram; Clough, Courtnee; Ochs, Hans D; Rawlings, David J; Scharenberg, Andrew M; Torgerson, Troy R

    2016-05-26

    Loss of CD40 ligand (CD40L) expression or function results in X-linked hyper-immunoglobulin (Ig)M syndrome (X-HIGM), characterized by recurrent infections due to impaired immunoglobulin class-switching and somatic hypermutation. Previous attempts using retroviral gene transfer to correct murine CD40L expression restored immune function; however, treated mice developed lymphoproliferative disease, likely due to viral-promoter-dependent constitutive CD40L expression. These observations highlight the importance of preserving endogenous gene regulation in order to safely correct this disorder. Here, we report efficient, on-target, homology-directed repair (HDR) editing of the CD40LG locus in primary human T cells using a combination of a transcription activator-like effector nuclease-induced double-strand break and a donor template delivered by recombinant adeno-associated virus. HDR-mediated insertion of a coding sequence (green fluorescent protein or CD40L) upstream of the translation start site within exon 1 allowed transgene expression to be regulated by endogenous CD40LG promoter/enhancer elements. Additionally, inclusion of the CD40LG 3'-untranslated region in the transgene preserved posttranscriptional regulation. Expression kinetics of the transgene paralleled that of endogenous CD40L in unedited T cells, both at rest and in response to T-cell stimulation. The use of this method to edit X-HIGM patient T cells restored normal expression of CD40L and CD40-murine IgG Fc fusion protein (CD40-muIg) binding, and rescued IgG class switching of naive B cells in vitro. These results demonstrate the feasibility of engineered nuclease-directed gene repair to restore endogenously regulated CD40L, and the potential for its use in T-cell therapy for X-HIGM syndrome.

  14. CD40 stimulation leads to effective therapy of CD40− tumors through induction of strong systemic cytotoxic T lymphocyte immunity

    PubMed Central

    van Mierlo, Geertje J. D.; den Boer, Annemieke Th.; Medema, Jan Paul; van der Voort, Ellen I. H.; Fransen, Marieke F.; Offringa, Rienk; Melief, Cornelis J. M.; Toes, Rene E. M.

    2002-01-01

    Adequate spontaneous activation of tumor-specific T lymphocytes in tumor-bearing hosts is rare, despite the expression of tumor antigens that are potentially highly immunogenic. For example, failure of the immune system to raise competent responses against established tumors expressing the human adenovirus E1A-antigen allows this tumor to grow in immunocompetent mice. We show that systemic in vivo administration of agonistic anti-CD40 antibodies into tumor-bearing mice results in tumor eradication mediated by CD8+ T cells. Treatment resulted in a strong expansion and systemic accumulation of E1A-specific CTL and depended on CD40 expression on host cells, as the tumor was CD40−, and therapy failed in CD40-deficient mice. Local intratumoral administration of anti-CD40 mAb is equally effective in licensing strong, systemic CTL immunity, resulting in the clearance of distant tumor nodules. Our data indicate that the immune response after cancer–host interactions can be directed toward competence, leading to the cure of established tumors merely by delivery of a CD40-dependent “license to kill” signal. PMID:11929985

  15. Blocking α5β1 Integrin Attenuates sCD40L-Mediated Platelet Activation.

    PubMed

    Simic, Damir; Bogdan, Nancy; Teng, Fang; Otieno, Monicah

    2017-09-01

    The soluble form of CD40L (sCD40L) is a platelet-derived mediator that links inflammation, hemostasis, and vascular dysfunction. Indeed, blockade of CD40L by neutralizing antibodies or genetic disruption in mice prevents atherosclerosis and atherothrombosis. Until recently, it was believed that CD40 and αIIbβ3 were the only receptors on platelets responsible for binding sCD40L, leading to platelet activation and initiation of thrombotic events. Recent findings showed α5β1 integrin as a novel platelet sCD40L receptor, with an unknown function. For the first time, using anti-α5β1 blocking antibodies, we show that sCD40L/α5β1 interaction leads to platelet activation as evaluated in the human whole blood. Establishing α5β1 integrin's role in platelet activation, and therefore thrombosis will help further shed light on the etiology of thrombotic disease.

  16. CD40L expression in plasma of volunteers following LPS administration: A comparison between assay of CD40L on platelet microvesicles and soluble CD40L.

    PubMed

    Mobarrez, Fariborz; Sjövik, Carolina; Soop, Anne; Hållström, Lars; Frostell, Claes; Pisetsky, David S; Wallén, Håkan

    2015-01-01

    CD40 ligand (CD40L) is a transmembrane protein that is mainly expressed on activated T cells and platelets. This protein, however, may also be shed from cells and circulate in the blood in a soluble form. "Soluble CD40L" has attracted interest as a biomarker as it can interact with CD40 and elicit cellular responses involved in the pathophysiology of various thrombotic and inflammatory conditions. As platelets can release microvesicles following activation, we investigated the expression of CD40L on circulating microvesicles as well as CD40L in plasma, in an experimental model of inflammation in healthy volunteers (i.e., intravenous lipopolysaccharide administration). We studied CD40L quantified as CD40L-positive platelet microvesicles by flow cytometry, and as CD40L in plasma ("soluble CD40L") by an ELISA. Results of these studies showed that levels of CD40L exposed on platelet microvesicles were significantly increased after lipopolysaccharide administration. ELISA measurements of CD40L in plasma ("soluble CD40L") did not show any significant increase in plasma levels over time. Separation of soluble and vesicle-bound CD40L by high-speed centrifugation indicated that the ELISA can also detect CD40L on microvesicles, as a trend toward increased concentrations were observed in the pellet of high-speed centrifuged samples (i.e., in samples in which microvesicles are enriched). Together, these findings suggest that platelet microvesicles are a source of CD40L in the circulation and that CD40L exposure on platelet microvesicles increases following experimentally induced inflammation. Our data also suggest that determining levels of CD40L on microvesicles in plasma samples may provide a more sensitive detection of changes in CD40L expression than measurement of "soluble CD40L" in plasma with an ELISA. In addition, information regarding the cellular source of CD40L can be obtained with a flow cytometry-based microvesicle assay in a way not possible with an ordinary

  17. Anti-CD40 ligand monoclonal antibody delays the progression of murine autoimmune cholangitis.

    PubMed

    Tanaka, H; Yang, G-X; Iwakoshi, N; Knechtle, S J; Kawata, K; Tsuneyama, K; Leung, P; Coppel, R L; Ansari, A A; Joh, T; Bowlus, C; Gershwin, M E

    2013-12-01

    While there have been significant advances in our understanding of the autoimmune responses and the molecular nature of the target autoantigens in primary biliary cirrhosis (PBC), unfortunately these data have yet to be translated into new therapeutic agents. We have taken advantage of a unique murine model of autoimmune cholangitis in which mice expressing a dominant negative form of transforming growth factor β receptor II (dnTGFβRII), under the control of the CD4 promoter, develop an intense autoimmune cholangitis associated with serological features similar to human PBC. CD40-CD40 ligand (CD40L) is a major receptor-ligand pair that provides key signals between cells of the adaptive immune system, prompting us to determine the therapeutic potential of treating autoimmune cholangitis with anti-CD40L antibody (anti-CD40L; MR-1). Four-week-old dnTGFβRII mice were injected intraperitoneally with either anti-CD40L or control immunoglobulin (Ig)G at days 0, 2, 4 and 7 and then weekly until 12 or 24 weeks of age and monitored for the progress of serological and histological features of PBC, including rigorous definition of liver cellular infiltrates and cytokine production. Administration of anti-CD40L reduced liver inflammation significantly to 12 weeks of age. In addition, anti-CD40L initially lowered the levels of anti-mitochondrial autoantibodies (AMA), but these reductions were not sustained. These data indicate that anti-CD40L delays autoimmune cholangitis, but the effect wanes over time. Further dissection of the mechanisms involved, and defining the events that lead to the reduction in therapeutic effectiveness will be critical to determining whether such efforts can be applied to PBC.

  18. CD40 deficiency in mice exacerbates obesity-induced adipose tissue inflammation, hepatic steatosis, and insulin resistance

    PubMed Central

    Guo, Chang-An; Kogan, Sophia; Amano, Shinya U.; Wang, Mengxi; Dagdeviren, Sezin; Friedline, Randall H.; Aouadi, Myriam; Kim, Jason K.

    2013-01-01

    The pathophysiology of obesity and type 2 diabetes in rodents and humans is characterized by low-grade inflammation in adipose tissue and liver. The CD40 receptor and its ligand CD40L initiate immune cell signaling promoting inflammation, but conflicting data on CD40L-null mice confound its role in obesity-associated insulin resistance. Here, we demonstrate that CD40 receptor-deficient mice on a high-fat diet display the expected decrease in hepatic cytokine levels but paradoxically exhibit liver steatosis, insulin resistance, and glucose intolerance compared with their age-matched wild-type controls. Hyperinsulinemic-euglycemic clamp studies also demonstrated insulin resistance in glucose utilization by the CD40-null mice compared with wild-type mice. In contrast to liver, adipose tissue in CD40-deficient animals harbors elevated cytokine levels and infiltration of inflammatory cells, particularly macrophages and CD8+ effector T cells. In addition, ex vivo explants of epididymal adipose tissue from CD40−/− mice display elevated basal and isoproterenol-stimulated lipolysis, suggesting a potential increase of lipid efflux from visceral fat to the liver. These findings reveal that 1) CD40-null mice represent an unusual model of hepatic steatosis with reduced hepatic inflammation, and 2) CD40 unexpectedly functions in adipose tissue to attenuate its inflammation in obesity, thereby protecting against hepatic steatosis. PMID:23482447

  19. BCR Ligation antagonizes the IL-21 enhancement of anti-CD40/IL-4 Plasma cell differentiation and IgE production found in low density human B cell cultures

    PubMed Central

    Caven, Timothy H.; Sturgill, Jamie L.; Conrad, Daniel H.

    2007-01-01

    We sought to discover the mechanisms explaining increased IgE production seen at low cell densities when IL-21 is added to human B cell cultures activated with anti-CD40 and IL-4. When cells were cultured in the absence of BCR ligation, qPCR demonstrated dramatic increases in mRNA for the plasma cell transcription factors BLIMP1 and XBP1. Furthermore, a majority of viable cells expressed high levels of CD38 while losing expression of surface IgD. In contrast, in the presence of BCR stimulation, both the XBP1 mRNA levels and CD38 cell surface expression were markedly reduced, and a large population of cells retained IgD expression, indicating reduced plasma cell differentiation. IgE levels were reduced in the BCR stimulated cultures by 90%, while IgG4 levels remained unchanged. In summary, IL-21 enhances IgE production at low densities through stimulating cell division and plasma cell differentiation and this activity is reduced upon BCR crosslinking. PMID:17888893

  20. BCR ligation antagonizes the IL-21 enhancement of anti-CD40/IL-4 plasma cell differentiation and IgE production found in low density human B cell cultures.

    PubMed

    Caven, Timothy H; Sturgill, Jamie L; Conrad, Daniel H

    2007-05-01

    We sought to discover the mechanisms explaining increased IgE production seen at low cell densities when IL-21 is added to human B cell cultures activated with anti-CD40 and IL-4. When cells were cultured in the absence of BCR ligation, qPCR demonstrated dramatic increases in mRNA for the plasma cell transcription factors BLIMP1 and XBP1. Furthermore, a majority of viable cells expressed high levels of CD38 while losing expression of surface IgD. In contrast, in the presence of BCR stimulation, both the XBP1 mRNA levels and CD38 cell surface expression were markedly reduced, and a large population of cells retained IgD expression, indicating reduced plasma cell differentiation. IgE levels were reduced in the BCR stimulated cultures by 90%, while IgG4 levels remained unchanged. In summary, IL-21 enhances IgE production at low densities through stimulating cell division and plasma cell differentiation and this activity is reduced upon BCR cross-linking.

  1. Enhanced Interleukin-12 and CD40 Ligand Activities but Reduced Staphylococcus aureus Cowan 1-Induced Responses Suggest a Generalized and Progressively Impaired Type 1 Cytokine Pattern for Human Schistosomiasis

    PubMed Central

    Montenegro, Silvia M. L.; Abath, Frederico G. C.; Domingues, Ana Lúcia C.; Melo, Wlademir G.; Morais, Clarice N. L.; Coutinho, Eridan M.; Mahanty, Siddhartha; Wynn, Thomas A.

    2002-01-01

    Whole-blood-cell cultures from schistosomiasis patients were stimulated with a variety of T-cell-dependent and T-cell-independent stimuli to determine whether the defect in type 1 cytokine expression observed following helminth infection is associated with alterations in interleukin-12 (IL-12) or CD40 ligand (CD40L) responsiveness. Cultures from uninfected individuals produced abundant gamma interferon in response to Staphylococcus aureus Cowan 1 (SAC), while patients with intestinal and hepatosplenic disease displayed intermediate and weak responses, respectively. Importantly, the decrease in type 1 cytokine expression was not attributed to defects in IL-12- or CD40L-induced activity. Indeed, schistosomiasis patients displayed heightened responses and even produced more biologically active IL-12 when stimulated with SAC and CD40L than did uninfected controls. Finally, additional studies suggested only a partial role for IL-10, since intestinal patients were the only group that overproduced this downregulatory cytokine. Together, these studies demonstrate that the type 1 deficiency in chronic hepatosplenic schistosomiasis is not related to specific defects in IL-12, IL-10, or CD40L activity, although changes in the functional status of antigen-presenting cells appear to be involved. PMID:12379664

  2. Synergistic effect of thrombin and CD40 ligand on endothelial matrix metalloproteinase-10 expression and microparticle generation in vitro and in vivo.

    PubMed

    Martínez de Lizarrondo, Sara; Roncal, Carmen; Calvayrac, Olivier; Rodríguez, Cristina; Varo, Nerea; Purroy, Ana; Lorente, Leonardo; Rodríguez, José A; Doeuvre, Loïc; Hervás-Stubbs, Sandra; Angles-Cano, Eduardo; Páramo, José A; Martínez-González, José; Orbe, Josune

    2012-06-01

    Thrombin induces CD40 ligand (CD40L) and matrix metalloproteinases (MMPs) under inflammatory/prothrombotic conditions. Thrombin and CD40L could modulate endothelial MMP-10 expression in vitro and in vivo. Human endothelial cells were stimulated with thrombin (0.1-10 U/mL), CD40L (0.25-1 μg/mL), or their combination (thrombin/CD40L) to assess MMP-10 expression and microparticle generation. Thrombin/CD40L elicited higher MMP-10 mRNA (5-fold; P<0.001) and protein levels (4.5-fold; P<0.001) than either stimulus alone. This effect was mimicked by a protease-activated receptor-1 agonist and antagonized by hirudin, a-protease-activated receptor-1, α-CD40L, and α-CD40 antibodies. The synergistic effect was dependent on p38 mitogen-activated protein kinase and c-Jun N-terminal kinase-1 pathways. Thrombin also upregulated the expression of CD40 in endothelial cell surface increasing its availability, thereby favoring its synergistic effects with CD40L. In mice, thrombin/CD40L further increased the aortic MMP-10 expression. Septic patients with systemic inflammation and enhanced thrombin generation (n=60) exhibited increased MMP-10 and soluble CD40L levels associated with adverse clinical outcome. Endothelial and systemic activation by thrombin/CD40L and lipopolysaccharide also increased microparticles harboring MMP-10 and CD40L. Thrombin/CD40L elicited a strong synergistic effect on endothelial MMP-10 expression and microparticles containing MMP-10 in vitro and in vivo, which may represent a new link between inflammation/thrombosis with prognostic implications.

  3. Activation of myeloid and endothelial cells by CD40L gene therapy supports T-cell expansion and migration into the tumor microenvironment.

    PubMed

    Eriksson, E; Moreno, R; Milenova, I; Liljenfeldt, L; Dieterich, L C; Christiansson, L; Karlsson, H; Ullenhag, G; Mangsbo, S M; Dimberg, A; Alemany, R; Loskog, A

    2017-02-01

    CD40 is an interesting target in cancer immunotherapy due to its ability to stimulate T-helper 1 immunity via maturation of dendritic cells and to drive M2 to M1 macrophage differentiation. Pancreatic cancer has a high M2 content that has shown responsive to anti-CD40 agonist therapy and CD40 may thus be a suitable target for immune activation in these patients. In this study, a novel oncolytic adenovirus armed with a trimerized membrane-bound extracellular CD40L (TMZ-CD40L) was evaluated as a treatment of pancreatic cancer. Further, the CD40L mechanisms of action were elucidated in cancer models. The results demonstrated that the virus transferring TMZ-CD40L had oncolytic capacity in pancreatic cancer cells and could control tumor progression. TMZ-CD40L was a potent stimulator of human myeloid cells and T-cell responses. Further, CD40L-mediated stimulation increased tumor-infiltrating T cells in vivo, which may be due to a direct activation of endothelial cells to upregulate receptors for lymphocyte attachment and transmigration. In conclusion, CD40L-mediated gene therapy is an interesting concept for the treatment of tumors with high levels of M2 macrophages, such as pancreatic cancer, and an oncolytic virus as carrier of CD40L may further boost tumor killing and immune activation.

  4. Interruption of classic CD40L-CD40 signalling but not of the novel CD40L-Mac-1 interaction limits arterial neointima formation in mice.

    PubMed

    Willecke, F; Tiwari, S; Rupprecht, B; Wolf, D; Hergeth, S; Hoppe, N; Dufner, B; Schulte, L; Anto Michel, N; Bukosza, N; Marchini, T; Jäckel, M; Stachon, P; Hilgendorf, I; Zeschky, K; Schleicher, R; Langer, H F; von Zur Muhlen, C; Bode, C; Peter, K; Zirlik, A

    2014-08-01

    The co-stimulatory immune molecule CD40L figures prominently in a variety of inflammatory conditions including arterial disease. Recently, we made the surprising finding that CD40L mediates atherogenesis independently of its classic receptor CD40 via a novel interaction with the leukocyte integrin Mac-1. Here, we hypothesised that selective blockade of the CD40L-Mac-1 interaction may also retard restenosis. We induced neointima formation in C57/BL6 mice by ligation of the left carotid artery. Mice were randomised to daily intraperitoneal injections of either cM7, a small peptide selectively inhibiting the CD40L-Mac-1 interaction, scM7, a scrambled control peptide, or saline for 28 days. Interestingly, cM7-treated mice developed neointima of similar size compared with mice receiving the control peptide or saline as assessed by computer-assisted analysis of histological cross sections. These data demonstrate that the CD40L-Mac-1 interaction is not required for the development of restenosis. In contrast, CD40-deficient mice subjected to carotid ligation in parallel, developed significantly reduced neointimal lesions compared with respective wild-type controls (2872 ± 843 µm² vs 35469 ± 11870 µm²). Flow cytometry in CD40-deficient mice revealed reduced formation of platelet-granulocyte and platelet-inflammatory monocyte- aggregates. In vitro, supernatants of CD40-deficient platelet-leukocyte aggregates attenuated proliferation and increased apoptosis of smooth muscle cells. Unlike in the setting of atherosclerosis, CD40L mediates neointima formation via its classic receptor CD40 rather than via its recently described novel interaction with Mac-1. Therefore, selective targeting of CD40L-Mac-1 binding does not appear to be a favorable strategy to fight restenosis.

  5. Tumor-induced senescent T cells promote the secretion of pro-inflammatory cytokines and angiogenic factors by human monocytes/macrophages through a mechanism that involves Tim-3 and CD40L

    PubMed Central

    Ramello, M C; Tosello Boari, J; Canale, F P; Mena, H A; Negrotto, S; Gastman, B; Gruppi, A; Acosta Rodríguez, E V; Montes, C L

    2014-01-01

    Solid tumors are infiltrated by immune cells where macrophages and senescent T cells are highly represented. Within the tumor microenvironment, a cross-talk between the infiltrating cells may occur conditioning the characteristic of the in situ immune response. Our previous work showed that tumors induce senescence of T cells, which are powerful suppressors of lympho-proliferation. In this study, we report that Tumor-Induced Senescent (TIS)-T cells may also modulate monocyte activation. To gain insight into this interaction, CD4+ or CD8+TIS-T or control-T cells were co-incubated with autologous monocytes under inflammatory conditions. After co-culture with CD4+ or CD8+TIS-T cells, CD14+ monocytes/macrophages (Mo/Ma) exhibit a higher expression of CD16+ cells and a reduced expression of CD206. These Mo/Ma produce nitric oxide and reactive oxygen species; however, TIS-T cells do not modify phagocyte capacity of Mo/Ma. TIS-T modulated-Mo/Ma show a higher production of pro-inflammatory cytokines (TNF, IL-1β and IL-6) and angiogenic factors (MMP-9, VEGF-A and IL-8) and a lower IL-10 and IP-10 secretion than monocytes co-cultured with controls. The mediator(s) present in the supernatant of TIS-T cell/monocyte-macrophage co-cultures promote(s) tubulogenesis and tumor-cell survival. Monocyte-modulation induced by TIS-T cells requires cell-to-cell contact. Although CD4+ shows different behavior from CD8+TIS-T cells, blocking mAbs against T-cell immunoglobulin and mucin protein 3 and CD40 ligand reduce pro-inflammatory cytokines and angiogenic factors production, indicating that these molecules are involved in monocyte/macrophage modulation by TIS-T cells. Our results revealed a novel role for TIS-T cells in human monocyte/macrophage modulation, which may have deleterious consequences for tumor progression. This modulation should be considered to best tailor the immunotherapy against cancer. PMID:25375372

  6. Giving blood: a new role for CD40 in tumorigenesis.

    PubMed

    Bergmann, Stephan; Pandolfi, Pier Paolo

    2006-10-30

    CD40 was initially identified as a receptor expressed by B cells that is crucial for inducing an effective adaptive immune response. CD40 was subsequently shown to be expressed by endothelial cells and to promote angiogenesis. New data now show that in tumor-prone transgenic mice, CD40-mediated neovascularization is essential for early stage tumorigenicity. This suggests, at least in this mouse model, that CD40 has an important role in the angiogenic process that is coupled to carcinogenesis, a finding that could lead to novel therapeutic opportunities.

  7. Adenovirus co-expressing CD40 ligand and interleukin (IL)-2 contributes to maturation of dendritic cells and production of IL-12

    PubMed Central

    Guo, Zhi; Gao, Hong-Yan; Zhang, Tian-Yang; Lou, Jin-Xing; Yang, Kai; Liu, Xiao-Dong; He, Xue-Peng; Chen, Hui-Ren

    2016-01-01

    The aim of the present study was to construct a chimeric adenovirus (Ad)5/F35 co-expressing human CD4O ligand (CD4OL) and interleukin (IL)-2 (Ad5/F35 CD40L-IL-2). The infection efficiency to human monocyte-derived dendritic cells (Mo-DCs), expression of genes, phenotype changes and IL-12 production of Mo-DC by Ad5/F35 CD40L-IL-2 were investigated. CD40L and IL-2 from total RNA extracted from human peripheral blood mononuclear cells (PBMCs) were cloned by reverse transcription-polymerase chain reaction and used to construct Ad5/F35 CD40L-IL-2. The infection efficiency, expression of CD40L, and phenotype changes of Mo-DC infected with Ad5/F35 CD40L-IL-2 were analyzed using flow cytometry. The quantities of IL-2 and IL-12 in the supernatants of Mo-DC following infection of Ad5/F35 CD40L-IL-2 were measured by enzyme-linked immunosorbent assay. The CD40L and IL-2 genes were successfully cloned and the Ad5/F35 CD40L-IL-2 was constructed. Ad5/F35 CD40L-IL-2 efficiently infected Mo-DCs with an infection efficiency of >75%, and the infected Mo-DCs expressed CD40L and secreted IL-2. The expression levels of cluster of differentiation (CD)80, CD86, CD40, and human leukocyte antigen-antigen D related on Mo-DC were moderate; however, CD83 was low prior to infection of Ad5/F35 CD40L-IL-2. Those molecules, particularly CD83, were markedly upregulated 24 h after the infection. Increasing quantities of IL-12 in the supernatants were detected subsequent to infection at different time points in a time-dependent manner. Thus, Ad5/F35 CD40L-IL-2 efficiently infected human Mo-DCs and its products, CD40L and IL-2, were subsequently expressed. In addition, infection with Ad5/F35 CD40L-IL-2 stimulated the maturation of Mo-DC and high levels of IL-12 production. PMID:27882218

  8. CD40 promotes MHC class II expression on adipose tissue macrophages and regulates adipose tissue CD4+ T cells with obesity.

    PubMed

    Morris, David L; Oatmen, Kelsie E; Mergian, Taleen A; Cho, Kae Won; DelProposto, Jennifer L; Singer, Kanakadurga; Evans-Molina, Carmella; O'Rourke, Robert W; Lumeng, Carey N

    2016-06-01

    Obesity activates both innate and adaptive immune responses in adipose tissue, but the mechanisms critical for regulating these responses remain unknown. CD40/CD40L signaling provides bidirectional costimulatory signals between antigen-presenting cells and CD4(+) T cells, and CD40L expression is increased in obese humans. Therefore, we examined the contribution of CD40 to the progression of obesity-induced inflammation in mice. CD40 was highly expressed on adipose tissue macrophages in mice, and CD40/CD40L signaling promoted the expression of antigen-presenting cell markers in adipose tissue macrophages. When fed a high fat diet, Cd40-deficient mice had reduced accumulation of conventional CD4(+) T cells (Tconv: CD3(+)CD4(+)Foxp3(-)) in visceral fat compared with wild-type mice. By contrast, the number of regulatory CD4(+) T cells (Treg: CD3(+)CD4(+)Foxp3(+)) in lean and obese fat was similar between wild-type and knockout mice. Adipose tissue macrophage content and inflammatory gene expression in fat did not differ between obese wild-type and knockout mice; however, major histocompatibility complex class II and CD86 expression on adipose tissue macrophages was reduced in visceral fat from knockout mice. Similar results were observed in chimeric mice with hematopoietic Cd40-deficiency. Nonetheless, neither whole body nor hematopoietic disruption of CD40 ameliorated obesity-induced insulin resistance in mice. In human adipose tissue, CD40 expression was positively correlated with CD80 and CD86 expression in obese patients with type 2 diabetes. These findings indicate that CD40 signaling in adipose tissue macrophages regulates major histocompatibility complex class II and CD86 expression to control the expansion of CD4(+) T cells; however, this is largely dispensable for the development of obesity-induced inflammation and insulin resistance in mice. © Society for Leukocyte Biology.

  9. The MS Risk Allele of CD40 Is Associated with Reduced Cell-Membrane Bound Expression in Antigen Presenting Cells: Implications for Gene Function.

    PubMed

    Field, Judith; Shahijanian, Fernando; Schibeci, Stephen; Johnson, Laura; Gresle, Melissa; Laverick, Louise; Parnell, Grant; Stewart, Graeme; McKay, Fiona; Kilpatrick, Trevor; Butzkueven, Helmut; Booth, David

    2015-01-01

    Human genetic and animal studies have implicated the costimulatory molecule CD40 in the development of multiple sclerosis (MS). We investigated the cell specific gene and protein expression variation controlled by the CD40 genetic variant(s) associated with MS, i.e. the T-allele at rs1883832. Previously we had shown that the risk allele is expressed at a lower level in whole blood, especially in people with MS. Here, we have defined the immune cell subsets responsible for genotype and disease effects on CD40 expression at the mRNA and protein level. In cell subsets in which CD40 is most highly expressed, B lymphocytes and dendritic cells, the MS-associated risk variant is associated with reduced CD40 cell-surface protein expression. In monocytes and dendritic cells, the risk allele additionally reduces the ratio of expression of full-length versus truncated CD40 mRNA, the latter encoding secreted CD40. We additionally show that MS patients, regardless of genotype, express significantly lower levels of CD40 cell-surface protein compared to unaffected controls in B lymphocytes. Thus, both genotype-dependent and independent down-regulation of cell-surface CD40 is a feature of MS. Lower expression of a co-stimulator of T cell activation, CD40, is therefore associated with increased MS risk despite the same CD40 variant being associated with reduced risk of other inflammatory autoimmune diseases. Our results highlight the complexity and likely individuality of autoimmune pathogenesis, and could be consistent with antiviral and/or immunoregulatory functions of CD40 playing an important role in protection from MS.

  10. The MS Risk Allele of CD40 Is Associated with Reduced Cell-Membrane Bound Expression in Antigen Presenting Cells: Implications for Gene Function

    PubMed Central

    Field, Judith; Shahijanian, Fernando; Schibeci, Stephen; Johnson, Laura; Gresle, Melissa; Laverick, Louise; Parnell, Grant; Stewart, Graeme; McKay, Fiona; Kilpatrick, Trevor; Butzkueven, Helmut; Booth, David

    2015-01-01

    Human genetic and animal studies have implicated the costimulatory molecule CD40 in the development of multiple sclerosis (MS). We investigated the cell specific gene and protein expression variation controlled by the CD40 genetic variant(s) associated with MS, i.e. the T-allele at rs1883832. Previously we had shown that the risk allele is expressed at a lower level in whole blood, especially in people with MS. Here, we have defined the immune cell subsets responsible for genotype and disease effects on CD40 expression at the mRNA and protein level. In cell subsets in which CD40 is most highly expressed, B lymphocytes and dendritic cells, the MS-associated risk variant is associated with reduced CD40 cell-surface protein expression. In monocytes and dendritic cells, the risk allele additionally reduces the ratio of expression of full-length versus truncated CD40 mRNA, the latter encoding secreted CD40. We additionally show that MS patients, regardless of genotype, express significantly lower levels of CD40 cell-surface protein compared to unaffected controls in B lymphocytes. Thus, both genotype-dependent and independent down-regulation of cell-surface CD40 is a feature of MS. Lower expression of a co-stimulator of T cell activation, CD40, is therefore associated with increased MS risk despite the same CD40 variant being associated with reduced risk of other inflammatory autoimmune diseases. Our results highlight the complexity and likely individuality of autoimmune pathogenesis, and could be consistent with antiviral and/or immunoregulatory functions of CD40 playing an important role in protection from MS. PMID:26068105

  11. Clinical disease upregulates expression of CD40 and CD40 ligand on peripheral blood mononuclear cells from cattle naturally infected with Mycobacterium avium subsp. paratuberculosis

    USDA-ARS?s Scientific Manuscript database

    CD40 and CD40L interactions have costimulatory effects that are part of a complex series of events in host cellular and humoral immune responses and inflammation. The purpose of this study was to examine the changes in expression of CD40 and CD40L on peripheral blood mononuclear cells (PBMCs) isolat...

  12. Differential peptide binding to CD40 evokes counteractive responses.

    PubMed

    Khan, Srijit; Alonso-Sarduy, Livan; Alonso, Livan; Roduit, Charles; Bandyopadhyay, Syamdas; Singh, Shailza; Saha, Shipra; Tacchini-Cottier, Fabienne; Roy, Somenath; Dietler, Giovanni; Kasas, Sandor; Das, Pradeep; Krishnasastry, M V; Saha, Bhaskar

    2012-05-01

    The antigen-presenting cell–expressed CD40 is implied in the regulation of counteractive immune responses such as induction of pro-inflammatory and anti-inflammatory cytokines interleukin (IL)–12 and IL-10, respectively. The mechanism of this duality in CD40 function remains unknown. Here, we investigated whether such duality depends on ligand binding. Based on CD40 binding, we identifed two dodecameric peptides, peptide-7 and peptide-19, from the phage peptide library. Peptide-7 induces IL-10 and increases Leishmania donovani infection in macrophages, whereas peptide-19 induces IL-12 and reduces L. donovani infection. CD40-peptide interaction analyses by surface plasmon resonance and atomic force microscopy suggest that the functional differences are not associated with the studied interaction parameters. The molecular dynamic simulation of the CD40-peptides interaction suggests that these two peptides bind to two different places on CD40. Thus, we suggest for the first time that differential binding of the ligands imparts functional duality to CD40.

  13. Platelet CD40L at the interface of adaptive immunity

    PubMed Central

    Elzey, Bennett D.; Ratliff, Timothy L.; Sowa, Jennifer M.; Crist, Scott A.

    2010-01-01

    Initiated by the finding that platelets express functional CD40 ligand (CD40L, CD154), many new roles for platelets have been discovered in unanticipated areas, including the immune response. When current literature is considered as a whole, the picture that is emerging begins to show that platelets are able to significantly affect, for better or worse, the overall health and condition of the mammalian host. Animal models have made significant contributions to our expanding knowledge of platelet function, much of which is anticipated to be clinically relevant. While still mostly circumstantial, the evidence supports a critical role for CD40L in many normal and disease processes. PMID:21075431

  14. HIV-1 induction of CD40 on endothelial cells promotes the outgrowth of AIDS-associated B-cell lymphomas.

    PubMed

    Moses, A V; Williams, S E; Strussenberg, J G; Heneveld, M L; Ruhl, R A; Bakke, A C; Bagby, G C; Nelson, J A

    1997-11-01

    Human immunodeficiency virus (HIV)-1 infection is associated with the development of aggressive extranodal B-cell non-Hodgkin's lymphomas. Using microvascular endothelial cell (MVEC)-enriched bone marrow stromal cultures, HIV infection of stromal MVECs from lymphoma patients induced the outgrowth of malignant B cells. MVECs were the only HIV-infected cells in the stroma, and purified brain MVECs also induced a phenotype supportive of neoplastic B-cell attachment and proliferation. HIV infection of MVECs stimulated surface expression of CD40 and allowed preferential induction of the vascular cell adhesion molecule VCAM-1 after CD40 triggering. B-lymphoma cells expressed the CD40 ligand (CD40L), and blocking of CD40-CD40L interactions between HIV-infected MVECs and B-lymphoma cells inhibited B-cell attachment and proliferation. These observations suggest that HIV promotes B-lymphoma cell growth through facilitating attachment of lymphoma cells to HIV-infected MVECs and represent a novel mechanism through which viruses may induce malignancies.

  15. CD40 ligand immunotherapy in cancer: an efficient approach.

    PubMed

    Kuwashima, N; Kageyama, S; Eto, Y; Urashima, M

    2001-01-01

    Cancer cells do not elicit a clinically sufficient anti-tumor immune response that results in tumor rejection. Recently, many investigators have been trying to enhance anti-tumor immunity and encouraging results have been reported. This review will discuss current anti-cancer immunotherapy; interleukin-2 therapy, tumor vaccine secreting Granulocyte macrophage-colony stimulating factor, dendritic cells fused with tumor cells, and CD40 ligand immunotherapy. Moreover, we introduce our two kinds of CD40 ligand immuno-genetherapy; (1) oral CD40 ligand gene therapy against lymphoma using attenuated Salmonella typhimurium (published in BLOOD 2000), (2) cancer vaccine transfected with CD40 ligand ex vivo for neuroblastoma (unpublished). Both approaches resulted in a high degree of protection against the tumor progression and they are simple and safe in the murine system.

  16. Increased CD40+ fibrocytes in patients with idiopathic orbital inflammation

    PubMed Central

    Lee, Brian J; Atkins, Stephen; Ginter, Anna; Elner, Victor M; Nelson, Christine C; Douglas, Raymond S

    2014-01-01

    Objective To investigate the phenotypic and functional characteristics of peripheral and tissue-infiltrating stem cells, called fibrocytes in patients with idiopathic orbital inflammation (IOI). Methods Seven patients with IOI were studied. In the three patients requiring orbital biopsy, fibrocytes were identified in orbital tissue from patients with IOI compared to healthy controls using immunohistochemistry. Fibrocytes from the peripheral blood of all seven patients and controls were quantified and phenotyped by flow cytometry and immunofluorescence for expression of CD34, alpha smooth muscle actin, CD40 and Collagen 1. Quantitation of CD40-mediated IL-6 production was measured using ELISA. Results Orbital biopsy specimens from patients with IOI demonstrate tissue infiltration by fibrocytes (n=3). Fibrocytes are present in the peripheral blood of IOI patients (n= 7) but are scarce in healthy donors (n=19). Fibrocytes from IOI patients express substantial levels of CD40 and ligation of CD40 increases IL-6 expression. Conclusions Fibrocytes are present in the peripheral blood and orbital tissues of patients with IOI and constitutively express CD40 and express IL-6 in response to ligation. This site-specific predilection of CD34+ fibrocytes to sites of orbital inflammation and fibrosis may suggest a role in IOI. Moreover CD40-mediated activation cytokine production may contribute to the proinflammatory and profibrotic features of IOI and may provide a mechanism for future targeted therapy. PMID:25098443

  17. Spontaneous Activation of Antigen-presenting Cells by Genes Encoding Truncated Homo-Oligomerizing Derivatives of CD40.

    PubMed

    Levin, Noam; Pato, Aviad; Cafri, Gal; Eisenberg, Galit; Peretz, Tamar; Margalit, Alon; Lotem, Michal; Gross, Gideon

    The interaction between the CD40 receptor on antigen-presenting cells (APCs) and its trimeric ligand on CD4 T cells is essential for the initiation and progression of the adaptive immune response. Here we undertook to endow CD40 with the capacity to trigger spontaneous APC activation through ligand-independent oligomerization. To this end we exploited the GCN4 yeast transcriptional activator, which contains a leucine zipper DNA-binding motif that induces homophilic interactions. We incorporated GCN4 variants forming homodimers, trimers, or tetramers at the intracellular domain of human and mouse CD40 and replaced the extracellular portion with peptide-β2m or other peptide tags. In parallel we examined similarly truncated CD40 monomers lacking a GCN4 motif. The oligomeric products appeared to arrange in high-molecular-weight aggregates and were considerably superior to the monomer in their ability to trigger nuclear factor kB signaling, substantiating the anticipated constitutively active (ca) phenotype. Cumulative results in human and mouse APC lines transfected with caCD40 mRNA revealed spontaneous upregulation of CD80, IL-1β, TNFα, IL-6, and IL-12, which could be further enhanced by caTLR4 mRNA. In mouse bone-marrow-derived dendritic cells caCD40 upregulated CD80, CD86, MHC-II, and IL-12 and in human monocyte-derived dendritic cells it elevated surface CD80, CD83 CD86, CCR7, and HLA-DR. Oligomeric products carrying the peptide-β2m extracellular portion could support MHC-I presentation of the linked peptide up to 4 days post-mRNA transfection. These findings demonstrate that the expression of a single caCD40 derivative in APCs can exert multiple immunostimulatory effects, offering a new powerful tool in the design of gene-based cancer vaccines.

  18. Dendritic cells induce Tc1 cell differentiation via the CD40/CD40L pathway in mice after exposure to cigarette smoke.

    PubMed

    Kuang, Liang-Jian; Deng, Ting-Ting; Wang, Qin; Qiu, Shi-Lin; Liang, Yi; He, Zhi-Yi; Zhang, Jian-Quan; Bai, Jing; Li, Mei-Hua; Deng, Jing-Min; Liu, Guang-Nan; Liu, Ji-Feng; Zhong, Xiao-Ning

    2016-09-01

    Dendritic cells and CD8(+) T cells participate in the pathology of chronic obstructive pulmonary disease, including emphysema, but little is known of the involvement of the CD40/CD40L pathway. We investigated the role of the CD40/CD40L pathway in Tc1 cell differentiation induced by dendritic cells in a mouse model of emphysema, and in vitro. C57BL/6J wild-type and CD40(-/-) mice were exposed to cigarette smoke (CS) or not (control), for 24 wk. In vitro experiments involved wild-type and CD40(-/-) dendritic cells treated with CS extract (CSE) or not. Compared with the control groups, the CS mice (both wild type and CD40(-/-)) had a greater percentage of lung dendritic cells and higher levels of major histocompatability complex (MHC) class I molecules and costimulatory molecules CD40 and CD80. Relative to the CS CD40(-/-) mice, the CS wild type showed greater signs of lung damage and Tc1 cell differentiation. In vitro, the CSE-treated wild-type cells evidenced more cytokine release (IL-12/p70) and Tc1 cell differentiation than did the CSE-treated CD40(-/-) cells. Exposure to cigarette smoke increases the percentage of lung dendritic cells and promotes Tc1 cell differentiation via the CD40/CD40L pathway. Blocking the CD40/CD40L pathway may suppress development of emphysema in mice exposed to cigarette smoke. Copyright © 2016 the American Physiological Society.

  19. CD40L is not involved in acute experimental pancreatitis.

    PubMed

    Abdulla, Aree; Awla, Darbaz; Jeppsson, Bengt; Regnér, Sara; Thorlacius, Henrik

    2011-05-20

    Recent data suggest that platelets not only control thrombosis and hemostasis but may also regulate inflammatory processes such as acute pancreatitis. However, the specific role of platelet-derived mediators in the pathophysiology of acute pancreatitis is not known. Herein, we examined the role of CD40 ligand (CD40L, CD154) in different models of acute pancreatitis. Acute pancreatitis was induced by repetitive caerulein administration (50μg/kg, i.p.) or infusion of sodium taurocholate (5%-10μl) into the pancreatic duct in wild-type C57BL/6 and CD40L-deficient mice. Neutrophil infiltration, myeloperoxidase (MPO), macrophage inflammatory protein-2 (MIP-2) levels, acinar cell necrosis, edema and hemorrhage in the pancreas as well as serum amylase activity and lung levels of MPO were quantified 24h after induction of acute pancreatitis. Caerulein and taurocholate challenge caused a clear-cut pancreatic damage characterized by increased acinar cell necrosis, neutrophil infiltration, focal hemorrhage, edema formation as well as increased levels of serum amylase and MIP-2 in the pancreas and lung MPO and histological damage. Notably, CD40L gene-deficient animals exhibited a similar phenotype as wild-type mice after challenge with caerulein and taurocholate. Similarly, administration of an antibody directed against CD40L had no effect against acute pancreatitis. Our data suggest that CD40L does not play a functional role in experimental acute pancreatitis. Thus, other candidates than CD40L needs to be explored in order to identify platelet-derived mediators in the pathophysiology of acute pancreatitis.

  20. CD40-directed scFv-TRAIL fusion proteins induce CD40-restricted tumor cell death and activate dendritic cells

    PubMed Central

    El-Mesery, M; Trebing, J; Schäfer, V; Weisenberger, D; Siegmund, D; Wajant, H

    2013-01-01

    Targeted cancer therapy concepts often aim at the induction of adjuvant antitumor immunity or stimulation of tumor cell apoptosis. There is further evidence that combined application of immune stimulating and tumor apoptosis-inducing compounds elicits a synergistic antitumor effect. Here, we describe the development and characterization of bifunctional fusion proteins consisting of a single-chain variable fragment (scFv) domain derived from the CD40-specific monoclonal antibody G28-5 that is fused to the N-terminus of stabilized trimeric soluble variants of the death ligand TNF-related apoptosis-inducing ligand (TRAIL). As shown before by us and others for other cell surface antigen-targeted scFv-TRAIL fusion proteins, scFv:G28-TRAIL displayed an enhanced capacity to induce apoptosis upon CD40 binding. Studies with scFv:G28 fusion proteins of TRAIL mutants that discriminate between the two TRAIL death receptors, TRAILR1 and TRAILR2, further revealed that the CD40 binding-dependent mode of apoptosis induction of scFv:G28-TRAIL is operable with each of the two TRAIL death receptors. Binding of scFv:G28-TRAIL fusion proteins to CD40 not only result in enhanced TRAIL death receptor signaling but also in activation of the targeted CD40 molecule. In accordance with the latter, the scFv:G28-TRAIL fusion proteins triggered strong CD40-mediated maturation of dendritic cells. The CD40-targeted TRAIL fusion proteins described in this study therefore represent a novel type of bifunctional fusion proteins that couple stimulation of antigen presenting cells and apoptosis induction. PMID:24232092

  1. Characterization of a Broadly Reactive Anti-CD40 Agonistic Monoclonal Antibody for Potential Use as an Adjuvant

    PubMed Central

    Waghela, Suryakant D.; Lokhandwala, Shehnaz; Ambrus, Andy; Bray, Jocelyn; Vuong, Christina; Vinodkumar, Vanitha; Dominowski, Paul J.; Rai, Sharath; Mwangi, Duncan; Foss, Dennis L.; Mwangi, Waithaka

    2017-01-01

    Lack of safe and effective adjuvants is a major hindrance to the development of efficacious vaccines. Signaling via CD40 pathway leads to enhanced antigen processing and presentation, nitric oxide expression, pro-inflammatory cytokine expression by antigen presenting cells, and stimulation of B-cells to undergo somatic hypermutation, immunoglobulin class switching, and proliferation. Agonistic anti-CD40 antibodies have shown promising adjuvant qualities in human and mouse vaccine studies. An anti-CD40 monoclonal antibody (mAb), designated 2E4E4, was identified and shown to have strong agonistic effects on primary cells from multiple livestock species. The mAb recognize swine, bovine, caprine, and ovine CD40, and evoked 25-fold or greater proliferation of peripheral blood mononuclear cells (PBMCs) from these species relative to cells incubated with an isotype control (p<0.001). In addition, the mAb induced significant nitric oxide (p<0.0001) release by bovine macrophages. Furthermore, the mAb upregulated the expression of MHC-II by PBMCs, and stimulated significant (p<0.0001) IL-1α, IL6, IL-8, and TNF-α expression by PBMCs. These results suggest that the mAb 2E4E4 can target and stimulate cells from multiple livestock species and thus, it is a potential candidate for adjuvant development. This is the first study to report an anti-swine CD40 agonistic mAb that is also broadly reactive against multiple species. PMID:28107431

  2. Dendritic cell based PSMA immunotherapy for prostate cancer using a CD40-targeted adenovirus vector.

    PubMed

    Williams, Briana Jill; Bhatia, Shilpa; Adams, Lisa K; Boling, Susan; Carroll, Jennifer L; Li, Xiao-Lin; Rogers, Donna L; Korokhov, Nikolay; Kovesdi, Imre; Pereboev, Alexander V; Curiel, David T; Mathis, J Michael

    2012-01-01

    Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells). To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ). Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.

  3. Dendritic Cell Based PSMA Immunotherapy for Prostate Cancer Using a CD40-Targeted Adenovirus Vector

    PubMed Central

    Williams, Briana Jill; Bhatia, Shilpa; Adams, Lisa K.; Boling, Susan; Carroll, Jennifer L.; Li, Xiao-Lin; Rogers, Donna L.; Korokhov, Nikolay; Kovesdi, Imre; Pereboev, Alexander V.; Curiel, David T.; Mathis, J. Michael

    2012-01-01

    Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells). To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ). Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy. PMID:23056548

  4. CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants.

    PubMed

    von Bergwelt-Baildon, Michael; Shimabukuro-Vornhagen, Alexander; Popov, Alexey; Klein-Gonzalez, Nela; Fiore, Francesca; Debey, Svenja; Draube, Andreas; Maecker, Britta; Menezes, Isaura; Nadler, Lee M; Schultze, Joachim L

    2006-04-01

    CD40-activated B cells (CD40-B cells) have previously been introduced as an alternative source of antigen-presenting cells for immunotherapy. CD40-B cells can prime naive and expand memory T cells, and they can be generated in large numbers from very small amounts of peripheral blood derived from healthy individuals or cancer patients alike. Administration of CD40-B cells as a cellular adjuvant would require these cells to migrate toward secondary lymphoid organs and attract T cells in situ, processes guided by specific chemokines and chemokine receptors. Here, we demonstrate that primary, human CD40-B cells express a pattern of adhesion molecules and chemokine receptors necessary for homing to secondary lymphoid organs and have the capacity to migrate to cognate ligands. Furthermore, we show that CD40-B cells express important T-cell attractants and induce strong T-cell chemotaxis. These findings further support the use of CD40-B cells as cellular adjuvant for cancer immunotherapy.

  5. RelB nuclear translocation regulates B cell MHC molecule, CD40 expression, and antigen-presenting cell function

    PubMed Central

    O'Sullivan, Brendan J.; MacDonald, Kelli P. A.; Pettit, Allison R.; Thomas, Ranjeny

    2000-01-01

    Mice with targeted RelB mutations demonstrated an essential role for RelB in immune responses and in myeloid dendritic cell differentiation. Human studies suggested a more global transcriptional role in antigen presentation. Burkitt lymphoma cell lines were used as a model to examine the role of RelB in antigen presentation. After transient transfection of BJAB with RelB, strong nuclear expression of RelB-p50 heterodimers was associated with increased APC function and expression of CD40 and MHC class I. Antisense RelB in DG75 reduced antigen-presenting capacity and CD40-mediated up-regulation of MHC molecules. The data indicate that RelB transcriptional activity directly affects antigen presentation and CD40 synthesis. Stimulation of RelB transcriptional activity may provide a positive feedback loop for facilitating productive APC/T cell interactions. PMID:11027342

  6. CD40/CD40L expression correlates with the survival of patients with glioblastomas and an augmentation in CD40 signaling enhances the efficacy of vaccinations against glioma models.

    PubMed

    Chonan, Masashi; Saito, Ryuta; Shoji, Takuhiro; Shibahara, Ichiyo; Kanamori, Masayuki; Sonoda, Yukihiko; Watanabe, Mika; Kikuchi, Toshiaki; Ishii, Naoto; Tominaga, Teiji

    2015-11-01

    The prognosis of glioblastoma (GBM) remains poor; therefore, effective therapeutic strategies need to be developed. CD40 is a costimulatory molecule whose agonistic antibody has been shown to activate antitumor effects. Recently, CD40 has been extensively targeted for immunotherapeutic purposes. Expressions of CD40/CD40L mRNAs were examined in 86 cases of World Health Organization grade IV GBM and 36 cases of grade III gliomas and correlated with outcomes. CD40 signaling was employed to augment the efficacy of immunotherapy against gliomas. The efficacy of FGK45, an agonistic antibody for CD40, was examined by adding it to a tumor lysate-based subcutaneous vaccination against a GL261 glioma model and an NSCL61 glioma-initiating cell-like cell tumor model. We demonstrated for the first time using quantitative PCR that grade III gliomas express higher levels of CD40/CD40L than does grade IV GBM. The higher expression of CD40/CD40L was associated with good prognoses in patients with GBM. Addition of FGK45 to the subcutaneous tumor cell lysate-based vaccination significantly prolonged survival in both tumor models. However, the efficacy was modest in NSCL61-model mice. Therefore, we established combination immunotherapeutic strategies using FGK45 and OX86, an agonistic antibody for OX40. Combination immunotherapy significantly prolonged survival with synergistic effects. Apoptosis increased and proliferation decreased in tumors treated with combination immunotherapy. The high expression of CD40/CD40L can be used as a biomarker for better prognoses in patients with gliomas. Immunotherapy using FGK45 significantly prolonged survival and represents a potential therapeutic strategy for gliomas including glioma-initiating cells. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. CD40CD40 Ligand Pathway Is a Major Component of Acute Neuroinflammation and Contributes to Long-term Cognitive Dysfunction after Sepsis

    PubMed Central

    Michels, Monique; Danieslki, Lucinéia Gainski; Vieira, Andriele; Florentino, Drielly; Dall’Igna, Dhébora; Galant, Letícia; Sonai, Beatriz; Vuolo, Francieli; Mina, Franciele; Pescador, Bruna; Dominguini, Diogo; Barichello, Tatiana; Quevedo, João; Dal-Pizzol, Felipe; Petronilho, Fabrícia

    2015-01-01

    Sepsis-associated encephalopathy (SAE) is associated with an increased rate of morbidity and mortality. It is not understood what the exact mechanism is for the brain dysfunction that occurs in septic patients, but brain inflammation and oxidative stress are a possible theory. Such events can occur through the alteration of molecules that perpetuate the inflammatory response. Thus, it is possible to postulate that CD40 may be involved in this process. The aim of this work is to evaluate the role of CD40CD40L pathway activation in brain dysfunction associated with sepsis in an animal model. Microglia activation induces the upregulation of CD40CD40L, both in vitro and in vivo. The inhibition of microglia activation decreases levels of CD40CD40L in the brain and decreases brain inflammation, oxidative damage and blood brain barrier dysfunction. Despite this, anti-CD40 treatment does not improve mortality in this model. However, it is able to improve long-term cognitive impairment in sepsis survivors. In conclusion, there is a major involvement of the CD40CD40L signaling pathway in long-term brain dysfunction in an animal model of sepsis. PMID:25822797

  8. CD40 activation: lessons for HIV immunotherapy from malignancies?

    PubMed

    Le Dieu, Rifca; Gribben, John

    2005-09-01

    In HIV, the immune defects seen are due not only to a decrease in T-cell numbers, but also to qualitative impairment in T-cell function as well as decreased antigen-presenting cell (APC) function. These defects in cell-mediated immunity lead to increased level of infection, contributing to inability to clear the HIV virus, and an increased incidence of tumours. One of the major defects in HIV appears to be the failure of CD4 T cells to provide CD 154 (CD40 ligand)-mediated help, which is required for APC function. In lymphomas, activation through CD40 leads to increased APC activity and induction of immune responses against tumours. Such an effect may also be useful in HIV to increase response against the virus and improve immune surveillance of tumours.

  9. Platelet CD40L mediates thrombotic and inflammatory processes in atherosclerosis

    PubMed Central

    Lievens, Dirk; Zernecke, Alma; Seijkens, Tom; Soehnlein, Oliver; Beckers, Linda; Munnix, Imke C. A.; Wijnands, Erwin; Goossens, Pieter; van Kruchten, Roger; Thevissen, Larissa; Boon, Louis; Flavell, Richard A.; Noelle, Randolph J.; Gerdes, Norbert; Biessen, Erik A.; Daemen, Mat J. A. P.; Heemskerk, Johan W. M.; Weber, Christian

    2010-01-01

    CD40 ligand (CD40L), identified as a costimulatory molecule expressed on T cells, is also expressed and functional on platelets. We investigated the thrombotic and inflammatory contributions of platelet CD40L in atherosclerosis. Although CD40L-deficient (Cd40l−/−) platelets exhibited impaired platelet aggregation and thrombus stability, the effects of platelet CD40L on inflammatory processes in atherosclerosis were more remarkable. Repeated injections of activated Cd40l−/− platelets into Apoe−/− mice strongly decreased both platelet and leukocyte adhesion to the endothelium and decreased plasma CCL2 levels compared with wild-type platelets. Moreover, Cd40l−/− platelets failed to form proinflammatory platelet-leukocyte aggregates. Expression of CD40L on platelets was required for platelet-induced atherosclerosis as injection of Cd40l−/− platelets in contrast to Cd40l+/+ platelets did not promote lesion formation. Remarkably, injection of Cd40l+/+, but not Cd40l−/−, platelets transiently decreased the amount of regulatory T cells (Tregs) in blood and spleen. Depletion of Tregs in mice injected with activated Cd40l−/− platelets abrogated the athero-protective effect, indicating that CD40L on platelets mediates the reduction of Tregs leading to accelerated atherosclerosis. We conclude that platelet CD40L plays a pivotal role in atherosclerosis, not only by affecting platelet-platelet interactions but especially by activating leukocytes, thereby increasing platelet-leukocyte and leukocyte-endothelium interactions. PMID:20705757

  10. Lipid rafts regulate cellular CD40 receptor localization in vascular endothelial cells

    SciTech Connect

    Xia Min; Wang Qing; Zhu Huilian; Ma Jing; Hou Mengjun; Tang Zhihong; Li Juanjuan; Ling Wenhua

    2007-09-28

    Cholesterol enriched lipid rafts are considered to function as platforms involved in the regulation of membrane receptor signaling complex through the clustering of signaling molecules. In this study, we tested whether these specialized membrane microdomains affect CD40 localization in vitro and in vivo. Here, we provide evidence that upon CD40 ligand stimulation, endogenous and exogenous CD40 receptor is rapidly mobilized into lipid rafts compared with unstimulated HAECs. Efficient binding between CD40L and CD40 receptor also increases amounts of CD40 protein levels in lipid rafts. Deficiency of intracellular conserved C terminus of the CD40 cytoplasmic tail impairs CD40 partitioning in raft. Raft disorganization after methyl-{beta}-cyclodextrin treatment diminishes CD40 localization into rafts. In vivo studies show that elevation of circulating cholesterol in high-cholesterol fed rabbits increases the cholesterol content and CD40 receptor localization in lipid rafts. These findings identify a physiological role for membrane lipid rafts as a critical regulator of CD40-mediated signal transduction and raise the possibility that certain pathologic conditions may be treated by altering CD40 signaling with drugs affecting its raft localization.

  11. CD40 agonist antibody mediated improvement of chronic Cryptosporidium infection in patients with X- linked hyper IgM syndrome

    PubMed Central

    Fan, Xiying; Upadhyaya, Bhaskar; Wu, Liming; Koh, Christopher; Santín-Durán, Mónica; Pittaluga, Stefania; Uzel, Gulbu; Kleiner, David; Williams, Ester; Ma, Chi A.; Bodansky, Aaron; Oliveira, Joao B.; Edmonds, Pamela; Hornung, Ronald; Wong, Duane W.; Fayer, Ronald; Fleisher, Tom; Heller, Theo; Prussin, Calman; Jain, Ashish

    2012-01-01

    X-linked hyper-IgM syndrome (XHM) is a combined immune deficiency disorder caused by mutations in CD40 ligand. We tested CP-870,893, a human CD40 agonist monoclonal antibody, in the treatment of two XHM patients with biliary Cryptosporidiosis. CP-870,893 activated B cells and APCs in vitro, restoring class switch recombination in XHM B cells and inducing cytokine secretion by monocytes. CP-870,893 infusions were well tolerated and showed significant activity in vivo, decreasing leukocyte concentration in peripheral blood. Although specific antibody responses were lacking, frequent dosing in one subject primed T cells to secrete IFN-g and suppressed oocyst shedding in the stool. Nevertheless, relapse occurred after discontinuation of therapy. The CD40 receptor was rapidly internalized following binding with CP-870,893, potentially explaining the limited capacity of CP-870,893 to mediate immune reconstitution. This study demonstrates that CP-870,893 suppressed oocysts shedding in XHM patients with biliary cryptosporidiosis. The continued study of CD40 agonists in XHM is warranted. PMID:22459705

  12. CD40L induces functional tunneling nanotube networks exclusively in dendritic cells programmed by mediators of type-1 immunity

    PubMed Central

    Zaccard, Colleen R.; Watkins, Simon C.; Kalinski, Pawel; Fecek, Ronald J.; Yates, Aarika L.; Salter, Russell D.; Ayyavoo, Velpandi; Rinaldo, Charles R.; Mailliard, Robbie B.

    2014-01-01

    The ability of dendritic cells (DC) to mediate CD4+ T cell help for cellular immunity is guided by instructive signals received during DC maturation, and the resulting pattern of DC responsiveness to the Th signal, CD40L. Furthermore, the professional transfer of antigenic information from migratory DC to lymph node-residing DC is critical for the effective induction of cellular immune responses. Here we report that, in addition to their enhanced IL-12p70 producing capacity, human DC matured in the presence of inflammatory mediators of type-1 immunity (DC1) are uniquely programmed to form networks of tunneling nanotube-like structures in response to CD40L-expressing Th cells or recombinant CD40L. This immunologic process of DC ‘reticulation’ facilitates intercellular trafficking of endosome-associated vesicles and Ag, but also pathogens such HIV-1, and is regulated by the opposing roles of IFN-γ and IL-4. The initiation of DC reticulation represents a novel helper function of CD40L and a superior mechanism of intercellular communication possessed by DC1, as well as a target for exploitation by pathogens to enhance direct cell-to-cell spread. PMID:25548234

  13. TLR7 and CD40 cooperate in IL-6 production via enhanced JNK and AP-1 activation.

    PubMed

    Vanden Bush, Tony J; Bishop, Gail A

    2008-02-01

    During vaccination or infection, adaptive and innate immune receptors of B cells are engaged by microbial antigens/ligands. A better understanding of how innate and adaptive signaling pathways interact could enlighten B lymphocyte biology as well as aid immunotherapy strategies and vaccine design. To address this goal, we examined the effects of TLR stimulation on BCR and CD40-induced B cell activation. Synergistic production of IL-6 was observed in both human and mouse primary B cells stimulated through B cell antigen receptors, CD40 and TLR7, and these two receptors also cooperated independently of BCR signals. The enhanced IL-6 production was dependent upon the activity of c-Jun kinase (JNK) and cFos. Dual stimulation through CD40 and TLR7 markedly enhanced JNK activity. The increased level of active JNK in dual-stimulated cells was accompanied by an increase in the level of active AP-1 monomers cJun and cFos. The stimulation of B cells through both CD40 and TLR7 therefore enhanced the production of cytokines through increased JNK signaling and AP-1 activity. In addition, the dual stimulation increased cFos/AP-1 species in stimulated cells, effectively expanding the repertoire of AP-1 dimers as compared to singly stimulated B cells.

  14. Influence of pre-analytical and analytical factors on soluble CD40L measurements.

    PubMed

    Varo, Nerea; Nuzzo, Rebecca; Natal, Cristina; Libby, Peter; Schönbeck, Uwe

    2006-11-01

    The soluble form of CD40L (CD40 ligand), a pro-atherogenic mediator, has emerged as a diagnostic and prognostic marker for cardiovascular events. However, as platelets can shed CD40L upon activation, accurate measurement has proved challenging. The present study addresses the controversy regarding the appropriate specimen and preparation for laboratory evaluation of blood sCD40L (soluble CD40L). Serum and plasma (collected in EDTA, citrate or heparin) were collected from healthy volunteers (n=20), and sCD40L was analysed by ELISA immediately or after one to three freeze-thaw cycles and at different centrifugation speeds. Urine sCD40L levels were measured in subjects with low- and high-plasma sCD40L levels. Serum sCD40L levels (5.45+/-4.55 ng/ml; P<0.001) were higher than in citrate, EDTA or heparin plasma (1.03+/-1.07, 1.43+/-1.03 or 1.80+/-1.25 ng/ml respectively), with no significant differences between plasma preparations. Increasing g values (200-13000 g), which gradually deplete plasma of platelets, yielded lower sCD40L levels. Repeated freeze-thaw cycles significantly (P<0.05) increased sCD40L concentrations in platelet-rich, but not platelet-depleted, plasma (up to 2.4-fold). Bilirubin and haemoglobin interfered positively, and triacylglycerols (triglycerides) and cholesterol quenched CD40L signalling. No sCD40L was detected in urine samples. In conclusion, serum yields higher sCD40L concentrations than plasma; accurate measurements of sCD40L require exclusion of platelets and avoiding their post-hoc activation. Samples with high concentrations of bilirubin, haemoglobin and/or triacylglycerols should be excluded, as these substances interfere with the assay.

  15. Gene-gene interaction between CD40 and CD40L reduces bone mineral density and increases osteoporosis risk in women.

    PubMed

    Pineda, B; Tarín, J J; Hermenegildo, C; Laporta, P; Cano, A; García-Pérez, M Á

    2011-05-01

    We have analysed the association of single-nucleotide polymorphisms (SNPs) in CD40 and CD40L genes with bone mineral density (BMD) in our women. Results showed that women with TT genotype for rs1883832 (CD40) and for rs1126535 (CD40L) SNPs displayed reduced BMD and increased risk for osteopenia/osteoporosis. Our data notwithstanding, the results need to be replicated. Recent data have revealed that the CD40/CD40L system can be implicated in bone metabolism regulation. Moreover, we previously demonstrated that rs1883832 in the CD40 gene was significantly associated with BMD and osteoporosis risk. The objective of the present work was to determine whether polymorphisms in CD40 and CD40L genes are associated with BMD and osteoporosis risk. We conducted an association study of BMD values with SNPs in CD40 and CD40L genes in a population of 811 women of which 693 and 711 had femoral neck (FN) and lumbar spine (LS) densitometric studies, respectively. Women with the TT genotype for rs1883832 (CD40) showed a reduction in FN-BMD (P = 0.005) and LS-BMD (P = 0.020) when compared with women with the CC/CT genotype. Moreover, we found that rs1126535 (CD40L) was significantly associated with LS-BMD so that women with the TT genotype displayed lower BMD (P = 0.014) than did women with the CC/CT genotype. Interestingly, we have found a strong interaction between polymorphisms in these genes. Thus, women with the TT genotype for both rs1883832 and rs1126535 SNPs (TT + TT women) showed a lower age-adjusted BMD (Z-score) for FN (P = 0.0007) and LS (0.007) after adjusting by years since menopause, body mass index, smoking and menopausal status, densitometer type, hormone replacement therapy (HRT) use and HRT duration and after making the Bonferroni adjustment for multiple comparisons than did the remaining women. Logistic regression analysis adjusted by these covariates showed that TT + TT women had increased risk for FN (odds ratio (OR) = 2.76; P = 0.006) and

  16. CD40L deficiency ameliorates adipose tissue inflammation and metabolic manifestations of obesity in mice.

    PubMed

    Poggi, Marjorie; Engel, David; Christ, Anette; Beckers, Linda; Wijnands, Erwin; Boon, Louis; Driessen, Ann; Cleutjens, Jack; Weber, Christian; Gerdes, Norbert; Lutgens, Esther

    2011-10-01

    Obese adipose tissue shows hallmarks of chronic inflammation, which promotes the development of metabolic disorders. The mechanisms by which immune cells interact with each other or with metabolism-associated cell types, and the players involved, are still unclear. The CD40-CD40L costimulatory dyad plays a pivotal role in immune responses and in diseases such as atherosclerosis and may therefore be a mediator of obesity. Here we investigated whether CD40L is involved in adipose tissue inflammation and its associated metabolic changes. To assess a putative role of CD40L in obesity in vivo, we evaluated metabolic and inflammatory consequences of 18 weeks of high-fat feeding in CD40L(+/+) and CD40L(-/-) mice. In addition, C57Bl6 mice were injected with neutralizing anti-CD40L (αCD40L) antibody for 12 weeks while being fed a high-fat diet. Genetic deficiency of CD40L attenuated the development of diet-induced obesity, hepatic steatosis, and increased systemic insulin sensitivity. In adipose tissue, it impaired obesity-induced immune cell infiltration and the associated deterioration of glucose and lipid metabolism. Accordingly, αCD40L treatment improved systemic insulin sensitivity, glucose tolerance, and CD4(+) T-cell infiltration in adipose tissue with limited effects on adipose tissue weight. CD40L plays a crucial role in the development of obesity-induced inflammation and metabolic complications.

  17. CD40 Gene Silencing Reduces the Progression of Experimental Lupus Nephritis Modulating Local Milieu and Systemic Mechanisms

    PubMed Central

    Ripoll, Èlia; Merino, Ana; Goma, Montse; Aran, Josep M.; Bolaños, Nuria; de Ramon, Laura; Herrero-Fresneda, Immaculada; Bestard, Oriol; Cruzado, Josep M.; Grinyó, Josep M.; Torras, Juan

    2013-01-01

    Lupus nephritis (LN) is an autoimmune disorder in which co-stimulatory signals have been involved. Here we tested a cholesterol-conjugated-anti-CD40-siRNA in dendritic cells (DC) in vitro and in a model of LPS to check its potency and tissue distribution. Then, we report the effects of Chol-siRNA in an experimental model of mice with established lupus nephritis. Our in vitro studies in DC show a 100%intracellular delivery of Chol-siRNA, with a significant reduction in CD40 after LPS stimuli. In vivo in ICR mice, the CD40-mRNA suppressive effects of our Chol-siRNA on renal tissue were remarkably sustained over a 5 days after a single preliminary dose of Chol-siRNA. The intra-peritoneal administration of Chol-siRNA to NZB/WF1 mice resulted in a reduction of anti-DNA antibody titers, and histopathological renal scores as compared to untreated animals. The higher dose of Chol-siRNA prevented the progression of proteinuria as effectively as cyclophosphamide, whereas the lower dose was as effective as CTLA4. Chol-siRNA markedly reduced insterstitialCD3+ and plasma cell infiltrates as well as glomerular deposits of IgG and C3. Circulating soluble CD40 and activated splenic lymphocyte subsets were also strikingly reduced by Chol-siRNA. Our data show the potency of our compound for the therapeutic use of anti-CD40-siRNA in human LN and other autoimmune disorders. PMID:23799000

  18. Trypanosoma cruzi infection induces the expression of CD40 in murine cardiomyocytes favoring CD40 ligation-dependent production of cardiopathogenic IL-6.

