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Sample records for activated antigen-presenting cells

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

    PubMed

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

    2016-05-01

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

  2. Carbohydrate-functionalized nanovaccines preserve HIV-1 antigen stability and activate antigen presenting cells

    PubMed Central

    Vela Ramirez, J.E.; Roychoudhury, R.; Habte, H.H.; Cho, M. W.; Pohl, N. L. B.; Narasimhan, B.

    2015-01-01

    The functionalization of polymeric nanoparticles with ligands that target specific receptors on immune cells offers the opportunity to tailor adjuvant properties by conferring pathogen mimicking attributes to the particles. Polyanhydride nanoparticles are promising vaccine adjuvants with desirable characteristics such as immunomodulation, sustained antigen release, activation of antigen presenting cells, and stabilization of protein antigens. These capabilities can be exploited to design nanovaccines against viral pathogens, such as HIV-1, due to the important role of dendritic cells and macrophages in viral spread. In this work, an optimized process was developed for carbohydrate functionalization of HIV-1 antigen-loaded polyanhydride nanoparticles. The carbohydrate-functionalized nanoparticles preserved antigenic properties upon release and also enabled sustained antigen release kinetics. Particle internalization was observed to be chemistry-dependent with positively charged nanoparticles being taken up more efficiently by dendritic cells. Up-regulation of the activation makers CD40 and CD206 was demonstrated with carboxymethyl-α-d-mannopyranosyl-(1,2)-d-mannopyranoside functionalized nanoparticles. The secretion of the cytokines IL-6 and TNF-α was shown to be chemistry-dependent upon stimulation with carbohydrate-functionalized nanoparticles. These results offer important new insights upon the interactions between carbohydrate-functionalized nanoparticles and antigen presenting cells and provide foundational information for the rational design of targeted nanovaccines against HIV-1. PMID:25068589

  3. Vaccine activation of the nutrient sensor GCN2 in dendritic cells enhances antigen presentation.

    PubMed

    Ravindran, Rajesh; Khan, Nooruddin; Nakaya, Helder I; Li, Shuzhao; Loebbermann, Jens; Maddur, Mohan S; Park, Youngja; Jones, Dean P; Chappert, Pascal; Davoust, Jean; Weiss, David S; Virgin, Herbert W; Ron, David; Pulendran, Bali

    2014-01-17

    The yellow fever vaccine YF-17D is one of the most successful vaccines ever developed in humans. Despite its efficacy and widespread use in more than 600 million people, the mechanisms by which it stimulates protective immunity remain poorly understood. Recent studies using systems biology approaches in humans have revealed that YF-17D-induced early expression of general control nonderepressible 2 kinase (GCN2) in the blood strongly correlates with the magnitude of the later CD8(+) T cell response. We demonstrate a key role for virus-induced GCN2 activation in programming dendritic cells to initiate autophagy and enhanced antigen presentation to both CD4(+) and CD8(+) T cells. These results reveal an unappreciated link between virus-induced integrated stress response in dendritic cells and the adaptive immune response. PMID:24310610

  4. A novel approach for reliable detection of cathepsin S activities in mouse antigen presenting cells.

    PubMed

    Steimle, Alex; Kalbacher, Hubert; Maurer, Andreas; Beifuss, Brigitte; Bender, Annika; Schäfer, Andrea; Müller, Ricarda; Autenrieth, Ingo B; Frick, Julia-Stefanie

    2016-05-01

    Cathepsin S (CTSS) is a eukaryotic protease mostly expressed in professional antigen presenting cells (APCs). Since CTSS activity regulation plays a role in the pathogenesis of various autoimmune diseases like multiple sclerosis, atherosclerosis, Sjögren's syndrome and psoriasis as well as in cancer progression, there is an ongoing interest in the reliable detection of cathepsin S activity. Various applications have been invented for specific detection of this enzyme. However, most of them have only been shown to be suitable for human samples, do not deliver quantitative results or the experimental procedure requires technical equipment that is not commonly available in a standard laboratory. We have tested a fluorogen substrate, Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2, that has been described to specifically detect CTSS activities in human APCs for its potential use for mouse samples. We have modified the protocol and thereby offer a cheap, easy, reproducible and quick activity assay to detect CTSS activities in mouse APCs. Since most of basic research on CTSS is performed in mice, this method closes a gap and offers a possibility for reliable and quantitative CTSS activity detection that can be performed in almost every laboratory. PMID:26899824

  5. Carbohydrate-functionalized nanovaccines preserve HIV-1 antigen stability and activate antigen presenting cells.

    PubMed

    Vela Ramirez, J E; Roychoudhury, R; Habte, H H; Cho, M W; Pohl, N L B; Narasimhan, B

    2014-01-01

    The functionalization of polymeric nanoparticles with ligands that target specific receptors on immune cells offers the opportunity to tailor adjuvant properties by conferring pathogen mimicking attributes to the particles. Polyanhydride nanoparticles are promising vaccine adjuvants with desirable characteristics such as immunomodulation, sustained antigen release, activation of antigen presenting cells (APCs), and stabilization of protein antigens. These capabilities can be exploited to design nanovaccines against viral pathogens, such as HIV-1, due to the important role of dendritic cells (DCs) and macrophages in viral spread. In this work, an optimized process was developed for carbohydrate functionalization of HIV-1 antigen-loaded polyanhydride nanoparticles. The carbohydrate-functionalized nanoparticles preserved antigenic properties upon release and also enabled sustained antigen release kinetics. Particle internalization was observed to be chemistry-dependent with positively charged nanoparticles being taken up more efficiently by DCs. Up-regulation of the activation makers CD40 and CD206 was demonstrated with carboxymethyl-α-d-mannopyranosyl-(1,2)-d-mannopyranoside functionalized nanoparticles. The secretion of the cytokines IL-6 and TNF-α was shown to be chemistry-dependent upon stimulation with carbohydrate-functionalized nanoparticles. These results offer important new insights upon the interactions between carbohydrate-functionalized nanoparticles and APCs and provide foundational information for the rational design of targeted nanovaccines against HIV-1. PMID:25068589

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

    PubMed Central

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

    2015-01-01

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

  7. PD-1 on Immature and PD-1 Ligands on Migratory Human Langerhans Cells Regulate Antigen-Presenting Cell Activity

    PubMed Central

    Peña-Cruz, Victor; McDonough, Sean M.; Diaz-Griffero, Felipe; Crum, Christopher P.; Carrasco, Ruben D.; Freeman, Gordon J.

    2010-01-01

    Langerhans cells (LCs) are known as “sentinels” of the immune system that function as professional antigen-presenting cells (APCs) after migration to draining lymph node. LCs are proposed to have a role in tolerance and the resolution of cutaneous immune responses. The Programmed Death-1 (PD-1) receptor and its ligands, PD-L1 and PD-L2, are a co-inhibitory pathway that contributes to the negative regulation of T-lymphocyte activation and peripheral tolerance. Surprisingly, we found PD-1 to be expressed on immature LCs (iLCs) in situ. PD-1 engagement on iLCs reduced IL-6 and macrophage inflammatory protein (MIP)-1α cytokine production in response to TLR2 signals but had no effect on LC maturation. PD-L1 and PD-L2 were expressed at very low levels on iLCs. Maturation of LCs upon migration from epidermis led to loss of PD-l expression and gain of high expression of PD-L1 and PD-L2 as well as co-stimulatory molecules. Blockade of PD-L1 and/or PD-L2 on migratory LCs (mLCs) and DDCs enhanced T-cell activation, as has been reported for other APCs. Thus the PD-1 pathway is active in iLCs and inhibits iLC activities, but expression of receptor and ligands reverses upon maturation and PD-L1 and PD-L2 on mLC function to inhibit T-cell responses. PMID:20445553

  8. P2X7 Receptor Activation Impairs Exogenous MHC Class I Oligopeptides Presentation in Antigen Presenting Cells

    PubMed Central

    Baroja-Mazo, Alberto; Barberà-Cremades, Maria; Pelegrín, Pablo

    2013-01-01

    Major histocompatibility complex class I (MHC I) on antigen presenting cells (APCs) is a potent molecule to activate CD8+ T cells and initiate immunity. P2X7 receptors (P2X7Rs) are present on the plasma membrane of APCs to sense the extracellular danger signal adenosine-5′-triphosphate (ATP). P2X7R activates the inflammasome and the release of IL-1β in macrophages and other immune cells to initiate the inflammatory response. Here we show that P2X7R stimulation by ATP in APCs decreased the amount of MHC I at the plasma membrane. Specific antagonism or genetic ablation of P2X7R inhibited the effects of ATP on levels of cellular MHC I. Furthermore, P2X7R stimulation was able to inhibit activation of CD8+ T cells via specific MHC I-oligopeptide complexes. Our study suggests that P2X7R activation on APCs is a novel inhibitor of adaptive CD8+ T cell immunity. PMID:23940597

  9. P2X7 receptor activation impairs exogenous MHC class I oligopeptides presentation in antigen presenting cells.

    PubMed

    Baroja-Mazo, Alberto; Barberà-Cremades, Maria; Pelegrín, Pablo

    2013-01-01

    Major histocompatibility complex class I (MHC I) on antigen presenting cells (APCs) is a potent molecule to activate CD8(+) T cells and initiate immunity. P2X7 receptors (P2X7Rs) are present on the plasma membrane of APCs to sense the extracellular danger signal adenosine-5'-triphosphate (ATP). P2X7R activates the inflammasome and the release of IL-1β in macrophages and other immune cells to initiate the inflammatory response. Here we show that P2X7R stimulation by ATP in APCs decreased the amount of MHC I at the plasma membrane. Specific antagonism or genetic ablation of P2X7R inhibited the effects of ATP on levels of cellular MHC I. Furthermore, P2X7R stimulation was able to inhibit activation of CD8(+) T cells via specific MHC I-oligopeptide complexes. Our study suggests that P2X7R activation on APCs is a novel inhibitor of adaptive CD8(+) T cell immunity. PMID:23940597

  10. Antigen-induced regulation of T-cell motility, interaction with antigen-presenting cells and activation through endogenous thrombospondin-1 and its receptors

    PubMed Central

    Bergström, Sten-Erik; Uzunel, Mehmet; Talme, Toomas; Bergdahl, Eva; Sundqvist, Karl-Gösta

    2015-01-01

    Antigen recognition reduces T-cell motility, and induces prolonged contact with antigen-presenting cells and activation through mechanisms that remain unclear. Here we show that the T-cell receptor (TCR) and CD28 regulate T-cell motility, contact with antigen-presenting cells and activation through endogenous thrombospondin-1 (TSP-1) and its receptors low-density lipoprotein receptor-related protein 1 (LRP1), calreticulin and CD47. Antigen stimulation induced a prominent up-regulation of TSP-1 expression, and transiently increased and subsequently decreased LRP1 expression whereas calreticulin was unaffected. This antigen-induced TSP-1/LRP1 response down-regulated a motogenic mechanism directed by LRP1-mediated processing of TSP-1 in cis within the same plasma membrane while promoting contact with antigen-presenting cells and activation through cis interaction of the C-terminal domain of TSP-1 with CD47 in response to N-terminal TSP-1 triggering by calreticulin. The antigen-induced TSP-1/LRP1 response maintained a reduced but significant motility level in activated cells. Blocking CD28 co-stimulation abrogated LRP1 and TSP-1 expression and motility. TCR/CD3 ligation alone enhanced TSP-1 expression whereas CD28 ligation alone enhanced LRP1 expression. Silencing of TSP-1 inhibited T-cell conjugation to antigen-presenting cells and T helper type 1 (Th1) and Th2 cytokine responses. The Th1 response enhanced motility and increased TSP-1 expression through interleukin-2, whereas the Th2 response weakened motility and reduced LRP1 expression through interleukin-4. Ligation of the TCR and CD28 therefore elicits a TSP-1/LRP1 response that stimulates prolonged contact with antigen-presenting cells and, although down-regulating motility, maintains a significant motility level to allow serial contacts and activation. Th1 and Th2 cytokine responses differentially regulate T-cell expression of TSP-1 and LRP1 and motility. PMID:25393517

  11. The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells

    PubMed Central

    Torreno-Pina, Juan A.; Manzo, Carlo; Salio, Mariolina; Aichinger, Michael C.; Oddone, Anna; Lakadamyali, Melike; Shepherd, Dawn; Besra, Gurdyal S.; Cerundolo, Vincenzo

    2016-01-01

    Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such “tonic” activation of iNKT cells remain unclear. Here, we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen-presenting cells (APCs) modulates activation of iNKT cells. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton. Dual-color single-particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton, preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the Toll-like receptor 7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a previously unidentified mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen-loaded CD1d nanoclusters. PMID:26798067

  12. The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells.

    PubMed

    Torreno-Pina, Juan A; Manzo, Carlo; Salio, Mariolina; Aichinger, Michael C; Oddone, Anna; Lakadamyali, Melike; Shepherd, Dawn; Besra, Gurdyal S; Cerundolo, Vincenzo; Garcia-Parajo, Maria F

    2016-02-01

    Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such "tonic" activation of iNKT cells remain unclear. Here, we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen-presenting cells (APCs) modulates activation of iNKT cells. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton. Dual-color single-particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton, preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the Toll-like receptor 7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a previously unidentified mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen-loaded CD1d nanoclusters. PMID:26798067

  13. Isolation and In vivo Transfer of Antigen Presenting Cells

    PubMed Central

    Arora, Pooja; Kharkwal, Shalu Sharma; Porcelli, Steven A.

    2016-01-01

    Transfer of antigen presenting cells in vivo is a method used by immunologists to examine the potency of antigen presentation by a selected population of cells. This method is most commonly used to analyze presentation of protein antigens to MHC class I or II restricted T cells, but it can also be used for studies of nonconventional antigens such as CD1-presented lipids. In a recent study focusing on CD1d-restricted glycolipid antigen presentation to Natural Killer T cells, we compared antigen presenting properties of splenic B cells, CD8αPos dendritc cells (DCs) and CD8αNeg DCs (Arora et al., 2014). This protocol describes the detailed method used for isolation of these cell populations, and their transfer into recipient mice to analyze their antigen presenting properties. PMID:27390759

  14. Role of the mononuclear phagocyte as an antigen-presenting cell for human gamma delta T cells activated by live Mycobacterium tuberculosis.

    PubMed Central

    Boom, W H; Chervenak, K A; Mincek, M A; Ellner, J J

    1992-01-01

    gamma delta T cells, both human and murine, have been found to be highly responsive to mycobacterial antigens. However, the role and function of gamma delta T cells in the immune response to Mycobacterium tuberculosis remain largely unknown. In earlier studies, we demonstrated that monocytes infected with live M. tuberculosis were particularly effective inducers of human peripheral blood gamma delta T cells. The present studies were performed to further characterize the interaction between human mononuclear phagocytes, gamma delta T cells, and live M. tuberculosis, in comparison with CD4+ T cells. First, we found that resting gamma delta T cells expanded in vitro by live M. tuberculosis were specific for M. tuberculosis, and that heat killing and washing the mycobacteria removed the antigen(s) for gamma delta T cells. In contrast, the heat-killed mycobacteria retained significant antigenicity for CD4+ T cells. Second, live M. tuberculosis-expanded gamma delta T cells from healthy tuberculin-positive donors did not respond significantly to the antigens in M. tuberculosis culture filtrate, including the 65- and 71-kDa mycobacterial heat shock proteins. Third, the activation of gamma delta T cells by live mycobacteria was dependent on antigen-presenting cells, and mononuclear phagocytes were found to be very efficient antigen-presenting cells both for resting peripheral blood gamma delta T cells and for activated expanded gamma delta T cells. The mononuclear phagocyte carried the necessary costimulatory factors necessary for gamma delta T-cell proliferation. Fourth, the antigen repertoire and HLA requirements for CD4+ memory T cells and those for gamma delta T cells appear to be quite distinct from each other. CD4+ T cells recognized both soluble protein antigens and whole organisms in a class II major histocompatibility complex-restricted manner, whereas gamma delta T cells appeared to recognize only constituents associated with the whole organism and were not

  15. Novel CD47: SIRPα Dependent Mechanism for the Activation of STAT3 in Antigen-Presenting Cell

    PubMed Central

    Toledano, Natan; Gur-Wahnon, Devorah; Ben-Yehuda, Adi; Rachmilewitz, Jacob

    2013-01-01

    Cell surface CD47 interacts with its receptor, signal-regulatory-protein α (SIRPα) that is expressed predominantly on macrophages, to inhibit phagocytosis of normal, healthy cells. This “don’t eat me” signal is mediated through tyrosine phosphorylation of SIRPα at the cytoplasmic ITIM motifs and the recruitment of the phosphatase, SHP-1. We previously revealed a novel mechanism for the activation of the STAT3 pathway and the regulation of human APC maturation and function that is based on cell:cell interaction. In this study, we present evidence supporting the notion that CD47:SIRPα serves as a cell surface receptor: ligand pair involved in this contact-dependent STAT3 activation and regulation of APC maturation. We show that upon co-culturing APC with various primary and tumor cell lines STAT3 phosphorylation and IL-10 expression are induced, and such regulation could be suppressed by specific CD47 siRNAs and shRNAs. Significantly, >50% reduction in CD47 expression abolished the contact-dependent inhibition of T cell activation. Furthermore, co-immunoprecipitation experiments revealed a physical association between SIRPα and STAT3. Thus, we suggest that in addition to signaling through the ITIM-SHP-1 complex that transmit an anti-phagocytotic, CD47:SIRPα also triggers STAT3 signaling that is linked to an immature APC phenotype and peripheral tolerance under steady state and pathological conditions. PMID:24073274

  16. Early Signaling in Primary T Cells Activated by Antigen Presenting Cells Is Associated with a Deep and Transient Lamellal Actin Network

    PubMed Central

    Roybal, Kole T.; Mace, Emily M.; Mantell, Judith M.; Verkade, Paul; Orange, Jordan S.; Wülfing, Christoph

    2015-01-01

    Cellular signaling transduction critically depends on molecular interactions that are in turn governed by dynamic subcellular distributions of the signaling system components. Comprehensive insight into signal transduction requires an understanding of such distributions and cellular structures driving them. To investigate the activation of primary murine T cells by antigen presenting cells (APC) we have imaged more than 60 signaling intermediates during T cell stimulation with microscopy across resolution limits. A substantial number of signaling intermediates associated with a transient, wide, and actin-associated lamellum extending from an interdigitated T cell:APC interface several micrometers into the T cell, as characterized in detail here. By mapping the more than 60 signaling intermediates onto the spatiotemporal features of cell biological structures, the lamellum and other ones previously described, we also define distinct spatial and temporal characteristics of T cell signal initiation, amplification, and core signaling in the activation of primary T cells by APCs. These characteristics differ substantially from ones seen when T cells are activated using common reductionist approaches. PMID:26237050

  17. Self-Antigen Presentation by Dendritic Cells in Autoimmunity

    PubMed Central

    Hopp, Ann-Katrin; Rupp, Anne; Lukacs-Kornek, Veronika

    2014-01-01

    The operation of both central and peripheral tolerance ensures the prevention of autoimmune diseases. The maintenance of peripheral tolerance requires self-antigen presentation by professional antigen presenting cells (APCs). Dendritic cells (DCs) are considered as major APCs involved in this process. The current review discusses the role of DCs in autoimmune diseases, the various factors involved in the induction and maintenance of tolerogenic DC phenotype, and pinpoints their therapeutic capacity as well as potential novel targets for future clinical studies. PMID:24592266

  18. Artificial antigen presenting cells for use in adoptive immunotherapy

    PubMed Central

    Turtle, Cameron J.; Riddell, Stanley R.

    2010-01-01

    The observation that T cells can recognize and specifically eliminate cancer cells has spurred interest in the development of efficient methods to generate large numbers of T cells with specificity for tumor antigens that can be harnessed for use in cancer therapy. Recent studies have demonstrated that during encounter with tumor antigen, the signals delivered to T cells by professional antigen presenting cells can affect T cell programming and their subsequent therapeutic efficacy. This has stimulated efforts to develop artificial antigen presenting cells that allow optimal control over the signals provided to T cells. In this review, we will discuss the advantages and disadvantages of cellular and acellular artificial antigen presenting cell systems and their use in T cell adoptive immunotherapy for cancer. PMID:20693850

  19. Mitochondrial H2O2 in Lung Antigen-Presenting Cells Blocks NF-κB Activation to Prevent Unwarranted Immune Activation.

    PubMed

    Khare, Anupriya; Raundhal, Mahesh; Chakraborty, Krishnendu; Das, Sudipta; Corey, Catherine; Kamga, Christelle K; Quesnelle, Kelly; St Croix, Claudette; Watkins, Simon C; Morse, Christina; Oriss, Timothy B; Huff, Rachael; Hannum, Rachel; Ray, Prabir; Shiva, Sruti; Ray, Anuradha

    2016-05-24

    Inhalation of environmental antigens such as allergens does not always induce inflammation in the respiratory tract. While antigen-presenting cells (APCs), including dendritic cells and macrophages, take up inhaled antigens, the cell-intrinsic molecular mechanisms that prevent an inflammatory response during this process, such as activation of the transcription factor NF-κB, are not well understood. Here, we show that the nuclear receptor PPARγ plays a critical role in blocking NF-κB activation in response to inhaled antigens to preserve immune tolerance. Tolerance induction promoted mitochondrial respiration, generation of H2O2, and suppression of NF-κB activation in WT, but not PPARγ-deficient, APCs. Forced restoration of H2O2 in PPARγ-deficient cells suppressed IκBα degradation and NF-κB activation. Conversely, scavenging reactive oxygen species from mitochondria promoted IκBα degradation with loss of regulatory and promotion of inflammatory T cell responses in vivo. Thus, communication between PPARγ and the mitochondria maintains immune quiescence in the airways. PMID:27184852

  20. Upregulation of Immunoproteasome Subunits in Myositis Indicates Active Inflammation with Involvement of Antigen Presenting Cells, CD8 T-Cells and IFNγ

    PubMed Central

    Ghannam, Khetam; Martinez-Gamboa, Lorena; Spengler, Lydia; Krause, Sabine; Smiljanovic, Biljana; Bonin, Marc; Bhattarai, Salyan; Grützkau, Andreas; Burmester, Gerd-R.

    2014-01-01

    Objective In idiopathic inflammatory myopathies (IIM) infiltration of immune cells into muscle and upregulation of MHC-I expression implies increased antigen presentation and involvement of the proteasome system. To decipher the role of immunoproteasomes in myositis, we investigated individual cell types and muscle tissues and focused on possible immune triggers. Methods Expression of constitutive (PSMB5, -6, -7) and corresponding immunoproteasomal subunits (PSMB8, -9, -10) was analyzed by real-time RT-PCR in muscle biopsies and sorted peripheral blood cells of patients with IIM, non-inflammatory myopathies (NIM) and healthy donors (HD). Protein analysis in muscle biopsies was performed by western blot. Affymetrix HG-U133 platform derived transcriptome data from biopsies of different muscle diseases and from immune cell types as well as monocyte stimulation experiments were used for validation, coregulation and coexpression analyses. Results Real-time RT-PCR revealed significantly increased expression of immunoproteasomal subunits (PSMB8/-9/-10) in DC, monocytes and CD8+ T-cells in IIM. In muscle biopsies, the immunosubunits were elevated in IIM compared to NIM and exceeded levels of matched blood samples. Proteins of PSMB8 and -9 were found only in IIM but not NIM muscle biopsies. Reanalysis of 78 myositis and 20 healthy muscle transcriptomes confirmed these results and revealed involvement of the antigen processing and presentation pathway. Comparison with reference profiles of sorted immune cells and healthy muscle confirmed upregulation of PSMB8 and -9 in myositis biopsies beyond infiltration related changes. This upregulation correlated highest with STAT1, IRF1 and IFNγ expression. Elevation of T-cell specific transcripts in active IIM muscles was accompanied by increased expression of DC and monocyte marker genes and thus reflects the cell type specific involvement observed in peripheral blood. Conclusions Immunoproteasomes seem to indicate IIM activity and

  1. The inhibition of antigen-presenting activity of dendritic cells resulting from UV irradiation of murine skin is restored by in vitro photorepair of cyclobutane pyrimidine dimers.