    PubMed

    Ayala, Mariela Alejandra Moreno; Casasco, Agustina; González, Mariela; Postan, Miriam; Corral, Ricardo Santiago; Petray, Patricia Beatriz

    2016-02-01

    The inflammatory response in the myocardium is an important aspect of the pathogenesis of Chagas' heart disease raised by Trypanosoma cruzi. CD40, a transmembrane type I receptor belonging to the tumor necrosis factor receptor (TNFR) family, is expressed in a broad spectrum of cell types and is crucial in several inflammatory and autoimmune diseases. Activation of CD40 through ligation to CD40L (CD154) induces multiple effects, including the secretion of proinflammatory molecules. In the present study, we examined the ability of T. cruzi to trigger the expression of CD40 in cardiac myocytes in vitro and in a murine model of chagasic cardiomyopathy. Our results indicate, for the first time, that T. cruzi is able to induce the expression of CD40 in HL-1 murine cardiomyocytes. Moreover, ligation of CD40 receptor upregulated interleukin-6 (IL-6), associated with inflammation. Furthermore, the induction of this costimulatory molecule was demonstrated in vivo in myocardium of mice infected with T. cruzi. This suggests that CD40-bearing cardiac muscle cells could interact with CD40L-expressing lymphocytes infiltrating the heart, thus contributing to inflammatory injury in chagasic cardiomyopathy.

  19. Expression of B7 (CD80) and CD40 antigens and the CD40 ligand in Hodgkin's disease is independent of latent Epstein—Barr virus infection

    PubMed Central

    Murray, P G; Oates, J; Reynolds, G M; Crocker, J; Young, L S

    1995-01-01

    Aim—To examine the expression of CD40 and B7 (CD80) antigens and the CD40 ligand in Hodgkin's disease. Methods—Antigen and ligand expression was studied in 17 cases of Hodgkin's disease using immunohistochemistry. The study included 11 cases of Hodgkin's disease in which latent Epstein-Barr virus (EBV) infection could be demonstrated within tumour cells by in situ hybridisation for the EBV encoded early RNAs (EBERs). Results—In all cases, irrespective of EBV status, Reed-Sternberg cells and their variants (HRS cells) showed strong expression of both B7 and CD40 antigens. CD40 ligand expression was not shown in HRS cells but was confined to a subset of small lymphocytes some of which were seen to be in intimate contact with HRS cells. Paraffin wax sections from a further 60 cases of Hodgkin's disease were examined for CD40 and EBER expression alone. The CD40 antigen was identified in HRS cells in all of these cases irrespective of EBER expression. Conclusions—As CD40 and B7 expression are features of professional antigen presenting cells, these results provide further evidence that HRS cells may have antigen presenting properties and that this may contribute to the characteristic recruitment and activation of non-malignant lymphocytes which is a feature of Hodgkin's disease. The ability of HRS cells to activate Th cells may in turn contribute to their own survival through the induction of the gp39/CD40 pathway. Images PMID:16695980

  20. Blocking CD40-TRAF6 signaling is a therapeutic target in obesity-associated insulin resistance

    PubMed Central

    Chatzigeorgiou, Antonios; Seijkens, Tom; Zarzycka, Barbara; Engel, David; Poggi, Marjorie; van den Berg, Susan; van den Berg, Sjoerd; Soehnlein, Oliver; Winkels, Holger; Beckers, Linda; Lievens, Dirk; Driessen, Ann; Kusters, Pascal; Biessen, Erik; Garcia-Martin, Ruben; Klotzsche-von Ameln, Anne; Gijbels, Marion; Noelle, Randolph; Boon, Louis; Hackeng, Tilman; Schulte, Klaus-Martin; Xu, Aimin; Vriend, Gert; Nabuurs, Sander; Chung, Kyoung-Jin; Willems van Dijk, Ko; Rensen, Patrick C. N.; Gerdes, Norbert; de Winther, Menno; Block, Norman L.; Schally, Andrew V.; Weber, Christian; Bornstein, Stefan R.; Nicolaes, Gerry; Chavakis, Triantafyllos; Lutgens, Esther

    2014-01-01

    The immune system plays an instrumental role in obesity and insulin resistance. Here, we unravel the role of the costimulatory molecule CD40 and its signaling intermediates, TNF receptor-associated factors (TRAFs), in diet-induced obesity (DIO). Although not exhibiting increased weight gain, male CD40−/− mice in DIO displayed worsened insulin resistance, compared with wild-type mice. This worsening was associated with excessive inflammation of adipose tissue (AT), characterized by increased accumulation of CD8+ T cells and M1 macrophages, and enhanced hepatosteatosis. Mice with deficient CD40-TRAF2/3/5 signaling in MHCII+ cells exhibited a similar phenotype in DIO as CD40−/− mice. In contrast, mice with deficient CD40-TRAF6 signaling in MHCII+ cells displayed no insulin resistance and showed a reduction in both AT inflammation and hepatosteatosis in DIO. To prove the therapeutic potential of inhibition of CD40-TRAF6 in obesity, DIO mice were treated with a small-molecule inhibitor that we designed to specifically block CD40-TRAF6 interactions; this compound improved insulin sensitivity, reduced AT inflammation, and decreased hepatosteatosis. Our study reveals that the CD40-TRAF2/3/5 signaling pathway in MHCII+ cells protects against AT inflammation and metabolic complications associated with obesity whereas CD40-TRAF6 interactions in MHCII+ cells aggravate these complications. Inhibition of CD40-TRAF6 signaling by our compound may provide a therapeutic option in obesity-associated insulin resistance. PMID:24492375

  1. Cancer immunotherapy: activating innate and adaptive immunity through CD40 agonists.

    PubMed

    Beatty, Gregory L; Li, Yan; Long, Kristen B

    2017-02-01

    CD40 is a promising therapeutic target for cancer immunotherapy. In patients with advanced solid malignancies, CD40 agonists have demonstrated some anti-tumor activity and a manageable toxicity profile. A 2(nd) generation of CD40 agonists has now been designed with optimized Fc receptor (FcR) binding based on preclinical evidence suggesting a critical role for FcR engagement in defining the potency of CD40 agonists in vivo. Areas covered: We provide a comprehensive review using PubMed and Google Patent databases on the current clinical status of CD40 agonists, strategies for applying CD40 agonists in cancer therapy, and the preclinical data that supports and is guiding the future development of CD40 agonists. Expert commentary: There is a wealth of preclinical data that provide rationale on several distinct approaches for using CD40 agonists in cancer immunotherapy. This data illustrates the need to strategically combine CD40 agonists with other clinically active treatment regimens in order to realize the full potential of activating CD40 in vivo. Thus, critical to the success of this class of immune-oncology drugs, which have the potential to restore both innate and adaptive immunosurveillance, will be the identification of biomarkers for monitoring and predicting responses as well as informing mechanisms of treatment resistance.

  2. Are polymorphisms of the immunoregulatory factor CD40LG implicated in acute transfusion reactions?

    PubMed Central

    Aloui, Chaker; Sut, Caroline; Prigent, Antoine; Fagan, Jocelyne; Cognasse, Fabrice; Granados-Herbepin, Viviana; Touraine, Renaud; Pozzetto, Bruno; Aouni, Mahjoub; Fendri, Chedlia; Hassine, Mohsen; Chakroun, Tahar; Jemni-Yacoub, Saloua; Garraud, Olivier; Laradi, Sandrine

    2014-01-01

    The CD40 ligand (CD40L/CD154), a member of TNF superfamily, is notably expressed on activated CD4+ T-cells and stimulated platelets. CD40L is linked to a variety of pathologies and to acute transfusion reactions (ATR). Mutations in this gene (CD40LG) lead to X-linked hyper-IgM syndrome. Some CD40LG polymorphisms are associated with variable protein expression. The rationale behind this study is that CD40L protein has been observed to be involved in ATR. We wondered whether genetic polymorphisms are implicated. We investigated genetic diversity in the CD40LG using DHPLC and capillary electrophoresis for screening and genotyping (n = 485 French and Tunisian blood donors). We identified significant difference in the CD40LG linkage pattern between the two populations. Variant minor alleles were significantly over-represented in Tunisian donors (P<0.0001 to 0.0270). We found higher heterogeneity in the Tunisian, including three novel low frequency variants. As there was not a particular pattern of CD40LG in single apheresis donors whose platelet components induced an ATR, we discuss how this information may be useful for future disease association studies on CD40LG. PMID:25430087

  3. CD40 Generation 2.5 Antisense Oligonucleotide Treatment Attenuates Doxorubicin-induced Nephropathy and Kidney Inflammation

    PubMed Central

    Donner, Aaron J; Yeh, Steve T; Hung, Gene; Graham, Mark J; Crooke, Rosanne M; Mullick, Adam E

    2015-01-01

    Preclinical and clinical data suggest CD40 activation contributes to renal inflammation and injury. We sought to test whether upregulation of CD40 in the kidney is a causative factor of renal pathology and if reduction of renal CD40 expression, using antisense oligonucleotides (ASOs) targeting CD40, would be beneficial in mouse models of glomerular injury and unilateral ureter obstruction. Administration of a Generation 2.5 CD40 ASO reduced CD40 mRNA and protein levels 75–90% in the kidney. CD40 ASO treatment mitigated functional, transcriptional, and pathological endpoints of doxorubicin-induced nephropathy. Experiments using an activating CD40 antibody revealed CD40 is primed in kidneys following doxorubicin injury or unilateral ureter obstruction and CD40 ASO treatment blunted CD40-dependent renal inflammation. Suborgan fractionation and imaging studies demonstrated CD40 in glomeruli before and after doxorubicin administration that becomes highly enriched within interstitial and glomerular foci following CD40 activation. Such foci were also sites of ASO distribution and activity and may be predominately comprised from myeloid cells as bone marrow CD40 deficiency sharply attenuated CD40 antibody responses. These studies suggest an important role of interstitial renal and/or glomerular CD40 to augment kidney injury and inflammation and demonstrate that ASO treatment could be an effective therapy in such disorders. PMID:26623936

  4. CD40-targeted adenoviral cancer vaccines: the long and winding road to the clinic

    PubMed Central

    Hangalapura, Basav N.; Timares, Laura; Oosterhoff, Dinja; Scheper, Rik J.; Curiel, David T.; de Gruijl, Tanja D.

    2012-01-01

    Summary The ability of Dendritic Cells (DC) to orchestrate innate and adaptive immune responses has been exploited to develop potent anti-cancer immunotherapies. Recent clinical trials exploring the efficacy of ex vivo modified autologous DC-based vaccines have reported some promising results. However, in vitro generation of autologous DC for clinical administration, their loading with tumor associated antigens (TAA) and their activation, is laborious and expensive, and, due to interindividual variability in the personalized vaccines, poorly standardized. An attractive alternative approach is to load resident DC in vivo by targeted delivery of TAA , using viral vectors and activating them simultaneously. To this end we have constructed genetically modified Adenoviral (Ad) vectors and bispecific adaptor molecules to retarget Ad vectors encoding TAA to the CD40 receptor on DC. Preclinical human and murine studies conducted so far have clearly demonstrated the suitability of a “two-component”, i.e. Ad and adaptor molecule, configuration for targeted modification of DC in vivo for cancer immunotherapy. This review summarizes recent progress in the development of CD40-targeted Ad-based cancer vaccines and highlights pre-clinical issues in clinical translation of this approach. PMID:22228547

  5. SHP-1 plays a crucial role in CD40 signaling reciprocity.

    PubMed

    Khan, Tabish Hasan; Srivastava, Neetu; Srivastava, Ankita; Sareen, Archana; Mathur, Ram K; Chande, Ajit G; Musti, Krishnasastry V; Roy, Somenath; Mukhopadhyaya, Robin; Saha, Bhaskar

    2014-10-01

    CD40 plays dual immunoregulatory roles in Leishmania major infection and tumor regression. The functional duality emerges from CD40-induced reciprocal p38MAPK and ERK-1/2 phosphorylations. Because phosphotyrosine-based signaling in hematopoietic cells is regulated by the phosphotyrosine phosphatase SHP-1, which is not implied in CD40 signaling, we examined whether SHP-1 played any roles in CD40-induced reciprocal signaling and anti-leishmanial function. We observed that a weaker CD40 stimulation increased SHP-1 activation. ERK-1/2 inhibition or p38MAPK overexpression inhibited CD40-induced SHP-1 activation. An ultra-low-dose, CD40-induced p38MAPK phosphorylation was enhanced by SHP-1 inhibition but reduced by SHP-1 overexpression. A reverse profile was observed with ERK-1/2 phosphorylation. SHP-1 inhibition reduced syk phosphorylation but increased lyn phosphorylation; syk inhibition reduced but lyn inhibition enhanced CD40-induced SHP-1 phosphorylation. Corroborating these findings, in L. major-infected macrophages, CD40-induced SHP-1 phosphorylation increased and SHP-1 inhibition enhanced CD40-induced p38MAPK activation and inducible NO synthase expression. IL-10 enhanced SHP-1 phosphorylation and CD40-induced ERK-1/2 phosphorylation but reduced the CD40-induced p38MAPK phosphorylation, whereas anti-IL-10 Ab exhibited reverse effects on these CD40-induced functions, identifying IL-10 as a crucial element in the SHP-1-MAPK feedback system. Lentivirally overexpressed SHP-1 rendered resistant C57BL/6 mice susceptible to the infection. Lentivirally expressed SHP-1 short hairpin RNA enhanced the CD40-induced L. major parasite killing in susceptible BALB/c mice. Thus, we establish an SHP-1-centered feedback system wherein SHP-1 modulates CD40-induced p38MAPK activation threshold and reciprocal ERK-1/2 activation, establishing itself as a critical regulator of CD40 signaling reciprocity and mechanistically re-emphasizing its role as a potential target against the

  6. Thymic medullary epithelium and thymocyte self tolerance require cooperation between CD28-CD80/86 and CD40-CD40L costimulatory pathways

    PubMed Central

    Williams, Joy A.; Zhang, Jingjing; Jeon, Hyein; Nitta, Takeshi; Ohigashi, Izumi; Klug, David; Kruhlak, Michael J.; Choudhury, Baishakhi; Sharrow, Susan O.; Granger, Larry; Adams, Anthony; Eckhaus, Michael A.; Jenkinson, S. Rhiannon; Richie, Ellen R.; Gress, Ronald E.; Takahama, Yousuke; Hodes, Richard J.

    2014-01-01

    A critical process during thymic development of the T cell repertoire is the induction of self-tolerance. Tolerance in developing T cells is highly dependent on medullary thymic epithelial cells (mTEC) and mTEC development in turn requires signals from mature single positive (SP) thymocytes, a bidirectional relationship termed thymus crosstalk. We show that CD28-CD80/86 and CD40-CD40L costimulatory interactions, which mediate negative selection and self-tolerance, upregulate expression of LTα, LTβ and RANK in the thymus and are necessary for medullary development. Combined absence of CD28-CD80/86 and CD40-CD40L results in profound deficiency in mTEC development comparable to that observed in the absence of SP thymocytes. This requirement for costimulatory signaling is maintained even in a TCR transgenic model of high affinity TCR-ligand interactions. CD4 thymocytes maturing in the altered thymic epithelial environment of CD40/CD80/86 KO mice are highly autoreactive in vitro and are lethal in congenic adoptive transfer in vivo, demonstrating a critical role for these costimulatory pathways in self-tolerance as well as thymic epithelial development. These findings demonstrate that cooperativity between CD28-CD80/86 and CD40-CD40L pathways is required for normal medullary epithelium and for maintenance of self-tolerance in thymocyte development. PMID:24337745

  7. A CD40 Kozak sequence polymorphism and susceptibility to antibody-mediated autoimmune conditions: the role of CD40 tissue-specific expression.

    PubMed

    Jacobson, E M; Huber, A K; Akeno, N; Sivak, M; Li, C W; Concepcion, E; Ho, K; Tomer, Y

    2007-04-01

    Previously, we and others have demonstrated the association of a C/T single nucleotide polymorphism (SNP), in the Kozak sequence of CD40, with Graves' disease (GD). Here, using an expanded data set of patients, we confirm the association of the CD40 SNP with GD (n=210, P=0.002, odds ratio (OR)=1.8). Subset analysis of patients with persistently elevated thyroid peroxidase (TPO) and/or thyroglobulin (Tg) antibodies (Abs), (TPO/Tg Abs), after treatment (n=126), revealed a significantly stronger association of the SNP with disease (P=5.2 x 10(-5), OR=2.5) than in GD patients who were thyroid antibody-negative. However, the CD40 SNP was not associated with TPO/Tg Abs in healthy individuals. Next, we tested the CD40 SNP for association with Myasthenia Gravis (MG), which, like GD is an antibody-mediated autoimmune condition. Analysis of 81 MG patients found no association of the SNP with disease. Functional studies revealed significant expression of CD40 mRNA and protein in the thyroid (target tissue in GD) but not in skeletal muscle (target tissue in MG). Combined, our genetic and tissue expression data suggest that the CD40 Kozak SNP is specific for thyroid antibody production involved in the etiology of GD. Increased thyroidal expression of CD40 driven by the SNP may contribute to this disease specificity.

  8. IN VITRO TESTING OF AN ANTI-CD40 MONOCLONAL ANTIBODY, CLONE 2C10, IN PRIMATES AND PIGS

    PubMed Central

    Lee, Whayoung; Satyananda, Vikas; Iwase, Hayato; Tanaka, Takayuki; Miyagawa, Yuko; Long, Cassandra; Ayares, David; Cooper, David KC; Hara, Hidetaka

    2015-01-01

    Background The CD40/CD154 and CD28/B7 pathways are important in allo- and xeno-transplantation. Owing to the thrombotic complications of anti-CD154mAb, anti-CD40mAb has emerged as a promising inhibitor of costimulation. Various clones of anti-CD40mAb have been developed against primate species, e.g., clone 2C10 against rhesus monkeys. We have compared the in vitro efficacy of 2C10 to prevent a T cell response in primates and pigs. Methods The binding of 2C10 to antigen-presenting cells (PBMCs [B cells]) of humans, rhesus and cynomolgus monkeys, baboons, and pigs was measured by flow cytometry, and was also tested indirectly by a blocking assay. The functional capacity of 2C10 was tested by mixed lymphocyte reaction (MLR) with polyclonal stimulation by phytohemagglutinin (PHA) and also with wild-type pig aortic endothelial cells (pAECs) as stimulators. Results There was a significant reduction in binding of 2C10 to baboon PBMCs compared to rhesus, cynomolgus, and human PBMCs, and minimal binding to pig PBMCs. The blocking assay confirmed that the binding of 2C10 was significantly lower to baboon PBMCs when compared to the other primate species tested. The functional assay with PHA showed significantly reduced inhibition of PBMC proliferation in humans, cynomolgus monkeys, and baboons compared to rhesus monkeys, which was confirmed on MLR with pAECs. Conclusions Since both the binding and functional activity of 2C10 in the baboon is lower than in rhesus monkeys, in vivo treatment using 2C10 in the baboon might require a higher dose or more frequent administration in comparison to rhesus monkeys. It may also be beneficial to develop species-specific clones of anti-CD40mAb. PMID:26458513

  9. Functional Specialty of CD40 and Dendritic Cell Surface Lectins for Exogenous Antigen Presentation to CD8(+) and CD4(+) T Cells.

    PubMed

    Yin, Wenjie; Gorvel, Laurent; Zurawski, Sandra; Li, Dapeng; Ni, Ling; Duluc, Dorothée; Upchurch, Katherine; Kim, JongRok; Gu, Chao; Ouedraogo, Richard; Wang, Zhiqing; Xue, Yaming; Joo, HyeMee; Gorvel, Jean-Pierre; Zurawski, Gerard; Oh, SangKon

    2016-03-01

    Dendritic cells (DCs) are major antigen-presenting cells that can efficiently prime and cross-prime antigen-specific T cells. Delivering antigen to DCs via surface receptors is thus an appealing strategy to evoke cellular immunity. Nonetheless, which DC surface receptor to target to yield the optimal CD8(+) and CD4(+) T cell responses remains elusive. Herein, we report the superiority of CD40 over 9 different lectins and scavenger receptors at evoking antigen-specific CD8(+) T cell responses. However, lectins (e.g., LOX-1 and Dectin-1) were more efficient than CD40 at eliciting CD4(+) T cell responses. Common and distinct patterns of subcellular and intracellular localization of receptor-bound αCD40, αLOX-1 and αDectin-1 further support their functional specialization at enhancing antigen presentation to either CD8(+) or CD4(+) T cells. Lastly, we demonstrate that antigen targeting to CD40 can evoke potent antigen-specific CD8(+) T cell responses in human CD40 transgenic mice. This study provides fundamental information for the rational design of vaccines against cancers and viral infections.

  10. Elevated serum soluble CD40 ligand in cancer patients may play an immunosuppressive role.

    PubMed

    Huang, Jianping; Jochems, Caroline; Talaie, Tara; Anderson, Austin; Jales, Alessandra; Tsang, Kwong Y; Madan, Ravi A; Gulley, James L; Schlom, Jeffrey

    2012-10-11

    Tumor cells can induce certain cytokines and soluble receptors that have a suppressive effect on the immune system. In this study, we showed that an extracellular portion of a membrane-bound ligand of CD40 (soluble CD40 ligand; sCD40L) was significantly elevated in the serum of cancer patients compared with healthy donors. In addition, PBMCs from cancer patients had a relatively larger population of myeloid-derived suppressor cells (MDSCs), defined as CD33(+)HLA-DR(-) cells, and these cells expressed higher levels of CD40. T-cell proliferation and IFN-γ production decreased when stimulated T cells were cocultured with an increased amount of autologous MDSCs. The addition of recombinant monomeric sCD40L enriched MDSCs and had an additive inhibitory effect on T-cell proliferation. PBMCs cultured in vitro with sCD40L also showed an expansion of regulatory T cells (CD4(+)CD25(high)Foxp3(+)), as well as induction of cytokines, such as IL-10 and IL-6. Moreover, sCD40L-induced enrichment of programmed death-1-expressing T cells was greater in cancer patients than in healthy donors. Preexisting sCD40L also inhibited IL-12 production from monocytes on activation. These data suggest that the higher levels of sCD40L seen in cancer patients may have an immunosuppressive effect.

  11. CD40 controls CXCR5-induced recruitment of myeloid-derived suppressor cells to gastric cancer.

    PubMed

    Ding, Yixin; Shen, Jin; Zhang, Guangbo; Chen, Xiaojuan; Wu, JiaMing; Chen, Weichang

    2015-11-17

    To explore the mechanisms of MDSC trafficking and accumulation during tumor progression. In this study, we report significant CD40 upregulation in tumor-infiltrating MDSC when compared with splenic MDSC. Microarray analyses comparing CD40(high) and CD40l(ow) MDSC revealed 1872 differentially expressed genes, including CD83, CXCR5, BTLA, CXCL9, TLR1, FLT3, NOD2 and CXCL10. In vivo experiments comparing wild-type (WT) and CD40 knockout (KO) mice demonstrated that CD40 critically regulates CXCR5 expression. Consistently, the transwell analysis confirmed the essential role of CXCR5-CXCL13 crosstalk in the migration of CD40+ MDSC toward gastric cancer. Furthermore, more MDSC accumulated in the gastric cancers of WT mice when compared with KO mice, and the WT tumors mostly contained CD40+ cells. Functionally, tumors grew faster in WT than KO mice. In conclusion, we demonstrate that CD40 expression upregulates the chemokine receptor CXCR5 and promotes MDSC migration toward and accumulation within cancer. Therefore, this study provides preliminary evidence that CD40 may stimulate tumor growth by enabling immune evasion via MDSC recruitment and inhibition of T cell expansion.

  12. Host CD40 Is Essential for DCG Treatment Against Metastatic Lung Cancer.

    PubMed

    Yamashita, Kimihiro; Hasegawa, Hiroshi; Fujita, Mitsugu; Nishi, Masayasu; Tanaka, Tomoko; Arimoto, Akira; Suzuki, Satoshi; Kamigaki, Takashi; Kakeji, Yoshihiro

    2016-07-01

    For the application of invariant natural killer T (iNKT) cells in cancer therapy, the CD40-CD40L interaction is indispensable in administering alpha-galactosylceramide (αGalCer). We hypothesized that CD40 plays an important role in dendritic cells (DC) pulsed with αGalCer (DCGs) in the treatment of lung metastases. Wild-type (WT) and CD40(-/-) mice were treated with DCGs isolated from WT or CD40(-/-) mice in a B16F10 lung metastases model and NK and NKT cell activity in lungs and the spleen were examined. DCG treatment improved WT mice survival but CD40(-/-) hosts received no survival benefit. Conversely, attenuation of a therapeutic effect in mice treated with CD40(-/-) DCGs was not observed. The functional activities of NK and NKT cells in DCG-treated CD40(-/-) mice were partially suppressed. Host CD40 is essential for DCG treatment to have a therapeutic effect on B16F10 lung metastases. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  13. Soluble CD40 Ligand in Aspirin-Treated Patients Undergoing Cardiac Catheterization

    PubMed Central

    Gremmel, Thomas; Frelinger, Andrew L.; Michelson, Alan D.

    2015-01-01

    Plasma soluble CD40 ligand (sCD40L) is mainly generated by cleavage of CD40L from the surface of activated platelets, and therefore considered a platelet activation marker. Although the predictive value of sCD40L for ischemic events has been demonstrated in patients with acute coronary syndromes (ACS), studies on the association of sCD40L with cardiovascular outcomes in lower risk populations yielded heterogeneous results. We therefore sought to investigate factors influencing sCD40L levels, and the predictive value of sCD40L for long-term ischemic events in unselected, aspirin-treated patients undergoing cardiac catheterization. sCD40L was determined by a commercially available enzyme-linked immunosorbent assay in 682 consecutive patients undergoing cardiac catheterization. Two-year follow-up data were obtained from 562 patients. Dual antiplatelet therapy with aspirin and clopidogrel was associated with significantly lower levels of sCD40L and lower platelet surface expressions of P-selectin and activated GPIIb/IIIa compared to aspirin monotherapy (all p≤0.01). Hypertension was linked to lower plasma concentrations of sCD40L, whereas female sex, increasing high-sensitivity C-reactive protein, and hematocrit were associated with higher sCD40L concentrations (all p<0.05). sCD40L levels were similar in patients without and with the primary endpoint in the overall study population (p = 0.4). Likewise, sCD40L levels did not differ significantly between patients without and with the secondary endpoints (both p≥0.4). Similar results were obtained when only patients with angiographically-proven coronary artery disease (n = 459), stent implantation (n = 205) or ACS (n = 125) were analyzed. The adjustment for differences in patient characteristics by multivariate regression analyses did not change the results. ROC curve analyses did not reveal cut-off values for sCD40L for the prediction of the primary or secondary endpoints. In conclusion, plasma sCD40L levels are

  14. Increased plasma soluble CD40 ligand concentration in pelvic inflammatory disease.

    PubMed

    Ho, Tsung-chin; Yang, Shun-Fa; Wang, Po-Hui; Lin, Long-Yau; Tee, Yi-Torng; Liao, Wen-Chun; Chang, Hsiu-Ju; Tsai, Hsiu-Ting

    2015-01-01

    The role of soluble CD40 ligand (sCD40L) in pelvic inflammatory disease (PID) remains unclear. We sought to determine whether sCD40L was an efficient serum marker as with WBC and CRP in PID patients. Enzyme-linked immunosorbent assay was used to measure the plasma levels of sCD40L before and after routine protocol treatments in sixty-four PID patients and seventy healthy controls. The level of plasma sCD40L (pg/ml) was significantly elevated in PID patients (1632.83±270.91) compared to that in normal controls (700.33±58.77; p=0.001) and decreased significantly as compared to that in the same patients (928.77±177.25; p=0.0001) after they received treatment. The concentration of sCD40L was significantly correlated with the level of plasma C-reactive protein (CRP) in the blood (r=0.202, p=0.01, n=134). When the cutoff level of plasma sCD40L levels was determined to be 1612.26pg/ml based on ROC, the sensitivity, specificity, and the area under the curve of plasma sCD40L level for predicting PID were 0.26, 0.97, and 0.58 (95% confidence interval: 0.48-0.68), respectively, while the adjusted odds ratio (AOR) with their 95% CI of plasma sCD40L for PID risk was 7.09 (95% CI=1.14-43.87, p=0.03). The expression of plasma sCD40L was increased in patients with PID and detection of plasma sCD40L could be useful for the diagnosis of PID. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Kick-starting the cancer-immunity cycle by targeting CD40

    PubMed Central

    Ellmark, P; Mangsbo, S M; Furebring, C; Tötterman, T H; Norlén, P

    2015-01-01

    Stimulation of CD40 on dendritic cells to expand and activate tumor-specific T cells and generate anticancer immunity is an attractive therapeutic approach. Since CD40 agonists exert their effects upstream of checkpoint inhibitors, including PD-1 or PD-L1 antagonists, they are ideal candidates for combination regimens. PMID:26140231

  16. Association between serum soluble CD40 ligand levels and mortality in patients with severe sepsis

    PubMed Central

    2011-01-01

    Introduction CD40 Ligand (CD40L) and its soluble counterpart (sCD40L) are proteins that exhibit prothrombotic and proinflammatory properties on binding to their cell surface receptor CD40. The results of small clinical studies suggest that sCD40L levels could play a role in sepsis; however, there are no data on the association between sCD40L levels and mortality of septic patients. Thus, the aim of this study was to determine whether circulating sCD40L levels could be a marker of adverse outcome in a large cohort of patients with severe sepsis. Methods This was a multicenter, observational and prospective study carried out in six Spanish intensive care units. Serum levels of sCD40L, tumour necrosis factor-alpha and interleukin-10, and plasma levels of tissue factor were measured in 186 patients with severe sepsis at the time of diagnosis. Serum sCD40L was also measured in 50 age- and sex-matched controls. Survival at 30 days was used as the endpoint. Results Circulating sCD40L levels were significantly higher in septic patients than in controls (P = 0.01), and in non-survivors (n = 62) compared to survivors (n = 124) (P = 0.04). However, the levels of CD40L were not different regarding sepsis severity. Logistic regression analysis showed that sCD40L levels >3.5 ng/mL were associated with higher mortality at 30 days (odds ratio = 2.89; 95% confidence interval = 1.37 to 6.07; P = 0.005). The area under the curve of sCD40L levels >3.5 ng/mL as predictor of mortality at 30 days was 0.58 (95% CI = 0.51 to 0.65; P = 0.03). Conclusions In conclusion, circulating sCD40L levels are increased in septic patients and are independently associated with mortality in these patients; thus, its modulation could represent an attractive therapeutic target. PMID:21406105

  17. Association of CD40L gene polymorphism with severe Plasmodium falciparum malaria in Indian population.

    PubMed

    Purohit, Prasanta; Mohanty, Pradeep Kumar; Patel, Siris; Das, Padmalaya; Das, Kishalaya; Panigrahi, Jogeswar

    2017-01-01

    Many host genetic factors are associated with the disease severity and fatal outcome of falciparum malaria. CD40L gene has been found to be one of the most important factors associated with malaria in African countries. This study was aimed to investigate the possible association of CD40L gene polymorphism in severe falciparum malaria in Indian adults. One hundred fifteen adult cases with severe falciparum malaria were included in the study. Two single- nucleotide polymorphisms (SNPs) of CD40L gene, CD40L-726(C/T) and CD40L+220(C/T) were investigated, and the possible association with different clinical sub-phenotypes of severe falciparum malaria were analyzed. Statistically no significant difference was observed in the incidence of CD40L-726C between the patients and control group. The incidence of CD40L+220C allele was found to be significantly higher (OR, 2.25; p = 0.03) in male patients compared to controls but no significant difference was observed in females. Haplotype data showed the susceptibility of -726T/+220C haplotype to severe malaria whereas -726C/+220T was associated with protection against severe malaria. CD40L+220C allele was associated with severe malarial anaemia in males (χ2 = 6.60; p = 0.01). CD40L gene polymorphism was found to be associated with severe falciparum malaria in Indian population especially in severe malarial anaemia. CD40L may be considered as a factor of immunity in understanding the pathophysiology of falciparum malaria.

  18. CD40 regulates the processing of NF-κB2 p100 to p52

    PubMed Central

    Coope, H.J.; Atkinson, P.G.P.; Huhse, B.; Belich, M.; Janzen, J.; Holman, M.J.; Klaus, G.G.B.; Johnston, L.H.; Ley, S.C.

    2002-01-01

    The nf-kb2 gene encodes the cytoplasmic NF-κB inhibitory protein p100 from which the active p52 NF-κB subunit is derived by proteasome-mediated proteolysis. Ligands which stimulate p100 processing to p52 have not been defined. Here, ligation of CD40 on transfected 293 cells is shown to trigger p52 production by stimulating p100 ubiquitylation and subsequent proteasome-mediated proteolysis. CD40-mediated p52 accumulation is dependent on de novo protein synthesis and triggers p52 translocation into the nucleus to generate active NF-κB dimers. Endogenous CD40 ligation on primary murine splenic B cells also stimulates p100 processing, which results in the delayed nuclear translocation of p52–RelB dimers. In both 293 cells and primary splenic B cells, the ability of CD40 to trigger p100 processing requires functional NF-κB-inducing kinase (NIK). In contrast, NIK activity is not required for CD40 to stimulate the degradation of IκBα in either cell type. The regulation of p100 processing by CD40 is likely to be important for the transcriptional regulation of CD40 target genes in adaptive immune responses. PMID:12374738

  19. Elevated serum soluble CD40 ligand in cancer patients may play an immunosuppressive role

    PubMed Central

    Huang, Jianping; Jochems, Caroline; Talaie, Tara; Anderson, Austin; Jales, Alessandra; Tsang, Kwong Y.; Madan, Ravi A.; Gulley, James L.

    2012-01-01

    Tumor cells can induce certain cytokines and soluble receptors that have a suppressive effect on the immune system. In this study, we showed that an extracellular portion of a membrane-bound ligand of CD40 (soluble CD40 ligand; sCD40L) was significantly elevated in the serum of cancer patients compared with healthy donors. In addition, PBMCs from cancer patients had a relatively larger population of myeloid-derived suppressor cells (MDSCs), defined as CD33+HLA-DR− cells, and these cells expressed higher levels of CD40. T-cell proliferation and IFN-γ production decreased when stimulated T cells were cocultured with an increased amount of autologous MDSCs. The addition of recombinant monomeric sCD40L enriched MDSCs and had an additive inhibitory effect on T-cell proliferation. PBMCs cultured in vitro with sCD40L also showed an expansion of regulatory T cells (CD4+CD25highFoxp3+), as well as induction of cytokines, such as IL-10 and IL-6. Moreover, sCD40L-induced enrichment of programmed death-1–expressing T cells was greater in cancer patients than in healthy donors. Preexisting sCD40L also inhibited IL-12 production from monocytes on activation. These data suggest that the higher levels of sCD40L seen in cancer patients may have an immunosuppressive effect. These studies were registered at www.clinicaltrials.gov as NCT00060528, NCT00019695, NCT00179309, NCT00514072, NCT00081848, and NCT00436956. PMID:22932804

  20. The role of CD40 expression in dendritic cells in cancer biology; a systematic review.

    PubMed

    Lee, Gui Han; Askari, Alan; Malietzis, George; Bernardo, David; Clark, Susan K; Knight, Stella C; Al-Hassi, Hafid Omar

    2014-01-01

    CD40 is a co-stimulatory molecule belonging to the tumor necrosis factor superfamily and is essential in activation of dendritic cells. Dendritic cells (DCs) are antigen-presenting cells capable of initiating cytotoxic T-lymphocyte immune response against cancer cells. However, there are few studies on the characterization of DCs in cancer, specifically their expression of CD40, despite its implication in cancer immunotherapy. We reviewed available data on the expression of CD40 on DCs in various cancers, and its implications for cancer immunotherapy. A systematic review on CD40 expression on DCs in cancer was performed with reference to preferred reporting items for systematic reviews and meta-analyses (PRISMA). Studies that satisfied the inclusion and exclusion criteria were 21 out of 927. Variations in type and status of the cancers, source of DCs and methodology for detecting CD40 expression amongst the studies resulted in contrasting results. DCs generally expressed low CD40 in tumor infiltrating DCs (tiDCs), in DCs derived by in vitro culture from blood monocytes using cytokine stimulation (MoDCs) and in DCs exposed in vitro to tumor cells lines; the studies suggested that CD40 expression in DCs is impaired in cancer particularly in metastatic disease. However, DCs identified in fresh peripheral blood mononuclear cells (PBMC) expressed higher numbers of CD40 positive cells in some cancer patients, which could be due to tumor-derived factors leading to partially-stimulated DCs. The results provide evidence that some cancer patients may show partial systemic DC activation and expression of increased CD40 in response to the presence of tumor but that such activity may become abortive in the presence of factors produced by the tumor. This review has thus identified key papers on CD40 expression on DCs in various cancers and discusses the limitations and contrasting results of these studies in relation to variations in methodology. The results highlight the need

  1. T Cell Cancer Therapy Requires CD40-CD40L Activation of Tumor Necrosis Factor and Inducible Nitric-Oxide-Synthase-Producing Dendritic Cells.

    PubMed

    Marigo, Ilaria; Zilio, Serena; Desantis, Giacomo; Mlecnik, Bernhard; Agnellini, Andrielly H R; Ugel, Stefano; Sasso, Maria Stella; Qualls, Joseph E; Kratochvill, Franz; Zanovello, Paola; Molon, Barbara; Ries, Carola H; Runza, Valeria; Hoves, Sabine; Bilocq, Amélie M; Bindea, Gabriela; Mazza, Emilia M C; Bicciato, Silvio; Galon, Jérôme; Murray, Peter J; Bronte, Vincenzo

    2016-09-12

    Effective cancer immunotherapy requires overcoming immunosuppressive tumor microenvironments. We found that local nitric oxide (NO) production by tumor-infiltrating myeloid cells is important for adoptively transferred CD8(+) cytotoxic T cells to destroy tumors. These myeloid cells are phenotypically similar to inducible nitric oxide synthase (NOS2)- and tumor necrosis factor (TNF)-producing dendritic cells (DC), or Tip-DCs. Depletion of immunosuppressive, colony stimulating factor 1 receptor (CSF-1R)-dependent arginase 1(+) myeloid cells enhanced NO-dependent tumor killing. Tumor elimination via NOS2 required the CD40-CD40L pathway. We also uncovered a strong correlation between survival of colorectal cancer patients and NOS2, CD40, and TNF expression in their tumors. Our results identify a network of pro-tumor factors that can be targeted to boost cancer immunotherapies.

  2. Silencing of CD40 in vivo reduces progression of experimental atherogenesis through an NF-κB/miR-125b axis and reveals new potential mediators in the pathogenesis of atherosclerosis.

    PubMed

    Hueso, Miguel; De Ramon, Laura; Navarro, Estanislao; Ripoll, Elia; Cruzado, Josep M; Grinyo, Josep M; Torras, Joan

    2016-12-01

    CD40/CD40L signaling exerts a critical role in the development of atherosclerosis, and microRNAs (miRNAs) are key regulators in vascular inflammation and plaque formation. In this work, we investigated mRNA/miRNA expression during progression of atherosclerotic lesions through CD40 silencing. We silenced CD40 with a specific siRNA in ApoE(-/-) mice and compared expression of mRNA/miRNA in ascending aorta with scrambled treated mice. siRNA-CD40 treated mice significantly reduced the extension and severity of atherosclerotic lesions, as well as the number of F4/80(+), galectin-3(+) macrophages and NF-κB(+) cells in the intima. Genome-wide mRNA/miRNA profiling allowed the identification of transcripts, which were significantly upregulated during atherosclerosis; among them, miR-125b and miR-30a, Xpr1, a regulator of macrophage differentiation, Taf3, a core transcription factor and the NF-κB activator Ikkβ, whereas, the NF-κB inhibitor Ikbα was downregulated during disease progression. All those changes were reversed upon CD40 silencing. Interestingly, TAF3, XPR1 and miR-125b were also overexpressed in human atherosclerotic plaques. Murine Taf3 and Xpr1 were detected in the perivascular adipose tissue (PVAT), and Taf3 also in intimal foam cells. Finally, expression of miR-125b was regulated by the CD40-NF-κB signaling axis in RAW264.7 macrophages. CD40 silencing with a specific siRNA ameliorates progression of experimental atherosclerosis in ApoE(-/-) mice, and evidences a role for NF-κB, Taf3, Xpr1, and miR-125b in the pathogenesis of atherosclerosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. Soluble CD40 ligand stimulates CD40-dependent activation of the β2 integrin Mac-1 and protein kinase C zeda (PKCζ) in neutrophils: implications for neutrophil-platelet interactions and neutrophil oxidative burst.

    PubMed

    Jin, Rong; Yu, Shiyong; Song, Zifang; Zhu, Xiaolei; Wang, Cuiping; Yan, Jinchuan; Wu, Fusheng; Nanda, Anil; Granger, D Neil; Li, Guohong

    2013-01-01

    Recent work has revealed an essential involvement of soluble CD40L (sCD40L) in inflammation and vascular disease. Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined. We aimed to determine the impact and the mechanisms of sCD40L on neutrophils. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte recruitment to the sites of injury. Our data show that CD40L contributes to neutrophil firm adhesion to and transmigration across activated surface-adherent platelets, possibly through two potential mechanisms. One involves the direct interaction of ligand-receptor (CD40L-CD40), i.e., platelet surface CD40L interaction with neutrophil CD40; another involves an indirect mechanism, i.e. soluble CD40L stimulates activation of the leukocyte-specific β2 integrin Mac-1 in neutrophils and thereby further promotes neutrophil adhesion and migration. Activation of the integrin Mac-1 is known to be critical for mediating neutrophil adhesion and migration. sCD40L activated Mac-1 in neutrophils and enhanced neutrophil-platelet interactions in wild-type neutrophils, but failed to elicit such responses in CD40-deficient neutrophils. Furthermore, our data show that the protein kinase C zeta (PKCζ) is critically required for sCD40L-induced Mac-1 activation and neutrophil adhesive function. sCD40L strongly stimulated the focal clustering of Mac-1 (CD11b) and the colocalization of Mac-1 with PKCζ in wild-type neutrophils, but had minimal effect in CD40-deficient neutrophils. Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion. Moreover, sCD40L strongly stimulates neutrophil oxidative burst via CD40-dependent activation of PI3K/NF-KB, but independent of Mac-1 and PKCζ. These findings may contribute to a better understanding of the

  4. CD40 agonist antibody mediated improvement of chronic Cryptosporidium infection in patients with X-linked hyper IgM syndrome.

    PubMed

    Fan, Xiying; Upadhyaya, Bhaskar; Wu, Liming; Koh, Christopher; Santín-Durán, Mónica; Pittaluga, Stefania; Uzel, Gulbu; Kleiner, David; Williams, Ester; Ma, Chi A; Bodansky, Aaron; Oliveira, Joao B; Edmonds, Pamela; Hornung, Ronald; Wong, Duane W; Fayer, Ronald; Fleisher, Tom; Heller, Theo; Prussin, Calman; Jain, Ashish

    2012-05-01

    X-linked hyper-IgM syndrome (XHM) is a combined immune deficiency disorder caused by mutations in CD40 ligand. We tested CP-870,893, a human CD40 agonist monoclonal antibody, in the treatment of two XHM patients with biliary Cryptosporidiosis. CP-870,893 activated B cells and APCs in vitro, restoring class switch recombination in XHM B cells and inducing cytokine secretion by monocytes. CP-870,893 infusions were well tolerated and showed significant activity in vivo, decreasing leukocyte concentration in peripheral blood. Although specific antibody responses were lacking, frequent dosing in one subject primed T cells to secrete IFN-g and suppressed oocyst shedding in the stool. Nevertheless, relapse occurred after discontinuation of therapy. The CD40 receptor was rapidly internalized following binding with CP-870,893, potentially explaining the limited capacity of CP-870,893 to mediate immune reconstitution. This study demonstrates that CP-870,893 suppressed oocysts shedding in XHM patients with biliary cryptosporidiosis. The continued study of CD40 agonists in XHM is warranted. Published by Elsevier Inc.

  5. Incorporation of Glycosylphosphatidylinositol-Anchored Granulocyte- Macrophage Colony-Stimulating Factor or CD40 Ligand Enhances Immunogenicity of Chimeric Simian Immunodeficiency Virus-Like Particles▿

    PubMed Central

    Skountzou, Ioanna; Quan, Fu-Shi; Gangadhara, Sailaja; Ye, Ling; Vzorov, Andrei; Selvaraj, Periasamy; Jacob, Joshy; Compans, Richard W.; Kang, Sang-Moo

    2007-01-01

    The rapid worldwide spread of human immunodeficiency virus (HIV) mandates the development of successful vaccination strategies. Since live attenuated HIV is not accepted as a vaccine due to safety concerns, virus-like particles (VLPs) offer an attractive safe alternative because they lack the viral genome yet they are perceived by the immune system as a virus particle. We hypothesized that adding immunostimulatory signals to VLPs would enhance their efficacy. To accomplish this we generated chimeric simian immunodeficiency virus (SIV) VLPs containing either glycosylphosphatidylinositol (GPI)-anchored granulocyte-macrophage colony-stimulating factor (GM-CSF) or CD40 ligand (CD40L) and investigated their biological activity and ability to enhance immune responses in vivo. Immunization of mice with chimeric SIV VLPs containing GM-CSF induced SIV Env-specific antibodies as well as neutralizing activity at significantly higher levels than those induced by standard SIV VLPs, SIV VLPs containing CD40L, or standard VLPs mixed with soluble GM-CSF. In addition, mice immunized with chimeric SIV VLPs containing either GM-CSF or CD40L showed significantly increased CD4+- and CD8+-T-cell responses to SIV Env, compared to standard SIV VLPs. Taken together, these results demonstrate that the incorporation of immunostimulatory molecules enhances humoral and cellular immune responses. We propose that anchoring immunostimulatory molecules into SIV VLPs can be a promising approach to augmenting the efficacy of VLP antigens. PMID:17108046

  6. Disruption of antigen-induced inflammatory responses in CD40 ligand knockout mice.

    PubMed Central

    Lei, X F; Ohkawara, Y; Stämpfli, M R; Mastruzzo, C; Marr, R A; Snider, D; Xing, Z; Jordana, M

    1998-01-01

    The objective of this study was to investigate the contribution of the interaction between CD40 and its ligand (CD40L) to antigen-induced airways inflammatory responses. To this end, we used a model involving ovalbumin (OVA) sensitization followed by OVA aerosol challenge in CD40L knockout (KO) mice. OVA-specific IgE and IgG1 were detected in the serum of the sensitized control, but not in CD40L-KO mice. After antigen challenge, sensitized control mice developed airway inflammation that was primarily eosinophilic. This inflammatory response was dramatically reduced in CD40L-KO mice. In contrast, similar numbers of eosinophils were observed in both the bone marrow and the peripheral blood in the sensitized controls and mutant strains after antigen challenge. To investigate the mechanisms underlying these findings, we examined levels of the cytokines IL-5, IL-4, and TNFalpha in both bronchoalveolar lavage (BAL) and serum. Similar levels of IL-5 were detected in BAL and serum of control and CD40L-KO mice; however, negligible levels of IL-4 in BAL and serum and of TNFalpha in BAL were detected in CD40L-KO mice when compared with control mice. Furthermore, we demonstrated that endothelial cell expression of vascular cell adhesion molecule 1 in OVA-sensitized and -challenged CD40L-KO mice was, as detected by immunohistochemistry, markedly decreased compared with that observed in similarly treated control mice. In addition, we locally overexpressed IL-4 and TNFalpha by using an adenoviral (Ad)-mediated gene transfer approach. Intranasal administration of either Ad/TNFalpha or Ad/IL-4 into OVA-sensitized and -challenged CD40L-KO mice did not reconstitute airway eosinophilia. However, concurrent administration of Ad/TNFalpha and Ad/IL-4 upregulated endothelial expression of vascular cell adhesion molecule 1, and resulted in full reconstitution of the inflammatory response in the airways. Together, these findings demonstrate the importance of the CD40-CD40L costimulatory

  7. Synergy of anti-CD40, CpG and MPL in activation of mouse macrophages.

    PubMed

    Shi, Yongyu; Felder, Mildred A R; Sondel, Paul M; Rakhmilevich, Alexander L

    2015-08-01

    Activation of macrophages is a prerequisite for their antitumor effects. Several reagents, including agonistic anti-CD40 monoclonal antibody (anti-CD40), CpG oligodeoxynucleotides (CpG) and monophosphoryl lipid A (MPL), can stimulate activation of macrophages. Our previous studies showed synergy between anti-CD40 and CpG and between anti-CD40 and MPL in macrophage activation and antitumor efficacy in mice. In the present study, we asked whether there was synergy among these three reagents. The activation of adherent peritoneal exudate cells (PEC) obtained from mice injected with anti-CD40 and then treated with CpG and/or MPL in vitro was determined by their ability to suppress proliferation of tumor cells and to produce various cytokines and chemokines in vitro. Cell sorting and histology followed by functional testing showed that macrophages were the main cell population in PEC activated by CD40 ligation in vivo. A combination of anti-CD40, CpG or MPL activated PEC to suppress proliferation of B16 cells and produce nitric oxide far greater than the single reagents or any of the double combinations of these reagents. In addition, the combination of all three reagents activated PEC to secrete IL-12, IFN-γ and MCP-1 to a greater degree than any single reagent or any two combined reagents. These results demonstrate that macrophages can be synergistically activated by anti-CD40, CpG and MPL, suggesting that this novel combined approach might be further investigated as potential cancer therapy.

  8. CD40 Negatively Regulates ATP-TLR4-Activated Inflammasome in Microglia.

    PubMed

    Gaikwad, Sagar; Patel, Divyesh; Agrawal-Rajput, Reena

    2017-03-01

    During acute brain injury and/or sterile inflammation, release of danger-associated molecular patterns (DAMPs) activates pattern recognition receptors (PRRs). Microglial toll-like receptor (TLR)-4 activated by DAMPs potentiates neuroinflammation through inflammasome-induced IL-1β and pathogenic Th17 polarization which critically influences brain injury. TLR4 activation accompanies increased CD40, a cognate costimulatory molecule, involved in microglia-mediated immune responses in the brain. During brain injury, excessive release of extracellular ATP (DAMPs) is involved in promoting the damage. However, the regulatory role of CD40 in microglia during ATP-TLR4-mediated inflammasome activation has never been explored. We report that CD40, in the absence of ATP, synergizes TLR4-induced proinflammatory cytokines but not IL-1β, suggesting that the response is independent of inflammasome. The presence of ATP during TLR4 activation leads to NLRP3 inflammasome activation and caspase-1-mediated IL-1β secretion which was inhibited during CD40 activation, accompanied with inhibition of ERK1/2 and reactive oxygen species (ROS), and elevation in p38 MAPK phosphorylation. Experiments using selective inhibitors prove indispensability of ERK 1/2 and ROS for inflammasome activation. The ATP-TLR4-primed macrophages polarize the immune response toward pathogenic Th17 cells, whereas CD40 activation mediates Th1 response. Exogenous supplementation of IFN-γ (a Th1 cytokine and CD40 inducer) results in decreased IL-1β, suggesting possible feedback loop mechanism of inflammasome inhibition, whereby IFN-γ-mediated increase in CD40 expression and activation suppress neurotoxic inflammasome activation required for Th17 response. Collectively, the findings indicate that CD40 is a novel negative regulator of ATP-TLR4-mediated inflammasome activation in microglia, thus providing a checkpoint to regulate excessive inflammasome activation and Th17 response during DAMP-mediated brain injury.

  9. Inhibitors of XIAP sensitize CD40-activated chronic lymphocytic leukemia cells to CD95-mediated apoptosis

    PubMed Central

    Kater, Arnon P.; Dicker, Frank; Mangiola, Massimo; Welsh, Kate; Houghten, Richard; Ostresh, John; Nefzi, Adel; Reed, John C.; Pinilla, Clemencia; Kipps, Thomas J.

    2005-01-01

    Patients with chronic lymphocytic leukemia (CLL) treated with adenovirus CD154 (Ad-CD154, CD40 ligand [CD40L]) gene therapy experienced rapid reductions in leukemia cell counts and lymph node size associated with the induced expression of Fas (CD95). However, CLL cells initially resist CD95-mediated apoptosis within the first 3 days after CD40 ligation in vitro. Thereafter, they become sensitive, which is associated with the CD40-induced expression of the proapoptotic protein B-cell leukemia 2 homology 3 (BH3) interacting domain death agonist (Bid). We hypothesized that the initial resistance to CD95-mediated apoptosis may be due to the high-level expression of X-linked inhibitor of apoptosis protein (XIAP) by CLL cells. Consistent with this, CLL cells from patients 1 day after treatment with autologous Ad-CD154-transduced CLL cells became sensitive to CD95-mediated apoptosis following treatment with a novel XIAP inhibitor, 1540-14. Similarly, 1540-14 specifically enhanced CD95-mediated apoptosis of CLL cells following CD40 ligation in vitro. Immunoblot analyses demonstrated that treatment with 1540-14 allowed CD40-stimulated CLL cells to experience high-level activation of caspases-8 and -3 and cleavage of poly(adenosine diphosphate [ADP]-ribose) polymerase following CD95 ligation. This study demonstrates that distal apoptosis regulators contribute to the initial resistance of CD40-activated CLL cells to CD95-mediated apoptosis and suggests that XIAP inhibitors might enhance the effectiveness of immune-based treatment strategies that target CD40, such as CD154 gene therapy. (Blood. 2005;106:1742-1748) PMID:15914559

  10. Identification and functional validation of PNAs that inhibit murine CD40 expression by redirection of splicing.

    PubMed

    Siwkowski, Andrew M; Malik, Leila; Esau, Christine C; Maier, Martin A; Wancewicz, Edward V; Albertshofer, Klaus; Monia, Brett P; Bennett, C Frank; Eldrup, Anne B

    2004-01-01

    Cognate recognition between the CD40 receptor and its ligand, CD154, is thought to play a central role in the initiation and propagation of immune responses. We describe the specific down regulation of cell surface associated CD40 protein expression by use of a peptide nucleic acid (PNA) antisense inhibitor, ISIS 208529, that is designed to bind to the 3' end of the exon 6 splice junction within the primary CD40 transcript. Binding of ISIS 208529 was found to alter constitutive splicing, leading to the accumulation of a transcript lacking exon 6. The resulting protein product lacks the transmembrane domain. ISIS 208529-mediated CD40 protein depletion was found to be sequence specific and dose dependent, and was dependent on the length of the PNA oligomer. CD40-dependent induction of IL-12 in primary murine macrophages was attenuated in cells treated with ISIS 208529. Oligolysine conjugation to the PNA inhibitor produced an inhibitor, ISIS 278647, which maintained its specificity and displayed efficacy in BCL1 cells and in primary murine macrophages in the absence of delivery agents. These results demonstrate that PNA oligomers can be effective inhibitors of CD40 expression and hence may be useful as novel immuno-modulatory agents.

  11. Cooperation of multiple signaling pathways in CD40-regulated gene expression in B lymphocytes

    PubMed Central

    Dadgostar, Hajir; Zarnegar, Brian; Hoffmann, Alexander; Qin, Xiao-Feng; Truong, Uyen; Rao, Govinda; Baltimore, David; Cheng, Genhong

    2002-01-01

    CD40/CD40L interaction is essential for multiple biological events in T dependent humoral immune responses, including B cell survival and proliferation, germinal center and memory B cell formation, and antibody isotype switching and affinity maturation. By using high-density microarrays, we examined gene expression in primary mouse B lymphocytes after multiple time points of CD40L stimulation. In addition to genes involved in cell survival and growth, which are also induced by other mitogens such as lipopolysaccharide, CD40L specifically activated genes involved in germinal center formation and T cell costimulatory molecules that facilitate T dependent humoral immunity. Next, by examining the roles of individual CD40-activated signal transduction pathways, we dissected the overall CD40-mediated response into genes independently regulated by the individual pathways or collectively by all pathways. We also found that gene down-regulation is a significant part of the overall response and that the p38 pathway plays an important role in this process, whereas the NF-κB pathway is important for the up-regulation of primary response genes. Our finding of overlapping independent control of gene expression modules by different pathways suggests, in principle, that distinct biological behaviors that depend on distinct gene expression subsets can be manipulated by targeting specific signaling pathways. PMID:11830667

  12. Elevated soluble CD40 ligand in diabetic patients with painless myocardial infarction.

    PubMed

    Huang, Yu; Qiu, Jianping; Zhang, Denghai; Qiu, Geng

    2011-01-01

    Because a useful biomarker for painless myocardial infarction (MI) has yet to be identified, the aim of this study was to identify a biomarker for diabetic patients with painless MI. A case-control design was used to compare inflammatory cytokine levels among 111 patients with diabetes mellitus, including 31 patients with stable coronary heart disease (CHD), 30 patients with painful MI, 20 patients with painless MI, and 30 age- and sex-matched patients without CHD (control group). In addition to baseline parameters, cytokine levels, including plasma high sensitivity C-reactive protein (HsCRP), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and soluble CD40 ligand (sCD40L) levels, were analyzed using enzyme-linked immunosorbent assays (ELISAs). No differences in baseline characteristics were observed for patients with painless MI as compared to the other patient groups. Significantly higher sCD40L, HsCRP, IL-6, and TNF-α levels were detected in patients with MI, and markedly elevated sCD40L and IL-6 levels were observed in patients with painless MI as compared to those with painful MI. sCD40L may be a useful biomarker for painless MI in diabetic patients, which could reduce misdiagnosis and expedite treatment. Further studies are required to validate the diagnostic utility of this putative biomarker as well as investigate the mechanism by which sCD40L is elevated in these patients.

  13. Non-survivor septic patients have persistently higher serum sCD40L levels than survivors.

    PubMed

    Lorente, Leonardo; Martín, María M; Pérez-Cejas, Antonia; Ferreres, José; Solé-Violán, Jordi; Labarta, Lorenzo; Díaz, César; Jiménez, Alejandro

    2017-10-01

    Soluble CD40 ligand (sCD40L) is a protein with proinflammatory and prothrombotic effects. Previously we found higher circulating sCD40L levels in non-survivor than in survivor patients at sepsis diagnosis. Now some questions arise such as how are serum sCD40L levels during the first week of severe sepsis?, is there an association between serum sCD40L levels during the first week and mortality?, and serum sCD40L levels during the first week could be used as sepsis mortality biomarker?. This study was developed to answer these asks. Study from 6 Spanish Intensive Care Units with 291 severe septic patients. There were determined serum levels of sCD40L and tumor necrosis factor (TNF)-alpha during the first week. The end-point study was 30-day mortality. We found that serum sCD40L at days 1, 4, and 8 could predict mortality at 30days, and are associated with mortality. The novel findings of our study were that there were higher serum sCD40L levels persistently during the first week in non-survivor than in survivor patients, that there is an association between serum sCD40L levels during the first week and sepsis mortality, and that serum sCD40L levels during the first week could be used as sepsis mortality biomarker. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. p62 regulates CD40-mediated NFκB activation in macrophages through interaction with TRAF6

    SciTech Connect

    Seibold, Kristina; Ehrenschwender, Martin

    2015-08-14

    CD40 is a member of the tumor necrosis factor (TNF) receptor family. Activation-induced recruitment of adapter proteins, so-called TNF-receptor-associated factors (TRAFs) to the cytoplasmic tail of CD40 triggers signaling cascades important in the immune system, but has also been associated with excessive inflammation in diseases such as atherosclerosis and rheumatoid arthritis. Especially, pro-inflammatory nuclear factor κB (NFκB) signaling emanating from CD40-associated TRAF6 appears to be a key pathogenic driving force. Consequently, targeting the CD40-TRAF6 interaction is emerging as a promising therapeutic strategy, but the underlying molecular machinery of this signaling axis is to date poorly understood. Here, we identified the multifunctional adaptor protein p62 as a critical regulator in CD40-mediated NFκB signaling via TRAF6. CD40 activation triggered formation of a TRAF6-p62 complex. Disturbing this interaction tremendously reduced CD40-mediated NFκB signaling in macrophages, while TRAF6-independent signaling pathways remained unaffected. This highlights p62 as a potential target in hyper-inflammatory, CD40-associated pathologies. - Highlights: • CD40 activation triggers interaction of the adapter protein TRAF6 with p62. • TRAF6-p62 interaction regulates CD40-mediated NFκB signaling in macrophages. • Defective TRAF6-p62 interaction reduces CD40-mediated NFκB activation in macrophages.

  15. A novel CD40LG deletion causes the hyper-IgM syndrome with normal CD40L expression in a 6-month-old child.

    PubMed

    López-Herrera, Gabriela; Maravillas-Montero, José Luis; Vargas-Hernández, Alexander; Berrón-Ruíz, Laura; Ramírez-Sánchez, Emmanuel; Yamazaki-Nakashimada, Marco Antonio; Espinosa-Rosales, Francisco Javier; Santos-Argumedo, Leopoldo

    2015-05-01

    The X-linked hyper-IgM syndrome (XHIGM) is the most common form of HIGM. Patients are clinically diagnosed on the basis of recurrent sinopulmonary infections, accompanied with low levels of IgG and IgA, normal to elevated levels of IgM, and the presence of peripheral B cells. Here, we have reported a novel deletion of four nucleotides in CD40LG exon 3, c.375_378delCAAA, which led to a frameshift mutation with a premature stop codon, p.Asn101*126. The deletion resulted in a truncated protein, in which majority of the extracellular domain was lost. However, detection of surface CD40L was still possible as the intracellular, transmembrane, and part of the extracellular domains were not affected. This indicated that this mutation did not affect protein stability and that immunodetection of CD40L expression is not enough for the diagnosis of XHIGM. Our study strongly suggests that genetic diagnosis for XHIGM should always be performed when clinical data support this diagnosis and CD40L protein is present.

  16. Processing and MHC class II presentation of exogenous soluble antigen involving a proteasome-dependent cytosolic pathway in CD40-activated B cells.

    PubMed

    Becker, Hans Jiro; Kondo, Eisei; Shimabukuro-Vornhagen, Alexander; Theurich, Sebastian; von Bergwelt-Baildon, Michael S

    2016-08-01

    Activated B cells have the capacity to present antigen and induce immune responses as potent antigen-presenting cells (APCs). As in other APCs, antigen presentation by B cells involves antigen internalization, antigen processing, and peptide loading onto MHC molecules. However, while the mechanism of antigen processing has been studied extensively in other APCs, this pathway remains elusive in B cells. The aim of this study was to investigate the MHC class II processing pathway in CD40-activated B cells (CD40Bs), as a model for activated, antigen-presenting B cells. Using CMV pp65 as a model antigen, we evaluated processing and presentation of the CD4 + T-cell epitope 509-523 (K509) by human CD40Bs in ELISPOT assays. As expected, stimulation of specific CD4 + T-cell clones was attenuated after pretreatment of CD40Bs with inhibitors of classic class II pathway components. However, proteasome inhibitors such as epoxomicin limited antigen presentation as well. This suggests that the antigen is processed in a non-classical, cytosolic MHC class II pathway. Further experiments with truncated protein variants revealed involvement of the proteasome in processing of the N and C extensions of the epitope. Access to the cytosol was shown to be size dependent. Epoxomicin sensitivity exclusively in CD40B cells, but not in dendritic cells, suggests a novel processing mechanism unique to this APC. Our data suggest that B cells process antigen using a distinct, non-classical class II pathway.

  17. miRNA-145 inhibits VSMC proliferation by targeting CD40

    PubMed Central

    Guo, Xin; Li, Dai; Chen, Min; Chen, Lei; Zhang, Bikui; Wu, Tian; Guo, Ren

    2016-01-01

    Recent studies have demonstrated functions of miR-145 in vascular smooth muscle cells (VSMCs) phenotypes and vascular diseases. In this study, we aim to determine whether CD40 is involved in miR-145 mediated switch of VSMC phenotypes. In cultured VSMCs, the effects of miR-145 and CD40 on TNF-α, TGF-β, and Homocysteine (Hcy) induced cell proliferation were evaluated by over-expression of miR-145 or by siRNA-mediated knockdown of CD40. We also used ultrasound imaging to explore the effect of miR-145 on carotid artery intima-media thickness (CIMT) in atherosclerotic cerebral infarction (ACI) patients. The results showed 50 ng/mL TNF-α, 5 ng/mL TGF-β, and 500 μmol/L Hcy significantly increased the expression of CD40, both at mRNA and protein levels, and also induced the proliferation of VSMCs. We found that over-expression of miR-145 significantly inhibited the expression of CD40 and the differentiation of VSMCs, and over-expression of miR-145 decreased IL-6 levels in VSMC supernatants. In ACI patients, the lower expression of miR-145 was associated with thicker CIMT and higher levels of plasma IL-6. Our results suggest that the miR-145/CD40 pathway is involved in regulating VSMC phenotypes in TNF-α, TGF-β, and Hcy induced VSMCs proliferation model. Targeting miR-145/CD40 might be a useful strategy for treating atherosclerosis. PMID:27731400

  18. CD40 ligand, Bcl-2 and apoptosis in B-chronic lymphocytic leukemia.

    PubMed

    Hussein, Ola A; Omran, Alaa A; Elnaggar, Amina M; Fathy, Ayman

    2009-01-01

    Chronic lymphocytic leukemia (CLL) is a haematopoetic neoplasm caused primarily by defects in apoptosis mechanisms and complicated by progressive marrow failure, immunosupression and increased resistance to chemotherapy. The CD40-CD40 ligand (CD40L) interaction has been shown to significantly increase antigen presentation in normal and malignant B-cells and it is a powerful regulator of cell survival. Bcl-2 expression is common in CLL and is associated with decreased overall survival. Our objective was to asses CD40 ligand (CD154) and Bcl-2 expressions and their correlation with clinical and laboratory features in CLL patients. This study was conducted on 40 subjects, including 10 healthy volunteers as the control group and 30 patients presented with de novo chronic lymphocytic leukemia (CLL), all of them were subjected to thorough history taking, full clinical examinations, routine laboratory investigations and flowcytometric assessment of CD40L and Bcl-2 on lymphocytes. There was a highly significant increase in TLC, absolute lymphocytic count, serum LDH, B2-microglobulin and Bcl-2 expression (P<0.001); there was a significant increase in CD40L expression (P<0.05); whereas there was a highly significant decrease in hemoglobin concentration and platelets count between the study group (P<0.001). There was no significant difference as regard direct Coombs' test between both groups. There was no significant relation between CD154 expression and clinical findings, Rai staging system and other laboratory parameters. CD40L expression is increased with staging of Modified Rai staging system but not reaching the significant level. There was no significant correlation between CD154 expression and some of clinical and laboratory parameters, whereas there was only significantly negative correlation between Bcl-2 expression and both haemoglobin concentration and platelets count (P<0.001). Combination of Bcl-2 antisense oligonucleotide with conventional chemotherapeutic drugs

  19. Aging murine B cells have decreased class switch induced by anti-CD40 or BAFF

    PubMed Central

    Frasca, Daniela; Riley, Richard L.; Blomberg, Bonnie B.

    2007-01-01

    We previously demonstrated that in vitro stimulated splenic B cells from senescent mice are deficient in production of multiple class switch isotypes, class switch recombination (CSR), induction of the E2A-encoded transcription factor E47, and activation-induced cytidine deaminase (AID) which is necessary for CSR and somatic hypermutation. Both anti-CD40 as well as BAFF have been shown to be able to induce CSR. We have investigated the ability of BAFF/IL-4, as compared to anti-CD40/IL-4, to induce CSR to γ1 in splenic B cells from young and old mice. We found that anti-CD40/IL-4 is a better CSR stimulus than BAFF/IL-4 in young B cells, as measured by RT-PCR of post-switch transcripts and flow cytometry. CSR is reduced in old B cells and this is independent of the stimulus. AID and γ1PSTs are significantly reduced in old B cells stimulated with anti-CD40/IL-4, but only slightly reduced with BAFF/IL-4. BAFF receptor mRNA expression (BAFF-R, TACI, BCMA) is not affected by aging. The age-related decrease in CSR induced by anti-CD40/IL-4 is primarily associated with a decrease in E47, whereas the less affected response to BAFF/IL-4 is associated with decreases in both E47 and NF-kB. Therefore, NF-kB is not involved in the decreased response of old B cells to anti-CD40/IL-4. These differences in B cell responses to CD40/IL-4 and BAFF/IL-4 may help to explain the maintenance of TI vs TD responses in senescent mice. PMID:17067770

  20. Critical role of microglial CD40 in the maintenance of mechanical hypersensitivity in a murine model of neuropathic pain

    PubMed Central

    Cao, Ling; Palmer, Christopher D.; Malon, Jennifer T.; De Leo, Joyce A.