    PubMed

    Vink, A A; Moodycliffe, A M; Shreedhar, V; Ullrich, S E; Roza, L; Yarosh, D B; Kripke, M L

    1997-05-13

    Exposing skin to UVB (280-320 nm) radiation suppresses contact hypersensitivity by a mechanism that involves an alteration in the activity of cutaneous antigen-presenting cells (APC). UV-induced DNA damage appears to be an important molecular trigger for this effect. The specific target cells in the skin that sustain DNA damage relevant to the immunosuppressive effect have yet to be identified. We tested the hypothesis that UV-induced DNA damage in the cutaneous APC was responsible for their impaired ability to present antigen after in vivo UV irradiation. Cutaneous APC were collected from the draining lymph nodes of UVB-irradiated, hapten-sensitized mice and incubated in vitro with liposomes containing a photolyase (Photosomes; Applied Genetics, Freeport, NY), which, upon absorption of photoreactivating light, splits UV-induced cyclobutane pyrimidine dimers. Photosome treatment followed by photoreactivating light reduced the number of dimer-containing APC, restored the in vivo antigen-presenting activity of the draining lymph node cells, and blocked the induction of suppressor T cells. Neither Photosomes nor photoreactivating light alone, nor photoreactivating light given before Photosomes, restored APC activity, and Photosome treatment did not reverse the impairment of APC function when isopsoralen plus UVA (320-400 nm) radiation was used instead of UVB. These controls indicate that the restoration of APC function matched the requirements of Photosome-mediated DNA repair for dimers and post-treatment photoreactivating light. These results provide compelling evidence that it is UV-induced DNA damage in cutaneous APC that leads to reduced immune function. PMID:9144224

  2. Immunomodulatory Glycan Lacto-N-Fucopentaose III Requires Clathrin-Mediated Endocytosis To Induce Alternative Activation of Antigen-Presenting Cells

    PubMed Central

    Srivastava, Leena; Tundup, Smanla; Choi, Beak-San; Norberg, Thomas

    2014-01-01

    The mechanism of alternative activation of antigen-presenting cells (APCs) is largely unknown. Lacto-N-fucopentaose III (LNFPIII) is a biologically conserved pentasaccharide that contains the Lewisx trisaccharide. LNFPIII conjugates and schistosome egg antigens, which contain the Lewisx trisaccharide, drive alternative activation of APCs and induce anti-inflammatory responses in vivo, preventing inflammation-based diseases, including psoriasis, transplant organ rejection, and metabolic disease. In this study, we show that LNFPIII conjugates and schistosome egg antigens interact with APCs via a receptor-mediated process, requiring internalization of these molecules through a clathrin/dynamin-dependent but caveolus-independent endocytic pathway. Using inhibitors/small interfering RNA (siRNA) against dynamin and clathrin, we show for the first time that endocytosis of Lewisx-containing glycans is required to drive alternative maturation of antigen-presenting cells and Th2 immune responses. We identified mouse SIGNR-1 as a cell surface receptor for LNFPIII conjugates. Elimination of SIGNR-1 showed no effect on uptake of LNFPIII conjugates, suggesting that other receptors bind to and facilitate uptake of LNFPIII conjugates. We demonstrate that disruption of actin filaments partially prevented the entry of LNFPIII conjugates into APCs and that LNFPIII colocalizes with both early and late endosomal markers and follows the classical endosomal pathway leading to lysosome maturation. The results of this study show that the ability of LNFPIII to induce alternative activation utilizes a receptor-mediated process that requires a dynamin-dependent endocytosis. Thus, key steps have been defined in the previously unknown mechanism of alternative activation that ultimately leads to induction of anti-inflammatory responses. PMID:24566617

  3. Antigen Export Reduces Antigen Presentation and Limits T Cell Control of M. tuberculosis.

    PubMed

    Srivastava, Smita; Grace, Patricia S; Ernst, Joel D

    2016-01-13

    Persistence of Mycobacterium tuberculosis results from bacterial strategies that manipulate host adaptive immune responses. Infected dendritic cells (DCs) transport M. tuberculosis to local lymph nodes but activate CD4 T cells poorly, suggesting bacterial manipulation of antigen presentation. However, M. tuberculosis antigens are also exported from infected DCs and taken up and presented by uninfected DCs, possibly overcoming this blockade of antigen presentation by infected cells. Here we show that the first stage of this antigen transfer, antigen export, benefits M. tuberculosis by diverting bacterial proteins from the antigen presentation pathway. Kinesin-2 is required for antigen export and depletion of this microtubule-based motor increases activation of antigen-specific CD4 T cells by infected cells and improves control of intracellular infection. Thus, although antigen transfer enables presentation by bystander cells, it does not compensate for reduced antigen presentation by infected cells and represents a bacterial strategy for CD4 T cell evasion. PMID:26764596

  4. Human antigen-presenting cells respond differently to gut-derived probiotic bacteria but mediate similar strain-dependent NK and T cell activation.

    PubMed

    Fink, Lisbeth N; Zeuthen, Louise H; Ferlazzo, Guido; Frøkiaer, Hanne

    2007-12-01

    The intestinal microbiota is essential for homeostasis of the local and systemic immune system, and particularly strains of lactic acid bacteria and Escherichia coli have been shown to have balancing effects on inflammatory conditions such as allergy and inflammatory bowel disease. However, in vitro assessment of the immunomodulatory effects of distinct strains may depend strongly on the cell type used as a model. To select the most appropriate model for screening of beneficial bacteria in human cells, the response to strains of intestinal bacteria of three types of antigen-presenting cells (APC) was compared; blood myeloid dendritic cells (DC), monocyte-derived DC and monocytes, and the effector response of natural killer cells and naïve T cells was characterized. Maturation induced by gut-derived bacteria differed between APC, with blood DC and monocytes responding with the production of IL-6 and tumour necrosis factor-alpha to bacteria, which elicited mainly IL-10 in monocyte-derived DC. In contrast, comparable IFN-gamma production patterns were found in both natural killer cells and T cells induced by all bacteria-matured APC. An inhibitory effect of certain strains on this IFN-gamma production was also mediated by all types of APC. The most potent responses were induced by monocyte-derived DC, which thus constitute a sensitive screening model. PMID:17903206

  5. Increase in a distinct pulmonary macrophage subset possessing an antigen-presenting cell phenotype and in vitro APC activity following silica exposure

    SciTech Connect

    Migliaccio, Christopher T. . E-mail: christopher.migliaccio@umontana.edu; Hamilton, Raymond F.; Holian, Andrij

    2005-06-01

    Silica inhalation results in chronic lung inflammation and fibrosis. While the role of the alveolar macrophage (AM) is considered key to the effects of silica on lung pathology, the etiology is not completely understood. Evidence suggests an increase in antigen presenting cell (APC) activity as a contributing factor to this process, as well as potential roles for both AM and interstitial macrophages (IM) in silicosis. In order to study the effects of crystalline silica on the APC activity of pulmonary macrophages, mice were exposed intranasally and changes in pulmonary macrophage populations were assessed using flow cytometry. Following intranasal instillation of silica, a significant increase in the APC activity of AM was observed, as well as a significant increase in a subset of IM expressing classic APC markers (MHC class II, CD11c). In addition, an in vitro system using bone marrow-derived macrophages (BMDM) was generated to assess the effects of silica on the APC activity of macrophages in vitro. Data using BMDM in the in vitro APC assay demonstrated a significant increase in APC activity following silica exposure, but not following exposure to saline or a control particle (TiO{sub 2}). Using a combination of in vivo and in vitro experiments, the current study describes a significant increase in an interstitial macrophage subset with an APC phenotype, as well as an increase in the APC activity of both AM and BMDM, as a direct result of exposure to crystalline silica. These studies suggest a specific mechanism, macrophage subset activation, by which crystalline silica exposure results in chronic pulmonary inflammation and, eventually, fibrosis.

  6. Phenotypic and functional profiling of mouse intestinal antigen presenting cells.

    PubMed

    Harusato, Akihito; Flannigan, Kyle L; Geem, Duke; Denning, Timothy L

    2015-06-01

    The microbiota that populates the mammalian intestine consists of hundreds of trillions of bacteria that are separated from underlying immune cells by a single layer of epithelial cells. The intestinal immune system effectively tolerates components of the microbiota that provide benefit to the host while remaining poised to eliminate those that are harmful. Antigen presenting cells, especially macrophages and dendritic cells, play important roles in maintaining intestinal homeostasis via their ability to orchestrate appropriate responses to the microbiota. Paramount to elucidating intestinal macrophage- and dendritic cell-mediated functions is the ability to effectively isolate and identify these cells from a complex cellular environment. In this review, we summarize methodology for the isolation and phenotypic characterization of macrophages and DCs from the mouse intestine and discuss how this may be useful for gaining insight into the mechanisms by which mucosal immune tolerance is maintained. PMID:25891794

  7. Tailored immunity by skin antigen-presenting cells

    PubMed Central

    Levin, Clement; Perrin, Helene; Combadiere, Behazine

    2014-01-01

    Skin vaccination aims at targeting epidermal and dermal antigen-presenting cells (APCs), indeed many subsets of different origin endowed with various functions populate the skin. The idea that the skin could represent a particularly potent site to induce adaptive and protective immune response emerged after the success of vaccinia virus vaccination by skin scarification. Recent advances have shown that multiple subsets of APCs coexist in the skin and participate in immunity to infectious diseases. Induction of an adaptive immune response depends on the initial recognition and capture of antigens by skin APCs and their transport to lymphoid organs. Innovative strategies of vaccination have thus been developed to target skin APCs for tailored immunity, hence this review will discuss recent insights into skin APC subsets characterization and how they can shape adaptive immune responses. PMID:25483512

  8. Multiphoton microscopy of antigen presenting cells in experimental cancer therapies

    NASA Astrophysics Data System (ADS)

    Watkins, Simon C.; Papworth, Glenn D.; Spencer, Lori A.; Larregina, Adriana T.; Hackstein, Holger

    2002-06-01

    The absence of effective conventional therapy for most cancer patients justifies the application of novel, experimental approaches. One alternative to conventional cytotoxic agents is a more defined molecular approach for cancer immune treatment; promotion of the immune system specifically to target and eliminate tumor cells on the basis of expression of tumor-associated antigens (TAA). TAA could be presented to T-cells by professional antigen-presenting cells (APC) that generate a more efficient and effective anti-tumor immune response. In fact, it has been well documented that dendritic cells, the most immunologically potent APC, are capable of recognizing, processing and presenting TAA, in turn initiating a specific antitumor immune response. Results from several laboratories and clinical trials suggested significant but still limited efficacy of TAA-pulsed dendritic cells administered to tumor-bearing hosts. Following such delivery, it is fundamentally necessary to dynamically assess cell abundance within the microenvironment of the tumor in the presence of the appropriate therapeutic agent. Multiphoton microscopy was used to assess the trafficking of pulsed dendritic cells and other APC in skin, lymph nodes and brain of several animal tumor models, following different routes of administration.

  9. Artificial antigen-presenting cells expressing AFP158-166 peptide and interleukin-15 activate AFP-specific cytotoxic T lymphocytes

    PubMed Central

    Sun, Longhao; Guo, Hao; Jiang, Ruoyu; Lu, Li; Liu, Tong; Zhang, Zhixiang; He, Xianghui

    2016-01-01

    Professional antigen-presenting cells (APCs) are potent generators of tumor antigen-specific cytotoxic T lymphocytes (CTLs) for adoptive immunotherapy; however, generation of APCs is cumbersome, expensive, and subject to the tumor microenvironment. Artificial APCs (aAPCs) have been developed as a cost-effective alternative to APCs. We developed a cellular aAPC that efficiently generated alpha-fetoprotein (AFP)-specific CTLs. We genetically modified the human B cell lymphoma cell line BJAB with a lentiviral vector to establish an aAPC called BA15. The expression of AFP158-166-HLA-A*02:01 complex, CD80, CD86, and interleukin (IL)-15 in BA15 cells was assessed. The efficiency of BA15 at generating AFP-specific CTLs and the specific cytotoxicity of CTLs against AFP+ cells were also determined. BA15 cells expressed high levels of AFP158-166 peptide, HLA-A2, CD80, CD86, and IL-15. BA15 cells also exhibited higher efficiency in generating AFP-specific CTLs than did dendritic cells. These CTLs had greater cytotoxicity against AFP+ hepatocellular carcinoma cells than did CTLs obtained from dendritic cells in vitro and in vivo. Our novel aAPC system could provide a robust platform for the generation of functional AFP-specific CTLs for adoptive immunotherapy of hepatocellular carcinoma. PMID:27007051

  10. Artificial antigen-presenting cells expressing AFP158-166 peptide and interleukin-15 activate AFP-specific cytotoxic T lymphocytes.

    PubMed

    Sun, Longhao; Guo, Hao; Jiang, Ruoyu; Lu, Li; Liu, Tong; Zhang, Zhixiang; He, Xianghui

    2016-04-01

    Professional antigen-presenting cells (APCs) are potent generators of tumor antigen-specific cytotoxic T lymphocytes (CTLs) for adoptive immunotherapy; however, generation of APCs is cumbersome, expensive, and subject to the tumor microenvironment. Artificial APCs (aAPCs) have been developed as a cost-effective alternative to APCs. We developed a cellular aAPC that efficiently generated alpha-fetoprotein (AFP)-specific CTLs. We genetically modified the human B cell lymphoma cell line BJAB with a lentiviral vector to establish an aAPC called BA15. The expression of AFP158-166-HLA-A*02:01 complex, CD80, CD86, and interleukin (IL)-15 in BA15 cells was assessed. The efficiency of BA15 at generating AFP-specific CTLs and the specific cytotoxicity of CTLs against AFP+ cells were also determined. BA15 cells expressed high levels of AFP158-166 peptide, HLA-A2, CD80, CD86, and IL-15. BA15 cells also exhibited higher efficiency in generating AFP-specific CTLs than did dendritic cells. These CTLs had greater cytotoxicity against AFP+ hepatocellular carcinoma cells than did CTLs obtained from dendritic cells in vitro and in vivo. Our novel aAPC system could provide a robust platform for the generation of functional AFP-specific CTLs for adoptive immunotherapy of hepatocellular carcinoma. PMID:27007051

  11. Antigen Presentation by Monocytes and Monocyte-derived Cells

    PubMed Central

    Randolph, Gwendalyn J.; Jakubzick, Claudia; Qu, Chunfeng

    2008-01-01

    Summary Monocytes are circulating mononuclear phagocytes with a fundamental capacity to differentiate into macrophages. This differentiation can, in the presence of the right environmental cues, be re-directed instead to dendritic cells (DCs). Recent advances have been made in understanding the role of monocytes and their derivatives in presenting antigen to drive immune responses, and we review this topic herein. We briefly discuss the heterogeneity of monocytes in the blood and subsequently raise the possibility that one of the major monocyte phenotypes in the blood corresponds with a population of “blood DCs” previously proposed to drive T-independent antibody reactions in the spleen. Then we evaluate the role of monocytes in T-dependent immunity, considering their role in acquiring antigens for presentation prior to exiting the bloodstream and their ability to differentiate into macrophages versus antigen-presenting DCs. Finally, we review recent literature on the role of monocyte-derived cells in cross-presentation and discuss the possibility that monocyte-derived cells participate critically in processing antigen for cross-priming, even if they do not present that antigen to T cells themselves. PMID:18160272

  12. Antigen-presenting cells containing multiple costimulatory molecules promote activation and expansion of antigen-specific memory CD8+ T cells

    PubMed Central

    Yang, Sixun; Schlom, Jeffrey

    2009-01-01

    We have previously demonstrated that multiple immunizations with vector-based vaccines containing transgenes for tumor Ags and a triad of costimulatory molecules (TRICOM) enhance the expansion and functional avidity of Ag-specific memory CD8+ T cells in a mouse model. However, the effect of enhanced costimulation on human memory CD8+ T cells is still unclear. The study reported here was an in vitro investigation of the proliferation and function of CEA-specific human memory CD8+ T cells following enhanced costimulation. Our results demonstrated that TRICOM costimulation enhanced production of multiple cytokines and expansion of CEA-specific memory CD8+ T cells. The lytic capacity of memory CTLs toward CEA+ tumors was also significantly enhanced. IL-2Rα (CD25) was upregulated dramatically following APC-TRICOM stimulation, suggesting that the enhanced expansion of memory CD8+ T cells may be mediated by increased expression of IL-2R on memory T cells. The enhanced cytokine production and proliferation following TRICOM signaling was completely blocked by the combination of neutralizing Abs against B7-1, ICAM-1, and LFA-3, the costimulatory molecules comprising TRICOM. No difference in T-cell apoptosis was observed between APC-TRICOM and APC-wild-type groups, as determined by annexin V, Bcl-2, and active caspase-3 staining. Results indicated that enhanced costimulation greatly expanded human CEA-specific CD8+ T cells and enhanced T-cell function, without inducing increased apoptosis of CEA-specific memory CD8+ T cells. PMID:18690438

  13. An Overview of B-1 Cells as Antigen-Presenting Cells

    PubMed Central

    Popi, Ana F.; Longo-Maugéri, Ieda M.; Mariano, Mario

    2016-01-01

    The role of B cells as antigen-presenting cells (APCs) has been extensively studied, mainly in relation to the activation of memory T cells. Considering the B cell subtypes, the role of B-1 cells as APCs is beginning to be explored. Initially, it was described that B-1 cells are activated preferentially by T-independent antigens. However, some reports demonstrated that these cells are also involved in a T-dependent response. The aim of this review is to summarize information about the ability of B-1 cells to play a role as APCs and to briefly discuss the role of the BCR and toll-like receptor signals in this process. Furthermore, some characteristics of B-1 cells, such as natural IgM production and phagocytic ability, could interfere in the participation of these cells in the onset of an adaptive response. PMID:27148259

  14. Calmodulin kinase II regulates the maturation and antigen presentation of human dendritic cells.

    PubMed

    Herrmann, Tara L; Morita, Craig T; Lee, Kelvin; Kusner, David J

    2005-12-01

    Dendritic cells (DC) are professional antigen-presenting cells, which activate the adaptive immune system. Upon receiving a danger signal, they undergo a maturation process, which increases their antigen presentation capacity, but the responsible regulatory mechanisms remain incompletely understood. A Ca2+-calmodulin (Cam)-Cam kinase II (CamK II) pathway regulates phagosome maturation in macrophages, and this pathway is inhibited by pathogenic microbes. Our hypothesis is that signal transduction events which control phagosome maturation also regulate antigen presentation. Stimulation of primary human DC or the human DC line KG-1, with particulate antigen, resulted in the activation of CamK II and its localization to the phagosome and plasma membrane. Two mechanistically distinct inhibitors of CamK II significantly reduced DC maturation, as determined by up-regulation of surface costimulatory and major histocompatibility complex (MHC) class II molecules and secretion of cytokines. Confocal microscopy demonstrated that the CamK II inhibitors blocked the antigen-induced increase in total cellular MHC class molecules as well as their trafficking to the plasma membrane. Inhibition of CamK II was associated with decreased presentation of particulate and soluble MHC class II-restricted antigen, with a greater effect on the former. These data support a model in which CamK II regulates critical stages of the maturation and antigen presentation capacity of human DC, particularly in response to stimulation via phagocytosis. PMID:16204647

  15. The Immunomodulator VacA Promotes Immune Tolerance and Persistent Helicobacter pylori Infection through Its Activities on T-Cells and Antigen-Presenting Cells

    PubMed Central

    Djekic, Aleksandra; Müller, Anne

    2016-01-01

    VacA is a pore-forming toxin that has long been known to induce vacuolization in gastric epithelial cells and to be linked to gastric disorders caused by H. pylori infection. Its role as a major colonization and persistence determinant of H. pylori is less well-understood. The purpose of this review is to discuss the various target cell types of VacA and its mechanism of action; specifically, we focus on the evidence showing that VacA targets myeloid cells and T-cells to directly and indirectly prevent H. pylori-specific T-cell responses and immune control of the infection. In particular, the ability of VacA-proficient H. pylori to skew T-cell responses towards regulatory T-cells and the effects of Tregs on H. pylori chronicity are highlighted. The by-stander effects of VacA-driven immunomodulation on extragastric diseases are discussed as well. PMID:27322319

  16. The Immunomodulator VacA Promotes Immune Tolerance and Persistent Helicobacter pylori Infection through Its Activities on T-Cells and Antigen-Presenting Cells.

    PubMed

    Djekic, Aleksandra; Müller, Anne

    2016-01-01

    VacA is a pore-forming toxin that has long been known to induce vacuolization in gastric epithelial cells and to be linked to gastric disorders caused by H. pylori infection. Its role as a major colonization and persistence determinant of H. pylori is less well-understood. The purpose of this review is to discuss the various target cell types of VacA and its mechanism of action; specifically, we focus on the evidence showing that VacA targets myeloid cells and T-cells to directly and indirectly prevent H. pylori-specific T-cell responses and immune control of the infection. In particular, the ability of VacA-proficient H. pylori to skew T-cell responses towards regulatory T-cells and the effects of Tregs on H. pylori chronicity are highlighted. The by-stander effects of VacA-driven immunomodulation on extragastric diseases are discussed as well. PMID:27322319

  17. Cimetidine modulates the antigen presenting capacity of dendritic cells from colorectal cancer patients.

    PubMed

    Kubota, T; Fujiwara, H; Ueda, Y; Itoh, T; Yamashita, T; Yoshimura, T; Okugawa, K; Yamamoto, Y; Yano, Y; Yamagishi, H

    2002-04-22

    Cimetidine, a H(2) receptor antagonist, has been reported to improve survival in gastrointestinal cancer patients. These effects have largely been attributed to the enhancing effects of cimetidine on the host's antitumour cell-mediated immune response, such as inhibition of suppressor T lymphocyte activity, stimulation of natural killer cell activity and increase of interleukin-2 production from helper T lymphocytes. We conducted an in vitro study on the effects of cimetidine on differentiation and antigen presenting capacity of monocyte-derived dendritic cells from advanced colorectal cancer patients and normal controls. As a result, an investigation of expression of surface molecules associated with dendritic cells by flow cytometric analyses showed that cimetidine had no enhancing effect on differentiation of dendritic cells from cancer patients and normal controls. An investigation of [(3)H]thymidine incorporation by allogeneic mixed lymphocyte reactions revealed that cimetidine increased the antigen presenting capacity of dendritic cells from both materials. Moreover, a higher antigen presenting capacity was observed in advanced cancer patients compared to normal controls. These effects might be mediated via specific action of cimetidine and not via H(2) receptors because famotidine did not show similar effects. Our results suggest that cimetidine may enhance the host's antitumour cell-mediated immunity by improving the suppressed dendritic cells function of advanced cancer patients. PMID:11953882

  18. SAMHD1 Limits HIV-1 Antigen Presentation by Monocyte-Derived Dendritic Cells

    PubMed Central

    Bruel, Timothée; Cardinaud, Sylvain; Porrot, Françoise; Prado, Julia G.; Moris, Arnaud

    2015-01-01

    ABSTRACT Monocyte-derived dendritic cells (MDDC) stimulate CD8+ cytotoxic T lymphocytes (CTL) by presenting endogenous and exogenous viral peptides via major histocompatibility complex class I (MHC-I) molecules. MDDC are poorly susceptible to HIV-1, in part due to the presence of SAMHD1, a cellular enzyme that depletes intracellular deoxynucleoside triphosphates (dNTPs) and degrades viral RNA. Vpx, an HIV-2/SIVsm protein absent from HIV-1, antagonizes SAMHD1 by inducing its degradation. The impact of SAMHD1 on the adaptive cellular immune response remains poorly characterized. Here, we asked whether SAMHD1 modulates MHC-I-restricted HIV-1 antigen presentation. Untreated MDDC or MDDC pretreated with Vpx were exposed to HIV-1, and antigen presentation was examined by monitoring the activation of an HIV-1 Gag-specific CTL clone. SAMHD1 depletion strongly enhanced productive infection of MDDC as well as endogenous HIV-1 antigen presentation. Time-lapse microscopy analysis demonstrated that in the absence of SAMHD1, the CTL rapidly killed infected MDDC. We also report that various transmitted/founder (T/F) HIV-1 strains poorly infected MDDC and, as a consequence, did not stimulate CTL. Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyping of T/F alleviated a block in viral entry and induced antigen presentation only in the absence of SAMHD1. Furthermore, by using another CTL clone that mostly recognizes incoming HIV-1 antigens, we demonstrate that SAMHD1 does not influence exogenous viral antigen presentation. Altogether, our results demonstrate that the antiviral activity of SAMHD1 impacts antigen presentation by DC, highlighting the link that exists between restriction factors and adaptive immune responses. IMPORTANCE Upon viral infection, DC may present antigens derived from incoming viral material in the absence of productive infection of DC or from newly synthesized viral proteins. In the case of HIV, productive infection of DC is blocked at an early

  19. Tubulin and actin interplay at the T cell and antigen-presenting cell interface.

    PubMed

    Martín-Cófreces, Noa Beatriz; Alarcón, Balbino; Sánchez-Madrid, Francisco

    2011-01-01

    T cells reorganize their actin and tubulin-based cytoskeletons to provide a physical basis to the immune synapse. However, growing evidence shows that their roles on T cell activation are more dynamic than merely serving as tracks or scaffold for different molecules. The crosstalk between both skeletons may be important for the formation and movement of the lamella at the immunological synapse by increasing the adhesion of the T cell to the antigen-presenting cells (APC), thus favoring the transport of components toward the plasma membrane and in turn regulating the T-APC intercellular communication. Microtubules and F-actin appear to be essential for the transport of the different signaling microclusters along the membrane, therefore facilitating the propagation of the signal. Finally, they can also be important for regulating the endocytosis, recycling, and degradation of the T cell receptor signaling machinery, thus helping both to sustain the activated state and to switch it off. PMID:22566814

  20. Tubulin and Actin Interplay at the T Cell and Antigen-Presenting Cell Interface

    PubMed Central

    Martín-Cófreces, Noa Beatriz; Alarcón, Balbino; Sánchez-Madrid, Francisco

    2011-01-01

    T cells reorganize their actin and tubulin-based cytoskeletons to provide a physical basis to the immune synapse. However, growing evidence shows that their roles on T cell activation are more dynamic than merely serving as tracks or scaffold for different molecules. The crosstalk between both skeletons may be important for the formation and movement of the lamella at the immunological synapse by increasing the adhesion of the T cell to the antigen-presenting cells (APC), thus favoring the transport of components toward the plasma membrane and in turn regulating the T-APC intercellular communication. Microtubules and F-actin appear to be essential for the transport of the different signaling microclusters along the membrane, therefore facilitating the propagation of the signal. Finally, they can also be important for regulating the endocytosis, recycling, and degradation of the T cell receptor signaling machinery, thus helping both to sustain the activated state and to switch it off. PMID:22566814

  1. A Neoglycoconjugate Containing the Human Milk Sugar LNFPIII Drives Anti-Inflammatory Activation of Antigen Presenting Cells in a CD14 Dependent Pathway

    PubMed Central

    Tundup, Smanla; Srivastava, Leena; Norberg, Thomas; Watford, Wendy; Harn, Donald

    2015-01-01

    The milk pentasaccharide LNFPIII has therapeutic action for metabolic and autoimmune diseases and prolongs transplant survival in mice when presented as a neoglycoconjugate. Within LNFPIII is the Lewisx trisaccharide, expressed by many helminth parasites. In humans, LNFPIII is found in human milk and also known as stage-specific embryonic antigen-1. LNFPIII-NGC drives alternative activation of macrophages and dendritic cells via NFκB activation in a TLR4 dependent mechanism. However, the connection between LNFPIII-NGC activation of APCs, TLR4 signaling and subsequent MAP kinase signaling leading to anti-inflammatory activation of APCs remains unknown. In this study we determined that the innate receptor CD14 was essential for LNFPIII-NGC induction of both ERK and NFkB activation in APCs. Induction of ERK activation by LNFPIII-NGC was completely dependent on CD14/TLR4-Ras-Raf1/TPL2-MEK axis in bone marrow derived dendritic cells (BMDCs). In addition, LNFPIII-NGC preferentially induced the production of Th2 “favoring” chemokines CCL22 and matrix metalloprotease protein-9 in a CD14 dependent manner in BMDCs. In contrast, LNFPIII-NGC induces significantly lower levels of Th1 “favoring” chemokines, MIP1α, MIP1β and MIP-2 compared to levels in LPS stimulated cells. Interestingly, NGC of the identical human milk sugar LNnT, minus the alpha 1–3 linked fucose, failed to activate APCs via TLR4/MD2/CD14 receptor complex, suggesting that the alpha 1–3 linked fucose in LNFPIII and not on LNnT, is required for this process. Using specific chemical inhibitors of the MAPK pathway, we found that LNFPIII-NGC induction of CCL22, MMP9 and IL-10 production was dependent on ERK activation. Over all, this study suggests that LNFPIII-NGC utilizes CD14/TLR4-MAPK (ERK) axis in modulating APC activation to produce anti-inflammatory chemokines and cytokines in a manner distinct from that seen for the pro-inflammatory PAMP LPS. These pathways may explain the in vivo therapeutic

  2. Recombinant Ov-ASP-1, a Th1-biased protein adjuvant derived from the helminth Onchocerca volvulus, can directly bind and activate antigen-presenting cells.