    2010-01-01

    We recently demonstrated a contributing role of spinal cord infiltrating CD4+ T lymphocytes in the maintenance of mechanical hypersensitivity in a rodent model of neuropathic pain, spinal nerve L5 transection (L5Tx). It has been demonstrated that microglia play a role in the etiology of pain states. We hypothesized that infiltrating CD4+ T lymphocytes communicate with microglia via a CD40-CD154 interaction. Here, we investigated the role of CD40 in the development of mechanical hypersensitivity post-L5Tx. CD40 KO mice displayed significantly decreased mechanical sensitivity compared with WT mice starting from day 5 post-L5Tx. Using bone marrow chimeric mice, we further identified a pro-nociceptive role of CNS microglial CD40 rather than the peripheral leukocytic CD40. Flow cytometric analysis determined a significant increase of CD40+ microglia in the ipsilateral side of lumbar spinal cord post-L5Tx. Further, spinal cord proinflammatory cytokine (IL-1β, IL-6, IL-12, and TNF-α) profiling demonstrated an induction of IL-6 in both WT and CD40 KO mice post-L5Tx prior to the increase of microglial CD40 expression, indicating a CD40-independent induction of IL-6 following L5Tx. These data establish a novel role of microglial CD40 in the maintenance of nerve injury-induced behavioral hypersensitivity, a behavioral sign of neuropathic pain. PMID:19750482

  1. [Relationship between serum level of CD40 ligand and persistent lone atrial fibrillation].

    PubMed

    Bozçalı, Evin; Polat, Veli; Kutlu, Gönül; Opan, Selçuk; Paker, Nurcan; Uygun, Turgut; Ökçün, Barış; Karakaya, Osman

    2016-07-01

    Inflammation is thought to play a role in the pathogenesis of atrial fibrillation. The relationship between CD40 ligand (CD40L), a prothrombotic and proinflammatory molecule, and lone atrial fibrillation was presently investigated for the first time. Levels of serum CD40L were also tested, regarding potential to distinguish patients with lone atrial fibrillation from healthy individuals. Presently included were 35 patients with lone persistent atrial fibrillation and a control group of 30 healthy individuals. Serum levels of CD40L and high-sensitive C-reactive protein (hs-CRP) were measured, and transthoracic echocardiography was performed. Mean serum CD40L, hs-CRP, left ventricular end-diastolic diameter, and left atrial diameter values were significantly higher in the group with lone persistent atrial fibrillation than in the control group (7.4±3.5 ng/mL vs 4.3±1.2 ng/mL, p<0.0001; 3.7±1.6 mg/L vs 1.7±0.8 mg/L, p<0.0001; 53.0±4.2 mm vs 46.0±3.8, p<0.0001; 43.5±3.5 mm vs 33.7±3.5, p<0.0001, respectively). Serum CD40L levels were positively correlated with left atrial diameter (r=0.81, p<0.0001) and hs-CRP (r=0.72, p<0.0001). Receiver operating characteristic curve analysis revealed that serum CD40L at the optimal cut-off level of >4.5 ng/mL successfully discriminated patients with lone atrial fibrillation from controls (area under the curve: 0.847; 95% confidence interval: 0.759-0.934; p<0.0001). The present findings suggest that CD40L levels play a crucial role in the development of lone atrial fibrillation. In addition, results support that regular clinical follow-up of these patients is necessary, due to increased cardiovascular disease risk, determined by elevated CD40L levels.

  2. Effective genetic vaccination with a widely shared endogenous retroviral tumor antigen requires CD40 stimulation during tumor rejection phase.

    PubMed

    Bronte, Vincenzo; Cingarlini, Sara; Apolloni, Elisa; Serafini, Paolo; Marigo, Ilaria; De Santo, Carmela; Macino, Beatrice; Marin, Oriano; Zanovello, Paola

    2003-12-15

    Endogenous retrovirus (ERV) products are recognized by T lymphocytes in mice and humans. As these Ags are preferentially expressed by neoplastic tissues, they might represent an ideal target for active immunization by genetic vaccination. However, i.m. inoculation of plasmid DNA encoding mouse gp70 or p15E, two products of the env gene of an endogenous murine leukemia virus, elicited a weak Ag-specific T lymphocyte response and resulted in partial protection from challenge with mouse tumors possessing these Ags. Depletion experiments showed that CD8(+), but not CD4(+), T lymphocytes were crucial for the antitumor activity of the vaccines. Systemic administration of agonistic anti-CD40 mAb increased the therapeutic potential of genetic vaccination, but only when given during the tumor rejection phase and not at the time of immunization. This effect correlated with a dramatic increase in the number of ERV-specific CD8(+) T lymphocytes. Adjuvant activity of CD40 agonists thus seems to be relevant to enhance the CD8(+) T cell-dependent response in tumor-bearing hosts, suggesting that sustaining tumor-specific T lymphocyte survival in subjects undergoing vaccination might be a key event in the successful vaccination with weak tumor Ags.

  3. APE/Ref-1 makes fine-tuning of CD40-induced B cell proliferation

    PubMed Central

    Merluzzi, Sonia; Gri, Giorgia; Gattei, Valter; Pagano, Michele; Pucillo, Carlo

    2009-01-01

    Apurinic/apyrimidinic endonuclease-1/Redox factor-1, a multifunctional DNA base excision repair and redox regulation enzyme, plays an important role in oxidative signalling, transcription factor regulation, and cell cycle control. Recently, we have demonstrated that following the triggering of CD40 on B cells, APE/Ref-1 translocates from the cytoplasm to the nucleus and regulates the activity of B cell-specific transcription factors. In the present paper we investigate whether APE/Ref-1 plays a role in controlling CD40-mediated B cell proliferation too. We demonstrate a concurrent increase in proliferation and decrease in apoptosis of primary mouse B cells activated by CD40 cross-linking and transfected with functional APE/Ref-1 antisense oligonucleotide. Moreover, we provide evidence that a redox-mediated signalling mechanism is involved in this process and we propose that APE/Ref-1, controlling the intracellular redox state, may also affect the cell cycle by inducing nucleus-cytoplasm redistribution of p21. Together, these findings suggest that APE/Ref-1 could act as a negative regulator in an adaptive response to elevated ROS levels following CD40 cross-linking. Considering the important role of ROS and APE/Ref-1 in CD40-mediated B cell proliferation, our data will contribute to understand the mechanisms of tumor escape and suggest APE/Ref-1 as a novel target for tumor therapeutic approaches. PMID:18617267

  4. APE/Ref-1 makes fine-tuning of CD40-induced B cell proliferation.

    PubMed

    Merluzzi, Sonia; Gri, Giorgia; Gattei, Valter; Pagano, Michele; Pucillo, Carlo

    2008-08-01

    Apurinic/apyrimidinic endonuclease-1/Redox factor-1, a multifunctional DNA base excision repair and redox regulation enzyme, plays an important role in oxidative signalling, transcription factor regulation, and cell cycle control. Recently, we have demonstrated that following the triggering of CD40 on B cells, APE/Ref-1 translocates from the cytoplasm to the nucleus and regulates the activity of B cell-specific transcription factors. In the present paper we investigate whether APE/Ref-1 plays a role in controlling CD40-mediated B cell proliferation too. We demonstrate a concurrent increase in proliferation and decrease in apoptosis of primary mouse B cells activated by CD40 cross-linking and transfected with functional APE/Ref-1 antisense oligonucleotide. Moreover, we provide evidence that a redox-mediated signalling mechanism is involved in this process and we propose that APE/Ref-1, controlling the intracellular redox state, may also affect the cell cycle by inducing nucleus-cytoplasm redistribution of p21. Together, these findings suggest that APE/Ref-1 could act as a negative regulator in an adaptive response to elevated ROS levels following CD40 cross-linking. Considering the important role of ROS and APE/Ref-1 in CD40-mediated B cell proliferation, our data will contribute to understand the mechanisms of tumor escape and suggest APE/Ref-1 as a novel target for tumor therapeutic approaches.

  5. Role of Different CD40 Polymorphisms in Graves' Disease and Hashimoto's Thyroiditis.

    PubMed

    Wang, Dongguo; Chen, Jiayu; Zhang, Huanyuan; Zhang, Fangfang; Yang, Linjun; Mou, Yonghua

    2017-08-01

    Genome-wide association studies have led to the discovery of several susceptibility genes related to autoimmune thyroid diseases (AITDs). However, controversial results have been reported regarding the role of single-nucleotide polymorphism (SNP) of CD40 in the disease susceptibility. The objective of this study was to identify the relationship of the polymorphisms of three sites of CD40 with the susceptibility to AITD in the Chinese population. We genotyped three polymorphisms of CD40: C/T -1 SNP, 58038T site of the third exon and C64610G site of the ninth exon in 196 GD cases, 121 HT cases and 122 control subjects. The three putative polymorphism sites were amplified by PCR for sequencing and analysis. The genotype frequencies of CD40 -1 C/C genotype and C allele were significantly higher in the GD group than those in normal control. For the C64610G polymorphism, the C/G genotype was significantly more frequent in HT group than in control group, and the G allele frequencies in the GD and HT group were both higher than those in control group. These results indicated that there exist different susceptibility loci for AITD within CD40, each contributing a different effect in the onset and development of AITDs.

  6. [CCL21-CD40L fusion gene induce augmented antitumor activity in colon cancer].

    PubMed

    Gong, Ting; Zhou, Hong-Li; Ba, Yi

    2013-09-01

    To investigate the anti-tumor activity of CCL21-exCD40L eukaryotic expression vector. CCL21-exCD40L fusion gene were constructed by overlap PCR connecting CCL21 and exCD40L through a flexible linker (Gly3Ser)4, and then was cloned into expression vector pcDNA3.1(+). pcDNA3.1(+)/CCL21 and pcDNA3.1(+)/exCD were constructed as negative control. Wsestern blot was used to identify the fusion protein. CHO cells was transfected with pcDNA3.1(+)/CCL21-exCD, pcDNA3.1(+)/CCL21 and pcDNA3.1(+), respectively. The chemotatic function of the expressed product was detected by Transwell method and its anti-tumor activity was tested with vivo transfection. Gene sequencing and restrictive digestion proved the successful construction of pcDNA3.1(+)/CCL21-exCD40L,and its expression was conformed by western blot. The transfectant supernantes of pcDNA3.1(+)/CCL21-exCD40 group had a significant chmotactic function to DCs, of which the cell numbers passing through the film was 14.95 times of blank control every high power microscope visual field. After tumor orthotoic injection of plasmid carrying fusion gene in Balb/c mouse, the tumor mass reduced remarkablely, and all the mouse in fusion gene group survived after 4 weeks. CCL21-exCD40L fusion protein had a remarkable function to DCs and it can inhibit tumor growth and prolong the mouse survival time, which is more effective than all control group.

  7. Small Molecule Inhibition of the TNF Family Cytokine CD40 Ligand Through a Subunit Fracture Mechanism

    SciTech Connect

    L Silvian; J Friedman; K Strauch; T Cachero; E Day; F Qian; B Cunningham; A Fung; L Sun; et al.

    2011-12-31

    BIO8898 is one of several synthetic organic molecules that have recently been reported to inhibit receptor binding and function of the constitutively trimeric tumor necrosis factor (TNF) family cytokine CD40 ligand (CD40L, aka CD154). Small molecule inhibitors of protein-protein interfaces are relatively rare, and their discovery is often very challenging. Therefore, to understand how BIO8898 achieves this feat, we characterized its mechanism of action using biochemical assays and X-ray crystallography. BIO8898 inhibited soluble CD40L binding to CD40-Ig with a potency of IC{sub 50} = 25 {mu}M and inhibited CD40L-dependent apoptosis in a cellular assay. A co-crystal structure of BIO8898 with CD40L revealed that one inhibitor molecule binds per protein trimer. Surprisingly, the compound binds not at the surface of the protein but by intercalating deeply between two subunits of the homotrimeric cytokine, disrupting a constitutive protein-protein interface and breaking the protein's 3-fold symmetry. The compound forms several hydrogen bonds with the protein, within an otherwise hydrophobic binding pocket. In addition to the translational splitting of the trimer, binding of BIO8898 was accompanied by additional local and longer-range conformational perturbations of the protein, both in the core and in a surface loop. Binding of BIO8898 is reversible, and the resulting complex is stable and does not lead to detectable dissociation of the protein trimer. Our results suggest that a set of core aromatic residues that are conserved across a subset of TNF family cytokines might represent a generic hot-spot for the induced-fit binding of trimer-disrupting small molecules.

  8. Overexpression of Cd40 Ligand in Murine Epidermis Results in Chronic Skin Inflammation and Systemic Autoimmunity

    PubMed Central

    Mehling, Annette; Loser, Karin; Varga, Georg; Metze, Dieter; Luger, Thomas A.; Schwarz, Thomas; Grabbe, Stephan; Beissert, Stefan

    2001-01-01

    CD40CD40 ligand (L) interactions play a pivotal role in immune-mediated inflammatory responses via the activation of antigen-presenting cells (APCs). To investigate the effects of continuous activation of resident tissue APCs, in this case the Langerhans cells (LCs) of the skin, CD40L expression was targeted to the basal keratinocytes of the epidermis of mice using the keratin-14 promoter. Approximately 80% of the transgenic (Tg) mice spontaneously developed dermatitis on the ears, face, tail, and/or paws. Compared with littermates, Tgs had a >90% decrease in epidermal LCs yet increased numbers within the dermis suggestive of enhanced emigration of CD40-activated LCs. Tgs also displayed massive regional lymphadenopathy with increased numbers of dendritic cells and B cells. Moreover, a decrease in IgM and an increase in IgG1/IgG2a/IgG2b/IgE serum concentrations was detectable. Screening for autoantibodies revealed the presence of antinuclear antibodies and anti-dsDNA antibodies implicative of systemic autoimmunity. Accordingly, renal Ig deposits, proteinuria, and lung fibrosis were observed. Adoptive transfer of T cells from Tgs to nonTg recipients evoked the development of skin lesions similar to those found in the Tgs. Dermatitis also developed in B cell–deficient CD40L Tg mice. These findings suggest that in situ activation of LCs by CD40L in the skin not only leads to chronic inflammatory dermatitis but also to systemic mixed-connective-tissue-like autoimmune disorders, possibly by breaking immune tolerance against the skin. PMID:11535630

  9. CD40-mediated NFκB activation in B cells is increased in multiple sclerosis and modulated by therapeutics1

    PubMed Central

    Chen, Ding; Ireland, Sara J.; Remington, Gina; Alvarez, Enrique; Racke, Michael K.; Greenberg, Benjamin; Frohman, Elliot M.; Monson, Nancy L.

    2017-01-01

    CD40 interacts with CD40 ligand and plays an essential role in immune regulation and homeostasis. Recent research findings, however, support a pathogenic role of CD40 in a number of autoimmune diseases. We previously showed that memory B cells from relapsing-remitting multiple sclerosis (RRMS) patients exhibited enhanced proliferation with CD40 stimulation compared to healthy donors. In this study, we used a multi-parameter phosflow approach to analyze the phosphorylation status of NFκB and three major MAP kinases (P38, ERK and JNK), the essential components of signaling pathways downstream of CD40 engagement in B cells from MS patients. We found that memory and naïve B cells from RRMS and secondary progressive MS (SPMS) patients exhibited a significantly elevated level of phosphorylated NFκB (p-P65) following CD40 stimulation compared to healthy donor controls. Combination therapy with interferon beta-1a (Avonex) and mycophenolate mofetil (Cellcept) modulated the hyper-phosphorylation of P65 in B cells of RRMS patients at levels similar to healthy donor controls. Lower disease activity after the combination therapy correlated with the reduced phosphorylation of P65 following CD40 stimulation in treated patients. In addition, glatiramer acetate (GA) treatment also significantly reduced CD40-mediated P65 phosphorylation in RRMS patients, suggesting that reducing CD40-mediated p-P65 induction may be a general mechanism by which some current therapies modulate MS disease. PMID:27798157

  10. Effect of CD40/CD40L signaling on IL-10-producing regulatory B cells in Chinese children with Henoch-Schönlein purpura nephritis.

    PubMed

    Yang, Baohui; Tan, Xiongjun; Xiong, Xiao; Wu, Daoqi; Zhang, Gaofu; Wang, Mo; Dong, Shifang; Liu, Wei; Yang, Haiping; Li, Qiu

    2016-11-11

    The aim of the present study was to examine the role and mechanism of interleukin-10 (IL-10)-producing regulatory B cells (B10 cells) in the pathogenesis of Henoch-Schönlein purpura nephritis (HSPN). We examined the percentage of B10 cells, CD19(+)CD24(hi)CD38(hi) B cells, CD19(+)CD24(hi)CD27(+) B cells, Th17 cells, and T regulatory (Treg) cells within the peripheral blood mononuclear cell (PBMC) population in healthy subjects and HSP/HSPN patients. The percentage of B10 cells and CD19(+)CD24(hi)CD38(hi) B cells was reduced in HSPN patients and that of CD19(+)CD24(hi)CD27(+) B cells was decreased only in HSPN patients with hematuria and proteinuria or massive proteinuria. The expression of IL-10 by B10 cells and their subsets was decreased in HSPN patients and returned to normal levels in HSP/HSPN patients in remission. B10 cells and their subsets negatively correlated with the Th17/Treg ratio. There was no difference in B10pro + B10 cells, Th17 cells, Treg cells, and the Th17/Treg ratio between children with HSP/HSPN and healthy controls after CD40L stimulation. On the other hand, the level of IL-10 expressed by CD19(+)CD40(+) B cells was decreased in HSPN, and the percentage of B10pro + B10 cells and Treg cells was reduced and that of Th17 cell was increased in the presence of anti-CD40L monoclonal antibody (mAb). Thus, decreased B10 cells and CD19(+)CD24(hi)CD38(hi) B cells may function as an early marker of renal impairment in HSPN. The dysfunction of B10 cells may play a role in the pathogenesis of HSPN by regulating the Th17/Treg balance. Moreover, the CD40/CD40L signaling pathway may play a role in B10 cell differentiation and functional maturation.

  11. CD40 Ligand enhances immunogenicity of vector-based vaccines in immunocompetent and CD4+ T cell deficient individuals

    PubMed Central

    Auten, Matthew W.; Huang, Weitao; Dai, Guixiang; Ramsay, Alistair J.

    2012-01-01

    Impairment of host immunity, particularly CD4+ T cell deficiency, presents significant complications for vaccine immunogenicity and efficacy. CD40 ligand (CD40L or CD154), a member of the tumor necrosis factor superfamily (TNFSF), is an important co-stimulatory molecule and, through interactions with its cognate receptor CD40, plays a pivotal role in the generation of host immune responses. Exploitation of CD40L and its receptor CD40 could provide a means to enhance and potentially restore protective immune responses in CD4+ T cell deficiency. To investigate the potential adjuvanticity of CD40L, we constructed recombinant plasmid DNA and adenoviral (Ad) vaccine vectors expressing murine CD40L and the mycobacterial protein antigen 85B (Ag85B). Co-immunization of mice with CD40L and Ag85B by intranasal or intramuscular prime-boosting led to route-dependent enhancement of the magnitude of vaccine-induced circulating and lung mucosal CD4+ and CD8+ T cell responses in both normal (CD4-replete) and CD4+ T cell deficient animals, including polyfunctional T cell responses. The presence of CD40L alone was insufficient to enhance or restore CD4+ T cell responses in CD4-ablated animals; however, in partially-depleted animals, co-immunization with Ag85B and CD40L was capable of eliciting enhanced T cell responses, similar to those observed in normal animals, when compared to those given vaccine antigen alone. In summary, these findings show that CD40L has the capacity to enhance the magnitude of vaccine-induced polyfunctional T cell responses in CD4+ T cell deficient mice, and warrants further study as an adjuvant for immunization against opportunistic pathogens in individuals with CD4+ T cell deficiency. PMID:22349523

  12. CD40 activation induces NREM sleep and modulates genes associated with sleep homeostasis.

    PubMed

    Gast, Heidemarie; Müller, Andreas; Lopez, Martin; Meier, Daniel; Huber, Reto; Dechent, Frieder; Prinz, Marco; Emmenegger, Yann; Franken, Paul; Birchler, Thomas; Fontana, Adriano

    2013-01-01

    The T-cell derived cytokine CD40 ligand is overexpressed in patients with autoimmune diseases. Through activation of its receptor, CD40 ligand leads to a tumor necrosis factor (TNF) receptor 1 (TNFR1) dependent impairment of locomotor activity in mice. Here we report that this effect is explained through a promotion of sleep, which was specific to non-rapid eye movement (NREM) sleep while REM sleep was suppressed. The increase in NREM sleep was accompanied by a decrease in EEG delta power during NREM sleep and by a decrease in the expression of transcripts in the cerebral cortex known to be associated with homeostatic sleep drive, such as Homer1a, Early growth response 2, Neuronal pentraxin 2, and Fos-like antigen 2. The effect of CD40 activation was mimicked by peripheral TNF injection and prevented by the TNF blocker etanercept. Our study indicates that sleep-wake dysregulation in autoimmune diseases may result from CD40 induced TNF:TNFR1 mediated alterations of molecular pathways, which regulate sleep-wake behavior.

  13. Simultaneous activation of viral antigen-specific memory CD4+ and CD8+ T-cells using mRNA-electroporated CD40-activated autologous B-cells.

    PubMed

    Van den Bosch, Glenn A; Van Gulck, Ellen; Ponsaerts, Peter; Nijs, Griet; Lenjou, Marc; Apers, Ludwig; Kint, Ilse; Heyndrickx, Leo; Vanham, Guido; Van Bockstaele, Dirk R; Berneman, Zwi N; Van Tendeloo, Viggo F I

    2006-01-01

    Recently, it has become obvious that not only CD8 T-cells, but also CD4 T-helper cells are required for the induction of an effective, long-lasting cellular immune response. In view of the clinical importance of cytomegalovirus (CMV) and human immunodeficiency virus (HIV) infection, we developed 2 strategies to simultaneously reactivate viral antigen-specific memory CD4 and CD8 T-cells of CMV-seropositive and HIV-seropositive subjects using mRNA-electroporated autologous CD40-activated B cells. In the setting of HIV, we provide evidence that CD40-activated B cells can be cultured from HAART-naive HIV-1 seropositive patients. These cells not only express and secrete the HIV p24 antigen after electroporation with codon-optimized HIV-1 gag mRNA, but can also be used to in vitro reactivate Gag antigen-specific interferon-gamma-producing CD4 and CD8 autologous T-cells. For the CMV-specific approach, we applied mRNA coding for the pp65 protein coupled to the lysosomal-associated membrane protein-1 to transfect CD40-activated B cells to induce CMV antigen-specific CD4 and CD8 T-cells. More detailed analysis of the activated interferon-gamma-producing CMV pp65 tetramer positive CD8 T-cells revealed an effector memory phenotype with the capacity to produce interleukin-2. Our findings clearly show that the concomitant activation of both CD4 and CD8 (memory) T-cells using mRNA-electroporated CD40-B cells is feasible in CMV and HIV-1-seropositive persons, which indicates the potential value of this approach for application in cellular immunotherapy of infectious diseases.

  14. Regulated Expansion and Survival of Chimeric Antigen Receptor-Modified T Cells Using Small Molecule-Dependent Inducible MyD88/CD40.

    PubMed

    Foster, Aaron E; Mahendravada, Aruna; Shinners, Nicholas P; Chang, Wei-Chun; Crisostomo, Jeannette; Lu, An; Khalil, Mariam; Morschl, Eva; Shaw, Joanne L; Saha, Sunandan; Duong, MyLinh T; Collinson-Pautz, Matthew R; Torres, David L; Rodriguez, Tania; Pentcheva-Hoang, Tsvetelina; Bayle, J Henri; Slawin, Kevin M; Spencer, David M

    2017-09-06

    Anti-tumor efficacy of T cells engineered to express chimeric antigen receptors (CARs) is dependent on their specificity, survival, and in vivo expansion following adoptive transfer. Toll-like receptor (TLR) and CD40 signaling in T cells can improve persistence and drive proliferation of antigen-specific CD4(+) and CD8(+) T cells following pathogen challenge or in graft-versus-host disease (GvHD) settings, suggesting that these costimulatory pathways may be co-opted to improve CAR-T cell persistence and function. Here, we present a novel strategy to activate TLR and CD40 signaling in human T cells using inducible MyD88/CD40 (iMC), which can be triggered in vivo via the synthetic dimerizing ligand, rimiducid, to provide potent costimulation to CAR-modified T cells. Importantly, the concurrent activation of iMC (with rimiducid) and CAR (by antigen recognition) is required for interleukin (IL)-2 production and robust CAR-T cell expansion and may provide a user-controlled mechanism to amplify CAR-T cell levels in vivo and augment anti-tumor efficacy. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  15. ERK1/2 has an essential role in B cell receptor- and CD40-induced signaling in an in vitro model of germinal center B cell selection.

    PubMed

    Adem, Jemal; Hämäläinen, Aleksi; Ropponen, Antti; Eeva, Jonna; Eray, Mine; Nuutinen, Ulla; Pelkonen, Jukka

    2015-10-01

    Germinal center (GC) B cells undergo apoptosis after B cell receptor (BCR) ligation, unless they receive CD40-mediated survival signal from helper T cells. In the present study, we used a human follicular lymphoma cell line HF1A3, as an in vitro model to study the selection process in germinal centers. We show here that BCR ligation led to immediate ERK1/2 activation and phosphorylations of its downstream targets, Bim EL/L and Bcl-2 (at Ser70) which resulted in short-term survival. On the other hand, during the late phase of BCR signaling, ERK1/2 phosphorylation was inhibited which resulted in apoptosis. In addition, CD40 signaling led to sustained ERK1/2 activation and up-regulation of Bcl-xL in BCR-primed HF1A3 GC B cells. In conclusion, MEK-ERK pathway and Bcl-2 family proteins are crucial players in BCR-mediated survival/apoptosis and CD40-mediated survival.

  16. Phase IA/II, multicentre, open-label study of the CD40 antagonistic monoclonal antibody lucatumumab in adult patients with advanced non-Hodgkin or Hodgkin lymphoma.

    PubMed

    Fanale, Michelle; Assouline, Sarit; Kuruvilla, John; Solal-Céligny, Philippe; Heo, Dae S; Verhoef, Gregor; Corradini, Paolo; Abramson, Jeremy S; Offner, Fritz; Engert, Andreas; Dyer, Martin J S; Carreon, Daniel; Ewald, Brett; Baeck, Johan; Younes, Anas; Freedman, Arnold S

    2014-01-01

    Despite advancements in the treatment of non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL), patients continue to relapse and thus a need for new targeted therapies remains. The CD40 receptor is highly expressed on neoplastic B cells and activation leads to enhanced proliferation and survival. Lucatumumab (HCD122) is a fully human antagonistic CD40 monoclonal antibody. A phase IA/II study was designed to determine the maximum tolerated dose (MTD) and activity of lucatumumab in patients with relapsed/refractory lymphoma. Determination of the MTD was the primary objective of the phase IA dose escalation portion and clinical response was the primary objective of the phase II dose expansion portion. Patients received escalating doses of lucatumumab administered intravenously once weekly for 4 weeks of an 8-week cycle. MTD was determined at 4 mg/kg of lucatumumab. A total of 111 patients with NHL (n = 74) and HL (n = 37) were enrolled. Responses were observed across various lymphoma subtypes. The overall response rate by computed tomography among patients with follicular lymphoma (FL) and marginal zone lymphoma of mucosa-associated lymphatic tissue (MZL/MALT) was 33·3% and 42·9%, respectively. Lucatumumab demonstrates modest activity in relapsed/refractory patients with advanced lymphoma, suggesting that targeting of CD40 warrants further investigation. © 2013 John Wiley & Sons Ltd.

  17. Soluble CD40 ligand: a novel biomarker in the pathogenesis of periodontal disease.

    PubMed

    Chaturvedi, Rashi; Gupta, Mili; Jain, Ashish; Das, Tarun; Prashar, Savita

    2015-01-01

    Periodontitis involves a complex interplay of micro-organisms and host immune response via numerous mediator molecules playing strategic roles in its pathogenesis. Soluble CD40L (sCD40L) is one such co-stimulatory molecule which is essential for T-helper cell activation and is a well-known risk indicator of cardiovascular diseases. The levels of this marker in crevicular fluid of patients of chronic periodontitis have been explored in the present study for the first time along with an analysis of its association with levels in serum in otherwise systemically healthy patients. Sixty patients 20 healthy and 40 of chronic periodontitis (18 moderate and 22 severe) participated in the study. Patients of the diseased group underwent non-surgical periodontal therapy. Clinical evaluation and collection of gingival crevicular fluid (GCF) and serum samples was done at baseline, and 6 weeks after phase I periodontal therapy. sCD40L levels were quantified in the fluids using ELISA. Levels of sCD40L in GCF were significantly higher in the diseased group (p ≤ 0.001) and strongly correlated not only with increasing severity of disease but also with levels in serum. In post-treatment, the levels decreased significantly in both the biological fluids (p ≤ 0.001). The present study brings to light the role of sCD40L as a novel marker in mediating periodontal destruction and disease progression. Evaluation of local treatment outcomes seems promising in minimizing these effects. Positive association of its local levels with those in serum further implicates the possibility of widespread systemic effects of this infection.

  18. Construction and immunological characterization of CD40L or GM-CSF incorporated Hantaan virus like particle

    PubMed Central

    Zhang, Xiaoxiao; Truax, Agnieszka D.; Ma, Ruixue; Liu, Ziyu; Lei, Yingfeng; Zhang, Liang; Ye, Wei; Zhang, Fanglin; Xu, Zhikai; Shang, Lei; Liu, Rongrong; Wang, Fang; Wu, Xingan

    2016-01-01

    Infection of Hantaan virus (HTNV) usually causes hemorrhagic fever with renal syndrome (HFRS). China has the worst epidemic incidence of HFRS as well as high fatality. Inactivated whole virus has been used for HFRS vaccination, however there are still problems such as safety concerns. CD40 ligand (CD40L) and granulocyte macrophage colony-stimulating factor (GM-CSF) are well-known immune stimulating molecules that can enhance antigen presenting, lymphocytes activation and maturation, incorporation of CD40L and GM-CSF to the surface of virus like particles (VLPs) can greatly improve the vaccination effect. We constructed eukaryotic vectors expressing HTNV M segment and S segment, as well as vectors expressing HTNV M segment with CD40L or GM-CSF, our results showed successful production of CD40L or GM-CSF incorporated HTNV VLPs. In vitro stimulation with CD40L or GM-CSF anchored HTNV VLP showed enhanced activation of macrophages and DCs. CD40L/GM-CSF incorporated VLP can induce higher level of HTNV specific antibody and neutralizing antibody in mice. Immunized mice splenocytes showed higher ability of secreting IFN-γ and IL-2, as well as enhancing CTL activity. These results suggest CD40L/GM-CSF incorporated VLP can serve as prospective vaccine candidate. PMID:27542281

  19. INVOLVEMENT OF TOLL-LIKE RECEPTOR 4 AND MAPK PATHWAYS IN LPS-INDUCED CD40 EXPRESSION IN MONOCYTIC CELLS

    EPA Science Inventory

    CD40 is a co-stimulatory surface molecule actively expressed on mature dendritic cells (DC). Recent studies suggest that endotoxin (LPS) inhalation induces DC maturation in the airways of healthy volunteers. To characterize the effect of LPS on CD40 expression and underlying mech...

  20. Reproducibility over time and effect of low-dose aspirin on soluble P-selectin and soluble CD40 ligand.

    PubMed

    Valdes, Vanessa; Nardi, Michael A; Elbaum, Lindsay; Berger, Jeffrey S

    2015-07-01

    Platelet markers [soluble CD40 ligand (sCD40L) and soluble p selectin (sPselectin)] are associated with platelet activation and cardiovascular events. We sought to investigate the reproducibility of these markers over time and the effect of low-dose aspirin on sCD40L and sPselectin in plasma and serum. Following an overnight fast, 40 healthy volunteers had weekly phlebotomy and were administered aspirin 81 mg/day between weeks 3 and 4. Reproducibility over time was assessed by coefficient of variation (CV) and inter-class correlation coefficient. Correlation between markers was assessed using Pearson r statistic. Difference between levels pre- and post-aspirin was measured with Wilcoxon signed-rank test. Data are presented as median (interquartile range). sCD40L and sPselectin measurements were reproducible over time in plasma and serum (CV < 10 %). Measurement of sCD40L and sPselectin in plasma correlated with levels in serum before aspirin and after aspirin. There was no significant correlation between sCD40L and sPselectin. After 1-week of aspirin 81 mg/day, there was a reduction in sCD40L and sPselectin in serum and plasma, respectively. Soluble CD40L and sPselectin are independent markers that are reproducible over time in both plasma and sera and are reduced by 1-week of low-dose aspirin.

  1. Production of canine soluble CD40 ligand to induce maturation of monocyte derived dendritic cells for cancer immunotherapy.

    PubMed

    Wijewardana, Viskam; Sugiura, Kikuya; Yahata, Mana; Akazawa, Takashi; Wijesekera, Daluthgamage Patsy H; Imamoto, Shigeki; Hatoya, Shingo; Inoue, Norimitsu; Inaba, Toshio

    2013-11-15

    CD40 ligand (CD40L) expressed by activated T cells is shown to induce maturation of immature dendritic cells (DCs) and this maturation is a vital part in DC based tumor immunotherapy. We constructed an expression vector by cloning the extracellular domain of canine CD40L fused to the signal sequence of canine IL-12p40. When PBMCs were incubated with canine granulocyte-macrophage (GM) -CSF and IL-4, expression of CD86 was significantly elevated, but the majority of cells displayed the morphology of immature DCs. Following addition of the expressed canine soluble CD40L (csCD40L) to the DC-inducing culture, the cell morphology shifted to that of mature DCs, and expression of CD80, CD86, MHC class II and CD1a was significantly enhanced. This morphological change and enhancement of expression was observed even when the csCD40L was present only in the second half period of the culture. Furthermore, the csCD40L caused a significant increase in IL-12 production from DCs. These results show that the csCD40L significantly promotes the maturation and activation of canine monocyte derived DCs. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. INVOLVEMENT OF TOLL-LIKE RECEPTOR 4 AND MAPK PATHWAYS IN LPS-INDUCED CD40 EXPRESSION IN MONOCYTIC CELLS

    EPA Science Inventory

    CD40 is a co-stimulatory surface molecule actively expressed on mature dendritic cells (DC). Recent studies suggest that endotoxin (LPS) inhalation induces DC maturation in the airways of healthy volunteers. To characterize the effect of LPS on CD40 expression and underlying mech...

  3. Nanovesicle-targeted Kv1.3 knockdown in memory T cells suppresses CD40L expression and memory phenotype.

    PubMed

    Chimote, Ameet A; Hajdu, Peter; Kottyan, Leah C; Harley, John B; Yun, Yeoheung; Conforti, Laura

    2016-05-01

    Ca(2+) signaling controls activation and effector functions of T lymphocytes. Ca(2+) levels also regulate NFAT activation and CD40 ligand (CD40L) expression in T cells. CD40L in activated memory T cells binds to its cognate receptor, CD40, on other cell types resulting in the production of antibodies and pro-inflammatory mediators. The CD40L/CD40 interaction is implicated in the pathogenesis of autoimmune disorders and CD40L is widely recognized as a therapeutic target. Ca(2+) signaling in T cells is regulated by Kv1.3 channels. We have developed lipid nanoparticles that deliver Kv1.3 siRNAs (Kv1.3-NPs) selectively to CD45RO(+) memory T cells and reduce the activation-induced Ca(2+) influx. Herein we report that Kv1.3-NPs reduced NFAT activation and CD40L expression exclusively in CD45RO(+) T cells. Furthermore, Kv1.3-NPs suppressed cytokine release and induced a phenotype switch of T cells from predominantly memory to naïve. These findings indicate that Kv1.3-NPs operate as targeted immune suppressive agents with promising therapeutic potentials.

  4. Involvement of the cytoplasmic cysteine-238 of CD40 in its up-regulation of CD23 expression and its enhancement of TLR4-triggered responses.

    PubMed

    Nadiri, Amal; Jundi, Malek; El Akoum, Souhad; Hassan, Ghada S; Yacoub, Daniel; Mourad, Walid

    2015-11-01

    CD40, a member of the tumor necrosis factor receptor superfamily, plays a key role in both adaptive and innate immunity. Engagement of CD40 with its natural trimeric ligand or with cross-linked antibodies results in disulfide-linked CD40 (dl-CD40) homodimer formation, a process mediated by the cysteine-238 residues of the cytoplasmic tail of CD40. The present study was designed to elucidate the biological relevance of cysteine-238-mediated dl-CD40 homodimers to the expression of CD23 on B cells and to investigate its possible involvement in the innate response. Our results indicate that cysteine-238-mediated dl-CD40 homodimerization is required for CD40-induced activation of PI3-kinase/Akt signaling and the subsequent CD23 expression, as inhibition of dl-CD40 homodimer formation through a point mutation-approach specifically impairs these responses. Interestingly, cysteine-238-mediated dl-CD40 homodimers are also shown to play a crucial role in Toll-like receptor 4-induced CD23 expression, further validating the importance of this system in bridging innate and adaptive immune responses. This process also necessitates the activation of the PI3-kinase/Akt cascade. Thus, our results highlight new roles for CD40 and cysteine-238-mediated CD40 homodimers in cell biology and identify a potential new target for therapeutic strategies against CD40-associated chronic inflammatory diseases.

  5. Canine progenitor epidermal keratinocytes express various inflammatory markers, including interleukin-8 and CD40, which are affected by certain antibiotics.

    PubMed

    White, Amelia G; Wolsic, Cassandra L; Campbell, Karen L; Lavergne, Sidonie N

    2014-12-01

    Bacterial skin infections are common in dogs and humans. Keratinocytes have phenotypic features of nonprofessional antigen-presenting cells and express various cytokines. However, little is known about the effects of antibiotics on inflammatory markers in canine keratinocytes. To investigate inflammatory markers in canine progenitor epidermal keratinocytes (CPEKs) and to determine the effects of selected antibiotics on these markers. The CPEKs were exposed for 2-24 h to three concentrations of amoxicillin, cefalexin, sulfadimethoxine, sulfamethoxazole (or its nitroso metabolite), amikacin or enrofloxacin. Enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry were used to detect major histocompatibility complex (MHC) II. CD40 and CXCR1 [interleukin (IL)-8 receptor] were detected using ELISA. Secreted cytokines/chemokines were quantified using a multiplex kit. No MHC II protein was detected. CD40 protein was found at 24 h, with levels being significantly increased by enrofloxacin. The CPEKs secreted no detectable monocyte chemotactic protein-1; undetectable to low (picogram per millilitre range) concentrations of IL-6, IL-7, IL-10, IL-15, tumour necrosis factor-α, interferon-γ and granulocyte-macrophage colony-stimulating factor; and high (nanogram per millilitre range) concentrations of IL-8. Levels of IL-8 increased over 24 h following cell proliferation. They were significantly increased by enrofloxacin after 8 h, and by cefalexin, sulfadimethoxine, sulfamethoxazole, its nitroso metabolite and enrofloxacin after 24 h. The CPEKs expressed CXCR1. Canine progenitor epidermal keratinocytes express various inflammatory proteins, with expression profiles being affected by certain antibiotics. This supports previous work showing keratinocytes to be mediators of inflammation and demonstrates the potential pro-inflammatory effects of certain antibiotics in the skin. © 2014 ESVD and ACVD.

  6. Determination of thromboxane formation, soluble CD40L release and thrombopoietin clearance in apheresis platelet concentrates.

    PubMed

    Wenzel, Folker; Baertl, Anja; Hohlfeld, Thomas; Zimmermann, Norbert; Weber, Artur Aron; Lorenz, Horst; Giers, Günther

    2012-01-01

    All deleterious changes in platelet morphology, structure and function that occur in platelet concentrates (PC) during storage are titled as the 'platelet storage lesion'. No single in vitro test currently available is sufficient in assessing these changes of platelet quality. The release of soluble CD40 Ligand (sCD40L), the formation of thromboxane (TXB2) and the thrombopoietin (TPO) clearance reflect different aspects of platelet metabolism and activitiy, and were used to examine platelet quality in apheresis platelet products. At days 1, 3 and 5, in single-donor apheresis platelet products (n = 10) under routine storage conditions, sCD40L (measured by ELISA) and TXB2 (measured by RIA) were determined after platelet stimulation (recalcification and clot formation). TPO (measured by ELISA) was determined after an incubation time of 5 h at 37°C with platelet-rich plasma (adjusted initial TPO concentration of about 500 pg/mL). Results were related to a therapeutic unit (TU = 2 × 10(11) platelets). Immediately after platelet preparation, sCD40L release was 41 ± 7.6 ng/TU, TXB2 formation 1688 ± 374 ng/TU and TPO clearance 1.22 ± 0.32 ng/h/TU. At days 1, 3 and 5, sCD40L was reduced to 89 ± 7%, 71 ± 12% and 57 ± 9%, TXB2 release to 91 ± 6%, 74 ± 12% and 58 ± 9% and TPO clearance to 90 ± 15%, 84 ± 5% and 79 ± 10% of the respective control values. In conclusion, in single-donor apheresis PC, sCD40L release and TXB2 formation as well as TPO clearance by the platelets were dependent on storage duration and reduced to about 60% to 80% of the respective control values after a storage period for 5 days. These findings are in line with literature data, indicating that a loss of platelet functionality of about 30% will occur after 5 days of storage.

  7. A Salmonella typhi OmpC fusion protein expressing the CD154 Trp140–Ser149 amino acid strand binds CD40 and activates a lymphoma B-cell line

    PubMed Central

    Vega, Mario I; Santos-Argumedo, Leopoldo; Huerta-Yepez, Sara; Luría-Perez, Rosendo; Ortiz-Navarrete, Vianney; Isibasi, Armado; González-Bonilla, Cesar R

    2003-01-01

    CD154 is a type II glycoprotein member of the tumour necrosis factor (TNF) ligand family, which is expressed mainly on the surface of activated T lymphocytes. The interaction with its receptor CD40, plays a central role in the control of several functions of the immune system. Structural models based on the homology of CD154 with TNF and lymphotoxin indicate that binding to CD40 involves three regions surrounding amino acids K143, R203 and Q220, and that strands W140–S149 and S198–A210 are critical for such interactions. Also, it has been reported that two recombinant CD154 fragments, including amino acid residues Y45–L261 or E108–L261 are biologically active, whereas other polypeptides, including S149–L261, are not. Therefore, we decided to construct a fusion protein inserting the W140-S149 amino acid strand (WAEKGYYTMS) in an external loop of the outer membrane protein C (OmpC) from Salmonella enterica serovar Typhi and assess its ability to bind CD40 and activate B cells. The sodium dodecyl sulphate–polyacrylamide gel electrophoresis demonstrated that the chimeric OmpC–gp39 protein conserved its ability to form trimers. Binding to CD40 was established by three variants of enzyme-linked immunosorbent assay, a direct binding assay by coating plates with a recombinant CD40–Fc protein and through two competition assays between OmpC–gp39 and recombinant CD154 or soluble CD40–Fc. Flow cytometry analysis demonstrated that OmpC–gp39 increased the expression levels of major histocompatibility complex II, CD23, and CD80, in Raji human B-cell lymphoma similarly to an antibody against CD40. These results further support that the CD154/CD40 interaction is similar to the TNF/TNF receptor. This is the first report of a bacterial fusion protein containing a small amino acid strand form a ligand that is able to activate its cognate receptor. PMID:14511234

  8. Interruption of CD40 Pathway Improves Efficacy of Transplanted Endothelial Progenitor Cells in Monocrotaline Induced Pulmonary Arterial Hypertension.

    PubMed

    YanYun, Pan; Wang, Shuai; Yang, JinXiu; Chen, Bin; Sun, ZeWei; Ye, LiFang; Zhu, JianHua; Wang, XingXiang

    2015-01-01

    Transplantation of endothelial progenitor cells (EPCs) plays a therapeutic role in pulmonary arterial hypertension (PAH). Meanwhile, recruitment of progenitors has potential inflammatory effects and exaggerates vascular injury. CD40 pathway is identified as a major player in vascular inflammatory events. In this study, we investigated the role of CD40 pathway in regulating early outgrowth EPC functions, and searched for improvements in PAH cell therapy. EPCs were isolated from rat bone marrow and cultured for 7 days. After treatment with soluble CD40 ligand (sCD40L) for 24 hours, EPC migration, adhesion, proliferation, paracrine and vasculogenesis functions were tested. Rat PAH model was founded by subcutaneous injection of monocrotaline (MCT). Control EPCs or lentivirus vectors (Lv)-shRNA-CD40 EPCs were infused via tail vein at day 7, 14, and 21 after MCT injection. Therapeutic effects were evaluated at day 28. sCD40L dose-dependently impaired EPC migration, adhesion, proliferation, and vasculogenesis functions. However, paracrine effects of soluble intercellular adhesion molecule-1, vascular endothelial growth factor and interleukin-6 were dose-dependently improved by sCD40L. Control EPC-derived conditioned medium protected endothelial cell in vitro vasculogenesis, while sCD40L-pretreated ones showed detrimental effects. After MCT injection, sCD40L levels in rat serum increased gradually. Other than in vitro results, benefits of both two EPC treatments were obvious, even taken at day 21. Benefits of control EPCs wore off over time, but those of Lv-shRNA-CD40 EPCs were more effective and enduring, as characterized by both ameliorated rat hemodynamic and reversed vascular remodeling. Furthermore, Lv-shRNA-CD40 EPCs integrated into endothelium better, rather than into adventitia and media. sCD40L impaired protective effects of EPCs. Traditional EPC treatments were limited in PAH, while interruption of CD40 pathway of transplanted cells could apparently improve the

  9. Identification of Signaling Pathways by Which CD40 Stimulates Autophagy and Antimicrobial Activity against Toxoplasma gondii in Macrophages

    PubMed Central

    Liu, Elizabeth; Lopez Corcino, Yalitza; Portillo, Jose-Andres C.; Miao, Yanling

    2016-01-01

    CD40 is an important stimulator of autophagy and autophagic killing of Toxoplasma gondii in host cells. In contrast to autophagy induced by nutrient deprivation or pattern recognition receptors, less is known about the effects of cell-mediated immunity on Beclin 1 and ULK1, key regulators of autophagy. Here we studied the molecular mechanisms by which CD40 stimulates autophagy in macrophages. CD40 ligation caused biphasic Jun N-terminal protein kinase (JNK) phosphorylation. The second phase of JNK phosphorylation was dependent on autocrine production of tumor necrosis factor alpha (TNF-α). TNF-α and JNK signaling were required for the CD40-induced increase in autophagy. JNK signaling downstream of CD40 caused Ser-87 phosphorylation of Bcl-2 and dissociation between Bcl-2 and Beclin 1, an event known to stimulate the autophagic function of Beclin 1. However, TNF-α alone was unable to stimulate autophagy. CD40 also stimulated autophagy via a pathway that included calcium/calmodulin-dependent kinase kinase β (CaMKKβ), AMP-activated protein kinase (AMPK), and ULK1. CD40 caused AMPK phosphorylation at its activating site, Thr-172. This effect was mediated by CaMKKβ and was not impaired by neutralization of TNF-α. CD40 triggered AMPK-dependent Ser-555 phosphorylation of ULK1. CaMKKβ, AMPK, and ULK1 were required for CD40-induced increase in autophagy. CD40-mediated autophagic killing of Toxoplasma gondii is known to require TNF-α. Knockdown of JNK, CaMKKβ, AMPK, or ULK1 prevented T. gondii killing in CD40-activated macrophages. The second phase of JNK phosphorylation—Bcl-2 phosphorylation—Bcl-2–Beclin 1 dissociation and AMPK phosphorylation-ULK1 phosphorylation occurred simultaneously at ∼4 h post-CD40 stimulation. Thus, CaMKKβ and TNF-α are upstream molecules by which CD40 acts on ULK1 and Beclin 1 to stimulate autophagy and killing of T. gondii. PMID:27354443

  10. Update on CD40 and CD154 blockade in transplant models

    PubMed Central

    Zhang, Tianshu; Pierson, Richard N; Azimzadeh, Agnes M

    2015-01-01

    Generation of an effective immune response against foreign antigens requires two distinct molecular signals: a primary signal provided by the binding of antigen-specific T-cell receptor to peptide-MHC on antigen-presenting cells and a secondary signal delivered via the engagement of costimulatory molecules. Among various costimulatory signaling pathways, the interactions between CD40 and its ligand CD154 have been extensively investigated given their essential roles in the modulation of adaptive immunity. Here, we review current understanding of the role CD40/CD154 costimulation pathway has in alloimmunity, and summarize recent mechanistic and preclinical advances in the evaluation of candidate therapeutic approaches to target this receptor-ligand pair in transplantation. PMID:26268734

  11. Stimulation through CD40 and TLR-4 Is an Effective Host Directed Therapy against Mycobacterium tuberculosis

    PubMed Central

    Khan, Nargis; Pahari, Susanta; Vidyarthi, Aurobind; Aqdas, Mohammad; Agrewala, Javed N.

    2016-01-01

    Tuberculosis (TB) is the leading cause of morbidity and mortality among all infectious diseases. Failure of Bacillus Calmette Guerin as a vaccine and serious side-effects and toxicity due to long-term TB drug regime are the major hurdles associated with TB control. The problem is further compounded by the emergence of drug-resistance strains of Mycobacterium tuberculosis (Mtb). Consequently, it demands a serious attempt to explore safer and superior treatment approaches. Recently, an improved understanding of host–pathogen interaction has opened up new avenues for immunotherapy for treating TB. Although, dendritic cells (DCs) show a profound role in generating immunity against Mtb, their immunotherapeutic potential needs to be precisely investigated in controlling TB. Here, we have devised an approach of bolstering DCs efficacy against Mtb by delivering signals through CD40 and TLR-4 molecules. We found that DCs triggered through CD40 and TLR-4 showed increased secretion of IL-12, IL-6, and TNF-α. It also augmented autophagy. Interestingly, CD40 and TLR-4 stimulation along with the suboptimal dose of anti-TB drugs significantly fortified their efficacy to kill Mtb. Importantly, animals treated with the agonists of CD40 and TLR-4 boosted Th1 and Th17 immunity. Furthermore, it amplified the pool of memory CD4 T cells as well as CD8 T cells. Furthermore, substantial reduction in the bacterial burden in the lungs was observed. Notably, this adjunct therapy employing immunomodulators and chemotherapy can reinvigorate host immunity suppressed due to drugs and Mtb. Moreover, it would strengthen the potency of drugs in curing TB. PMID:27729911

  12. Dendritic and tumor cell fusions transduced with adenovirus encoding CD40L eradicate B-cell lymphoma and induce a Th17-type response.

    PubMed

    Alvarez, E; Moga, E; Barquinero, J; Sierra, J; Briones, J

    2010-04-01

    Fusion of dendritic cells and tumor cells (FCs) constitutes a promising tool for generating an antitumor response because of their capacity to present tumor antigens and provide appropriate costimulatory signals. CD40-CD40L interaction has an important role in the maturation and survival of dendritic cells and provides critical help for T-cell priming. In this study, we sought to improve the effectiveness of FC vaccines in a murine model of B-cell lymphoma by engineering FCs to express CD40L by means of an adenovirus encoding CD40L (Adv-CD40L). Before transduction with Adv-CD40L, no CD40L expression was detected in FCs, DCs or tumor cells. The surface expression of CD40L in FC transduced with Adv-CD40L (FC-CD40L) ranged between 50 and 60%. FC-CD40L showed an enhanced expression of CD80, CD86, CD54 and MHC class II molecules and elicited a strong in vitro immune response in a syngeneic mixed lymphocyte reaction. Furthermore, FC-CD40L showed enhanced migration to secondary lymphoid organs. Splenocytes from mice treated with FC-CD40L had a dramatic increase in the production of IL-17, IL-6 and IFN-gamma, compared with controls. Treatment with the FC-CD40L vaccine induced regression of established tumors and increased survival. Our data demonstrate that FC transduced with Adv-CD40L enhances the antitumor effect of FC vaccines in a murine lymphoma model and this is associated with an increased Th17-type immune response.

  13. Potential predictive role of chemotherapy-induced changes of soluble CD40 ligand in untreated advanced pancreatic ductal adenocarcinoma

    PubMed Central

    Azzariti, Amalia; Brunetti, Oronzo; Porcelli, Letizia; Graziano, Giusi; Iacobazzi, Rosa Maria; Signorile, Michele; Scarpa, Aldo; Lorusso, Vito; Silvestris, Nicola

    2016-01-01

    Pancreas ductal adenocarcinoma lacks predictive biomarkers. CD40 is a member of the tumor necrosis factor superfamily. CD40–sCD40L interaction is considered to contribute to the promotion of tumor cell growth and angiogenesis. The aim of the present study was to investigate the role of serum sCD40L as a predictor in metastatic pancreatic cancer. We evaluated 27 consecutive pancreatic cancer patients treated with FOLFIRINOX (21 patients) or gemcitabine plus nab-paclitaxel combination (six patients). The sCD40L level was measured in serum by enzyme-linked immunosorbent assay at baseline, at first evaluation (all patients), and at time to progression (18 patients). The radiological response was evaluated according to the Response Evaluation Criteria in Solid Tumors, Version 1.1. The Wilcoxon signed-rank test was used to compare pre–post treatment sCD40L levels with respect to clinical response, while Pearson’s correlation coefficient was used for the correlation between sCD40L and CA19.9 pre- and post-treatment. The Kruskal–Wallis test was also conducted for further comparisons. We observed a statistically significant reduction in the sCD40L level after 3 months of treatment in patients with partial response (11,718.05±7,097.13 pg/mL vs 4,689.42±5,409.96 pg/mL; P<0.01). Conversely, in patients with progressive disease, the biomarker statistically increased in the same time (9,351.51±7,356.91 pg/mL vs 22,282.92±11,629.35 pg/mL; P<0.01). This trend of sCD40L was confirmed in 18 patients at time to progression after the first evaluation. No differences were recorded within the stable disease group. Moreover, there was a positive correlation between the sCD40L and CA19.9 pre–post treatment variation percentage (Pearson’s correlation coefficient =0.52; P<0.05). Our data suggest a possible predictive role of sCD40L in pancreatic cancer patients, similar to CA19.9. PMID:27555786

  14. Characterisation of the TNF superfamily members CD40L and BAFF in the small-spotted catshark (Scyliorhinus canicula).

    PubMed

    Li, Ronggai; Redmond, Anthony K; Wang, Tiehui; Bird, Steve; Dooley, Helen; Secombes, Chris J

    2015-11-01

    The tumour necrosis factor superfamily (TNFSF) members CD40L and BAFF play critical roles in mammalian B cell survival, proliferation and maturation, however little is known about these key cytokines in the oldest jawed vertebrates, the cartilaginous fishes. Here we report the cloning of CD40L and BAFF orthologues (designated ScCD40L and ScBAFF) in the small-spotted catshark (Scyliorhinus canicula). As predicted both proteins are type II membrane-bound proteins with a TNF homology domain in their extracellular region and both are highly expressed in shark immune tissues. ScCD40L transcript levels correlate with those of TCRα and transcription of both genes is modulated in peripheral blood leukocytes following in vitro stimulation. Although a putative CD40L orthologue was identified in the elephant shark genome the work herein is the first molecular characterisation and transcriptional analysis of CD40L in a cartilaginous fish. ScBAFF was also cloned and its transcription characterised in an attempt to resolve the discrepancies observed between spiny dogfish BAFF and bamboo shark BAFF in previously published studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Ribavirin increases mitogen- and antigen-induced expression of CD40L on CD4+ T cells in vivo

    PubMed Central

    Bergamini, A; Cepparulo, M; Bolacchi, F; Araco, A; Tisone, G; Ombres, D; Rocchi, G; Angelico, M

    2002-01-01

    Here, CD40L expression and cytokine production have been analysed in peripheral blood cells from orthotopic liver transplantation (OLT) recipients treated with ribavirin for recurrent chronic hepatitis C. The study included 18 OLT recipients treated with ribavirin, eight control OLT recipients and 10 healthy controls. FACS analysis showed that baseline expression of CD40L was not different between ribavirin-treated patients and controls. In contrast, after stimulation with both HCV core antigen and phorbol myristate acetate (PMA) plus ionomycin (IO), the expression of CD40L on CD4 lymphocytes was significantly higher in the ribavirin group compared with controls. In the ribavirin group, the increased expression of CD40L significantly correlated with reduction of HCV RNA levels with respect to pretreatment values. Finally, ribavirin treatment was not associated with modification of PMA-IO-induced cytokine production by T lymphocytes and interleukin (IL)-1β and tumour necrosis-α (TNF)-α production by CD40L-stimulated monocytes. In conclusion, these data indicate that ribavirin upmodulates CD40L expression on CD4 T cells, a property which may account in part for its ability to enhance the antiviral activity of interferon-α in the treatment of chronic HCV infection. PMID:12390318

  16. Autocrine stimulation of IL-10 is critical to the enrichment of IL-10-producing CD40(hi)CD5(+) regulatory B cells in vitro and in vivo.

    PubMed

    Kim, Hyuk Soon; Lee, Jun Ho; Han, Hee Dong; Kim, A-Ram; Nam, Seung Taek; Kim, Hyun Woo; Park, Young Hwan; Lee, Dajeong; Lee, Min Bum; Park, Yeong Min; Kim, Hyung Sik; Kim, Young Mi; You, Ji Chang; Choi, Wahn Soo

    2015-01-01

    IL-10-producing B (Breg) cells regulate various immune responses. However, their phenotype remains unclear. CD40 expression was significantly increased in B cells by LPS, and the Breg cells were also enriched in CD40(hi)CD5(+) B cells. Furthermore, CD40 expression on Breg cells was increased by IL-10, CD40 ligand, and B cell-activating factor, suggesting that CD40(hi) is a common phenotype of Breg cells. LPS-induced CD40 expression was largely suppressed by an anti-IL-10 receptor antibody and in IL-10(-/-)CD5(+)CD19(+) B cells. The autocrine effect of IL-10 on the CD40 expression was largely suppressed by an inhibitor of JAK/STAT3. In vivo, the LPS treatment increased the population of CD40(hi)CD5(+) Breg cells in mice. However, the population of CD40(hi)CD5(+) B cells was minimal in IL-10(-/-) mice by LPS. Altogether, our findings show that Breg cells are largely enriched in CD40(hi)CD5(+) B cells and the autocrine effect of IL-10 is critical to the formation of CD40(hi)CD5(+) Breg cells.

  17. A Genetically Engineered Adenovirus Vector Targeted to CD40 Mediates Transduction of Canine Dendritic Cells and Promotes Antigen-Specific Immune Responses In Vivo

    PubMed Central

    Thacker, Erin E.; Nakayama, Masaharu; Smith, Bruce F.; Bird, R. Curtis; Muminova, Zhanat; Strong, Theresa; Timares, Laura; Korokhov, Nikolay; O'Neill, Ann Marie; de Gruijl, Tanja D.; Glasgow, Joel N.; Tani, Kenzaburo; Curiel, David T.

    2009-01-01

    Targeting viral vectors encoding tumor-associated antigens to dendritic cells (DCs) in vivo is likely to enhance the effectiveness of immunotherapeutic cancer vaccines. We have previously shown that genetic modification of adenovirus (Ad) 5 to incorporate CD40 ligand (CD40L) rather than native fiber allows selective transduction and activation of DCs in vitro. Here, we examine the capacity of this targeted vector to induce immune responses to the tumor antigen CEA in a stringent in vivo canine model. CD40-targeted Ad5 transduced canine DCs via the CD40-CD40L pathway in vitro, and following vaccination of healthy dogs, CD40-targeted Ad5 induced strong anti-CEA cellular and humoral responses. These data validate the canine model for future translational studies and suggest targeting of Ad5 vectors to CD40 for in vivo delivery of tumor antigens to DCs is a feasible approach for successful cancer therapy. PMID:19786146

  18. Phase I study of the CD40 agonist antibody CP-870,893 combined with carboplatin and paclitaxel in patients with advanced solid tumors.

    PubMed

    Vonderheide, Robert H; Burg, Jennifer M; Mick, Rosemarie; Trosko, Jennifer A; Li, Dongguang; Shaik, M Naveed; Tolcher, Anthony W; Hamid, Omid

    2013-01-01

    CD40 is a cell-surface molecule that critically regulates immune responses. CP-870,893 is a fully human, CD40-specific agonist monoclonal antibody (mAb) exerting clinical antineoplastic activity. Here, the safety of CP-870,893 combined with carboplatin and paclitaxel was assessed in a Phase I study. Patients with advanced solid tumors received standard doses of paclitaxel and carboplatin on day 1 followed by either 0.1 mg/Kg or 0.2 mg/Kg CP-870,893 on day 3 (Schedule A) or day 8 (Schedule B), repeated every 21 d. The primary objective was to determine safety and maximum-tolerated dose (MTD) of CP-870,893. Secondary objectives included the evaluation of antitumor responses, pharmacokinetics and immune modulation. Thirty-two patients were treated with CP-870,893, 16 patients on each schedule. Two dose-limiting toxicities were observed (grade 3 cytokine release and transient ischemic attack), each at the 0.2 mg/Kg dose level, which was estimated to be the MTD. The most common treatment-related adverse event was fatigue (81%). Of 30 evaluable patients, 6 (20%) exhibited partial responses constituting best responses as defined by RECIST. Following CP-870,893 infusion, the peripheral blood manifested an acute depletion of B cells associated with upregulation of immune co-stimulatory molecules. T-cell numbers did not change significantly from baseline, but transient tumor-specific T-cell responses were observed in a small number of evaluable patients. The CD40 agonist mAb CP-870,893, given on either of two schedules in combination with paclitaxel and carboplatin, was safe for patients affected with advanced solid tumors. Biological and clinical responses were observed, providing a rationale for Phase II studies.

  19. The interleukin-2 receptor α chain (CD25) plays an important role in regulating monocyte-derived CD40 expression during anti-porcine cellular responses.

    PubMed

    Sun, Z-G; Wang, Z; Zhu, L-M; Fang, Y-S; Yu, L-Z; Xu, H

    2012-05-01

    Long-term xenograft survival is limited by delayed xenograft rejection, and monocytes are thought to play an important role in this process. Although typically considered a T cell surface marker, interleukin 2 the receptor chain CD25 is also functional on monocytes. We hypothesized that CD25 expression on monocytes functions to augment monocyte activation in xeno-specific cellular responses. Xenogeneic mixed lymphocyte-endothelial cell reactions were used to study the role of CD25 in facilitating xenogeneic cell-mediated immune responses an in vitro. We also tested the effect of the anti-CD25 antibody daclizumab on monocyte-mediated T cell activation during xeno-specific cellular responses. Co-culture with porcine endothelial cells (PEC) elicited a pronounced proliferative response by human peripheral blood mononuclear cells (PBMC) that was accompanied by upregulation of CD25 and CD40 on CD14(+) monocytes. CD4(+) cells proliferated in response to PEC-conditioned monocytes, while blockade of CD25 with daclizumab reduced CD4(+) cell proliferation in the presence of PEC-conditioned monocytes. In addition, daclizumab inhibited proliferation of PBMC in responses to PEC. Analysis of monocytes from PBMC-PEC cocultures by flow cytometry indicated that daclizumab inhibited CD40 upregulation on PEC-activated monocytes. These data demonstrate that CD25 blockade prevents xenogeneic cellular responses by directly blocking CD25 expression on both activated T cells and monocytes. CD25 blockade on T cells or monocytes may indirectly affect upregulation of CD40 on xenoreactive monocytes. Our data strengthen the rationale for incorporating CD25 directed therapy in discordant xenotransplantation.

  20. IgG-mediated anaphylaxis via Fcγ receptor in CD40-deficient mice

    PubMed Central

    Wakayama, H; Hasegawa, Y; Kawabe, T; Saito, H; Kikutani, H; Shimokata, K

    1998-01-01

    Anaphylaxis denotes an immediate hypersensitivity reaction to allergen, exclusively mediated by IgE antibodies. However, IgE antibodies do not explain all the syndromes that are encountered. We investigated potent IgG-mediated anaphylaxis in CD40-deficient mice that lack the immunoglobulin class switching for T cell-dependent antigens. Immunization with ovalbumin did not induce either humoral responses of IgG, IgA, and IgE, or systemic anaphylaxis in CD40-deficient mice. Although systemic anaphylaxis by active immunization was not observed in CD40-deficient mice, both passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis assessed by mouse blood pressure monitoring with cervical artery catheterization did take place when antigen-specific IgG was transferred and then antigen challenge given. Further, to investigate the inflammatory pathway of IgG-mediated immediate hypersensitivity reactions, we focused on the Fcγ receptor (FcγR) function. Pretreatment of the mice with the anti-FcγRII/FcγRIII MoAb clearly blocked the response of PCA and passive systemic anaphylaxis, suggesting that they were initiated through FcγR. In conclusion, we directly demonstrate the IgG-mediated anaphylaxis and its triggering mechanism through FcγR in in vivo conditions. In addition to IgE-mediated anaphylaxis, IgG-mediated anaphylaxis should be considered and the blocking of FcγR would provide one of the therapeutic targets for the control of IgG-mediated hypersensitivity diseases. PMID:9822270

  1. CD40: Novel Association with Crohn's Disease and Replication in Multiple Sclerosis Susceptibility

    PubMed Central

    Alcina, Antonio; Teruel, María; Díaz-Gallo, Lina M.; Gómez-García, María; López-Nevot, Miguel A.; Rodrigo, Luis; Nieto, Antonio; Cardeña, Carlos; Alcain, Guillermo; Díaz-Rubio, Manuel; de la Concha, Emilio G.; Fernandez, Oscar; Arroyo, Rafael

    2010-01-01

    Background A functional polymorphism located at −1 from the start codon of the CD40 gene, rs1883832, was previously reported to disrupt a Kozak sequence essential for translation. It has been consistently associated with Graves' disease risk in populations of different ethnicity and genetic proxies of this variant evaluated in genome-wide association studies have shown evidence of an effect in rheumatoid arthritis and multiple sclerosis (MS) susceptibility. However, the protective allele associated with Graves' disease or rheumatoid arthritis has shown a risk role in MS, an effect that we aimed to replicate in the present work. We hypothesized that this functional polymorphism might also show an association with other complex autoimmune condition such as inflammatory bowel disease, given the CD40 overexpression previously observed in Crohn's disease (CD) lesions. Methodology Genotyping of rs1883832C>T was performed in 1564 MS, 1102 CD and 969 ulcerative colitis (UC) Spanish patients and in 2948 ethnically matched controls by TaqMan chemistry. Principal Findings The observed effect of the minor allele rs1883832T was replicated in our independent Spanish MS cohort [p = 0.025; OR (95% CI) = 1.12 (1.01–1.23)]. The frequency of the minor allele was also significantly higher in CD patients than in controls [p = 0.002; OR (95% CI) = 1.19 (1.06–1.33)]. This increased predisposition was not detected in UC patients [p = 0.5; OR (95% CI) = 1.04 (0.93–1.17)]. Conclusion The impact of CD40 rs1883832 on MS and CD risk points to a common signaling shared by these autoimmune conditions. PMID:20634952

  2. Excess soluble CD40L contributes to blood brain barrier permeability in vivo: implications for HIV-associated neurocognitive disorders.