    PubMed

    He, Yuxian; Barker, Sophie J; MacDonald, Angus J; Yu, Yu; Cao, Long; Li, Jingjing; Parhar, Ranjit; Heck, Susanne; Hartmann, Susanne; Golenbock, Douglas T; Jiang, Shibo; Libri, Nathan A; Semper, Amanda E; Rosenberg, William M; Lustigman, Sara

    2009-04-01

    We previously reported that rOv-ASP-1, a recombinant Onchocerca volvulus activation associated protein-1, was a potent adjuvant for recombinant protein or synthetic peptide-based Ags. In this study, we further evaluated the adjuvanticity of rOv-ASP-1 and explored its mechanism of action. Consistently, recombinant full-length spike protein of SARS-CoV or its receptor-binding domain in the presence of rOv-ASP-1 could effectively induce a mixed but Th1-skewed immune response in immunized mice. It appears that rOv-ASP-1 primarily bound to the APCs among human PBMCs and triggered Th1-biased proinflammatory cytokine production probably via the activation of monocyte-derived dendritic cells and the TLR, TLR2, and TLR4, thus suggesting that rOv-ASP-1 is a novel potent innate adjuvant. PMID:19299698

  3. Modes of Antigen Presentation by Lymph Node Stromal Cells and Their Immunological Implications

    PubMed Central

    Hirosue, Sachiko; Dubrot, Juan

    2015-01-01

    Antigen presentation is no longer the exclusive domain of cells of hematopoietic origin. Recent works have demonstrated that lymph node stromal cell (LNSC) populations, such as fibroblastic reticular cells, lymphatic and blood endothelial cells, not only provide a scaffold for lymphocyte interactions but also exhibit active immunomodulatory roles that are critical to mounting and resolving effective immune responses. Importantly, LNSCs possess the ability to present antigens and establish antigen-specific interactions with T cells. One example is the expression of peripheral tissue antigens, which are presented on major histocompatibility complex (MHC)-I molecules with tolerogenic consequences on T cells. Additionally, exogenous antigens, including self and tumor antigens, can be processed and presented on MHC-I complexes, which result in dysfunctional activation of antigen-specific CD8+ T cells. While MHC-I is widely expressed on cells of both hematopoietic and non-hematopoietic origins, antigen presentation via MHC-II is more precisely regulated. Nevertheless, LNSCs are capable of endogenously expressing, or alternatively, acquiring MHC-II molecules. Transfer of antigen between LNSC and dendritic cells in both directions has been recently suggested to promote tolerogenic roles of LNSCs on the CD4+ T cell compartment. Thus, antigen presentation by LNSCs is thought to be a mechanism that promotes the maintenance of peripheral tolerance as well as generates a pool of diverse antigen-experienced T cells for protective immunity. This review aims to integrate the current and emerging literature to highlight the importance of LNSCs in immune responses, and emphasize their role in antigen trafficking, retention, and presentation. PMID:26441957

  4. Dendritic cell function and antigen presentation in malaria.

    PubMed

    Cockburn, Ian A; Zavala, Fidel

    2016-06-01

    Due to the diverse roles T cells play in protection against malaria as well as pathogenesis it is critical to know which cells present antigen and the nature of the antigens they present. During pre-erythrocytic stages of infection, cutting-edge imaging studies have shown how Plasmodium antigens are presented during both the priming and effector phases of the protective CD8+ T cell response. During blood stages, pathology is in part due to the loss of DC function and the action of pathogenic T cells in the brain. Recently endothelial cells presenting malaria antigen to cognate T cells have emerged as critical players in malaria pathogenesis. Manipulating these processes may inform both vaccine design and the development of therapies for cerebral malaria. PMID:26845735

  5. Nanoengineering approaches to the design of artificial antigen-presenting cells

    PubMed Central

    Sunshine, Joel C; Green, Jordan J

    2014-01-01

    Artificial antigen-presenting cells (aAPCs) have shown great initial promise for ex vivo activation of cytotoxic T cells. The development of aAPCs has focused mainly on the choice of proteins to use for surface presentation to T cells when conjugated to various spherical, microscale particles. We review here biomimetic nanoengineering approaches that have been applied to the development of aAPCs that move beyond initial concepts about aAPC development. This article also discusses key technologies that may be enabling for the development of nano- and micro-scale aAPCs with nanoscale features, and suggests several future directions for the field. PMID:23837856

  6. Survival and signaling changes in antigen presenting cell subsets after radiation

    NASA Astrophysics Data System (ADS)

    Parker, Jennifer Janell

    Radiation therapy is a widely used cancer treatment that has the potential to influence anti-tumor immune responses. Both myeloablative and non-myeloablative radiation are often used as part of preparatory regimens for hematopoetic stem cell transplantation, in combination with other chemotherapy or immuno-modulatory (e.g. Anti-thymocyte globulin (ATG)) therapies for both cytotoxic and immune modulatory purposes. However, the mechanisms responsible for the effect of radiation on antigen presenting cell (APC) responsiveness and radioresistance are poorly understood. The first studies described in this thesis were designed to identify and characterize early radiation-induced signaling changes in antigen presenting cells and to determine the effects of these signaling changes on APC receptor expression and function. The NFkappaB pathway in antigen presenting cells was chosen for study because it is activated by radiation in a wide range of other cell types and plays a vital role in the maintenance and regulation of the immune system. The effects of therapeutically relevant doses radiation (2 and 20 Gy) were compared at various timepoints in the human monocytic cell line (U937) using phospho-flow cytometry staining methods and cytometric analysis. These studies demonstrated that radiation-induced changes in the phosphorylation state of NFkappaB family members that were p53 independent. However, these changes were dependent upon activation of ATM in response to single or double-stranded breaks in DNA, as shown in experiments using an inhibitor of ATM and ATM siRNA knockdown U937 cells. In addition, studies examining the effect of radiation on co-stimulatory receptors with and without inhibition of the NFkappaB pathway via phospho-flow cytometry revealed that radiation-induced phosphorylation of NEMO promoted the activation and functional maturation of U937 cells. Furthermore, functional studies using both phospho-flow cytometry and/or mixed lymphocyte reactions to

  7. Dendritic Cells Are the Major Antigen Presenting Cells in Inflammatory Lesions of Murine Mycoplasma Respiratory Disease

    PubMed Central

    Sun, Xiangle; Jones, Harlan P.; Dobbs, Nicole; Bodhankar, Sheetal; Simecka, Jerry W.

    2013-01-01

    Mycoplasmas cause chronic respiratory diseases in animals and humans, and to date, development of vaccines have been problematic. Using a murine model of mycoplasma pneumonia, lymphocyte responses, specifically T cells, were shown to confer protection as well as promote immunopathology in mycoplasma disease. Because T cells play such a critical role, it is important to define the role of antigen presenting cells (APC) as these cells may influence either exacerbation of mycoplasma disease pathogenesis or enhancement of protective immunity. The roles of APC, such as dendritic cells and/or macrophages, and their ability to modulate adaptive immunity in mycoplasma disease are currently unknown. Therefore, the purpose of this study was to identify individual pulmonary APC populations that may contribute to the activation of T cell responses during mycoplasma disease pathogenesis. The present study indeed demonstrates increasing numbers of CD11c− F4/80+ cells, which contain macrophages, and more mature/activated CD11c+ F4/80− cells, containing DC, in the lungs after infection. CD11c− F4/80+ macrophage-enriched cells and CD11c+ F4/80− dendritic cell-enriched populations showed different patterns of cytokine mRNA expression, supporting the idea that these cells have different impacts on immunity in response to infection. In fact, DC containing CD11c+ F4/80− cell populations from the lungs of infected mice were most capable of stimulating mycoplasma-specific CD4+ Th cell responses in vitro. In vivo, these CD11c+F4/80− cells were co-localized with CD4+ Th cells in inflammatory infiltrates in the lungs of mycoplasma-infected mice. Thus, CD11c+F4/80− dendritic cells appear to be the major APC population responsible for pulmonary T cell stimulation in mycoplasma-infected mice, and these dendritic cells likely contribute to responses impacting disease pathogenesis. PMID:23390557

  8. HIV-1 Trans Infection of CD4+ T Cells by Professional Antigen Presenting Cells

    PubMed Central

    Rinaldo, Charles R.

    2013-01-01

    Since the 1990s we have known of the fascinating ability of a complex set of professional antigen presenting cells (APCs; dendritic cells, monocytes/macrophages, and B lymphocytes) to mediate HIV-1 trans infection of CD4+ T cells. This results in a burst of virus replication in the T cells that is much greater than that resulting from direct, cis infection of either APC or T cells, or trans infection between T cells. Such APC-to-T cell trans infection first involves a complex set of virus subtype, attachment, entry, and replication patterns that have many similarities among APC, as well as distinct differences related to virus receptors, intracellular trafficking, and productive and nonproductive replication pathways. The end result is that HIV-1 can sequester within the APC for several days and be transmitted via membrane extensions intracellularly and extracellularly to T cells across the virologic synapse. Virus replication requires activated T cells that can develop concurrently with the events of virus transmission. Further research is essential to fill the many gaps in our understanding of these trans infection processes and their role in natural HIV-1 infection. PMID:24278768

  9. Human Peritoneal Mesothelial Cells Display Phagocytic and Antigen-Presenting Functions to Contribute to Intraperitoneal Immunity.

    PubMed

    Shaw, Tanya J; Zhang, Xiang Y; Huo, Zhiming; Robertson, David; Lovell, Patricia A; Dalgleish, Angus G; Barton, Desmond P J

    2016-06-01

    Mesothelial cells lining the peritoneal cavity are strategically positioned to respond to and counter intraperitoneal infections, cancer cells, and other challenges. We have investigated human peritoneal mesothelial cells (HPMCs) for phagocytic activity, expression of surface Major Histocompatibility Complex (MHC) class II and accessory molecules involved in antigen presentation, and the ability to present recall antigens to T cells. Phagocytosis of dextran, latex beads, and Escherichia coli was observed by flow cytometry, and internalization was visualized using confocal and electron microscopy. Flow cytometry and/or cellular enzyme-linked immunosorbent assay showed constitutive expression of ICAM-1, LFA-3, and B7-1, but not B7-2 or MHC class II. Interferon-gamma induced MHC II and ICAM-1 expression in a dose- and time-dependent manner. Importantly, HPMCs induced autologous CD3 T-lymphocyte proliferation (H incorporation) after pulse with recall antigen. Human peritoneal mesothelial cells equipped with phagocytic and antigen-presenting machinery are anticipated to have an integral role in intraperitoneal immune surveillance. PMID:27120688

  10. Role of antigen presenting cell invariant chain in the development of hepatic steatosis in mouse model.

    PubMed

    Mishra, Alaknanda; Iyer, Srikanth; Kesarwani, Ashwani; Baligar, Prakash; Arya, Satya Pal; Arindkar, Shailendra; Kumar, M J Mahesh; Upadhyay, Pramod; Majumdar, Subeer S; Nagarajan, Perumal

    2016-08-15

    The role of Invariant chain (CD74 or Ii) in antigen presentation via Antigen Presenting Cells (APC), macrophage recruitment as well as survival, T cell activation and B cell differentiation has been well recognized. However, the aspect of CD74 which is involved in the development of hepatic steatosis and the pathways through which it acts remain to be studied. In this study, we investigated the role of CD74 in the inflammatory pathway and its contribution to development of hepatic steatosis. For this, wild type C57BL/6J and CD74 deficient mice (Ii(-/-) mice) were fed with high fat high fructose (HFHF) diet for 12 weeks. Chronic consumption of this feed did not develop hepatic steatosis, glucose intolerance or change in the level of immune cells in Ii(-/-) mice. Moreover, there was relatively delayed expression of genes involved in development of non alcoholic fatty liver disease (NAFLD) in HFHF fed Ii(-/-) mice as compared to that of C57BL/6J phenotype. Taken together, the data suggest that HFHF diet fed Ii(-/-) mice fail to develop hepatic steatosis, suggesting that Ii mediated pathways play a vital role in the initiation and propagation of liver inflammation. PMID:27371158

  11. Vaccinia virus infection induces dendritic cell maturation but inhibits antigen presentation by MHC class II

    PubMed Central

    Yao, Yongxue; Li, Ping; Singh, Pratibha; Thiele, Allison T.; Wilkes, David S.; Renukaradhya, Gourapura J.; Brutkiewicz, Randy R.; Travers, Jeffrey B.; Luker, Gary D.; Hong, Soon-Cheol; Blum, Janice S.; Chang, Cheong-Hee

    2007-01-01

    Vaccinia virus (VV) infection is known to inhibit dendritic cells (DC) functions in vitro. Paradoxically, VV is also highly immunogenic and thus has been used as a vaccine. In the present study, we investigated the effects of an in vivo VV infection on DC function by focusing on early innate immunity. Our data indicated that DC are activated upon in vivo VV infection of mice. Splenic DC from VV-infected mice expressed elevated levels of MHC class I and co-stimulatory molecules on their cell surface and exhibited the enhanced potential to produce cytokines upon LPS stimulation. DC from VV-infected mice also expressed a high level of interferon-β. However, a VV infection resulted in the down-regulation of MHC class II expression and the impairment of antigen presentation to CD4 T cells by DC. Thus, during the early stage of a VV infection, although DC are impaired in some of the critical antigen presentation functions, they can promote innate immune defenses against viral infection. PMID:17678637

  12. MHC Class II Antigen Presentation by Dendritic Cells Regulated through Endosomal Sorting

    PubMed Central

    ten Broeke, Toine; Wubbolts, Richard; Stoorvogel, Willem

    2013-01-01

    For the initiation of adaptive immune responses, dendritic cells present antigenic peptides in association with major histocompatibility complex class II (MHCII) to naïve CD4+ T lymphocytes. In this review, we discuss how antigen presentation is regulated through intracellular processing and trafficking of MHCII. Newly synthesized MHCII is chaperoned by the invariant chain to endosomes, where peptides from endocytosed pathogens can bind. In nonactivated dendritic cells, peptide-loaded MHCII is ubiquitinated and consequently sorted by the ESCRT machinery to intraluminal vesicles of multivesicular bodies, ultimately leading to lysosomal degradation. Ubiquitination of newly synthesized MHCII is blocked when dendritic cells are activated, now allowing its transfer to the cell surface. This mode of regulation for MHCII is a prime example of how molecular processing and sorting at multivesicular bodies can determine the expression of signaling receptors at the plasma membrane. PMID:24296169

  13. Modulation of liver tolerance by conventional and nonconventional antigen-presenting cells and regulatory immune cells

    PubMed Central

    Horst, Andrea Kristina; Neumann, Katrin; Diehl, Linda; Tiegs, Gisa

    2016-01-01

    The liver is a tolerogenic organ with exquisite mechanisms of immune regulation that ensure upkeep of local and systemic immune tolerance to self and foreign antigens, but that is also able to mount effective immune responses against pathogens. The immune privilege of liver allografts was recognized first in pigs in spite of major histo-compatibility complex mismatch, and termed the “liver tolerance effect”. Furthermore, liver transplants are spontaneously accepted with only low-dose immunosuppression, and induce tolerance for non-hepatic co-transplanted allografts of the same donor. Although this immunotolerogenic environment is favorable in the setting of organ transplantation, it is detrimental in chronic infectious liver diseases like hepatitis B or C, malaria, schistosomiasis or tumorigenesis, leading to pathogen persistence and weak anti-tumor effects. The liver is a primary site of T-cell activation, but it elicits poor or incomplete activation of T cells, leading to their abortive activation, exhaustion, suppression of their effector function and early death. This is exploited by pathogens and can impair pathogen control and clearance or allow tumor growth. Hepatic priming of T cells is mediated by a number of local conventional and nonconventional antigen-presenting cells (APCs), which promote tolerance by immune deviation, induction of T-cell anergy or apoptosis, and generating and expanding regulatory T cells. This review will focus on the communication between classical and nonclassical APCs and lymphocytes in the liver in tolerance induction and will discuss recent insights into the role of innate lymphocytes in this process. PMID:27041638

  14. CD1d-restricted antigen presentation by Vγ9Vδ2-T cells requires trogocytosis.

    PubMed

    Schneiders, Famke L; Prodöhl, Jan; Ruben, Jurjen M; O'Toole, Tom; Scheper, Rik J; Bonneville, Marc; Scotet, Emmanuel; Verheul, Henk M W; de Gruijl, Tanja D; van der Vliet, Hans J

    2014-08-01

    CD1d-restricted invariant natural killer T cells (iNKT) constitute an important immunoregulatory T-cell subset that can be activated by the synthetic glycolipid α-galactosylceramide (α-GalCer) and play a dominant role in antitumor immunity. Clinical trials with α-GalCer-pulsed monocyte-derived dendritic cells (moDC) have shown anecdotal antitumor activity in advanced cancer. It was reported that phosphoantigen (pAg)-activated Vγ9Vδ2-T cells can acquire characteristics of professional antigen-presenting cells (APC). Considering the clinical immunotherapeutic applications, Vγ9Vδ2-T APC can offer important advantages over moDC, potentially constituting an attractive novel APC platform. Here, we demonstrate that Vγ9Vδ2-T APC can present antigens to iNKT. However, this does not result from de novo synthesis of CD1d by Vγ9Vδ2-T, but critically depends on trogocytosis of CD1d-containing membrane fragments from pAg-expressing cells. CD1d-expressing Vγ9Vδ2-T cells were able to activate iNKT in a CD1d-restricted and α-GalCer-dependent fashion. Although α-GalCer-loaded moDC outperformed Vγ9Vδ2-T APC on a per cell basis, Vγ9Vδ2-T APC possess unique features with respect to clinical immunotherapeutic application that make them an interesting platform for consideration in future clinical trials. PMID:24934445

  15. Modulation of Th1/Th2 Immune Responses by Killed Propionibacterium acnes and Its Soluble Polysaccharide Fraction in a Type I Hypersensitivity Murine Model: Induction of Different Activation Status of Antigen-Presenting Cells

    PubMed Central

    Mussalem, Juliana Sekeres; Ishimura, Mayari Eika; Longo-Maugéri, Ieda Maria

    2015-01-01

    Propionibacterium acnes (P. acnes) is a gram-positive anaerobic bacillus present in normal human skin microbiota, which exerts important immunomodulatory effects, when used as heat- or phenol-killed suspensions. We previously demonstrated that heat-killed P. acnes or its soluble polysaccharide (PS), extracted from the bacterium cell wall, suppressed or potentiated the Th2 response to ovalbumin (OVA) in an immediate hypersensitivity model, depending on the treatment protocol. Herein, we investigated the mechanisms responsible for these effects, using the same model and focusing on the activation status of antigen-presenting cells (APCs). We verified that higher numbers of APCs expressing costimulatory molecules and higher expression levels of these molecules are probably related to potentiation of the Th2 response to OVA induced by P. acnes or PS, while higher expression of toll-like receptors (TLRs) seems to be related to Th2 suppression. In vitro cytokines production in cocultures of dendritic cells and T lymphocytes indicated that P. acnes and PS seem to perform their effects by acting directly on APCs. Our data suggest that P. acnes and PS directly act on APCs, modulating the expression of costimulatory molecules and TLRs, and these differently activated APCs drive distinct T helper patterns to OVA in our model. PMID:25973430

  16. Modulation of Th1/Th2 immune responses by killed Propionibacterium acnes and its soluble polysaccharide fraction in a type I hypersensitivity murine model: induction of different activation status of antigen-presenting cells.

    PubMed

    Squaiella-Baptistão, Carla Cristina; Teixeira, Daniela; Mussalem, Juliana Sekeres; Ishimura, Mayari Eika; Longo-Maugéri, Ieda Maria

    2015-01-01

    Propionibacterium acnes (P. acnes) is a gram-positive anaerobic bacillus present in normal human skin microbiota, which exerts important immunomodulatory effects, when used as heat- or phenol-killed suspensions. We previously demonstrated that heat-killed P. acnes or its soluble polysaccharide (PS), extracted from the bacterium cell wall, suppressed or potentiated the Th2 response to ovalbumin (OVA) in an immediate hypersensitivity model, depending on the treatment protocol. Herein, we investigated the mechanisms responsible for these effects, using the same model and focusing on the activation status of antigen-presenting cells (APCs). We verified that higher numbers of APCs expressing costimulatory molecules and higher expression levels of these molecules are probably related to potentiation of the Th2 response to OVA induced by P. acnes or PS, while higher expression of toll-like receptors (TLRs) seems to be related to Th2 suppression. In vitro cytokines production in cocultures of dendritic cells and T lymphocytes indicated that P. acnes and PS seem to perform their effects by acting directly on APCs. Our data suggest that P. acnes and PS directly act on APCs, modulating the expression of costimulatory molecules and TLRs, and these differently activated APCs drive distinct T helper patterns to OVA in our model. PMID:25973430

  17. Pros and Cons of Antigen-Presenting Cell Targeted Tumor Vaccines

    PubMed Central

    Goyvaerts, Cleo; Breckpot, Karine

    2015-01-01

    In therapeutic antitumor vaccination, dendritic cells play the leading role since they decide if, how, when, and where a potent antitumor immune response will take place. Since the disentanglement of the complexity and merit of different antigen-presenting cell subtypes, antitumor immunotherapeutic research started to investigate the potential benefit of targeting these subtypes in situ. This review will discuss which antigen-presenting cell subtypes are at play and how they have been targeted and finally question the true meaning of targeting antitumor-based vaccines. PMID:26583156

  18. Extracts from presumed "reduced harm" cigarettes induce equivalent or greater toxicity in antigen-presenting cells.

    PubMed

    Vassallo, Robert; Wang, Lei; Hirano, Yoshimi; Walters, Paula; Grill, Diane

    2015-09-01

    The tobacco industry has promoted certain cigarette products with claims that their use may be less harmful to the smoker as they purportedly deliver lower amounts of toxic chemicals compared to conventional cigarettes. This study was designed to compare the relative antigen presenting cellular toxicity of Eclipse, a presumed reduced exposure product (PREP) cigarette, when compared with the reference research 3R4F cigarettes (Kentucky University). Utilizing a murine macrophage cell line, murine bone marrow derived dendritic cells (DCs) and human monocyte-derived DCs incubated with extracts generated from Eclipse and Kentucky reference 3R4F cigarettes, we determined the relative toxic effects of the different cigarette smoke extracts on cellular viability, oxidative stress, T-helper-1 (Th-1) polarizing cytokine production and general gene expression. Eclipse and 3R4F cigarette smoke extracts induced equivalent oxidatively-mediated cellular heme oxygenase-1 (HO-1) protein levels in macrophages and DCs. Cellular viability determination demonstrated greater induction of cell death by apoptosis and necrosis by Eclipse extracts in DCs. The production of the key Th-1 polarizing cytokine interleukin-12 (IL-12) by activated DCs or macrophages was suppressed to an equivalent or greater extent by Eclipse extracts. Microarray studies performed on bone marrow derived murine DCs incubated with Eclispe or 3R4F cigarette extracts showed identical genotoxic profiles. These studies imply that presumed reduced harm Eclipse cigarettes induce equivalent or greater antigen presenting cell dysfunction relative to 3R4F cigarettes and illustrate the importance of independent validation and testing of similar products claimed to be associated with reduced toxicity relative to other cigarettes. PMID:26169828

  19. Deficiency of Antigen Presenting Cell Invariant Chain Reduces Atherosclerosis in Mice

    PubMed Central

    Sun, Jiusong; Hartvigsen, Karsten; Chou, Meng-Yun; Zhang, Yadong; Sukhova, Galina K.; Zhang, Jie; Lopez-Ilasaca, Marco; Diehl, Cody J.; Yakov, Niva; Harats, Dror; George, Jacob; Witztum, Joseph L.; Libby, Peter; Ploegh, Hidde; Shi, Guo-Ping

    2010-01-01

    Background Adaptive and innate immunity play important roles in atherogenesis. Invariant chain (CD74) mediates antigen presenting cell (APC) antigen presentation and T cell activation. This study tested the hypothesis that CD74-deficient mice have reduced numbers of active T cells and resist atherogenesis. Methods and Results In low-density lipoprotein receptor-deficient mice (Ldlr−/−), CD74 deficiency (Ldlr−/−Cd74−/−) significantly reduced atherosclerosis and CD25+ activated T cells in the atheromata. While Ldlr−/−Cd74−/− mice had decreased levels of plasma IgG1, IgG2b, and IgG2c against malondialdehyde-modified LDL (MDA-LDL), presumably due to impaired APC function, Ldlr−/−Cd74−/− mice showed higher levels of anti-MDA-LDL IgM and IgG3. After immunization with MDA-LDL, Ldlr−/−Cd74−/− mice had lower levels of all anti-MDA-LDL immunoglobulin (Ig) isotypes compared with Ldlr−/− mice. As anticipated, only Ldlr−/− splenocytes responded to in vitro stimulation with MDA-LDL, producing Th1/Th2 cytokines. Heat shock protein-65 (HSP65) immunization enhanced atherogenesis in Ldlr−/− mice, but Ldlr−/−Cd74−/− mice remained protected. Compared with Ldlr−/− mice, Ldlr−/−Cd74−/− mice had higher anti-MDA-LDL autoantibody titers, fewer lesion CD25+ activated T cells, impaired release of Th1/Th2 cytokines from APC after HSP65-stimulation, and reduced levels of all plasma anti-HSP65 Ig isotypes. Cytofluorimetry of splenocytes and peritoneal cavity cells of MDA-LDL- or HSP65-immunized mice showed increased percentages of autoantibody-producing marginal zone-B and B-1 cells in Ldlr−/−Cd74−/− mice compared to Ldlr−/− mice. Conclusion Invariant chain deficiency in Ldlr−/− mice reduced atherosclerosis. This finding was associated with an impaired adaptive immune response to disease-specific antigens. Concomitantly, there occurred an unexpected increase in the number of innate-like peripheral B-1 cell