    PubMed

    Davidson, Donna C; Hirschman, Michael P; Sun, Anita; Singh, Meera V; Kasischke, Karl; Maggirwar, Sanjay B

    2012-01-01

    Despite the use of anti-retroviral therapies, a majority of HIV-infected individuals still develop HIV-Associated Neurocognitive Disorders (HAND), indicating that host inflammatory mediators, in addition to viral proteins, may be contributing to these disorders. Consistently, we have previously shown that levels of the inflammatory mediator soluble CD40L (sCD40L) are elevated in the circulation of HIV-infected, cognitively impaired individuals as compared to their infected, non-impaired counterparts. Recent studies from our group suggest a role for the CD40/CD40L dyad in blood brain barrier (BBB) permeability and interestingly, sCD40L is thought to regulate BBB permeability in other inflammatory disorders of the CNS. Using complementary multiphoton microscopy and quantitative analyses in wild-type and CD40L deficient mice, we now reveal that the HIV transactivator of transcription (Tat) can induce BBB permeability in a CD40L-dependent manner. This permeability of the BBB was found to be the result of aberrant platelet activation induced by Tat, since depletion of platelets prior to treatment reversed Tat-induced BBB permeability. Furthermore, Tat treatment led to an increase in granulocyte antigen 1 (Gr1) positive monocytes, indicating an expansion of the inflammatory subset of cells in these mice, which were found to adhere more readily to the brain microvasculature in Tat treated animals. Exploring the mechanisms by which the BBB becomes compromised during HIV infection has the potential to reveal novel therapeutic targets, thereby aiding in the development of adjunct therapies for the management of HAND, which are currently lacking.

  3. Excess Soluble CD40L Contributes to Blood Brain Barrier Permeability In Vivo: Implications for HIV-Associated Neurocognitive Disorders

    PubMed Central

    Davidson, Donna C.; Hirschman, Michael P.; Sun, Anita; Singh, Meera V.; Kasischke, Karl; Maggirwar, Sanjay B.

    2012-01-01

    Despite the use of anti-retroviral therapies, a majority of HIV-infected individuals still develop HIV-Associated Neurocognitive Disorders (HAND), indicating that host inflammatory mediators, in addition to viral proteins, may be contributing to these disorders. Consistently, we have previously shown that levels of the inflammatory mediator soluble CD40L (sCD40L) are elevated in the circulation of HIV-infected, cognitively impaired individuals as compared to their infected, non-impaired counterparts. Recent studies from our group suggest a role for the CD40/CD40L dyad in blood brain barrier (BBB) permeability and interestingly, sCD40L is thought to regulate BBB permeability in other inflammatory disorders of the CNS. Using complementary multiphoton microscopy and quantitative analyses in wild-type and CD40L deficient mice, we now reveal that the HIV transactivator of transcription (Tat) can induce BBB permeability in a CD40L-dependent manner. This permeability of the BBB was found to be the result of aberrant platelet activation induced by Tat, since depletion of platelets prior to treatment reversed Tat-induced BBB permeability. Furthermore, Tat treatment led to an increase in granulocyte antigen 1 (Gr1) positive monocytes, indicating an expansion of the inflammatory subset of cells in these mice, which were found to adhere more readily to the brain microvasculature in Tat treated animals. Exploring the mechanisms by which the BBB becomes compromised during HIV infection has the potential to reveal novel therapeutic targets, thereby aiding in the development of adjunct therapies for the management of HAND, which are currently lacking. PMID:23251626

  4. Metabolism-associated danger signal-induced immune response and reverse immune checkpoint-activated CD40(+) monocyte differentiation.

    PubMed

    Dai, Jin; Fang, Pu; Saredy, Jason; Xi, Hang; Ramon, Cueto; Yang, William; Choi, Eric T; Ji, Yong; Mao, Wei; Yang, Xiaofeng; Wang, Hong

    2017-07-24

    Adaptive immunity is critical for disease progression and modulates T cell (TC) and antigen-presenting cell (APC) functions. Three signals were initially proposed for adaptive immune activation: signal 1 antigen recognition, signal 2 co-stimulation or co-inhibition, and signal 3 cytokine stimulation. In this article, we propose to term signal 2 as an immune checkpoint, which describes interactions of paired molecules leading to stimulation (stimulatory immune checkpoint) or inhibition (inhibitory immune checkpoint) of an immune response. We classify immune checkpoint into two categories: one-way immune checkpoint for forward signaling towards TC only, and two-way immune checkpoint for both forward and reverse signaling towards TC and APC, respectively. Recently, we and others provided evidence suggesting that metabolic risk factors (RF) activate innate and adaptive immunity, involving the induction of immune checkpoint molecules. We summarize these findings and suggest a novel theory, metabolism-associated danger signal (MADS) recognition, by which metabolic RF activate innate and adaptive immunity. We emphasize that MADS activates the reverse immune checkpoint which leads to APC inflammation in innate and adaptive immunity. Our recent evidence is shown that metabolic RF, such as uremic toxin or hyperhomocysteinemia, induced immune checkpoint molecule CD40 expression in monocytes (MC) and elevated serum soluble CD40 ligand (sCD40L) resulting in CD40(+) MC differentiation. We propose that CD40(+) MC is a novel pro-inflammatory MC subset and a reliable biomarker for chronic kidney disease severity. We summarize that CD40:CD40L immune checkpoint can induce TC and APC activation via forward stimulatory, reverse stimulatory, and TC contact-independent immune checkpoints. Finally, we modeled metabolic RF-induced two-way stimulatory immune checkpoint amplification and discussed potential signaling pathways including AP-1, NF-κB, NFAT, STAT, and DNA methylation and their

  5. Vaccination Produces CD4 T Cells with a Novel CD154-CD40-Dependent Cytolytic Mechanism.

    PubMed

    Coler, Rhea N; Hudson, Thomas; Hughes, Sean; Huang, Po-Wei D; Beebe, Elyse A; Orr, Mark T

    2015-10-01

    The discovery of new vaccines against infectious diseases and cancer requires the development of novel adjuvants with well-defined activities. The TLR4 agonist adjuvant GLA-SE elicits robust Th1 responses to a variety of vaccine Ags and is in clinical development for both infectious diseases and cancer. We demonstrate that immunization with a recombinant protein Ag and GLA-SE also induces granzyme A expression in CD4 T cells and produces cytolytic cells that can be detected in vivo. Surprisingly, these in vivo CTLs were CD4 T cells, not CD8 T cells, and this cytolytic activity was not dependent on granzyme A/B or perforin. Unlike previously reported CD4 CTLs, the transcription factors Tbet and Eomes were not necessary for their development. CTL activity was also independent of the Fas ligand-Fas, TRAIL-DR5, and canonical death pathways, indicating a novel mechanism of CTL activity. Rather, the in vivo CD4 CTL activity induced by vaccination required T cell expression of CD154 (CD40L) and target cell expression of CD40. Thus, vaccination with a TLR4 agonist adjuvant induces CD4 CTLs, which kill through a previously unknown CD154-dependent mechanism. Copyright © 2015 by The American Association of Immunologists, Inc.

  6. CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

    PubMed

    Jain, Shweta; Chodisetti, Sathi Babu; Agrewala, Javed N

    2011-01-01

    Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB) cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID), signifying class switch recombination (CSR). Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

  7. Dendritic cell secretion of IL-15 is induced by recombinant huCD40LT and augments the stimulation of antigen-specific cytolytic T cells.

    PubMed

    Kuniyoshi, J S; Kuniyoshi, C J; Lim, A M; Wang, F Y; Bade, E R; Lau, R; Thomas, E K; Weber, J S

    1999-04-10

    Dendritic cells (DC) are professional antigen-presenting cells which stimulate strong proliferative and cytolytic T cell responses. Stimulation of CD40 on dendritic cells by its ligands and anti-CD40 antibodies induces maturation and enhances DC stimulatory ability. In order to understand the mechanism by which ligand:CD40 interactions augment DC function, we assessed the role of T cell stimulatory cytokines IL-12 and IL-15 in the function of DC stimulated with soluble trimeric CD40L, a recombinant fusion protein incorporating three covalently linked extracellular CD40L domains (huCD40LT). Peripheral blood derived DC treated with huCD40LT and/or IFN-gamma were used to stimulate T cell responses in vitro to specific antigens. DC treated with huCD40LT or IFN-gamma/huCD40LT stimulated enhanced T cell proliferation to CASTA, a soluble protein from C. albicans, induced T cells with augmented antigen-specific lysis, and increased the yield of antigen-specific IFN-gamma-producing T cells. IL-15 production by DC was enhanced in cultures treated with huCD40LT and correlated with expansion of antigen-specific cytolytic T cells. Addition of a neutralizing anti-IL-15 monoclonal antibody inhibited the expansion of viral and tumor antigen-specific T cells stimulated by IFN-gamma and huCD40LT-treated DC. In contrast, this enhanced stimulatory ability of DC did not appear to depend on synthesis of IL-12 since huCD40LT treatment stimulated the generation of antigen-specific cytokine producing and cytolytic T cells without increased IL-12 production. Addition of anti-IL-12 monoclonal antibody did not inhibit expansion of these cells. These data suggest that production of IL-15 but not IL-12 is an important factor in the enhanced immunostimulatory ability of huCD40LT-treated DC.

  8. Synergy between CD40 and MyD88 Does Not Influence Host Survival to Salmonella Infection.

    PubMed

    Wenzel, Ulf Alexander; Fernandez-Santoscoy, Maria; Tam, Miguel A; Tegtmeyer, Pia; Wick, Mary Jo

    2015-01-01

    Previous studies using purified toll-like receptor (TLR) ligands plus agonistic anti-CD40 antibodies showed that TLRs and CD40 can act synergistically on dendritic cells (DCs) to optimize T cell activation and Th1 differentiation. However, a synergistic effect of TLRs and CD40 during bacterial infection is not known. Here, we show that mice lacking the TLR adaptor MyD88 alone, or lacking both MyD88 and CD40 [double knockout (DKO) mice], are compromised in survival to Salmonella infection but have intact recruitment of neutrophils and inflammatory monocytes as well as unaltered abundance of DC subsets and DC activation in infected tissues. In contrast to infected wildtype and CD40(-/-) mice, both MyD88(-/-) mice and DKO mice lack detectable serum IFN-γ and have elevated IL-10. A synergistic effect of TLRs and CD40 was revealed in co-culture experiments where OT-II T cell proliferation was compromised when DKO DCs were pulsed with OVA protein and OVA323-339 peptide, but not with heat-killed Salmonella expressing OVA (HKSOVA), relative to MyD88(-/-) DCs. By contrast, MyD88(-/-) or DKO DCs pulsed with any of the antigens had a similar ability to induce IFN-γ that was lower than WT or CD40(-/-) DCs. DKO DCs pulsed with HKSOVA, but not with OVA or OVA323-339, had increased IL-10 relative to MyD88(-/-) DCs. Finally, HKSOVA-pulsed MyD88(-/-) and DKO DCs had similar and low induction of NFκB-dependent and -independent genes upon co-culture with OT-II cells. Overall, our data revealed that synergistic effects of CD40 and MyD88 do not influence host survival to Salmonella infection or serum levels of IFN-γ or IL-10. However, synergistic effects of MyD88 and CD40 may be apparent on some (IL-10 production) but not all (OT-II proliferation and IFN-γ production) DC functions and depend on the complexity of the antigen. Indeed, synergistic effects observed using purified ligands and well-defined antigens may not necessarily apply when complex antigens, such as live bacteria

  9. [Effect of CD40 knock out on cytotoxic effector function in CD8(+) T cell of mice with cigarette smoke-induced emphysema].

    PubMed

    Wang, Q; Deng, T T; Kuang, L J; Qiu, S L; Liang, Y; Zhong, X N; He, Z Y; Zhang, J Q; Bai, J; Li, M H

    2016-05-31

    To explore the effect of CD40 knock out on the cytotoxic function of CD8(+) T cell of mice with cigarette smoke-induced emphysema. A total of 40 male C57 mice were divided into four groups according to the random number table, including CD40(+ /+) control group, CD40(+ /+) smoke-exposure group, CD40(-/-)control group, CD40(-/-)smoke-exposure group. The smoke-exposure groups were exposed to cigarette smoke for 24 weeks to establish emphysema model. Morphological changes were evaluated by linear intercepts. The percentages of CD8, perforin, granzyme B positive cells were evaluated by immunohistochemistry. The mRNA expressions of perforin, granzyme B, interleukin (IL) -27 were measured by fluorescent real time quantitative polymerase chain reaction (RT-PCR). The IL-27 cytokine level was tested by enzyme-linked immunosorbent assay (ELISA). The mean linear intercepts in CD40(+ /+) smoke-exposure group was significantly higher than CD40(+ /+) control group, CD40(-/-)control group, and CD40(-/-)smoke-exposure group [(37.2±3.6) vs (24.0±3.4), (22.5±2.4), (29.9±1.7) μm] (all P<0.05). CD40(-/-)smoke-exposure group was higher than CD40(+ /+) control group, CD40(-/-)control group (all P<0.05). The percentages of CD8 positive, perforin positive and granzyme B positive cells in CD40(+ /+) smoke-exposure group [(16.3±2.3)%, (11.4±2.1)%, (10.7±1.9)%] were significantly higher than CD40(+ /+) control group [(8.3±1.6)%, (5.1±1.2)%, (4.6±1.0)%], CD40(-/-)control group [ (6.4±1.5)%, (4.3±1.0)%, (4.2±1.0)%] and CD40(-/-)smoke-exposure group [(8.6±1.7)%, (5.6±1.3)%, (5.5±1.3)%] (all P<0.05). RT-PCR results showed that the mRNA expressions of perforin, granzyme B and IL-27 in CD40(+ /+) smoke-exposure group [(20.3±7.3), (18.3±12.3), (2.2±0.7)] were significantly higher than CD40(+ /+) control group [(9.4±4.8), (10.6±3.8), (1.3±0.6)], CD40(-/-)control group [ (8.1±3.1), (7.7±3.5), (1.1±0.5)] and CD40(-/-)smoke-exposure group [(12.9±6.2), (10.4±4.6), (1.5±0

  10. Immunosuppression With CD40 Costimulatory Blockade Plus Rapamycin for Simultaneous Islet-Kidney Transplantation in Nonhuman Primates.

    PubMed

    Oura, T; Hotta, K; Lei, J; Markmann, J; Rosales, I; Dehnadi, A; Kawai, K; Ndishabandi, D; Smith, R-N; Cosimi, A B; Kawai, T

    2017-03-01

    The lack of a reliable immunosuppressive regimen that effectively suppresses both renal and islet allograft rejection without islet toxicity hampers a wider clinical application of simultaneous islet-kidney transplantation (SIK). Seven MHC-mismatched SIKs were performed in diabetic cynomolgus monkeys. Two recipients received rabbit antithymocyte globulin (ATG) induction followed by daily tacrolimus and rapamycin (ATG/Tac/Rapa), and five recipients were treated with anti-CD40 monoclonal antibody (mAb) and rapamycin (aCD40/Rapa). Anti-inflammatory therapy, including anti-interleukin-6 receptor mAb and anti-tumor necrosis factor-α mAb, was given in both groups. The ATG/Tac/Rapa recipients failed to achieve long-term islet allograft survival (19 and 26 days) due to poor islet engraftment and cytomegalovirus pneumonia. In contrast, the aCD40/Rapa regimen provided long-term islet and kidney allograft survival (90, 94, >120, >120, and >120 days), with only one recipient developing evidence of allograft rejection. The aCD40/Rapa regimen was also tested in four kidney-alone transplant recipients. All four recipients achieved long-term renal allograft survival (100% at day 120), which was superior to renal allograft survival (62.9% at day 120) with triple immunosuppressive regimen (tacrolimus, mycophenolate mofetil, and steroids). The combination of anti-CD40 mAb and rapamycin is an effective and nontoxic immunosuppressive regimen that uses only clinically available agents for kidney and islet recipients.

  11. CD40-signalling abrogates induction of RORγt+ Treg cells by intestinal CD103+ DCs and causes fatal colitis

    PubMed Central

    Barthels, Christian; Ogrinc, Ana; Steyer, Verena; Meier, Stefanie; Simon, Ferdinand; Wimmer, Maria; Blutke, Andreas; Straub, Tobias; Zimber-Strobl, Ursula; Lutgens, Esther; Marconi, Peggy; Ohnmacht, Caspar; Garzetti, Debora; Stecher, Bärbel; Brocker, Thomas

    2017-01-01

    Immune homeostasis in intestinal tissues depends on the generation of regulatory T (Treg) cells. CD103+ dendritic cells (DCs) acquire microbiota-derived material from the gut lumen for transport to draining lymph nodes and generation of receptor-related orphan γt+ (RORγt+) Helios−-induced Treg (iTreg) cells. Here we show CD40-signalling as a microbe-independent signal that can induce migration of CD103+ DCs from the lamina propria (LP) to the mesenteric lymph nodes. Transgenic mice with constitutive CD11c-specific CD40-signalling have reduced numbers of CD103+ DCs in LP and a low frequency of RORγt+Helios− iTreg cells, exacerbated inflammatory Th1/Th17 responses, high titres of microbiota-specific immunoglobulins, dysbiosis and fatal colitis, but no pathology is detected in other tissues. Our data demonstrate a CD40-dependent mechanism capable of abrogating iTreg cell induction by DCs, and suggest that the CD40L/CD40-signalling axis might be able to intervene in the generation of new iTreg cells in order to counter-regulate immune suppression to enhance immunity. PMID:28276457

  12. Soluble CD40 ligand contributes to blood-brain barrier breakdown and central nervous system inflammation in multiple sclerosis and neuromyelitis optica spectrum disorder.

    PubMed

    Masuda, Hiroki; Mori, Masahiro; Uchida, Tomohiko; Uzawa, Akiyuki; Ohtani, Ryohei; Kuwabara, Satoshi

    2017-04-15

    Soluble CD40 ligand (sCD40L) is reported to disrupt the blood-brain barrier (BBB). Cerebrospinal fluid (CSF) and serum sCD40L levels were measured in 29 multiple sclerosis (MS), 29 neuromyelitis optica spectrum disorder (NMOSD), and 27 disease control (DC) patients. In MS, serum sCD40L levels were higher than in DCs and positively correlated with the CSF/serum albumin ratio (Qalb). In NMOSD, CSF sCD40L levels were significantly increased compared to DCs, and were correlated to Qalb, CSF cell counts, protein concentrations, and interleukin-6 levels. sCD40L could be involved in BBB disruption in MS, whereas it may contribute to CNS inflammation in NMOSD. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. The TNFRs OX40, 4-1BB, and CD40 as targets for cancer immunotherapy

    PubMed Central

    Moran, Amy E.; Kovacsovics-Bankowski, Magdalena; Weinberg, Andrew D.

    2013-01-01

    T cell-mediated rejection of tumors requires signals from the T cell receptor and co-stimulatory molecules to license effector functions of tumor-antigen specific T cells. There is also an array of immune suppressive mechanisms within the tumor microenvironment that can suppress anti-tumor immunity. The use of monoclonal antibodies to overcome this suppression and/or enhance tumor-antigen specific T cell responses has shown promise in clinical trials. In particular, targeting co-stimulatory members of the tumor necrosis factor receptor (TNFR) family with agonist Abs enhances T cell function, which has led to encouraging therapeutic results in cancer-bearing hosts. These encouraging data establish TNFRs as important targets for enhancing tumor-specific immune responses in mice and man. This review will focus on agonists that target the TNFRs OX40, 4-1BB, and CD40. PMID:23414607

  14. Stromal endothelial cells establish a bidirectional crosstalk with chronic lymphocytic leukemia cells through the TNF-related factors BAFF, APRIL and CD40L

    PubMed Central

    Cols, Montserrat; Barra, Carolina M.; He, Bing; Puga, Irene; Xu, Weifeng; Chiu, April; Tam, Wayne; Knowles, Daniel M.; Dillon, Stacey R.; Leonard, John P.; Furman, Richard R.; Chen, Kang; Cerutti, Andrea

    2012-01-01

    Chronic lymphocytic leukemia (CLL) is a clonal B cell disorder of unknown origin. Accessory signals from the microenvironment are critical for the survival, expansion and progression of malignant B cells. We found that the CLL stroma included microvascular endothelial cells (MVECs) expressing BAFF and APRIL, two TNF family members related to the T cell-associated B cell-stimulating molecule CD40 ligand (CD40L). Constitutive release of soluble BAFF and APRIL increased upon engagement of CD40 on MVECs by CD40L aberrantly expressed on CLL cells. In addition to enhancing MVEC expression of the CD40 receptor, leukemic CD40L induced cleavases that elicited intracellular processing of pro-BAFF and pro-APRIL proteins in MVECs. The resulting soluble BAFF and APRIL proteins delivered survival, proliferation, Ig gene-remodeling and differentiation signals by activating CLL cells through TACI, BAFF-R and BCMA receptors. BAFF and APRIL further amplified CLL cell survival by up-regulating the expression of leukemic CD40L. Inhibition of TACI, BCMA and BAFF-R expression on CLL cells, abrogation of CD40 expression in MVECs, or suppression of BAFF and APRIL cleavases in MVECs reduced the survival and diversification of malignant B cells. These data indicate that BAFF, APRIL and CD40L form a CLL-enhancing bidirectional signaling network linking neoplastic B cells with the microvascular stroma. PMID:22593611

  15. A Graves' disease-associated Kozak sequence single-nucleotide polymorphism enhances the efficiency of CD40 gene translation: a case for translational pathophysiology.

    PubMed

    Jacobson, Eric M; Concepcion, Erlinda; Oashi, Taiji; Tomer, Yaron

    2005-06-01

    We analyzed the mechanism by which a Graves' disease-associated C/T polymorphism in the Kozak sequence of CD40 affects CD40 expression. CD40 expression levels on B cells in individuals with CT and TT genotypes were decreased by 13.3 and 39.4%, respectively, compared with the levels in CC genotypes (P = 0.012). Similarly, Rat-2 fibroblasts transfected with T-allele cDNA expressed 32.2% less CD40 compared with their C-allele-transfected counterparts (P = 0.004). Additionally, an in vitro transcription/translation system showed that the T-allele makes 15.5% less CD40 than the C-allele (P < 0.001), demonstrating that the effect of the single-nucleotide polymorphism (SNP) on CD40 expression is at the level of translation. However, the SNP did not affect transcription, because the mRNA levels of CD40, as measured by quantitative RT-PCR, were independent of genotype. Therefore, our results may suggest that the C allele of the CD40 Kozak SNP, which is associated with Graves' disease, could predispose to disease by increasing the efficiency of translation of CD40 mRNA.

  16. CD40 ligand exhibits a direct antiviral effect on Herpes Simplex Virus type-1 infection via a PI3K-dependent, autophagy-independent mechanism.

    PubMed

    Vlahava, Virginia-Maria; Eliopoulos, Aristides G; Sourvinos, George

    2015-06-01

    The interaction between CD40 and its ligand, CD40L/CD154, is crucial for the efficient initiation and regulation of immune responses against viruses. Herpes Simplex Virus type-1 (HSV-1) is a neurotropic virus capable of manipulating host responses and exploiting host proteins to establish productive infection. Herein we have examined the impact of CD40L-mediated CD40 activation on HSV-1 replication in U2OS cells stably expressing the CD40 receptor. Treatment of these cells with CD40L significantly reduced the HSV-1 progeny virus compared to non-treated cells. The activation of CD40 signaling did not affect the binding of HSV-1 virions on the cell surface but rather delayed the translocation of VP16 to the nucleus, affecting all stages of viral life cycle. Using pharmacological inhibitors and RNAi we show that inhibition of PI3 kinase but not autophagy reverses the effects of CD40L on HSV-1 replication. Collectively, these data demonstrate that CD40 activation exerts a direct inhibitory effect on HSV-1, initiating from the very early stages of the infection by exploiting PI3 kinase-dependent but autophagy-independent mechanisms.

  17. Expression of CD40 ligand on CD4+ T-cells and platelets correlated to the coronary artery lesion and disease progress in Kawasaki disease.

    PubMed

    Wang, Chih-Lu; Wu, Yu-Tsun; Liu, Chieh-An; Lin, Mei-Wei; Lee, Chia-Jung; Huang, Li-Tung; Yang, Kuender D

    2003-02-01

    Kawasaki disease (KD) is an acute febrile vasculitic syndrome in children. CD40 ligand (CD40L) has been implicated in certain types of vasculitis. We proposed that CD40L expression might be correlated with coronary artery lesions in KD. Blood samples were collected from 43 patients with KD before intravenous immunoglobulin (IVIG) treatment and 3 days afterward. Forty-three age-matched febrile children with various diseases were studied in parallel as controls. CD40L expression on T-cells and platelets were detected by flow cytometry, and soluble CD40L (sCD40L) levels were measured by enzyme-linked immunosorbent assay. We found that CD40L expression on CD4(+) T-cells was significantly higher in patients with KD than in the febrile control (FC) group (28.69 +/- 1.17% vs 4.37 +/- 0.36%). CD40L expression decreased significantly 3 days after IVIG administration (28.69 +/- 1.17% vs 13.53 +/- 0.55%). CD40L expression on platelets from patients with KD was also significantly higher than in the FC group (8.20 +/- 0.41% vs 1.26 +/- 0.12%) and decreased after IVIG therapy. sCD40L levels were also significantly higher in KD patients with those of FC (9.69 +/- 0.45 ng/mL vs 2.25 +/- 0.19 ng/mL) but were not affected by IVIG treatment 3 days afterward (9.69 +/- 0.45 ng/mL vs 9.03 +/- 0.32 ng/mL). More interesting, we found that in KD patients, CD40L expression on CD4(+) T-cells and platelets but not on CD8(+) T-cells or sCD40L was correlated with the occurrence of coronary artery lesions. CD40L might play a role in the immunopathogenesis of KD. IVIG therapy might downregulate CD40L expression, resulting in decrease of CD40L-mediated vascular damage in KD. This implicates that modulation of CD40L expression may benefit to treat KD vasculitis.

  18. The 3'-UTR (CA)n microsatellite on CD40LG gene as a possible genetic marker for rheumatoid arthritis in Mexican population: impact on CD40LG mRNA expression.

    PubMed

    Román-Fernández, I V; Sánchez-Zuno, G A; Padilla-Gutiérrez, J R; Cerpa-Cruz, S; Hernández-Bello, J; Valle, Y; Ramírez-Dueñas, M G; Carrillo, C; Muñoz-Valle, J F

    2017-09-30

    The objective of this study was to determine the association of the CD40LG 3'-UTR (CA)n microsatellite with rheumatoid arthritis (RA) and CD40LG mRNA levels in females from western Mexico. A case-control study with 219 RA patients and 175 control subjects (CS) was conducted. Genotyping was performed by polymerase chain reaction (PCR), X (2) test was used to compare genotype and allele frequencies, and odds ratios and 95% confidence intervals were calculated to evaluate the association between RA and the microsatellite. CD40LG mRNA expression was assessed by real-time quantitative PCR. For comparisons between groups, Kruskal-Wallis or Mann-Whitney U tests for non-parametric data and ANOVA test for parametric data were performed. Among the 13 different alleles identified, CA25 was the most represented (45.4% RA and 46.3% CS). Stratification according to CA repeats as CA25 showed a tendency towards a higher frequency of >CA25 alleles in RA patients (29%) compared to CS (23.4%). There was no association between any genotype and the clinical parameters of RA patients. According to the 2(-∆∆Cq) method, CD40LG mRNA expression in RA patients was 4.5-fold higher compared to CS; this difference was significant when assessed by the 2(-∆Cq) method (p = 0.028). Compared to carriers of the CA25/CA25 genotype, CS carrying the CD40LG mRNA expression (9.97-fold), unlike RA patients, where expression was 2.55-fold higher for >CA25/>CA25 carriers. The 3'-UTR CD40LG (CA)n microsatellite is not a genetic marker for RA in western Mexican population; however, results suggest that it plays a role in the CD40LG mRNA expression.

  19. Constitutive CD40L expression on B cells prematurely terminates germinal center response and leads to augmented plasma cell production in T cell areas.

    PubMed

    Bolduc, Anna; Long, Eugene; Stapler, Dale; Cascalho, Marilia; Tsubata, Takeshi; Koni, Pandelakis A; Shimoda, Michiko

    2010-07-01

    CD40/CD40L engagement is essential to T cell-dependent B cell proliferation and differentiation. However, the precise role of CD40 signaling through cognate T-B interaction in the generation of germinal center and memory B cells is still incompletely understood. To address this issue, a B cell-specific CD40L transgene (CD40LBTg) was introduced into mice with B cell-restricted MHC class II deficiency. Using this mouse model, we show that constitutive CD40L expression on B cells alone could not induce germinal center differentiation of MHC class II-deficient B cells after immunization with T cell-dependent Ag. Thus, some other MHC class II-dependent T cell-derived signals are essential for the generation of germinal center B cells in response to T cell-dependent Ag. In fact, CD40LBTg mice generated a complex Ag-specific IgG1 response, which was greatly enhanced in early, but reduced in late, primary response compared with control mice. We also found that the frequency of Ag-specific germinal center B cells in CD40LBTg mice was abruptly reduced 1 wk after immunization. As a result, the numbers of Ag-specific IgG1 long-lived plasma cells and memory B cells were reduced. By histology, large numbers of Ag-specific plasma cells were found in T cell areas adjacent to Ag-specific germinal centers of CD40LBTg mice, temporarily during the second week of primary response. These results indicate that CD40L expression on B cells prematurely terminated their ongoing germinal center response and produced plasma cells. Our results support the notion that CD40 signaling is an active termination signal for germinal center reaction.

  20. Direct T Cell Activation via CD40 Ligand Generates High Avidity CD8+ T Cells Capable of Breaking Immunological Tolerance for the Control of Tumors

    PubMed Central

    Soong, Ruey-Shyang; Song, Liwen; Trieu, Janson; Lee, Sung Yong; He, Liangmei; Tsai, Ya-Chea; Wu, T.-C.; Hung, Chien-Fu

    2014-01-01

    CD40 and CD40 ligand (CD40L) are costimulatory molecules that play a pivotal role in the proinflammatory immune response. Primarily expressed by activated CD4+ T cells, CD40L binds to CD40 on antigen presenting cells (APCs), thereby inducing APC activation. APCs, in turn, prime cytotoxic T lymphocytes (CTLs). Here, two tumor-associated antigen (TAA) animal models, p53-based and GP100-based, were utilized to examine the ability of CD40-CD40L to improve antigen-specific CTL-mediated antitumor immune responses. Although p53 and GP100 are self-antigens that generate low affinity antigen-specific CD8+ T cells, studies have shown that their functional avidity can be improved with CD40L-expressing APCs. Therefore, in the current study, we immunized mice with a DNA construct encoding a TAA in conjunction with another construct encoding CD40L via intramuscular injection followed by electroporation. We observed a significant increase in the antigen-specific CTL-mediated immune responses as well as the potent antitumor effects in both models. Antibody depletion experiments demonstrated that CD8+ T cells play a crucial role in eliciting antitumor effects in vaccinated mice. Furthermore, we showed that in vitro stimulation with irradiated tumor cells expressing both TAA and CD40L improved the functional avidity of antigen-specific CD8+ T cells. Thus, our data show that vaccination with TAA/CD40L DNA can induce potent antitumor effects against TAA-expressing tumors through the generation of better functioning antigen-specific CD8+ T cells. Our study serves as an important foundation for future clinical translation. PMID:24664420

  1. PU.1 Expression in T Follicular Helper Cells Limits CD40L-Dependent Germinal Center B Cell Development.

    PubMed

    Awe, Olufolakemi; Hufford, Matthew M; Wu, Hao; Pham, Duy; Chang, Hua-Chen; Jabeen, Rukhsana; Dent, Alexander L; Kaplan, Mark H

    2015-10-15

    PU.1 is an ETS family transcription factor that is important for the development of multiple hematopoietic cell lineages. Previous work demonstrated a critical role for PU.1 in promoting Th9 development and in limiting Th2 cytokine production. Whether PU.1 has functions in other Th lineages is not clear. In this study, we examined the effects of ectopic expression of PU.1 in CD4(+) T cells and observed decreased expression of genes involved with the function of T follicular helper (Tfh) cells, including Il21 and Tnfsf5 (encoding CD40L). T cells from conditional mutant mice that lack expression of PU.1 in T cells (Sfpi1(lck-/-)) demonstrated increased production of CD40L and IL-21 in vitro. Following adjuvant-dependent or adjuvant-independent immunization, we observed that Sfpi1(lck-/-) mice had increased numbers of Tfh cells, increased germinal center B cells (GCB cells), and increased Ab production in vivo. This correlated with increased expression of IL-21 and CD40L in Tfh cells from Sfpi1(lck-/-) mice compared with control mice. Finally, although blockade of IL-21 did not affect GCB cells in Sfpi1(lck-/-) mice, anti-CD40L treatment of immunized Sfpi1(lck-/-) mice decreased GCB cell numbers and Ag-specific Ig concentrations. Together, these data indicate an inhibitory role for PU.1 in the function of Tfh cells, germinal centers, and Tfh-dependent humoral immunity.

  2. Incorporation of GM-CSF or CD40L Enhances the Immunogenicity of Hantaan Virus-Like Particles

    PubMed Central

    Cheng, Lin-Feng; Wang, Fang; Zhang, Liang; Yu, Lan; Ye, Wei; Liu, Zi-Yu; Ying, Qi-Kang; Wu, Xing-An; Xu, Zhi-Kai; Zhang, Fang-Lin

    2016-01-01

    A safe and effective Hantaan virus (HTNV) vaccine is highly desirable because HTNV causes an acute and often fatal disease (hemorrhagic fever with renal syndrome, HFRS). Since the immunity of the inactivated vaccine is weak and the safety is poor, HTNV virus-like particles (VLPs) offer an attractive and safe alternative. These particles lack the viral genome but are perceived by the immune system as virus particles. We hypothesized that adding immunostimulatory signals to VLPs would enhance their efficacy. To accomplish this enhancement, we generated chimeric HTNV VLPs containing glycosylphosphatidylinositol (GPI)-anchored granulocyte macrophage colony-stimulating factor (GM-CSF) or CD40 ligand (CD40L) and investigated their biological activity in vitro. The immunization of mice with chimeric HTNV VLPs containing GM-CSF or CD40L induced stronger humoral immune responses and cellular immune responses compared to the HTNV VLPs and Chinese commercial inactivated hantavirus vaccine. Chimeric HTNV VLPs containing GM-CSF or CD40L also protected mice from an HTNV challenge. Altogether, our results suggest that anchoring immunostimulatory molecules into HTNV VLPs can be a potential approach for the control and prevention of HFRS. PMID:28066721

  3. Role of platelet CD40 ligand for endothelial cell-monocyte interaction in the presence of flow

    NASA Astrophysics Data System (ADS)

    Wagner, Andreas H.; Schwarz, Manuel; König, Gerd; Hecker, Markus

    2014-11-01

    CD40 ligand (CD154)-induced ultra-large von Willebrand factor (vWF) multimer-mediated endothelial cell-platelet-monocyte interaction may play an important role in adaptive and maladaptive vascular remodeling processes. Here we analyzed the impact of and conditions favouring the deposition of these multimers on the endothelial cell (EC) surface by way of CD40-CD154 co-stimulation in settings mimicking different forms of blood flow. Upon exposure to low oscillatory shear stress and sCD154, a release of vWF multimers comparable to histamine stimulation was monitored on the EC surface in a string-like fashion. Moreover, ex vivo perfused carotid arteries of wild type mice at low laminar shear stress rates showed a luminal release of vWF as ultra-large vWF multimers (ULVWF) upon stimulation with sCD154 which was absent in blood vessels of CD40 knockout mice. The observed CD40- and flow-dependent vWF release from intact endothelial cells and subsequent vWF multimer formation may facilitate adhesion and subsequent activation of circulating platelets at atherosclerotic predilection sites, which are characterized by disturbed flow patterns. This in turn may amplify endothelial cell-monocyte interaction, thus possibly initiating or promoting early atherosclerotic lesion formation.

  4. Macrophages and Fc-receptor interactions contribute to the antitumour activities of the anti-CD40 antibody SGN-40.

    PubMed

    Oflazoglu, E; Stone, I J; Brown, L; Gordon, K A; van Rooijen, N; Jonas, M; Law, C-L; Grewal, I S; Gerber, H-P

    2009-01-13

    SGN-40 is a therapeutic antibody targeting CD40, which induces potent anti-lymphoma activities via direct apoptotic signalling cells and by cell-mediated cytotoxicity. Here we show antibody-dependent cellular phagocytosis (ADCP) by macrophages to contribute significantly to the therapeutic activities and that the antitumour effects of SGN-40 depend on Fc interactions.

  5. Acute Suppression of Circulating sCD40L during Hyperglycemia and Euglycemic-Hyperinsulinemia in Healthy Young Males

    PubMed Central

    Oliver, Stacy R.; Flores, Rebecca L.; Pontello, Andria M.; Rosa, Jaime S.; Zaldivar, Frank P.; Galassetti, Pietro R.

    2013-01-01

    sCD40L is a pro-atherogenic cytokine, part of the TNF superfamily and consistently associated with obesity, diabetes, and increased cardiovascular (CV) risk. While the role of sCD40L in the onset/progression of CV complications of dysmetabolic diseases may be modulated by acute and/or chronic fluctuations of plasma insulin and glucose, very little has been done to clarify this interaction. The kinetic profile of sCD40L (and, in an exploratory manner, of several immuno-modulatory factors), were measured during hyperglycemia and euglycemic-hyperinsulinemia in a group of ten healthy young males (26.8±1.4 yrs). After an overnight fast, i.v. catheters were placed in antecubital veins of both arms for blood drawing and dextrose/insulin i.v. infusions. Procedures lasted 240 min, including baseline (t = 0–60), hyperglycemia (t = 60–150; plasma glucose ~220 mg/dL via i.v. dextrose infusion); and euglycemic-hyperinsulinemia (t = 150–240; glucose infusion continued to clamp glycemic levels between 80 and 110 mg/dL; constant insulin infusion @ 1.5 mU/kg/min). Plasma for cytokine assays was sampled at 12 separate time-points. Plasma levels of sCD40L were significantly reduced (p<0.01) during hyperglycemia and euglycemic-hyperinsulinemia, paralleling the kinetic profiles of FFA and ketone bodies. This pattern was also observed in other immuno-moduatory factors (notably cortisol and EGF), while (IL-1α, -4, -6, -9, -10, TNF-α, Eotaxin) did not change significantly. Significant reductions of the pro-atherogenic cytokine sCD40L were observed during endogenous and exogenous hyperinsulinemia, independent of prevailing glucose concentration, in young healthy males. Our data suggest a mechanism by which correct insulin action may exert a beneficial protective role against inflammation independent of its immediate glucose-lowering effect. PMID:18797414

  6. Phosphatidylinositol 3-kinase and NF-kappa B/Rel are at the divergence of CD40-mediated proliferation and survival pathways.

    PubMed

    Andjelic, S; Hsia, C; Suzuki, H; Kadowaki, T; Koyasu, S; Liou, H C

    2000-10-01

    CD40 receptor ligation evokes several crucial outcomes for the fate of an activated B cell, including proliferation and survival. Although multiple signaling molecules in the CD40 pathways have been identified, their specific roles in regulating proliferation and maintaining cell viability are still obscure. In this report, we demonstrate that the activation of both phosphatidylinositol 3-kinase (PI-3K) and NF-kappaB/Rel transcription factors is crucial for CD40-mediated proliferation. Furthermore, our data indicate that PI-3K is indispensable for CD40-mediated NF-kappaB/Rel activation. This is achieved via activation of AKT and the degradation of IkappaBalpha. Furthermore, we show that PI-3K activity is necessary for the degradation of cyclin-dependent kinase inhibitor p27kip. Therefore, both of these events comprise the mechanism by which PI-3K controls cell proliferation. In contrast to the absolute requirement of PI-3K and NF-kappaB/Rel for proliferation, these signaling molecules are only partially responsible for CD40-mediated survival, as blocking of PI-3K activity did not lead to apoptosis of anti-CD40-treated cells. However, the PI-3K/NF-kappaB pathway is still required for CD40-induced Bcl-X gene expression. Taken together, our data indicate that multiple survival pathways are triggered via this receptor, whereas NF-kappaB/Rel and PI-3K are crucial for CD40-induced proliferation.

  7. Immunogenicity and protective efficacy of an Eimeria vaccine candidate based on Eimeria tenella immune mapped protein 1 and chicken CD40 ligand.

    PubMed

    Yin, Guangwen; Lin, Qian; Qiu, Jianhan; Qin, Mei; Tang, Xinming; Suo, Xun; Huang, Zhijian; Liu, Xianyong

    2015-05-30

    The CD40 ligand (CD40L) has shown potential as a powerful immunological adjuvant in various studies. Here, the efficacy of a chimeric subunit vaccine, consisting of Eimeria tenella immune mapped protein 1 (EtIMP1) and chicken CD40L, was evaluated against E. tenella infection. The recombinant EtIMP1-CD40L was purified from E. coli over-expressing this protein. Chickens were vaccinated with EtIMP1-CD40L without adjuvant or EtIMP1 with Freund's adjuvant. Immunization of chickens with EtIMP1-CD40L fusion protein resulted in stronger IFN-γ secretion and IgA response than that with only recombinant EtIMP1 with Freund's adjuvant. The clinical effect (cecal lesions, body weights gain, and oocysts shedding) of the EtIMP1-CD40L without adjuvant was also better than that of the EtIMP1 with adjuvant, as evidenced by the difference between the two groups in the oocyst output of E. tenella-challenged chickens. The results suggest that the EtIMP1-CD40L fusion protein can be used as an effective immunogen in the development of subunit vaccines against Eimeria infection.

  8. Multivalent Porous Silicon Nanoparticles Enhance the Immune Activation Potency of Agonistic CD40 Antibody

    PubMed Central

    Gu, Luo; Ruff, Laura E.; Qin, Zhengtao; Corr, Maripat P.; Hedrick, Stephen M.; Sailor, Michael J.

    2012-01-01

    One of the fundamental paradigms in the use of nanoparticles to treat disease is to evade or suppress the immune system in order to minimize systemic side effects and deliver sufficient nanoparticle quantities to the intended tissues. However, the immune system is the body's most important and effective defense against diseases. It protects the host by identifying and eliminating foreign pathogens as well as selfmalignancies. Here we report a nanoparticle engineered to work with the immune system, enhancing the intended activation of antigen presenting cells (APCs). We show that luminescent porous silicon nanoparticles (LPSiNPs), each containing multiple copies of an agonistic antibody (FGK45) to the APC receptor CD40, greatly enhance activation of B cells. The cellular response to the nanoparticle-based stimulators is equivalent to a 30–40 fold larger concentration of free FGK45. The intrinsic near-infrared photoluminescence of LPSiNPs is used to monitor degradation and track the nanoparticles inside APCs. PMID:22689074

  9. A Role for CD154, the CD40 Ligand, in Granulomatous Inflammation

    PubMed Central

    Desmoulière, Alexis; Dewitte, Antoine; Bordeau, Nelly; Costet, Pierre; Bassaganyas, Laia; Fricain, Jean-Christophe; Ripoche, Jean; Lepreux, Sébastien

    2017-01-01

    Granulomatous inflammation is a distinctive form of chronic inflammation in which predominant cells include macrophages, epithelioid cells, and multinucleated giant cells. Mechanisms regulating granulomatous inflammation remain ill-understood. CD154, the ligand of CD40, is a key mediator of inflammation. CD154 confers a proinflammatory phenotype to macrophages and controls several macrophagic functions. Here, we studied the contribution of CD154 in a mouse model of toxic liver injury with carbon tetrachloride and a model of absorbable suture graft. In both models, granulomas are triggered in response to endogenous persistent liver calcified necrotic lesions or by grafted sutures. CD154-deficient mice showed delayed clearance of carbon tetrachloride-induced liver calcified necrotic lesions and impaired progression of suture-induced granuloma. In vitro, CD154 stimulated phagocytosis of opsonized erythrocytes by macrophages, suggesting a potential mechanism for the altered granulomatous inflammation in CD154KO mice. These results suggest that CD154 may contribute to the natural history of granulomatous inflammation. PMID:28785137

  10. Selective IAP inhibition results in sensitization of unstimulated but not CD40-stimulated chronic lymphocytic leukaemia cells to TRAIL-induced apoptosis

    PubMed Central

    Zhuang, Jianguo; Laing, Naomi; Oates, Melanie; Lin, Ke; Johnson, Gillian; Pettitt, Andrew R

    2014-01-01

    Despite recent advances in therapy, chronic lymphocytic leukaemia (CLL) remains incurable and new treatment strategies are therefore urgently required. Inhibitor of apoptosis proteins (IAPs) are over-expressed in CLL, suggesting both a role in disease pathogenesis and the potential for therapeutic targeting. To explore these questions, we evaluated the effects on primary CLL cells of AZD5582, a novel potent and selective inhibitor of IAPs. AZD5582 at nanomolar concentrations induced extensive degradation of cIAP-1 and cIAP-2, but minimally of X chromosome-linked IAP (XIAP). However, these effects of AZD5582 produced little or no direct cytotoxicity, nor did they sensitize CLL cells to p53-dependent killing by fludarabine or p53-independent killing by dexamethasone. In contrast, AZD5582 significantly enhanced apoptosis induced by the death receptor (DR) agonist tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Importantly, killing by TRAIL plus AZD5582 was independent of adverse prognostic features including TP53 deletion which is strongly associated with chemoresistance in CLL. Coculture experiments involving transfected mouse fibroblasts expressing human CD40L (CD154) to mimic the effect of T cells at sites of tissue involvement showed that CD40 stimulation almost completely prevented the killing of CLL cells by TRAIL plus AZD5582 despite up-regulating TRAIL receptors 1 and 2. In conclusion, our findings confirm the rate-limiting, upstream involvement of IAPs in the extrinsic but not intrinsic apoptotic pathway of CLL cells and suggest that drug combinations that simultaneously activate DRs and inhibit IAPs may have therapeutic potential in patients with CLL who have failed T-cell-depleting chemotherapy. PMID:25505620

  11. A High Level of Soluble CD40L Is Associated with P. aeruginosa Infection in Patients with Cystic Fibrosis.

    PubMed

    Bustamante, Adriana Ester; Jaime-Pérez, José Carlos; Cordero-Pérez, Paula; Galindo-Rodríguez, Gabriela; Muñoz-Espinosa, Linda Elsa; Villarreal-Villarreal, César Daniel; Mercado-Longoria, Roberto

    2016-01-01

    The aim of this study was to evaluate whether the concentration of sCD40L, a product of platelet activation, correlates with the presence of Pseudomonas aeruginosa in the airway of patients with cystic fibrosis (CF) and to determine its possible clinical association. Sixty patients with CF, ranging in age from 2 months to 36 years, were studied. The demographics, cystic fibrosis transmembrane conductance regulator (CFTR) genotype, spirometry measurements, radiographic and tomographic scans, platelet count in peripheral blood, sCD40L, IL-6, TNF-α and ICAM1 data were collected. Infection-colonization of the airway was evaluated using sputum and throat swab cultures; the levels of anti-Pseudomonas aeruginosa antibodies (Anti-PaAb) were evaluated. Patients with CF and chronic colonization had anti-PaAb values of 11.6 ± 2.1 ELISA units (EU) and sCD40L in plasma of 1530.9 ±1162.3 pg/mL; those with intermittent infection had 5.7 ± 2.7 EU and 2243.6 ± 1475.9 pg/mL; and those who were never infected had 3.46 ± 1.8 EU (p≤0.001) and 1008.1 ± 746.8 pg/mL (p≤0.01), respectively. The cutoff value of sCD40L of 1255 pg/mL was associated with an area under the ROC (receiver operating characteristic curve) of 0.84 (95% CI, 0.71 to 0.97), reflecting P. aeruginosa infection with a sensitivity of 73% and a specificity of 89%. Lung damage was determined using the Brasfield Score, the Bhalla Score, and spirometry (FVC%, FEV1%) and found to be significantly different among the groups (p≤0.001). Circulating sCD40L levels are increased in patients with cystic fibrosis and P. aeruginosa infection. Soluble CD40L appears to reflect infection and provides a tool for monitoring the evolution of lung deterioration.

  12. A High Level of Soluble CD40L Is Associated with P. aeruginosa Infection in Patients with Cystic Fibrosis

    PubMed Central

    Jaime-Pérez, José Carlos; Cordero-Pérez, Paula; Galindo-Rodríguez, Gabriela; Muñoz-Espinosa, Linda Elsa; Villarreal-Villarreal, César Daniel; Mercado-Longoria, Roberto

    2016-01-01

    Objective The aim of this study was to evaluate whether the concentration of sCD40L, a product of platelet activation, correlates with the presence of Pseudomonas aeruginosa in the airway of patients with cystic fibrosis (CF) and to determine its possible clinical association. Methods Sixty patients with CF, ranging in age from 2 months to 36 years, were studied. The demographics, cystic fibrosis transmembrane conductance regulator (CFTR) genotype, spirometry measurements, radiographic and tomographic scans, platelet count in peripheral blood, sCD40L, IL-6, TNF-α and ICAM1 data were collected. Infection-colonization of the airway was evaluated using sputum and throat swab cultures; the levels of anti-Pseudomonas aeruginosa antibodies (Anti-PaAb) were evaluated. Results Patients with CF and chronic colonization had anti-PaAb values of 11.6 ± 2.1 ELISA units (EU) and sCD40L in plasma of 1530.9 ±1162.3 pg/mL; those with intermittent infection had 5.7 ± 2.7 EU and 2243.6 ± 1475.9 pg/mL; and those who were never infected had 3.46 ± 1.8 EU (p≤0.001) and 1008.1 ± 746.8 pg/mL (p≤0.01), respectively. The cutoff value of sCD40L of 1255 pg/mL was associated with an area under the ROC (receiver operating characteristic curve) of 0.84 (95% CI, 0.71 to 0.97), reflecting P. aeruginosa infection with a sensitivity of 73% and a specificity of 89%. Lung damage was determined using the Brasfield Score, the Bhalla Score, and spirometry (FVC%, FEV1%) and found to be significantly different among the groups (p≤0.001). Conclusion Circulating sCD40L levels are increased in patients with cystic fibrosis and P. aeruginosa infection. Soluble CD40L appears to reflect infection and provides a tool for monitoring the evolution of lung deterioration. PMID:28030642

  13. Increased CD40 Expression Enhances Early STING-Mediated Type I Interferon Response and Host Survival in a Rodent Malaria Model

    PubMed Central

    Yao, Xiangyu; Wu, Jian; Lin, Meng; Sun, Wenxiang; He, Xiao; Gowda, Channe; Bolland, Silvia; Long, Carole A.; Wang, Rongfu; Su, Xin-zhuan

    2016-01-01

    Both type I interferon (IFN-I) and CD40 play a significant role in various infectious diseases, including malaria and autoimmune disorders. CD40 is mostly known to function in adaptive immunity, but previous observations of elevated CD40 levels early after malaria infection of mice led us to investigate its roles in innate IFN-I responses and disease control. Using a Plasmodium yoelii nigeriensis N67 and C57BL/6 mouse model, we showed that infected CD40-/- mice had reduced STING and serum IFN-β levels day-2 post infection, higher day-4 parasitemia, and earlier deaths. CD40 could greatly enhance STING-stimulated luciferase signals driven by the IFN-β promoter in vitro, which was mediated by increased STING protein levels. The ability of CD40 to influence STING expression was confirmed in CD40-/- mice after malaria infection. Substitutions at CD40 TRAF binding domains significantly decreased the IFN-β signals and STING protein level, which was likely mediated by changes in STING ubiquitination and degradation. Increased levels of CD40, STING, and ISRE driven luciferase signal in RAW Lucia were observed after phagocytosis of N67-infected red blood cells (iRBCs), stimulation with parasite DNA/RNA, or with selected TLR ligands [LPS, poly(I:C), and Pam3CSK4]. The results suggest stimulation of CD40 expression by parasite materials through TLR signaling pathways, which was further confirmed in bone marrow derived dendritic cells/macrophages (BMDCs/BMDMs) and splenic DCs from CD40-/-, TLR3-/- TLR4-/-, TRIF-/-, and MyD88-/- mice after iRBC stimulation or parasite infection. Our data connect several signaling pathways consisting of phagocytosis of iRBCs, recognition of parasite DNA/RNA (possibly GPI) by TLRs, elevated levels of CD40 and STING proteins, increased IFN-I production, and longer host survival time. This study reveals previously unrecognized CD40 function in innate IFN-I responses and protective pathways in infections with malaria strains that induce a strong

  14. CD40-independent help by memory CD4 T cells induces pathogenic alloantibody but does not lead to long-lasting humoral immunity

    PubMed Central

    Fan, Ran; Yu, Hong; Valujskikh, Anna

    2014-01-01

    CD40/CD154 interactions are essential for productive antibody responses to T-dependent antigens. Memory CD4 T cells express accelerated helper functions and are less dependent on costimulation when compared to naïve T cells. Here we report that donor-reactive memory CD4 T cells can deliver help to CD40-deficient B cells and induce high titers of IgG alloantibodies that contribute to heart allograft rejection in CD40−/− heart recipients. While cognate interactions between memory helper T cells and B cells are crucial for CD40-independent help, this process is not accompanied by germinal center formation and occurs despite ICOS blockade. Consistent with the extrafollicular nature of T/B cell interactions, CD40-independent help fails to maintain stable levels of serum alloantibody and induce differentiation of long-lived plasma cells and memory B cells. In summary, our data suggest that while CD40-independent help by memory CD4 T cells is sufficient to induce high levels of pathogenic alloantibody, it does not sustain long-lasting anti-donor humoral immunity and B cell memory responses. This information may guide the future use of CD40/CD154 targeting therapies in transplant recipients containing donor-reactive memory T cells. PMID:24102790

  15. Increased CD40 ligation and reduced BCR signalling leads to higher IL-10 production in B-cells from tolerant kidney transplant patients

    PubMed Central

    Nova-Lamperti, Estefania; Chana, Prabhjoat; Mobillo, Paula; Runglall, Manohursingh; Kamra, Yogesh; McGregor, Reuben; Lord, Graham M.; Lechler, Robert I.; Lombardi, Giovanna; Hernandez-Fuentes, Maria P.

    2016-01-01

    Background An increased percentage of peripheral transitional B-cells producing IL-10 has been observed in patients tolerant to kidney allografts. In healthy volunteers, the balance between the CD40 and B-cell receptor (BCR) signalling modulated IL-10 production by B-cells, with stimulation via the BCR decreasing CD40-mediated-IL-10 production. In this study, we evaluate whether in tolerant kidney transplant patients the increased IL-10 production by B-cells was due to an altered CD40 and/or BCR signalling. Methods B-cells obtained from a new cohort of tolerant renal transplant recipients and those from age- and gender-matched healthy volunteers, were activated via CD40 and BCR, either alone or in combination. Results In tolerant patients we observed higher percentages of B-cells producing IL-10 after CD40 ligation and higher expression of CD40L on activated T-cells, compared to healthy controls. Furthermore, B-cells from tolerant recipients had reduced ERK signalling following BCR-mediated activation compared to healthy controls. In keeping with this, combining BCR signalling with CD40 ligation did not reduce IL-10 secretion as was observed in healthy control transitional B-cells. Conclusion Altogether our data suggests that the altered response of B-cells in tolerant recipients may contribute to long-term stable graft acceptance. PMID:27472092

  16. Antitumor effects of anti-CD40/CpG immunotherapy combined with gemcitabine or 5-fluorouracil chemotherapy in the B16 melanoma model.

    PubMed

    Qu, Xiaoyi; Felder, Mildred A R; Perez Horta, Zulmarie; Sondel, Paul M; Rakhmilevich, Alexander L

    2013-12-01

    Our previous studies demonstrated that anti-CD40 mAb (anti-CD40) can synergize with CpG oligodeoxynucleotides (CpG) to mediate antitumor effects by activating myeloid cells, such as macrophages in tumor-bearing mice. Separate teams have shown that chemotherapy with gemcitabine (GEM) or 5-fluorouracil (5-FU) can reduce tumor-induced myeloid-derived suppressor cells (MDSC) in mice. In this study we asked if the same chemotherapy regimens with GEM or 5-FU will enhance the antitumor effect of anti-CD40 and CpG. Using the model of B16 melanoma growing intraperitoneally in syngeneic C57BL/6 mice, we show that these GEM or 5-FU treatment regimens reduced MDSC in the peritoneal cavity of tumor-bearing mice. Treatment of mice with GEM or 5-FU did not significantly affect the antitumor function of macrophages as assessed in vitro. In vivo, treatment with these GEM or 5-FU regimens followed by anti-CD40/CpG resulted in antitumor effects similar to those of anti-CD40/CpG in the absence of GEM or 5-FU. Likewise, reduction of MDSC by in vivo anti-Gr-1 mAb treatment did not significantly affect anti-CD40/CpG antitumor responses. Together, the results show that the GEM or 5-FU chemotherapy regimens did not substantially affect the antitumor effects induced by anti-CD40/CpG immunotherapy.

  17. Immune stimulatory receptor CD40 is required for T-cell suppression and T regulatory cell activation mediated by myeloid-derived suppressor cells in cancer.

    PubMed

    Pan, Ping-Ying; Ma, Ge; Weber, Kaare J; Ozao-Choy, Junko; Wang, George; Yin, Bingjiao; Divino, Celia M; Chen, Shu-Hsia

    2010-01-01

    Immune tolerance to tumors is often associated with accumulation of myeloid-derived suppressor cells (MDSC) and an increase in the number of T-regulatory cells (Treg). In tumor-bearing mice, MDSCs can themselves facilitate the generation of tumor-specific Tregs. In this study, we demonstrate that expression of the immune stimulatory receptor CD40 on MDSCs is required to induce T-cell tolerance and Treg accumulation. In an immune reconstitution model, adoptive transfer of Gr-1+CD115+ monocytic MDSCs derived from CD40-deficient mice failed to recapitulate the ability of wild-type MDSCs to induce tolerance and Treg development in vivo. Agonistic anti-CD40 antibodies phenocopied the effect of CD40 deficiency and also improved the therapeutic efficacy of IL-12 and 4-1BB immunotherapy in the treatment of advanced tumors. Our findings suggest that CD40 is essential not only for MDSC-mediated immune suppression but also for tumor-specific Treg expansion. Blockade of CD40-CD40L interaction between MDSC and Treg may provide a new strategy to ablate tumoral immune suppression and thereby heighten responses to immunotherapy.

  18. Targeting the HA2 subunit of influenza A virus hemagglutinin via CD40L provides universal protection against diverse subtypes

    PubMed Central

    Fan, X; Hashem, A M; Chen, Z; Li, C; Doyle, T; Zhang, Y; Yi, Y; Farnsworth, A; Xu, K; Li, Z; He, R; Li, X; Wang, J

    2015-01-01

    The influenza viral hemagglutinin (HA) is comprised of two subunits. Current influenza vaccine predominantly induces neutralizing antibodies (Abs) against the HA1 subunit, which is constantly evolving in unpredictable fashion. The other subunit, HA2, however, is highly conserved but largely shielded by the HA head domain. Thus, enhancing immune response against HA2 could potentially elicit broadly inhibitory Abs. We generated a recombinant adenovirus (rAd) encoding secreted fusion protein, consisting of codon-optimized HA2 subunit of influenza A/California/7/2009(H1N1) virus fused to a trimerized form of murine CD40L, and determined its ability of inducing protective immunity upon intranasal administration. We found that mice immunized with this recombinant viral vaccine were completely protected against lethal challenge with divergent influenza A virus subtypes including H1N1, H3N2, and H9N2. Codon-optimization of HA2 as well as the use of CD40L as a targeting ligand/molecular adjuvant were indispensable to enhance HA2-specific mucosal IgA and serum IgG levels. Moreover, induction of HA2-specific T-cell responses was dependent on CD40L, as rAd secreting HA2 subunit without CD40L failed to induce any significant levels of T-cell cytokines. Finally, sera obtained from immunized mice were capable of inhibiting 13 subtypes of influenza A viruses in vitro. These results provide proof of concept for a prototype HA2-based universal influenza vaccine. PMID:25052763

  19. Extracellular vesicles released by CD40/IL-4-stimulated CLL cells confer altered functional properties to CD4+ T cells.

    PubMed

    Smallwood, Dawn T; Apollonio, Benedetta; Willimott, Shaun; Lezina, Larissa; Alharthi, Afaf; Ambrose, Ashley R; De Rossi, Giulia; Ramsay, Alan G; Wagner, Simon D

    2016-07-28

    The complex interplay between cancer cells, stromal cells, and immune cells in the tumor microenvironment (TME) regulates tumorigenesis and provides emerging targets for immunotherapies. Crosstalk between CD4(+) T cells and proliferating chronic lymphocytic leukemia (CLL) tumor B cells occurs within lymphoid tissue pseudofollicles, and investigating these interactions is essential to understand both disease pathogenesis and the effects of immunotherapy. Tumor-derived extracellular vesicle (EV) shedding is emerging as an important mode of intercellular communication in the TME. In order to characterize tumor EVs released in response to T-cell-derived TME signals, we performed microRNA (miRNA [miR]) profiling of EVs released from CLL cells stimulated with CD40 and interleukin-4 (IL-4). Our results reveal an enrichment of specific cellular miRNAs including miR-363 within EVs derived from CD40/IL-4-stimulated CLL cells compared with parental cell miRNA content and control EVs from unstimulated CLL cells. We demonstrate that autologous patient CD4(+) T cells internalize CLL-EVs containing miR-363 that targets the immunomodulatory molecule CD69. We further reveal that autologous CD4(+) T cells that are exposed to EVs from CD40/IL-4-stimulated CLL cells exhibit enhanced migration, immunological synapse signaling, and interactions with tumor cells. Knockdown of miR-363 in CLL cells prior to CD40/IL-4 stimulation prevented the ability of CLL-EVs to induce increased synapse signaling and confer altered functional properties to CD4(+) T cells. Taken together, these data reveal a novel role for CLL-EVs in modifying T-cell function that highlights unanticipated complexity of intercellular communication that may have implications for bidirectional CD4(+) T-cell:tumor interactions within the TME. © 2016 by The American Society of Hematology.

  20. The expression and concentration of CD40 ligand in normal pregnancy, preeclampsia, and hemolytic anemia, elevated liver enzymes and low platelet count (HELLP) syndrome.

    PubMed

    Azzam, Hanan A G; Abousamra, Nashwa K; Goda, Hossam; El-Shouky, Reda; El-Gilany, Abdel-Hady

    2013-01-01

    Preeclampsia has been associated with increased platelet activation detected before disease onset. Inappropriate activation of platelets may be involved in pathogenesis in preeclampsia by promoting coagulation and thrombosis and also as a mediator of inflammation. The exaggerated platelet activation and inflammation leading to endothelial damage in preeclampsia can be explained by the CD40-CD40 ligand (CD40L) system. Expression of CD40L on platelets was determined by whole-blood flow cytometry, and serum levels of soluble CD40L (sCD40L) were measured by enzyme-linked immunosorbent assay in 11 women with mild preeclampsia, 11 women with severe preeclampsia, and six women with hemolytic anemia, elevated liver enzymes and low platelet count (HELLP) syndrome compared with 13 normotensive pregnant women as a control group. The platelet surface expression of CD40L was significantly higher in women with mild and severe preeclampsia and HELLP compared with normal pregnancy group (P = 0.001; P ≤ 0.001; P = 0.003, respectively), with no significant difference being found between women with mild preeclampsia compared with HELLP and severe preeclampsia compared with HELLP (P = 0.2; P = 0.8, respectively). The serum concentration of sCD40L was significantly higher in women with mild and severe preeclampsia and HELLP compared with the normal pregnancy group (P = 0.001; P ≤ 0.001; P = 0.022, respectively), with no significant difference being found between women with mild compared with severe preeclampsia or HELLP and severe preeclampsia compared with HELLP (P = 0.7; P = 0.6; P = 0.6, respectively). In conclusion, the higher expression and concentration of CD40L in women with preeclampsia and HELLP syndrome compared with normal pregnant women may indicate an exaggerated activation of platelets and endothelial cells in the disorder.

  1. CD40-TNF activation in mice induces extended sickness behavior syndrome co-incident with but not dependent on activation of the kynurenine pathway.

    PubMed

    Cathomas, Flurin; Fuertig, Rene; Sigrist, Hannes; Newman, Gregory N; Hoop, Vanessa; Bizzozzero, Manuela; Mueller, Andreas; Luippold, Andreas; Ceci, Angelo; Hengerer, Bastian; Seifritz, Erich; Fontana, Adriano; Pryce, Christopher R

    2015-11-01

    The similarity between sickness behavior syndrome (SBS) in infection and autoimmune disorders and certain symptoms in major depressive disorder (MDD), and the high co-morbidity of autoimmune disorders and MDD, constitutes some of the major evidence for the immune-inflammation hypothesis of MDD. CD40 ligand-CD40 immune-activation is important in host response to infection and in development of autoimmunity. Mice given a single intra-peritoneal injection of CD40 agonist antibody (CD40AB) develop SBS for 2-3days characterized by weight loss and increased sleep, effects that are dependent on the cytokine, tumor necrosis factor (TNF). Here we report that CD40AB also induces behavioral effects that extend beyond acute SBS and co-occur with but are not mediated by kynurenine pathway activation and recovery. CD40AB led to decreased saccharin drinking (days 1-7) and decreased Pavlovian fear conditioning (days 5-6), and was without effect on physical fatigue (day 5). These behavioral effects co-occurred with increased plasma and brain levels of kynurenine and its metabolites (days 1-7/8). Co-injection of TNF blocker etanercept with CD40AB prevented each of SBS, reduced saccharin drinking, and kynurenine pathway activation in plasma and brain. Repeated oral administration of a selective indoleamine 2,3-dioxygenase (IDO) inhibitor blocked activation of the kynurenine pathway but was without effect on SBS and saccharin drinking. This study provides novel evidence that CD40-TNF activation induces deficits in saccharin drinking and Pavlovian fear learning and activates the kynurenine pathway, and that CD40-TNF activation of the kynurenine pathway is not necessary for induction of the acute or extended SBS effects. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. TNF-α promotes IFN-γ-induced CD40 expression and antigen process in Myb-transformed hematological cells.

    PubMed

    Gu, Wenyi; Chen, Jiezhong; Yang, Lei; Zhao, Kong-Nan

    2012-01-01

    Tumour necrosis factor-α, interferon-γ and interleukin-4 are critical cytokines in regulating the immune responses against infections and tumours. In this study, we investigated the effects of three cytokines on CD40 expression in Myb-transformed hematological cells and their regulatory roles in promoting these cells into dendritic cells. We observed that both interleukin-4 and interferon-γ increased CD40 expression in these hematological cells in a dose-dependent manner, although the concentration required for interleukin-4 was significantly higher than that for interferon-γ. We found that tumour necrosis factor-α promoted CD40 expression induced by interferon-γ, but not by interleukin-4. Our data showed that tumour necrosis factor-α plus interferon-γ-treated Myb-transformed hematological cells had the greatest ability to take up and process the model antigen DQ-Ovalbumin. Tumour necrosis factor-α also increased the ability of interferon-γ to produce the mixed lymphocyte reaction to allogenic T cells. Furthermore, only cotreatment with tumour necrosis factor-α and interferon-γ induced Myb-transformed hematological cells to express interleukin-6. These results suggest that tumour necrosis factor-α plays a key regulatory role in the development of dendritic cells from hematological progenitor cells induced by interferon-γ.

  3. Killing of normal melanocytes, combined with heat shock protein 70 and CD40L expression, cures large established melanomas.

    PubMed

    Sanchez-Perez, Luis; Kottke, Timothy; Daniels, Gregory A; Diaz, Rosa Maria; Thompson, Jill; Pulido, Jose; Melcher, Alan; Vile, Richard G

    2006-09-15

    Previously, we showed that nine intradermal injections of a plasmid in which the HSVtk suicide gene is expressed from a melanocyte-specific promoter (Tyr-HSVtk), combined with a plasmid expressing heat shock protein 70 (CMV-hsp70), along with systemic ganciclovir, kills normal melanocytes and raises a CD8+ T cell response that is potent enough to eradicate small, 3-day established B16 tumors. We show in this study that, in that regimen, hsp70 acts as a potent immune adjuvant through TLR-4 signaling and local induction of TNF-alpha. hsp70 is required for migration of APC resident in the skin to the draining lymph nodes to present Ags, derived from the killing of normal melanocytes, to naive T cells. The addition of a plasmid expressing CD40L increased therapeutic efficacy, such that only six plasmid injections were now required to cure large, 9-day established tumors. Generation of potent immunological memory against rechallenge in cured mice accompanied these therapeutic gains, as did induction of aggressive autoimmune symptoms. Expression of CD40L, along with hsp70, increased both the frequency and activity of T cells activated against melanocyte-derived Ags. In this way, addition of CD40L to the hsp70-induced inflammatory killing of melanocytes can be used to cure large established tumors and to confer immunological memory against tumor cells, although a concomitant increase in autoimmune sequelae also is produced.

  4. Binding of CD40L to Mac-1’s I-domain involves the EQLKKSKTL motif and mediates leukocyte recruitment and atherosclerosis – but does not affect immunity and thrombosis in mice

    PubMed Central

    Wolf, Dennis; Hohmann, Jan-David; Wiedemann, Ansgar; Bledzka, Kamila; Blankenbach, Hermann; Marchini, Timoteo; Gutte, Katharina; Zeschky, Katharina; Bassler, Nicole; Hoppe, Natalie; Rodriguez, Alexandra Ortiz; Herr, Nadine; Hilgendorf, Ingo; Stachon, Peter; Willecke, Florian; Dürschmied, Daniel; von zur Mühlen, Constantin; Soloviev, Dmitry A.; Zhang, Li; Bode, Christoph; Plow, Edward F.; Libby, Peter; Peter, Karlheinz; Zirlik, Andreas

    2012-01-01

    Rationale CD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and haemostasis by interaction with CD40 receptor and the platelet integrin GPIIb/IIIa, its global inhibition compromises host defense and generated thromboembolic complications in clinical trials. We recently reported that CD40L mediates atherogenesis independently of CD40 and proposed Mac-1 as an alternate receptor. Objective Here, we molecularly characterized the CD40L-Mac-1 interaction and tested whether its selective inhibition by a small peptide modulates inflammation and atherogenesis in vivo. Methods and Results CD40L concentration-dependently bound to Mac-1 I-domain in solid phase binding assays, and a high affinity interaction was revealed by surface-plasmon-resonance analysis. We identified the motif EQLKKSKTL, an exposed loop between the α1 helix and the β-sheet B, on Mac-1 as binding site for CD40L. A linear peptide mimicking this sequence, M7, specifically inhibited the interaction of CD40L and Mac-1. cM7, a cyclisized version optimized for in vivo use, decreased peritoneal inflammation and inflammatory cell recruitment in vivo. Finally, LDLr-/- mice treated with intraperitoneal injections of cM7 developed smaller, less inflamed atherosclerotic lesions featuring characteristics of stability. However, cM7 did not interfere with CD40L-CD40 binding in vitro and CD40L-GPIIb/IIIa-mediated thrombus formation in vivo. Conclusions We present the novel finding that CD40L binds to the EQLKKSKTL motif on Mac-1 mediating leukocyte recruitment and atherogenesis. Specific inhibition of CD40L-Mac-1 binding may represent an attractive anti-inflammatory treatment strategy for atherosclerosis and other inflammatory conditions, potentially avoiding the unwanted immunologic and thrombotic effects of global inhibition of CD40L. PMID:21998326

  5. Clinical Trials Using Adenovector-transduced AP1903-inducible MyD88/CD40-expressing Autologous PSMA-specific Prostate Cancer Vaccine BPX-201

    Cancer.gov

    NCI supports clinical trials that test new and more effective ways to treat cancer. Find clinical trials studying adenovector-transduced ap1903-inducible myd88/cd40-expressing autologous psma-specific prostate cancer vaccine bpx-201.

  6. Superiority in Rhesus Macaques of Targeting HIV-1 Env Gp140 to CD40 Versus LOX-1 in Combination with Replication Competent NYVAC-KC for Induction of Env-Specific Antibody and T Cell Responses.

    PubMed

    Zurawski, Gerard; Shen, Xiaoying; Zurawski, Sandra; Tomaras, Georgia D; Montefiori, David C; Roederer, Mario; Ferrari, Guido; Lacabaratz, Christine; Klucar, Peter; Wang, Zhiqing; Foulds, Kathryn E; Kao, Shing-Fen; Yu, Xuesong; Sato, Alicia; Yates, Nicole L; LaBranche, Celia; Stanfield-Oakley, Sherry; Kibler, Karen; Jacobs, Bertram; Salazar, Andres; Self, Steve; Fulp, Jimmy; Gottardo, Raphael; Galmin, Lindsey; Weiss, Deborah; Cristillo, Anthony; Pantaleo, Giuseppe; Levy, Yves

    2017-02-15

    We compared the HIV-1-specific immune responses generated by targeting HIV-1 envelope protein (Env gp140) to either CD40 or LOX-1, two endocytic receptors on dendritic cells (DCs), in Rhesus macaques primed with a poxvirus vector (NYVAC-KC) expressing Env gp140. The DC-targeting vaccines, humanized recombinant monoclonal antibodies fused to Env gp140, were administered as a boost with poly ICLC adjuvant either alone or co-administered with the NYVAC-KC vector. All the DC-targeting vaccine administrations with poly ICLC increased the low-level serum anti-Env IgG responses elicited by NYVAC-KC priming significantly more (up to P =0.01) than a group without poly ICLC. The responses were robust, cross-reactive, and contained antibodies specific to multiple epitopes within gp140 including the C1, C2, V1-3, C4, C5, and gp41 immuno-dominant regions. The DC-targeting vaccines also elicited modest serum Env-specific IgA responses. All groups gave serum neutralization activity limited to Tier 1 viruses and antibody dependent cytotoxicity responses (ADCC) after DC-targeting boosts. Furthermore, CD4(+) and CD8(+) T cell responses specific to multiple Env epitopes were strongly boosted by the DC-targeting vaccines + poly ICLC. Together, these results indicate that prime/boost immunization via NYVAC-KC and either αCD40.Env gp140/poly ICLC or αLOX-1.Env gp140/poly ICLC induced balanced antibody and T cell responses against HIV-1 Env. Co-administration of NYVAC-KC with the DC-targeting vaccines increased T cell responses, but had minimal effects on antibody responses except for suppressing serum IgA responses. Overall, compared to LOX-1, targeting Env to CD40 gave more robust T cell and serum antibody responses with broader epitope representation and greater durability.IMPORTANCE An effective vaccine to prevent HIV-1 infection does not yet exist. An approach to elicit strong protective antibody development is to direct virus protein antigens specifically to dendritic cells

  7. High sCD40L levels early after trauma are associated with enhanced shock, sympathoadrenal activation, tissue and endothelial damage, coagulopathy and mortality.

    PubMed

    Johansson, P I; Sørensen, A M; Perner, A; Welling, K-L; Wanscher, M; Larsen, C F; Ostrowski, S R

    2012-02-01

    Severe injury activates the sympathoadrenal, hemostatic and inflammatory systems, but a maladapted response may contribute to a poor outcome. Soluble CD40L is a platelet-derived mediator that links inflammation, hemostasis and vascular dysfunction. To investigate the association between the sCD40L level and tissue injury, shock, coagulopathy and mortality in trauma patients. A prospective, observational study of 80 trauma patients admitted to a Level I Trauma Center. Data on demography, biochemistry, Injury Severity Score (ISS) and 30-day mortality were recorded and admission plasma/serum analyzed for sCD40L and biomarkers reflecting sympathoadrenal activation (adrenaline, noradrenaline), tissue/endothelial cell/glycocalyx damage (histone-complexed DNA fragments [hcDNA], Annexin V, thrombomodulin and syndecan-1), coagulation activation/inhibition (PF1.2, TAT-complex, antithrombin, protein C, activated protein C, sEPCR, TFPI, von Willebrand factor [VWF], fibrinogen and factor [F] XIII), fibrinolysis (D-dimer, tissue plasminogen activator [tPA] and plasminogen activator inhibitor-1 [PAI-1]) and inflammation (interleukin-6 [IL-6] and sC5b-9). We compared patients stratified by median sCD40L level and investigated predictive values of sCD40L for mortality. High circulating sCD40L was associated with enhanced tissue and endothelial damage (ISS, hcDNA, Annexin V, syndecan-1 and sTM), shock (pH, standard base excess), sympathoadrenal activation (adrenaline) and coagulopathy evidenced by reduced thrombin generation (PF1.2), hyperfibrinolysis (D-dimer), increased activated partial thromboplastin time (APTT) and inflammation (IL-6) (all P < 0.05). A higher ISS (P = 0.017), adrenaline (P = 0.049) and platelet count (P = 0.012) and lower pH (P =0.002) were associated with higher sCD40L by multivariate linear regression analysis. High circulating sCD40L (odds ratio [OR] 1.84 [95% CI 1.05-3.23], P = 0.034), high age (P = 0.002) and low Glasgow Coma Score (GCS) pre-hospital (P

  8. Correlation between Intravoxel Incoherent Motion Magnetic Resonance Imaging Derived Metrics and Serum Soluble CD40 Ligand Level in an Embolic Canine Stroke Model.

    PubMed

    Xu, Xiao-Quan; Wu, Chen-Jiang; Lu, Shan-Shan; Gao, Qian-Qian; Zu, Qing-Quan; Liu, Xing-Long; Shi, Hai-Bin; Liu, Sheng

    2017-01-01

    To determine the relationship between intravoxel incoherent motion (IVIM) imaging derived quantitative metrics and serum soluble CD40 ligand (sCD40L) level in an embolic canine stroke model. A middle cerebral artery occlusion model was established in 24 beagle dogs. Experimental dogs were divided into low- and high-sCD40L group according to serum sCD40L level at 4.5 hours after establishing the model. IVIM imaging was scanned at 4.5 hours after model establishment using 10 b values ranging from 0 to 900 s/mm(2). Quantitative metrics diffusion coefficient (D), pseudodiffusion coefficient (D(*)), and perfusion fraction (f) of ischemic lesions were calculated. Quantitative metrics of ischemic lesions were normalized by contralateral hemisphere using the following formula: normalized D = Dstroke / Dcontralateral. Differences in IVIM metrics between the low- and high-sCD40L groups were compared using t test. Pearson's correlation analyses were performed to determine the relationship between IVIM metrics and serum sCD40L level. The high-sCD40L group showed significantly lower f and normalized f values than the low-sCD40L group (f, p < 0.001; normalized f, p < 0.001). There was no significant difference in D(*), normalized D(*), D, or normalized D value between the two groups (All p > 0.05). Both f and normalized f values were negatively correlated with serum sCD40L level (f, r = -0.789, p < 0.001; normalized f, r = -0.823, p < 0.001). However, serum sCD40L level had no significant correlation with D(*), normalized D(*), D, or normalized D (All p > 0.05). The f value derived from IVIM imaging was negatively correlated with serum sCD40L level. f value might serve as a potential imaging biomarker to assess the formation of microvascular thrombosis in hyperacute period of ischemic stroke.

  9. CD40 ligand-mediated activation of the de novo RelB NF-kappaB synthesis pathway in transformed B cells promotes rescue from apoptosis.

    PubMed

    Mineva, Nora D; Rothstein, Thomas L; Meyers, John A; Lerner, Adam; Sonenshein, Gail E

    2007-06-15

    CD40, a tumor necrosis factor receptor family member, is expressed on B lymphocytes. Interaction between CD40 and its ligand (CD40L), expressed on activated T lymphocytes, is critical for B cell survival. Here, we demonstrate that CD40 signals B cell survival in part via transcriptional activation of the RelB NF-kappaB subunit. CD40L treatment of chronic lymphocytic leukemia cells induced levels of relB mRNA. Similarly, CD40L-mediated rescue of WEHI 231 B lymphoma cells from apoptosis induced upon B cell receptor (surface IgM) engagement led to increased relB mRNA levels. Recently, we characterized a new de novo synthesis pathway for the RelB NF-kappaB subunit, induced by the cytomegalovirus IE1 protein, in which binding of p50/p65 NF-kappaB and c-Jun/Fra-2 AP-1 complexes to the relB promoter works in synergy to potently activate transcription (Wang, X., and Sonenshein, G. E. (2005) J. Virol. 79, 95-105). CD40L treatment of WEHI 231 cells caused induction of AP-1 family members Fra-2, c-Jun, JunD, and JunB. Cotransfection of Fra-2 with the Jun AP-1 subunits and p50/c-Rel NF-kappaB led to synergistic activation of the relB promoter. Ectopic expression of relB or RelB knockdown using small interfering RNA demonstrated the important role of this subunit in control of WEHI 231 cell survival and implicated activation of the anti-apoptotic factors Survivin and manganese superoxide dismutase. Thus, CD40 engagement of transformed B cells activates relB gene transcription via a process we have termed the de novo RelB synthesis pathway, which protects these cells from apoptosis.

  10. Generation of a soluble recombinant trimeric form of bovine CD40L and its potential use as a vaccine adjuvant in cows.

    PubMed

    Pujol, Julien; Bouillenne, Fabrice; Farnir, Frédéric; Dufrasne, Isabelle; Mainil, Jacques; Galleni, Moreno; Lekeux, Pierre; Bureau, Fabrice; Fiévez, Laurence

    2015-11-15

    Vaccination is the most cost-effective way to control infectious diseases in cattle. However, many infectious diseases leading to severe economical losses worldwide still remain for which a really effective and safe vaccine is not available. These diseases are most often due to intracellular pathogens such as bacteria or viruses, which are, by their localization, protected from antibiotics and/or CD4(+) T cell-dependent humoral responses. We therefore postulated that strategies leading to induction of not only CD4(+) T cell responses but also CD8(+) cytotoxic T lymphocyte (CTL) responses against infected cells should be privileged in the development of new vaccines against problematic intracellular pathogens in bovines. CD40 signaling in antigen-presenting cells may lead to the induction of robust CD4-independent CTL responses and several studies, especially in mice, have used CD40 stimulation to promote CD8(+) T cell-mediated immunity. For example, we have recently shown that immunization of mice with heat-killed Staphylococcus aureus (HKSA) and agonistic anti-CD40 monoclonal antibodies elicits strong CTL responses capable of protecting mice from subsequent staphylococcal mastitis. Unfortunately, there is at present no tool available to efficiently stimulate CD40 in cattle. In this study, we therefore first produced a soluble recombinant trimeric form of the natural bovine CD40 ligand (sboCD40LT). We then observed that sboCD40LT was able to potently stimulate bovine cells in vitro. Finally, we provide evidence that immunization of cows with sboCD40LT combined with HKSA was able to significantly increase the number of both HKSA-specific CD4(+) and CD8(+) T cells in the draining lymph nodes. In conclusion, we suggest that this new molecular tool could help in the development of vaccine strategies against bovine diseases caused by intracellular pathogens. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Blockade of CD40 ligand suppresses chronic experimental myasthenia gravis by down-regulation of Th1 differentiation and up-regulation of CTLA-4.

    PubMed

    Im, S H; Barchan, D; Maiti, P K; Fuchs, S; Souroujon, M C

    2001-06-01

    Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are T cell-dependent Ab-mediated autoimmune disorders, in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. Th1-type cells and costimulatory factors such as CD40 ligand (CD40L) contribute to disease pathogenesis by producing proinflammatory cytokines and by activating autoreactive B cells. In this study we demonstrate the capacity of CD40L blockade to modulate EAMG, and analyze the mechanism underlying this disease suppression. Anti-CD40L Abs given to rats at the chronic stage of EAMG suppress the clinical progression of the autoimmune process and lead to a decrease in the AChR-specific humoral response and delayed-type hypersensitivity. The cytokine profile of treated rats suggests that the underlying mechanism involves down-regulation of AChR-specific Th1-regulated responses with no significant effect on Th2- and Th3-regulated AChR-specific responses. EAMG suppression is also accompanied by a significant up-regulation of CTLA-4, whereas a series of costimulatory factors remain unchanged. Adoptive transfer of splenocytes from anti-CD40L-treated rats does not protect recipient rats against subsequently induced EAMG. Thus it seems that the suppressed progression of chronic EAMG by anti-CD40L treatment does not induce a switch from Th1 to Th2/Th3 regulation of the AChR-specific immune response and does not induce generation of regulatory cells. The ability of anti-CD40L treatment to suppress ongoing chronic EAMG suggests that blockade of CD40L may serve as a potential approach for the immunotherapy of MG and other Ab-mediated autoimmune diseases.

  12. A recombinant fusion protein consisting of West Nile virus envelope domain III fused in-frame with equine CD40 ligand induces antiviral immune responses in horses.

    PubMed

    Liu, Shiliang A; Haque, Muzammel; Stanfield, Brent; Andrews, Frank M; Roy, Alma A; Kousoulas, Konstantin G

    2017-01-01

    West Nile Virus (WNV) is endemic in the US and causes severe neurologic disease in horses since its introduction in 1999. There is no effective pharmaceutical treatment for WNV infection rendering vaccination as the only approach to prevention and control of disease. The purpose of this study was to evaluate a recombinant vaccine containing domain III (DIII) of the WNV envelope glycoprotein with and without a natural adjuvant equine (CD40L) in producing virus neutralizing antibodies in horses. Serum IgG1 concentration in the groups of horses vaccinated with the DIII-CD40L+TiterMax and DIII-CD40L proteins were significantly increased (p<0.05) after the second booster vaccination compared to other groups. Serum IgG4 and IgG7, IgG3 and IgG5 concentrations were not significantly increased among all groups. Western blot results showed that animals immunized with the DIII-CD40L protein (with or without TiterMax) exhibited the highest specific anti-DIII antibody activities after vaccinations. Moreover, animals immunized with the DIII-CD40L protein (with or without TiterMax) exhibited significantly stronger neutralization activity (p<0.05) compared to other groups starting at week eight. The DIII-CD40L protein (with or without TiterMax) stimulated more CD8(+)T cells, but not CD4(+)T cells in equine PMBCs. The results demonstrated that vaccination with recombinant WNV E DIII-CD40L protein induced superior humoral and cellular immune response in healthy horses that may be protective against WNV-associated disease in infected animals. CD40L could be utilized as a non-toxic, alternative adjuvant to boost the immunogenicity of subunit vaccines in horses. Copyright © 2016. Published by Elsevier B.V.

  13. Polypyrimidine tract-binding protein is critical for the turnover and subcellular distribution of CD40 ligand mRNA in CD4+ T cells.

    PubMed

    Matus-Nicodemos, Rodrigo; Vavassori, Stefano; Castro-Faix, Moraima; Valentin-Acevedo, Anibal; Singh, Karnail; Marcelli, Valentina; Covey, Lori R

    2011-02-15

    CD40L (CD154) is regulated at the posttranscriptional level by an activation-induced process that results in a highly stable transcript at extended times of T cell activation. Transcript stability is mediated by polypyrimidine tract-binding protein (PTB)-containing complexes (complex I and II) that bind to three adjacent CU-rich sequences within the 3' untranslated region. To assess the role of PTB in the expression and distribution of CD40L mRNA, PTB was targeted using short hairpin RNA in both primary T cells and a T cell line that recapitulates the stability phase of regulated CD40L mRNA decay. PTB knockdown resulted in a marked decrease in the mRNA stability that resulted in lowered CD40L surface expression. PTB was also critical for appropriate distribution of CD40L mRNA between the nucleus and cytoplasm and in the cytoplasm between the cytosol and the translating polysomes. The activation-induced formation of PTB-specific ribonucleoprotein complexes was observed only with cytoplasmic and not nuclear PTB indicating functional differences in the protein defined by cellular localization. Finally, we observed that cytoplasmic and nuclear PTB isoforms were differentially modified relative to each other and that the changes in cytoplasmic PTB were consistent with activation-induced phosphorylation. Together this work suggests that differentially modified PTB regulates CD40L expression at multiple steps by 1) retaining CD40L mRNA in the nucleus, 2) directly regulating mRNA stability at late times of activation, and 3) forming a ribonuclear complex that preferentially associates with translating ribosomes thus leading to an enhanced level of CD40L protein.

  14. AdCD40L gene therapy counteracts T regulatory cells and cures aggressive tumors in an orthotopic bladder cancer model.

    PubMed

    Loskog, Angelica S I; Fransson, Moa E; Totterman, Thomas T H

    2005-12-15

    The aim of this study was to develop an immunostimulating gene therapy for the treatment of orthotopic bladder carcinoma by transferring the gene for CD40L into the tumor site. CD40L stimulation of dendritic cells induces interleukin-12 expression that drives Th1 type of immune responses with activation of cytotoxic T cells. The gene for murine CD40L was transferred into bladders of tumor-bearing mice using an adenoviral vector construct. To facilitate viral uptake, the bladders were pretreated with Clorpactin. Survival of mice as well as transgene expression and immunologic effect, such as resistance to tumor challenge and presence of T regulatory cells, were monitored. On viral vector instillation, CD40L expression could be detected by reverse transcription-PCR. As a sign of transgene function, interleukin-12 (IL-12) expression was significantly increased. AdCD40L gene therapy cured 60% of mice with preestablished tumors. The cured mice were completely resistant to subcutaneous challenge with MB49 tumor cells, whereas the growth of a syngeneic irrelevant tumor was unaltered. Furthermore, the mRNA expression level of the T regulatory cell transcription factor Foxp3 was evaluated both in tumor biopsies and lymph nodes. There were no differences within the tumors of the different treatment groups. However, Foxp3 mRNA levels were down-regulated in the lymph nodes of AdCD40L-treated mice. Correspondingly, T cells from AdCD40L-treated mice were not able to inhibit proliferation of naive T cells as opposed to T cells from control-treated, tumor-bearing mice. AdCD40L gene therapy evokes Th1 cytokine responses and counteracts T regulatory cell development and/or function.

  15. Involvement of calcitonin gene-related peptide and CCL2 production in CD40-mediated behavioral hypersensitivity in a model of neuropathic pain

    PubMed Central

    MALON, JENNIFER T.; MADDULA, SWATHI; BELL, HARMONY; CAO, LING

    2014-01-01

    The neuropeptide calcitonin gene-related peptide (CGRP) is known to play a pro-nociceptive role after peripheral nerve injury upon its release from primary afferent neurons in preclinical models of neuropathic pain. We previously demonstrated a critical role for spinal cord microglial CD40 in the development of spinal nerve L5 transection (L5Tx)-induced mechanical hypersensitivity. Herein, we investigated whether CGRP is involved in the CD40-mediated behavioral hypersensitivity. First, L5Tx was found to significantly induce CGRP expression in wild-type (WT) mice up to 14 days post-L5Tx. This increase in CGRP expression was reduced in CD40 knockout (KO) mice at day 14 post-L5Tx. Intrathecal injection of the CGRP antagonist CGRP8–37 significantly blocked L5Tx-induced mechanical hypersensitivity. In vitro, CGRP induced glial IL-6 and CCL2 production, and CD40 stimulation added to the effects of CGRP in neonatal glia. Further, there was decreased CCL2 production in CD40 KO mice compared to WT mice 21 days post-L5Tx. However, CGRP8–37 did not significantly affect spinal cord CCL2 production following L5Tx in WT mice. Altogether, these data suggest that CD40 contributes to the maintenance of behavioral hypersensitivity following peripheral nerve injury in part through two distinct pathways, the enhancement of CGRP expression and spinal cord CCL2 production. PMID:22377050

  16. Vaccination with a Fusion Protein That Introduces HIV-1 Gag Antigen into a Multitrimer CD40L Construct Results in Enhanced CD8+ T Cell Responses and Protection from Viral Challenge by Vaccinia-Gag

    PubMed Central

    Gupta, Sachin; Termini, James M.; Raffa, Francesca N.; Williams, Cindi-Ann; Kornbluth, Richard S.

    2014-01-01

    CD40 ligand (CD40L, CD154) is a membrane protein that is important for the activation of dendritic cells (DCs) and DC-induced CD8+ T cell responses. To be active, CD40L must cluster CD40 receptors on responding cells. To produce a soluble form of CD40L that clusters CD40 receptors necessitates the use of a multitrimer construct. With this in mind, a tripartite fusion protein was made from surfactant protein D (SPD), HIV-1 Gag as a test antigen, and CD40L, where SPD serves as a scaffold for the multitrimer protein complex. This SPD-Gag-CD40L protein activated CD40-bearing cells and bone marrow-derived DCs in vitro. Compared to a plasmid for Gag antigen alone (pGag), DNA vaccination of mice with pSPD-Gag-CD40L induced an increased number of Gag-specific CD8+ T cells with increased avidity for major histocompatibility complex class I-restricted Gag peptide and improved vaccine-induced protection from challenge by vaccinia-Gag virus. The importance of the multitrimeric nature of the complex was shown using a plasmid lacking the N terminus of SPD that produced a single trimer fusion protein. This plasmid, pTrimer-Gag-CD40L, was only weakly active on CD40-bearing cells and did not elicit strong CD8+ T cell responses or improve protection from vaccinia-Gag challenge. An adenovirus 5 (Ad5) vaccine incorporating SPD-Gag-CD40L was much stronger than Ad5 expressing Gag alone (Ad5-Gag) and induced complete protection (i.e., sterilizing immunity) from vaccinia-Gag challenge. Overall, these results show the potential of a new vaccine design in which antigen is introduced into a construct that expresses a multitrimer soluble form of CD40L, leading to strongly protective CD8+ T cell responses. PMID:24227853

  17. Interleukin 21 Controls mRNA and MicroRNA Expression in CD40-Activated Chronic Lymphocytic Leukemia Cells

    PubMed Central

    De Cecco, Loris; Capaia, Matteo; Zupo, Simona; Cutrona, Giovanna; Matis, Serena; Brizzolara, Antonella; Orengo, Anna Maria; Croce, Michela; Marchesi, Edoardo; Ferrarini, Manlio; Canevari, Silvana; Ferrini, Silvano

    2015-01-01

    Several factors support CLL cell survival in the microenvironment. Under different experimental conditions, IL21 can either induce apoptosis or promote CLL cell survival. To investigate mechanisms involved in the effects of IL21, we studied the ability of IL21 to modulate gene and miRNA expressions in CD40-activated CLL cells. IL21 was a major regulator of chemokine production in CLL cells and it modulated the expression of genes involved in cell movement, metabolism, survival and apoptosis. In particular, IL21 down-regulated the expression of the chemokine genes CCL4, CCL3, CCL3L1, CCL17, and CCL2, while it up-regulated the Th1-related CXCL9 and CXCL10. In addition, IL21 down-regulated the expression of genes encoding signaling molecules, such as CD40, DDR1 and PIK3CD. IL21 modulated a similar set of genes in CLL and normal B-cells (e.g. chemokine genes), whereas other genes, including MYC, TNF, E2F1, EGR2 and GAS-6, were regulated only in CLL cells. An integrated analysis of the miRNome and gene expression indicated that several miRNAs were under IL21 control and these could, in turn, influence the expression of potential target genes. We focused on hsa-miR-663b predicted to down-regulate several relevant genes. Transfection of hsa-miR-663b or its specific antagonist showed that this miRNA regulated CCL17, DDR1, PIK3CD and CD40 gene expression. Our data indicated that IL21 modulates the expression of genes mediating the crosstalk between CLL cells and their microenvironment and miRNAs may take part in this process. PMID:26305332

  18. Effects of representative glucocorticoids on TNFα- and CD40L-induced NF-κB activation in sensor cells.

    PubMed

    Cechin, Sirlene R; Buchwald, Peter

    2014-07-01

    Glucocorticoids are an important class of anti-inflammatory/immunosuppressive drugs. A crucial part of their anti-inflammatory action results from their ability to repress proinflammatory transcription factors such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) upon binding to the glucocorticoid receptor (GR). Accordingly, sensor cells quantifying their effect on inflammatory signal-induced NF-κB activation can provide useful information regarding their potencies as well as their transrepression abilities. Here, we report results obtained on their effect in suppressing both the TNFα- and the CD40L-induced activation of NF-κB in sensor cells that contain an NF-κB-inducible SEAP construct. In these cells, we confirmed concentration-dependent NF-κB activation for both TNFα and CD40L at low nanomolar concentrations (EC50). Glucocorticoids tested included hydrocortisone, prednisolone, dexamethasone, loteprednol etabonate, triamcinolone acetonide, beclomethasone dipropionate, and clobetasol propionate. They all caused significant, but only partial inhibition of these activations in concentration-dependent manners that could be well described by sigmoid response-functions. Despite the limitations of only partial maximum inhibitions, this cell-based assay could be used to quantitate the suppressing ability of glucocorticoids (transrepression potency) on the expression of proinflammatory transcription factors caused by two different cytokines in parallel both in a detailed, full dose-response format as well as in a simpler single-dose format. Whereas inhibitory potencies obtained in the TNF assay correlated well with consensus glucocorticoid potencies (receptor-binding affinities, Kd, RBA, at the GR) for all compounds, the non-halogenated steroids (hydrocortisone, prednisolone, and loteprednol etabonate) were about an order of magnitude more potent than expected in the CD40 assay in this system.

  19. Interleukin 21 Controls mRNA and MicroRNA Expression in CD40-Activated Chronic Lymphocytic Leukemia Cells.

    PubMed

    De Cecco, Loris; Capaia, Matteo; Zupo, Simona; Cutrona, Giovanna; Matis, Serena; Brizzolara, Antonella; Orengo, Anna Maria; Croce, Michela; Marchesi, Edoardo; Ferrarini, Manlio; Canevari, Silvana; Ferrini, Silvano

    2015-01-01

    Several factors support CLL cell survival in the microenvironment. Under different experimental conditions, IL21 can either induce apoptosis or promote CLL cell survival. To investigate mechanisms involved in the effects of IL21, we studied the ability of IL21 to modulate gene and miRNA expressions in CD40-activated CLL cells. IL21 was a major regulator of chemokine production in CLL cells and it modulated the expression of genes involved in cell movement, metabolism, survival and apoptosis. In particular, IL21 down-regulated the expression of the chemokine genes CCL4, CCL3, CCL3L1, CCL17, and CCL2, while it up-regulated the Th1-related CXCL9 and CXCL10. In addition, IL21 down-regulated the expression of genes encoding signaling molecules, such as CD40, DDR1 and PIK3CD. IL21 modulated a similar set of genes in CLL and normal B-cells (e.g. chemokine genes), whereas other genes, including MYC, TNF, E2F1, EGR2 and GAS-6, were regulated only in CLL cells. An integrated analysis of the miRNome and gene expression indicated that several miRNAs were under IL21 control and these could, in turn, influence the expression of potential target genes. We focused on hsa-miR-663b predicted to down-regulate several relevant genes. Transfection of hsa-miR-663b or its specific antagonist showed that this miRNA regulated CCL17, DDR1, PIK3CD and CD40 gene expression. Our data indicated that IL21 modulates the expression of genes mediating the crosstalk between CLL cells and their microenvironment and miRNAs may take part in this process.

  20. An autologous in situ tumor vaccination approach for hepatocellular carcinoma. 2. Tumor-specific immunity and cure after radio-inducible suicide gene therapy and systemic CD40-ligand and Flt3-ligand gene therapy in an orthotopic tumor model.

    PubMed

    Kawashita, Yujo; Deb, Niloy J; Garg, Madhur K; Kabarriti, Rafi; Fan, Zuoheng; Alfieri, Alan A; Roy-Chowdhury, Jayanta; Guha, Chandan

    2014-08-01

    Diffuse hepatocellular carcinoma (HCC) is a lethal disease that radiation therapy (RT) currently has a limited role in treating because of the potential for developing fatal radiation-induced liver disease. However, recently diffuse HCC, "radio-inducible suicide gene therapy" has been shown to enhance local tumor control and residual microscopic disease within the liver for diffuse HCC, by using a combination of chemoactivation and molecular radiosensitization. We have demonstrated that the addition of recombinant adenovirus-expressing human Flt3 ligand (Adeno-Flt3L) after radio-inducible suicide gene therapy induced a Th1-biased, immune response and enhanced tumor control in an ectopic model of HCC. We hypothesized that sequential administration of recombinant adenovirus-expressing CD40L (Adeno-CD40L) could further potentiate the efficacy of our trimodal therapy with RT + HSV-TK + Adeno-Flt3L. We examined our hypothesis in an orthotopic model of diffuse HCC using BNL1ME A.7R.1 (BNL) cells in Balb/c mice. BNL murine hepatoma cells (5 × 10(4)) transfected with an expression vector of HSV-TK under the control of a radiation-inducible promoter were injected intraportally into BALB/cJ mice. Fourteen days after the HCC injection, mice were treated with a 25 Gy dose of radiation to the whole liver, followed by ganciclovir (GCV) treatment and systemic adenoviral cytokine gene therapy (Flt3L or CD40L or both). Untreated mice died in 27 ± 4 days. Radiation therapy alone had a marginal effect on survival (median = 35 ± 7 days) and the addition of HSV-TK/GCV gene therapy improved the median survival to 47 ± 6 days. However, the addition of Adeno-Flt3L to radiation therapy and HSV-TK/GCV therapy significantly (P = 0.0005) increased survival to a median of 63 ± 20 days with 44% (7/16) of the animals still alive 116 days after tumor implantation. The curative effect of Flt3L was completely abolished when using immunodeficient nude mice or mice depleted for CD4, CD8 and

  1. Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft.

    PubMed

    Mohiuddin, Muhammad M; Singh, Avneesh K; Corcoran, Philip C; Thomas, Marvin L; Clark, Tannia; Lewis, Billeta G; Hoyt, Robert F; Eckhaus, Michael; Pierson, Richard N; Belli, Aaron J; Wolf, Eckhard; Klymiuk, Nikolai; Phelps, Carol; Reimann, Keith A; Ayares, David; Horvath, Keith A

    2016-04-05

    Preventing xenograft rejection is one of the greatest challenges of transplantation medicine. Here, we describe a reproducible, long-term survival of cardiac xenografts from alpha 1-3 galactosyltransferase gene knockout pigs, which express human complement regulatory protein CD46 and human thrombomodulin (GTKO.hCD46.hTBM), that were transplanted into baboons. Our immunomodulatory drug regimen includes induction with anti-thymocyte globulin and αCD20 antibody, followed by maintenance with mycophenolate mofetil and an intensively dosed αCD40 (2C10R4) antibody. Median (298 days) and longest (945 days) graft survival in five consecutive recipients using this regimen is significantly prolonged over our recently established survival benchmarks (180 and 500 days, respectively). Remarkably, the reduction of αCD40 antibody dose on day 100 or after 1 year resulted in recrudescence of anti-pig antibody and graft failure. In conclusion, genetic modifications (GTKO.hCD46.hTBM) combined with the treatment regimen tested here consistently prevent humoral rejection and systemic coagulation pathway dysregulation, sustaining long-term cardiac xenograft survival beyond 900 days.

  2. Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft

    PubMed Central

    Mohiuddin, Muhammad M.; Singh, Avneesh K.; Corcoran, Philip C.; Thomas III, Marvin L.; Clark, Tannia; Lewis, Billeta G.; Hoyt, Robert F.; Eckhaus, Michael; Pierson III, Richard N.; Belli, Aaron J.; Wolf, Eckhard; Klymiuk, Nikolai; Phelps, Carol; Reimann, Keith A.; Ayares, David; Horvath, Keith A.

    2016-01-01

    Preventing xenograft rejection is one of the greatest challenges of transplantation medicine. Here, we describe a reproducible, long-term survival of cardiac xenografts from alpha 1-3 galactosyltransferase gene knockout pigs, which express human complement regulatory protein CD46 and human thrombomodulin (GTKO.hCD46.hTBM), that were transplanted into baboons. Our immunomodulatory drug regimen includes induction with anti-thymocyte globulin and αCD20 antibody, followed by maintenance with mycophenolate mofetil and an intensively dosed αCD40 (2C10R4) antibody. Median (298 days) and longest (945 days) graft survival in five consecutive recipients using this regimen is significantly prolonged over our recently established survival benchmarks (180 and 500 days, respectively). Remarkably, the reduction of αCD40 antibody dose on day 100 or after 1 year resulted in recrudescence of anti-pig antibody and graft failure. In conclusion, genetic modifications (GTKO.hCD46.hTBM) combined with the treatment regimen tested here consistently prevent humoral rejection and systemic coagulation pathway dysregulation, sustaining long-term cardiac xenograft survival beyond 900 days. PMID:27045379

  3. MiR-21 Regulates TNF-α-Induced CD40 Expression via the SIRT1-NF-κB Pathway in Renal Inner Medullary Collecting Duct Cells.

    PubMed

    Lin, Qinqin; Geng, Yuanwen; Zhao, Meng; Lin, Shuaishuai; Zhu, Qing; Tian, Zhenjun

    2017-01-01

    Recent studies have indicated that microRNA-21 (miR-21) is involved in the inflammatory response in relation to renal disease. Sirtuin1 (SIRT1) exerts renoprotective properties by counteracting inflammation. The activation of CD40 triggers inflammation that participates in renal inflammation and injury. The relationship between miR-21, SIRT1 and CD40, however, remains elusive. Immunohistochemistry, small-interfering RNA (siRNA) transfection, quantitative real-time PCR and western blotting were applied to assess the morphological, functional and molecular mechanisms in primary cultured renal inner medullary collecting duct (IMCD) cells. TNF-α induced miR-21, CD40 and acetylated-NF-κBp65 (Ac-p65) expressions and reduced SIRT1 expression in IMCD cells. miR-21 mimics increased SIRT1 expression and attenuated Ac-p65 and CD40 expressions in TNF-α-induced IMCD cells, and the corresponding changes were observed with a miR-21 inhibitor. SIRT1 overexpression or activation by SRT1720 diminished TNF-α-induced CD40 and Ac-p65 expressions, which was reversed by SIRT1 siRNA or the inhibitors Ex527 and sirtinol and augmented by pretreatment with NF-κB siRNA. Further study found that the inhibitory effect of miR-21 on Ac-p65 and CD40 expressions was impeded by pretreatment with SIRT1 siRNA. The present study indicates that miR-21 inhibits TNF-α-induced CD40 expression in IMCD cells via the SIRT1-NF-κB signalling pathway, which provides new insight in understanding the anti-inflammatory effect of miR-21. © 2017 The Author(s)Published by S. Karger AG, Basel.

  4. Tumor Necrosis Factor-α/CD40 Ligand-Engineered Mesenchymal Stem Cells Greatly Enhanced the Antitumor Immune Response and Lifespan in Mice

    PubMed Central

    Daneshmandi, Saeed; Menaa, Farid

    2014-01-01

    Abstract The interaction between mesenchymal stem cells (MSCs) and dendritic cells (DCs) affects T cell development and function. Further, the chemotactic capacity of MSCs, their interaction with the tumor microenvironment, and the intervention of immune-stimulatory molecules suggest possible exploitation of tumor necrosis factor-α (TNF-α) and CD40 ligand (CD40L) to genetically modify MSCs for enhanced cancer therapy. Both DCs and MSCs were isolated from BALB/c mice. DCs were then cocultured with MSCs transduced with TNF-α and/or CD40L [(TNF-α/CD40L)-MSCs]. Major DCs' maturation markers, DC and T cell cytokines such as interleukin-4, -6, -10, -12, TNF-α, tumor growth factor-β, as well as T cell proliferation, were assessed. Meantime, a BALB/c mouse breast tumor model was inducted by injecting 4T1 cells subcutaneously. Mice (n=10) in each well-defined test groups (n=13) were cotreated with DCs and/or (TNF-α/CD40L)-MSCs. The controls included untreated, empty vector-MSC, DC-lipopolysaccharide, and immature DC mouse groups. Eventually, cytokine levels from murine splenocytes, as well as tumor volume and survival of mice, were assessed. Compared with the corresponding controls, both in vitro and in vivo analyses showed induction of T helper 1 (Th1) as well as suppression of Th2 and Treg responses in test groups, which led to a valuable antitumor immune response. Further, the longest mouse survival was observed in mouse groups that were administered with DCs plus (TNF-α/CD40L)-MSCs. In our experimental setting, the present pioneered study demonstrates that concomitant genetic modification of MSCs with TNF-α and CD40L optimized the antitumor immunity response in the presence of DCs, meantime increasing the mouse lifespan. PMID:24372569

  5. Atorvastatin effect on circulating and leukocyte-produced CD40 ligand concentrations in people with normal cholesterol levels: a pilot study.

    PubMed

    Zineh, Issam; Welder, Gregory J; DeBella, Amy E; Arant, Christopher B; Wessel, Timothy R; Schofield, Richard S

    2006-11-01

    To investigate whether atorvastatin decreases serum or leukocyte-produced CD40 ligand (CD40L) levels and whether these effects are dependent on reduction in low-density lipoprotein cholesterol (LDL) levels in people without overt dyslipidemia. Prospective pilot study. University research center. Twenty-five normocholesterolemic volunteers (mean age 32 +/- 11 yrs; 15 women, 10 men) without cardiovascular disease. After a 2-week drug-free run-in period, subjects received atorvastatin 80 mg/day orally for 16 weeks. All lipoprotein level measurements were performed with the subject in the fasting state. The CD40L concentrations were measured by immunofluorescence detection in serum and leukocyte culture supernates after 24-hour incubation, and treatment effect was analyzed. Baseline mean +/- SD total cholesterol, LDL, high-density lipoprotein cholesterol, and triglyceride levels were 179 +/- 33, 97 +/- 29, 62 +/- 20, and 102 +/- 69 mg/dl, respectively. Mean changes in each of these levels, respectively, after 16 weeks of atorvastatin were -34%, -59%, +3%, and -23%. The median serum CD40L level was lower at 16 weeks (2.3 ng/ml, interquartile range [IQR] 1.2-5.0 ng/ml) than at baseline (3.0 ng/ml, IQR 2.1-3.7 ng/ml), but the change was not significant (p=0.24). However, atorvastatin significantly lowered CD40L produced from leukocytes by 57% (21 pg/mg of protein [IQR 10-38 pg/mg] vs 49 pg/mg [IQR 21-149 pg/mg], p=0.045). Effects were independent of reduction in cholesterol levels. Although atorvastatin did not significantly lower serum CD40L levels, significant reduction in leukocyte production was seen independent of degree of LDL reduction. These pilot data suggest a potential benefit in normocholesterolemic individuals that should be further investigated, and that leukocyte CD40L concentrations should be considered in the drug response.

  6. Developmentally regulated availability of RANKL and CD40 ligand reveals distinct mechanisms of fetal and adult cross-talk in the thymus medulla.

    PubMed

    Desanti, Guillaume E; Cowan, Jennifer E; Baik, Song; Parnell, Sonia M; White, Andrea J; Penninger, Josef M; Lane, Peter J L; Jenkinson, Eric J; Jenkinson, William E; Anderson, Graham

    2012-12-15

    T cell tolerance in the thymus is a key step in shaping the developing T cell repertoire. Thymic medullary epithelial cells play multiple roles in this process, including negative selection of autoreactive thymocytes, influencing thymic dendritic cell positioning, and the generation of Foxp3(+) regulatory T cells. Previous studies show that medullary thymic epithelial cell (mTEC) development involves hemopoietic cross-talk, and numerous TNFR superfamily members have been implicated in this process. Whereas CD40 and RANK represent key examples, interplay between these receptors, and the individual cell types providing their ligands at both fetal and adult stages of thymus development, remain unclear. In this study, by analysis of the cellular sources of receptor activator for NF-κB ligand (RANKL) and CD40L during fetal and adult cross-talk in the mouse, we show that the innate immune cell system drives initial fetal mTEC development via expression of RANKL, but not CD40L. In contrast, cross-talk involving the adaptive immune system involves both RANKL and CD40L, with analysis of distinct subsets of intrathymic CD4(+) T cells revealing a differential contribution of CD40L by conventional, but not Foxp3(+) regulatory, T cells. We also provide evidence for a stepwise involvement of TNFRs in mTEC development, with CD40 upregulation induced by initial RANK signaling subsequently controlling proliferation within the mTEC compartment. Collectively, our findings show how multiple hemopoietic cell types regulate mTEC development through differential provision of RANKL/CD40L during ontogeny, revealing molecular differences in fetal and adult hemopoietic cross-talk. They also suggest a stepwise process of mTEC development, in which RANK is a master player in controlling the availability of other TNFR family members.

  7. HLA-DR3 transgenic mice immunized with adenovirus encoding the thyrotropin receptor: T cell epitopes and functional analysis of the CD40 Graves' polymorphism.

    PubMed

    Pichurin, Pavel; Pham, Nancy; David, Chella S; Rapoport, Basil; McLachlan, Sandra M

    2006-12-01

    The major histocompatibility (MHC) molecule HLA-DR3 is a susceptibility gene for Graves' disease (GD) in Caucasians. Mice lacking murine MHC and expressing human HLA-DR3 develop thyrotropin receptor (TSHR) antibodies and sometimes hyperthyroidism after vaccination with TSHR-DNA. MHC molecules present peptides processed from antigens to T cells. Therefore, we used DR3-transgenic mice to investigate recognition of TSHR ectodomain peptides. After immunization with TSHR A-subunit adenovirus (A-subunit-Ad) but not control-adenovirus (Control-Ad), splenocytes from DR3 mice responded to A-subunit protein in culture by producing interferon-gamma (IFN-gamma). When challenged with 29 overlapping TSHR peptides, splenocytes from A-subunit-Ad- or Control-Ad-immunized mice responded to several peptides. However, in splenocytes from A-subunit-Ad but not Control-Ad mice, a peptide containing TSHR residues 142-161 induced significantly more IFN-gamma than the same splenocytes in medium alone. Immunized DR3 mice also permitted testing the TSHR-specific function of the CD40 single nucleotide polymorphism (C vs. T) associated with GD. Of three human DR3 human Epstein-Barr virus lines (EBVL), two had C in both alleles (CC) and one was CT. However, these EBVL presented peptides poorly and there was no difference between CC vs. CT EBVL in peptide presentation to splenocytes from immunized mice. A peptide corresponding to residues 145-163 is one of seven suggested to be important in GD based on HLA-binding affinities, T-epitope algorithms, and human studies. Consequently, as in human GD, TSHR amino acids 142-161 appear to include a major T cell epitope in HLA-DR3 transgenic mice immunized with A-subunit-Ad.

  8. Leishmania amazonensis impairs DC function by inhibiting CD40 expression via A2B adenosine receptor activation.

    PubMed

    Figueiredo, Amanda B; Serafim, Tiago D; Marques-da-Silva, Eduardo A; Meyer-Fernandes, José R; Afonso, Luís C C

    2012-05-01

    Dendritic cells (DCs) play an essential role in the modulation of immune responses and several studies have evaluated the interactions between Leishmania parasites and DCs. While extracellular ATP exhibits proinflammatory properties, adenosine is an important anti-inflammatory mediator. Here we investigated the effects of Leishmania infection on DC responses and the participation of purinergic signalling in this process. Bone marrow-derived dendritic cells (BMDCs) from C57BL/6J mice infected with Leishmania amazonensis, Leishmania braziliensis or Leishmania major metacyclic promastigotes showed decreased major histocompatibility complex (MHC) class II and CD86 expression and increased ectonucleotidase expression as compared with uninfected cells. In addition, L. amazonensis-infected DCs, which had lower CD40 expression, exhibited a decreased ability to induce T-cell proliferation. The presence of MRS1754, a highly selective A(2B) adenosine receptor antagonist at the time of infection increased MHC class II, CD86 and CD40 expression in L. amazonensis-infected DCs and restored the ability of the infected DCs to induce T-cell proliferation. Similar results were obtained through the inhibition of extracellular ATP hydrolysis using suramin. In conclusion, we propose that A(2B) receptor activation may be used by L. amazonensis to inhibit DC function and evade the immune response. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Vaccination produces CD4 T cells with a novel CD154-CD40 dependent cytolytic mechanism ¶

    PubMed Central

    Coler, Rhea N.; Hudson, Thomas; Hughes, Sean; Huang, Po-wei D.; Beebe, Elyse A.; Orr, Mark T.

    2015-01-01

    The discovery of new vaccines against infectious diseases and cancer requires the development of novel adjuvants with well-defined activities. The TLR4 agonist adjuvant GLA-SE elicits robust TH1 responses to a variety of vaccine antigens and is in clinical development for both infectious diseases and cancer. We demonstrate that immunization with a recombinant protein antigen and GLA-SE also induces granzyme A expression in CD4 T cells and produces cytolytic cells that can be detected in vivo. Surprisingly these in vivo CTLs were CD4 T cells, not CD8 T cells and this cytolytic activity was not dependent on granzyme A/B or perforin. Unlike previously reported CD4 CTLs the transcription factors Tbet and Eomes were not necessary for their development. CTL activity was also independent of the FasL-Fas, TRAIL-DR5, and canonical death pathways, indicating a novel mechanism of CTL activity. Rather, the in vivo CD4 CTL activity induced by vaccination required T cell expression of CD154 (CD40 ligand) and target cell expression of CD40. Thus, vaccination with a TLR4 agonist adjuvant induces CD4 CTLs which kill through a previously unknown CD154-dependent mechanism. PMID:26297758

  10. SIRT1 regulates lipopolysaccharide-induced CD40 expression in renal medullary collecting duct cells by suppressing the TLR4-NF-κB signaling pathway.

    PubMed

    Lin, Qin-Qin; Geng, Yuan-Wen; Jiang, Zhong-Wei; Tian, Zhen-Jun

    2017-02-01

    Recent evidence indicates that sirtuin1 (SIRT1), an NAD(+)-dependent deacetylase, exerts a protective effect against inflammatory kidney injury by suppressing pro-inflammatory cytokines production. The co-stimulatory molecule, CD40, is expressed in a variety of inflammatory diseases in the kidney. Here, we aimed to investigate the potential effect of SIRT1 on CD40 expression induced by lipopolysaccharide (LPS) and to disclose the underlying mechanisms in renal inner medullary collecting duct (IMCD) cells. mRNA and protein expressions were identified by quantitative real-time PCR and Western blot respectively. Subcellular localization of SIRT1 and CD40 were respectively detected by immunofluorescence and immunohistochemical staining. Small-interfering RNA (siRNA) was carried out for mechanism study. LPS reduced SIRT1 expression and up-regulated the expression of CD40, Toll-like receptor 4 (TLR4) and phospho-NF-κBp65 (p-NF-κBp65) in time- and concentration-dependent manners. Moreover, SIRT1 overexpression or activation by SRT1720 diminished the expression of CD40, TLR4 and p-NF-κBp65, which was reversed by SIRT1 siRNA or inhibitors Ex527 and sirtinol in LPS-stimulated IMCD cells. In addition, knockdown of TLR4 decreased the expression of CD40 and p-NF-κBp65 in IMCD cells exposed to LPS. Knockdown of NF-κBp65 or NF-κBp65 inhibition by pyrrolidine dithiocarbamate (PDTC) reduced LPS-induced CD40 expression in IMCD cells. Importantly, the inhibitory effect of SIRT1 on the expression of CD40 and p-NF-κBp65 was augmented by pre-treating with TLR4 siRNA. Our data indicate that SIRT1 inhibits LPS-induced CD40 expression in IMCD cells by suppressing the TLR4-NF-κB signaling pathway, which might provide novel insight into understanding the protective effect of SIRT1 in kidney. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Soluble CD40 ligand is elevated in type 1 diabetic nephropathy but not predictive of mortality, cardiovascular events or kidney function.

    PubMed

    Lajer, Maria; Tarnow, Inge; Michelson, Alan D; Jorsal, Anders; Frelinger, Andrew L; Parving, Hans-Henrik; Rossing, Peter; Tarnow, Lise

    2010-01-01

    Soluble CD40 ligand (sCD40L) derived from platelets mediates atherothrombosis, leading to proinflammatory and proatherosclerotic responses. We investigated the predictive value of plasma sCD40L for all-cause mortality, cardiovascular mortality and morbidity, progression towards end-stage renal disease (ESRD) and rate of decline in glomerular filtration rate (GFR) in patients with type 1 diabetes (T1DM) and nephropathy. The study was a prospective, observational follow-up study of 443 T1DM patients with diabetic nephropathy (274 men; age 42.1 ± 10.5 years [mean ± SD], duration of diabetes 28.3 ± 8.9 years, GFR 76 ± 33 ml/min/1.73 m2) and a control group of 421 patients with longstanding type 1 diabetes and persistent normoalbuminuria (232 men; age 45.4 ± 11.5 years, duration of diabetes 27.7 ± 10.1 years) at baseline. sCD40L was measured by ELISA. Plasma sCD40L levels were higher in patients with diabetic nephropathy compared to normoalbuminuric patients (median (range) 1.54 (0.02-13.38) vs. 1.30 (0.04-20.65) µg/L, respectively p = 0.004). The patients were followed for 8.1 (0.0-12.9) years (median (range)). Among normoalbuminuric patients, sCD40L levels did not predict all-cause mortality (p = 0.33) or combined fatal and non-fatal cardiovascular disease (CVD) (p = 0.27). Similarly, among patients with diabetic nephropathy, the covariate adjusted sCD40L levels did not predict all-cause mortality (p = 0.86) or risk of fatal and non-fatal CVD (p = 0.08). Furthermore, high levels of sCD40L did not predict development of ESRD (p = 0.85) nor rate of decline in GFR (p = 0.69). Plasma sCD40L is elevated in T1DM nephropathy but is not a predictor of all-cause mortality, cardiovascular mortality and morbidity or deterioration of kidney function

  12. The protective effect of fenofibrate against TNF-α-induced CD40 expression through SIRT1-mediated deacetylation of NF-κB in endothelial cells.

    PubMed

    Wang, Weirong; Bai, Ling; Qiao, Hu; Lu, Yanxiang; Yang, Lina; Zhang, Jiye; Lin, Rong; Ren, Feng; Zhang, Jianfeng; Ji, Meixi

    2014-02-01

    Fenofibrate, as a lipid-lowering drug in clinic, participates in the regulation of inflammatory response. Recently, increasing studies have indicated that sirtuin1 (SIRT1), a NAD+-dependent deacetylase, has potential anti-inflammatory effect in endothelial cells. However, whether the regulatory effect of fenofibrate on inflammation response is mediated by SIRT1 remains unclear. The aim of this study was to investigate the effect of fenofibrate on the expressions of SIRT1 and pro-inflammatory cytokine CD40 in endothelial cells and explore the underlying mechanisms. The results showed that fenofibrate upregulated SIRT1 expression and inhibited CD40 expression in TNF-α-stimulated endothelial cells, but these effects were reversed by peroxisome proliferator-activated receptor-α (PPARα) antagonist GW6471. Furthermore, SIRT1 inhibitors sirtinol/nicotinamide (NAM) or SIRT1 knockdown could attenuate the effect of fenofibrate on CD40 expression in endothelial cells. Importantly, NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) augmented the effect of fenofibrate on CD40 expression. Further study found that fenofibrate decreased the expression of acetylated-NF-κB p65 (Ac-NF-κB p65) in TNF-α-stimulated endothelial cells, which was abolished by SIRT1 knockdown. These results indicate that fenofibrate has protective effect against TNF-α-induced CD40 expression through SIRT1-mediated deacetylation of the p65 subunit of NF-κB.

  13. Sunitinib enhances the antitumor responses of agonistic CD40-antibody by reducing MDSCs and synergistically improving endothelial activation and T-cell recruitment

    PubMed Central

    van Hooren, Luuk; Georganaki, Maria; Huang, Hua; Mangsbo, Sara M.; Dimberg, Anna

    2016-01-01

    CD40-activating immunotherapy has potent antitumor effects due to its ability to activate dendritic cells and induce cytotoxic T-cell responses. However, its efficacy is limited by immunosuppressive cells in the tumor and by endothelial anergy inhibiting recruitment of T-cells. Here, we show that combining agonistic CD40 monoclonal antibody (mAb) therapy with vascular targeting using the tyrosine kinase inhibitor sunitinib decreased tumor growth and improved survival in B16.F10 melanoma and T241 fibrosarcoma. Treatment of tumor-bearing mice with anti-CD40 mAb led to increased activation of CD11c+ dendritic cells in the tumor draining lymph node, while sunitinib treatment reduced vessel density and decreased accumulation of CD11b+Gr1+ myeloid derived suppressor cells. The expression of ICAM-1 and VCAM-1 adhesion molecules was up-regulated on tumor endothelial cells only when anti-CD40 mAb treatment was combined with sunitinib. This was associated with enhanced intratumoral infiltration of CD8+ cytotoxic T-cells. Our results show that combining CD40-stimulating immunotherapy with sunitinib treatment exerts potent complementary antitumor effects mediated by dendritic cell activation, a reduction in myeloid derived suppressor cells and increased endothelial activation, resulting in enhanced recruitment of cytotoxic T-cells. PMID:27385210

  14. Defective expression of the CD40 ligand in X chromosome-linked immunoglobulin deficiency with normal or elevated IgM.

    PubMed Central

    Fuleihan, R; Ramesh, N; Loh, R; Jabara, H; Rosen, R S; Chatila, T; Fu, S M; Stamenkovic, I; Geha, R S

    1993-01-01

    B lymphocytes from patients with X chromosome-linked immunoglobulin deficiency with normal or elevated serum IgM are unable to switch from the synthesis of IgM/IgD to that of other immunoglobulin isotypes. Isotype switch recombination was evaluated in three affected males by examining interleukin 4-driven IgE synthesis. T-cell-dependent IgE synthesis was completely absent in the B lymphocytes of the patients. In contrast, CD40 mAb plus interleukin 4 induced the patients' B cells to synthesize IgE and to undergo deletional switch recombination. Because interaction between CD40 and its ligand on activated T cells is critical for T-cell-driven isotype switching, we examined CD40 ligand expression. In contrast to normal T cells, lymphocytes from the patients expressed no detectable CD40 ligand on their surface after stimulation with phorbol 12-myristate 13-acetate and ionomycin, although the mRNA of the ligand was expressed normally. These results suggest that defective expression of the CD40 ligand underlies the failure of isotype switching in this disease. Images Fig. 1 Fig. 3 PMID:7681587

  15. CD154-CD40 T-cell co-stimulation pathway is a key mechanism in kidney ischemia-reperfusion injury

    PubMed Central

    de Ramon, Laura; Ripoll, Elia; Merino, Ana; Lúcia, Marc; Aran, Josep M; Pérez-Rentero, S; Lloberas, Nuria; Cruzado, Josep M; Grinyó, Josep M; Torras, Juan

    2015-01-01

    Ischemia-reperfusion occurs in a great many clinical settings and contributes to organ failure or dysfunction. CD154-CD40 signaling in leukocyte–endothelial cell interactions or T-cell activation facilitates tissue inflammation and injury. Here we tested a siRNA anti-CD40 in rodent warm and cold ischemia models to check the therapeutic efficacy and anti-inflammatory outcome of in vivo gene silencing. In the warm ischemia model different doses were used, resulting in clear renal function improvement and a structural renoprotective effect. Renal ischemia activated the CD40 gene and protein expression, which was inhibited by intravenous siRNA administration. CD40 gene silencing improved renal inflammatory status, as seen by the reduction of CD68 and CD3 T-cell infiltrates, attenuated pro-inflammatory, and enhanced anti-inflammatory mediators. Furthermore, siRNA administration decreased a spleen pro-inflammatory monocyte subset and reduced TNFα secretion by splenic T cells. In the cold ischemia model with syngeneic and allogeneic renal transplantation, the most effective dose induced similar functional and structural renoprotective effects. Our data show the efficacy of our siRNA in modulating both the local and the systemic inflammatory milieu after an ischemic insult. Thus, CD40 silencing could emerge as a novel therapeutic strategy in solid organ transplantation. PMID:25993320

  16. [The determination of content of circulating CD32+CD40+ micro particles: the modification of method of assessment of destruction of endothelial cells].

    PubMed

    Kutsenko, N L; Savchenko, L G; Kaĭdasheva, E I; Kutsenko, L A; Solokhina, I L; Kaĭdashev, I P

    2011-07-01

    The purpose of study was to develop and test the method of determination of destruction of endotheliocytes in blood-vascular system. The level of circulating endothelial cells was determined by enumeration of CD32+CD40+ micro particles by means of flow cytofluorometry. During the application of the modified method it is demonstrated that in patients with metabolic syndrome the amount of CD32+CD40+ micro particles is twice higher than in the control group (p < 0.05). The research data revealed the increase of content of big endothelin and atherogenic lipoproteids. The method of determination of the level of circulating CD32+CD40+ micro particles can be applied to assess the desquamationed endotheliocytes.

  17. Concurrent evaluation of novel cardiac biomarkers in acute coronary syndrome: myeloperoxidase and soluble CD40 ligand and the risk of recurrent ischaemic events in TACTICS-TIMI 18

    PubMed Central

    Morrow, David A.; Sabatine, Marc S.; Brennan, Marie-Luise; de Lemos, James A.; Murphy, Sabina A.; Ruff, Christian T.; Rifai, Nader; Cannon, Christopher P.; Hazen, Stanley L.

    2010-01-01

    Aims We investigated the prognostic performance of myeloperoxidase (MPO), and soluble CD40 ligand (sCD40L) along with B-type natriuretic peptide (BNP), high-sensitivity C-reactive protein (hsCRP), and cardiac troponin I (cTnI) for non-fatal recurrent ischaemic events in non-ST elevation acute coronary syndrome (ACS). Methods and results We measured plasma MPO and sCD40L in 1524 patients with ACS treated with tirofiban and randomized to early invasive vs. conservative management in the TACTICS-TIMI 18 trial who survived to 180 days. Patients with elevated baseline MPO (>884 pM) were at higher risk of non-fatal myocardial infarction or rehospitalization for ACS at 30 days (9.3 vs. 4.6%, P < 0.001). In contrast, no difference was observed with higher sCD40L (>989 pg/mL, 7.6 vs. 6.3%, P = 0.31). MPO remained associated with recurrent ischaemic events after adjustment for age, ST-deviation, diabetes, prior coronary artery disease, heart failure, cTnI, hsCRP, and sCD40L (OR 2.10; 95% CI 1.36–3.23, P = 0.001). This association was attenuated by 180 days (OR 1.26; 0.95–1.68). Stratification using baseline MPO, BNP, and cTnI identified a >3-fold gradient of risk. Conclusion MPO adds to BNP and cTnI for short-term risk assessment for recurrent ischaemic events in non-ST elevation ACS. sCD40L was not associated with risk in this population treated with a platelet GPIIb/IIIa receptor antagonist. PMID:18339606

  18. OX40 ligand-transduced tumor cell vaccine synergizes with GM-CSF and requires CD40-Apc signaling to boost the host T cell antitumor response.

    PubMed

    Gri, Giorgia; Gallo, Elena; Di Carlo, Emma; Musiani, Piero; Colombo, Mario P

    2003-01-01

    Efficient T cell priming by GM-CSF and CD40 ligand double-transduced C26 murine colon carcinoma is not sufficient to cure metastases in a therapeutic setting. To determine whether a cellular vaccine that interacts directly with both APC and T cells in vivo might be superior, we generated C26 carcinoma cells transduced with the T cell costimulatory molecule OX40 ligand (OX40L) either alone (C26/OX40L) or together with GM-CSF (C26/GM/OX40L), which is known to activate APC. Mice injected with C26/OX40L cells displayed only a delay in tumor growth, while the C26/GM/OX40L tumor regressed in 85% of mice. Tumor rejection required granulocytes, CD4+, CD8+ T cells, and APC-mediated CD40-CD40 ligand cosignaling, but not IFN-gamma or IL-12 as shown using subset-depleted and knockout (KO) mice. CD40KO mice primed with C26/GM/OX40L cells failed to mount a CTL response, and T cells infiltrating the C26/GM/OX40L tumor were OX40 negative, suggesting an impairment in APC-T cell cross-talk in CD40KO mice. Indeed, CD4+ T cell-depleted mice failed to mount any CTL activity against the C26 tumor, while treatment with agonistic mAb to CD40, which acts on APC, bypassed the requirement for CD4+ T cells and restored CTL activation. C26/GM/OX40L cells cured 83% of mice bearing lung metastases, whereas C26/OX40L or C26/GM vaccination cured only 28 and 16% of mice, respectively. These results indicate the synergistic activity of OX40L and GM-CSF in a therapeutic setting.

  19. Molecular Mechanisms of TNFR-associated Factor 6 (TRAF6) Utilization by the Oncogenic Viral Mimic of CD40, Latent Membrane Protein 1 (LMP1)*

    PubMed Central

    Arcipowski, Kelly M.; Stunz, Laura L.; Graham, John P.; Kraus, Zachary J.; Bush, Tony J. Vanden; Bishop, Gail A.

    2011-01-01

    Latent membrane protein 1 (LMP1), encoded by Epstein-Barr virus, is required for EBV-mediated B cell transformation and plays a significant role in the development of posttransplant B cell lymphomas. LMP1 has also been implicated in exacerbation of autoimmune diseases such as systemic lupus erythematosus. LMP1 is a constitutively active functional mimic of the tumor necrosis factor receptor superfamily member CD40, utilizing tumor necrosis factor receptor-associated factor (TRAF) adaptor proteins to induce signaling. However, LMP1-mediated B cell activation is amplified and sustained compared with CD40. We have previously shown that LMP1 and CD40 use TRAFs 1, 2, 3, and 5 differently. TRAF6 is important for CD40 signaling, but the role of TRAF6 in LMP1 signaling in B cells is not clear. Although TRAF6 binds directly to CD40, TRAF6 interaction with LMP1 in B cells has not been characterized. Here we tested the hypothesis that TRAF6 is a critical regulator of LMP1 signaling in B cells, either as part of a receptor-associated complex and/or as a cytoplasmic adaptor protein. Using TRAF6-deficient B cells, we determined that TRAF6 was critical for LMP1-mediated B cell activation. Although CD40-mediated TRAF6-dependent signaling does not require the TRAF6 receptor-binding domain, we found that LMP1 signaling required the presence of this domain. Furthermore, TRAF6 was recruited to the LMP1 signaling complex via the TRAF1/2/3/5 binding site within the cytoplasmic domain of LMP1. PMID:21262968

  20. Effect of the hepatitis B virus S‑ecdCD40L vaccine therapy in HBV transgenic mice: A vaccine‑induced activation of antigen presenting dendritic cells.

    PubMed

    Guan, Huaqin; Lan, Songsong; Wu, Jinming; Tang, Binbin; Xu, Yin

    2017-08-22

    The classical hepatitis B virus (HBV) DNA vaccination plasmid only encodes for a single viral antigen, either the S or the PreS2/S antigen. Many strategies have been employed to improve the effect of these DNA vaccines. Our previous study identified that the fusion gene, HBV S‑ecd cluster of differentiation 40 ligand (CD40L), may promote the activation of dendritic cells (DCs) and enhance their function in vitro. In the current study, the effect of HBV S‑ecdCD40L vaccine therapy on liver DCs was investigated, and its therapeutic potential in HBV transgenic (HBV‑Tg) mice was evaluated. The eukaryotic expression plasmid, pcDNA3.1‑S‑ecdCD40L, was constructed by inserting the HBV S gene and mouse CD40L gene into the vector, pcDNA3.1 (+). HBV‑Tg mice were immunized with pcDNA3.1‑S‑ecdCD40L, pcDNA3.1‑S, pcDNA3.1 or PBS. Following this, immunophenotyping, cytokine production and T‑cell activation were analyzed in the CD11c‑enriched DC population obtained from the liver. Vaccine efficacy was further assessed by the detection of serological and biochemical parameters. When comparing with other control groups, DCs from HBV‑Tg mice immunized with pcDNA3.1‑S‑ecdCD40L exhibited increased expression of immunologically important cell molecules (CD86 and major histocompatibility complex class II), pro‑inflammatory cytokines (interleukin‑12), and enhanced capacity to promote allogeneic T‑cell proliferation. Furthermore, the HBV S‑ecdCD40L vaccine resulted in a significant inhibition of HBV DNA replication and downregulation of the hepatitis B virus surface antigen (HBsAg) in HBV‑Tg mice, without obvious liver injury. In conclusion, the HBV S‑ecdCD40L vaccine may be a feasible strategy for chronic HBV immunotherapy via promoting DC activation and function.