  20. The Exonuclease Domain of Lassa Virus Nucleoprotein Is Involved in Antigen-Presenting-Cell-Mediated NK Cell Responses

    PubMed Central

    Russier, Marion; Reynard, Stéphanie; Carnec, Xavier

    2014-01-01

    ABSTRACT Lassa virus is an Old World Arenavirus which causes Lassa hemorrhagic fever in humans, mostly in West Africa. Lassa fever is an important public health problem, and a safe and effective vaccine is urgently needed. The infection causes immunosuppression, probably due to the absence of activation of antigen-presenting cells (dendritic cells and macrophages), low type I interferon (IFN) production, and deficient NK cell function. However, a recombinant Lassa virus carrying D389A and G392A substitutions in the nucleoprotein that abolish the exonuclease activity and IFN activation loses its inhibitory activity and induces strong type I IFN production by dendritic cells and macrophages. We show here that during infection by this mutant Lassa virus, antigen-presenting cells trigger efficient human NK cell responses in vitro, including production of IFN-γ and cytotoxicity. NK cell activation involves close contact with both antigen-presenting cells and soluble factors. We report that infected dendritic cells and macrophages express the NKG2D ligands major histocompatibility complex (MHC) class I-related chains A and B and that they may produce interleukin-12 (IL-12), IL-15, and IL-18, all involved in NK cell functions. NK cell degranulation is significantly increased in cocultures, suggesting that NK cells seem to kill infected dendritic cells and macrophages. This work confirms the inhibitory function of Lassa virus nucleoprotein. Importantly, we demonstrate for the first time that Lassa virus nucleoprotein is involved in the inhibition of antigen-presenting cell-mediated NK cell responses. IMPORTANCE The pathogenesis and immune responses induced by Lassa virus are poorly known. Recently, an exonuclease domain contained in the viral nucleoprotein has been shown to be able to inhibit the type I IFN response by avoiding the recognition of viral RNA by cell sensors. Here, we studied the responses of NK cells to dendritic cells and macrophages infected with a

  1. Vaccine delivery by penetratin: mechanism of antigen presentation by dendritic cells.

    PubMed

    Pouniotis, Dodie; Tang, Choon-Kit; Apostolopoulos, Vasso; Pietersz, Geoffrey

    2016-08-01

    Cell-penetrating peptides (CPP) or membrane-translocating peptides such as penetratin from Antennapedia homeodomain or TAT from human immunodeficiency virus are useful vectors for the delivery of protein antigens or their cytotoxic (Tc) or helper (Th) T cell epitopes to antigen-presenting cells. Mice immunized with CPP containing immunogens elicit antigen-specific Tc and/or Th responses and could be protected from tumor challenges. In the present paper, we investigate the mechanism of class I and class II antigen presentation of ovalbumin covalently linked to penetratin (AntpOVA) by bone marrow-derived dendritic cells with the use of biochemical inhibitors of various pathways of antigen processing and presentation. Results from our study suggested that uptake of AntpOVA is via a combination of energy-independent (membrane fusion) and energy-dependent pathways (endocytosis). Once internalized by either mechanism, multiple tap-dependent or independent antigen presentation pathways are accessed while not completely dependent on proteasomal processing but involving proteolytic trimming in the ER and Golgi compartments. Our study provides an understanding on the mechanism of antigen presentation mediated by CPP and leads to greater insights into future development of vaccine formulations. PMID:27138940

  2. Neutrophil elastase enhances antigen presentation by upregulating human leukocyte antigen class I expression on tumor cells.

    PubMed

    Chawla, Akhil; Alatrash, Gheath; Philips, Anne V; Qiao, Na; Sukhumalchandra, Pariya; Kerros, Celine; Diaconu, Iulia; Gall, Victor; Neal, Samantha; Peters, Haley L; Clise-Dwyer, Karen; Molldrem, Jeffrey J; Mittendorf, Elizabeth A

    2016-06-01

    Neutrophil elastase (NE) is an innate immune cell-derived inflammatory mediator that we have shown increases the presentation of tumor-associated peptide antigens in breast cancer. In this study, we extend these observations to show that NE uptake has a broad effect on enhancing antigen presentation by breast cancer cells. We show that NE increases human leukocyte antigen (HLA) class I expression on the surface of breast cancer cells in a concentration and time-dependent manner. HLA class I upregulation requires internalization of enzymatically active NE. Western blots of NE-treated breast cancer cells confirm that the expression of total HLA class I as well as the antigen-processing machinery proteins TAP1, LMP2, and calnexin does not change following NE treatment. This suggests that NE does not increase the efficiency of antigen processing; rather, it mediates the upregulation of HLA class I by stabilizing and reducing membrane recycling of HLA class I molecules. Furthermore, the effects of NE extend beyond breast cancer since the uptake of NE by EBV-LCL increases the presentation of HLA class I-restricted viral peptides, as shown by their increased sensitivity to lysis by EBV-specific CD8+ T cells. Together, our results show that NE uptake increases the responsiveness of breast cancer cells to adaptive immunity by broad upregulation of membrane HLA class I and support the conclusion that the innate inflammatory mediator NE enhances tumor cell recognition and increases tumor sensitivity to the host adaptive immune response. PMID:27129972

  3. Regulation of Hemichannels and Gap Junction Channels by Cytokines in Antigen-Presenting Cells

    PubMed Central

    Shoji, Kenji F.; Aguirre, Adam; Sáez, Juan C.

    2014-01-01

    Autocrine and paracrine signals coordinate responses of several cell types of the immune system that provide efficient protection against different challenges. Antigen-presenting cells (APCs) coordinate activation of this system via homocellular and heterocellular interactions. Cytokines constitute chemical intercellular signals among immune cells and might promote pro- or anti-inflammatory effects. During the last two decades, two membrane pathways for intercellular communication have been demonstrated in cells of the immune system. They are called hemichannels (HCs) and gap junction channels (GJCs) and provide new insights into the mechanisms of the orchestrated response of immune cells. GJCs and HCs are permeable to ions and small molecules, including signaling molecules. The direct intercellular transfer between contacting cells can be mediated by GJCs, whereas the release to or uptake from the extracellular milieu can be mediated by HCs. GJCs and HCs can be constituted by two protein families: connexins (Cxs) or pannexins (Panxs), which are present in almost all APCs, being Cx43 and Panx1 the most ubiquitous members of each protein family. In this review, we focus on the effects of different cytokines on the intercellular communication mediated by HCs and GJCs in APCs and their impact on purinergic signaling. PMID:25301274

  4. Engineering tolerance using biomaterials to target and control antigen presenting cells.

    PubMed

    Tostanoski, Lisa H; Gosselin, Emily A; Jewell, Christopher M

    2016-05-01

    Autoimmune diseases occur when cells of the adaptive immune system incorrectly recognize and attack "self" tissues. Importantly, the proliferation and differentiation of these cells is triggered and controlled by interactions with antigen presenting cells (APCs), such as dendritic cells. Thus, modulating the signals transduced by APCs (e.g., cytokines, costimulatory surface proteins) has emerged as a promising strategy to promote tolerance for diseases such as multiple sclerosis, type 1 diabetes, and lupus. However, many approaches have been hindered by non-specific activity of immunosuppressive or immunoregulatory cues, following systemic administration of soluble factors via traditional injections routes (e.g., subcutaneous, intravenous). Biomaterials offer a unique opportunity to control the delivery of tolerogenic signals in vivo via properties such as controlled particle size, tunable release kinetics, and co-delivery of multiple classes of cargo. In this review, we highlight recent reports that exploit these properties of biomaterials to target APCs and promote tolerance via three strategies, i) passive or active targeting of particulate carriers to APCs, ii) biomaterial-mediated control over antigen localization and processing, and iii) targeted delivery of encapsulated or adsorbed immunomodulatory signals. These reports represent exciting advances toward the goal of more effective therapies for autoimmune diseases, without the broad suppressive effects associated with current clinically-approved therapies. PMID:27355336

  5. mTOR controls lysosome tubulation and antigen presentation in macrophages and dendritic cells

    PubMed Central

    Saric, Amra; Hipolito, Victoria E. B.; Kay, Jason G.; Canton, Johnathan; Antonescu, Costin N.; Botelho, Roberto J.

    2016-01-01

    Macrophages and dendritic cells exposed to lipopolysaccharide (LPS) convert their lysosomes from small, punctate organelles into a network of tubules. Tubular lysosomes have been implicated in phagosome maturation, retention of fluid phase, and antigen presentation. There is a growing appreciation that lysosomes act as sensors of stress and the metabolic state of the cell through the kinase mTOR. Here we show that LPS stimulates mTOR and that mTOR is required for LPS-induced lysosome tubulation and secretion of major histocompatibility complex II in macrophages and dendritic cells. Specifically, we show that the canonical phosphatidylinositol 3-kinase–Akt–mTOR signaling pathway regulates LPS-induced lysosome tubulation independently of IRAK1/4 and TBK. Of note, we find that LPS treatment augmented the levels of membrane-associated Arl8b, a lysosomal GTPase required for tubulation that promotes kinesin-dependent lysosome movement to the cell periphery, in an mTOR-dependent manner. This suggests that mTOR may interface with the Arl8b-kinesin machinery. To further support this notion, we show that mTOR antagonists can block outward movement of lysosomes in cells treated with acetate but have no effect in retrograde movement upon acetate removal. Overall our work provides tantalizing evidence that mTOR plays a role in controlling lysosome morphology and trafficking by modulating microtubule-based motor activity in leukocytes. PMID:26582390

  6. mTOR controls lysosome tubulation and antigen presentation in macrophages and dendritic cells.

    PubMed

    Saric, Amra; Hipolito, Victoria E B; Kay, Jason G; Canton, Johnathan; Antonescu, Costin N; Botelho, Roberto J

    2016-01-15

    Macrophages and dendritic cells exposed to lipopolysaccharide (LPS) convert their lysosomes from small, punctate organelles into a network of tubules. Tubular lysosomes have been implicated in phagosome maturation, retention of fluid phase, and antigen presentation. There is a growing appreciation that lysosomes act as sensors of stress and the metabolic state of the cell through the kinase mTOR. Here we show that LPS stimulates mTOR and that mTOR is required for LPS-induced lysosome tubulation and secretion of major histocompatibility complex II in macrophages and dendritic cells. Specifically, we show that the canonical phosphatidylinositol 3-kinase-Akt-mTOR signaling pathway regulates LPS-induced lysosome tubulation independently of IRAK1/4 and TBK. Of note, we find that LPS treatment augmented the levels of membrane-associated Arl8b, a lysosomal GTPase required for tubulation that promotes kinesin-dependent lysosome movement to the cell periphery, in an mTOR-dependent manner. This suggests that mTOR may interface with the Arl8b-kinesin machinery. To further support this notion, we show that mTOR antagonists can block outward movement of lysosomes in cells treated with acetate but have no effect in retrograde movement upon acetate removal. Overall our work provides tantalizing evidence that mTOR plays a role in controlling lysosome morphology and trafficking by modulating microtubule-based motor activity in leukocytes. PMID:26582390

  7. Killer artificial antigen-presenting cells: the synthetic embodiment of a 'guided missile'.

    PubMed

    Schütz, Christian; Oelke, Mathias; Schneck, Jonathan P; Mackensen, Andreas; Fleck, Martin

    2010-07-01

    At present, the treatment of T-cell-dependent autoimmune diseases relies exclusively on strategies leading to nonspecific suppression of the immune systems causing a substantial reduced ability to control concomitant infections or malignancies. Furthermore, long-term treatment with most drugs is accompanied by several serious adverse effects and does not consequently result in cure of the primary immunological malfunction. By contrast, antigen-specific immunotherapy offers the potential to achieve the highest therapeutic efficiency in accordance with minimal adverse effects. Therefore, several studies have been performed utilizing antigen-presenting cells specifically engineered to deplete allo- or antigen-specific T cells ('guided missiles'). Many of these strategies take advantage of the Fas/Fas ligand signaling pathway to efficiently induce antigen-presenting cell-mediated apoptosis in targeted T cells. In this article, we discuss the advantages and shortcomings of a novel non-cell-based 'killer artificial antigen-presenting cell' strategy, developed to overcome obstacles related to current cell-based approaches for the treatment of T-cell-mediated autoimmunity. PMID:20636007

  8. Equine infectious anemia virus-infected dendritic cells retain antigen presentation capability

    SciTech Connect

    Rivera, Julie A.; McGuire, Travis C. . E-mail: mcguiret@vetmed.wsu.edu

    2005-05-10

    To determine if equine monocyte-derived dendritic cells (DC) were susceptible to equine infectious anemia virus (EIAV) infection, ex vivo-generated DC were infected with virus in vitro. EIAV antigen was detected by immunofluorescence 3 days post-infection with maximum antigen being detected on day 4, whereas there was no antigen detected in DC incubated with the same amount of heat-inactivated EIAV. No cytolytic activity was observed after EIAV{sub WSU5} infection of DC. These monocyte-derived DC were more effective than macrophages and B cells in stimulating allogenic T lymphocytes. Both infected macrophages and DC stimulated similar levels of memory CTL responses in mixtures of CD8+ and CD4+ cells as detected with {sup 51}Cr-release assays indicating that EIAV infection of DC did not alter antigen presentation. However, EIAV-infected DC were more effective than infected macrophages when used to stimulate memory CTL in isolated CD8+ cells. The maintenance of antigen processing and presenting function by EIAV-infected DC in vitro suggests that this function is maintained during in vivo infection.

  9. Chronic HIV Infection Enhances the Responsiveness of Antigen Presenting Cells to Commensal Lactobacillus

    PubMed Central

    Nagy, Lauren H.; Grishina, Irina; Macal, Monica; Hirao, Lauren A.; Hu, William K.; Sankaran-Walters, Sumathi; Gaulke, Christopher A.; Pollard, Richard; Brown, Jennifer; Suni, Maria; Baumler, Andreas J.; Ghanekar, Smita; Marco, Maria L.; Dandekar, Satya

    2013-01-01

    Chronic immune activation despite long-term therapy poses an obstacle to immune recovery in HIV infection. The role of antigen presenting cells (APCs) in chronic immune activation during HIV infection remains to be fully determined. APCs, the frontline of immune defense against pathogens, are capable of distinguishing between pathogens and non-pathogenic, commensal bacteria. We hypothesized that HIV infection induces dysfunction in APC immune recognition and response to some commensal bacteria and that this may promote chronic immune activation. Therefore we examined APC inflammatory cytokine responses to commensal lactobacilli. We found that APCs from HIV-infected patients produced an enhanced inflammatory response to Lactobacillus plantarum WCFS1 as compared to APCs from healthy, HIV-negative controls. Increased APC expression of TLR2 and CD36, signaling through p38-MAPK, and decreased expression of MAP kinase phosphatase-1 (MKP-1) in HIV infection was associated with this heightened immune response. Our findings suggest that chronic HIV infection enhances the responsiveness of APCs to commensal lactobacilli, a mechanism that may partly contribute to chronic immune activation. PMID:24023646

  10. Peripheral blood and synovial fluid T cells differ in their response to alloantigens and recall antigens presented by dendritic cells.

    PubMed Central

    Stagg, A J; Harding, B; Hughes, R A; Keat, A; Knight, S C

    1991-01-01

    Properties of T cells from inflammatory lesions were analysed by comparing the response of peripheral blood (PB) and synovial fluid (SF) T cells from 19 patients with a range of arthropathies to enriched allogeneic dendritic cells (DC) in a primary mixed leucocyte reaction (MLR). In 17 patients the proliferative response of SF T cells was significantly (P less than 0.05) less than that of PB lymphocytes. The reduced response of SF T cells was observed in all disease categories studied and could not be attributed to differences in cell number requirements or response kinetics. Addition of recombinant interleukin-2 enhanced the response of SF T cells in a dose-dependent manner. Cell mixing experiments suggested that active suppression was not the underlying mechanism of the poor MLR response of SF T cells. In contrast to the MLR response. SF T cells were able to mount vigorous proliferative responses to recall antigen presented by autologous antigen-presenting cells. The possibility is discussed that T cells compartmentalized at inflammatory lesions are a unique population with a diminished ability to interact with DC and respond to primary stimuli but an ability to respond to secondary antigenic challenge. PMID:1826648

  11. Mannosylated poly(beta-amino esters) for targeted antigen presenting cell immune modulation.

    PubMed

    Jones, Charles H; Chen, Mingfu; Ravikrishnan, Anitha; Reddinger, Ryan; Zhang, Guojian; Hakansson, Anders P; Pfeifer, Blaine A

    2015-01-01

    Given the rise of antibiotic resistance and other difficult-to-treat diseases, genetic vaccination is a promising preventative approach that can be tailored and scaled according to the vector chosen for gene delivery. However, most vectors currently utilized rely on ubiquitous delivery mechanisms that ineffectively target important immune effectors such as antigen presenting cells (APCs). As such, APC targeting allows the option for tuning the direction (humoral vs cell-mediated) and strength of the resulting immune responses. In this work, we present the development and assessment of a library of mannosylated poly(beta-amino esters) (PBAEs) that represent a new class of easily synthesized APC-targeting cationic polymers. Polymeric characterization and assessment methodologies were designed to provide a more realistic physiochemical profile prior to in vivo evaluation. Gene delivery assessment in vitro showed significant improvement upon PBAE mannosylation and suggested that mannose-mediated uptake and processing influence the magnitude of gene delivery. Furthermore, mannosylated PBAEs demonstrated a strong, efficient, and safe in vivo humoral immune response without use of adjuvants when compared to genetic and protein control antigens. In summary, the gene delivery effectiveness provided by mannosylated PBAE vectors offers specificity and potency in directing APC activation and subsequent immune responses. PMID:25453962

  12. Gatekeeper role of brain antigen-presenting CD11c+ cells in neuroinflammation.

    PubMed

    Paterka, Magdalena; Siffrin, Volker; Voss, Jan O; Werr, Johannes; Hoppmann, Nicola; Gollan, René; Belikan, Patrick; Bruttger, Julia; Birkenstock, Jérôme; Jung, Steffen; Esplugues, Enric; Yogev, Nir; Flavell, Richard A; Bopp, Tobias; Zipp, Frauke

    2016-01-01

    Multiple sclerosis is the most frequent chronic inflammatory disease of the CNS. The entry and survival of pathogenic T cells in the CNS are crucial for the initiation and persistence of autoimmune neuroinflammation. In this respect, contradictory evidence exists on the role of the most potent type of antigen-presenting cells, dendritic cells. Applying intravital two-photon microscopy, we demonstrate the gatekeeper function of CNS professional antigen-presenting CD11c(+) cells, which preferentially interact with Th17 cells. IL-17 expression correlates with expression of GM-CSF by T cells and with accumulation of CNS CD11c(+) cells. These CD11c(+) cells are organized in perivascular clusters, targeted by T cells, and strongly express the inflammatory chemokines Ccl5, Cxcl9, and Cxcl10. Our findings demonstrate a fundamental role of CNS CD11c(+) cells in the attraction of pathogenic T cells into and their survival within the CNS. Depletion of CD11c(+) cells markedly reduced disease severity due to impaired enrichment of pathogenic T cells within the CNS. PMID:26612827

  13. Therapeutic antiviral T cells noncytopathically clear persistently infected microglia after conversion into antigen-presenting cells

    PubMed Central

    Herz, Jasmin; Johnson, Kory R.

    2015-01-01

    Several viruses can infect the mammalian nervous system and induce neurological dysfunction. Adoptive immunotherapy is an approach that involves administration of antiviral T cells and has shown promise in clinical studies for the treatment of peripheral virus infections in humans such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus, among others. In contrast, clearance of neurotropic infections is particularly challenging because the central nervous system (CNS) is relatively intolerant of immunopathological reactions. Therefore, it is essential to develop and mechanistically understand therapies that noncytopathically eradicate pathogens from the CNS. Here, we used mice persistently infected from birth with lymphocytic choriomeningitis virus (LCMV) to demonstrate that therapeutic antiviral T cells can completely purge the persistently infected brain without causing blood–brain barrier breakdown or tissue damage. Mechanistically, this is accomplished through a tailored release of chemoattractants that recruit antiviral T cells, but few pathogenic innate immune cells such as neutrophils and inflammatory monocytes. Upon arrival, T cells enlisted the support of nearly all brain-resident myeloid cells (microglia) by inducing proliferation and converting them into CD11c+ antigen-presenting cells (APCs). Two-photon imaging experiments revealed that antiviral CD8+ and CD4+ T cells interacted directly with CD11c+ microglia and induced STAT1 signaling but did not initiate programmed cell death. We propose that noncytopathic CNS viral clearance can be achieved by therapeutic antiviral T cells reliant on restricted chemoattractant production and interactions with apoptosis-resistant microglia. PMID:26122661

  14. Therapeutic antiviral T cells noncytopathically clear persistently infected microglia after conversion into antigen-presenting cells.

    PubMed

    Herz, Jasmin; Johnson, Kory R; McGavern, Dorian B

    2015-07-27

    Several viruses can infect the mammalian nervous system and induce neurological dysfunction. Adoptive immunotherapy is an approach that involves administration of antiviral T cells and has shown promise in clinical studies for the treatment of peripheral virus infections in humans such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus, among others. In contrast, clearance of neurotropic infections is particularly challenging because the central nervous system (CNS) is relatively intolerant of immunopathological reactions. Therefore, it is essential to develop and mechanistically understand therapies that noncytopathically eradicate pathogens from the CNS. Here, we used mice persistently infected from birth with lymphocytic choriomeningitis virus (LCMV) to demonstrate that therapeutic antiviral T cells can completely purge the persistently infected brain without causing blood-brain barrier breakdown or tissue damage. Mechanistically, this is accomplished through a tailored release of chemoattractants that recruit antiviral T cells, but few pathogenic innate immune cells such as neutrophils and inflammatory monocytes. Upon arrival, T cells enlisted the support of nearly all brain-resident myeloid cells (microglia) by inducing proliferation and converting them into CD11c(+) antigen-presenting cells (APCs). Two-photon imaging experiments revealed that antiviral CD8(+) and CD4(+) T cells interacted directly with CD11c(+) microglia and induced STAT1 signaling but did not initiate programmed cell death. We propose that noncytopathic CNS viral clearance can be achieved by therapeutic antiviral T cells reliant on restricted chemoattractant production and interactions with apoptosis-resistant microglia. PMID:26122661

  15. Antigen presenting cell-selective drug delivery by glycan-decorated nanocarriers.

    PubMed

    Frenz, Theresa; Grabski, Elena; Durán, Verónica; Hozsa, Constantin; Stępczyńska, Anna; Furch, Marcus; Gieseler, Robert K; Kalinke, Ulrich

    2015-09-01

    Targeted drug delivery systems hold promise for selective provision of active compounds to distinct tissues or cell subsets. Thus, locally enhanced drug concentrations are obtained that would confer improved efficacy. As a consequence adverse effects should be diminished, as innocent bystander cells are less affected. Currently, several controlled drug delivery systems based on diverse materials are being developed. Some systems exhibit material-associated toxic effects and/or show low drug loading capacity. In contrast, liposomal nanocarriers are particularly favorable because they are well tolerated, poorly immunogenic, can be produced in defined sizes, and offer a reasonable payload capacity. Compared with other immune cells, professional antigen-presenting cells (APCs) demonstrate enhanced liposome uptake mediated by macropinocytosis, phagocytosis and presumably also by clathrin- and caveolae-mediated endocytosis. In order to further enhance the targeting efficacy toward APCs, receptor-mediated uptake appears advisable. Since APC subsets generally do not express single linage-specific receptors, members of the C-type lectin receptor (CLR) family are compelling targets. Examples of CLR expressed by APCs include DEC-205 (CD205) expressed by myeloid dendritic cells (DC) and monocytes, the mannose receptor C type 1 (MR, CD206) expressed by DC, monocytes and macrophages, DC-SIGN (CD209) expressed by DC, and several others. These receptors bind glycans, which are typically displayed by pathogens and thus support pathogen uptake and endocytosis. Further research will elucidate whether glycan-decorated liposomes will not only enhance APCs targeting but also enable preferential delivery of their payload to discrete subcellular compartments. PMID:25701806

  16. Autoreactive natural killer T cells: promoting immune protection and immune tolerance through varied interactions with myeloid antigen-presenting cells

    PubMed Central

    Hegde, Subramanya; Fox, Lisa; Wang, Xiaohua; Gumperz, Jenny E

    2010-01-01

    Natural killer T (NKT) cells are innate T lymphocytes that are restricted by CD1d antigen-presenting molecules and recognize lipids and glycolipids as antigens. NKT cells have attracted attention for their potent immunoregulatory effects. Like other types of regulatory lymphocytes, a high proportion of NKT cells appear to be autoreactive to self antigens. Thus, as myeloid antigen-presenting cells (APCs) such as monocytes, dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) constitutively express CD1d, NKT cells are able to interact with these APCs not only during times of immune activation but also in immunologically quiescent periods. The interactions of NKT cells with myeloid APCs can have either pro-inflammatory or tolerizing outcomes, and a central question is how the ensuing response is determined. Here we bring together published results from a variety of model systems to highlight three critical factors that influence the outcome of the NKT–APC interaction: (i) the strength of the antigenic signal delivered to the NKT cell, as determined by antigen abundance and/or T-cell receptor (TCR) affinity; (ii) the presence or absence of cytokines that costimulate NKT cells [e.g. interleukin (IL)-12, IL-18 and interferon (IFN)-α]; (iii) APC intrinsic factors such as differentiation state (e.g. monocyte versus DC) and Toll-like receptor (TLR) stimulation. Together with recent findings that demonstrate new links between NKT cell activation and endogenous lipid metabolism, these results outline a picture in which the functions of NKT cells are closely attuned to the existing biological context. Thus, NKT cells may actively promote tolerance until a critical level of danger signals arises, at which point they switch to activating pro-inflammatory immune responses. PMID:20465577