  1. PPARα agonist fenofibrate attenuates TNF-α-induced CD40 expression in 3T3-L1 adipocytes via the SIRT1-dependent signaling pathway

    SciTech Connect

    Wang, Weirong; Lin, Qinqin; Lin, Rong; Zhang, Jiye; Ren, Feng; Zhang, Jianfeng; Ji, Meixi; Li, Yanxiang

    2013-06-10

    The ligand-activated transcription factor peroxisome proliferator-activated receptor-α (PPARα) participates in the regulation of cellular inflammation. More recent studies indicated that sirtuin1 (SIRT1), a NAD{sup +}-dependent deacetylase, regulates the inflammatory response in adipocytes. However, whether the role of PPARα in inflammation is mediated by SIRT1 remains unclear. In this study, we aimed to determine the effect of PPARα agonist fenofibrate on the expressions of SIRT1 and pro-inflammatory cytokine CD40 and underlying mechanisms in 3T3-L1 adipocytes. We found that fenofibrate inhibited CD40 expression and up-regulated SIRT1 expression in tumor necrosis factor-α (TNF-α)-stimulated adipocytes, and these effects of fenofibrate were reversed by PPARα antagonist GW6471. Moreover, SIRT1 inhibitors sirtinol/nicotinamide (NAM) or knockdown of SIRT1 could attenuate the effect of fenofibrate on TNF-α-induced CD40 expression in adipocytes. Importantly, NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) augmented the effect of fenofibrate on CD40 expression in adipocytes. Further study found that fenofibrate decreased the expression of acetylated-NF-κB p65 (Ac-NF-κB p65) in TNF-α-stimulated adipocytes, and the effect of fenofibrate was abolished by SIRT1 inhibition. In addition, fenofibrate up-regulated SIRT1 expression through AMPK in TNF-α-stimulated adipocytes. Taken together, these findings indicate that PPARα agonist fenofibrate inhibits TNF-α-induced CD40 expression in 3T3-L1 adipocytes via the SIRT1-dependent signaling pathway. -- Highlights: • Fenofibrate up-regulates SIRT1 expression in TNF-α-stimulated adipocytes. • Fenofibrate inhibits CD40 expression through SIRT1 in adipocytes. • The effects of fenofibrate on CD40 and SIRT1 expressions are dependent on PPARα. • Fenofibrate inhibits CD40 expression via SIRT1-dependent deacetylation of NF-κB. • Fenofibrate increases SIRT1 expression through PPARα and AMPK in adipocytes.

  2. Irradiation up-regulates CD80 expression through induction of tumour necrosis factor-α and CD40 ligand expression on B lymphoma cells

    PubMed Central

    Ishikawa, Fumio; Nakano, Hideki; Seo, Akira; Okada, Yayoi; Torihata, Hideko; Tanaka, Yuriko; Uchida, Tetsuya; Miyake, Hidekazu; Kakiuchi, Terutaka

    2002-01-01

    Previously, we reported that 100 Gy X-ray irradiation followed by 24 hr incubation up-regulates CD80 expression in murine B lymphoma cells, A20-2J. In the present study, we analysed the underlying mechanisms of such up-regulation using A20-HL cells derived from A20-2J cells. Irradiation of A20-HL cells with 100 Gy enhanced CD80 expression. Incubation of untreated A20-HL cells with those 100 Gy irradiated induced up-regulation of CD80 expression. Irradiation of A20-HL cells also up-regulated the expression of tumour necrosis factor-α (TNF-α) and CD40 ligand (CD40L), and the amount of immunoprecipitable TNF-α and CD40L in cell lysates. The addition of anti-TNF-α or anti-CD40L monoclonal antibody (mAb) to the incubation of irradiated A20-HL cells partially inhibited up-regulation of CD80 expression, and the addition of both antibodies together almost completely inhibited the up-regulation, suggesting that irradiation up-regulated the CD80 expression through the induction of TNF-α and CD40L expression. Irradiation also increased the accumulation of CD80, TNF-α and CD40L mRNA. n-tosyl-l-phenylalanine chloromethyl ketone (TPCK), a nuclear factor (NF)-κB inhibitor, markedly decreased irradiation-induced accumulation of CD80 mRNA and CD80 expression. FK506, a calcineurin inhibitor, and nifedipine, a calcium channel inhibitor, inhibited not only the expression of TNF-α and CD40L, but also the up-regulation of CD80 on irradiated A20-HL cells. These results strongly suggested that irradiation induced TNF-α and CD40L expression, which then up-regulated CD80 mRNA and CD80 expression through activation of NF-κB transcription factor in A20-HL cells. PMID:12100723

  3. Urocortin-1 Mediated Cardioprotection Involves XIAP and CD40-Ligand Recovery: Role of EPAC2 and ERK1/2

    PubMed Central

    Ordóñez, Antonio; Smani, Tarik

    2016-01-01

    Aims Urocortin-1 (Ucn-1) is an endogenous peptide that protects heart from ischemia and reperfusion (I/R) injuries. Ucn-1 is known to prevent cardiac cell death, but its role in the transcription of specific genes related to survival signaling pathway has not been fully defined. The aim of this study was to investigate the molecular signaling implicated in the improvement of cardiac myocytes survival induced by Ucn-1. Methods and Results Ucn-1 administration before ischemia and at the onset of reperfusion, in rat hearts perfused in Langendorff system, fully recovered heart contractility and other hemodynamic parameters. Ucn-1 enhanced cell viability and decreased lactate dehydrogenase (LDH) release in adult cardiac myocytes subjected to simulated I/R. Annexin V-FITC/PI staining indicated that Ucn-1 promoted cell survival and decreased cell necrosis through Epac2 (exchange protein directly activated by cAMP) and ERK1/2 (extracellular signal–regulated kinases 1/2) activation. We determined that Ucn-1 shifted cell death from necrosis to apoptosis and activated caspases 9 and 3/7. Furthermore, mini-array, RT-qPCR and protein analyses of apoptotic genes showed that Ucn-1 upregulated the expression of CD40lg, Xiap and BAD in cells undergoing I/R, involving Epac2 and ERK1/2 activation. Conclusions Our data indicate that Ucn-1 efficiently protected hearts from I/R damage by increasing the cell survival and stimulated apoptotic genes, CD40lg, Xiap and BAD, overexpression through the activation of Epac2 and ERK1/2. PMID:26840743

  4. Urocortin-1 Mediated Cardioprotection Involves XIAP and CD40-Ligand Recovery: Role of EPAC2 and ERK1/2.

    PubMed

    Calderón-Sánchez, Eva; Díaz, Ignacio; Ordóñez, Antonio; Smani, Tarik

    2016-01-01

    Urocortin-1 (Ucn-1) is an endogenous peptide that protects heart from ischemia and reperfusion (I/R) injuries. Ucn-1 is known to prevent cardiac cell death, but its role in the transcription of specific genes related to survival signaling pathway has not been fully defined. The aim of this study was to investigate the molecular signaling implicated in the improvement of cardiac myocytes survival induced by Ucn-1. Ucn-1 administration before ischemia and at the onset of reperfusion, in rat hearts perfused in Langendorff system, fully recovered heart contractility and other hemodynamic parameters. Ucn-1 enhanced cell viability and decreased lactate dehydrogenase (LDH) release in adult cardiac myocytes subjected to simulated I/R. Annexin V-FITC/PI staining indicated that Ucn-1 promoted cell survival and decreased cell necrosis through Epac2 (exchange protein directly activated by cAMP) and ERK1/2 (extracellular signal-regulated kinases 1/2) activation. We determined that Ucn-1 shifted cell death from necrosis to apoptosis and activated caspases 9 and 3/7. Furthermore, mini-array, RT-qPCR and protein analyses of apoptotic genes showed that Ucn-1 upregulated the expression of CD40lg, Xiap and BAD in cells undergoing I/R, involving Epac2 and ERK1/2 activation. Our data indicate that Ucn-1 efficiently protected hearts from I/R damage by increasing the cell survival and stimulated apoptotic genes, CD40lg, Xiap and BAD, overexpression through the activation of Epac2 and ERK1/2.

  5. A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model.

    PubMed

    Gupta, Sachin; Termini, James M; Rivas, Yaelis; Otero, Miguel; Raffa, Francesca N; Bhat, Vikas; Farooq, Amjad; Stone, Geoffrey W

    2015-09-11

    Vaccination with tumor-associated antigens can induce cancer-specific CD8+ T cells. A recent improvement has been the targeting of antigen to dendritic cells (DC) using antibodies that bind DC surface molecules. This study explored the use of multi-trimers of CD40L to target the gp100 melanoma tumor antigen to DC. The spontaneously-multimerizing gene Surfactant Protein D (SPD) was used to fuse gp100 tumor antigen and CD40L, creating the recombinant protein SPD-gp100-CD40L. This "third generation" DC-targeting vaccine was designed to both target antigen to DC and optimally activate dendritic cells by aggregating CD40 trimers on the DC membrane surface. SPD-gp100-CD40L expressed as a 110kDa protein. Analytical light scattering analysis gave elution data corresponding to 4-trimer and multi-trimer SPD-gp100-CD40L oligomers. The protein was biologically active on dendritic cells and induced CD40-mediated NF-κB signaling. DNA vaccination with SPD-gp100-CD40L plasmid, together with plasmids encoding IL-12p70 and GM-CSF, significantly enhanced survival and inhibited tumor growth in a B16-F10 melanoma model. Expression of gp100 and SPD-CD40L as separate molecules did not enhance survival, highlighting the requirement to encode gp100 within SPD-CD40L for optimal vaccine activity. These data support a model where DNA vaccination with SPD-gp100-CD40L targets gp100 to DC in situ, induces activation of these DC, and generates a protective anti-tumor response when given in combination with IL-12p70 and GM-CSF plasmids.

  6. ZNT7 binds to CD40 and influences CD154-triggered p38 MAPK activity in B lymphocytes-a possible regulatory mechanism for zinc in immune function

    USDA-ARS?s Scientific Manuscript database

    Zinc deficiency impairs immune system leading to frequent infections. Although it is known that zinc plays critical roles in maintaining healthy immune function, the underlying molecular targets are largely unknown. In this study, we showed that zinc is important for the CD154-CD40-mediated activati...

  7. Retinoic acid promotes mouse splenic B cell surface IgG expression and maturation stimulated by CD40 and IL-4

    PubMed Central

    Chen, Qiuyan; Ross, A Catharine

    2008-01-01

    Retinoic acid (RA) increases antibody production in vivo but its role in B-cell activation is unclear. In a model of purified mouse splenic B cells stimulated by CD40 coreceptor (as a surrogate of T cell co-stimulation), IL-4, a principal Th-2 cytokine, and ligation of the B-cell antigen receptor, CD40 engagement or IL-4 alone induced B-cell activation indicated by increased Igγ1 germline transcripts, cell proliferation, and surface (s)IgG1 expression, while triple stimulation with the combination of anti-CD40/IL-4/anti-μ synergized to heighten B-cell activation. Although RA was growth inhibitory for anti-CD40-activated B cells, RA increased the proportion of B cells that had more differentiated phenotypes, such as expression of higher level of activation-induced deaminase, Blimp-1, CD138/syndecan-1 and sIgG1. Overall, RA can promote B-cell maturation at the population level by increasing the number of sIgG1 and CD138 expressing cells, which may be related to the potentiation of humoral immunity in vivo. PMID:18082674

  8. Tumor-induced CD11b(+) Gr-1(+) myeloid-derived suppressor cells exacerbate immune-mediated hepatitis in mice in a CD40-dependent manner.

    PubMed

    Kapanadze, Tamar; Medina-Echeverz, José; Gamrekelashvili, Jaba; Weiss, Jonathan M; Wiltrout, Robert H; Kapoor, Veena; Hawk, Nga; Terabe, Masaki; Berzofsky, Jay A; Manns, Michael P; Wang, Ena; Marincola, Francesco M; Korangy, Firouzeh; Greten, Tim F

    2015-04-01

    Immunosuppressive CD11b(+) Gr-1(+) myeloid-derived suppressor cells (MDSCs) accumulate in the livers of tumor-bearing (TB) mice. We studied hepatic MDSCs in two murine models of immune-mediated hepatitis. Unexpectedly, treatment of TB mice with Concanavalin A (Con A) or α-galactosylceramide resulted in increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels in comparison to tumor-free mice. Adoptive transfer of hepatic MDSCs into naïve mice exacerbated Con A induced liver damage. Hepatic CD11b(+) Gr-1(+) cells revealed a polarized proinflammatory gene signature after Con A treatment. An IFN-γ-dependent upregulation of CD40 on hepatic CD11b(+) Gr-1(+) cells along with an upregulation of CD80, CD86, and CD1d after Con A treatment was observed. Con A treatment resulted in a loss of suppressor function by tumor-induced CD11b(+) Gr-1(+) MDSCs as well as enhanced reactive oxygen species (ROS)-mediated hepatotoxicity. CD40 knockdown in hepatic MDSCs led to increased arginase activity upon Con A treatment and lower ALT/AST serum levels. Finally, blockade of arginase activity in Cd40(-/-) tumor-induced myeloid cells resulted in exacerbation of hepatitis and increased ROS production in vivo. Our findings indicate that in a setting of acute hepatitis, tumor-induced hepatic MDSCs act as proinflammatory immune effector cells capable of killing hepatocytes in a CD40-dependent manner.

  9. PPARα agonist fenofibrate attenuates TNF-α-induced CD40 expression in 3T3-L1 adipocytes via the SIRT1-dependent signaling pathway.

    PubMed

    Wang, Weirong; Lin, Qinqin; Lin, Rong; Zhang, Jiye; Ren, Feng; Zhang, Jianfeng; Ji, Meixi; Li, Yanxiang

    2013-06-10

    The ligand-activated transcription factor peroxisome proliferator-activated receptor-α (PPARα) participates in the regulation of cellular inflammation. More recent studies indicated that sirtuin1 (SIRT1), a NAD(+)-dependent deacetylase, regulates the inflammatory response in adipocytes. However, whether the role of PPARα in inflammation is mediated by SIRT1 remains unclear. In this study, we aimed to determine the effect of PPARα agonist fenofibrate on the expressions of SIRT1 and pro-inflammatory cytokine CD40 and underlying mechanisms in 3T3-L1 adipocytes. We found that fenofibrate inhibited CD40 expression and up-regulated SIRT1 expression in tumor necrosis factor-α (TNF-α)-stimulated adipocytes, and these effects of fenofibrate were reversed by PPARα antagonist GW6471. Moreover, SIRT1 inhibitors sirtinol/nicotinamide (NAM) or knockdown of SIRT1 could attenuate the effect of fenofibrate on TNF-α-induced CD40 expression in adipocytes. Importantly, NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) augmented the effect of fenofibrate on CD40 expression in adipocytes. Further study found that fenofibrate decreased the expression of acetylated-NF-κB p65 (Ac-NF-κB p65) in TNF-α-stimulated adipocytes, and the effect of fenofibrate was abolished by SIRT1 inhibition. In addition, fenofibrate up-regulated SIRT1 expression through AMPK in TNF-α-stimulated adipocytes. Taken together, these findings indicate that PPARα agonist fenofibrate inhibits TNF-α-induced CD40 expression in 3T3-L1 adipocytes via the SIRT1-dependent signaling pathway. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Upregulation of CD72 expression on CD19(+) CD27(+) memory B cells by CD40L in primary immune thrombocytopenia.

    PubMed

    Lyu, Mingen; Hao, Yating; Li, Yang; Lyu, Cuicui; Liu, Wenjie; Li, Huiyuan; Xue, Feng; Liu, Xiaofan; Yang, Renchi

    2017-07-01

    CD72 is a co-receptor of B cells and regulates B cell activation. Although aberrant expression of CD72 has been reported in primary immune thrombocytopenia (ITP), it is uncertain whether this aberrant expression is restricted to specific B cell subsets. Furthermore, the mechanisms that regulate CD72 expression are unknown. In this study, we found higher frequency of CD19(+) B cells, CD19(+) CD27(+) memory B cells and lower frequency of CD19(+) CD27(-) naive B cells in active ITP patients compared with controls and patients in remission. CD72 expression on CD19(+) CD27(+) cells was upregulated in active ITP patients and correlated with platelet count and anti-platelet autoantibodies. In vitro, CD40L could specifically induce CD72 upregulation on CD19(+) CD27(+) B cells. In combination with CD40L, interleukin (IL) 10 and BAFF (also termed TNFSF13B) further enhanced CD72 expression on CD19(+) CD27(+) B cells, whereas IL21 reduced CD72 upregulation. CD72mRNA expression after CD40L stimulation was increased in ITP patients and controls. Significant increase of CD40L on CD4(+) T cells was correlated with CD72 expression on CD19(+) CD27(+) B cells in ITP patients. In conclusion, upregulation of CD72 expression on CD27(+) memory B cells might take part in the pathogenesis of ITP. Elevated CD40L on CD4(+) cells combined with cytokines might contribute to the upregulation of CD72 expression on CD27(+) memory B cells. © 2017 John Wiley & Sons Ltd.

  11. Roles of the kinase TAK1 in CD40-mediated effects on vascular oxidative stress and neointima formation after vascular injury.

    PubMed

    Song, Zifang; Zhu, Xiaolei; Jin, Rong; Wang, Cuiping; Yan, Jinchuan; Zheng, Qichang; Nanda, Anil; Granger, D Neil; Li, Guohong

    2014-01-01

    Although TAK1 has been implicated in inflammation and oxidative stress, its roles in vascular smooth muscle cells (VSMCs) and in response to vascular injury have not been investigated. The present study aimed to investigate the role of TAK1 in modulating oxidative stress in VSMCs and its involvement in neointima formation after vascular injury. Double immunostaining reveals that vascular injury induces a robust phosphorylation of TAK1 (Thr187) in the medial VSMCs of injured arteries in wildtype mice, but this effect is blocked in CD40-deficient mice. Upregulation of TAK1 in VSMCs is functionally important, as it is critically involved in pro-oxidative and pro-inflammatory effects on VSMCs and eventual neointima formation. In vivo, pharmacological inhibition of TAK1 with 5Z-7-oxozeaenol blocked the injury-induced phosphorylation of both TAK1 (Thr187) and NF-kB/p65 (Ser536), associated with marked inhibition of superoxide production, 3-nitrotyrosine, and MCP-1 in the injured arteries. Cell culture experiments demonstrated that either siRNA knockdown or 5Z-7-oxozeaenol inhibition of TAK1 significantly attenuated NADPH oxidase activation and superoxide production induced by CD40L/CD40 stimulation. Co-immunoprecipitation experiments indicate that blockade of TAK1 disrupted the CD40L-induced complex formation of p22phox with p47phox, p67phox, or Nox4. Blockade of TAK1 also inhibited CD40L-induced NF-kB activation by modulating IKKα/β and NF-kB p65 phosphorylation and this was related to reduced expression of proinflammatory genes (IL-6, MCP-1 and ICAM-1) in VSMCs. Lastly, treatment with 5Z-7-oxozeaenol attenuated neointimal formation in wire-injured femoral arteries. Our findings demonstrate previously uncharacterized roles of TAK1 in vascular oxidative stress and the contribution to neointima formation after vascular injury.

  12. Regulatory T Cells and Myeloid-Derived Suppressor Cells in the Tumor Microenvironment Undergo Fas-Dependent Cell Death during IL-2/αCD40 Therapy

    PubMed Central

    Weiss, Jonathan M.; Subleski, Jeff J.; Back, Tim; Chen, Xin; Watkins, Stephanie K.; Yagita, Hideo; Sayers, Thomas J.; Murphy, William J.

    2014-01-01

    Fas ligand expression in certain tumors has been proposed to contribute to immunosuppression and poor prognosis. However, immunotherapeutic approaches may elicit the Fas-mediated elimination of immunosuppressive regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) within tumors that represent major obstacles for cancer immunotherapy. Previously, we showed that IL-2 and agonistic CD40 Ab (αCD40) elicited synergistic antitumor responses coincident with the efficient removal of Tregs and MDSCs. We demonstrate in this study in two murine tumor models that Treg and MDSC loss within the tumor microenvironment after IL-2/αCD40 occurs through a Fas-dependent cell death pathway. Among tumor-infiltrating leukocytes, CD8+ T cells, neutrophils, and immature myeloid cells expressed Fas ligand after treatment. Fas was expressed by tumor-associated Tregs and immature myeloid cells, including MDSCs. Tregs and MDSCs in the tumor microenvironment expressed active caspases after IL-2/αCD40 therapy and, in contrast with effector T cells, Tregs significantly downregulated Bcl-2 expression. In contrast, Tregs and MDSCs proliferated and expanded in the spleen after treatment. Adoptive transfer of Fas-deficient Tregs or MDSCs into wild-type, Treg-, or MDSC-depleted hosts resulted in the persistence of Tregs or MDSCs and the loss of antitumor efficacy in response to IL-2/αCD40. These results demonstrate the importance of Fas-mediated Treg/MDSC removal for successful antitumor immunotherapy. Our results suggest that immunotherapeutic strategies that include exploiting Treg and MDSC susceptibility to Fas-mediated apoptosis hold promise for treatment of cancer. PMID:24808361

  13. Separate cis-trans Pathways Post-transcriptionally Regulate Murine CD154 (CD40 Ligand) Expression

    PubMed Central

    Hamilton, B. JoNell; Wang, Xiao-Wei; Collins, Jane; Bloch, Donald; Bergeron, Alan; Henry, Brian; Terry, Benjamin M.; Zan, Moe; Mouland, Andrew J.; Rigby, William F. C.

    2008-01-01

    We report a role for CA repeats in the 3′-untranslated region (3′-UTR) in regulating CD154 expression. Human CD154 is encoded by an unstable mRNA; this instability is conferred in cis by a portion of its 3′-UTR that includes a polypyrimidine-rich region and CA dinucleotide repeat. We demonstrate similar instability activity with the murine CD154 3′-UTR. This instability element mapped solely to a conserved 100-base CU-rich region alone, which we call a CU-rich response element. Surprisingly, the CA dinucleotide-rich region also regulated reporter expression but at the level of translation. This activity was associated with poly(A) tail shortening and regulated by heterogeneous nuclear ribonucleoprotein L levels. We conclude that the CD154 3′-UTR contains dual cis-acting elements, one of which defines a novel function for exonic CA dinucleotide repeats. These findings suggest a mechanism for the association of 3′-UTR CA-rich response element polymorphisms with CD154 overexpression and the subsequent risk of autoimmune disease. PMID:18640985

  14. Critical role for CD8 T cells in allograft acceptance induced by DST and CD40/CD154 costimulatory blockade.

    PubMed

    Gao, Donghong; Lunsford, Keri E; Eiring, Anna M; Bumgardner, Ginny L

    2004-07-01

    Donor-specific transfusion (DST) and CD40/CD154 costimulation blockade is a powerful immunosuppressive strategy which prolongs survival of many allografts. The efficacy of DST and anti-CD154 mAb for prolongation of hepatocellular allograft survival was only realized in C57BL/6 mice that have both CD4- and CD8-dependent pathways available (median survival time, MST, 82 days). Hepatocyte rejection in CD8 KO mice which is CD4-dependent was not suppressed by DST and anti-CD154 mAb treatment (MST, 7 days); unexpectedly DST abrogated the beneficial effects of anti-CD154 mAb for suppression of hepatocyte rejection (MST, 42 days) and on donor-reactive alloantibody production. Hepatocyte rejection in CD4 KO mice which is CD8-dependent was suppressed by treatment with DST and anti-CD154 mAb therapy (MST, 35 days) but did not differ significantly from immunotherapy with anti-CD154 mAb alone (MST, 32 days). Induction of hepatocellular allograft acceptance by DST and anti-CD154 mAb immunotherapy was dependent on host CD8(+) T cells, as demonstrated by CD8 depletion studies in C57BL/6 mice (MST, 14 days) and CD8 reconstitution of CD8 KO mice (MST, 56 days). These studies demonstrate that both CD4(+) and CD8(+) T-cell subsets contribute to induction of hepatocellular allograft acceptance by this immunotherapeutic strategy.

  15. Safety and biodistribution of a double-deleted oncolytic vaccinia virus encoding CD40 ligand in laboratory Beagles

    PubMed Central

    Autio, Karoliina; Knuuttila, Anna; Kipar, Anja; Pesonen, Sari; Guse, Kilian; Parviainen, Suvi; Rajamäki, Minna; Laitinen-Vapaavuori, Outi; Vähä-Koskela, Markus; Kanerva, Anna; Hemminki, Akseli

    2014-01-01

    We evaluated adverse events, biodistribution and shedding of oncolytic vaccinia virus encoding CD40 ligand in two Beagles, in preparation for a phase 1 trial in canine cancer patients. Dog 1 received one dose of vaccinia virus and was euthanized 24 hours afterwards, while dog 2 received virus four times once weekly and was euthanized 7 days after that. Dogs were monitored for adverse events and underwent a detailed postmortem examination. Blood, saliva, urine, feces, and organs were collected for virus detection. Dog 1 had mild fever and lethargy while dog 2 experienced a possible seizure 5.5 hours after first virus administration. Viral DNA declined quickly in the blood after virus administration in both dogs but was still detectable 1 week later by quantitative polymerase chain reaction. Only samples taken directly after virus infusion contained infectious virus. Small amounts of viral DNA, but no infectious virus, were detected in a few saliva and urine samples. Necropsies did not reveal any relevant pathological changes and virus DNA was detected mainly in the spleen. The dogs in the study did not have cancer, and thus adverse events could be more common and viral load higher in dogs with tumors which allow viral amplification. PMID:27119092

  16. CD40 agonist converting CTL exhaustion via the activation of the mTORC1 pathway enhances PD-1 antagonist action in rescuing exhausted CTLs in chronic infection.

    PubMed

    Xu, Aizhang; Wang, Rong; Freywald, Andrew; Stewart, Kristoffor; Tikoo, Suresh; Xu, Jianqing; Zheng, Changyu; Xiang, Jim

    2017-03-11

    Expansion of PD-1-expressing CD8(+) cytotoxic T lymphocytes (CTLs) and associated CTL exhaustion are chief issues for ineffective virus-elimination in chronic infectious diseases. PD-1 blockade using antagonistic anti-PD-L1 antibodies results in a moderate conversion of CTL exhaustion. We previously demonstrated that CD40L signaling of ovalbumin (OVA)-specific vaccine, OVA-Texo, converts CTL exhaustion via the activation of the mTORC1 pathway in OVA-expressing adenovirus (AdVova)-infected B6 mice showing CTL inflation and exhaustion. Here, we developed AdVova-infected B6 and transgenic CD11c-DTR (termed AdVova-B6 and AdVova-CD11c-DTR) mice with chronic infection, and assessed a potential effect of CD40 agonist on the conversion of CTL exhaustion and on a potential enhancement of PD-1 antagonist action in rescuing exhausted CTLs in our chronic infection models. We demonstrate that a single dose of anti-CD40 alone can effectively convert CTL exhaustion by activating the mTORC1 pathway, leading to CTL proliferation, up-regulation of an effector-cytokine IFN-γ and the cytolytic effect in AdVova-B6 mice. Using anti-CD4 antibody and diphtheria toxin (DT) to deplete CD4(+) T-cells and dendritic cells (DCs), we discovered that the CD40 agonist-induced conversion in AdVova-B6 and AdVova-CD11c-DTR mice is dependent upon host CD4(+) T-cell and DC involvements. Moreover, CD40 agonist significantly enhances PD-1 antagonist effectiveness in rescuing exhausted CTLs in chronic infection. Taken together, our data demonstrate the importance of CD40 signaling in the conversion of CTL exhaustion and its ability to enhance PD-1 antagonist action in rescuing exhausted CTLs in chronic infection. Therefore, our findings may positively impact the design of new therapeutic strategies for chronic infectious diseases.

  17. IgE and IgA produced by OX40-OX40L or CD40-CD40L interaction in B cells-mast cells re-activate FcεRI or FcαRI on mast cells in mouse allergic asthma.

    PubMed

    Hong, Gwan Ui; Lim, Ji Yeun; Kim, Nam Goo; Shin, Joo-Ho; Ro, Jai Youl

    2015-05-05

    Mast cells are major effector cells of allergic diseases related to IgE. This study was undertaken to determine whether IgE or IgA, produced by CD40-CD40L or OX40-OX40L interactions between B cells and mast cells, re-activate FcεRI or FcαRI on mast cell surface. C57BL mice were sensitized and subjected to OVA challenge to induce asthma. Bone marrow-derived mast cells (BMMCs) and primary B cells were co-cultured. Mast cell recruitment into airways was stained by May-Grünwald Giemsa, the expression of markers or signaling molecules were determined by immunohistochemistry or Western blotting, and co-localization of B cells and mast cells by immunofluorescence. Anti-CD40 plus anti-OX40L Abs synergistically reduced IgE and IgA production, and mediators (histamine, LTs and cytokines) released in mast cells, and additively reduced other responses, such as, numbers of mast cells, the expression of markers (tryptase, mMCP5, B220 and CD19), surface molecules (CD40, CD40L, OX40 and OX40L), FcεRI or FcαRI and the co-localization of BMMCs and B cells, and IgE- or IgA-producing cells, as compared with individual blocking Ab treatment which reducedresponses in BAL cells or lung tissues of OVA-challenged mice or in co-culture of B and mast cells. The data suggest that IgE and IgA, produced by OX40-OX40L or CD40-CD40L interaction between B cells and mast cells, may re-activate receptors of FCεRI and FcαRI on mast cell surfaces, followed by more mediator release, and furthermore, that treatment with anti-CD40 plus anti-OX40L Abs offers a potential treatment for allergic asthma.

  18. TTRAP, a novel protein that associates with CD40, tumor necrosis factor (TNF) receptor-75 and TNF receptor-associated factors (TRAFs), and that inhibits nuclear factor-kappa B activation.

    PubMed

    Pype, S; Declercq, W; Ibrahimi, A; Michiels, C; Van Rietschoten, J G; Dewulf, N; de Boer, M; Vandenabeele, P; Huylebroeck, D; Remacle, J E

    2000-06-16

    CD40 belongs to the tumor necrosis factor (TNF) receptor family. CD40 signaling involves the recruitment of TNF receptor-associated factors (TRAFs) to its cytoplasmic domain. We have identified a novel intracellular CD40-binding protein termed TRAF and TNF receptor-associated protein (TTRAP) that also interacts with TNF-R75 and CD30. The region of the CD40 cytoplasmic domain that is required for TTRAP association overlaps with the TRAF6 recognition motif. Association of TTRAP with CD40 increases profoundly in response to treatment of cells with CD40L. Interestingly, TTRAP also associates with TRAFs, with the highest affinity for TRAF6. In transfected cells, TTRAP inhibits in a dose-dependent manner the transcriptional activation of a nuclear factor-kappaB (NF-kappaB)-dependent reporter mediated by CD40, TNF-R75 or Phorbol 12-myristate 13-acetate (PMA) and to a lesser extent by TRAF2, TRAF6, TNF-alpha, or interleukin-1beta (IL-1beta). TTRAP does not affect stimulation of NF-kappaB induced by overexpression of the NF-kappaB-inducing kinase (NIK), the IkappaB kinase alpha (IKKalpha), or the NF-kappaB subunit P65/RelA, suggesting it acts upstream of the latter proteins. Our results indicate that we have isolated a novel regulatory factor that is involved in signal transduction by distinct members of the TNF receptor family.

  19. Innate CD8αα+ lymphocytes enhance anti‐CD40 antibody‐mediated colitis in mice

    PubMed Central

    Kumar, Aaram A.; Delgado, Alberto G.; Piazuelo, M. Blanca; Van Kaer, Luc

    2017-01-01

    Abstract Introduction Immune responses in the intestines require tight regulation to avoid uncontrolled inflammation. We previously described an innate lymphocyte population in the intestinal epithelium (referred to as innate CD8αα+, or iCD8α cells) that can protect against gastrointestinal infections such as those mediated by Citrobacter rodentium. Methods Here, we have evaluated the potential contribution of these cells to intestinal inflammation by analyzing inflammation development in mice with decreased numbers of iCD8α cells. We also determined the potential of iCD8α cells to secrete granzymes and their potential role during inflammatory processes. Results We found that iCD8α cells play a pro‐inflammatory role in the development of disease in a colitis model induced by anti‐CD40 antibodies. We further found that the effects of iCD8α cells correlated with their capacity to secrete granzymes. We also observed that the pro‐inflammatory properties of iCD8α cells were controlled by interactions of CD8αα homodimers on these cells with the thymus leukemia antigen expressed by intestinal epithelial cells. Conclusions Our findings suggest that iCD8α cells modulate inflammatory responses in the intestinal epithelium, and that dysregulation of iCD8α cells effector functions may enhance disease. We propose that one of the mechanism by which iCD8α cells enhance inflammation is by the secretion of granzymes, which may promote recruitment of infiltrating cells into the epithelium. PMID:28474503

  20. Innate CD8αα(+) lymphocytes enhance anti-CD40 antibody-mediated colitis in mice.

    PubMed

    Kumar, Aaram A; Delgado, Alberto G; Piazuelo, M Blanca; Van Kaer, Luc; Olivares-Villagómez, Danyvid

    2017-06-01

    Immune responses in the intestines require tight regulation to avoid uncontrolled inflammation. We previously described an innate lymphocyte population in the intestinal epithelium (referred to as innate CD8αα(+) , or iCD8α cells) that can protect against gastrointestinal infections such as those mediated by Citrobacter rodentium. Here, we have evaluated the potential contribution of these cells to intestinal inflammation by analyzing inflammation development in mice with decreased numbers of iCD8α cells. We also determined the potential of iCD8α cells to secrete granzymes and their potential role during inflammatory processes. We found that iCD8α cells play a pro-inflammatory role in the development of disease in a colitis model induced by anti-CD40 antibodies. We further found that the effects of iCD8α cells correlated with their capacity to secrete granzymes. We also observed that the pro-inflammatory properties of iCD8α cells were controlled by interactions of CD8αα homodimers on these cells with the thymus leukemia antigen expressed by intestinal epithelial cells. Our findings suggest that iCD8α cells modulate inflammatory responses in the intestinal epithelium, and that dysregulation of iCD8α cells effector functions may enhance disease. We propose that one of the mechanism by which iCD8α cells enhance inflammation is by the secretion of granzymes, which may promote recruitment of infiltrating cells into the epithelium. © 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.

  1. MiR-145-5p regulates hypoxia-induced inflammatory response and apoptosis in cardiomyocytes by targeting CD40.

    PubMed

    Yuan, Ming; Zhang, Liwei; You, Fei; Zhou, Jingyu; Ma, Yongjiang; Yang, Feifei; Tao, Ling

    2017-03-09

    An increasing body of evidence indicates that inflammation and apoptosis are involved in the development of acute myocardial infarction (AMI). In this study, we sought to investigate the specific role and the underlying regulatory mechanism of miR-145-5p in myocardial ischemic injury. H9c2 cardiac cells were exposed to hypoxia to establish a model of myocardial hypoxic/ischemic injury. We found that miR-145-5p was notably down-regulated, while CD40 expression was highly elevated in H9c2 cells following exposure to acute hypoxia. Additionally, hypoxia markedly enhanced the inflammatory response, as reflected by an increase in the secretion of the cytokines IL-1β, TNF-α, and IL-6, whereas the introduction of miR-145-5p effectively suppressed inflammatory factor production triggered by hypoxia. Furthermore, we observed hypoxia stimulation significantly augmented apoptosis accompanied by a decrease in the expression of Bcl-2 and an increase in the expression of Bax, Caspase-3, and Caspase-9. However, augmentation of miR-145-5p led to a dramatic prevention of hypoxia-induced apoptosis. Importantly, we identified CD40 as a direct target of miR-145-5p. Interestingly, the depletion of CD40 with small interfering RNAs (siRNAs) apparently repressed the production of inflammatory cytokines and apoptosis in the setting of acute hypoxic treated. Taken together, these data demonstrated that miR-145-5p may function as a cardiac-protective molecule in myocardial ischemic injury by ameliorating inflammation and apoptosis via negative regulation of CD40. The study gives evidence that miR-145-5p provides an interesting strategy for protecting cardiomyocytes from hypoxia-induced inflammatory response and apoptosis.

  2. A20 overexpression inhibits lipopolysaccharide-induced NF-κB activation, TRAF6 and CD40 expression in rat peritoneal mesothelial cells.

    PubMed

    Zou, Xun-Liang; Pei, De-An; Yan, Ju-Zhen; Xu, Gang; Wu, Ping

    2014-04-17

    Zinc finger protein A20 is a key negative regulator of inflammation. However, whether A20 may affect inflammation during peritoneal dialysis (PD)-associated peritonitis is still unclear. This study was aimed to investigate the effect of A20 overexpression on lipopolysaccharide (LPS)-induced inflammatory response in rat peritoneal mesothelial cells (RPMCs). Isolated and cultured RPMCs in vitro. Plasmid pGEM-T easy-A20 was transfected into RPMCs by Lipofectamine™2000. The protein expression of A20, phospho-IκBα, IκBα, TNF receptor-associated factor (TRAF) 6 and CD40 were analyzed by Western blot. The mRNA expression of TRAF6, CD40, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined by real time-PCR. NF-κB p65 DNA binding activity, IL-6 and TNF-α levels in cells culture supernatant were determined by ELISA. Our results revealed that RPMCs overexpression of A20 lead to significant decrease of LPS-induced IκBα phosphorylation and NF-κB DNA binding activity (all p<0.01). In addition, A20 also attenuated the expression of TRAF6, CD40, IL-6 and TNF-α as well as levels of IL-6 and TNF-α in cells culture supernatant (all p<0.05). However, A20 only partly inhibited CD40 expression. Our study indicated that A20 overexpression may depress the inflammatory response induced by LPS in cultured RPMCs through negatively regulated the relevant function of adaptors in LPS signaling pathway.

  3. B-cell activation with CD40L or CpG measures the function of B-cell subsets and identifies specific defects in immunodeficient patients.

    PubMed

    Marasco, Emiliano; Farroni, Chiara; Cascioli, Simona; Marcellini, Valentina; Scarsella, Marco; Giorda, Ezio; Piano Mortari, Eva; Leonardi, Lucia; Scarselli, Alessia; Valentini, Diletta; Cancrini, Caterina; Duse, Marzia; Grimsholm, Ola; Carsetti, Rita

    2017-01-01

    Around 65% of primary immunodeficiencies are antibody deficiencies. Functional tests are useful tools to study B-cell functions in vitro. However, no accepted guidelines for performing and evaluating functional tests have been issued yet. Here, we report our experience on the study of B-cell functions in infancy and throughout childhood. We show that T-independent stimulation with CpG measures proliferation and differentiation potential of memory B cells. Switched memory B cells respond better than IgM memory B cells. On the other hand, CD40L, a T-dependent stimulus, does not induce plasma cell differentiation, but causes proliferation of naïve and memory B cells. During childhood, the production of plasmablasts in response to CpG increases with age mirroring the development of memory B cells. The response to CD40L does not change with age. In patients with selective IgA deficiency (SIgAD), we observed that switched memory B cells are reduced due to the absence of IgA memory B cells. In agreement, IgA plasma cells are not generated in response to CpG. Unexpectedly, B cells from SIgAD patients show a reduced proliferative response to CD40L. Our results demonstrate that functional tests are an important tool to assess the functions of the humoral immune system.

  4. Inducible activation of MyD88 and CD40 in CAR T-cells results in controllable and potent antitumor activity in preclinical solid tumor models.

    PubMed

    Mata, Melinda; Gerken, Claudia; Nguyen, Phuong; Krenciute, Giedre; Spencer, David M; Gottschalk, Stephen

    2017-08-11

    Adoptive immunotherapy with T-cells expressing chimeric antigen receptors (CARs) has had limited success for solid tumors in early phase clinical studies. We reasoned that introducing into CAR T-cells an inducible co-stimulatory (iCO) molecule consisting of a chemical inducer of dimerization (CID)-binding domain and the MyD88 and CD40 signaling domains would improve and control CAR T-cell activation. In the presence of CID, T-cells expressing HER2-CARζ and a MyD88/CD40-based iCO molecule (HER2ζ.iCO T-cells) had superior T-cell proliferation, cytokine production, and ability to sequentially kill targets in vitro relative to HER2ζ.iCO T-cells without CID and T-cells expressing HER2-CAR.CD28ζ. HER2ζ.iCO T-cells with CID also significantly improved survival in vivo in two xenograft models. Repeat injections of CID were able to further increase the antitumor activity of HER2ζ.iCO T-cells in vivo. Thus, expressing MyD88/CD40-based iCO molecules in CAR T-cells has the potential to improve the efficacy of CAR T-cell therapy approaches for solid tumors. Copyright ©2017, American Association for Cancer Research.

  5. Agreement of skin test with IL-4 production and CD40L expression by T cells upon immunotherapy of subjects with systemic reactions to Hymenoptera stings.

    PubMed

    Urra, José M; Cabrera, Carmen M; Alfaya, Teresa; Feo-Brito, Francisco

    2016-02-01

    Venom immunotherapy is the only curative intervention for subjects with Hymenoptera venom allergy who suffering systemic reactions upon bee or wasp stings. Venom immunotherapy can restore normal immunity against venom allergens, as well as providing to allergic subjects a lifetime tolerance against venoms. Nevertheless, it is necessary using safety assays to monitoring the development of tolerance in the VIT protocols to avoid fatal anaphylactic reactions. The purpose of this study was to assess the modifications in several markers of tolerance induction in subjects with Hymenoptera venom allergy undergoing immunotherapy. The studies were performed at baseline time and after six month of VIT. Intradermal skin tests, basophil activation tests, specific IgE levels; and the T-cell markers (IL-4 and IFN-γ producing cells; and expression of the surface activation markers CD40L and CTLA-4) were assayed. At six month of immunotherapy all parameters studied had significant alterations. All decreased, except the IFN-γ producing cells. In addition, modifications in intradermal skin test showed a significant correlation with both, CD40L expression on CD4 T lymphocytes (p=0.043) and IL-4 producing T lymphocytes (p=0.012). Neither basophil activation test nor serum levels of sIgE demonstrated any correlation with the immunological parameters studied nor among them. These results suggest that both IL-4 production and CD40L expression could be two good indicators of the beneficial effects of venom immunotherapy which translate into skin tests.

  6. Earlier low-dose TBI or DST overcomes CD8+ T-cell-mediated alloresistance to allogeneic marrow in recipients of anti-CD40L.

    PubMed

    Takeuchi, Yasuo; Ito, Hiroshi; Kurtz, Josef; Wekerle, Thomas; Ho, Leon; Sykes, Megan

    2004-01-01

    Treatment with a single injection of anti-CD40L (CD154) monoclonal antibody (mAb) and fully mismatched allogeneic bone marrow transplant (BMT) allows rapid tolerization of CD4+ T cells to the donor. The addition of in vivo CD8 T-cell depletion leads to permanent mixed hematopoietic chimerism and tolerance. We now describe two approaches that obviate the requirement for CD8 T-cell depletion by rapidly tolerizing recipient CD8 T cells in addition to CD4 cells. Administration of donor-specific transfusion (DST) to mice receiving 3 Gy total body irradiation (TBI), BMT and anti-CD40L mAb on day 0 uniformly led to permanent mixed chimerism and tolerance, compared with only 40% of mice receiving similar treatment without DST. In the absence of DST, moving the timing of 3 Gy TBI to day -1 or day -2 instead of day 0 led to rapid (by 2 weeks) induction of CD8+ cell tolerance, and also permitted uniform achievement of permanent mixed chimerism and donor-specific tolerance in recipients of anti-CD40L and BMT on day 0. These nontoxic regimens overcome CD8+ and CD4+ T-cell-mediated alloresistance without requiring host T-cell depletion, permitting the induction of permanent mixed chimerism and tolerance.

  7. A GM-CSF and CD40L bystander vaccine is effective in a murine breast cancer model

    PubMed Central

    Soliman, Hatem; Mediavilla-Varela, Melanie; Antonia, Scott J

    2015-01-01

    Background There is increasing interest in using cancer vaccines to treat breast cancer patients in the adjuvant setting to prevent recurrence in high risk situations or in combination with other immunomodulators in the advanced setting. Current peptide vaccines are limited by immunologic compatibility issues, and engineered autologous cellular vaccines are difficult to produce on a large scale. Using standardized bystander cell lines modified to secrete immune stimulating adjuvant substances can greatly enhance the ability to produce immunogenic cellular vaccines using unmodified autologous cells or allogeneic medical grade tumor cell lines as targets. We investigated the efficacy of a cellular vaccine using B78H1 bystander cell lines engineered to secrete granulocyte macrophage-colony stimulating factor and CD40 ligand (BCG) in a murine model of breast cancer. Methods Five-week-old female BALB/c mice were injected orthotopically in the mammary fat pad with 4T1 tumor cells. Treatment consisted of irradiated 4T1 ± BCG cells given subcutaneously every 4 days and was repeated three times per mouse when tumors became palpable. Tumors were measured two to three times per week for 25 days. The vaccine’s activity was confirmed in a second experiment using Lewis lung carcinoma (LLC) cells in C57BL/6 mice to exclude a model specific effect. Interferon-γ (IFN-γ) and interleukin-2 (IL-2) enzyme-linked immunospots (ELISPOTS) were performed on splenic lymphocytes incubated with 4T1 lysates along with immunohistochemistry for CD3 on tumor sections. Results Tumor growth was significantly inhibited in the 4T1-BCG and LLC-BCG treatment groups when compared to 4T1 and LLC treatment groups. There were higher levels of IL-2 and IFN-γ secreting T-cells on ELISPOT for BCG treated groups, and a trend for higher numbers of tumor infiltrating CD3+ lymphocytes. Some tumors in the 4T1-BCG demonstrated organized lymphoid structures within the tumor microenvironment as well. Conclusion

  8. Targeting nanoparticles to CD40, DEC-205 or CD11c molecules on dendritic cells for efficient CD8(+) T cell response: a comparative study.

    PubMed

    Cruz, Luis J; Rosalia, Rodney A; Kleinovink, Jan Willem; Rueda, Felix; Löwik, Clemens W G M; Ossendorp, Ferry

    2014-10-28

    Here we demonstrated the importance of targeting antigens (Ags) to dendritic cell (DC) receptors to achieve an efficient cytotoxic T cell response which was associated with a strong activation of DC. Pegylated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) were used to encapsulate ovalbumin (OVA) as a model Ag. This PLGA complex, together with Toll like receptor (TLR) 3 and 7 ligands, was then targeted to distinct DC cell-surface molecules. These cell-surface molecules, including CD40, a TNF-α family receptor, DEC-205, a C-type lectin receptor and CD11c, an integrin receptor, were targeted by means of specific monoclonal antibodies (mAbs) coupled to the NP. The efficiency of these different targeting strategies to activate DC and elicit a potent CD8(+) T cell response was studied. PLGA-(Ag/TLR3+7L) NP was more efficiently targeted to and internalized by DC in vitro compared to the control non-targeted NP. We observed a small but significantly improved internalization of CD40-targeted NP compared to DEC-205 or CD11c targeted NP. In contrast to non-targeted NP, all targeted NPs equally stimulated IL-12 production and expression of co-stimulatory molecules by DC, inducing strong proliferation and IFN-y production by T cells in vitro. Moreover, subcutaneous vaccination with CD40, DEC-205 and CD11c-targeted NP consistently showed higher efficacy than non-targeted NP in stimulating CD8+ T cell responses. However, all targeted NP vaccines showed an equal capacity to prime cytotoxic CD8+ T cells, which subsequently were able to induce targeted cell lysis. In conclusion, delivery of NP-vaccines to DC by targeting via cell-surface molecules leads to strong enhancement of vaccine potency and induction of T cell responses compared to non-specific delivery of NP to DC. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Cerebral toxoplasmosis in a middle-aged man as first presentation of primary immunodeficiency due to a hypomorphic mutation in the CD40 ligand gene.

    PubMed

    Yong, P F K; Post, F A; Gilmour, K C; Grosse-Kreul, D; King, A; Easterbrook, P; Ibrahim, M A A

    2008-11-01

    Cerebral toxoplasmosis can occur outside the setting of advanced HIV immunodeficiency or drug-induced immunosuppression. A case of cerebral toxoplasmosis is reported in a previously healthy 41-year-old man who was found to have a genetic defect in CD40 ligand, resulting in the X linked hyper-IgM syndrome despite normal surface protein expression on flow cytometry. This highlights the fact that primary immunodeficiencies can first present late in life with a relatively mild phenotype and should be considered in the differential diagnosis of opportunistic infections in non-HIV infected patients; in addition, normal protein expression does not necessarily rule out hypomorphic mutations.

  10. First Case of CD40LG Deficiency in Ecuador, Diagnosed after Whole Exome Sequencing in a Patient with Severe Cutaneous Histoplasmosis

    PubMed Central

    Pedroza, Luis Alberto; Guerrero, Nina; Stray-Pedersen, Asbjørg; Tafur, Cristina; Macias, Roque; Muñoz, Greta; Akdemir, Zeynep Coban; Jhangiani, Shalini N.; Watkin, Levi B.; Chinn, Ivan K.; Lupski, James R.; Orange, Jordan S.

    2017-01-01

    Severe infections with Histoplasma capsulatum are commonly observed in patient with secondary immunodeficiency disorders. We report a two and a half years old boy previously healthy with disseminated cutaneous histoplasmosis. Using whole exome sequencing, we found an indel mutation at the CD40LG gene, suggesting a diagnosis of hyper-IgM (HIGM) syndrome, even in the absence of the usual features for the disease. Interestingly, the patient lives in a region endemic for histoplasmosis. The unusual infections in our case suggest that in children with severe histoplasmosis and resident in endemic areas, HIGM syndrome should be considered as a diagnosis. PMID:28239602

  11. CD40 triggering induces strong cytotoxic T lymphocyte responses to heat-killed Staphylococcus aureus immunization in mice: a new vaccine strategy for staphylococcal mastitis.

    PubMed

    Wallemacq, Hugues; Bedoret, Denis; Pujol, Julien; Desmet, Christophe; Drion, Pierre-Vincent; Farnir, Frédéric; Mainil, Jacques; Lekeux, Pierre; Bureau, Fabrice; Fiévez, Laurence

    2012-03-09

    Staphylococcus (S.) aureus is a major pathogen involved in chronic bovine mastitis. Staphylococcal mastitis is difficult to control due to the ability of S. aureus to invade and survive within host cells. We therefore postulated that induction of CD8(+) cytotoxic T lymphocyte (CTL) responses leading to destruction of infected cells could help in the control of S. aureus mastitis. We demonstrate that immunization of mice with heat-killed S. aureus together with agonistic anti-CD40 monoclonal antibodies elicits strong CTL responses capable of reducing the severity of subsequent staphylococcal mastitis. Our study shows promise for CTL-dependent vaccination against S. aureus mastitis.

  12. Serial immunomonitoring of cancer patients receiving combined antagonistic anti-CD40 and chemotherapy reveals consistent and cyclical modulation of T cell and dendritic cell parameters.

    PubMed

    McDonnell, Alison M; Cook, Alistair; Robinson, Bruce W S; Lake, Richard A; Nowak, Anna K

    2017-06-15

    CD40 signalling can synergise with chemotherapy in preclinical cancer models, and early clinical studies are promising. We set out to define the immunological changes associated with this therapeutic combination to identify biomarkers for a response to the therapy. Here, we present serial immunomonitoring examining dendritic cell and T cell subpopulations over sequential courses of chemoimmunotherapy. Fifteen patients with mesothelioma received up to six 21-day cycles of pemetrexed plus cisplatin chemotherapy and anti-CD40 (CP-870,893). Peripheral blood was collected weekly, and analysed by flow cytometry. Longitudinal immunophenotyping data was analysed by linear mixed modelling, allowing for variation between patients. Exploratory analyses testing for any correlation between overall survival and immunophenotyping data were undertaken up to the third cycle of treatment. Large statistically significant cyclical variations in the proportions of BDCA-1+, BDCA-2+ and BDCA-3+ dendritic cells were observed, although all subsets returned to baseline levels after each cycle and no significant changes were observed between start and end of treatment. Expression levels of CD40 and HLA-DR on dendritic cells were also cyclically modulated, again without significant change between start and end of treatment. CD8 and CD4 T cell populations, along with regulatory T cells, effector T cells, and markers of proliferation and activation, showed similar patterns of statistically significant cyclical modulation in response to therapy without changes between start and end of treatment. Exploratory analysis of endpoints revealed that patients with a higher than average proportion of BDCA-2+ dendritic cells (p = 0.010) or a higher than average proportion of activated (ICOS+) CD8 T cells (0.022) in pretreatment blood samples had better overall survival. A higher than average proportion of BDCA-3+ dendritic cells was associated with poorer overall survival at both the second (p = 0

  13. First Case of CD40LG Deficiency in Ecuador, Diagnosed after Whole Exome Sequencing in a Patient with Severe Cutaneous Histoplasmosis.

    PubMed

    Pedroza, Luis Alberto; Guerrero, Nina; Stray-Pedersen, Asbjørg; Tafur, Cristina; Macias, Roque; Muñoz, Greta; Akdemir, Zeynep Coban; Jhangiani, Shalini N; Watkin, Levi B; Chinn, Ivan K; Lupski, James R; Orange, Jordan S

    2017-01-01

    Severe infections with Histoplasma capsulatum are commonly observed in patient with secondary immunodeficiency disorders. We report a two and a half years old boy previously healthy with disseminated cutaneous histoplasmosis. Using whole exome sequencing, we found an indel mutation at the CD40LG gene, suggesting a diagnosis of hyper-IgM (HIGM) syndrome, even in the absence of the usual features for the disease. Interestingly, the patient lives in a region endemic for histoplasmosis. The unusual infections in our case suggest that in children with severe histoplasmosis and resident in endemic areas, HIGM syndrome should be considered as a diagnosis.

  14. A SNP in 5′ untranslated region of CD40 gene is associated with an increased risk of ischemic stroke in a Chinese population: a case-control study

    PubMed Central

    Huang, Hua-Tuo; Guo, Jing; Xiang, Yang; Chen, Jian-Ming; Luo, Hong-Cheng; Meng, Lan-Qing; Wei, Ye-Sheng

    2017-01-01

    Abstract Cluster of differentiation 40 (CD40), the receptor for CD154, is a member of the tumor necrosis factor (TNF) receptor superfamily. Several studies have been conducted to investigate the effect of the CD40 rs1883832 polymorphism on atherosclerotic disease in different population; however, inconsistent results were obtained. In this study, we investigated the association of four polymorphisms (rs1883832, rs13040307, rs752118 and rs3765459) of CD40 gene and their effect on CD40 expression with the risk of ischemic stroke (IS) in a Chinese population. Three hundred and eighty patients with IS and 450 control subjects were included in the study. The CD40 polymorphisms were discriminated by Snapshot SNP genotyping assay. Serum soluble CD40 (sCD40) levels were detected by ELISA. We found that the rs1883832CT and rs1883832TT genotypes were associated with an increased risk of IS compared with the rs1883832CC genotype (OR = 1.42, 95% CI: 1.03–1.95, p = 0.030 and OR = 1.91, 95% CI: 1.29–2.82, P = 0.001, respectively), and the rs1883832T allele was associated with a significantly increased risk of IS compared with rs1883832C allele (OR = 1.40, 95% CI: 1.15–1.70, P = 0.001). Elevated serum sCD40 levels were observed in patients with IS compared with the control gropu (P < 0.01). Individuals carrying the rs1883832TT or rs1883832CT genotypes showed significantly higher sCD40 levels compared with the rs1883832CC genotype in the IS group [(64.8 ± 25.4 pg/mL, TT = 94); (63.9 ± 24.3 pg/mL, CT = 185) vs (53.3 ± 22.5 pg/mL, CC = 101), P < 0.01]. The TCCA haplotype was associated with an increased risk of IS compared with the control group (OR = 2.10, 95% CI: 1.23–3.58, p = 0.005). However, we did not find a significant association between the other three polymorphisms and IS risk. In conclusion, after a comprehensive comparison with other studies, we confirmed that the rs1883832T allele but not the rs1883832C allele is associated with an increased risk of IS. The

  15. Biological rational for sequential targeting of Bruton tyrosine kinase and Bcl-2 to overcome CD40-induced ABT-199 resistance in mantle cell lymphoma

    PubMed Central

    Chiron, David; Touzeau, Cyrille; Maïga, Sophie; Moreau, Philippe; Pellat-Deceunynck, Catherine; Le Gouill, Steven; Amiot, Martine

    2015-01-01

    The aggressive biological behavior of mantle cell lymphoma (MCL) and its short response to current treatment highlight a great need for better rational therapy. Herein, we investigate the ability of ABT-199, the Bcl-2-selective BH3 mimetic, to kill MCL cells. Among MCL cell lines tested (n = 8), only three were sensitive (LD50 < 200 nM). In contrast, all primary MCL samples tested (n = 11) were highly sensitive to ABT-199 (LD50 < 10 nM). Mcl-1 and Bcl-xL both confer resistance to ABT-199-specific killing and BCL2/(BCLXL + MCL1) mRNA ratio is a strong predictor of sensitivity. By mimicking the microenvironment through CD40 stimulation, we show that ABT-199 sensitivity is impaired through activation of NF-kB pathway and Bcl-xL up-regulation. We further demonstrate that resistance is rapidly lost when MCL cells detach from CD40L-expressing fibroblasts. It has been reported that ibrutinib induces lymphocytosis in vivo holding off malignant cells from their protective microenvironment. We show here for two patients undergoing ibrutinib therapy that mobilized MCL cells are highly sensitive to ABT-199. These results provide evidence that in situ ABT-199 resistance can be overcome when MCL cells escape from the lymph nodes. Altogether, our data support the clinical application of ABT-199 therapy both as a single agent and in sequential combination with BTK inhibitors. PMID:25797245

  16. B cell activating factor (BAFF) and a proliferation inducing ligand (APRIL) mediate CD40-independent help by memory CD4 T cells.

    PubMed

    Gorbacheva, V; Ayasoufi, K; Fan, R; Baldwin, W M; Valujskikh, A

    2015-02-01

    Donor-reactive memory T cells undermine organ transplant survival and are poorly controlled by immunosuppression or costimulatory blockade. Memory CD4 T cells provide CD40-independent help for the generation of donor-reactive effector CD8 T cells and alloantibodies (alloAbs) that rapidly mediate allograft rejection. The goal of this study was to investigate the role of B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) in alloresponses driven by memory CD4 T cells. The short-term neutralization of BAFF alone or BAFF plus APRIL synergized with anti-CD154 mAb to prolong heart allograft survival in recipients containing donor-reactive memory CD4 T cells. The prolongation was associated with reduction in antidonor alloAb responses and with inhibited reactivation and helper functions of memory CD4 T cells. Additional depletion of CD8 T cells did not enhance the prolonged allograft survival suggesting that donor-reactive alloAbs mediate late graft rejection in these recipients. This is the first report that targeting the BAFF cytokine network inhibits both humoral and cellular immune responses induced by memory CD4 T cells. Our results suggest that reagents neutralizing BAFF and APRIL may be used to enhance the efficacy of CD40/CD154 costimulatory blockade and improve allograft survival in T cell-sensitized recipients. © Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.

  17. Bezafibrate and medroxyprogesterone acetate target resting and CD40L-stimulated primary marginal zone lymphoma and show promise in indolent B-cell non-Hodgkin lymphomas.

    PubMed

    Hayden, Rachel E; Kussaibati, Racha; Cronin, Laura M; Pratt, Guy; Roberts, Claudia; Drayson, Mark T; Bunce, Christopher M

    2015-04-01

    B cell non-Hodgkin lymphomas (B-NHLs) are the most common adult hematological cancers and many remain incurable. Development of chemotherapy regimens is confounded by the prevalence of B-NHL in older, frailer patients and the chemo-protective tumor microenvironment. Although biological therapies such as rituximab have significantly improved outcomes and selective kinase inhibitors are showing promise, the rate of new drug discovery remains disappointing, the treatments expensive and long-term benefits uncertain. An alternative strategy is redeployment of available, inexpensive and non-toxic drugs. Here, we demonstrate the antiproliferative and mitochondrial superoxide (MSO) driven pro-apoptotic activities of bezafibrate (BEZ) and medroxyprogesterone acetate (MPA) against B-NHL cells, with a bias toward MZL, in the presence and absence of the microenvironmental signal CD40L. Our study is the first to confirm the presence of CD40L within the lymph node of B-NHL and its capacity to drive B-NHL proliferation. These findings implicate BEZ + MPA as a potential therapeutic strategy in B-NHL.

  18. CD40 ligand is necessary and sufficient to support primary diffuse large B-cell lymphoma cells in culture: a tool for in vitro preclinical studies with primary B-cell malignancies

    PubMed Central

    Ito, Daisuke; Frantz, Aric M.; Williams, Christina; Thomas, Rachael; Burnett, Robert C.; Avery, Anne C.; Breen, Matthew; Mason, Nicola J.; O’Brien, Timothy D.; Modiano, Jaime F.

    2013-01-01

    Established cell lines are utilized extensively to study tumor biology and preclinical therapeutic development; however, they may not accurately recapitulate the heterogeneity of their corresponding primary disease. B-cell tumor cells are especially difficult to maintain under conventional culture conditions, limiting access to samples that faithfully represent this disease for preclinical studies. Here, we used primary canine diffuse large B-cell lymphoma to establish a culture system that reliably supports the growth of these cells. CD40 ligand, either expressed by feeder cells or provided as a soluble two-trimeric form, was sufficient to support primary lymphoma cells in vitro. The tumor cells retained their original phenotype, clonality and known karyotypic abnormalities after extended expansion in culture. Finally, we illustrate the utility of the feeder cell-free culture system for comparable assessment of cytotoxicity using dog and human B-cell malignancies. We conclude this system has broad applications for in vitro preclinical development for B-cell malignancies. PMID:22229753

  19. CD40 ligand is necessary and sufficient to support primary diffuse large B-cell lymphoma cells in culture: a tool for in vitro preclinical studies with primary B-cell malignancies.

    PubMed

    Ito, Daisuke; Frantz, Aric M; Williams, Christina; Thomas, Rachael; Burnett, Robert C; Avery, Anne C; Breen, Matthew; Mason, Nicola J; O'Brien, Timothy D; Modiano, Jaime F

    2012-07-01

    Established cell lines are utilized extensively to study tumor biology and preclinical therapeutic development. However, they may not accurately recapitulate the heterogeneity of their corresponding primary disease. B-cell tumor cells are especially difficult to maintain under conventional culture conditions, limiting access to samples that faithfully represent this disease for preclinical studies. Here, we used primary canine diffuse large B-cell lymphoma to establish a culture system that reliably supports the growth of these cells. CD40 ligand, either expressed by feeder cells or provided as a soluble two-trimeric form, was sufficient to support primary lymphoma cells in vitro. The tumor cells retained their original phenotype, clonality and known karyotypic abnormalities after extended expansion in culture. Finally, we illustrate the utility of the feeder cell-free culture system for comparable assessment of cytotoxicity using dog and human B-cell malignancies. We conclude that this system has broad applications for in vitro preclinical development for B-cell malignancies.

  20. A phase 1b clinical trial of the CD40-activating antibody CP-870,893 in combination with cisplatin and pemetrexed in malignant pleural mesothelioma.

    PubMed

    Nowak, A K; Cook, A M; McDonnell, A M; Millward, M J; Creaney, J; Francis, R J; Hasani, A; Segal, A; Musk, A W; Turlach, B A; McCoy, M J; Robinson, B W S; Lake, R A

    2015-12-01

    Data from murine models suggest that CD40 activation may synergize with cytotoxic chemotherapy. We aimed to determine the maximum tolerated dose (MTD) and toxicity profile and to explore immunological biomarkers of the CD40-activating antibody CP-870,893 with cisplatin and pemetrexed in patients with malignant pleural mesothelioma (MPM). Eligible patients had confirmed MPM, ECOG performance status 0-1, and measurable disease. Patients received cisplatin 75 mg/m(2) and pemetrexed 500 mg/m(2) on day 1 and CP-870,893 on day 8 of a 21-day cycle for maximum 6 cycles with up to 6 subsequent cycles single-agent CP-870,893. Immune cell subset changes were examined weekly by flow cytometry. Fifteen patients were treated at three dose levels. The MTD of CP-870,893 was 0.15 mg/kg, and was exceeded at 0.2 mg/kg with one grade 4 splenic infarction and one grade 3 confusion and hyponatraemia. Cytokine release syndrome (CRS) occurred in most patients (80%) following CP-870,893. Haematological toxicities were consistent with cisplatin and pemetrexed chemotherapy. Six partial responses (40%) and 9 stable disease (53%) as best response were observed. The median overall survival was 16.5 months; the median progression-free survival was 6.3 months. Three patients survived beyond 30 months. CD19+ B cells decreased over 6 cycles of chemoimmunotherapy (P < 0.001) with a concomitant increase in the proportion of CD27+ memory B cells (P < 0.001) and activated CD86+CD27+ memory B cells (P < 0.001), as an immunopharmacodynamic marker of CD40 activation. CP-870,893 with cisplatin and pemetrexed is safe and tolerable at 0.15 mg/kg, although most patients experience CRS. While objective response rates are similar to chemotherapy alone, three patients achieved long-term survival. ACTRN12609000294257. © The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  1. Loss of cooperativity of secreted CD40L and increased dose-response to IL4 on CLL cell viability correlates with enhanced activation of NF-kB and STAT6.

    PubMed

    Bhattacharya, Nupur; Reichenzeller, Michaela; Caudron-Herger, Maiwen; Haebe, Sarah; Brady, Nathan; Diener, Susanne; Nothing, Maria; Döhner, Hartmut; Stilgenbauer, Stephan; Rippe, Karsten; Mertens, Daniel

    2015-01-01

    Chronic lymphocytic leukemia (CLL) cells fail to enter apoptosis in vivo as opposed to their non-malignant B-lymphocyte counterparts. The ability of CLL cells to escape apoptosis is highly dependent on their microenvironment. Compared to non-malignant B cells, CLL cells are more responsive to complex stimuli that can be reproduced in vitro by the addition of cytokines. To understand the molecular mechanism of the environment-dependent anti-apoptotic signaling circuitry of CLL cells, we quantified the effect of the SDF-1, BAFF, APRIL, anti-IgM, interleukin-4 (IL4) and secreted CD40L (sCD40L) on the survival of in vitro cultured CLL cells and found IL4 and sCD40L to be most efficient in rescuing CLL cells from apoptosis. In quantitative dose-response experiments using cell survival as readout, the binding affinity of IL4 to its receptor was similar between malignant and non-malignant cells. However, the downstream signaling in terms of the amount of STAT6 and its degree of phosphorylation was highly stimulated in CLL cells. In contrast, the response to sCD40L showed a loss of cooperative binding in CLL cells but displayed a largely increased ligand binding affinity. Although a high-throughput microscopy analysis did not reveal a significant difference in the spatial CD40 receptor organization, the downstream signaling showed an enhanced activation of the NF-kB pathway in the malignant cells. Thus, we propose that the anti-apoptotic phenotype of CLL involves a sensitized response for IL4 dependent STAT6 phosphorylation, and an activation of NF-kB signaling due to an increased affinity of sCD40L to its receptor.

  2. Association Study of Exon Variants in the NF-κB and TGFβ Pathways Identifies CD40 as a Modifier of Duchenne Muscular Dystrophy.

    PubMed

    Bello, Luca; Flanigan, Kevin M; Weiss, Robert B; Spitali, Pietro; Aartsma-Rus, Annemieke; Muntoni, Francesco; Zaharieva, Irina; Ferlini, Alessandra; Mercuri, Eugenio; Tuffery-Giraud, Sylvie; Claustres, Mireille; Straub, Volker; Lochmüller, Hanns; Barp, Andrea; Vianello, Sara; Pegoraro, Elena; Punetha, Jaya; Gordish-Dressman, Heather; Giri, Mamta; McDonald, Craig M; Hoffman, Eric P

    2016-11-03

    The expressivity of Mendelian diseases can be influenced by factors independent from the pathogenic mutation: in Duchenne muscular dystrophy (DMD), for instance, age at loss of ambulation (LoA) varies between individuals whose DMD mutations all abolish dystrophin expression. This suggests the existence of trans-acting variants in modifier genes. Common single nucleotide polymorphisms (SNPs) in candidate genes (SPP1, encoding osteopontin, and LTBP4, encoding latent transforming growth factor β [TGFβ]-binding protein 4) have been established as DMD modifiers. We performed a genome-wide association study of age at LoA in a sub-cohort of European or European American ancestry (n = 109) from the Cooperative International Research Group Duchenne Natural History Study (CINRG-DNHS). We focused on protein-altering variants (Exome Chip) and included glucocorticoid treatment as a covariate. As expected, due to the small population size, no SNPs displayed an exome-wide significant p value (< 1.8 × 10(-6)). Subsequently, we prioritized 438 SNPs in the vicinities of 384 genes implicated in DMD-related pathways, i.e., the nuclear-factor-κB and TGFβ pathways. The minor allele at rs1883832, in the 5'-untranslated region of CD40, was associated with earlier LoA (p = 3.5 × 10(-5)). This allele diminishes the expression of CD40, a co-stimulatory molecule for T cell polarization. We validated this association in multiple independent DMD cohorts (United Dystrophinopathy Project, Bio-NMD, and Padova, total n = 660), establishing this locus as a DMD modifier. This finding points to cell-mediated immunity as a relevant pathogenetic mechanism and potential therapeutic target in DMD. Copyright © 2016. Published by Elsevier Inc.