  17. Effects of topical corticosteroid therapy on Langerhans cell antigen presenting function in human skin.

    PubMed

    Ashworth, J; Booker, J; Breathnach, S M

    1988-04-01

    We have investigated the mechanisms by which topical corticosteroids modulate cutaneous immune reactions in man. Volunteers applied clobetasone butyrate 0.05% (Eumovate; EV), betamethasone valerate 0.1% (Betnovate; BV), clobetasol propionate 0.05% (Dermovate; DV), and control vehicles twice daily to forearm skin for 7 days. Steroid therapy significantly decreased the number of HLA-DR/T6 (CD1a) positive Langerhans cells (LCs) per mm2 in suction blister-derived epidermal sheets, expressed as a mean percentage of controls, as follows: EV 69.2%; BV 67.3%; DV 37.8%. LC antigen presenting capacity was determined in the allogeneic and autologous epidermal cell-lymphocyte reactions. The LC-dependent allostimulatory capacity of epidermal cells, expressed as a mean percentage of controls, was also significantly reduced by steroid therapy: EV 45.1%; BV 41.9%; DV 23.4%. Following therapy with clobetasol propionate 0.05%, the capacity of epidermal cells to present tetanus toxoid to, and to augment concanavalin A mediated lymphocyte stimulation of, autologous lymphocytes was reduced to 33.6% and 19.7% respectively of controls. Depression of epidermal cell allostimulatory capacity was not the result of a steroid-induced decrease in the production of epidermal cell-derived thymocyte activating factor (ETAF)/interleukin 1 by keratinocytes, since it could not be reversed by addition of exogenous interleukin 1. Indomethacin, added to block any potential prostaglandin synthesis during the culture period, did not restore the allostimulatory capacity of epidermal cells from steroid-treated sites. Addition of epidermal cells from DV-treated sites depressed the capacity of control epidermal cells to stimulate lymphocytes in the allogeneic epidermal-lymphocyte reaction. Our results demonstrate that the anti-inflammatory action of topical corticosteroids in man is associated not only with a reduction in the number of HLA-DR/T6 positive LCs, but also with a marked decrease in Langerhans cell

  18. Antigenically Modified Human Pluripotent Stem Cells Generate Antigen-Presenting Dendritic Cells

    PubMed Central

    Zeng, Jieming; Wu, Chunxiao; Wang, Shu

    2015-01-01

    Human pluripotent stem cells (hPSCs) provide a promising platform to produce dendritic cell (DC) vaccine. To streamline the production process, we investigated a unique antigen-loading strategy that suits this novel platform. Specifically, we stably modified hPSCs using tumour antigen genes in the form of a full-length tumour antigen gene or an artificial tumour antigen epitope-coding minigene. Such antigenically modified hPSCs were able to differentiate into tumour antigen-presenting DCs. Without conventional antigen-loading, DCs derived from the minigene-modified hPSCs were ready to prime a tumour antigen-specific T cell response and further expand these specific T cells in restimulation processes. These expanded tumour antigen-specific T cells were potent effectors with central memory or effector memory phenotype. Thus, we demonstrated that immunocompetent tumour antigen-loaded DCs can be directly generated from antigenically modified hPSCs. Using such strategy, we can completely eliminate the conventional antigen-loading step and significantly simplify the production of DC vaccine from hPSCs. PMID:26471005

  19. The effect of CpG-ODN on antigen presenting cells of the foal

    PubMed Central

    Flaminio, M Julia BF; Borges, Alexandre S; Nydam, Daryl V; Horohov, David W; Hecker, Rolf; Matychak, Mary Beth

    2007-01-01

    Background Cytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN) has been used successfully to induce immune responses against viral and intracellular organisms in mammals. The main objective of this study was to test the effect of CpG-ODN on antigen presenting cells of young foals. Methods Peripheral blood monocytes of foals (n = 7) were isolated in the first day of life and monthly thereafter up to 3 months of life. Adult horse (n = 7) monocytes were isolated and tested once for comparison. Isolated monocytes were stimulated with IL-4 and GM-CSF (to obtain dendritic cells, DC) or not stimulated (to obtain macrophages). Macrophages and DCs were stimulated for 14–16 hours with either CpG-ODN, LPS or not stimulated. The stimulated and non-stimulated cells were tested for cell surface markers (CD86 and MHC class II) using flow cytometry, mRNA expression of cytokines (IL-12, IFNα, IL-10) and TLR-9 using real time quantitative RT-PCR, and for the activation of the transcription factor NF-κB p65 using a chemiluminescence assay. Results The median fluorescence of the MHC class II molecule in non-stimulated foal macrophages and DCs at birth were 12.5 times and 11.2 times inferior, respectively, than adult horse cells (p = 0.009). That difference subsided at 3 months of life (p = 0.3). The expression of the CD86 co-stimulatory molecule was comparable in adult horse and foal macrophages and DCs, independent of treatment. CpG-ODN stimulation induced IL-12p40 (53 times) and IFNα (23 times) mRNA expression in CpG-ODN-treated adult horse DCs (p = 0.078), but not macrophages, in comparison to non-stimulated cells. In contrast, foal APCs did not respond to CpG-ODN stimulation with increased cytokine mRNA expression up to 3 months of age. TLR-9 mRNA expression and NF-kB activation (NF-kB p65) in foal DCs and macrophages were comparable (p > 0.05) to adult horse cells. Conclusion CpG-ODN treatment did not induce specific maturation and cytokine expression in foal

  20. Antigen presenting cells in situ: their identification and involvement in immunopathology.

    PubMed Central

    Poulter, L W

    1983-01-01

    Macrophages and other dendritic non-lymphoid cells have been shown to be functionally capable of presenting antigen to induce lymphocyte responses. These cells can now be studied in situ and distinguished, one from another, within normal tissues and sites of cellular infiltration. Analysis of the microenvironment within which these cells are found can be made with immunohistological methods using monoclonal antibodies (McAbs) and cytochemical techniques. In some cases McAbs are specific for particular types of antigen presenting cell. Using such reagents, evidence is accumulating that these cells may be intimately involved in the pathogenesis of immunoregulatory disorders. What is now required is a more definitive correlation between functional capacity and cell phenotype established with cells isolated from blood, and from normal and pathological tissues. If this is possible the immunopathologist may be able, not only to analyse complex microenvironments but also directly determine the interactions and mechanisms at play within the diseased tissues. PMID:6352095

  1. The proteasome activator 11 S REG (PA28) and class I antigen presentation.

    PubMed Central

    Rechsteiner, M; Realini, C; Ustrell, V

    2000-01-01

    There are two immune responses in vertebrates: humoral immunity is mediated by circulating antibodies, whereas cytotoxic T lymphocytes (CTL) confer cellular immunity. CTL lyse infected cells upon recognition of cell-surface MHC Class I molecules complexed with foreign peptides. The displayed peptides are produced in the cytosol by degradation of host proteins or proteins from intracellular pathogens that might be present. Proteasomes are cylindrical multisubunit proteases that generate many of the peptides eventually transferred to the cell surface for immune surveillance. In mammalian proteasomes, six active sites face a central chamber. As this chamber is sealed off from the enzyme's surface, there must be mechanisms to promote entry of substrates. Two protein complexes have been found to bind the ends of the proteasome and activate it. One of the activators is the 19 S regulatory complex of the 26 S proteasome; the other activator is '11 S REG' [Dubiel, Pratt, Ferrell and Rechsteiner (1992) J. Biol. Chem. 267, 22369-22377] or 'PA28' [Ma, Slaughter and DeMartino (1992) J. Biol. Chem. 267, 10515-10523]. During the past 7 years, our understanding of the structure of REG molecules has increased significantly, but much less is known about their biological functions. There are three REG subunits, namely alpha, beta and gamma. Recombinant REGalpha forms a ring-shaped heptamer of known crystal structure. 11 S REG is a heteroheptamer of alpha and beta subunits. REGgamma is also presumably a heptameric ring, and it is found in the nuclei of the nematode work Caenorhabditis elegans and higher organisms, where it may couple proteasomes to other nuclear components. REGalpha and REGbeta, which are abundant in vertebrate immune tissues, are located mostly in the cytoplasm. Synthesis of REG alpha and beta subunits is induced by interferon-gamma, and this has led to the prevalent hypothesis that REG alpha/beta hetero-oligomers play an important role in Class I antigen

  2. Binding of recombinant T cell receptor ligands (RTL) to antigen presenting cells prevents upregulation of CD11b and inhibits T cell activation and transfer of experimental autoimmune encephalomyelitis.

    PubMed

    Sinha, Sushmita; Miller, Lisa; Subramanian, Sandhya; McCarty, Owen J T; Proctor, Thomas; Meza-Romero, Roberto; Huan, Jianya; Burrows, Gregory G; Vandenbark, Arthur A; Offner, Halina

    2010-08-25

    Recombinant T cell ligands (RTLs) ameliorate experimental autoimmune encephalomyelitis (EAE) in an antigen-specific manner. We evaluated effects of RTL401 (I-A(s) alpha1beta1+PLP-139-151) on splenocytes from SJL/J mice with EAE to study RTL-T cell tolerance-inducing mechanisms. RTLs bound to B, macrophages and DCs, through RTL-MHC-alpha1beta1 moiety. RTL binding reduced CD11b expression on splenic macrophages/DC, and RTL401-conditioned macrophages/DC, not B cells, inhibited T cell activation. Reduced ability of RTL- incubated splenocytes to transfer EAE was likely mediated through macrophages/DC, since B cells were unnecessary for RTL treatment of EAE. These results demonstrate a novel pathway of T cell regulation by RTL-bound APCs. PMID:20546940

  3. Binding of recombinant T cell receptor ligands (RTL) to antigen presenting cells prevents upregulation of CD11b and inhibits T cell activation and transfer of experimental autoimmune encephalomyelitis

    PubMed Central

    Sinha, Sushmita; Miller, Lisa; Subramanian, Sandhya; McCarty, Owen; Proctor, Thomas; Meza-Romero, Roberto; Burrows, Gregory G.; Vandenbark, Arthur A.; Offner, Halina

    2010-01-01

    Recombinant T cell ligands (RTLs) ameliorate experimental autoimmune encephalomyelitis (EAE) in antigen specific manner. We evaluated effects of RTL401 (I-As α1β1 + PLP-139-151) on splenocytes from mice with EAE to study RTL- T cell-tolerance-inducing mechanisms. RTLs bound to B, macrophages and DCs, through RTL-MHC-α1β1 moiety. RTL binding reduced CD11b expression on splenic macrophages/DC, and RTL401-conditioned macrophages/DC, not B cells, inhibited T cell activation. Reduced ability of RTL- incubated splenocytes to transfer EAE was likely mediated through macrophages/DC, since B cells were unnecessary for RTL treatment of EAE. These results demonstrate novel pathway of T cell regulation by RTL bound APCs. PMID:20546940

  4. Pollen-induced antigen presentation by mesenchymal stem cells and T cells from allergic rhinitis.

    PubMed

    Desai, Mauli B; Gavrilova, Tatyana; Liu, Jianjun; Patel, Shyam A; Kartan, Saritha; Greco, Steven J; Capitle, Eugenio; Rameshwar, Pranela

    2013-10-01

    Mesenchymal stem cells (MSCs) are promising cellular suppressor of inflammation. This function of MSCs is partly due to their licensing by inflammatory mediators. In cases with reduced inflammation, MSCs could become immune-enhancer cells. MSCs can suppress the inflammatory response of antigen-challenged lymphocytes from allergic asthma. Although allergic rhinitis (AR) is also an inflammatory response, it is unclear if MSCs can exert similar suppression. This study investigated the immune effects (suppressor vs enhancer) of MSCs on allergen-stimulated lymphocytes from AR subjects (grass or weed allergy). In contrast to subjects with allergic asthma, MSCs caused a significant (P<0.05) increase in the proliferation of antigen-challenged lymphocytes from AR subjects. The increase in lymphocyte proliferation was caused by the MSCs presenting the allergens to CD4(+) T cells (antigen-presenting cells (APCs)). This correlated with increased production of inflammatory cytokines from T cells, and increased expressions of major histocompatibility complex (MHC)-II and CD86 on MSCs. The specificity of APC function was demonstrated in APC assay using MSCs that were knocked down for the master regulator of MHC-II transcription, CIITA. The difference in the effects of MSCs on allergic asthma and AR could not be explained by the sensitivity to the allergen, based on skin tests. Thus, we deduced that the contrasting immune effects of MSCs for antigen-challenged lymphocytes on AR and allergic asthma could be disease specific. It is possible that the enhanced inflammation from asthma might be required to license the MSCs to become suppressor cells. This study underscores the need for robust preclinical studies to effectively translate MSCs for any inflammatory disorder. PMID:25505949

  5. [Use of heat shock of antigen-presenting cells for functional testing of allospecificity memory T-cells].

    PubMed

    Kazanskiĭ, D B; Petrishchev, V N; Shtil', A A; Chernysheva, A D; Sernova, N V; Abronina, I F; Pobezinskiĭ, L A; Agafonova, E L

    1999-02-01

    For many years, the search for the appropriate method of testing the functional activity of the memory T-cells was an urgent problem and determined progress in the study of immunological memory. We proposed simple methods of functional testing the memory of CD8+ T-cells specific to the H-2Kb alloantigen based on measuring their proliferation in response to heat-treated allogenic splenocytes and cells of allogenic tumors in vitro. Primary proliferative response to the alloantigen was shown not to develop when the allogenic antigen-presenting cells were subjected to an acute (45 degrees C, 1 h) or moderate (42 degrees C, 30 min) heat shock. The block of the primary allogenic response of naive T-lymphocytes to the heated splenocytes could not be abrogated by the addition of exogenous IL-2 and was not due to deletion or suppression of antigen-reactive clones. On the contrary, the long-lived memory CD8+ T-cells induced in the course of the primary in vivo response were capable of proliferation in response to heat-treated allogenic stimulators carrying the same immunizing antigen. The different response of the naive T-cells and memory T-cells to the allogenic stimulators subjected to a heat shock might be due to a strict dependence of the naive T-cells on the inducing co-stimulation provided by the B7 ligand, whose expression was suppressed in the cultures containing the heat-treated stimulator cells. These results probably suggest that a specific immunoregulatory mechanism exists that is based on a disorder in costimulatory functions due to the cellular stress-response induced in the antigen-presenting cells. PMID:10495901

  6. HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

    PubMed Central

    Chiu, Yen-Ling; Schneck, Jonathan P.; Oelke, Mathias

    2011-01-01

    CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy

  7. Interleukin-15-Induced CD56+ Myeloid Dendritic Cells Combine Potent Tumor Antigen Presentation with Direct Tumoricidal Potential

    PubMed Central

    Anguille, Sébastien; Lion, Eva; Tel, Jurjen; de Vries, I. Jolanda M; Couderé, Karen; Fromm, Phillip D.; Van Tendeloo, Viggo F.

    2012-01-01

    Dendritic cells (DCs) are the quintessential antigen-presenting cells of the human immune system and play a prime role in coordinating innate and adaptive immune responses, explaining the strong and still growing interest in their application for cancer immunotherapy. Much current research in the field of DC-based immunotherapy focuses on optimizing the culture conditions for in vitro DC generation in order to assure that DCs with the best possible immunogenic qualities are being used for immunotherapy. In this context, monocyte-derived DCs that are alternatively induced by interleukin-15 (IL-15 DCs) have attracted recent attention due to their superior immunostimulatory characteristics. In this study, we show that IL-15 DCs, in addition to potent tumor antigen-presenting function, possess tumoricidal potential and thus qualify for the designation of killer DCs. Notwithstanding marked expression of the natural killer (NK) cell marker CD56 on a subset of IL-15 DCs, we found no evidence of a further phenotypic overlap between IL-15 DCs and NK cells. Allostimulation and antigen presentation assays confirmed that IL-15 DCs should be regarded as bona fide myeloid DCs not only from the phenotypic but also from the functional point of view. Concerning their cytotoxic activity, we demonstrate that IL-15 DCs are able to induce apoptotic cell death of the human K562 tumor cell line, while sparing tumor antigen-specific T cells. The cytotoxicity of IL-15 DCs is predominantly mediated by granzyme B and, to a small extent, by tumor necrosis factor-α (TNF-α)-related apoptosis-inducing ligand (TRAIL) but is independent of perforin, Fas ligand and TNF-α. In conclusion, our data provide evidence of a previously unappreciated role for IL-15 in the differentiation of human monocytes towards killer DCs. The observation that IL-15 DCs have killer DC capacity lends further support to their implementation in DC-based immunotherapy protocols. PMID:23284789

  8. Unique Transcompartmental Bridge: Antigen-Presenting Cells Sampling across Endothelial and Mucosal Barriers

    PubMed Central

    Allen, Frederick; Tong, Alexander A.; Huang, Alex Y.

    2016-01-01

    Potentially harmful pathogens can gain access to tissues and organ systems through body sites that are in direct contact with the outside environment, such as the skin, the gut, and the airway mucosa. Antigen-presenting cells (APCs) represent a bridge between the innate and adaptive immunity, and their capacity for constant immune surveillance and rapid sampling of incoming pathogens and other potentially harmful antigens is central for mounting an effective and robust protective host response. The classical view is that APCs perform this task efficiently within the tissue to sense invading agents intra-compartmentally. However, recent data based on high resolution imaging support an additional transcompartmental surveillance behavior by APC by reaching across intact physical barriers. In this review, we summarize intravital microscopic evidences of APC to sample antigens transcompartmentally at the gut mucosa and other body sites. PMID:27375624

  9. HAM56 and CD68 antigen presenting cells surrounding a sarcoidal granulomatous tattoo

    PubMed Central

    Velez, Ana Maria Abreu; DeJoseph, Louis M.; Howard, Michael S.

    2011-01-01

    Context Tattoos are produced by introducing colorants of various compositions into the skin, either accidentally or for cosmetic purposes. Case Report: A 62-year-old male presented with a cosmetic tattoo and requested a total excision of the lesion. Dermatopathologic analysis of the excised tissue with hematoxylin and eosin examination, as well as immunohistochemistry was performed. H&E staining demonstrated classic histologic features of a tattoo. Utilizing immunohistochemistry, dermal histiocytic antigen presenting cells stained with HAM56 and CD68 antibodies; the staining was present surrounding the tattoo pigment. Conclusions We identified two macrophage markers (HAM56 and CD68) surrounding dermal tattoo pigment. A minimal dermal inflammatory immune was noted to the tattoo pigment. Moreover, the immune response and/or tolerance to tattoos is not well characterized. We suggest that tattoo materials and techniques could be utilized in therapeutic delivery for diseases such recessive dystrophic epidermolysis bullosa, potentially preventing immune rejection of gene therapy agents. PMID:22363088

  10. Network-based gene expression analysis of intracranial aneurysm tissue reveals role of antigen presenting cells.

    PubMed

    Krischek, B; Kasuya, H; Tajima, A; Akagawa, H; Sasaki, T; Yoneyama, T; Ujiie, H; Kubo, O; Bonin, M; Takakura, K; Hori, T; Inoue, I

    2008-07-17

    Little is known about the pathology and pathogenesis of the rupture of intracranial aneurysms. For a better understanding of the molecular processes involved in intracranial aneurysm (IA) formation we performed a gene expression analysis comparing ruptured and unruptured aneurysm tissue to a control artery. Tissue samples of six ruptured and four unruptured aneurysms, and four cerebral arteries serving as controls, were profiled using oligonucleotide microarrays. Gene ontology classification of the differentially expressed genes was analyzed and regulatory functional networks and canonical pathways were identified with a network-based computational pathway analysis tool. Real time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining were performed as confirmation. Analysis of aneurysmal and control tissue revealed 521 differentially expressed genes. The most significantly associated gene ontology term was antigen processing (P=1.64E-16). Further network-based analysis showed the top scoring regulatory functional network to be built around overexpressed major histocompatibility class (MHC) I and II complex related genes and confirmed the canonical pathway "Antigen Presentation" to have the highest upregulation in IA tissue (P=7.3E-10). Real time RT-PCR showed significant overexpression of MHC class II genes. Immunohistochemical staining showed strong positivity for MHC II molecule specific antibody (HLA II), for CD68 (macrophages, monocytes), for CD45RO (T-cells) and HLA I antibody. Our results offer strong evidence for MHC class II gene overexpression in human IA tissue and that antigen presenting cells (macrophages, monocytes) play a key role in IA formation. PMID:18538937

  11. Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity.

    PubMed

    Omokoko, Tana A; Luxemburger, Uli; Bardissi, Shaheer; Simon, Petra; Utsch, Magdalena; Breitkreuz, Andrea; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard (51)Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the (51)Cr release and further confirmed the assay's ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay's combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies. PMID:27057556

  12. Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity

    PubMed Central

    Omokoko, Tana A.; Luxemburger, Uli; Bardissi, Shaheer; Simon, Petra; Utsch, Magdalena; Breitkreuz, Andrea; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard 51Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the 51Cr release and further confirmed the assay's ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay's combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies. PMID:27057556

  13. Antibody-Functionalized Peptidic Membranes for Neutralization of Allogeneic Skin Antigen-Presenting Cells

    PubMed Central

    Wen, Yi; Liu, Wen; Bagia, Christina; Zhang, Shaojuan; Bai, Mingfeng; Janjic, Jelena M.; Giannoukakis, Nick; Gawalt, Ellen S.; Meng, Wilson S.

    2014-01-01

    We report herein application of an in situ material strategy to attenuate allograft T cell responses in a skin transplant mouse model. Functionalized peptidic membranes were used to impede trafficking of donor antigen-presenting cells (dAPCs) from skin allografts in recipient mice. Membranes formed by self-assembling peptides (SAPs) presenting antibodies were found to remain underneath grafted skins for up to 6 days. At the host-graft interface, dAPCs were targeted by using a monoclonal antibody that binds to a class II MHC molecule (IAd) expressed exclusively by donor cells. Using a novel cell labeling near-infrared nanoemulsion, we found more dAPCs remained in allografts treated with membranes loaded with aI-Ad than without. In vitro, dAPCs released from skin explants were found adsorbed preferentially on aI-Ad membranes. Recipient T cells from these mice produced lower concentrations of interferon-gamma cultured ex vivo with donor cells. Taken together, the data indicate that the strategy has the potential to alter the natural course of rejection immune mechanisms in stringent allogeneic models. PMID:25117952

  14. Antigen presenting cell abnormalities in the Cln3(-/-) mouse model of juvenile neuronal ceroid lipofuscinosis.

    PubMed

    Hersrud, Samantha L; Kovács, Attila D; Pearce, David A

    2016-07-01

    Mutations of the CLN3 gene lead to juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive lysosomal storage disorder that causes progressive neurodegeneration in children and adolescents. There is evidence of immune system involvement in pathology that has been only minimally investigated. We characterized bone marrow stem cell-derived antigen presenting cells (APCs), peritoneal macrophages, and leukocytes from spleen and blood, harvested from the Cln3(-/-) mouse model of JNCL. We detected dramatically elevated CD11c surface levels and increased total CD11c protein in Cln3(-/-) cell samples compared to wild type. This phenotype was specific to APCs and also to a loss of CLN3, as surface levels did not differ from wild type in other leukocyte subtypes nor in cells from two other NCL mouse models. Subcellularly, CD11c was localized to lipid rafts, indicating that perturbation of surface levels is attributable to derangement of raft dynamics, which has previously been shown in Cln3 mutant cells. Interrogation of APC function revealed that Cln3(-/-) cells have increased adhesiveness to CD11c ligands as well as an abnormal secretory pattern that closely mimics what has been previously reported for Cln3 mutant microglia. Our results show that CLN3 deficiency alters APCs, which can be a major contributor to the autoimmune response in JNCL. PMID:27101989

  15. Dermal dendrocytes FXIIIa+ are essential antigen-presenting cells in indeterminate leprosy.

    PubMed

    de Alvarenga Lira, Marcia Lanzoni; Pagliari, Carla; de Lima Silva, Aline Alves; de Andrade, Heitor Franco; Duarte, Maria Irma Seixas

    2015-04-01

    Indeterminate leprosy (IL) is the early phase of Hansen disease and reword (APCs). Langerhans cells and dermal dendrocytes FXIIIa positive (DDFXIIIa) are the major APCs in the skin and can be identified by the expression of CD1a and FXIIIa, respectively, by immunohistochemical techniques. Plasmacytoid dendritic cells (PDCs) are another type of dermal dendrocytes with a questionable antigen-presenting function and can be highlighted by anti-CD123 expression. To our knowledge, there are no studies evaluating DDFXIIIa and PDC in IL. The purpose was to investigate the involvement of these cells in the pathogenesis of IL. The authors performed a retrospective study on 18 cases of IL (10 confirmed and 8 suspected) to investigate expression of FXIIIa, CD1a, and CD123. The results were compared with normal skin (for CD1a and FXIIIa only). A higher amount of FXIIIa-positive cells (P , 0.05) in confirmed and suspected IL cases was noted when comparing with normal skin. However, CD1a showed no quantitative differences in the epidermis of IL lesions when comparing with normal skin and CD123 expression was negligible. Based on these findings, the authors postulate that Langerhans cells and PDCs do not have a major role in IL and that DDFXIIIa may be the main APCs in IL. Further study is required to establish this. PMID:25365500

  16. Development of the Nanobody display technology to target lentiviral vectors to antigen-presenting cells

    PubMed Central

    Goyvaerts, C; De Groeve, K; Dingemans, J; Van Lint, S; Robays, L; Heirman, C; Reiser, J; Zhang, X-Y; Thielemans, K; De Baetselier, P; Raes, G; Breckpot, K

    2012-01-01

    Lentiviral vectors (LVs) provide unique opportunities for the development of immunotherapeutic strategies, as they transduce a variety of cells in situ, including antigen-presenting cells (APCs). Engineering LVs to specifically transduce APCs is required to promote their translation towards the clinic. We report on the Nanobody (Nb) display technology to target LVs to dendritic cells (DCs) and macrophages. This innovative approach exploits the budding mechanism of LVs to incorporate an APC-specific Nb and a binding-defective, fusion-competent form of VSV.G in the viral envelope. In addition to production of high titer LVs, we demonstrated selective, Nb-dependent transduction of mouse DCs and macrophages both in vitro and in situ. Moreover, this strategy was translated to a human model in which selective transduction of in vitro generated or lymph node (LN)-derived DCs and macrophages, was demonstrated. In conclusion, the Nb display technology is an attractive approach to generate LVs targeted to specific cell types. PMID:22241177

  17. Fas-mediated elimination of antigen-presenting cells and autoreactive T cells contribute to prevention of autoimmunity

    PubMed Central

    Stranges, Peter. B.; Watson, Jessica; Cooper, Cristie J.; Choisy-Rossi, Caroline-Morgane; Stonebraker, Austin C.; Beighton, Ryan A.; Hartig, Heather; Sundberg, John P.; Servick, Stein; Kaufmann, Gunnar; Fink, Pamela J.; Chervonsky, Alexander V.

    2008-01-01

    Summary Fas (Apo-1, CD95) receptor has been suggested to control T cell expansion by triggering T cell-autonomous apoptosis. This paradigm is based on the extensive lymphoproliferation and systemic autoimmunity in mice and humans lacking Fas or its ligand. However, with systemic loss of Fas, it is unclear whether T cell-extrinsic mechanisms contribute to autoimmunity. We found that tissue-specific deletion of Fas in mouse antigen presenting cells (APC) was sufficient to cause systemic autoimmunity, implying that normally APC are destroyed during immune responses via a Fas-mediated mechanism. Fas expression by APC was increased by exposure to microbial stimuli. Analysis of mice with Fas loss restricted to T cells revealed that Fas indeed controls autoimmune T cells, but not T cells responding to strong antigenic stimulation. Thus, Fas-dependent elimination of APC is a major regulatory mechanism curbing autoimmune responses and acts in concert with Fas-mediated regulation of chronically activated autoimmune T cells. PMID:17509906

  18. Generation of competent bone marrow-derived antigen presenting cells from the deer mouse (Peromyscus maniculatus)

    PubMed Central

    Davenport, Bennett J; Willis, Derall G; Prescott, Joseph; Farrell, Regina M; Coons, Teresa A; Schountz, Tony

    2004-01-01

    Background Human infections with Sin Nombre virus (SNV) and related New World hantaviruses often lead to hantavirus cardiopulmonary syndrome (HCPS), a sometimes fatal illness. Lungs of patients who die from HCPS exhibit cytokine-producing mononuclear infiltrates and pronounced pulmonary inflammation. Deer mice (Peromyscus maniculatus) are the principal natural hosts of SNV, in which the virus establishes life-long persistence without conspicuous pathology. Little is known about the mechanisms SNV employs to evade the immune response of deer mice, and experimental examination of this question has been difficult because of a lack of methodologies for examining such responses during infection. One such deficiency is our inability to characterize T cell responses because susceptible syngeneic deer mice are not available. Results To solve this problem, we have developed an in vitro method of expanding and generating competent antigen presenting cells (APC) from deer mouse bone marrow using commercially-available house mouse (Mus musculus) granulocyte-macrophage colony stimulating factor. These cells are capable of processing and presenting soluble protein to antigen-specific autologous helper T cells in vitro. Inclusion of antigen-specific deer mouse antibody augments T cell stimulation, presumably through Fc receptor-mediated endocytosis. Conclusions The use of these APC has allowed us to dramatically expand deer mouse helper T cells in culture and should permit extensive characterization of T cell epitopes. Considering the evolutionary divergence between deer mice and house mice, it is probable that this method will be useful to other investigators using unconventional models of rodent-borne diseases. PMID:15458574

  19. Aluminum hydroxide adjuvant induces macrophage differentiation towards a specialized antigen-presenting cell type.

    PubMed

    Rimaniol, Anne-Cécile; Gras, Gabriel; Verdier, François; Capel, Francis; Grigoriev, Vladimir B; Porcheray, Fabrice; Sauzeat, Elisabeth; Fournier, Jean-Guy; Clayette, Pascal; Siegrist, Claire-Anne; Dormont, Dominique

    2004-08-13

    Aluminum hydroxide (AlOOH) has been used for many years as a vaccine adjuvant, but little is known about its mechanism of action. We investigated in this study the in vitro effect of aluminum hydroxide adjuvant on isolated macrophages. We showed that AlOOH-stimulated macrophages contain large and persistent intracellular crystalline inclusions, a characteristic property of muscle infiltrated macrophages described in animal models of vaccine injection, as well as in the recently described macrophagic myofasciitis (MMF) histological reaction in humans. AlOOH-loaded macrophages exhibited phenotypical and functional modifications, as they expressed the classical markers of myeloid dendritic cells (HLA-DR(high)/CD86(high)/CD83(+)/CD1a(-)/CD14(-)) and displayed potent ability to induce MHC-II-restricted antigen specific memory responses, but kept a macrophage morphology. This suggests a key role of macrophages, in the reaction to AlOOH-adjuvanted vaccines and these mature antigen-presenting macrophages may therefore be of particular importance in the establishment of memory responses and in vaccination mechanisms leading to long-lasting protection. PMID:15297065

  20. A fusion DNA vaccine that targets antigen-presenting cells increases protection from viral challenge

    NASA Astrophysics Data System (ADS)

    Deliyannis, Georgia; Boyle, Jefferey S.; Brady, Jamie L.; Brown, Lorena E.; Lew, Andrew M.

    2000-06-01

    Improving the immunological potency, particularly the Ab response, is a serious hurdle for the protective efficacy and hence broad application of DNA vaccines. We examined the immunogenicity and protective efficacy of a hemagglutinin-based influenza DNA vaccine that was targeted to antigen-presenting cells (APCs) by fusion to CTLA4. The targeted vaccine was shown to induce an accelerated and increased Ab response (as compared with those receiving the nontargeted control) that was predominated by IgG1 and recognized conformationally dependent viral epitopes. Moreover, mice receiving the APC-targeted DNA vaccine had significantly reduced viral titers (100-fold) after a nonlethal virus challenge. The increased protective efficacy was most likely because of increased Ab responses, as cytotoxic T lymphocyte responses were not enhanced. Targeting was demonstrated by direct binding studies of CTLA4 fusion proteins to the cognate ligand (B7; expressed on APCs in vivo). In addition, a targeted protein was detected at 4-fold higher levels in draining lymph nodes within 2-24 h of administration. Therefore, this study demonstrates that targeting DNA-encoded antigen to APCs results in enhanced immunity and strongly suggests that this approach may be useful in improving the protective efficacy of DNA vaccines.

  1. A fusion DNA vaccine that targets antigen-presenting cells increases protection from viral challenge

    PubMed Central

    Deliyannis, Georgia; Boyle, Jefferey S.; Brady, Jamie L.; Brown, Lorena E.; Lew, Andrew M.

    2000-01-01

    Improving the immunological potency, particularly the Ab response, is a serious hurdle for the protective efficacy and hence broad application of DNA vaccines. We examined the immunogenicity and protective efficacy of a hemagglutinin-based influenza DNA vaccine that was targeted to antigen-presenting cells (APCs) by fusion to CTLA4. The targeted vaccine was shown to induce an accelerated and increased Ab response (as compared with those receiving the nontargeted control) that was predominated by IgG1 and recognized conformationally dependent viral epitopes. Moreover, mice receiving the APC-targeted DNA vaccine had significantly reduced viral titers (100-fold) after a nonlethal virus challenge. The increased protective efficacy was most likely because of increased Ab responses, as cytotoxic T lymphocyte responses were not enhanced. Targeting was demonstrated by direct binding studies of CTLA4 fusion proteins to the cognate ligand (B7; expressed on APCs in vivo). In addition, a targeted protein was detected at 4-fold higher levels in draining lymph nodes within 2–24 h of administration. Therefore, this study demonstrates that targeting DNA-encoded antigen to APCs results in enhanced immunity and strongly suggests that this approach may be useful in improving the protective efficacy of DNA vaccines. PMID:10823919

  2. A Novel Laser Vaccine Adjuvant Increases the Motility of Antigen Presenting Cells

    PubMed Central

    Chen, Xinyuan; Kim, Pilhan; Farinelli, Bill; Doukas, Apostolos; Yun, Seok-Hyun; Gelfand, Jeffrey A.; Anderson, Richard R.; Wu, Mei X.

    2010-01-01

    Background Development of a potent vaccine adjuvant without introduction of any side effects remains an unmet challenge in the field of the vaccine research. Methodology/Principal Findings We found that laser at a specific setting increased the motility of antigen presenting cells (APCs) and immune responses, with few local or systemic side effects. This laser vaccine adjuvant (LVA) effect was induced by brief illumination of a small area of the skin or muscle with a nondestructive, 532 nm green laser prior to intradermal (i.d.) or intramuscular (i.m.) administration of vaccines at the site of laser illumination. The pre-illumination accelerated the motility of APCs as shown by intravital confocal microscopy, leading to sufficient antigen (Ag)-uptake at the site of vaccine injection and transportation of the Ag-captured APCs to the draining lymph nodes. As a result, the number of Ag+ dendritic cells (DCs) in draining lymph nodes was significantly higher in both the 1° and 2° draining lymph nodes in the presence than in the absence of LVA. Laser-mediated increases in the motility and lymphatic transportation of APCs augmented significantly humoral immune responses directed against a model vaccine ovalbumin (OVA) or influenza vaccine i.d. injected in both primary and booster vaccinations as compared to the vaccine itself. Strikingly, when the laser was delivered by a hair-like diffusing optical fiber into muscle, laser illumination greatly boosted not only humoral but also cell-mediated immune responses provoked by i.m. immunization with OVA relative to OVA alone. Conclusion/Significance The results demonstrate the ability of this safe LVA to augment both humoral and cell-mediated immune responses. In comparison with all current vaccine adjuvants that are either chemical compounds or biological agents, LVA is novel in both its form and mechanism; it is risk-free and has distinct advantages over traditional vaccine adjuvants. PMID:21048884

  3. Nano-clustering of ligands on surrogate antigen presenting cells modulates T cell membrane adhesion and organization.

    PubMed

    Dillard, Pierre; Pi, Fuwei; Lellouch, Annemarie C; Limozin, Laurent; Sengupta, Kheya

    2016-03-14

    We investigate the adhesion and molecular organization of the plasma membrane of T lymphocytes interacting with a surrogate antigen presenting cell comprising glass supported ordered arrays of antibody (α-CD3) nano-dots dispersed in a non-adhesive matrix of polyethylene glycol (PEG). The local membrane adhesion and topography, as well as the distribution of the T cell receptors (TCRs) and the kinase ZAP-70, are influenced by dot-geometry, whereas the cell spreading area is determined by the overall average density of the ligands rather than specific characteristics of the dots. TCR clusters are recruited preferentially to the nano-dots and the TCR cluster size distribution has a weak dot-size dependence. On the patterns, the clusters are larger, more numerous, and more enriched in TCRs, as compared to the homogeneously distributed ligands at comparable concentrations. These observations support the idea that non-ligated TCRs residing in the non-adhered parts of the proximal membrane are able to diffuse and enrich the existing clusters at the ligand dots. However, long distance transport is impaired and cluster centralization in the form of a central supramolecular cluster (cSMAC) is not observed. Time-lapse imaging of early cell-surface contacts indicates that the ZAP-70 microclusters are directly recruited to the site of the antibody dots and this process is concomitant with membrane adhesion. These results together point to a complex interplay of adhesion, molecular organization and activation in response to spatially modulated stimulation. PMID:26887857

  4. Evidence of Microbial Translocation Associated with Perturbations in T Cell and Antigen-Presenting Cell Homeostasis in Hookworm Infections

    PubMed Central

    George, Palakkal Jovvian; Anuradha, Rajamanickam; Kumar, Nathella Pavan; Kumaraswami, Vasanthapuram; Nutman, Thomas B.; Babu, Subash

    2012-01-01

    Background Microbial translocation (MT) is the process by which microbes or microbial products translocate from the intestine to the systemic circulation. MT is a common cause of systemic immune activation in HIV infection and is associated with reduced frequencies of CD4+ T cells; no data exist, however, on the role of MT in intestinal helminth infections. Methods We measured the plasma levels of MT markers, acute-phase proteins, and pro- and anti - inflammatory cytokines in individuals with or without hookworm infections. We also estimated the absolute counts of CD4+ and CD8+ T cells as well as the frequencies of memory T cell and dendritic cell subsets. Finally, we also measured the levels of all of these parameters in a subset of individuals following treatment of hookworm infection. Results Our data suggest that hookworm infection is characterized by increased levels of markers associated with MT but not acute-phase proteins nor pro-inflammatory cytokines. Hookworm infections were also associated with increased levels of the anti – inflammatory cytokine – IL-10, which was positively correlated with levels of lipopolysaccharide (LPS). In addition, MT was associated with decreased numbers of CD8+ T cells and diminished frequencies of particular dendritic cell subsets. Antihelmintic treatment of hookworm infection resulted in reversal of some of the hematologic and microbiologic alterations. Conclusions Our data provide compelling evidence for MT in a human intestinal helminth infection and its association with perturbations in the T cell and antigen-presenting cell compartments of the immune system. Our data also reveal that at least one dominant counter-regulatory mechanism i.e. increased IL-10 production might potentially protect against systemic immune activation in hookworm infections. PMID:23056659

  5. Effect of dendritic cell state and antigen-presentation conditions on resulting T-cell phenotypes and Th cytokine profiles.

    PubMed

    de Lastic, Anne-Lise; Rodi, Maria; Mouzaki, Athanasia

    2016-08-01

    T cells play a pivotal role in controlling the immune response and have been the focus of extensive research. We studied the process of in vitro generation of antigen-specific T effector cells (Teffs) to assess the dynamics of antigen presentation and determine the best conditions for cell therapy. We used a peptidic construct consisting of combined HLA class I and II epitopes of the tumor antigen MAGE-3 as an antigen. Monocytes were isolated from healthy donors and were differentiated to dendritic cells (DCs) in vitro. The peptide was added to the DC culture, the pulsed cells were transferred to a co-culture with lymphocytes from the same donor, either as irradiated feeders or untreated, and were cultured in the presence or absence of IL-2. Several rounds of restimulation followed. The cells were analyzed by Flow Cytometry, and cytokine levels were measured by ELISA and Cytometric Bead Array for Th1/Th2/Th17 profiling. The results showed that the lymphocytes in culture upregulated their activation markers and produced Th1 proinflammatory cytokines in response to the peptide, optimally when it was presented by non-irradiated dendritic cells in the presence of IL-2. In contrast, DC irradiation resulted in low activation potential and a shift toward a suppressive phenotype. After prolonged antigenic stimulation, the culture displayed Th17 polarization. In conclusion, the functional integrity of DCs is necessary for the development of antigen-specific Teffs, and culture conditions can be developed to create Teffs with specific properties for eventual use in cell therapy applications. PMID:27130240

  6. Anthrax lethal toxin impairs CD1d-mediated antigen presentation by targeting the extracellular signal-related kinase 1/2 mitogen-activated protein kinase pathway.

    PubMed

    Khan, Masood A; Gallo, Richard M; Brutkiewicz, Randy R

    2010-05-01

    Lethal toxin (LT) is a critical virulence factor of Bacillus anthracis and an important means by which this bacterium evades the host's immune system. In this study, we demonstrate that CD1d-expressing cells treated with LT have reduced CD1d-mediated antigen presentation. We earlier showed an important role for the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 (ERK1/2) in the regulation of CD1d-mediated antigen presentation, and we report here that LT impairs antigen presentation by CD1d in an ERK1/2-dependent manner. Similarly, LT and the ERK1/2 pathway-specific inhibitor U0126 caused a decrease in major histocompatibility complex (MHC) class II-mediated antigen presentation. Confocal microscopy analyses revealed altered intracellular distribution of CD1d and LAMP-1 in LT-treated cells, similar to the case for ERK1/2-inhibited cells. These results suggest that Bacillus anthracis has the ability to evade the host's innate immune system by reducing CD1d-mediated antigen presentation through targeting the ERK1/2 pathway. PMID:20194602

  7. Tolerance is dependent on complement C3 fragment iC3b binding to antigen-presenting cells

    PubMed Central

    Sohn, Jeong-Hyeon; Bora, Puran S.; Suk, Hye-Jung; Molina, Hector; Kaplan, Henry J.; Bora, Nalini S.

    2007-01-01

    Systemic tolerance can be induced by the introduction of antigen into an immune-privileged site. Here we investigated the role of complement in the induction of tolerance after intraocular injection. We found that the development of antigen-specific tolerance is dependent on a complement activation product. The ligation of the complement C3 activation product iC3b to complement receptor type 3 (the iC3b receptor) on antigen-presenting cells resulted in the sequential production of transforming growth factor-β2 and interleukin-10, which is essential for the induction of tolerance. These observations may extend to the development of both neonatal tolerance and other forms of acquired tolerance. PMID:12514742

  8. Characterization of antigen presenting cells and T-cells in progressing scabietic skin lesions.

    PubMed

    Stemmer, B L; Arlian, L G; Morgan, M S; Rapp, C M; Moore, P F

    1996-12-31

    Experimentally infested dogs expressed successful adaptive immunity and self-cured of scabies after previously having scabies that required treatment to cure. A biphasic increase and decrease of CD1a+ Langerhans cells (LCs) in the epidermis of hosts infested the first time (sensitized) and infested a second time (challenged) suggested that these cells were actively involved in the hosts' early immune response to scabies. In contrast, in the dermis CD1a+ cell densities during both infestations increased to a single peak that followed the first peak of these cells in the epidermis. In addition, there was an influx of T-lymphocytes (CD3 epsilon + cells) and CD11c+ cells into the dermis following the first peak of LCs in the epidermis. The influx of T-lymphocytes in the dermis coincided with the peak density of CD1a+ cells in the dermis and epidermis during the second infestation. In both the epidermis and dermis, MHC Class II+ cell density profiles were similar to that of CD1a during the first infestation and then exhibited single peaks during the second infestation. The increases in CD1a+, CD3 epsilon + (T-lymphocytes), CD11c+, and MHC Class II+ cell responses in the dermis occurred earlier and were more intense in the challenge infestation compared with the first infestation. These data indicate that T-lymphocytes (CD3 epsilon +), CD11c+, MHC Class II+, and CD1a+ cells in the dermis played a major role in the successful immune response to scabies mites. PMID:9017872

  9. Immune Tolerance Maintained by Cooperative Interactions between T Cells and Antigen Presenting Cells Shapes a Diverse TCR Repertoire.

    PubMed

    Best, Katharine; Chain, Benny; Watkins, Chris

    2015-01-01

    The T cell population in an individual needs to avoid harmful activation by self peptides while maintaining the ability to respond to an unknown set of foreign peptides. This property is acquired by a combination of thymic and extra-thymic mechanisms. We extend current models for the development of self/non-self discrimination to consider the acquisition of self-tolerance as an emergent system level property of the overall T cell receptor repertoire. We propose that tolerance is established at the level of the antigen presenting cell/T cell cluster, which facilitates and integrates cooperative interactions between T cells of different specificities. The threshold for self-reactivity is therefore imposed at a population level, and not at the level of the individual T cell/antigen encounter. Mathematically, the model can be formulated as a linear programing optimization problem that can be implemented as a multiplicative update algorithm, which shows a rapid convergence to a stable state. The model constrains self-reactivity within a predefined threshold, but maintains repertoire diversity and cross reactivity which are key characteristics of human T cell immunity. We show further that the size of individual clones in the model repertoire becomes heterogeneous, and that new clones can establish themselves even when the repertoire has stabilized. Our study combines the salient features of the "danger" model of self/non-self discrimination with the concepts of quorum sensing, and extends repertoire generation models to encompass the establishment of tolerance. Furthermore, the dynamic and continuous repertoire reshaping, which underlies tolerance in this model, suggests opportunities for therapeutic intervention to achieve long-term tolerance following transplantation. PMID:26300880

  10. Immune Tolerance Maintained by Cooperative Interactions between T Cells and Antigen Presenting Cells Shapes a Diverse TCR Repertoire

    PubMed Central

    Best, Katharine; Chain, Benny; Watkins, Chris

    2015-01-01

    The T cell population in an individual needs to avoid harmful activation by self peptides while maintaining the ability to respond to an unknown set of foreign peptides. This property is acquired by a combination of thymic and extra-thymic mechanisms. We extend current models for the development of self/non-self discrimination to consider the acquisition of self-tolerance as an emergent system level property of the overall T cell receptor repertoire. We propose that tolerance is established at the level of the antigen presenting cell/T cell cluster, which facilitates and integrates cooperative interactions between T cells of different specificities. The threshold for self-reactivity is therefore imposed at a population level, and not at the level of the individual T cell/antigen encounter. Mathematically, the model can be formulated as a linear programing optimization problem that can be implemented as a multiplicative update algorithm, which shows a rapid convergence to a stable state. The model constrains self-reactivity within a predefined threshold, but maintains repertoire diversity and cross reactivity which are key characteristics of human T cell immunity. We show further that the size of individual clones in the model repertoire becomes heterogeneous, and that new clones can establish themselves even when the repertoire has stabilized. Our study combines the salient features of the “danger” model of self/non-self discrimination with the concepts of quorum sensing, and extends repertoire generation models to encompass the establishment of tolerance. Furthermore, the dynamic and continuous repertoire reshaping, which underlies tolerance in this model, suggests opportunities for therapeutic intervention to achieve long-term tolerance following transplantation. PMID:26300880

  11. Inflammatory environment and oxidized LDL convert circulating human proangiogenic cells into functional antigen-presenting cells.

    PubMed

    Vinci, Maria Cristina; Piacentini, Luca; Chiesa, Mattia; Saporiti, Federica; Colombo, Gualtiero I; Pesce, Maurizio

    2015-09-01

    The function of human circulating PACs has been described extensively. However, little focus has been placed on understanding how these cells differ in their functions in the presence of microenvironments mimicking vascular inflammation. We hypothesized that exposure to proinflammatory cytokines or the oxLDL, an autoantigen abundant in advanced atherosclerotic plaques, converts PACs into immune-modulating/proinflammatory cells. Hence, we examined the effect of oxLDL and inflammatory stimuli on their phenotype by use of a functional genomics model based on secretome and whole genome transcriptome profiling. PACs obtained from culturing a PBMC fraction in angiogenic medium were primed with DC differentiation cytokines and then exposed to proinflammatory cytokines or oxLDL. Under these conditions, PACs converted into APCs, expressed maturation markers CD80 and CD83, and showed an increased up-regulation of CD86. APCcy and APCox induced a robust T cell BrdU incorporation. Despite a similar ability to induce lymphocyte proliferation, APCcy and APCox differed for the secretory pathway and mRNA expression. Analysis of the differentially expressed genes identified 4 gene "clusters," showing reciprocal modulation in APCcy vs. APCox, justifying, according to functional genomics analyses, a different putative function of the cells in antigen processing. Together, these data show that treatment with inflammatory cytokines or oxLDL converts human PAC phenotypes and functions into that of APCs with similar lymphocyte-activating ability but distinct maturation degree and paracrine functions. PMID:25990243

  12. Neoantigen Load, Antigen Presentation Machinery, and Immune Signatures Determine Prognosis in Clear Cell Renal Cell Carcinoma.

    PubMed

    Matsushita, Hirokazu; Sato, Yusuke; Karasaki, Takahiro; Nakagawa, Tohru; Kume, Haruki; Ogawa, Seishi; Homma, Yukio; Kakimi, Kazuhiro

    2016-05-01

    Tumors commonly harbor multiple genetic alterations, some of which initiate tumorigenesis. Among these, some tumor-specific somatic mutations resulting in mutated protein have the potential to induce antitumor immune responses. To examine the relevance of the latter to immune responses in the tumor and to patient outcomes, we used datasets of whole-exome and RNA sequencing from 97 clear cell renal cell carcinoma (ccRCC) patients to identify neoepitopes predicted to be presented by each patient's autologous HLA molecules. We found that the number of nonsilent or missense mutations did not correlate with patient prognosis. However, combining the number of HLA-restricted neoepitopes with the cell surface expression of HLA or β2-microglobulin(β2M) revealed that an A-neo(hi)/HLA-A(hi) or ABC-neo(hi)/β2M(hi) phenotype correlated with better clinical outcomes. Higher expression of immune-related genes from CD8 T cells and their effector molecules [CD8A, perforin (PRF1) and granzyme A (GZMA)], however, did not correlate with prognosis. This may have been due to the observed correlation of these genes with the expression of other genes that were associated with immunosuppression in the tumor microenvironment (CTLA-4, PD-1, LAG-3, PD-L1, PD-L2, IDO1, and IL10). This suggested that abundant neoepitopes associated with greater antitumor effector immune responses were counterbalanced by a strongly immunosuppressive microenvironment. Therefore, immunosuppressive molecules should be considered high-priority targets for modulating immune responses in patients with ccRCC. Blockade of these molecular pathways could be combined with immunotherapies targeting neoantigens to achieve synergistic antitumor activity. Cancer Immunol Res; 4(5); 463-71. ©2016 AACR. PMID:26980598

  13. Immunomodulation of Lactobacillus rhamnosus GG (LGG)-derived soluble factors on antigen-presenting cells of healthy blood donors.