  3. Th40 cells (CD4+CD40+ Tcells) drive a more severe form of Experimental Autoimmune Encephalomyelitis than conventional CD4 T cells

    PubMed Central

    Vaitaitis, Gisela M.; Yussman, Martin G.; Waid, Dan M.; Wagner, David H.

    2017-01-01

    CD40-CD154 interaction is critically involved in autoimmune diseases, and CD4 T cells play a dominant role in the Experimental Autoimmune Encephalomyelitis (EAE) model of Multiple Sclerosis (MS). CD4 T cells expressing CD40 (Th40) are pathogenic in type I diabetes but have not been evaluated in EAE. We demonstrate here that Th40 cells drive a rapid, more severe EAE disease course than conventional CD4 T cells. Adoptively transferred Th40 cells are present in lesions in the CNS and are associated with wide spread demyelination. Primary Th40 cells from EAE-induced donors adoptively transfer EAE without further in-vitro expansion and without requiring the administration of the EAE induction regimen to the recipient animals. This has not been accomplished with primary, non-TCR-transgenic donor cells previously. If co-injection of Th40 donor cells with Freund’s adjuvant (CFA) in the recipient animals is done, the disease course is more severe. The CFA component of the EAE induction regimen causes generalized inflammation, promoting expansion of Th40 cells and infiltration of the CNS, while MOG-antigen shapes the antigen-specific TCR repertoire. Those events are both necessary to precipitate disease. In MS, viral infections or trauma may induce generalized inflammation in susceptible individuals with subsequent disease onset. It will be important to further understand the events leading up to disease onset and to elucidate the contributions of the Th40 T cell subset. Also, evaluating Th40 levels as predictors of disease onset would be highly useful because if either the generalized inflammation event or the TCR-honing can be interrupted, disease onset may be prevented. PMID:28192476

  4. Tumour-necrosis-factor-receptor-associated factor 6, NF-kappaB-inducing kinase and IkappaB kinases mediate IgE isotype switching in response to CD40.

    PubMed Central

    Brady, K; Fitzgerald, S; Moynagh, P N

    2000-01-01

    The process of IgE switching requires the prior transcription of the unrearranged Cepsilon gene, which leads to its recombination with the VDJ region. The activation of NF-kappaB by CD40 is a key process in facilitating this transcription by promoting the activation of the Cepsilon promoter. The present study explores the uncharacterized signalling pathways employed by CD40 in activating NF-kappaB by the overexpression of genes encoding wild-type and dominant-negative forms of the signalling components tumour-necrosis-factor-receptor-associated factor 6 (TRAF-6), NF-kappaB-inducing kinase (NIK), IkappaB kinase (IKK)-1 and IKK-2 in the BJAB B-cell line. The overexpression of TRAF-6 or NIK was sufficient to activate NF-kappaB and the Cepsilon promoter, whereas their dominant-negative counterparts decreased the ability of CD40 to activate NF-kappaB and the Cepsilon promoter. The overexpression of wild-type IKK-1 or IKK-2 seemed to cause toxic effects on the cells, whereas the dominant-negative forms were selective in their blockade of NF-kappaB and the Cepsilon promoter. These results suggest that CD40 employs TRAF-6, which presumably recruits NIK, which in turn employs IKK-1/IKK-2 to activate NF-kappaB and the Cepsilon promoter, the prologue to IgE switching. Thus the findings define a crucially important pathway in the generation of allergic states. PMID:10970786

  5. [Effects of essential periodontal treatment on serum level of sCD40L and periodontal clinical parameters in patients with moderate to severe periodontitis at high risk of stroke].

    PubMed

    Gao, Lei; Sun, Xiao-Ju; Xie, Hong; Nan, Shun-Hua; Xie, Hui-Xin

    2016-10-01

    To investigate the effect of periodontal treatment on patients with moderate to severe periodontitis at high risk of stroke, by detecting the level of serum soluble cell differentiation antigen 40 ligand (sCD40L) before and after periodontal non-surgical treatment. Seventy-six patients with moderate to severe periodontitis at high risk of stroke were collected and randomly divided into 2 groups, 40 patients in group A received essential periodontal treatment + routine maintenance therapy, 36 cases in group B only received routine maintenance therapy. Another 36 patients with moderate and severe periodontitis were selected as group C, and received essential periodontal treatment. Bleeding on probing (BOP), periodontal probing depth (PD) and attachment loss (AL) in 6 loci were examined by the same dentists, and enzyme linked immunosorbent assay (ELISA) was used to detect the level of serum sCD40L before treatment and 3 months after treatment. The data were analyzed by SPSS 17.0 software package. Compared with pre-treatment, serum level of sCD40L and periodontal clinical indexes of the three groups decreased. Compared with group B, serum SCD40L in group A significantly decreased(P<0.05). Periodontal treatment can reduce the serum level of sCD40L in patients with moderate to severe periodontitis at high risk of stroke, and improve the patient's inflammatory state. To a certain extent, periodontal treatment may reduce the risk of high-risk stroke population to develop stroke.

  6. Increased Levels of Oxidative Stress Markers, Soluble CD40 Ligand, and Carotid Intima-Media Thickness Reflect Acceleration of Atherosclerosis in Male Patients with Ankylosing Spondylitis in Active Phase and without the Classical Cardiovascular Risk Factors

    PubMed Central

    Cholewka, Armand; Sieroń, Karolina; Sieroń, Aleksander

    2017-01-01

    Objective The primary aim of the study was to assess levels of oxidative stress markers, soluble CD40 ligand (sCD40L), serum pregnancy-associated plasma protein-A (PAPP-A), and placental growth factor (PlGF) as well as carotid intima-media thickness (IMT) in patients with ankylosing spondylitis (AS) with active phase without concomitant classical cardiovascular risk factors. Material and methods The observational study involved 96 male subjects: 48 AS patients and 48 healthy ones, who did not differ significantly regarding age, BMI, comorbid disorders, and distribution of classical cardiovascular risk factors. In both groups, we estimated levels of oxidative stress markers, lipid profile, and inflammation parameters as well as sCD40L, serum PAPP-A, and PlGF. In addition, we estimated carotid IMT in each subject. Results The study showed that markers of oxidative stress, lipid profile, and inflammation, as well as sCD40L, PlGF, and IMT, were significantly higher in the AS group compared to the healthy group. Conclusion Our results demonstrate that ankylosing spondylitis may be associated with increased risk for atherosclerosis. PMID:28883908

  7. The protein tyrosine phosphatase PTP1B is a negative regulator of CD40 and BAFF-R signaling and controls B cell autoimmunity

    PubMed Central

    Hobeika, Elias; Biesen, Robert; Kollert, Florian; Taddeo, Adriano; Voll, Reinhard E.; Hiepe, Falk

    2014-01-01

    Tyrosine phosphorylation of signaling molecules that mediate B cell activation in response to various stimuli is tightly regulated by protein tyrosine phosphatases (PTPs). PTP1B is a ubiquitously expressed tyrosine phosphatase with well-characterized functions in metabolic signaling pathways. We show here that PTP1B negatively regulates CD40, B cell activating factor receptor (BAFF-R), and TLR4 signaling in B cells. Specifically, PTP1B counteracts p38 mitogen-activated protein kinase (MAPK) activation by directly dephosphorylating Tyr182 of this kinase. Mice with a B cell–specific PTP1B deficiency show increased T cell–dependent immune responses and elevated total serum IgG. Furthermore, aged animals develop systemic autoimmunity with elevated serum anti-dsDNA, spontaneous germinal centers in the spleen, and deposition of IgG immune complexes and C3 in the kidney. In a clinical setting, we observed that B cells of rheumatoid arthritis patients have significantly reduced PTP1B expression. Our data suggest that PTP1B plays an important role in the control of B cell activation and the maintenance of immunological tolerance. PMID:24590766

  8. CD40-independent natural killer-cell help promotes dendritic cell vaccine-induced T-cell immunity against endogenous B-cell lymphoma.

    PubMed

    Hömberg, Nadine; Adam, Christian; Riedel, Tanja; Brenner, Christoph; Flatley, Andrew; Röcken, Martin; Mocikat, Ralph

    2014-12-15

    It is well established that an interplay between natural killer (NK) cells and dendritic cells (DCs) gives rise to their reciprocal activation and provides a Th1-biased cytokine milieu that fosters antitumor T-cell responses. Ex vivo-differentiated DCs transferred into mice strongly stimulate endogenous NK cells to produce interferon (IFN)-γ and initiate a cascade that eventually leads to cytotoxic T-lymphocyte responses. We show that the ability of exogenous DCs to trigger this pathway obviates CD40 signaling and CD4(+) T-cell help and depends on a preceding maturation step. Importantly, this mechanism was also effective in endogenously arising tumors where IFN-γ production is compromised in contrast to transplantable tumors. In c-myc-transgenic mice developing spontaneous lymphomas, injection of unpulsed DCs caused NK-cell activation and induced CD8(+) T cells capable of recognizing the lymphoma cells. Animals treated with unpulsed DCs showed a survival benefit compared to untreated myc mice. Hence, tumor immunity induced by DC-based vaccines not only depends on specific antigens loaded on the DCs. Rather, DC vaccines generate broader immune responses, because endogenous DCs presenting tumor antigens may also become stimulated by NK cells that were activated by exogenous DCs. Thus, the DC/NK-cell/cytotoxic T lymphocyte axis may commonly have relevance for DC-based vaccination protocols in clinical settings. © 2014 UICC.

  9. Cancer Immunology Miniatures: Immune activation and a 9-year ongoing complete remission following CD40 antibody therapy and metastasectomy in a patient with metastatic melanoma

    PubMed Central

    Bajor, David L.; Xu, Xiaowei; Torigian, Drew A.; Mick, Rosemarie; Garcia, Laura R.; Richman, Lee P.; Desmarais, Cindy; Nathanson, Katherine L.; Schuchter, Lynn M.; Kalos, Michael; Vonderheide, Robert H.

    2014-01-01

    Direct immune activation via agonistic monoclonal antibodies (mAb) is a potentially complementary approach to therapeutic blockade of inhibitory immune receptors in cancer. Here, we provide genetic analysis of the immunological consequences associated with the use of an agonistic CD40 mAb in a patient with metastatic melanoma who responded, underwent a single metastasectomy, and then achieved a complete remission ongoing for more than 9 years after starting therapy. Tumor microenvironment after immunotherapy was associated with pro-inflammatory modulations and emergence of a de novo T-cell repertoire as detected by next-generation sequencing of T-cell receptors (TCR) in the tumor and blood. The de-novo T-cell repertoire identified in the post-treatment metastasectomy sample was also present – and in some cases expanded – in the circulation years after completion of therapy. Comprehensive study of this “exceptional responder” highlights the emerging potential of direct immune agonists in the next wave of cancer immunotherapies and a potential role for TCR deep sequencing in cancer immune assessment. PMID:25252722

  10. Glucolipotoxicity initiates pancreatic β-cell death through TNFR5/CD40-mediated STAT1 and NF-κB activation

    PubMed Central

    Bagnati, Marta; Ogunkolade, Babatunji W; Marshall, Catriona; Tucci, Carmen; Hanna, Katie; Jones, Tania A; Bugliani, Marco; Nedjai, Belinda; Caton, Paul W; Kieswich, Julius; Yaqoob, Muhammed M; Ball, Graham R; Marchetti, Piero; Hitman, Graham A; Turner, Mark D

    2016-01-01

    Type 2 diabetes is a chronic metabolic disorder, where failure to maintain normal glucose homoeostasis is associated with, and exacerbated by, obesity and the concomitant-elevated free fatty acid concentrations typically found in these patients. Hyperglycaemia and hyperlipidaemia together contribute to a decline in insulin-producing β-cell mass through activation of the transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT)-1. There are however a large number of molecules potentially able to modulate NF-κB and STAT1 activity, and the mechanism(s) by which glucolipotoxicity initially induces NF-κB and STAT1 activation is currently poorly defined. Using high-density microarray analysis of the β-cell transcritptome, we have identified those genes and proteins most sensitive to glucose and fatty acid environment. Our data show that of those potentially able to activate STAT1 or NF-κB pathways, tumour necrosis factor receptor (TNFR)-5 is the most highly upregulated by glucolipotoxicity. Importantly, our data also show that the physiological ligand for TNFR5, CD40L, elicits NF-κB activity in β-cells, whereas selective knockdown of TNFR5 ameliorates glucolipotoxic induction of STAT1 expression and NF-κB activity. This data indicate for the first time that TNFR5 signalling has a major role in triggering glucolipotoxic islet cell death. PMID:27512950

  11. MyD88/CD40 Genetic Adjuvant Function in Cutaneous Atypical Antigen-Presenting Cells Contributes to DNA Vaccine Immunogenicity

    PubMed Central

    Slawin, Kevin M.; Levitt, Jonathan M.; Spencer, David M.

    2016-01-01

    Therapeutic DNA-based vaccines aim to prime an adaptive host immune response against tumor-associated antigens, eliminating cancer cells primarily through CD8+ cytotoxic T cell-mediated destruction. To be optimally effective, immunological adjuvants are required for the activation of tumor-specific CD8+ T cells responses by DNA vaccination. Here, we describe enhanced anti-tumor efficacy of an in vivo electroporation-delivered DNA vaccine by inclusion of a genetically encoded chimeric MyD88/CD40 (MC) adjuvant, which integrates both innate and adaptive immune signaling pathways. When incorporated into a DNA vaccine, signaling by the MC adjuvant increased antigen-specific CD8+ T cells and promoted elimination of pre-established tumors. Interestingly, MC-enhanced vaccine efficacy did not require direct-expression of either antigen or adjuvant by local antigen-presenting cells, but rather our data supports a key role for MC function in “atypical” antigen-presenting cells of skin. In particular, MC adjuvant-modified keratinocytes increased inflammatory cytokine secretion, upregulated surface MHC class I, and were able to increase in vitro and in vivo priming of antigen-specific CD8+ T cells. Furthermore, in the absence of critical CD8α+/CD103+ cross-priming dendritic cells, MC was still able to promote immune priming in vivo, albeit at a reduced level. Altogether, our data support a mechanism by which MC signaling activates an inflammatory phenotype in atypical antigen-presenting cells within the cutaneous vaccination site, leading to an enhanced CD8+ T cell response against DNA vaccine-encoded antigens, through both CD8α+/CD103+ dendritic cell-dependent and independent pathways. PMID:27741278

  12. Effects of gene transfer CTLA4Ig and anti-CD40L monoclonal antibody on islet xenograft rejection in mice.

    PubMed

    Zhang, J; Li, H; Jiang, N; Zhang, Q; Wang, G-S; Yi, H-M; Fu, B-S; Wang, G-Y; Yang, Y; Chen, G-H

    2010-06-01

    Blockade of a costimulatory pathway by adenovirus-mediated cytotoxic T lymphocyte associated antigen 4 immunoglobulin (CTLA4-Ig) gene transfer and anti-CD40L mAb(MR1) have been reported to enhance graft survival in several experimental transplantation models. In this study, we investigated the effects of gene transfer of CTLA4Ig and MR1 on islet xenograft rejection in mice. Recombinant adenovirus AdCTLA4Ig was constructed to express CTLA4Ig. Islet grafts from adult male DA rats transferred with AdCTLA4Ig were transplanted to streptozocin-induced diabetic Balb/c mice. The diabetic mice were treated with MR1 after transplantation. We evaluated the islet xenograft mean survival time as well as changes in interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha) levels in transplanted mice. The mean survival of islet xenografts in the MR1 treatment group was 34.9 +/- 5.62 days, in the AdCTLA4Ig treatment group it was 56.5 +/- 10.64 days, and in the AdCTLA4Ig plus MR1 treatment group it was 112.9 +/- 19.26 days, all significantly prolonged compared with an untreated group (8.1 +/- 0.83 days). Within 1 week after transplantation the levels of IL-2 and TNF-alpha showed sharp increases in the untreated group, being significantly higher than those observed prior to transplantation. In conclusion, using both AdCTLA4Ig and MR1 can improve the islet xenograft survival. The beneficial effects of the combined use of the 2 reagents were superior to either 1 alone, possibly related to down-regulated expression of Th1 cell-related cytokines.

  13. Immunization of chickens with an agonistic monoclonal anti-chicken CD40 antibody-hapten complex: rapid and robust IgG response induced by a single subcutaneous injection.

    PubMed

    Chen, Chang-Hsin; Abi-Ghanem, Daad; Waghela, Suryakant D; Chou, Wen-Ko; Farnell, Morgan B; Mwangi, Waithaka; Berghman, Luc R

    2012-04-30

    Producing diagnostic antibodies in chicken egg yolk represents an alternate animal system that offers many advantages including high productivity at low cost. Despite being an excellent counterpart to mammalian antibodies, chicken IgG from yolk still represents an underused resource. The potential of agonistic monoclonal anti-CD40 antibodies (mAb) as a powerful immunological adjuvant has been demonstrated in mammals, but not in chickens. We recently reported an agonistic anti-chicken CD40 mAb (designated mAb 2C5) and showed that it may have potential as an immunological adjuvant. In this study, we examined the efficacy of targeting a short peptide to chicken CD40 [expressed by the antigen-presenting cells (APCs)] in enhancing an effective IgG response in chickens. For this purpose, an immune complex consisting of one streptavidin molecule, two directionally biotinylated mAb 2C5 molecules, and two biotinylated peptide molecules was produced. Chickens were immunized subcutaneously with doses of this complex ranging from 10 to 90 μg per injection once, and relative quantification of the peptide-specific IgG response showed that the mAb 2C5-based complex was able to elicit a strong IgG response as early as four days post-immunization. This demonstrates that CD40-targeting antigen to chicken APCs can significantly enhance antibody responses and induce immunoglobulin isotype-switching. This immunization strategy holds promise for rapid production of hapten-specific IgG in chickens.

  14. Datasets for the validation of the "in vivo" siRNA-silencing of CD40 and for the detection of new markers of atherosclerosis progression in ApoE-deficient mice.

    PubMed

    Hueso, Miguel; De Ramon, Laura; Navarro, Estanislao; Ripoll, Elia; Cruzado, Josep M; Grinyo, Josep M; Torras, Joan

    2016-12-01

    Data presented in this Data in Brief article correspond to the article "in vivo" silencing of CD40 reduces progression of experimental atherogenesis through a NFκB/miR-125b axis and reveals new potential mediators in the pathogenesis of atherosclerosis" (M. Hueso, L. De Ramon, E. Navarro, E. Ripoll, J.M. Cruzado, J.M. Grinyo, J. Torras, 2016) [1]. Here, we describe the validation of the silencing of CD40 expression with a specific siRNA in ApoE(-/-) mouse aortas, and its systemic effects on splenic lymphocytic subpopulations as well as on the infiltration of aortic intima by F4/80(+), galectin-3(+) macrophages or by NF-κB(+) cells. We also show the output of a Gene Ontology and TLDA analysis which allowed the detection of potential mediators of atherosclerosis progression. We provide the scientific community with a set of genes whose expression is increased during atherosclerosis progression but downregulated upon CD40 silencing.

  15. Tumor Necrosis Factor Receptor Associated Factors (TRAFs) 2 and 3 Form a Transcriptional Complex with Phosho-RNA Polymerase II and p65 in CD40 Ligand Activated Neuro2a Cells.

    PubMed

    El Hokayem, Jimmy; Brittain, George C; Nawaz, Zafar; Bethea, John R

    2017-03-01

    The tumor necrosis factor receptor-associated factors (TRAFs) have been classically described as adaptor proteins that function as solely cytosolic signaling intermediates for the TNF receptor superfamily, Toll-like receptors (TLRs), NOD, like receptors (NLRs), cytokine receptors, and others. In this study, we show for the first time that TRAFs are present within the cytoplasm and nucleus of Neuro2a cells and primary cortical neurons, and that TRAF2 and TRAF3 translocate into the nucleus within minutes of CD40L stimulation. Analysis of the transcriptional regulatory potential of TRAFs by luciferase assay revealed that each of the TRAFs differentially functions as a transcriptional activator or repressor in a cell-specific manner. Interestingly, ChIP-qPCR data demonstrate that TRAFs 2/3, p65, and pRNAPol II form part of a transcriptional complex on the Icam-1 gene promoter upon CD40L stimulation. We further determined that TRAF2 recruitment to the nucleus is critical for the ubiquitination of H2b, a transcription permissive epigenetic modification. Our findings demonstrate for the first time that TRAFs 2/3 participate in the formation of a CD40L-induced transcriptional complex in neuronal cells.

  16. Soluble CD40 ligand, plasminogen activator inhibitor-1 and thrombin-activatable fibrinolysis inhibitor-1-antigen in normotensive type 2 diabetic subjects without diabetic complications. Effects of metformin and rosiglitazone.

    PubMed

    Yener, Serkan; Comlekci, Abdurrahman; Akinci, Baris; Demir, Tevfik; Yuksel, Faize; Ozcan, Mehmet Ali; Bayraktar, Firat; Yesil, Sena

    2009-01-01

    To evaluate subclinical inflammation and fibrinolysis in low-risk type 2 diabetic subjects and to assess the efficacy of metformin and rosiglitazone in this group. Sixty-one normotensive, normoalbuminuric type 2 diabetic subjects without diabetes-related complications were included in a 4-week standardization period with glimepiride. After the standardization period, 21 subjects were excluded and the remaining 40 were randomly divided into two groups matched for age, gender, body mass index and disease duration. The first group (n = 20) received metformin (1,700 mg/day), the second group (n = 20) rosiglitazone (4 mg/day) for 12 weeks. Patients with low-density lipoprotein-cholesterol higher than 130 mg/dl at the beginning of the randomization period were treated with simvastatin (maximum dose 20 mg/day). Twenty-three healthy controls were also recruited. Cytokine measurements were performed with ELISA kits. Baseline plasma plasminogen activator inhibitor-1 (PAI-1) level of type 2 diabetic subjects was significantly elevated (p = 0.038), but baseline levels of soluble CD40 ligand (sCD40L) and thrombin-activatable fibrinolysis inhibitor-1 (TAFI) antigen did not differ from healthy controls. Twelve weeks of metformin or rosiglitazone therapy did not cause significant changes in sCD40L, PAI-1 and TAFI antigen levels. In simvastatin-treated subjects (n = 9) significant reductions of PAI-1 were achieved (p = 0.028), while sCD40L and TAFI-Ag did not differ from baseline values. Our results showed that nonobese diabetic patients at low cardiovascular risk had similar levels of subclinical markers of inflammation and fibrinolysis as matched healthy controls. Neither metformin nor rosiglitazone caused marked changes in sCD40L, PAI-1 and TAFI antigen levels. A subset of patients who received simvastatin showed a modest decrease in PAI-1 level and could contribute to beneficial vasculoprotective effect of the drug in type 2 diabetics. Copyright (c) 2009 S. Karger AG, Basel.

  17. Neuroprotective effect of salvianolic acid B against cerebral ischemic injury in rats via the CD40/NF-κB pathway associated with suppression of platelets activation and neuroinflammation.

    PubMed

    Xu, Shixin; Zhong, Aiqin; Ma, Huining; Li, Dan; Hu, Yue; Xu, Yingzhi; Zhang, Junping

    2017-04-15

    Neuroinflammation plays a critical role in the pathogenesis of ischemia/reperfusion (I/R) injury. Activated platelets are increasingly regarded as initiators and/or amplifiers of inflammatory processes in cerebral I/R injury. Salvianolic acid B (SAB) is the most abundant bioactive compound of Salviae miltiorrhizae, a well-known Chinese herb used to promote blood circulation and eliminating blood stasis. S. miltiorrhizae has been used clinically in Asia for the treatment of ischemic cerebrovascular diseases. In the present study, a rat model of transient middle cerebral artery occlusion (tMCAO) was established to investigate the neuroprotective effects and mechanisms of SAB treatment against focal cerebral I/R insult. The results showed that SAB treatment (3mg/kg, 6mg/kg and 12mg/kg, i.p.) dose-dependently decreased I/R-induced neurological deficits at 24, 48, and 72h after reperfusion and decreased plasma-soluble P-selectin and soluble CD40 ligand as early as 6h after onset of I/R insult. At 24h after reperfusion, SAB treatment significantly reduced neuronal and DNA damage in the hippocampal CA1 region and decreased neural cell loss in the ischemic core. The I/R-induced pro-inflammatory mediator mRNA and protein overexpression in the penumbra cortex, including ICAM-1, IL-1β, IL-6, IL-8, and MCP-1, were significantly inhibited by SAB in a dose-dependent manner. Further studies suggested SAB treatment attenuated CD40 expression and NF-κB activation, which involved NF-κB/p65 phosphorylation and IκBα phosphorylation and degradation. In conclusion, our findings indicated that the neuroprotective effects of SAB post cerebral I/R injury are associated with the inhibition of both platelets activation and production of pro-inflammatory mediators and the downregulation of the CD40/NF-κB pathway.

  18. Differential role of reactive oxygen species in the activation of mitogen-activated protein kinases and Akt by key receptors on B-lymphocytes: CD40, the B cell antigen receptor, and CXCR4

    PubMed Central

    Lee, Rosaline L.; Westendorf, Jens

    2007-01-01

    Background Antibodies produced by B-lymphocytes play a key role in the host defense against infection. The development, survival, and activation of B cell is regulated by multiple receptors including the B cell antigen receptor (BCR), which detects the presence of pathogens, CD40, which binds co-stimulatory molecules on activated T cells, and chemokines such as SDF-1 (CXCL12) that play key roles in B cell development and trafficking. Signaling by many receptors results in the generation of reactive oxygen species (ROS) that function as second messengers by regulating the activity of redox-sensitive kinases and phosphatases. We investigated the role of ROS in signaling by the BCR, CD40, and CXCR4, the receptor for SDF-1. We focused on activation of ERK, JNK, p38, and Akt, kinases that regulate multiple processes including cell survival, proliferation, and migration. Results Using the anti-oxidants N-acetyl L-cysteine (NAC) and ebselen to deplete intracellular ROS, we identified a differential requirement for ROS in the activation of ERK, JNK, p38, and Akt by these receptors. We found that CD40 activated JNK, p38, and Akt via redox-dependent pathways that were sensitive to ROS depletion by NAC and ebselen. In contrast, BCR-induced activation of ERK, JNK, p38, and Akt was not affected by ROS depletion. We also found that CXCR4-induced Akt activation was ROS-dependent even though activation of the ERK, JNK, and p38 MAP kinases by CXCR4 occurred via ROS-independent pathways. Conclusion The differential requirement for ROS in the activation of ERK, JNK, p38, and Akt by the BCR, CD40, and CXCR4 likely reflects the multiplicity of upstream activators for each of these kinases, only some of which may be regulated in a redox-dependent manner. These findings support the idea that ROS are important second messengers in B cells and suggest that oxidants or anti-oxidants could be used to modulate B cell activation. PMID:18481208

  19. B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses.

    PubMed

    Pone, Egest J; Lou, Zheng; Lam, Tonika; Greenberg, Milton L; Wang, Rui; Xu, Zhenming; Casali, Paolo

    2015-02-01

    Ig class switch DNA recombination (CSR) in B cells is crucial to the maturation of antibody responses. It requires IgH germline IH-CH transcription and expression of AID, both of which are induced by engagement of CD40 or dual engagement of a Toll-like receptor (TLR) and B cell receptor (BCR). Here, we have addressed cross-regulation between two different TLRs or between a TLR and CD40 in CSR induction by using a B cell stimulation system involving lipopolysaccharides (LPS). LPS-mediated long-term primary class-switched antibody responses and memory-like antibody responses in vivo and induced generation of class-switched B cells and plasma cells in vitro. Consistent with the requirement for dual TLR and BCR engagement in CSR induction, LPS, which engages TLR4 through its lipid A moiety, triggered cytosolic Ca2+ flux in B cells through its BCR-engaging polysaccharidic moiety. In the presence of BCR crosslinking, LPS synergized with a TLR1/2 ligand (Pam3CSK4) in CSR induction, but much less efficiently with a TLR7 (R-848) or TLR9 (CpG) ligand. In the absence of BCR crosslinking, R-848 and CpG, which per se induced marginal CSR, virtually abrogated CSR to IgG1, IgG2a, IgG2b, IgG3 and/or IgA, as induced by LPS or CD154 (CD40 ligand) plus IL-4, IFN-γ or TGF-β, and reduced secretion of class-switched Igs, without affecting B cell proliferation or IgM expression. The CSR inhibition by TLR9 was associated with the reduction in AID expression and/or IgH germline IH-S-CH transcription, and required co-stimulation of B cells by CpG with LPS or CD154. Unexpectedly, B cells also failed to undergo CSR or plasma cell differentiation when co-stimulated by LPS and CD154. Overall, by addressing the interaction of TLR1/2, TLR4, TLR7 and TLR9 in the induction of CSR and modulation of TLR-dependent CSR by BCR and CD40, our study suggests the complexity of how different stimuli cross-regulate an important B cell differentiation process and an important role of TLRs in inducing

  20. B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses

    PubMed Central

    Pone, Egest J.; Lou, Zheng; Lam, Tonika; Greenberg, Milton L.; Wang, Rui; Xu, Zhenming; Casali, Paolo

    2015-01-01

    Ig class switch DNA recombination (CSR) in B cells is crucial to the maturation of antibody responses. It requires IgH germline IH-CH transcription and expression of AID, both of which are induced by engagement of CD40 or dual engagement of a Toll-like receptor (TLR) and B cell receptor (BCR). Here, we have addressed cross-regulation between two different TLRs or between a TLR and CD40 in CSR induction by using a B cell stimulation system involving lipopolysaccharides (LPS). LPS mediated long-term primary class-switched antibody responses and memory-like antibody responses in vivo and induced generation of class-switched B cells and plasma cells in vitro. Consistent with the requirement for dual TLR and BCR engagement in CSR induction, LPS, which engages TLR4 through its lipid A moiety, triggered cytosolic Ca2+ flux in B cells through its BCR-engaging polysaccharidic moiety. In the presence of BCR crosslinking, LPS synergized with a TLR1/2 ligand (Pam3CSK4) in CSR induction, but much less efficiently with a TLR7 (R-848) or TLR9 (CpG) ligand. In the absence of BCR crosslinking, R-848 and CpG, which per se induced marginal CSR, virtually abrogated CSR to IgG1, IgG2a, IgG2b, IgG3 and/or IgA, as induced by LPS or CD154 (CD40 ligand) plus IL-4, IFN-γ or TGF-β, and reduced secretion of class-switched Igs, without affecting B cell proliferation or IgM expression. The CSR inhibition by TLR9 was associated with the reduction in AID expression and/or IgH germline IH-S-CH transcription, and required co-stimulation of B cells by CpG with LPS or CD154. Unexpectedly, B cells also failed to undergo CSR or plasma cell differentiation when co-stimulated by LPS and CD154. Overall, by addressing the interaction of TLR1/2, TLR4, TLR7 and TLR9 in the induction of CSR and modulation of TLR-dependent CSR by BCR and CD40, our study suggests the complexity of how different stimuli cross-regulate an important B cell differentiation process and an important role of TLRs in inducing

  1. Plasmacytoid dendritic cells resident in human thymus drive natural Treg cell development.

    PubMed

    Martín-Gayo, Enrique; Sierra-Filardi, Elena; Corbí, Angel L; Toribio, María L

    2010-07-01

    The generation of natural regulatory T cells (nTregs) is crucial for the establishment of immunologic self-tolerance and the prevention of autoimmunity. Still, the origin of nTregs and the mechanisms governing their differentiation within the thymus are poorly understood, particularly in humans. It was recently shown that conventional dendritic cells (cDCs) in human thymus were capable of inducing nTreg differentiation. However, the function of plasmacytoid DCs (pDCs), the other major subset of thymic DCs, remains unknown. Here we report that pDCs resident in the human thymus, when activated with CD40 ligand (CD40L) plus interleukin-3, efficiently promoted the generation of CD4(+)CD25(+)Foxp3(+) nTregs from autologous thymocytes. The progenitors of these nTregs were selectively found within CD4(+)CD8(+) thymocytes that had accomplished positive selection, as judged by their CD69(hi)TCR(hi) phenotype. Supporting the involvement of the CD40-CD40L pathway in pDC-induced nTreg generation, we show that positively selected CD4(+)CD8(+) progenitors specifically transcribed CD40L in vivo and up-regulated CD40L expression on T-cell receptor engagement, thereby promoting the activation of pDCs. Finally, evidence is provided that nTregs primed by pDCs displayed reciprocal interleukin-10/transforming growth factor-beta cytokine expression profiles compared with nTregs primed by cDCs. This functional diversity further supports a nonredundant tolerogenic role for thymic pDCs in the human thymus.

  2. TLR7 and TLR9 responsive human B cells share phenotypic and genetic characteristics

    PubMed Central

    Simchoni, Noa; Cunningham-Rundles, Charlotte

    2015-01-01

    B cells activated by nucleic-acid sensing Toll-like receptor 7 and TLR9 proliferate and secrete immune globulins. Memory B cells are presumably more responsive due to higher TLR expression levels, but selectivity and differential outcomes remain largely unknown. In this study, peripheral blood human B cells were stimulated by TLR7 or TLR9 ligands, with or without IFNα, and compared to activators CD40L plus IL-21, to identify differentially responsive cell populations, defined phenotypically and by BCR characteristics. While all activators induced differentiation and antibody secretion, TLR stimulation expanded IgM+ memory and plasma cell lineage committed populations and favored secretion of IgM, unlike CD40L/IL-21 which drove IgM and IgG more evenly. Patterns of proliferation similarly differed, with CD40L/IL-21 inducing proliferation of most memory and naïve B cells, in contrast to TLRs which induced robust proliferation in a subset of these cells. On deep sequencing of the IgH locus, TLR responsive B cells shared patterns of IgHV and IgHJ usage, clustering apart from CD40L/IL-21 and control conditions. TLR activators, but not CD40L/IL-21, similarly promoted increased sharing of CDR3 sequences. TLR responsive B cells were characterized by more somatic hypermutation, shorter CDR3 segments, and less negative charges. TLR activation also induced long positively charged CDR3 segments, suggestive of autoreactive antibodies. Testing this, culture supernatants from TLR stimulated B cells were found to bind HEp-2 cells, while those from CD40L/IL-21 stimulated cells did not. Human B cells possess selective sensitivity to TLR stimulation, with distinctive phenotypic and genetic signatures. PMID:25740945

  3. Cutting edge: IL-21 is a switch factor for the production of IgG1 and IgG3 by human B cells.

    PubMed

    Pène, Jérôme; Gauchat, Jean-François; Lécart, Sandrine; Drouet, Elodie; Guglielmi, Paul; Boulay, Vera; Delwail, Adriana; Foster, Don; Lecron, Jean-Claude; Yssel, Hans

    2004-05-01

    IL-21 is a cytokine that regulates the activation of T and NK cells and promotes the proliferation of B cells activated via CD40. In this study, we show that rIL-21 strongly induces the production of all IgG isotypes by purified CD19(+) human spleen or peripheral blood B cells stimulated with anti-CD40 mAb. Moreover, it was found to specifically induce the production of IgG(1) and IgG(3) by CD40-activated CD19(+)CD27(-) naive human B cells. Although stimulation of CD19(+) B cells via CD40 alone induced gamma 1 and gamma 3 germline transcripts, as well as the expression of activation-induced cytidine deaminase, only stimulation with both anti-CD40 mAb and rIL-21 resulted in the production of S gamma/S mu switch circular DNA. These results show that IL-21, in addition to promoting growth and differentiation of committed B cells, is a specific switch factor for the production of IgG(1) and IgG(3).

  4. 2,3,7,8-Tetrachlorodibenzo-p-dioxin suppresses tumor necrosis factor-alpha and anti-CD40-induced activation of NF-kappaB/Rel in dendritic cells: p50 homodimer activation is not affected.

    PubMed

    Ruby, Carl E; Leid, Mark; Kerkvliet, Nancy I

    2002-09-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) suppresses many immune responses, both innate and adaptive. Suppression is mediated by the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. The AhR mediates TCDD toxicity presumably through the alteration of transcriptional events, either by promoting gene expression or potentially by physically interacting with other transcription factors. Another transcription factor, NF-kappaB/Rel, is involved in several signaling pathways in immune cells and is crucial for generating effective immune responses. Dendritic cells (DCs), considered to be the "pacemakers" of the immune system, were recently recognized as targets of TCDD and are also dependent on NF-kappaB/Rel for activation and survival. In these studies, we investigated whether TCDD would alter the activation of NF-kappaB/Rel in DCs. The dendritic cell line DC2.4 was exposed to TCDD before treatment with tumor necrosis factor alpha (TNF-alpha) or anti-CD40, and NF-kappaB/Rel activation was measured by electrophoretic mobility shift assay and immunoblotting. TCDD suppressed the binding of NF-kappaB/Rel to its cognate response element in TNF-alpha- and anti-CD40-treated cells and blocked translocation to the nucleus. The AhR was shown to associate with RelA, after coimmunoprecipitation, and seemed to block its binding to DNA. It is noteworthy that p50 homodimers freely bound to DNA. These results suggest that TCDD may alter the balance between NF-kappaB/Rel heterodimers and transcriptional inhibitory p50 homodimers in DCs, leading to defects in the DCs and suppression of the immune response.

  5. Distinct Toll-like receptor-mediated cytokine production and immunoglobulin secretion in human newborn naïve B cells

    PubMed Central

    Pettengill, Matthew A; van Haren, Simon D; Li, Ning; Dowling, David J; Bergelson, Ilana; Jans, Jop; Ferwerda, Gerben; Levy, Ofer

    2016-01-01

    Neonatal innate immunity is distinct from that of adults, which may contribute to increased susceptibility to infection and limit vaccine responses. B cells play critical roles in protection from infection and detect PAMPs via TLRs, that, when co-activated with CD40, can drive B cell proliferation and antibody production. We characterized the expression of TLRs in circulating B cells from newborns and adults, and evaluated TLR- and CD40-mediated naïve B cell class-switch recombination (CSR) and cytokine production. Gene expression levels of most TLRs was similar between newborn and adult B cells, except newborn naïve B cells expressed more TLR9 than adult naïve B cells. Neonatal naïve B cells demonstrated impaired TLR2- and TLR7- but enhanced TLR9-mediated cytokine production. Significantly fewer newborn naïve B cells underwent CSR to produce IgG, an impairment also noted with IL-21 stimulation. Additionally, co-stimulation via CD40 and TLRs induced greater cytokine production in adult B cells. Thus, while newborn naïve B cells demonstrate adult-level expression of TLRs and CD40, the responses to stimulation of these receptors are distinct. Relatively high expression of TLR9 and impaired CD40-mediated Ig secretion contributes to distinct innate and adaptive immunity of human newborns and may inform novel approaches to early life immunization. PMID:27252169

  6. Co-culture of primary CLL cells with bone marrow mesenchymal cells, CD40 ligand and CpG ODN promotes proliferation of chemoresistant CLL cells phenotypically comparable to those proliferating in vivo

    PubMed Central

    Purroy, Noelia; Abrisqueta, Pau; Carabia, Júlia; Carpio, Cecilia; Palacio, Carles

    2015-01-01

    Chronic lymphocytic leukemia (CLL) cells residing in the bone marrow (BM) and in secondary lymphoid tissues receive survival and proliferative signals from the microenvironment, resulting in persistence of residual disease after treatment. In this study, we characterized primary CLL cells cultured with BM stromal cells, CD40 ligand and CpG ODN to partially mimic the microenvironment in the proliferative centers. This co-culture system induced proliferation and chemoresistance in primary CLL cells. Importantly, co-cultured primary CLL cells shared many phenotypical features with circulating proliferative CLL cells, such as upregulation of ZAP-70 and CD38 and higher CD49d and CD62L expression. This indicates aggressiveness and capability to interact with surrounding cells, respectively. In addition, levels of CXCR4 were decreased due to CXCR4 internalization after CXCL12 stimulation by BM stromal cells. We suggest that this co-culture system can be used to test drugs and their combinations that target the proliferative and drug resistant CLL cells. PMID:25544766

  7. Nasal mast cells in perennial allergic rhinitics exhibit increased expression of the Fc epsilonRI, CD40L, IL-4, and IL-13, and can induce IgE synthesis in B cells.

    PubMed Central

    Pawankar, R; Okuda, M; Yssel, H; Okumura, K; Ra, C

    1997-01-01

    Cross-linking of allergen specific IgE bound to the high affinity IgE receptor (FC epsilonRI) on the surface of mast cells with multivalent allergens results in the release of both pre-formed and newly generated mediators, and in the manifestation of allergic symptoms. The expression of Fc epsilonRI, and the synthesis of IgE are therefore critical for the development of allergic diseases. In this study, we report that nasal mast cells (NMC) from patients with perennial allergic rhinitis (PAR) expressed significantly greater levels of the Fc epsilonRI, CD40L, IL-4, and IL-13 as compared to NMC from patients with chronic infective rhinitis (CIR). The level of Fc epsilonRI expression in NMC of PAR patients strongly correlated with the levels of serum total (r = 0.8, P < 0.003) and specific IgE (r = 0.89, P < 0.0004) antibodies. In addition, stimulation of NMC with IL-4, upregulated the Fc epsilonRIalpha chain expression both at the protein and mRNA levels, as detected by flow cytometry and reverse transcriptase-polymerase chain reaction. Furthermore, NMC from PAR, but not CIR, patients induced IgE synthesis by purified B cells in the presence of Der fII (mite antigen). These results suggest novel and critical roles for mast cells in promoting the allergic reaction through the increased expression of Fc epsilonRI and by enhancing and amplifying the IgE production, within the local microenvironment. PMID:9119992

  8. Medroxyprogesterone acetate impairs human dendritic cell activation and function.

    PubMed

    Quispe Calla, N E; Ghonime, M G; Cherpes, T L; Vicetti Miguel, R D

    2015-05-01

    Does medroxyprogesterone acetate (MPA) impair human dendritic cell (DC) activation and function? In vitro MPA treatment suppressed expression of CD40 and CD80 by human primary DCs responding to Toll-like receptor 3 (TLR3) agonist stimulation (i.e. DC activation). Moreover, this MPA-mediated decrease in CD40 expression impaired DC capacity to stimulate T cell proliferation (i.e. DC function). MPA is the active molecule in Depo-Provera(®) (DMPA), a commonly used injectable hormonal contraceptive (HC). Although DMPA treatment of mice prior to viral mucosal tissue infection impaired the capacity of DCs to up-regulate CD40 and CD80 and prime virus-specific T cell proliferation, neither DC activation marker expression nor the ability of DCs to promote T cell proliferation were affected by in vitro progesterone treatment of human DCs generated from peripheral blood monocytes. This cross-sectional study examined MPA-mediated effects on the activation and function of human primary untouched peripheral blood DCs. Human DCs isolated from peripheral blood mononuclear cells by negative immunomagnetic selection were incubated for 24 h with various concentrations of MPA. After an additional 24 h incubation with the TLR3 agonist polyinosinic:polycytidylic acid (poly I:C), flow cytometry was used to evaluate DC phenotype (i.e. expression of CD40, CD80, CD86, and HLA-DR). In separate experiments, primary untouched human DCs were sequentially MPA-treated, poly I:C-activated, and incubated for 7 days with fluorescently labeled naïve allogeneic T cells. Flow cytometry was then used to quantify allogeneic T cell proliferation. Several pharmacologically relevant concentrations of MPA dramatically reduced CD40 and CD80 expression in human primary DCs responding to the immunostimulant poly I:C. In addition, MPA-treated DCs displayed a reduced capacity to promote allogeneic CD4(+) and CD8(+) T cell proliferation. In other DC: T cell co-cultures, the addition of antibody blocking the CD40

  9. Activation of Langerhans-Type Dendritic Cells Alters Human Cytomegalovirus Infection and Reactivation in a Stimulus-Dependent Manner

    PubMed Central

    Coronel, Roxanne; Jesus, Desyree M.; Dalle Ore, Lucia; Mymryk, Joe S.; Hertel, Laura

    2016-01-01

    Oral mucosal Langerhans cells (LC) are likely to play important roles in host defense against infection by human cytomegalovirus (CMV). We previously showed that in vitro-differentiated immature LC (iLC) populations contain smaller amounts of infected cells but produce higher yields than mature LC (mLC) cultures, obtained by iLC stimulation with fetal bovine serum (FBS), CD40 ligand (CD40L) and lipopolysaccharide (LPS). Here, we sought to determine if exposure to select stimuli can improve LC permissiveness to infection, if specific components of the mLC cocktail are responsible for lowering viral yields, if this is due to defects in progeny production or release, and if these restrictions are also effective against reactivated virus. None of the stimuli tested extended the proportion of infected cells to 100%, suggesting that the block to infection onset cannot be fully removed. While CD40L and FBS exerted positive effects on viral progeny production per cell, stimulation with LPS alone or in combination with CD40L was detrimental. Reductions in viral titers were not due to defects in progeny release, and the permissive or restrictive intracellular environment established upon exposure to each stimulus appeared to act in a somewhat similar way toward lytic and latent infections. PMID:27683575

  10. Disodium cromoglycate inhibits S mu-->S epsilon deletional switch recombination and IgE synthesis in human B cells

    PubMed Central

    1994-01-01

    IgE synthesis requires interleukin 4 (IL-4) and a T-B cell interaction that involves the B cell antigen CD40 and its ligand expressed on activated T cells. IL-4 induces epsilon germline transcription whereas ligation of CD40 results in deletional S mu-->S epsilon switch recombination, expression of mature epsilon transcripts, and IgE synthesis and secretion. We demonstrate that disodium cromoglycate (DSCG), a drug commonly used for the prophylactic treatment of allergic disease, inhibits T cell-driven IgE synthesis by human B cells at concentrations readily achievable in the course of inhaled therapy for asthma. Inhibition of IgE synthesis by DSCG was not the result of drug toxicity because DSCG did not affect the viability of T and B cells or their proliferation to mitogens. DSCG did not interfere with CD40 ligand expression by T cells but clearly targeted the B cells because it inhibited IgE synthesis induced by anti-CD40 and IL-4 in populations of highly purified B cells. DSCG had no effect on the induction of epsilon germline transcripts by IL-4 but strongly inhibited CD40 mediated S mu-->S epsilon deletional switch recombination in IL-4- treated B cells as assayed by nested primer PCR. The effect of DSCG was not specific for CD40-mediated induction of IgE isotype switching because DSCG inhibited IgE synthesis as well as S mu-->S epsilon deletional switch recombination induced by hydrocortisone and IL-4 in B cells. Moreover, the effect of DSCG was not specific for IgE isotype switching because DSCG inhibited the synthesis of IgG4 by B cells sorted for lack of surface expression of IgG4 and stimulated with anti- CD40 and IL-4. DSCG caused only minimal inhibition (< 15%) of spontaneous IgE synthesis by lymphocytes from patients with the hyper- IgE syndrome and did not affect pokeweed mitogen-induced IgG and IgA synthesis by lymphocytes suggesting that it has little effect on B cells that have already undergone isotype switching. These results indicate that DSCG

  11. Human CD100, a novel leukocyte semaphorin that promotes B-cell aggregation and differentiation.

    PubMed Central

    Hall, K T; Boumsell, L; Schultze, J L; Boussiotis, V A; Dorfman, D M; Cardoso, A A; Bensussan, A; Nadler, L M; Freeman, G J

    1996-01-01

    Herein we describe the molecular characterization of the human leukocyte activation antigen CD100 and identify it as the first semaphorin, to our knowledge, in the immune system. Semaphorins have recently been described as neuronal chemorepellants that direct pioneering neurons during nervous system development. In this study we demonstrate that CD100 induces B cells to aggregate and improves their viability in vitro. We show that CD100 modifies CD40-CD40L B-cell signaling by augmenting B-cell aggregation and survival and down-regulating CD23 expression. Thus, these results suggest that semaphorins as exemplified by CD100 also play a functional role in the immune system. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8876214

  12. Activation status of human microglia is dependent on lesion formation stage and remyelination in multiple sclerosis.

    PubMed

    Peferoen, Laura A N; Vogel, Daphne Y S; Ummenthum, Kimberley; Breur, Marjolein; Heijnen, Priscilla D A M; Gerritsen, Wouter H; Peferoen-Baert, Regina M B; van der Valk, Paul; Dijkstra, Christine D; Amor, Sandra

    2015-01-01

    Similar to macrophages, microglia adopt diverse activation states and contribute to repair and tissue damage in multiple sclerosis. Using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, we show that in vitro M1-polarized (proinflammatory) human adult microglia express the distinctive markers CD74, CD40, CD86, and CCR7, whereas M2 (anti-inflammatory) microglia express mannose receptor and the anti-inflammatory cytokine CCL22. The expression of these markers was assessed in clusters of activated microglia in normal-appearing white matter (preactive lesions) and areas of remyelination, representing reparative multiple sclerosis lesions. We show that activated microglia in preactive and remyelinating lesions express CD74, CD40, CD86, and the M2 markers CCL22 and CD209, but not mannose receptor. To examine whether this intermediate microglia profile is static or dynamic and thus susceptible to changes in the microenvironment, we polarized microglia into M1 or M2 phenotype in vitro and then subsequently treated them with the opposing polarization regimen. These studies revealed that expression of CD40, CXCL10, and mannose receptor is dynamic and that microglia, like macrophages, can switch between M1 and M2 phenotypic profiles. Taken together, our data define the differential activation states of microglia during lesion development in multiple sclerosis-affected CNS tissues and underscore the plasticity of human adult microglia in vitro.

  13. [Towards an industrial control of the cloning of lymphocytes B human for the manufacturing of monoclonal antibodies stemming from the human repertoire].

    PubMed

    Guillot-Chene, P; Lebecque, S; Rigal, D

    2009-05-01

    Monoclonal antibodies (mAbs) are efficient drugs for treating infectious, inflammatory and cancer diseases. Antibodies secreted by human lymphocytes that have been isolated from either peripheral blood or tissues present the definite interest of being part of the physiological or disease-related response to antigens present in the human body. However, attempts to generate hybridomas with human B cells have been largely unsuccessful, and cloning of human B cells has been achieved only via their inefficient immortalization with Epstein Barr Virus (EBV). However, recent progress in our understanding of the molecular mechanisms of polyclonal B cell activation has dramatically increased the capacity to clone human B cells. In particular, activation of human naïve and memory B cells through CD40 or memory B cells only through TLR9 was shown to greatly facilitate their immortalization by EBV. Industrial development based on these observations will soon provide large collections of high affinity human mAbs of every isotype directly selected by the human immune system directed to recognize epitopes relevant for individual patients. Moreover, after CD40 activation, these mAbs will cover the full human repertoire, including the natural auto-immune repertoire. Full characterization of the biological activity of these mAbs will in turn bring useful information for selecting vaccine epitopes. This breakthrough in human B cell cloning opens the way into new areas for therapeutic use of mAbs.

  14. Detection of epsilon class switching and IgE synthesis in human B cells.

    PubMed

    Pène, Jérôme; Guilhot, Florence; Cognet, Isabelle; Guglielmi, Paul; Guay-Giroux, Angélique; Bonnefoy, Jean-Yves; Elson, Greg C; Yssel, Hans; Gauchat, Jean-François

    2006-01-01

    We observed that mast cells, as other cells expressing the CD40 ligand CD154, can trigger IgE synthesis in B cells in the presence of interleukin (IL)-4. Numerous complementary techniques can be used to follow the succession of molecular events leading to IgE synthesis. This chapter will illustrate how human B cells (naïve or memory) can be purified, stored, and cultivated in medium that is permissive for IgE synthesis and stimulated with IL-4 or IL-13 and CD40 activation, the latter being induced by soluble CD154, anti-CD40 antibodies, or CD154-expressing cells. All these molecules are expressed by mast cells. The quantification of the epsilon-sterile transcript synthesis by polymerase chain reaction or Northern blot, the epsilon excision circles produced during immunoglobulin heavy chain locus rearrangement by polymerase chain reaction, and the IgE production by enzyme-linked immunosorbent assay will be described.

  15. Characterization of candidate anti-allergic probiotic strains in a model of th2-skewed human peripheral blood mononuclear cells.

    PubMed

    Holvoet, Sébastien; Zuercher, Adrian W; Julien-Javaux, Françoise; Perrot, Marie; Mercenier, Annick

    2013-01-01

    Pre-clinical and clinical studies have evaluated the efficacy of probiotics in allergy. However, predictive in vitro systems for rational strain selection are still missing. We developed a novel in vitro screening system for the characterization of probiotics with anti-allergic potential. In this model, human peripheral blood mononuclear cells (PBMC) from healthy donors (n = 68) were skewed towards a Th2 cytokine phenotype by culture with IL-4 and anti-CD40, to resemble cells from allergic donors. Th2-skewed cells were then co-cultured with probiotics; a total of 35 strains were tested. Levels of IFN-γ, IL-10, IL-5 and 7 additional cytokines in culture supernatants were determined by ELISA or multiplex assay. Gene expression was assessed by real-time PCR. For validation, splenocytes from ovalbumin-primed mice and PBMC from grass-allergic donors were restimulated with respective antigen and co-cultured with probiotics, and cytokine profiles were correlated. Culture with IL-4 and anti-CD40 antibody induced secretion of IL-5 from PBMC, indicative of induction of a Th2 phenotype. Cytokine profiles induced by probiotics were strain specific even though species- and genus-specific clustering was observed for many strains by principal component analysis. This was paralleled by mRNA levels of the corresponding genes such as increased Tbet and reduced GATA-3 gene expression. Cytokine profiles induced by probiotics in PBMC stimulated with IL-4 and anti-CD40 correlated with those obtained from allergen-stimulated murine splenocytes or human PBMC from grass-allergic donors. Cytokine profiling of probiotic strains with IL-4-/anti-CD40-stimulated PBMC allowed to determine the effect of probiotics on Th2-skewed cells and thus to classify probiotic strains with anti-allergic potential. Copyright © 2013 S. Karger AG, Basel.

  16. Peroxisome proliferator-activated receptor gamma activators affect the maturation of human monocyte-derived dendritic cells.

    PubMed

    Gosset, P; Charbonnier, A S; Delerive, P; Fontaine, J; Staels, B; Pestel, J; Tonnel, A B; Trottein, F

    2001-10-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma ), a member of the nuclear receptor superfamily, has recently been described as a modulator of macrophage functions and as an inhibitor of T cell proliferation. Here, we investigated the role of PPARgamma in dendritic cells (DC), the most potent antigen-presenting cells. We showed that PPARgamma is highly expressed in immature human monocyte-derived DC (MDDC) and that it may affect the immunostimulatory function of MDDC stimulated with lipopolysaccharide (LPS) or via CD40 ligand (CD40L). We found that the synthetic PPARgamma agonist rosiglitazone (as well as pioglitazone and troglitazone) significantly increases on LPS- and CD40L-activated MDDC, the surface expression of CD36 (by 184% and 104%, respectively) and CD86 (by 54% and 48%), whereas it reduces the synthesis of CD80 (by 42% and 42%). Moreover, activation of PPARgamma resulted in a dramatic decreased secretion of the Th1-promoting factor IL-12 in LPS- and CD40L-stimulated cells (by 47% and 62%), while the production of IL-1beta, TNF-alpha, IL-6 and IL-10 was unaffected. Finally, PPARgamma ligands down-modulate the synthesis of IFN-gamma -inducible protein-10 (recently termed as CXCL10) and RANTES (CCL5), both chemokines involved in the recruitment of Th1 lymphocytes (by 49% and 30%), but not the levels of the Th2 cell-attracting chemokines,macrophage-derived chemokine (CCL22) and thymus and activation regulated chemokine (CCL17), in mature MDDC. Taken together, our data suggest that activation of PPARgamma in human DC may have an impact in the orientation of primary and secondary immune responses by favoring type 2 responses.

  17. The induction of CD80 and apoptosis on B cells and CD40L in CD4+ T cells in response to seasonal influenza vaccination distinguishes responders versus non-responders in healthy controls and aviremic ART-treated HIV-infected individuals

    PubMed Central

    Powell, Anna M.; Luo, Zhenwu; Martin, Lisa; Wan, Zhuang; Ma, Lei; Liao, Guoyang; Song, Yuxia; Li, Xiaochun; Kilby, J. Michael; Huang, Lei; Jiang, Wei

    2016-01-01

    Background Studies have shown that HIV infection is associated with an impaired influenza vaccine response. We examined the role of cellular phenotypes and function in influenza vaccine responsiveness in healthy controls and aviremic HIV-infected subjects on antiretroviral treatment (ART). Methods 16 healthy controls and 26 ART+ aviremic HIV+ subjects were enrolled in the current study. Blood was collected at pre-vaccination (D0), and on days 7–10 (D7) and 14–21 (D14) following the 2013–2014 seasonal influenza vaccine administrations. Subjects were classified as responders if neutralizing titers against H1N1 virus increased ≥ 4-fold at D14 compared to D0. A serial analysis of B and CD4+ T cell frequencies and activation was performed on D0 and D7 by flow cytometry. Results 9 of 26 (34.6%) HIV-infected individuals and 7 of 16 (43.8%) healthy controls were classified as responders to influenza vaccines. Total B cell apoptosis (annexin V) was increased on D7 post-vaccination in non-responders but not in responders among both controls and HIV+ subjects. Surface CD80 expression on memory B cells and intracellular CD40L expression on memory CD4+ T cells were induced on D7 in responders of controls but not in non-responders. The CD80 and CD40L induction was not demonstrable in HIV-infected subjects regardless of responders and non-responders. Memory CD4+ T cell cycling tended to increase on D7 in the four study groups but did not achieve significance. All the other parameters were indistinguishable between responders and non-responders, regardless of HIV-infection status. Conclusion The perturbation of activation and apoptotic induction on B cells or CD4+ T cells after seasonal influenza vaccination in non-responders and HIV-infected subjects may help understand the mechanism of impaired vaccine responsiveness. PMID:28017428

  18. Btk inhibition suppresses agonist-induced human macrophage activation and inflammatory gene expression in RA synovial tissue explants.

    PubMed

    Hartkamp, Linda M; Fine, Jay S; van Es, Inge E; Tang, Man Wai; Smith, Michael; Woods, John; Narula, Satwant; DeMartino, Julie; Tak, Paul P; Reedquist, Kris A

    2015-08-01

    Bruton's tyrosine kinase (Btk) is required for B lymphocyte and myeloid cell contributions to pathology in murine models of arthritis. Here, we examined the potential contributions of synovial Btk expression and activation to inflammation in rheumatoid arthritis (RA). Btk was detected by immunohistochemistry and digital image analysis in synovial tissue from biologically naive RA (n=16) and psoriatic arthritis (PsA) (n=12) patients. Cell populations expressing Btk were identified by immunofluorescent double labelling confocal microscopy, quantitative (q-) PCR and immunoblotting. The effects of a Btk-specific inhibitor, RN486, on gene expression in human macrophages and RA synovial tissue explants (n=8) were assessed by qPCR, ELISA and single-plex assays. Btk was expressed at equivalent levels in RA and PsA synovial tissue, restricted to B lymphocytes, monocytes, macrophages and mast cells. RN486 significantly inhibited macrophage IL-6 production induced by Fc receptor and CD40 ligation. RN486 also reduced mRNA expression of overlapping gene sets induced by IgG, CD40 ligand (CD40L) and RA synovial fluid, and significantly suppressed macrophage production of CD40L-induced IL-8, TNF, MMP-1 and MMP-10, LPS-induced MMP-1, MMP-7 and MMP-10 production, and spontaneous production of IL-6, PDGF, CXCL-9 and MMP-1 by RA synovial explants. Btk is expressed equivalently in RA and PsA synovial tissue, primarily in macrophages. Btk activity is needed to drive macrophage activation in response to multiple agonists relevant to inflammatory arthritis, and promotes RA synovial tissue cytokine and MMP production. Pharmacological targeting of Btk may be of therapeutic benefit in the treatment of RA and other inflammatory diseases. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  19. Human IDO-competent, long-lived immunoregulatory dendritic cells induced by intracellular pathogen, and their fate in humanized mice.

    PubMed

    Tyagi, Rajeev K; Miles, Brodie; Parmar, Rajesh; Garg, Neeraj K; Dalai, Sarat K; Baban, Babak; Cutler, Christopher W

    2017-02-15

    Targeting of myeloid-dendritic cell receptor DC-SIGN by numerous chronic infectious agents, including Porphyromonas gingivalis, is shown to drive-differentiation of monocytes into dysfunctional mDCs. These mDCs exhibit alterations of their fine-tuned homeostatic function and contribute to dysregulated immune-responses. Here, we utilize P. gingivalis mutant strains to show that pathogen-differentiated mDCs from primary human-monocytes display anti-apoptotic profile, exhibited by elevated phosphorylated-Foxo1, phosphorylated-Akt1, and decreased Bim-expression. This results in an overall inhibition of DC-apoptosis. Direct stimulation of complex component CD40 on DCs leads to activation of Akt1, suggesting CD40 involvement in anti-apoptotic effects observed. Further, these DCs drove dampened CD8(+) T-cell and Th1/Th17 effector-responses while inducing CD25(+)Foxp3(+)CD127(-) Tregs. In vitro Treg induction was mediated by DC expression of indoleamine 2,3-dioxygenase, and was confirmed in IDO-KO mouse model. Pathogen-infected &CMFDA-labeled MoDCs long-lasting survival was confirmed in a huMoDC reconstituted humanized mice. In conclusion, our data implicate PDDCs as an important target for resolution of chronic infection.

  20. Human IDO-competent, long-lived immunoregulatory dendritic cells induced by intracellular pathogen, and their fate in humanized mice

    PubMed Central

    Tyagi, Rajeev K.; Miles, Brodie; Parmar, Rajesh; Garg, Neeraj K.; Dalai, Sarat K.; Baban, Babak; Cutler, Christopher W.

    2017-01-01

    Targeting of myeloid-dendritic cell receptor DC-SIGN by numerous chronic infectious agents, including Porphyromonas gingivalis, is shown to drive-differentiation of monocytes into dysfunctional mDCs. These mDCs exhibit alterations of their fine-tuned homeostatic function and contribute to dysregulated immune-responses. Here, we utilize P. gingivalis mutant strains to show that pathogen-differentiated mDCs from primary human-monocytes display anti-apoptotic profile, exhibited by elevated phosphorylated-Foxo1, phosphorylated-Akt1, and decreased Bim-expression. This results in an overall inhibition of DC-apoptosis. Direct stimulation of complex component CD40 on DCs leads to activation of Akt1, suggesting CD40 involvement in anti-apoptotic effects observed. Further, these DCs drove dampened CD8+ T-cell and Th1/Th17 effector-responses while inducing CD25+Foxp3+CD127− Tregs. In vitro Treg induction was mediated by DC expression of indoleamine 2,3-dioxygenase, and was confirmed in IDO-KO mouse model. Pathogen-infected & CMFDA-labeled MoDCs long-lasting survival was confirmed in a huMoDC reconstituted humanized mice. In conclusion, our data implicate PDDCs as an important target for resolution of chronic infection. PMID:28198424

  1. 1,25-dihydroxyvitamin D{sub 3} impairs NF-{kappa}B activation in human naive B cells

    SciTech Connect

    Geldmeyer-Hilt, Kerstin; Heine, Guido; Hartmann, Bjoern; Baumgrass, Ria; Radbruch, Andreas; Worm, Margitta

    2011-04-22

    Highlights: {yields} In naive B cells, VDR activation by calcitriol results in reduced NF-{kappa}B p105 and p50 protein expression. {yields} Ligating the VDR with calcitriol causes reduced nuclear translocation of NF-{kappa}B p65. {yields} Reduced nuclear amount of p65 after calcitriol incubation results in reduced binding of p65 on the p105 promoter. {yields} Thus, vitamin D receptor signaling may reduce or prevent activation of B cells and unwanted immune responses, e.g. in IgE dependent diseases such as allergic asthma. -- Abstract: 1{alpha},25-dihydroxyvitamin D{sub 3} (calcitriol), the bioactive metabolite of vitamin D, modulates the activation and inhibits IgE production of anti-CD40 and IL-4 stimulated human peripheral B cells. Engagement of CD40 results in NF-{kappa}B p50 activation, which is essential for the class switch to IgE. Herein, we investigated by which mechanism calcitriol modulates NF-{kappa}B mediated activation of human naive B cells. Naive B cells were predominantly targeted by calcitriol in comparison with memory B cells as shown by pronounced induction of the VDR target gene cyp24a1. Vitamin D receptor activation resulted in a strongly reduced p105/p50 protein and mRNA expression in human naive B cells. This effect is mediated by impaired nuclear translocation of p65 and consequently reduced binding of p65 to its binding site in the p105 promoter. Our data indicate that the vitamin D receptor reduces NF-{kappa}B activation by interference with NF-{kappa}B p65 and p105. Thus, the vitamin D receptor inhibits costimulatory signal transduction in naive B cells, namely by reducing CD40 signaling.

  2. [Surface markers and functions of human dendritic cells exposed to mobile phone 1800 MHz electromagnetic fields].

    PubMed

    Zhou, Zhi-dong; Zeng, Qun-li; Zheng, Yun; Zhang, Jian-bin; Chen, Hai-yang; Lu, De-qiang; Shao, Chuan-sen; Xia, Da-jing

    2008-01-01

    To investigate the effects of mobile phone 1800 MHz electromagnetic fields (EMF) on the surface markers and the functions of human dendritic cells (DC). Human DCs were exposed to intermittent 5 min on/10 min off EMF with specific absorption rates (SAR) 4 W/kg for 0 h, 1 h, 12 h or 24 h, respectively. FACS analysis was used to detect the positive percentage of DC surface markers including HLA-DR and co-stimulatory molecules such as CD80, CD86, CD40 and CD11c. CCK-8 kit was adopted to examine the function of allo-mixed lymphocyte reaction (allo-MLR) of DC, and enzyme linked immunosorbent assay (ELISA) to identify the levels of IL-12p70 and TNF-alpha secreted by DC. Compared with the sham radiation group, after exposure to the electromagnetic fields for 1 h, 12 h, or 24 h, HLA-DR, CD80,CD86 and CD40 were all declined except CD11c. The ability of DC allo-MLR in each exposure group was decreased significantly (P<0.05), especially in the 24 h exposure group. However, the secreted levels of IL-12p70 and TNF-alpha of DC in each exposure group remained no changed. The study showed that EMF exposure could down-regulate the surface molecules and stimulation ability of human DC.

  3. Safety Study of SEA-CD40 in Cancer Patients

    ClinicalTrials.gov

    2017-09-01

    Cancer; Carcinoma; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Hematologic Malignancies; Hodgkin Disease; Lymphoma; Lymphoma, B-Cell; Lymphoma, Follicular; Lymphoma, Large B-Cell, Diffuse; Melanoma; Neoplasms; Neoplasm Metastasis; Neoplasms, Head and Neck; Neoplasms, Squamous Cell; Non-Small Cell Lung Cancer; Non-Small Cell Lung Cancer Metastatic; Non-small Cell Carcinoma; Squamous Cell Cancer; Squamous Cell Carcinoma; Squamous Cell Carcinoma of the Head and Neck; Squamous Cell Neoplasm; Lymphoma, Non-Hodgkin

  4. Altered human gut dendritic cell properties in ulcerative colitis are reversed by Lactobacillus plantarum extracellular encrypted peptide STp.