    PubMed

    Fong, Fiona Long Yan; Kirjavainen, Pirkka V; El-Nezami, Hani

    2016-01-01

    Lactobacillus rhamnosus GG (LGG) cells have been shown to promote type-1 immune responsiveness; however knowledge of immunomodulation of soluble factors secreted by LGG is limited. This is the first study to investigate whether LGG soluble factors promote a comparable immune responsiveness as the bacterial cells. Both treatments - LGG conditioned medium with (CM + LGG) or without (CM) LGG cells, in this study increased expression of several toll-like receptors (TLRs) in all studied cell types and antigen presentation-associated receptor HLA-DR in macrophages and "intermediate" monocytes; but decreased that of activation markers on monocytes and macrophages and production of IL-10, IL-12 and TNFα in macrophages. In co-culture with mononuclear cells, CM increased Th1-type cytokine profile but not as pronounced as CM + LGG. This study suggests that LGG soluble factors exert similar immunomodulatory effects as the intact cells, but cells may be required for optimal type-1 immune responsiveness polarizing capacity of this probiotic strain. PMID:26961406

  14. Immunomodulation of Lactobacillus rhamnosus GG (LGG)-derived soluble factors on antigen-presenting cells of healthy blood donors

    PubMed Central

    Fong, Fiona Long Yan; Kirjavainen, Pirkka V.; El-Nezami, Hani

    2016-01-01

    Lactobacillus rhamnosus GG (LGG) cells have been shown to promote type-1 immune responsiveness; however knowledge of immunomodulation of soluble factors secreted by LGG is limited. This is the first study to investigate whether LGG soluble factors promote a comparable immune responsiveness as the bacterial cells. Both treatments − LGG conditioned medium with (CM + LGG) or without (CM) LGG cells, in this study increased expression of several toll-like receptors (TLRs) in all studied cell types and antigen presentation-associated receptor HLA-DR in macrophages and “intermediate” monocytes; but decreased that of activation markers on monocytes and macrophages and production of IL-10, IL-12 and TNFα in macrophages. In co-culture with mononuclear cells, CM increased Th1-type cytokine profile but not as pronounced as CM + LGG. This study suggests that LGG soluble factors exert similar immunomodulatory effects as the intact cells, but cells may be required for optimal type-1 immune responsiveness polarizing capacity of this probiotic strain. PMID:26961406

  15. Interleukin-10 Modulates Antigen Presentation by Dendritic Cells through Regulation of NLRP3 Inflammasome Assembly during Chlamydia Infection

    PubMed Central

    Omosun, Yusuf; McKeithen, Danielle; Ryans, Khamia; Kibakaya, Caroline; Blas-Machado, Uriel; Li, Duo; Singh, Rajesh; Inoue, Koichi; Xiong, Zhi-Gang; Eko, Francis; Black, Carolyn; Igietseme, Joseph

    2015-01-01

    Interleukin-10 (IL-10) has been implicated in susceptibility to genital chlamydial infection and the development of tubal pathologies. IL-10 limitation also resulted in the rapid elicitation of immune responses against Chlamydia, and decreased levels of IL-10 correlated with protective anti-Chlamydia immunity. To investigate the molecular basis for these effects, we compared the reproductive pathologies and fertility rates in Chlamydia-infected wild-type (WT) and IL-10-knockout (IL-10−/−) mice; we also analyzed the expression of the Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) superfamily, IL-1β production, NLRP3 inflammasome assembly and activation, and the immunostimulatory capacity and apoptotic predilection of Chlamydia-exposed dendritic cells (DCs) from WT and IL-10−/− mice. Our results revealed that, in addition to the rapid clearance of infection, genitally infected IL-10−/− mice were protected from tubal pathologies and infertility, whereas WT (IL-10+/+) mice were not. Chlamydia-pulsed IL-10−/− DCs expressed larger numbers of TLR4/IL-1R molecules and had enhanced IL-1β production. In addition, NLRP3 inflammasome assembly was suppressed in IL-10−/− DCs through the inhibition of the P2X purinoceptor 7 (P2X7) receptor (P2X7R), an ATP-gated ion channel, and a decrease in intracellular Ca2+ levels, which inhibited DC apoptosis. Thus, the potent immunostimulatory capacity of IL-10-deficient DCs is due, at least in part, to the suppression of the intracellular inflammasome assembly, which prevents DC apoptosis, allowing efficient antigen presentation. The results indicate that IL-10 deficiency enables efficient antigen presentation by DCs for rapid and enhanced immune activation against Chlamydia, which results in rapid microbial clearance, which prevents tubal pathologies during infection. Our finding has important implications for the induction of protective immunity against Chlamydia and other infectious and noninfectious

  16. Interleukin-10 modulates antigen presentation by dendritic cells through regulation of NLRP3 inflammasome assembly during Chlamydia infection.

    PubMed

    Omosun, Yusuf; McKeithen, Danielle; Ryans, Khamia; Kibakaya, Caroline; Blas-Machado, Uriel; Li, Duo; Singh, Rajesh; Inoue, Koichi; Xiong, Zhi-Gang; Eko, Francis; Black, Carolyn; Igietseme, Joseph; He, Qing

    2015-12-01

    Interleukin-10 (IL-10) has been implicated in susceptibility to genital chlamydial infection and the development of tubal pathologies. IL-10 limitation also resulted in the rapid elicitation of immune responses against Chlamydia, and decreased levels of IL-10 correlated with protective anti-Chlamydia immunity. To investigate the molecular basis for these effects, we compared the reproductive pathologies and fertility rates in Chlamydia-infected wild-type (WT) and IL-10-knockout (IL-10(-/-)) mice; we also analyzed the expression of the Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) superfamily, IL-1β production, NLRP3 inflammasome assembly and activation, and the immunostimulatory capacity and apoptotic predilection of Chlamydia-exposed dendritic cells (DCs) from WT and IL-10(-/-) mice. Our results revealed that, in addition to the rapid clearance of infection, genitally infected IL-10(-/-) mice were protected from tubal pathologies and infertility, whereas WT (IL-10(+/+)) mice were not. Chlamydia-pulsed IL-10(-/-) DCs expressed larger numbers of TLR4/IL-1R molecules and had enhanced IL-1β production. In addition, NLRP3 inflammasome assembly was suppressed in IL-10(-/-) DCs through the inhibition of the P2X purinoceptor 7 (P2X7) receptor (P2X7R), an ATP-gated ion channel, and a decrease in intracellular Ca(2+) levels, which inhibited DC apoptosis. Thus, the potent immunostimulatory capacity of IL-10-deficient DCs is due, at least in part, to the suppression of the intracellular inflammasome assembly, which prevents DC apoptosis, allowing efficient antigen presentation. The results indicate that IL-10 deficiency enables efficient antigen presentation by DCs for rapid and enhanced immune activation against Chlamydia, which results in rapid microbial clearance, which prevents tubal pathologies during infection. Our finding has important implications for the induction of protective immunity against Chlamydia and other infectious and noninfectious diseases by

  17. Antigen Presenting Cells and Stromal Cells Trigger Human Natural Killer Lymphocytes to Autoreactivity: Evidence for the Involvement of Natural Cytotoxicity Receptors (NCR) and NKG2D

    PubMed Central

    Poggi, Alessandro; Zocchi, Maria Raffaella

    2006-01-01

    Human natural killer (NK) lymphocytes should not damage autologous cells due to the engagement of inhibitory receptor superfamily (IRS) members by HLA-I. Nevertheless, NK cells kill self cells expressing low levels or lacking HLA-I, as it may occur during viral infections (missing-self hypothesis). Herein, we show that human NK cells can be activated upon binding with self antigen presenting cells or stromal cells despite the expression of HLA-I. Indeed, NK cells can kill and produce pro-inflammatory and regulating cytokines as IFN-γ, TNF-α and IL10 during interaction with autologous dendritic cells or bone marrow stromal cells or skin fibroblasts. The killing of antigen presenting and stromal cells is dependent on LFA1/ICAM1 interaction. Further, the natural cytotoxicity receptors (NCR) NKp30 and NKp46 are responsible for the delivery of lethal hit to DC, whereas NKG2D activating receptor, the ligand of the MHC-related molecule MIC-A and the UL16 binding protein, is involved in stromal cell killing. These findings indicate that different activating receptors are involved in cell to self cell interaction. Finally, NK cells can revert the veto effect of stromal cells on mixed lymphocyte reaction further supporting the idea that NK cells may alter the interaction between T lymphocytes and microenvironment leading to autoreactivity. PMID:17162374

  18. Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4{sup +} intestinal intraepithelial lymphocytes

    SciTech Connect

    Hatano, Ryo; Yamada, Kiyoshi; Iwamoto, Taku; Maeda, Nana; Emoto, Tetsuro; Shimizu, Makoto; Totsuka, Mamoru

    2013-06-14

    Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4{sup +} IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4{sup +} IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4{sup +} IELs. -- Abstract: Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4{sup +} IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4{sup +} LPLs and primed splenic CD4{sup +} T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4{sup +} IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo.

  19. The Effect of the Nonionic Block Copolymer Pluronic P85 on Gene Expression in Mouse Muscle and Antigen Presenting Cells

    PubMed Central

    Gaymalov, Zagit Z.; Yang, Zhihui; Pisarev, Vladimir M.; Alakhov, Valery Yu.; Kabanov, Alexander V.

    2008-01-01

    DNA vaccines can be greatly improved by polymer agents that simultaneously increase transgene expression and activate immunity. We describe here Pluronic P85 (P85), a triblock copolymer of ethylene oxide (EO) and propylene oxide (PO) EO26-PO40-EO26,. Using a mouse model we demonstrate that co-administration of a bacterial plasmid DNA with P85 in a skeletal muscle greatly increases gene expression in the injection site and distant organs, especially the draining lymph nodes and spleen. The reporter expression colocalizes with the specific markers of myocytes and keratinocytes in the muscle, as well as dendritic cells (DC) and macrophages in the muscle, lymph nodes and spleen. Furthermore, DNA/P85 and P85 alone increase the systemic expansion of CD11c+ (DC), and local expansion of CD11c+, CD14+ (macrophages) and CD49b+ (natural killer) cell populations. DNA/P85 (but not P85) also increases maturation of local DC (CD11c+CD86+, CD11c+CD80+, and CD11c+CD40+). We suggest that DNA/P85 promotes the activation and recruitment of the antigen-presenting cells, which further incorporate, express and carry the transgene to the immune system organs. PMID:19064283

  20. A new TLR2 agonist promotes cross-presentation by mouse and human antigen presenting cells

    PubMed Central

    Santone, Melissa; Aprea, Susanna; Wu, Tom Y H; Cooke, Michael P; Mbow, M Lamine; Valiante, Nicholas M; Rush, James S; Dougan, Stephanie; Avalos, Ana; Ploegh, Hidde; De Gregorio, Ennio; Buonsanti, Cecilia; D'Oro, Ugo

    2015-01-01

    Cross-presentation is the process by which professional APCs load peptides from an extracellularly derived protein onto class I MHC molecules to trigger a CD8+ T cell response. The ability to enhance this process is therefore relevant for the development of antitumor and antiviral vaccines. We investigated a new TLR2-based adjuvant, Small Molecule Immune Potentiator (SMIP) 2.1, for its ability to stimulate cross-presentation. Using OVA as model antigen, we demonstrated that a SMIP2.1-adjuvanted vaccine formulation induced a greater CD8+ T cell response, in terms of proliferation, cytokine production and cytolytic activity, than a non-adjuvanted vaccine. Moreover, using an OVA-expressing tumor model, we showed that the CTLs induced by the SMIP2.1 formulated vaccine inhibits tumor growth in vivo. Using a BCR transgenic mouse model we found that B cells could cross-present the OVA antigen when stimulated with SMIP2.1. We also used a flow cytometry assay to detect activation of human CD8+ T cells isolated from human PBMCs of cytomegalovirus-seropositive donors. Stimulation with SMIP2.1 increased the capacity of human APCs, pulsed in vitro with the pp65 CMV protein, to activate CMV-specific CD8+ T cells. Therefore, vaccination with an exogenous antigen formulated with SMIP2.1 is a successful strategy for the induction of a cytotoxic T cell response along with antibody production. PMID:26024409

  1. Molecular basis of mycobacterial lipid antigen presentation by CD1c and its recognition by αβ T cells.

    PubMed

    Roy, Sobhan; Ly, Dalam; Li, Nan-Sheng; Altman, John D; Piccirilli, Joseph A; Moody, D Branch; Adams, Erin J

    2014-10-28

    CD1c is a member of the group 1 CD1 family of proteins that are specialized for lipid antigen presentation. Despite high cell surface expression of CD1c on key antigen-presenting cells and the discovery of its mycobacterial lipid antigen presentation capability, the molecular basis of CD1c recognition by T cells is unknown. Here we present a comprehensive functional and molecular analysis of αβ T-cell receptor (TCR) recognition of CD1c presenting mycobacterial phosphomycoketide antigens. Our structure of CD1c with the mycobacterial phosphomycoketide (PM) shows similarities to that of CD1c-mannosyl-β1-phosphomycoketide in that the A' pocket accommodates the mycoketide alkyl chain; however, the phosphate head-group of PM is shifted ∼6 Å in relation to that of mannosyl-β1-PM. We also demonstrate a bona fide interaction between six human TCRs and CD1c-mycoketide complexes, measuring high to moderate affinities. The crystal structure of the DN6 TCR and mutagenic studies reveal a requirement of five complementarity determining region (CDR) loops for CD1c recognition. Furthermore, mutagenesis of CD1c reveals residues in both the α1 and α2 helices involved in TCR recognition, yet not entirely overlapping among the examined TCRs. Unlike patterns for MHC I, no archetypical binding footprint is predicted to be shared by CD1c-reactive TCRs, even when recognizing the same or similar antigens. PMID:25298532

  2. Molecular basis of mycobacterial lipid antigen presentation by CD1c and its recognition by αβ T cells

    PubMed Central

    Roy, Sobhan; Ly, Dalam; Li, Nan-Sheng; Altman, John D.; Piccirilli, Joseph A.; Moody, D. Branch; Adams, Erin J.

    2014-01-01

    CD1c is a member of the group 1 CD1 family of proteins that are specialized for lipid antigen presentation. Despite high cell surface expression of CD1c on key antigen-presenting cells and the discovery of its mycobacterial lipid antigen presentation capability, the molecular basis of CD1c recognition by T cells is unknown. Here we present a comprehensive functional and molecular analysis of αβ T-cell receptor (TCR) recognition of CD1c presenting mycobacterial phosphomycoketide antigens. Our structure of CD1c with the mycobacterial phosphomycoketide (PM) shows similarities to that of CD1c-mannosyl-β1-phosphomycoketide in that the A' pocket accommodates the mycoketide alkyl chain; however, the phosphate head-group of PM is shifted ∼6 Å in relation to that of mannosyl-β1-PM. We also demonstrate a bona fide interaction between six human TCRs and CD1c-mycoketide complexes, measuring high to moderate affinities. The crystal structure of the DN6 TCR and mutagenic studies reveal a requirement of five complementarity determining region (CDR) loops for CD1c recognition. Furthermore, mutagenesis of CD1c reveals residues in both the α1 and α2 helices involved in TCR recognition, yet not entirely overlapping among the examined TCRs. Unlike patterns for MHC I, no archetypical binding footprint is predicted to be shared by CD1c-reactive TCRs, even when recognizing the same or similar antigens. PMID:25298532

  3. Corruption of dendritic cell antigen presentation during acute GVHD leads to regulatory T-cell failure and chronic GVHD.

    PubMed

    Leveque-El Mouttie, Lucie; Koyama, Motoko; Le Texier, Laetitia; Markey, Kate A; Cheong, Melody; Kuns, Rachel D; Lineburg, Katie E; Teal, Bianca E; Alexander, Kylie A; Clouston, Andrew D; Blazar, Bruce R; Hill, Geoffrey R; MacDonald, Kelli P A

    2016-08-11

    Chronic graft-versus-host disease (cGVHD) is a major cause of late mortality following allogeneic bone marrow transplantation (BMT) and is characterized by tissue fibrosis manifesting as scleroderma and bronchiolitis obliterans. The development of acute GVHD (aGVHD) is a powerful clinical predictor of subsequent cGVHD, suggesting that aGVHD may invoke the immunologic pathways responsible for cGVHD. In preclinical models in which sclerodermatous cGVHD develops after a preceding period of mild aGVHD, we show that antigen presentation within major histocompatibility complex (MHC) class II of donor dendritic cells (DCs) is markedly impaired early after BMT. This is associated with a failure of regulatory T-cell (Treg) homeostasis and cGVHD. Donor DC-restricted deletion of MHC class II phenocopied this Treg deficiency and cGVHD. Moreover, specific depletion of donor Tregs after BMT also induced cGVHD, whereas adoptive transfer of Tregs ameliorated it. These data demonstrate that the defect in Treg homeostasis seen in cGVHD is a causative lesion and is downstream of defective antigen presentation within MHC class II that is induced by aGVHD. PMID:27338097

  4. Cytokines Regulate Proteolysis in Major Histocompatibility Complex Class II–Dependent Antigen Presentation by Dendritic Cells

    PubMed Central

    Fiebiger, Edda; Meraner, Paul; Weber, Ekkehard; Fang, I-Fei; Stingl, Georg; Ploegh, Hidde; Maurer, Dieter

    2001-01-01

    Endo/lysosomal proteases control two key events in antigen (Ag) presentation: the degradation of protein Ag and the generation of peptide-receptive major histocompatibility complex (MHC) class II molecules. Here we show that the proinflammatory cytokines tumor necrosis factor α and interleukin (IL)-1β rapidly increase the activity of cathepsin (cat) S and catB in human dendritic cells (DCs). As a consequence, a wave of MHC class II sodium dodecyl sulfate stable dimer formation ensues in a catS-dependent fashion. In contrast, the antiinflammatory cytokine IL-10 renders DCs incapable of upregulating catS and catB activity and in fact, attenuates the level of both enzymes. Suppressed catS and catB activity delays MHC class II sodium dodecyl sulfate stable dimer formation and impairs Ag degradation. In DCs exposed to tetanus toxoid, IL-10 accordingly reduces the number of MHC class II–peptide complexes accessible to tetanus toxoid–specific T cell receptors, as analyzed by measuring T cell receptor downregulation in Ag-specific T cell clones. Thus, the control of protease activity by pro- and antiinflammatory cytokines is an essential feature of the Ag presentation properties of DCs. PMID:11304549

  5. Rationally designed inhibitor targeting antigen-trimming aminopeptidases enhances antigen presentation and cytotoxic T-cell responses.

    PubMed

    Zervoudi, Efthalia; Saridakis, Emmanuel; Birtley, James R; Seregin, Sergey S; Reeves, Emma; Kokkala, Paraskevi; Aldhamen, Yasser A; Amalfitano, Andrea; Mavridis, Irene M; James, Edward; Georgiadis, Dimitris; Stratikos, Efstratios

    2013-12-01

    Intracellular aminopeptidases endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2), and as well as insulin-regulated aminopeptidase (IRAP) process antigenic epitope precursors for loading onto MHC class I molecules and regulate the adaptive immune response. Their activity greatly affects the antigenic peptide repertoire presented to cytotoxic T lymphocytes and as a result can regulate cytotoxic cellular responses contributing to autoimmunity or immune evasion by viruses and cancer cells. Therefore, pharmacological regulation of their activity is a promising avenue for modulating the adaptive immune response with possible applications in controlling autoimmunity, in boosting immune responses to pathogens, and in cancer immunotherapy. In this study we exploited recent structural and biochemical analysis of ERAP1 and ERAP2 to design and develop phosphinic pseudopeptide transition state analogs that can inhibit this family of enzymes with nM affinity. X-ray crystallographic analysis of one such inhibitor in complex with ERAP2 validated our design, revealing a canonical mode of binding in the active site of the enzyme, and highlighted the importance of the S2' pocket for achieving inhibitor potency. Antigen processing and presentation assays in HeLa and murine colon carcinoma (CT26) cells showed that these inhibitors induce increased cell-surface antigen presentation of transfected and endogenous antigens and enhance cytotoxic T-cell responses, indicating that these enzymes primarily destroy epitopes in those systems. This class of inhibitors constitutes a promising tool for controlling the cellular adaptive immune response in humans by modulating the antigen processing and presentation pathway. PMID:24248368

  6. The HIV-1 gp120/V3 modifies the response of uninfected CD4 T cells to antigen presentation: mapping of the specific transcriptional signature

    PubMed Central

    2011-01-01

    Background The asymptomatic phase of HIV-1 infection is characterized by a progressive depletion of uninfected peripheral effector/memory CD4+ T cells that subsequently leads to immune dysfunction and AIDS symptoms. We have previously demonstrated that the presence of specific gp120/V3 peptides during antigen presentation can modify the activation of normal T-cells leading to altered immune function. The aim of the present study was to map the specific transcriptional profile invoked by an HIV-1/V3 epitope in uninfected T cells during antigen presentation. Methods We exposed primary human peripheral blood monocytes to V3 lipopeptides using a liposome delivery system followed by a superantigen-mediated antigen presentation system. We then evaluated the changes in the T-cell transcriptional profile using oligonucleotide microarrays and performed Ingenuity Pathway Analysis (IPA) and DAVID analysis. The results were validated using realtime PCR, FACS, Western blotting and immunofluorescence. Results Our results revealed that the most highly modulated transcripts could almost entirely be categorized as related to the cell cycle or transcriptional regulation. The most statistically significant enriched categories and networks identified by IPA were associated with cell cycle, gene expression, immune response, infection mechanisms, cellular growth, proliferation and antigen presentation. Canonical pathways involved in energy and cell cycle regulation, and in the co-activation of T cells were also enriched. Conclusions Taken together, these results document a distinct transcriptional profile invoked by the HIV-1/V3 epitope. These data could be invaluable to determine the underlying mechanism by which HIV-1 epitopes interfere with uninfected CD4+ T-cell function causing hyper proliferation and AICD. PMID:21943198

  7. Disruption of HLA class II antigen presentation in Burkitt lymphoma: implication of a 47 000 MW acid labile protein in CD4+ T-cell recognition

    PubMed Central

    God, Jason M; Zhao, Dan; Cameron, Christine A; Amria, Shereen; Bethard, Jennifer R; Haque, Azizul

    2014-01-01

    While Burkitt lymphoma (BL) has a well-known defect in HLA class I-mediated antigen presentation, the exact role of BL-associated HLA class II in generating a poor CD4+ T-cell response remains unresolved. Here, we found that BL cells are deficient in their ability to optimally stimulate CD4+ T cells via the HLA class II pathway. This defect in CD4+ T-cell recognition was not associated with low levels of co-stimulatory molecules on BL cells, as addition of external co-stimulation failed to elicit CD4+ T-cell activation by BL. Further, the defect was not caused by faulty antigen/class II interaction, because antigenic peptides bound with measurable affinity to BL-associated class II molecules. Interestingly, functional class II–peptide complexes were formed at acidic pH 5·5, which restored immune recognition. Acidic buffer (pH 5·5) eluate from BL cells contained molecules that impaired class II-mediated antigen presentation and CD4+ T-cell recognition. Biochemical analysis showed that these molecules were greater than 30 000 molecular weight in size, and proteinaceous in nature. In addition, BL was found to have decreased expression of a 47 000 molecular weight enolase-like molecule that enhances class II-mediated antigen presentation in B cells, macrophages and dendritic cells, but not in BL cells. These findings demonstrate that BL likely has multiple defects in HLA class II-mediated antigen presentation and immune recognition, which may be exploited for future immunotherapies. PMID:24628049

  8. Induction of Cell Signaling Events by the Cholera Toxin B Subunit in Antigen-Presenting Cells▿

    PubMed Central

    Schnitzler, Aletta C.; Burke, Jennifer M.; Wetzler, Lee M.

    2007-01-01

    Cholera toxin (CT) is one of the most effective and widely studied mucosal adjuvants. Although the ADP-ribosylating A subunit has been implicated in augmenting immune responses, the receptor-binding B subunit (CT-B) has greater immunogenicity and may be a repository of adjuvant activity without potential toxicity. In order to elucidate mechanisms of immune modulation by CT-B alone, primary B cells and macrophages were assessed for responses to CT-B in vitro, as measured by the expression of cell surface markers, cellular signaling events, and cytokine secretion. Increased phosphorylation of multiple signaling molecules, including Erk1/2 and p38, was detected. CT-B also induced transactivation of the transcription elements cyclic AMP-responsive element and NF-κB, the latter of which was inhibited by phosphotyrosine inhibition. While specific inhibition of MEK1/2 did not reduce CT-B induction of cell surface marker expression, it did attenuate CT-B-mediated interleukin-6 secretion. These data show that CT-B induces a set of signaling events related to cellular activation, surface molecule expression, and cytokine production that has potential implications for elucidating CT-B adjuvant activity in the absence of enzymatically active holotoxin. PMID:17353279

  9. Peripheral blood antigen presenting cell responses in otitis-prone and non-otitis-prone infants.

    PubMed

    Surendran, Naveen; Nicolosi, Ted; Kaur, Ravinder; Pichichero, Michael E

    2016-01-01

    Stringently defined otitis-prone (sOP) children represent a new classification of the otitis-prone condition. Previous studies showed dysfunction in Ab, B-cell memory and T-cell memory responses. We sought to determine whether there are defects in numbers, phenotype and/or function of professional APC in the peripheral blood of sOP infants. APC phenotypic counts, MHC II expression and intracellular cytokine levels were determined in response to TLR7/8 (R848) stimulation by flow cytometry. Innate immune mRNA expression was measured using RT-PCR and cytokines were measured using Luminex technology. Significant (P < 0.05) increases in the phenotypic counts of monocytes and conventional dendritic cells but not plasmacytoid DCs were observed in sOP compared with non-otitis-prone (NOP) age-matched infants. No significant differences in APC activation or function were observed. Expression of various TLRs, intracellular signaling molecules and downstream cytokines was also not found to be significantly different between sOP and NOP infants. Higher numbers of APCs in sOP infants suggest the possibility of a persistent mucosal inflammatory status. Transcriptional and cytokine profiles of PBMCs among sOP infants suggest their systemic innate responses are not different compared to NOP infants. PMID:26566651

  10. CD80 and CD86 differentially regulate mechanical interactions of T-cells with antigen-presenting dendritic cells and B-cells.