    PubMed

    Al-Hassi, Hafid O; Mann, Elizabeth R; Sanchez, Borja; English, Nicholas R; Peake, Simon T C; Landy, Jonathan; Man, Ripple; Urdaci, Maria; Hart, Ailsa L; Fernandez-Salazar, Luis; Lee, Gui Han; Garrote, Jose A; Arranz, Eduardo; Margolles, Abelardo; Stagg, Andrew J; Knight, Stella C; Bernardo, David

    2014-05-01

    The human/microbiota cross-talk is partially mediated by bacteria-derived peptides like Serine-Threonine peptide (STp), which is resistant to gut proteolysis, is found in the human healthy colon and induces regulatory properties on gut dendritic cells (DCs); here we characterized human gut DC in ulcerative colitis (UC) patients and studied the effect of STp on their properties. Human colonic DC from healthy controls and UC patients were isolated, conditioned for 24 h +/- STp and characterized by flow cytometry, immunohistochemistry, and electron microscopy. Expression of immature DC markers DC-SIGN and ILT3, and Toll-like receptors were increased on gut UC-DC. Langerin (involved in phagocytosis), lymph node homing marker CCR7, and activation markers CD40/CD80/CD86 were decreased in UC. Gut DC had restricted stimulatory capacity for T-cells in UC. Conditioning of DC with STp in vitro reduced Toll-like receptor expression, increased CD40 and CD80 expression, and restored their stimulatory capacity. Colonic DCs display an abnormal immature phenotype in UC, which was partially restored following STp treatment. Bacteria-derived metabolites, like STp, seem to have a role in gut homeostasis that is missing in UC so they might lead a new era of probiotic products setting the basis for nondrug dietary therapy in inflammatory bowel disease. © 2013 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA Weinheim.

  5. Human regulatory B cells control the TFH cell response.

    PubMed

    Achour, Achouak; Simon, Quentin; Mohr, Audrey; Séité, Jean-François; Youinou, Pierre; Bendaoud, Boutahar; Ghedira, Ibtissem; Pers, Jacques-Olivier; Jamin, Christophe

    2017-07-01

    Follicular helper T (TFH) cells support terminal B-cell differentiation. Human regulatory B (Breg) cells modulate cellular responses, but their control of TFH cell-dependent humoral immune responses is unknown. We sought to assess the role of Breg cells on TFH cell development and function. Human T cells were polyclonally stimulated in the presence of IL-12 and IL-21 to generate TFH cells. They were cocultured with B cells to induce their terminal differentiation. Breg cells were included in these cultures, and their effects were evaluated by using flow cytometry and ELISA. B-cell lymphoma 6, IL-21, inducible costimulator, CXCR5, and programmed cell death protein 1 (PD-1) expressions increased on stimulated human T cells, characterizing TFH cell maturation. In cocultures they differentiated B cells into CD138(+) plasma and IgD(-)CD27(+) memory cells and triggered immunoglobulin secretions. Breg cells obtained by Toll-like receptor 9 and CD40 activation of B cells prevented TFH cell development. Added to TFH cell and B-cell cocultures, they inhibited B-cell differentiation, impeded immunoglobulin secretions, and expanded Foxp3(+)CXCR5(+)PD-1(+) follicular regulatory T cells. Breg cells modulated IL-21 receptor expressions on TFH cells and B cells, and their suppressive activities involved CD40, CD80, CD86, and intercellular adhesion molecule interactions and required production of IL-10 and TGF-β. Human Breg cells control TFH cell maturation, expand follicular regulatory T cells, and inhibit the TFH cell-mediated antibody secretion. These novel observations demonstrate a role for the Breg cell in germinal center reactions and suggest that deficient activities might impair the TFH cell-dependent control of humoral immunity and might lead to the development of aberrant autoimmune responses. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  6. Bcl6 and Maf cooperate to instruct human follicular helper CD4 T cell differentiation.

    PubMed

    Kroenke, Mark A; Eto, Danelle; Locci, Michela; Cho, Michael; Davidson, Terence; Haddad, Elias K; Crotty, Shane

    2012-04-15

    Follicular helper CD4 T (Tfh) cells provide B cells with signals that are important for the generation of high-affinity Abs and immunological memory and, therefore, are critical for the protective immunity elicited by most human vaccines. Transcriptional regulators of human Tfh cell differentiation are poorly understood. In this article, we demonstrate that Bcl6 controls specific gene modules for human Tfh cell differentiation. The introduction of Bcl6 expression in primary human CD4 T cells resulted in the regulation of a core set of migration genes that enable trafficking to germinal centers: CXCR4, CXCR5, CCR7, and EBI2. Bcl6 expression also induced a module of protein expression critical for T-B interactions, including SAP, CD40L, PD-1, ICOS, and CXCL13. This constitutes direct evidence for Bcl6 control of most of these functions and includes three genes known to be loci of severe human genetic immunodeficiencies (CD40L, SH2D1A, and ICOS). Introduction of Bcl6 did not alter the expression of IL-21 or IL-4, the primary cytokines of human Tfh cells. We show in this article that introduction of Maf (c-Maf) does induce the capacity to express IL-21. Surprisingly, Maf also induced CXCR5 expression. Coexpression of Bcl6 and Maf revealed that Bcl6 and Maf cooperate in the induction of CXCR4, PD-1, and ICOS. Altogether, these findings reveal that Bcl6 and Maf collaborate to orchestrate a suite of genes that define core characteristics of human Tfh cell biology.

  7. Bcl6 and Maf cooperate to instruct human follicular helper CD4 T cell (Tfh) differentiation

    PubMed Central

    Kroenke, Mark A.; Eto, Danelle; Locci, Michela; Cho, Michael; Davidson, Terence; Haddad, Elias; Crotty, Shane

    2012-01-01

    Follicular helper CD4 T cells (Tfh) provide B cells with signals important for the generation of high-affinity antibodies and immunological memory, and are therefore critical for the protective immunity elicited by most human vaccines. Transcriptional regulators of human Tfh cell differentiation are poorly understood. Here we demonstrate that Bcl6 controls specific gene modules for human Tfh differentiation. The introduction of Bcl6 expression in primary human CD4 T cells resulted in regulation of a core set of migration genes that enable trafficking to germinal centers: CXCR4, CXCR5, CCR7, and EBI2. Bcl6 expression also induced a module of protein expression critical for T:B interactions, including SAP, CD40L, PD-1, ICOS, and CXCL13. This is the first direct evidence for Bcl6 control of most of these functions, and includes three genes known to be loci of severe human genetic immunodeficiencies (CD40L, SH2D1A, ICOS). Introduction of Bcl6 did not alter expression of IL-21 or IL-4, the primary cytokines of human Tfh cells. We show here that introduction of Maf (c-Maf) does induce the capacity to express IL-21. Surprisingly, Maf also induced CXCR5 expression. Co-expression of Bcl6 and Maf together revealed that Bcl6 and Maf cooperate in the induction of CXCR4, PD-1, and ICOS. Altogether, these findings reveal that Bcl6 and Maf collaborate to orchestrate a suite of genes that define core characteristics of human Tfh cell biology. PMID:22427637

  8. Insights from the Molecular Docking of Hydrolytic Products of Methyl Isocyanate (MIC) to Inhibition of Human Immune Proteins.

    PubMed

    Tripathi, Manish Kumar; Yasir, Mohammad; Gurjar, Vikram Singh; Bose, Protiti; Dubey, Amit; Shrivastava, Rahul

    2015-09-01

    This study is an attempt to find the reason for immunological suppression in victims of Bhopal gas tragedy during 1984 against Mycobacterium tuberculosis (Mtb) infection. Here, we tried to understand this problem by studying interactions between immune proteins associated with susceptibility to tuberculosis and hydrolytic products of methyl isocyanate (MIC) released during the tragedy. The hydrolytic products of MIC i.e. dimethyl urea, trimethyl urea and trimethyl isocyanurate were docked to different human immune proteins against Mtb using AutoDock 4.0. Results shows that all hydrolytic products (dimethyl urea, trimethyl urea and trimethylisocyanurate) strongly inhibit to CD40 ligand, and their binding energies were found to be [Formula: see text] G [Formula: see text]3.51, [Formula: see text]3.79, [Formula: see text]4.55 (Kcal/mole), respectively. Further, to check the stability of docked complex, we performed the molecular dynamics simulation study which also shows that CD40 Ligand was maximally inhibited by trimethylisocyanurate and has a role in the macrophage activation for the destruction of M. tuberculosis. The present study may lead to better understanding of human immune protein inhibition by hydrolytic product of MIC.

  9. Induction, binding specificity and function of human ICOS.

    PubMed

    Beier, K C; Hutloff, A; Dittrich, A M; Heuck, C; Rauch, A; Büchner, K; Ludewig, B; Ochs, H D; Mages, H W; Kroczek, R A

    2000-12-01

    Recently, we have identified the inducible co-stimulator (ICOS), an activation-dependent, T cell-specific cell surface molecule related to CD28 and CTLA-4. Detailed analysis of human ICOS presented here shows that it is a 55-60-kDa homodimer with differently N-glycosylated subunits of 27 and 29 kDa. ICOS requires both phorbol 12-myristate 13-acetate and ionomycin for full induction, and is sensitive to Cyclosporin A. ICOS is up-regulated early on all T cells, including the CD28- subset, and continues to be expressed into later phases of T cell activation. On stimulation of T cells by antigen-presenting cells, the CD28/B7, but not the CD40 ligand/CD40 pathway is critically involved in the induction of ICOS. ICOS does not bind to B7-1 or B7-2, and CD28 does not bind to ICOS ligand; thus the CD28 and ICOS pathways do not cross-interact on the cell surface. In vivo, ICOS is expressed in the medulla of the fetal and newborn thymus, in the T cell zones of tonsils and lymph nodes, and in the apical light zones of germinal centers (predominant expression). Functionally, ICOS co-induces a variety of cytokines including IL-4, IL-5, IL-6, IFN-gamma, TNF-alpha, GM-CSF, but not IL-2, and superinduces IL-10. Furthermore, ICOS co-stimulation prevents the apoptosis of pre-activated T cells. The human ICOS gene maps to chromosome 2q33 - 34.

  10. Resveratrol Protects against TNF-α-Induced Injury in Human Umbilical Endothelial Cells through Promoting Sirtuin-1-Induced Repression of NF-KB and p38 MAPK

    PubMed Central

    Huang, Shujie; Zhu, Pengli

    2016-01-01

    Inflammation and reactive oxygen species (ROS) play important roles in the pathogenesis of atherosclerosis. Resveratrol has been shown to possess anti-inflammatory and antioxidative stress activities, but the underlying mechanisms are not fully understood. In the present study, we investigated the molecular basis associated with the protective effects of resveratrol on tumor necrosis factor-alpha (TNF-α)-induced injury in human umbilical endothelial cells (HUVECs) using a variety of approaches including a cell viability assay, reverse transcription and quantitative polymerase chain reaction, western blot, and immunofluorescence staining. We showed that TNF-α induced CD40 expression and ROS production in cultured HUVECs, which were attenuated by resveratrol treatment. Also, resveratrol increased the expression of sirtuin 1 (SIRT1); and repression of SIRT1 by small-interfering RNA (siRNA) and the SIRT1 inhibitor Ex527 reduced the inhibitory effects of resveratrol on CD40 expression and ROS generation. In addition, resveratrol downregulated the levels of p65 and phospho-p38 MAPK, but this inhibitory effect was attenuated by the suppression of SIRT1 activity. Moreover, the p38 MAPK inhibitor SD203580 and the nuclear factor (NF)-κB inhibitor pyrrolidine dithiocarbamate (PDTC) achieved similar repressive effects as resveratrol on TNF-α-induced ROS generation and CD40 expression. Thus, our study provides a mechanistic link between resveratrol and the activation of SIRT1, the latter of which is involved in resveratrol-mediated repression of the p38 MAPK/NF-κB pathway and ROS production in TNF-α-treated HUVECs. PMID:26799794

  11. Human, Humanities, Humanitarian, Humanism.

    ERIC Educational Resources Information Center

    Penman, Kenneth A.; Adams, Samuel H.

    1982-01-01

    Traces the development of secular humanism in education and calls for educators to present their students with a "real" picture of the world, including the values upon which the Unites States was founded. (FL)

  12. Enhancement of terminal B lymphocyte differentiation in vitro by fibroblast-like stromal cells from human spleen.

    PubMed

    Skibinski, G; Skibinska, A; Stewart, G D; James, K

    1998-12-01

    Stromal elements are major components of lymphoid tissues contributing to both tissue architecture and function. In this study we report on the phenotype and function of fibroblast-like stromal cells obtained from human spleen. These cells express high levels of CD44 and ICAM-1 and moderate levels of VLA-4, VCAM, CD40 and CD21. They fail to express endothelial, epithelial, lymphocyte and monocyte/macrophage markers. We show that these cells interact with B cell blasts induced in vitro by anti-CD40 and anti-mu stimulation. As a result of these interactions both IL-6 and IgG secretion into culture medium is increased. The enhanced secretion of IgG is partly inhibited by abolishing B cell blaststromal cell contact or by anti-IL-6, anti-VCAM or anti-CD49d antibodies. Our studies also suggest that the ability of stromal cells to promote B cell survival is most likely the underlying mechanism of the enhanced immunoglobulin secretion. Comparison of stromal cells from different lymphoid and non-lymphoid organs revealed that bone marrow- and spleen-derived stromal cells are the most effective in promoting B cell blast differentiation.

  13. GILZ expression in human dendritic cells redirects their maturation and prevents antigen-specific T lymphocyte response.

    PubMed

    Cohen, Nicolas; Mouly, Enguerran; Hamdi, Haifa; Maillot, Marie-Christine; Pallardy, Marc; Godot, Véronique; Capel, Francis; Balian, Axel; Naveau, Sylvie; Galanaud, Pierre; Lemoine, François M; Emilie, Dominique

    2006-03-01

    Interleukin (IL)-10 and glucocorticoids (GCs) inhibit the ability of antigen-presenting dendritic cells (DCs) to stimulate T lymphocytes. We show that induction of GILZ (GC-induced leucine zipper) is involved in this phenomenon. IL-10, dexamethasone (DEX), and transforming growth factor (TGF)beta stimulate GILZ production in human immature DCs derived from monocytes and from CD34+ cells. GILZ is necessary and sufficient for DEX, IL-10, and TGFbeta modulation of CD80, CD83, CD86, immunoglobulin-like transcript (ILT)-3, and B7-H1 expression by DCs, and alteration of DC functions. GILZ stimulates the production of IL-10 by immature DCs and prevents the production of inflammatory chemokines by CD40L-activated DCs. In contrast, GILZ does not prevent CD40 ligand-mediated inhibition of phagocytosis, indicating that it affects some but not all aspects of DC maturation. GILZ prevents DCs from activating antigen-specific T lymphocyte responses. Administration of GCs to patients stimulates GILZ expression in their circulating antigen-presenting cells, and this contributes to the weak lymphocyte responses of GC-treated patients. Thus, regulation of GILZ expression is an important factor determining the decision of DCs whether or not to stimulate T lymphocytes, and IL-10, GCs, and TGFbeta share this mechanism for influencing DC functions and the balance between immune response and tolerance.

  14. IL-27 induces the production of IgG1 by human B cells.

    PubMed

    Boumendjel, Amel; Tawk, Lina; Malefijt, René de Waal; Boulay, Vera; Yssel, Hans; Pène, Jérôme

    2006-12-01

    It has been reported that IL-27 specifically induces the production of IgG2a by mouse B cells and inhibits IL-4-induced IgG1 synthesis. Here, we show that human naïve cord blood expresses a functional IL-27 receptor, consisting of the TCCR and gp130 subunits, although at lower levels as compared to naïve and memory splenic B cells. IL-27 does not induce proliferative responses and does not increase IgG1 production by CD19(+)CD27(+) memory B cells. However, it induces a low, but significant production of IgG1 by naïve CD19(+)CD27(-)IgD(+)IgG(-) spleen and cord blood B cells, activated via CD40, whereas it has no effect on the production of the other IgG subclasses. In addition, IL-27 induces the differentiation of a population of B cells that express high levels of CD38, in association with a down-regulation of surface IgD expression, and that are surface IgG(+/int), CD20(low), CD27(high), indicating that IL-27 promotes isotype switching and plasma cell differentiation of naive B cells. However, as compared to the effects of IL-21 and IL-10, both switch factors for human IgG1 and IgG3, those of IL-27 are modest and regulate exclusively the production of IgG1. Finally, although IL-27 has no effect on IL-4 and anti-CD40-induced Cepsilon germline promoter activity, it up-regulates IL-4-induced IgE production by naive B cells. These results point to a partial redundancy of switch factors regulating the production of IgG1 in humans, and furthermore indicate the existence of a common regulation of the human IgG1and murine IgG2a isotypes by IL-27.

  15. Amplification of IL-21 signalling pathway through Bruton's tyrosine kinase in human B cell activation.

    PubMed

    Wang, Sheau-Pey; Iwata, Shigeru; Nakayamada, Shingo; Niiro, Hiroaki; Jabbarzadeh-Tabrizi, Siamak; Kondo, Masahiro; Kubo, Satoshi; Yoshikawa, Maiko; Tanaka, Yoshiya

    2015-08-01

    B cells play an important role in the pathogenesis of autoimmune diseases. The role of Bruton's tyrosine kinase (Btk) in cytokine-induced human B cell differentiation and class-switch recombination remains incompletely defined. This study analysed the effect of Btk on human activated B cells. Purified B cells from healthy subjects were stimulated with B cell receptor (BCR) and other stimuli with or without a Btk inhibitor and gene expression was measured. The B cell line BJAB was used to assess Btk-associated signalling cascades. Phosphorylated Btk (p-Btk) in peripheral blood B cells obtained from 10 healthy subjects and 41 patients with RA was measured by flow cytometry and compared with patient backgrounds. IL-21 signalling, in concert with BCR, CD40 and BAFF signals, led to robust expression of differentiation- and class-switch DNA recombination-related genes and IgG production in human B cells, all of which were significantly suppressed by the Btk inhibitor. Although phosphorylation of STAT1 and STAT3 was induced by co-stimulation with IL-21, BCR and CD40, STAT1 phosphorylation in the nucleus, but not in the cytoplasm, was exclusively impaired by Btk blockade. High levels of p-Btk were noted in B cells of RA patients compared with controls and they correlated significantly with titres of RF among RF-positive patients. The findings elucidate a model in which Btk not only plays a fundamental role in the regulation of BCR signalling, but may also mediate crosstalk with cytokine signalling pathways through regulation of IL-21-induced phosphorylation of STAT1 in the nuclei of human B cells. Btk appears to have pathological relevance in RA. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  16. Suppression by Δ(9)-tetrahydrocannabinol of the primary immunoglobulin M response by human peripheral blood B cells is associated with impaired STAT3 activation.

    PubMed

    Ngaotepprutaram, Thitirat; Kaplan, Barbara L F; Carney, Stephen; Crawford, Robert; Kaminski, Norbert E

    2013-08-09

    This study was undertaken to gain insights into the mechanism for Δ(9)-tetrahydrocannabinol (Δ(9)-THC)-mediated suppression of primary immunoglobulin M (IgM) responses in humans. An in vitro activation model, which employs cell surface-expressed CD40 ligand (CD40L) and recombinant cytokines (interleukin (IL)-2, -6, and -10), was used to differentiate human peripheral blood (HPB) naïve B cells into IgM secreting cells. Pretreatment with Δ(9)-THC significantly decreased the number of IgM secreting cells as determined by ELISPOT. The attenuation of IgM secretion by Δ(9)-THC involved, at least in part, the impairment of plasma cell differentiation as evidenced by suppression of immunoglobulin joining chain (IgJ) mRNA expression. The analysis at each of two different stages critically involved in plasma cell differentiation indicates that Δ(9)-THC impaired both the primary activation stage and proliferation of B cells. Interestingly, Δ(9)-THC selectively suppressed the surface expression of CD80, but not other measured B-cell activation markers (CD69, CD86, and ICAM1). Furthermore, pretreatment with Δ(9)-THC was accompanied by a robust decrease of STAT3 phosphorylation, whereas the phosphorylation of the p65 NFκB subunit was not affected. Collectively, these data provide new insights into the mechanisms for impaired B cell function by Δ(9)-THC.

  17. IL-21 dependent IgE production in human and mouse in vitro culture systems is cell density and cell division dependent and is augmented by IL-10.

    PubMed

    Caven, Timothy H; Shelburne, Anne; Sato, Jun; Chan-Li, Yee; Becker, Steve; Conrad, Daniel H

    2005-12-01

    IL-21 is known to enhance immunoglobulin production using human in vitro models. Using either PBMC or purified tonsilar B cells both stimulated with anti-CD40, IL-4+/-IL-21, this enhancement was shown to correlate with increased cell division especially for IgE and to a lesser extent for IgM and total IgG. Cell division was monitored by CFSE staining and maximum cell division was found at low initial cell plating densities. A correlation between increased cell division and IL-10-mediated enhancement of IgE production was also seen; however, increased cell division plays a smaller role with IL-10 than IL-21. This is further emphasized in that when IL-10 and IL-21 were added together there was a further synergistic increase in IgE seen, but no accompanying further increase in cell division. The mouse system was also examined for IL-21 effects as a function of cell concentration, and as in humans, IL-21 added to murine cells increased IgE production over IL-4/CD40 stimulated cells at lower cell concentrations; however, IL-21 significantly reduced IgE at higher plated cell concentrations.

  18. Efficient generation of antigen-specific CTLs by the BAFF-activated human B Lymphocytes as APCs: a novel approach for immunotherapy

    PubMed Central

    Yingshi, Chen; Lishi, Su; Baohong, Luo; Chao, Liu; Linghua, Li; Ting, Pan; Hui, Zhang

    2016-01-01

    Efficient antigen presentation is indispensable for cytotoxic T lymphocyte (CTL)-mediated immunotherapy. B-lymphocytes propagated with CD40L have been developed as antigen-presenting cells (APCs), but this capacity needs further optimization. Here, we aimed to expand human B-lymphocytes on a large scale while maintaining their antigen-presenting ability by using both CD40L and B-cell activating factor (BAFF). The addition of BAFF enhanced the expansion efficiency and prolonged the culture time without causing apoptosis of the expanded B-cells. This method thus provided an almost unlimited source of cellular adjuvant to achieve sufficient expansion of CTLs in cases where several rounds of stimulation are required. We also showed that the addition of BAFF significantly enhanced the expression of major costimulatory molecules, CD80 and CD86. Subsequently, the antigen-presenting ability of the B-lymphocytes also increased. Consequently, these B-lymphocytes showed robust CTL responses to inhibit tumor growth after tumor-specific peptide pulses. A similar method induced potent antigen-specific CTL responses, which effectively eradicated human immunodeficiency virus type 1 (HIV-1) latency in CD4 T-lymphocytes isolated from patients receiving suppressive anti-retroviral therapy (ART). Together, our findings indicate that potent antigen-specific CTLs can be generated using BAFF-activated B-lymphocytes as APCs ex vivo. This approach can be applied for CTL-mediated immunotherapy in patients with cancers or chronic viral infections. PMID:27780916

  19. Human activation-induced cytidine deaminase is induced by IL-4 and negatively regulated by CD45: implication of CD45 as a Janus kinase phosphatase in antibody diversification.

    PubMed

    Zhou, Cheng; Saxon, Andrew; Zhang, Ke

    2003-02-15

    Activation-induced cytidine deaminase (AID) plays critical roles in Ig class switch recombination and V(H) gene somatic hypermutation. We investigated the role of IL-4 in AID mRNA induction, the signaling transduction involved in IL-4-mediated AID induction, and the effect of CD45 on IL-4-dependent AID expression in human B cells. IL-4 was able to induce AID expression in human primary B cells and B cell lines, and IL-4-induced AID expression was further enhanced by CD40 signaling. IL-4-dependent AID induction was inhibited by a dominant-negative STAT6, indicating that IL-4 induced AID expression via the Janus kinase (JAK)/STAT6 signaling pathway. Moreover, triggering of CD45 with anti-CD45 Abs can inhibit IL-4-induced AID expression, and this CD45-mediated AID inhibition correlated with the ability of anti-CD45 to suppress IL-4-activated JAK1, JAK3, and STAT6 phosphorylations. Thus, in humans, IL-4 alone is sufficient to drive AID expression, and CD40 signaling is required for optimal AID production; IL-4-induced AID expression is mediated via the JAK/STAT signaling pathway, and can be negatively regulated by the JAK phosphatase activity of CD45. This study indicates that the JAK phosphatase activity of CD45 can be induced by anti-CD45 Ab treatment, and this principle may find clinical application in modulation of JAK activation in immune-mediated diseases.

  20. Parallel T-cell cloning and deep sequencing of human MAIT cells reveal stable oligoclonal TCRβ repertoire.

    PubMed

    Lepore, Marco; Kalinichenko, Artem; Kalinicenko, Artem; Colone, Alessia; Paleja, Bhairav; Singhal, Amit; Tschumi, Andreas; Lee, Bernett; Poidinger, Michael; Zolezzi, Francesca; Quagliata, Luca; Sander, Peter; Newell, Evan; Bertoletti, Antonio; Terracciano, Luigi; De Libero, Gennaro; Mori, Lucia

    2014-05-15

    Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize conserved bacterial antigens derived from riboflavin precursors, presented by the non-polymorphic MHC class I-like molecule MR1. Here we show that human MAIT cells are remarkably oligoclonal in both the blood and liver, display high inter-individual homology and exhibit a restricted length CDR3β domain of the TCRVβ chain. We extend this analysis to a second sub-population of MAIT cells expressing a semi-invariant TCR conserved between individuals. Similar to 'conventional' MAIT cells, these lymphocytes react to riboflavin-synthesizing microbes in an MR1-restricted manner and infiltrate solid tissues. Both MAIT cell types release Th0, Th1 and Th2 cytokines, and sCD40L in response to bacterial infection, show cytotoxic capacity against infected cells and promote killing of intracellular bacteria, thus suggesting important protective and immunoregulatory functions of these lymphocytes.

  1. Inhibitive effect of purple sweet potato leaf extract and its components on cell adhesion and inflammatory response in human aortic endothelial cells.

    PubMed

    Chao, Pi-Yu; Huang, Ya-Ping; Hsieh, Wen-Bin

    2013-01-01

    This study investigated the effects of purple sweet potato leaf extract (PSPLE) and its components, cyanidin and quercetin, on human aortic endothelial cells (HAECs) during the inflammatory process. HAECs were pretreated with 100 μg/mL PSPLE or 10 μM quercetin, cyanidin or aspirin for 18 h followed by TNF-α (2 ng/mL) for 6 h, and U937 cell adhesion was determined. Adhesion molecule expression and CD40 were evaluated; NFκB p65 protein localization and DNA binding were assessed. PSPLE, aspirin, cyanidin and quercetin significantly inhibited TNF-α-induced monocyte-endothelial cell adhesion (p < 0.05). Cyanidin, quercetin and PSPLE also significantly attenuated VCAM-1, IL-8 and CD40 expression, and quercetin significantly attenuated ICAM-1 and E-selectin expression (p < 0.05). Significant reductions in NFκB expression and DNA binding by aspirin, cyanidin and quercetin were also observed in addition to decreased expression of ERK1, ERK2 and p38 MAPK (p < 0.05). Thus, PSPLE and its components, cyanidin and quercetin, have anti-inflammatory effects through modulation of NFκB and MAPK signaling. Further in vivo studies are necessary to explore the possible therapeutic effects of PSPLE on atherosclerosis.

  2. The PANE1 gene encodes a novel human minor histocompatibility antigen that is selectively expressed in B-lymphoid cells and B-CLL

    PubMed Central

    Brickner, Anthony G.; Evans, Anne M.; Mito, Jeffrey K.; Xuereb, Suzanne M.; Feng, Xin; Nishida, Tetsuya; Fairfull, Liane; Ferrell, Robert E.; Foon, Kenneth A.; Hunt, Donald F.; Shabanowitz, Jeffrey; Engelhard, Victor H.; Riddell, Stanley R.; Warren, Edus H.

    2006-01-01

    Minor histocompatibility antigens (mHAg's) are peptides encoded by polymorphic genes that are presented by major histocompatibility complex (MHC) molecules and recognized by T cells in recipients of allogeneic hematopoietic cell transplants. Here we report that an alternative transcript of the proliferation-associated nuclear element 1 (PANE1) gene encodes a novel human leukocyte antigen (HLA)-A*0301-restricted mHAg that is selectively expressed in B-lymphoid cells. The antigenic peptide is entirely encoded within a unique exon not present in other PANE1 transcripts. Sequencing of PANE1 alleles in mHAg-positive and mHAg-negative cells demonstrates that differential T-cell recognition is due to a single nucleotide polymorphism within the variant exon that replaces an arginine codon with a translation termination codon. The PANE1 transcript that encodes the mHAg is expressed at high levels in resting CD19+ B cells and B-lineage chronic lymphocytic leukemia (B-CLL) cells, and at significantly lower levels in activated B cells. Activation of B-CLL cells through CD40 ligand (CD40L) stimulation decreases expression of the mHAg-encoding PANE1 transcript and reciprocally increases expression of PANE1 transcripts lacking the mHAg-encoding exon. These studies suggest distinct roles for different PANE1 isoforms in resting compared with activated CD19+ cells, and identify PANE1 as a potential therapeutic target in B-CLL. PMID:16391015

  3. Monogenic mutations differentially impact the quantity and quality of T follicular helper cells in human primary immunodeficiencies

    PubMed Central

    Ma, Cindy S; Wong, Natalie; Rao, Geetha; Avery, Danielle T; Torpy, James; Hambridge, Thomas; Bustamante, Jacinta; Okada, Satoshi; Stoddard, Jennifer L; Deenick, Elissa K; Pelham, Simon J; Payne, Katherine; Boisson-Dupuis, Stéphanie; Puel, Anne; Kobayashi, Masao; Arkwright, Peter D; Kilic, Sara Sebnem; Baghdadi, Jamila El; Nonoyama, Shigeaki; Minegishi, Yoshiyuki; Mahdaviani, Seyed Alireza; Mansouri, Davood; Bousfiha, Aziz; Blincoe, Annaliesse K; French, Martyn A; Hsu, Peter; Campbell, Dianne E.; Stormon, Michael O; Wong, Melanie; Adelstein, Stephen; Smart, Joanne M; Fulcher, David A; Cook, Matthew C; Phan, Tri G; Stepensky, Polina; Boztug, Kaan; Kansu, Aydan; Ikincioğullari, Aydan; Baumann, Ulrich; Beier, Rita; Roscioli, Tony; Ziegler, John B; Gray, Paul; Picard, Capucine; Grimbacher, Bodo; Warnatz, Klaus; Holland, Steven M; Casanova, Jean-Laurent; Uzel, Gulbu; Tangye, Stuart G

    2016-01-01

    Background T follicular helper (Tfh) cells underpin T-cell dependent humoral immunity and the success of most vaccines. Tfh cells also contribute to human immune disorders such as autoimmunity, immunodeficiency and malignancy. Understanding the molecular requirements for the generation and function of Tfh cells will provide strategies for targeting these cells to modulate their behavior in the setting of these immunological abnormalities. Objective To determine the signaling pathways and cellular interactions required for the development and function of Tfh cells in humans. Methods Human primary immunodeficiencies (PIDs) resulting from monogenic mutations provide a unique opportunity to assess the requirement for particular molecules in regulating human lymphocyte function. Circulating Tfh (cTfh) cell subsets, memory B cells and serum Ig levels were quantified and functionally assessed in healthy controls as well as patients with PIDs resulting from mutations in STAT3, STAT1, TYK2, IL21, IL21R, IL10R, IFNGR1/2, IL12RB1, CD40LG, NEMO, ICOS or BTK. Results Loss-of function (LOF) mutations in STAT3, IL10R, CD40LG, NEMO, ICOS or BTK reduced cTfh frequencies. STAT3, IL21/R LOF and STAT1 gain-of function mutations skewed cTfh differentiation towards a phenotype characterized by over-expression of IFNγ and programmed death -1 (PD-1). IFNγ inhibited cTfh function in vitro and in vivo, corroborated by hypergammaglobulinemia in patients with IFNGR1/2, STAT1 and IL12RB1 LOF mutations. Conclusion Specific mutations impact the quantity and quality of cTfh cells, highlighting the need to assess Tfh cells in patients by multiple criteria, including phenotype and function. Furthermore, IFNγ functions in vivo to restrain Tfh-induced B cell differentiation. These findings shed new light on Tfh biology and the integrated signaling pathways required for their generation, maintenance and effector function, and explain compromised humoral immunity in some PIDs. PMID:26162572

  4. A Single-Cell Gene-Expression Profile Reveals Inter-Cellular Heterogeneity within Human Monocyte Subsets

    PubMed Central

    Gren, Susanne T.; Rasmussen, Thomas B.; Janciauskiene, Sabina; Håkansson, Katarina; Gerwien, Jens G.; Grip, Olof

    2015-01-01

    Human monocytes are a heterogeneous cell population classified into three different subsets: Classical CD14++CD16-, intermediate CD14++CD16+, and non-classical CD14+CD16++ monocytes. These subsets are distinguished by their differential expression of CD14 and CD16, and unique gene expression profile. So far, the variation in inter-cellular gene expression within the monocyte subsets is largely unknown. In this study, the cellular variation within each human monocyte subset from a single healthy donor was described by using a novel single-cell PCR gene-expression analysis tool. We investigated 86 different genes mainly encoding cell surface markers, and proteins involved in immune regulation. Within the three human monocyte subsets, our descriptive findings show multimodal expression of key immune response genes, such as CD40, NFⱪB1, RELA, TLR4, TLR8 and TLR9. Furthermore, we discovered one subgroup of cells within the classical monocytes, which showed alterations of 22 genes e.g. IRF8, CD40, CSF1R, NFⱪB1, RELA and TNF. Additionally one subgroup within the intermediate and non-classical monocytes also displayed distinct gene signatures by altered expression of 8 and 6 genes, respectively. Hence the three monocyte subsets can be further subdivided according to activation status and differentiation, independently of the traditional classification based on cell surface markers. Demonstrating the use and the ability to discover cell heterogeneity within defined populations of human monocytes is of great importance, and can be useful in unravelling inter-cellular variation in leukocyte populations, identifying subpopulations involved in disease pathogenesis and help tailor new therapies. PMID:26650546

  5. Generation of Human Antigen-Specific Monoclonal IgM Antibodies Using Vaccinated “Human Immune System” Mice

    PubMed Central

    van Geelen, Caroline M. M.; Noerder, Miriam; Huntington, Nicholas D.; Lim, Annick; Yasuda, Etsuko; Diehl, Sean A.; Scheeren, Ferenc A.; Ott, Michael; Weijer, Kees; Wedemeyer, Heiner; Di Santo, James P.; Beaumont, Tim; Guzman, Carlos A.; Spits, Hergen

    2010-01-01

    Background Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the ‘humanization’ of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique. Methodology/Principal Findings After transplantation with CD34+CD38− human hematopoietic progenitor cells, BALB/c Rag2−/−IL-2Rγc−/− mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. “Human Immune System” mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19+CD27+ B cells were retrovirally transduced with the human B cell lymphoma (BCL)-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively. Conclusion/Significance This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigens. PMID:20957227

  6. Salvianolic acid B suppresses maturation of human monocyte-derived dendritic cells by activating PPARγ

    PubMed Central

    Sun, Aijun; Liu, Hongying; Wang, Shijun; Shi, Dazhuo; Xu, Lei; Cheng, Yong; Wang, Keqiang; Chen, Keji; Zou, Yunzeng; Ge, Junbo

    2011-01-01

    BACKGROUND AND PURPOSE Salvianolic acid B (Sal B), a water-soluble antioxidant derived from a Chinese medicinal herb, is known to be effective in the prevention of atherosclerosis. Here, we tested the hypothesis that the anti-atherosclerotic effect of Sal B might be mediated by suppressing maturation of human monocyte-derived dendritic cells (h-monDC). EXPERIMENTAL APPROACH h-monDC were derived by incubating purified human monocytes with GM-CSF and IL-4. h-monDC were pre-incubated with or without Sal B and stimulated by oxidized low-density lipoprotein (ox-LDL) in the presence or absence of PPARγ siRNA. Expression of h-monDC membrane molecules (CD40, CD86, CD1a, HLA-DR) were analysed by FACS, cytokines were measured by elisa and the TLR4-associated signalling pathway was determined by Western blotting. KEY RESULTS Ox-LDL promoted h-monDC maturation, stimulated CD40, CD86, CD1a, HLA-DR expression and IL-12, IL-10, TNF-α production; and up-regulated TLR4 signalling. These effects were inhibited by Sal B. Sal B also triggered PPARγ activation and promoted PPARγ nuclear translocation, attenuated ox-LDL-induced up-regulation of TLR4 and myeloid differentiation primary-response protein 88 and inhibited the downstream p38-MAPK signalling cascade. Knocking down PPARγ with the corresponding siRNA blocked these effects of Sal B. CONCLUSIONS AND IMPLICATIONS Our data suggested that Sal B effectively suppressed maturation of h-monDC induced by ox-LDL through PPARγ activation. PMID:21649636

  7. Lactodifucotetraose, a human milk oligosaccharide, attenuates platelet function and inflammatory cytokine release.

    PubMed

    Newburg, David S; Tanritanir, Ayse C; Chakrabarti, Subrata

    2016-07-01

    Human milk strongly quenches inflammatory processes in vitro, and breastfed infants have lower incidence of inflammatory diseases than those fed artificially. Platelets from neonates, in contrast to those from adults, are less responsive to platelet agonists such as collagen, thrombin, ADP, and epinephrine. Breastfed infants absorb oligosaccharides intact from the human milk in their gut to the circulation. This study was to determine whether these oligosaccharides can attenuate platelet function and platelet secretion of pro-inflammatory proteins, and to identify the active component. The natural mixture of oligosaccharides from human milk and pure individual human milk oligosaccharides were tested for their ability to modulate responses of platelets isolated from human blood following exposure to thrombin, ADP, and collagen. Human milk and the natural mixture of human milk oligosaccharides inhibited platelet release of inflammatory proteins. Of the purified human milk oligosaccharides tested, only lactodifucotetraose (LDFT) significantly inhibited thrombin induced release of the pro-inflammatory proteins RANTES and sCD40L. LDFT also inhibited platelet adhesion to a collagen-coated surface, as well as platelet aggregation induced by ADP or collagen. These data indicate that LDFT may help modulate hemostasis by suppressing platelet-induced inflammatory processes in breastfed infants. This activity suggests further study of LDFT for its potential as a therapeutic agent in infants and adults.

  8. Jet exhaust particles alter human dendritic cell maturation.

    PubMed

    Ferry, D; Rolland, C; Delhaye, D; Barlesi, F; Robert, P; Bongrand, P; Vitte, Joana

    2011-03-01

    Among combustion-derived air pollutants, little is known about jet kerosene characteristics and effects. Particles yielded by experimental kerosene combustion in a jet engine were characterized with electron microscopy and X-ray energy dispersive spectroscopy. Immature human monocyte-derived dendritic cells were exposed for 18 h to 10, 25 or 100 μg/mL jet exhaust particles and/or Escherichia coli-derived endotoxin. Antigen-presenting and costimulation molecules (HLA DR, CD40, CD80, CD86, CD11c), tumor necrosis factor-α and interleukin-10 production were measured. The primary particles of jet exhaust are spherical (9.9 nm), carbonaceous and exert an adjuvant effect on human monocyte-derived dendritic cell maturation in vitro. Concomitant particle and endotoxin stimulation induced a high cytokine production with low antigen-presenting molecules; particle contact prior to endotoxin contact led to an opposite phenotype. Finally, low cytokine production and high costimulation molecules were present when particle adjunction followed endotoxin contact. Jet exhaust particles act as adjuvants to endotoxin-induced dendritic cell maturation, suggesting possible implications for human health and a role for the time pattern of infectious and pollutant interplay.

  9. Expression of Surface Molecules in Human Mesenchymal Stromal Cells Co-Cultured with Nucleated Umbilical Cord Blood Cells.

    PubMed

    Romanov, Yu A; Balashova, E E; Volgina, N E; Kabaeva, N V; Dugina, T N; Sukhikh, G T

    2017-02-01

    We studied the expression of different classes of surface molecules (CD13, CD29, CD40, CD44, CD54, CD71, CD73, CD80, CD86, CD90, CD105, CD106, CD146, HLA-I, and HLA-DR) in mesenchymal stromal cells from human umbilical cord and bone marrow during co-culturing with nucleated umbilical cord blood cells. Expression of the majority of surface markers in both types of mesenchymal stromal cells was stable and did not depend on the presence of the blood cells. Significant differences were found only for cell adhesion molecules CD54 (ICAM-1) and CD106 (VCAM-1) responsible for direct cell-cell contacts with leukocytes and only for bone marrow derived cells.

  10. IFN-gamma-mediated inhibition of human IgE synthesis by IL-21 is associated with a polymorphism in the IL-21R gene.

    PubMed

    Pène, Jérôme; Guglielmi, Laurence; Gauchat, Jean-François; Harrer, Nathalie; Woisetschläger, Maximilian; Boulay, Vera; Fabre, Jean-Michel; Demoly, Pascal; Yssel, Hans

    2006-10-15

    IL-21 is a cytokine produced by CD4+ T cells that has been reported to regulate human, as well as, mouse T and NK cell function and to inhibit Ag-induced IgE production by mouse B cells. In the present study, we show that human rIL-21 strongly enhances IgE production by both CD19+ CD27- naive, and CD19+ CD27+ memory B cells, stimulated with anti-CD40 mAb and rIL-4 and that it promotes the proliferative responses of these cells. However, rIL-21 does not significantly affect anti-CD40 mAb and rIL-4-induced Cepsilon promoter activation in a gene reporter assay, nor germline Cepsilon mRNA expression in purified human spleen or peripheral blood B cells. In contrast, rIL-21 inhibits rIL-4-induced IgE production in cultures of PBMC or total splenocytes by an IFN-gamma-dependent mechanism. The presence of a polymorphism (T-83C), in donors heterozygous for this mutation was found to be associated not only with lower rIL-21-induced IFN-gamma production levels, but also with a lower sensitivity to the inhibitory effects of IL-21 on the production of IgE, compared with those in donors expressing the wild-type IL-21R. Taken together, these results show that IL-21 differentially regulates IL-4-induced human IgE production, via its growth- and differentiation-promoting capacities on isotype-, including IgE-, committed B cells, as well as via its ability to induce IFN-gamma production, most likely by T and NK cells, whereas the outcome of these IL-21-mediated effects is dependent on the presence of a polymorphism in the IL-21R.

  11. Functional Analysis of CD28/B7 and CD40/CD40L Costimulation During the in vivo Type 2 Immune Response

    DTIC Science & Technology

    1995-10-06

    while suppressing cell-mediated immunity. Transplantation 58, 1082..1090. 177 Balzano, C., Buonavista, N., Rouvier , E., and Golstein, P. (1992). CTLA4 and...ofanergy in T cell clones. Nature 356, 607·609. Harper, K., Balzano, C., Rouvier , E., Mattei, M.G., Luciani, M.F., and Goistein, P. (1991). CTLA-4 and CD28

  12. Characteristics of Human Amniotic Fluid Mesenchymal Stem Cells and Their Tropism to Human Ovarian Cancer

    PubMed Central

    Li, Liru; Wang, Dejun; Zhou, Jun; Cheng, Yan; Liang, Tian; Zhang, Guangmei

    2015-01-01

    The mesenchymal stem cells (MSCs) derived from amniotic fluid (AF) have become an attractive stem cells source for cell-based therapy because they can be harvested at low cost and avoid ethical disputes. In human research, stem cells derived from AF gradually became a hot research direction for disease treatment, specifically for their plasticity, their reduced immunogenicity and their tumor tropism regardless of the tumor size, location and source. Our work aimed to obtain and characterize human amniotic fluid mesenchymal stem cells (AFMSCs) and detect their ovarian cancer tropsim in nude mice model. Ten milliliters of twenty independent amniotic fluid samples were collected from 16-20 week pregnant women who underwent amniocentesis for fetal genetic determination in routine prenatal diagnosis in the first affiliated hospital of Harbin medical university. We successfully isolated the AFMSCs from thirteen of twenty amniotic fluid samples. AFMSCs presented a fibroblastic-like morphology during the culture. Flow cytometry analyses showed that the cells were positive for specific stem cell markers CD73,CD90, CD105, CD166 and HLA-ABC (MHC class I), but negative for CD 45,CD40, CD34, CD14 and HLA-DR (MHC class II). RT-PCR results showed that the AFMSCs expressed stem cell marker OCT4. AFMSCs could differentiate into bone cells, fat cells and chondrocytes under certain conditions. AFMSCs had the high motility to migrate to ovarian cancer site but didn’t have the tumorigenicity. This study enhances the possibility of AFMSCs as drug carrier in human cell-based therapy. Meanwhile, the research emphasis in the future can also put in targeting therapy of ovarian cancer. PMID:25880317

  13. Characteristics of human amniotic fluid mesenchymal stem cells and their tropism to human ovarian cancer.

    PubMed

    Li, Liru; Wang, Dejun; Zhou, Jun; Cheng, Yan; Liang, Tian; Zhang, Guangmei

    2015-01-01

    The mesenchymal stem cells (MSCs) derived from amniotic fluid (AF) have become an attractive stem cells source for cell-based therapy because they can be harvested at low cost and avoid ethical disputes. In human research, stem cells derived from AF gradually became a hot research direction for disease treatment, specifically for their plasticity, their reduced immunogenicity and their tumor tropism regardless of the tumor size, location and source. Our work aimed to obtain and characterize human amniotic fluid mesenchymal stem cells (AFMSCs) and detect their ovarian cancer tropsim in nude mice model. Ten milliliters of twenty independent amniotic fluid samples were collected from 16-20 week pregnant women who underwent amniocentesis for fetal genetic determination in routine prenatal diagnosis in the first affiliated hospital of Harbin medical university. We successfully isolated the AFMSCs from thirteen of twenty amniotic fluid samples. AFMSCs presented a fibroblastic-like morphology during the culture. Flow cytometry analyses showed that the cells were positive for specific stem cell markers CD73,CD90, CD105, CD166 and HLA-ABC (MHC class I), but negative for CD 45,CD40, CD34, CD14 and HLA-DR (MHC class II). RT-PCR results showed that the AFMSCs expressed stem cell marker OCT4. AFMSCs could differentiate into bone cells, fat cells and chondrocytes under certain conditions. AFMSCs had the high motility to migrate to ovarian cancer site but didn't have the tumorigenicity. This study enhances the possibility of AFMSCs as drug carrier in human cell-based therapy. Meanwhile, the research emphasis in the future can also put in targeting therapy of ovarian cancer.

  14. NF-κB Links TLR2 and PAR1 to Soluble Immunomodulator Factor Secretion in Human Platelets

    PubMed Central

    Damien, Pauline; Cognasse, Fabrice; Payrastre, Bernard; Spinelli, Sherry L.; Blumberg, Neil; Arthaud, Charles-Antoine; Eyraud, Marie-Ange; Phipps, Richard P.; McNicol, Archibald; Pozzetto, Bruno; Garraud, Olivier; Hamzeh-Cognasse, Hind

    2017-01-01

    The primary toll-like receptor (TLR)-mediated immune cell response pathway common for all TLRs is MyD88-dependent activation of NF-κB, a seminal transcription factor for many chemokines and cytokines. Remarkably, anucleate platelets express the NF-κB machinery, whose role in platelets remains poorly understood. Here, we investigated the contribution of NF-κB in the release of cytokines and serotonin by human platelets, following selective stimulation of TLR2 and protease activated receptor 1 (PAR1), a classical and non-classical pattern-recognition receptor, respectively, able to participate to the innate immune system. We discovered that platelet PAR1 activation drives the process of NF-κB phosphorylation, in contrast to TLR2 activation, which induces a slower phosphorylation process. Conversely, platelet PAR1 and TLR2 activation induces similar ERK1/2, p38, and AKT phosphorylation. Moreover, we found that engagement of platelet TLR2 with its ligand, Pam3CSK4, significantly increases the release of sCD62P, RANTES, and sCD40L; this effect was attenuated by incubating platelets with a blocking anti-TLR2 antibody. This effect appeared selective since no modulation of serotonin secretion was observed following platelet TLR2 activation. Platelet release of sCD62P, RANTES, and sCD40L following TLR2 or PAR1 triggering was abolished in the presence of the NF-κB inhibitor Bay11-7082, while serotonin release following PAR1 activation was significantly decreased. These new findings support the concept that NF-κB is an important player in platelet immunoregulations and functions. PMID:28220122

  15. LMP1 and LMP2A collaborate to promote Epstein-Barr virus (EBV)-induced B cell lymphomas in a cord blood-humanized mouse model but are not essential.

    PubMed

    Ma, Shi-Dong; Tsai, Ming-Han; Romero-Masters, James C; Ranheim, Erik A; Huebner, Shane M; Bristol, Jillian; Delecluse, Henri-Jacques; Kenney, Shannon C

    2017-01-11

    Epstein-Barr virus (EBV) infection is associated with B cell lymphomas in humans. The ability of EBV to convert human B cells into long-lived lymphoblastoid cell lines (LCLs) in vitro requires the collaborative effects of EBNA2 (which hijacks notch signaling), LMP1 (which mimics CD40 signaling), and EBNA 3A/3C (which inhibit oncogene-induced senescence and apoptosis). However, we recently showed that an LMP1-deleted EBV mutant induces B cell lymphomas in a newly developed cord blood-humanized mouse model that allows EBV-infected B cells to interact with CD4 T cells (the major source of CD40 ligand). Here we examined whether the EBV LMP2A protein, which mimics constitutively active B cell receptor signaling, is required for EBV-induced lymphomas in this model. We find that deletion of LMP2A delays the onset of EBV-induced lymphomas, but does not affect the tumor phenotype or the number of tumors. Simultaneous deletion of both LMP1 and LMP2A results in fewer tumors, and a further delay in tumor onset. Nevertheless, the double LMP1/LMP2A mutant induces lymphomas in approximately half of the infected animals. These results indicate that neither LMP1 nor LMP2A is absolutely essential for the ability of EBV to induce B cell lymphomas in the cord blood-humanized mouse model, although simultaneous loss of both LMP1/LMP2A decreases the proportion of animals developing tumors and increases the time to tumor onset. Thus, either LMP1 or LMP2A expression may be sufficient to promote early-onset EBV-induced tumors in this model.

  16. Contribution of enhanced engagement of antigen presentation machinery to the clinical immunogenicity of a human interleukin (IL)-21 receptor-blocking therapeutic antibody.

    PubMed

    Xue, L; Hickling, T; Song, R; Nowak, J; Rup, B

    2016-01-01

    Reliable risk assessment for biotherapeutics requires accurate evaluation of risk factors associated with immunogenicity. Immunogenicity risk assessment tools were developed and applied to investigate the immunogenicity of a fully human therapeutic monoclonal antibody, ATR-107 [anti-interleukin (IL)-21 receptor] that elicited anti-drug antibodies (ADA) in 76% of healthy subjects in a Phase 1 study. Because the ATR-107 target is expressed on dendritic cells (DCs), the immunogenicity risk related to engagement with DC and antigen presentation pathways was studied. Despite the presence of IL-21R on DCs, ATR-107 did not bind to the DCs more extensively than the control therapeutic antibody (PF-1) that had elicited low clinical ADA incidence. However, ATR-107, but not the control therapeutic antibody, was translocated to the DC late endosomes, co-localized with intracellular antigen-D related (HLA-DR) molecules and presented a dominant T cell epitope overlapping the complementarity determining region 2 (CDR2) of the light chain. ATR-107 induced increased DC activation exemplified by up-regulation of DC surface expression of CD86, CD274 (PD-L1) and CD40, increased expansion of activated DC populations expressing CD86(hi), CD40(hi), CD83(hi), programmed death ligand 1 (PD-L1)(hi), HLA-DR(hi) or CCR7(hi), as well as elevated secretion of tumour necrosis factor (TNF)-α by DCs. DCs exposed to ATR-107 stimulated an autologous T cell proliferative response in human donor cells, in concert with the detection of immunoglobulin (Ig)G-type anti-ATR-107 antibody response in clinical samples. Collectively, the enhanced engagement of antigen presentation machinery by ATR-107 was suggested. The approaches and findings described in this study may be relevant to identifying lower immunogenicity risk targets and therapeutic molecules. © 2015 British Society for Immunology.

  17. Thymic epithelial cell development and its dysfunction in human diseases.

    PubMed

    Sun, Lina; Li, Hongran; Luo, Haiying; Zhao, Yong

    2014-01-01

    Thymic epithelial cells (TECs) are the key components in thymic microenvironment for T cells development. TECs, composed of cortical and medullary TECs, are derived from a common bipotent progenitor and undergo a stepwise development controlled by multiple levels of signals to be functionally mature for supporting thymocyte development. Tumor necrosis factor receptor (TNFR) family members including the receptor activator for NF κ B (RANK), CD40, and lymphotoxin β receptor (LT β R) cooperatively control the thymic medullary microenvironment and self-tolerance establishment. In addition, fibroblast growth factors (FGFs), Wnt, and Notch signals are essential for establishment of functional thymic microenvironment. Transcription factors Foxn1 and autoimmune regulator (Aire) are powerful modulators of TEC development, differentiation, and self-tolerance. Dysfunction in thymic microenvironment including defects of TEC and thymocyte development would cause physiological disorders such as tumor, infectious diseases, and autoimmune diseases. In the present review, we will summarize our current understanding on TEC development and the underlying molecular signals pathways and the involvement of thymus dysfunction in human diseases.

  18. In Vitro Treatment of Human Monocytes/Macrophages with Myristoylated Recombinant Nef of Human Immunodeficiency Virus Type 1 Leads to the Activation of Mitogen-Activated Protein Kinases, IκB Kinases, and Interferon Regulatory Factor 3 and to the Release of Beta Interferon▿

    PubMed Central

    Mangino, Giorgio; Percario, Zulema A.; Fiorucci, Gianna; Vaccari, Gabriele; Manrique, Santiago; Romeo, Giovanna; Federico, Maurizio; Geyer, Matthias; Affabris, Elisabetta

    2007-01-01

    The viral protein Nef is a virulence factor that plays multiple roles during the early and late phases of human immunodeficiency virus (HIV) replication. Nef regulates the cell surface expression of critical proteins (including down-regulation of CD4 and major histocompatibility complex class I), T-cell receptor signaling, and apoptosis, inducing proapoptotic effects in uninfected bystander cells and antiapoptotic effects in infected cells. It has been proposed that Nef intersects the CD40 ligand signaling pathway in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit and activate T lymphocytes, rendering them susceptible to HIV infection. There is also increasing evidence that in vitro cell treatment with Nef induces signaling effects. Exogenous Nef treatment is able to induce apoptosis in uninfected T cells, maturation in dendritic cells, and suppression of CD40-dependent immunoglobulin class switching in B cells. Previously, we reported that Nef treatment of primary human monocyte-derived macrophages (MDMs) induces a cycloheximide-independent activation of NF-κB and the synthesis and secretion of a set of chemokines/cytokines that activate STAT1 and STAT3. Here, we show that Nef treatment is capable of hijacking cellular signaling pathways, inducing a very rapid regulatory response in MDMs that is characterized by the rapid and transient phosphorylation of the α and β subunits of the IκB kinase complex and of JNK, ERK1/2, and p38 mitogen-activated protein kinase family members. In addition, we have observed the activation of interferon regulatory factor 3, leading to the synthesis of beta interferon mRNA and protein, which in turn induces STAT2 phosphorylation. All of these effects require Nef myristoylation. PMID:17182689

  19. Muramyl dipeptide-Lys stimulates the function of human dendritic cells.

    PubMed

    Todate, A; Suda, T; Kuwata, H; Chida, K; Nakamura, H

    2001-11-01

    Muramyl dipeptide (MDP)-Lys (L18), a synthetic MDP analogue derived from bacterial cell walls, has been reported to be a potent immunoadjuvant that enhances protective immunity against pathogens and tumors by stimulating immune-competent cells, such as monocytes and macrophages. However, it is not known whether MDP-Lys modulates the function of dendritic cells (DCs), which are the most potent antigen-presenting cells and play a crucial role in initiating T cell-mediated immunity. Therefore, we examined the effects of MDP-Lys on the expression of surface molecules, cytokine production, and antigen-presenting function of human DCs generated from peripheral blood cells in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor. We found that MDP-Lys markedly up-regulated the expression of CD80, CD83, CD86, and CD40, but not human leukocyte antigen-DR, and stimulated the production of tumor necrosis factor-alpha, IL-6, IL-8, IL-10, and IL-12 (p40) by human DCs in a dose-dependent manner. Furthermore, MDP-Lys-treated DCs showed enhanced antigen-presenting function compared with untreated DCs, as assessed by an allogeneic mixed lymphocyte reaction. These results suggested that the immunoadjuvant activity of MDP-Lys in vivo is mediated, in part, by its stimulation of DC function.

  20. Human HOIP and LUBAC deficiency underlies autoinflammation, immunodeficiency, amylopectinosis, and lymphangiectasia

    PubMed Central

    Boisson, Bertrand; Laplantine, Emmanuel; Dobbs, Kerry; Cobat, Aurélie; Tarantino, Nadine; Hazen, Melissa; Lidov, Hart G.W.; Hopkins, Gregory; Du, Likun; Belkadi, Aziz; Chrabieh, Maya; Itan, Yuval; Picard, Capucine; Fournet, Jean-Christophe; Eibel, Hermann; Tsitsikov, Erdyni; Pai, Sung-Yun; Abel, Laurent; Al-Herz, Waleed; Israel, Alain

    2015-01-01

    Inherited, complete deficiency of human HOIL-1, a component of the linear ubiquitination chain assembly complex (LUBAC), underlies autoinflammation, infections, and amylopectinosis. We report the clinical description and molecular analysis of a novel inherited disorder of the human LUBAC complex. A patient with multiorgan autoinflammation, combined immunodeficiency, subclinical amylopectinosis, and systemic lymphangiectasia, is homozygous for a mutation in HOIP, the gene encoding the catalytic component of LUBAC. The missense allele (L72P, in the PUB domain) is at least severely hypomorphic, as it impairs HOIP expression and destabilizes the whole LUBAC complex. Linear ubiquitination and NF-κB activation are impaired in the patient’s fibroblasts stimulated by IL-1β or TNF. In contrast, the patient’s monocytes respond to IL-1β more vigorously than control monocytes. However, the activation and differentiation of the patient’s B cells are impaired in response to CD40 engagement. These cellular and clinical phenotypes largely overlap those of HOIL-1-deficient patients. Clinical differences between HOIL-1- and HOIP-mutated patients may result from differences between the mutations, the loci, or other factors. Our findings show that human HOIP is essential for the assembly and function of LUBAC and for various processes governing inflammation and immunity in both hematopoietic and nonhematopoietic cells. PMID:26008899

  1. Plant-based Food Cyanidin-3-Glucoside Modulates Human Platelet Glycoprotein VI Signaling and Inhibits Platelet Activation and Thrombus Formation.

    PubMed

    Yao, Yanling; Chen, Yanqiu; Adili, Reheman; McKeown, Thomas; Chen, Pingguo; Zhu, Guangheng; Li, Dan; Ling, Wenhua; Ni, Heyu; Yang, Yan

    2017-08-30

    Background: Platelets play an important role in hemostasis, thrombosis, and atherosclerosis. Glycoprotein VI (GPVI) is a major platelet receptor that interacts with exposed collagen on injured vessel walls. Our previous studies have shown that anthocyanins (a type of natural plant pigment) attenuate platelet function; however, whether anthocyanins affect collagen-induced GPVI signaling remains unknown.Objective: The objective of this study was to explore the effects of cyanidin-3-glucoside (Cy-3-g, one of the major bioactive compounds in anthocyanins) on platelet activation and thrombosis and the GPVI signaling pathway.Methods: Platelets from healthy men and women were isolated and incubated with different concentrations (0, 0.5, 5, and 50 μM) of Cy-3-g. The expression of activated integrin αIIbβ3, P-selectin, CD63, and CD40L, fibrinogen binding to platelets, and platelet aggregation were evaluated in vitro. Platelet adhesion and aggregation in whole blood under flow conditions were assessed in collagen-coated perfusion chambers. Thrombosis and hemostasis were assessed in 3-4-wk-old male C57BL/6J mice through FeCl3-induced intravital microscopy and tail bleeding time. The effect of Cy-3-g on collagen-induced human platelet GPVI signaling was explored with Western blot.Results: Cy-3-g attenuated platelet function in a dose-dependent manner. The 0.5-μM dose of Cy-3-g inhibited (P < 0.05) human platelet adhesion and aggregation to collagen at both venous (-54.02%) and arterial (-22.90%) shear stresses. The 5-μM dose inhibited (P < 0.05) collagen-induced human platelet activation (PAC-1: -48.21%, P-selectin: -50.63%), secretion (CD63: -73.89%, CD40L: -43.70%), fibrinogen binding (-56.79%), and aggregation (-17.81%). The 5-μM dose attenuated (P < 0.01) thrombus growth (-66.67%) without prolonging bleeding time in mice. The 50-μM dose downregulated (P < 0.05) collagen-induced GPVI signaling in human platelets and significantly decreased phosphorylation of Syk

  2. TLR-4 and -6 agonists reverse apoptosis and promote maturation of simian virus 5-infected human dendritic cells through NFkB-dependent pathways.

    PubMed

    Arimilli, Subhashini; Johnson, John B; Alexander-Miller, Martha A; Parks, Griffith D

    2007-08-15

    Infection of primary cultures of human immature monocyte-derived dendritic cells (moDC) with the paramyxovirus Simian Virus 5 (SV5) results in extensive cytopathic effect (CPE) and induction of apoptosis, but DC maturation pathways are not activated. In this study, we investigated the relationship between SV5-induced apoptosis and the lack of DC maturation. Reducing CPE and apoptosis in SV5-infected immature DC by the addition of a pancaspase inhibitor resulted in only low level expression of maturation markers CD40, CD80 and CD86, suggesting that SV5 infection either actively blocked maturation pathways or failed to provide sufficient signals to activate maturation. To distinguish between these hypotheses, SV5-infected immature DC were challenged with agonists that stimulate toll-like receptors (TLRs). Treatment with the TLR-4 agonist LPS or TLR-6 agonist FSL1 enhanced cell surface expression of CD40, CD80 and CD86 on SV5-infected cells to levels approaching that of mock-infected TLR-treated moDC, but treatment with agonists for TLR-2, -3, -5 or -8 had little effect. Addition of TLR-4 or -6 agonists to SV5-infected DC also dramatically reduced CPE and apoptosis, but the levels of viral protein and virus yield were not affected. Similarly, SV5-infected immature moDC were matured by treatment with IL-1beta, and these mature infected cells also showed reduced CPE and apoptosis. In the presence of NFkB inhibitors, TLR-4 and -6 agonists did not promote maturation or reduce apoptosis of SV5-infected DC, indicating that maturation and cell survival were both dependent on signaling through NFkB-dependent pathways. Our results suggest a model whereby SV5 replication induces apoptosis in immature DC but fails to provide strong maturation signals, while activation of NFkB-dependent pathways by exogenous ligands can lead to moDC maturation and override SV5-induced cell death.

  3. CD43-mediated signals induce DNA binding activity of AP-1, NF-AT, and NFkappa B transcription factors in human T lymphocytes.

    PubMed

    Santana, M A; Pedraza-Alva, G; Olivares-Zavaleta, N; Madrid-Marina, V; Horejsi, V; Burakoff, S J; Rosenstein, Y

    2000-10-06

    Although numerous reports document a role for CD43 in T cell signaling, the direct participation of this molecule in cell activation has been questioned. In this study we show that CD43 ligation on human normal peripheral T cells was sufficient to induce interleukin-2, CD69, and CD40-L gene expression, without requiring signals provided by additional receptor molecules. This response was partially inhibited by cyclosporin A and staurosporine, suggesting the participation of both the Ca(2+) and the protein kinase C pathways in CD43 signaling. Consistent with the transient CD43-dependent intracellular Ca(2+) peaks reported by others, signals generated through the CD43 molecule resulted in the induction of NF-AT DNA binding activity. CD43-dependent signals resulted also in AP-1 and NFkappaB activation, probably as a result of protein kinase C involvement. AP-1 complexes bound to the AP-1 sequence contained c-Jun, and those bound to the NF-AT-AP-1 composite site contained c-Jun and Fos. NFkappaB complexes containing p65 could be found as early as 1 h after CD43 cross-linking, suggesting that CD43 participates in early events of T cell activation. The induction of the interleukin-2, CD69, and CD-40L genes and the participation of AP-1, NF-AT, and NFkappaB in the CD43-mediated signaling cascade implicate an important role for this molecule in the regulation of gene expression and cell function.

  4. Natural mannosylation of HIV-1 gp120 imposes no immunoregulatory effects in primary human plasmacytoid dendritic cells.

    PubMed

    Søndergaard, Jonas Nørskov; Vinner, Lasse; Brix, Susanne

    2014-06-01

    Plasmacytoid dendritic cells (pDCs) play a vital role in activation of anti-HIV-1 immunity, and suppression of pDCs might mitigate immune responses against HIV-1. HIV-1 gp120 high-mannose has been attributed immunosuppressive roles in human myeloid DCs, but no receptors for high-mannose have so far been reported on human pDCs. Here we show that upon activation with HIV-1 or by a synthetic compound triggering the same receptor in human pDCs as single-stranded RNA, human pDCs upregulate the mannose receptor (MR, CD206). To examine the functional outcome of this upregulation, inactivated intact or viable HIV-1 particles with various degrees of mannosylation were cultured with pDCs. Activation of pDCs was determined by assaying secretion of IFN-alpha, viability, and upregulation of several pDC-activation markers: CD40, CD86, HLA-DR, CCR7, and PD-L1. The level of activation negatively correlated with degree of mannosylation, however, subsequent reduction in the original mannosylation level had no effect on the pDC phenotype. Furthermore, two of the infectious HIV-1 strains induced profound necrosis in pDCs, also in a mannose-independent manner. We therefore conclude that natural mannosylation of HIV-1 is not involved in HIV-1-mediated immune suppression of pDCs.

  5. Monogenic mutations differentially affect the quantity and quality of T follicular helper cells in patients with human primary immunodeficiencies.

    PubMed

    Ma, Cindy S; Wong, Natalie; Rao, Geetha; Avery, Danielle T; Torpy, James; Hambridge, Thomas; Bustamante, Jacinta; Okada, Satoshi; Stoddard, Jennifer L; Deenick, Elissa K; Pelham, Simon J; Payne, Kathryn; Boisson-Dupuis, Stéphanie; Puel, Anne; Kobayashi, Masao; Arkwright, Peter D; Kilic, Sara Sebnem; El Baghdadi, Jamila; Nonoyama, Shigeaki; Minegishi, Yoshiyuki; Mahdaviani, Seyed Alireza; Mansouri, Davood; Bousfiha, Aziz; Blincoe, Annaliesse K; French, Martyn A; Hsu, Peter; Campbell, Dianne E; Stormon, Michael O; Wong, Melanie; Adelstein, Stephen; Smart, Joanne M; Fulcher, David A; Cook, Matthew C; Phan, Tri Giang; Stepensky, Polina; Boztug, Kaan; Kansu, Aydan; İkincioğullari, Aydan; Baumann, Ulrich; Beier, Rita; Roscioli, Tony; Ziegler, John B; Gray, Paul; Picard, Capucine; Grimbacher, Bodo; Warnatz, Klaus; Holland, Steven M; Casanova, Jean-Laurent; Uzel, Gulbu; Tangye, Stuart G

    2015-10-01

    Follicular helper T (TFH) cells underpin T cell-dependent humoral immunity and the success of most vaccines. TFH cells also contribute to human immune disorders, such as autoimmunity, immunodeficiency, and malignancy. Understanding the molecular requirements for the generation and function of TFH cells will provide strategies for targeting these cells to modulate their behavior in the setting of these immunologic abnormalities. We sought to determine the signaling pathways and cellular interactions required for the development and function of TFH cells in human subjects. Human primary immunodeficiencies (PIDs) resulting from monogenic mutations provide a unique opportunity to assess the requirement for particular molecules in regulating human lymphocyte function. Circulating follicular helper T (cTFH) cell subsets,