    PubMed

    Lim, Tong Seng; Goh, James Kang Hao; Mortellaro, Alessandra; Lim, Chwee Teck; Hämmerling, Günter J; Ricciardi-Castagnoli, Paola

    2012-01-01

    Functional T-cell responses are initiated by physical interactions between T-cells and antigen-presenting cells (APCs), including dendritic cells (DCs) and B-cells. T-cells are activated more effectively by DCs than by B-cells, but little is known about the key molecular mechanisms that underpin the particular potency of DC in triggering T-cell responses. To better understand the influence of physical intercellular interactions on APC efficacy in activating T-cells, we used single cell force spectroscopy to characterize and compare the mechanical forces of interactions between DC:T-cells and B:T-cells. Following antigen stimulation, intercellular interactions of DC:T-cell conjugates were stronger than B:T-cell interactions. DCs induced higher levels of T-cell calcium mobilization and production of IL-2 and IFNγ than were elicited by B-cells, thus suggesting that tight intercellular contacts are important in providing mechanically stable environment to initiate T-cell activation. Blocking antibodies targeting surface co-stimulatory molecules CD80 or CD86 weakened intercellular interactions and dampen T-cell activation, highlighting the amplificatory roles of CD80/86 in regulating APC:T-cell interactions and T-cell functional activation. The variable strength of mechanical forces between DC:T-cells and B:T-cell interactions were not solely dependent on differential APC expression of CD80/86, since DCs were superior to B-cells in promoting strong interactions with T-cells even when CD80 and CD86 were inhibited. These data provide mechanical insights into the effects of co-stimulatory molecules in regulating APC:T-cell interactions. PMID:23024807

  11. CD80 and CD86 Differentially Regulate Mechanical Interactions of T-Cells with Antigen-Presenting Dendritic Cells and B-Cells

    PubMed Central

    Lim, Tong Seng; Goh, James Kang Hao; Mortellaro, Alessandra; Lim, Chwee Teck; Hämmerling, Günter J.; Ricciardi-Castagnoli, Paola

    2012-01-01

    Functional T-cell responses are initiated by physical interactions between T-cells and antigen-presenting cells (APCs), including dendritic cells (DCs) and B-cells. T-cells are activated more effectively by DCs than by B-cells, but little is known about the key molecular mechanisms that underpin the particular potency of DC in triggering T-cell responses. To better understand the influence of physical intercellular interactions on APC efficacy in activating T-cells, we used single cell force spectroscopy to characterize and compare the mechanical forces of interactions between DC:T-cells and B:T-cells. Following antigen stimulation, intercellular interactions of DC:T-cell conjugates were stronger than B:T-cell interactions. DCs induced higher levels of T-cell calcium mobilization and production of IL-2 and IFNγ than were elicited by B-cells, thus suggesting that tight intercellular contacts are important in providing mechanically stable environment to initiate T-cell activation. Blocking antibodies targeting surface co-stimulatory molecules CD80 or CD86 weakened intercellular interactions and dampen T-cell activation, highlighting the amplificatory roles of CD80/86 in regulating APC:T-cell interactions and T-cell functional activation. The variable strength of mechanical forces between DC:T-cells and B:T-cell interactions were not solely dependent on differential APC expression of CD80/86, since DCs were superior to B-cells in promoting strong interactions with T-cells even when CD80 and CD86 were inhibited. These data provide mechanical insights into the effects of co-stimulatory molecules in regulating APC:T-cell interactions. PMID:23024807

  12. A Lipid Based Antigen Delivery System Efficiently Facilitates MHC Class-I Antigen Presentation in Dendritic Cells to Stimulate CD8+ T Cells

    PubMed Central

    Maji, Mithun; Mazumder, Saumyabrata; Bhattacharya, Souparno; Choudhury, Somsubhra Thakur; Sabur, Abdus; Shadab, Md.; Bhattacharya, Pradyot; Ali, Nahid

    2016-01-01

    The most effective strategy for protection against intracellular infections such as Leishmania is vaccination with live parasites. Use of recombinant proteins avoids the risks associated with live vaccines. However, due to low immunogenicity, they fail to trigger T cell responses particularly of CD8+ cells requisite for persistent immunity. Previously we showed the importance of protein entrapment in cationic liposomes and MPL as adjuvant for elicitation of CD4+ and CD8+ T cell responses for long-term protection. In this study we investigated the role of cationic liposomes on maturation and antigen presentation capacity of dendritic cells (DCs). We observed that cationic liposomes were taken up very efficiently by DCs and transported to different cellular sites. DCs activated with liposomal rgp63 led to efficient presentation of antigen to specific CD4+ and CD8+ T cells. Furthermore, lymphoid CD8+ T cells from liposomal rgp63 immunized mice demonstrated better proliferative ability when co-cultured ex vivo with stimulated DCs. Addition of MPL to vaccine enhanced the antigen presentation by DCs and induced more efficient antigen specific CD8+ T cell responses when compared to free and liposomal antigen. These liposomal formulations presented to CD8+ T cells through TAP-dependent MHC-I pathway offer new possibilities for a safe subunit vaccine. PMID:27251373

  13. A Lipid Based Antigen Delivery System Efficiently Facilitates MHC Class-I Antigen Presentation in Dendritic Cells to Stimulate CD8(+) T Cells.

    PubMed

    Maji, Mithun; Mazumder, Saumyabrata; Bhattacharya, Souparno; Choudhury, Somsubhra Thakur; Sabur, Abdus; Shadab, Md; Bhattacharya, Pradyot; Ali, Nahid

    2016-01-01

    The most effective strategy for protection against intracellular infections such as Leishmania is vaccination with live parasites. Use of recombinant proteins avoids the risks associated with live vaccines. However, due to low immunogenicity, they fail to trigger T cell responses particularly of CD8(+) cells requisite for persistent immunity. Previously we showed the importance of protein entrapment in cationic liposomes and MPL as adjuvant for elicitation of CD4(+) and CD8(+) T cell responses for long-term protection. In this study we investigated the role of cationic liposomes on maturation and antigen presentation capacity of dendritic cells (DCs). We observed that cationic liposomes were taken up very efficiently by DCs and transported to different cellular sites. DCs activated with liposomal rgp63 led to efficient presentation of antigen to specific CD4(+) and CD8(+) T cells. Furthermore, lymphoid CD8(+) T cells from liposomal rgp63 immunized mice demonstrated better proliferative ability when co-cultured ex vivo with stimulated DCs. Addition of MPL to vaccine enhanced the antigen presentation by DCs and induced more efficient antigen specific CD8(+) T cell responses when compared to free and liposomal antigen. These liposomal formulations presented to CD8(+) T cells through TAP-dependent MHC-I pathway offer new possibilities for a safe subunit vaccine. PMID:27251373

  14. A Lipid Based Antigen Delivery System Efficiently Facilitates MHC Class-I Antigen Presentation in Dendritic Cells to Stimulate CD8+ T Cells

    NASA Astrophysics Data System (ADS)

    Maji, Mithun; Mazumder, Saumyabrata; Bhattacharya, Souparno; Choudhury, Somsubhra Thakur; Sabur, Abdus; Shadab, Md.; Bhattacharya, Pradyot; Ali, Nahid

    2016-06-01

    The most effective strategy for protection against intracellular infections such as Leishmania is vaccination with live parasites. Use of recombinant proteins avoids the risks associated with live vaccines. However, due to low immunogenicity, they fail to trigger T cell responses particularly of CD8+ cells requisite for persistent immunity. Previously we showed the importance of protein entrapment in cationic liposomes and MPL as adjuvant for elicitation of CD4+ and CD8+ T cell responses for long-term protection. In this study we investigated the role of cationic liposomes on maturation and antigen presentation capacity of dendritic cells (DCs). We observed that cationic liposomes were taken up very efficiently by DCs and transported to different cellular sites. DCs activated with liposomal rgp63 led to efficient presentation of antigen to specific CD4+ and CD8+ T cells. Furthermore, lymphoid CD8+ T cells from liposomal rgp63 immunized mice demonstrated better proliferative ability when co-cultured ex vivo with stimulated DCs. Addition of MPL to vaccine enhanced the antigen presentation by DCs and induced more efficient antigen specific CD8+ T cell responses when compared to free and liposomal antigen. These liposomal formulations presented to CD8+ T cells through TAP-dependent MHC-I pathway offer new possibilities for a safe subunit vaccine.

  15. Cholera Toxin Promotes Th17 Cell Differentiation by Modulating Expression of Polarizing Cytokines and the Antigen-Presenting Potential of Dendritic Cells

    PubMed Central

    Kang, Jung-Ok; Lee, Jee-Boong

    2016-01-01

    Cholera toxin (CT), an exotoxin produced by Vibrio cholera, acts as a mucosal adjuvant. In a previous study, we showed that CT skews differentiation of CD4 T cells to IL-17-producing Th17 cells. Here, we found that intranasal administration of CT induced migration of migratory dendritic cell (DC) populations, CD103+ DCs and CD11bhi DCs, to the lung draining mediastinal lymph nodes (medLN). Among those DC subsets, CD11bhi DCs that were relatively immature had a major role in Th17 cell differentiation after administration of CT. CT-treated BMDCs showed reduced expression of MHC class II and CD86, similar to CD11bhi DCs in medLN, and these BMDCs promoted Th17 cell differentiation more potently than other BMDCs expressing higher levels of MHC class II and CD86. By analyzing the expression of activation markers such as CD25 and CD69, proliferation and IL-2 production, we determined that CT-treated BMDCs showed diminished antigen-presenting potential to CD4+ T cells compared with normal BMDCs. We also found that CT-stimulated BMDCs promote activin A expression as well as IL-6 and IL-1β, and activin A had a synergic role with TGF-β1 in CT-mediated Th17 cell differentiation. Taken together, our results suggest that CT-stimulated DCs promote Th17 cell differentiation by not only modulating antigen-presenting potential but also inducing Th polarizing cytokines. PMID:27271559

  16. Use of human antigen presenting cell gene array profiling to examine the effect of human T-cell leukemia virus type 1 Tax on primary human dendritic cells.

    PubMed

    Ahuja, Jaya; Kampani, Karan; Datta, Suman; Wigdahl, Brian; Flaig, Katherine E; Jain, Pooja

    2006-02-01

    Human T-cell leukemia virus type 1 (HTLV-1) is etiologically linked to adult T-cell leukemia and a progressive demyelinating disorder termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). One of the most striking features of the immune response in HAM/TSP centers on the expansion of HTLV-1-specific CD8(+) cytotoxic T lymphocyte (CTL) compartment in the peripheral blood and cerebrospinal fluid. More than 90% of the HTLV-1-specific CTLs are directed against the viral Tax (11-19) peptide implying that Tax is available for immune recognition by antigen presenting cells, such as dendritic cells (DCs). DCs obtained from HAM/TSP patients have been shown to be infected with HTLV-1 and exhibit rapid maturation. Therefore, we hypothesized that presentation of Tax peptides by activated DCs to naIve CD8(+) T cells may play an important role in the induction of a Tax-specific CTL response and neurologic dysfunction. In this study, a pathway-specific antigen presenting cell gene array was used to study transcriptional changes induced by exposure of monocyte-derived DCs to extracellular HTLV-1 Tax protein. Approximately 100 genes were differentially expressed including genes encoding toll-like receptors, cell surface receptors, proteins involved in antigen uptake and presentation and adhesion molecules. The differential regulation of chemokines and cytokines characteristic of functional DC activation was also observed by the gene array analyses. Furthermore, the expression pattern of signal transduction genes was also significantly altered. These results have suggested that Tax-mediated DC gene regulation might play a critical role in cellular activation and the mechanisms resulting in HTLV-1-induced disease. PMID:16595374

  17. Synthesis of Dideoxymycobactin Antigens Presented by CD1a Reveals T Cell Fine Specificity for Natural Lipopeptide Structures*

    PubMed Central

    Young, David C.; Kasmar, Anne; Moraski, Garrett; Cheng, Tan-Yun; Walz, Andrew J.; Hu, Jingdan; Xu, Yanping; Endres, Gregory W.; Uzieblo, Adam; Zajonc, Dirk; Costello, Catherine E.; Miller, Marvin J.; Moody, D. Branch

    2009-01-01

    Mycobacterium tuberculosis survival in cells requires mycobactin siderophores. Recently, the search for lipid antigens presented by the CD1a antigen-presenting protein led to the discovery of a mycobactin-like compound, dideoxymycobactin (DDM). Here we synthesize DDMs using solution phase and solid phase peptide synthesis chemistry. Comparison of synthetic standards to natural mycobacterial mycobactins by nuclear magnetic resonance and mass spectrometry allowed identification of an unexpected α-methyl serine unit in natural DDM. This finding further distinguishes these pre-siderophores as foreign compounds distinct from conventional peptides, and we provide evidence that this chemical variation influences the T cell response. One synthetic DDM recapitulated natural structures and potently stimulated T cells, making it suitable for patient studies of CD1a in infectious disease. DDM analogs differing in the stereochemistry of their butyrate or oxazoline moieties were not recognized by human T cells. Therefore, we conclude that T cells show precise specificity for both arms of the peptide, which are predicted to lie at the CD1a-T cell receptor interface. PMID:19605355

  18. Ectopic ATP synthase facilitates transfer of HIV-1 from antigen-presenting cells to CD4+ target cells

    PubMed Central

    Yavlovich, Amichai; Viard, Mathias; Zhou, Ming; Veenstra, Timothy D.; Wang, Ji Ming; Gong, Wanghua; Heldman, Eliahu; Blumenthal, Robert

    2012-01-01

    Antigen-presenting cells (APCs) act as vehicles that transfer HIV to their target CD4+ cells through an intercellular junction, termed the virologic synapse. The molecules that are involved in this process remain largely unidentified. In this study, we used photoaffinity labeling and a proteomic approach to identify new proteins that facilitate HIV-1 transfer. We identified ectopic mitochondrial ATP synthase as a factor that mediates HIV-1 transfer between APCs and CD4+ target cells. Monoclonal antibodies against the β-subunit of ATP synthase inhibited APC-mediated transfer of multiple strains HIV-1 to CD4+ target cells. Likewise, the specific inhibitors of ATPase, citreoviridin and IF1, completely blocked APC-mediated transfer of HIV-1 at the APC-target cell interaction step. Confocal fluorescent microscopy showed localization of extracellular ATP synthase at junctions between APC and CD4+ target cells. We conclude that ectopic ATP synthase could be an accessible molecular target for inhibiting HIV-1 proliferation in vivo. PMID:22753871

  19. Distribution of Primed T Cells and Antigen-Loaded Antigen Presenting Cells Following Intranasal Immunization in Mice

    PubMed Central

    Ciabattini, Annalisa; Pettini, Elena; Fiorino, Fabio; Prota, Gennaro; Pozzi, Gianni; Medaglini, Donata

    2011-01-01

    Priming of T cells is a key event in vaccination, since it bears a decisive influence on the type and magnitude of the immune response. T-cell priming after mucosal immunization via the nasal route was studied by investigating the distribution of antigen-loaded antigen presenting cells (APCs) and primed antigen-specific T cells. Nasal immunization studies were conducted using the model protein antigen ovalbumin (OVA) plus CpG oligodeoxynucleotide adjuvant. Trafficking of antigen-specific primed T cells was analyzed in vivo after adoptive transfer of OVA-specific transgenic T cells in the presence or absence of fingolimod, a drug that causes lymphocytes sequestration within lymph nodes. Antigen-loaded APCs were observed in mediastinal lymph nodes, draining the respiratory tract, but not in distal lymph nodes. Antigen-specific proliferating T cells were first observed within draining lymph nodes, and later in distal iliac and mesenteric lymph nodes and in the spleen. The presence at distal sites was due to migration of locally primed T cells as shown by fingolimod treatment that caused a drastic reduction of proliferated T cells in non-draining lymph nodes and an accumulation of extensively divided T cells within draining lymph nodes. Homing of nasally primed T cells in distal iliac lymph nodes was CD62L-dependent, while entry into mesenteric lymph nodes depended on both CD62L and α4β7, as shown by in vivo antibody-mediated inhibition of T-cell trafficking. These data, elucidating the trafficking of antigen-specific primed T cells to non-draining peripheral and mucosa-associated lymph nodes following nasal immunization, provide relevant insights for the design of vaccination strategies based on mucosal priming. PMID:21559409

  20. Role of the mannose receptor in phagocytosis of Enterococcus faecalis strain EC-12 by antigen-presenting cells

    PubMed Central

    Tsuruta, Takeshi; Inoue, Ryo; Nagino, Takayuki; Nishibayashi, Ryoichiro; Makioka, Yuko; Ushida, Kazunari

    2013-01-01

    Abstract The aim of this study was to clarify the phagocytic mechanisms of a heat-killed cell preparation of Enterococcus faecalis strain EC-12 (EC-12) by antigen-presenting cells (APCs). Fluorescein isothiocyanate (FITC)-labeled EC-12 was cocultured with peritoneal macrophage and the amount of EC-12 phagocytosed by peritoneal macrophages was measured using a microplate fluorometer. Peritoneal macrophages from toll-like receptor (TLR)2-, TLR7-, and MyD88-deficient knockout (KO) mice exhibited similar levels of EC-12 phagocytosis to those from wild-type mice. Similarly, dectin-1 neutralization of peritoneal macrophages had no effect on EC-12 phagocytosis. However, blockade of the mannose receptor (MR) significantly decreased the amount of EC-12 phagocytosed by peritoneal macrophages; the same effect was observed in bone marrow-derived macrophages and dendritic cells. Our findings suggest that MR plays a major role in EC-12 phagocytosis by the APCs. This aim of this study was to clarify the phagocytic mechanisms of a heat-killed cell preparation of Enterococcus faecalis strain EC-12 (EC-12) by antigen-presenting cells (APCs). Our findings suggest that mannose receptor (MR) plays a major role in EC-12 phagocytosis by the APCs. PMID:23801521

  1. T-cell brain infiltration and immature antigen-presenting cells in transgenic models of Alzheimer's disease-like cerebral amyloidosis.

    PubMed

    Ferretti, M T; Merlini, M; Späni, C; Gericke, C; Schweizer, N; Enzmann, G; Engelhardt, B; Kulic, L; Suter, T; Nitsch, R M

    2016-05-01

    Cerebral beta-amyloidosis, one of the pathological hallmarks of Alzheimer's disease (AD), elicits a well-characterised, microglia-mediated local innate immune response. In contrast, it is not clear whether cells of the adaptive immune system, in particular T-cells, react to cerebral amyloidosis in AD. Even though parenchymal T-cells have been described in post-mortem brains of AD patients, it is not known whether infiltrating T-cells are specifically recruited to the extracellular deposits of beta-amyloid, and whether they are locally activated into proliferating, effector cells upon interaction with antigen-presenting cells (APCs). To address these issues we have analysed by confocal microscopy and flow-cytometry the localisation and activation status of both T-cells and APCs in transgenic (tg) mice models of AD-like cerebral amyloidosis. Increased numbers of infiltrating T-cells were found in amyloid-burdened brain regions of tg mice, with concomitant up-regulation of endothelial adhesion molecules ICAM-1 and VCAM-1, compared to non-tg littermates. The infiltrating T-cells in tg brains did not co-localise with amyloid plaques, produced less interferon-gamma than those in controls and did not proliferate locally. Bona-fide dendritic cells were virtually absent from the brain parenchyma of both non-tg and tg mice, and APCs from tg brains showed an immature phenotype, with accumulation of MHC-II in intracellular compartments. These results indicate that cerebral amyloidosis promotes T-cell infiltration but interferes with local antigen presentation and T-cell activation. The inability of the brain immune surveillance to orchestrate a protective immune response to amyloid-beta peptide might contribute to the accumulation of amyloid in the progression of the disease. PMID:26872418

  2. Regulation of Lipid Specific and Vitamin Specific Non-MHC Restricted T Cells by Antigen Presenting Cells and Their Therapeutic Potentials

    PubMed Central

    Salio, Mariolina; Cerundolo, Vincenzo

    2015-01-01

    Since initial reports, more than 25 years ago, that T cells recognize lipids in the context on non-polymorphic CD1 molecules, our understanding of antigen presentation to non-peptide-specific T cell populations has deepened. It is now clear that αβ T cells bearing semi-invariant T cell receptor, as well as subsets of γδ T cells, recognize a variety of self and non-self lipids and contribute to shaping immune responses via cross talk with dendritic cells and B cells. Furthermore, it has been demonstrated that small molecules derived from the microbial riboflavin biosynthetic pathway (vitamin B2) bind monomorphic MR1 molecules and activate mucosal-associated invariant T cells, another population of semi-invariant T cells. Novel insights in the biological relevance of non-peptide-specific T cells have emerged with the development of tetrameric CD1 and MR1 molecules, which has allowed accurate enumeration and functional analysis of CD1- and MR1-restricted T cells in humans and discovery of novel populations of semi-invariant T cells. The phenotype and function of non-peptide-specific T cells will be discussed in the context of the known distribution of CD1 and MR1 molecules by different subsets of antigen-presenting cells at steady state and following infection. Concurrent modulation of CD1 transcription and lipid biosynthetic pathways upon TLR stimulation, coupled with efficient lipid antigen processing, result in the increased cell surface expression of antigenic CD1–lipid complexes. Similarly, MR1 expression is almost undetectable in resting APC and it is upregulated following bacterial infection, likely due to stabilization of MR1 molecules by microbial antigens. The tight regulation of CD1 and MR1 expression at steady state and during infection may represent an important mechanism to limit autoreactivity, while promoting T cell responses to foreign antigens. PMID:26284072

  3. Hepatitis B virus-like particles access major histocompatibility class I and II antigen presentation pathways in primary dendritic cells.

    PubMed

    Moffat, Jessica M; Cheong, Wan-Shoo; Villadangos, José A; Mintern, Justine D; Netter, Hans J

    2013-04-26

    Virus-like particles (VLPs) represent high density displays of viral proteins that efficiently trigger immunity. VLPs composed of the small hepatitis B virus envelope protein (HBsAgS) are useful vaccine platforms that induce humoral and cellular immune responses. Notably, however, some studies suggest HBsAgS VLPs impair dendritic cell (DC) function. Here we investigated HBsAgS VLP interaction with DC subsets and antigen access to major histocompatibility complex (MHC) class I and II antigen presentation pathways in primary DCs. HBsAgS VLPs impaired plasmacytoid DC (pDC) interferon alpha (IFNα) production in response to CpG in vitro, but did not alter conventional DC (cDC) or pDC phenotype when administered in vivo. To assess cellular immune responses, HBsAgS VLPs were generated containing the ovalbumin (OVA) model epitopes OVA(257-264) and OVA(323-339) to access MHCI and MHCII antigen presentation pathways, respectively; both in vitro and following immunisation in vivo. HBsAgS VLP-OVA(257-264) elicited CTL responses in vivo that were not enhanced by inclusion of an additional MHCII helper epitope. HBsAgS VLP-OVA(257-264) administered in vivo was cross-presented by CD8(+) DCs, but not CD8(-) DCs. Therefore, HBsAgS VLPs can deliver antigen to both MHCI and MHCII antigen presentation pathways in primary DCs and promote cytotoxic and helper T cell priming despite their suppressive effect on pDCs. PMID:23473776

  4. Increased generation of Foxp3(+) regulatory T cells by manipulating antigen presentation in the thymus.

    PubMed

    Lin, Jiqiang; Yang, Lu; Silva, Hernandez Moura; Trzeciak, Alissa; Choi, Yongwon; Schwab, Susan R; Dustin, Michael L; Lafaille, Juan J

    2016-01-01

    Regulatory T-cell (Treg) selection in the thymus is essential to prevent autoimmune diseases. Although important rules for Treg selection have been established, there is controversy regarding the degree of self-reactivity displayed by T-cell receptors expressed by Treg cells. In this study we have developed a model of autoimmune skin inflammation, to determine key parameters in the generation of skin-reactive Treg cells in the thymus (tTreg). tTreg development is predominantly AIRE dependent, with an AIRE-independent component. Without the knowledge of antigen recognized by skin-reactive Treg cells, we are able to enhance skin-specific tTreg cell generation using three approaches. First, we increase medullary thymic epithelial cells by using mice lacking osteoprotegerin or by adding TRANCE (RANKL, Tnfsf11). Second, we inject intrathymically peripheral dendritic cells from skin-draining sites. Finally, we inject skin tissue lysates intrathymically. These findings have implications for enhancing the generation of organ-specific Treg cells in autoimmune diseases. PMID:26923114

  5. Increased generation of Foxp3+ regulatory T cells by manipulating antigen presentation in the thymus

    PubMed Central

    Lin, Jiqiang; Yang, Lu; Silva, Hernandez Moura; Trzeciak, Alissa; Choi, Yongwon; Schwab, Susan R.; Dustin, Michael L.; Lafaille, Juan J.

    2016-01-01

    Regulatory T-cell (Treg) selection in the thymus is essential to prevent autoimmune diseases. Although important rules for Treg selection have been established, there is controversy regarding the degree of self-reactivity displayed by T-cell receptors expressed by Treg cells. In this study we have developed a model of autoimmune skin inflammation, to determine key parameters in the generation of skin-reactive Treg cells in the thymus (tTreg). tTreg development is predominantly AIRE dependent, with an AIRE-independent component. Without the knowledge of antigen recognized by skin-reactive Treg cells, we are able to enhance skin-specific tTreg cell generation using three approaches. First, we increase medullary thymic epithelial cells by using mice lacking osteoprotegerin or by adding TRANCE (RANKL, Tnfsf11). Second, we inject intrathymically peripheral dendritic cells from skin-draining sites. Finally, we inject skin tissue lysates intrathymically. These findings have implications for enhancing the generation of organ-specific Treg cells in autoimmune diseases. PMID:26923114

  6. Carbon monoxide impairs mitochondria-dependent endosomal maturation and antigen presentation in dendritic cells.

    PubMed

    Riquelme, Sebastián A; Pogu, Julien; Anegon, Ignacio; Bueno, Susan M; Kalergis, Alexis M

    2015-12-01

    Heme-oxygenase 1 (HO-1) prevents T cell-mediated inflammatory disease by producing carbon monoxide (CO) and impairing DC immunogenicity. However, the cellular mechanisms causing this inhibition are unknown. Here, we show that CO impairs mitochondrial function in DCs by reducing both the mitochondrial membrane potential and ATP production, and resembling the effect of a nonlethal dose of a classical mitochondria uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Moreover, both CO and CCCP reduced cargo transport, endosome-to-lysosome fusion, and antigen processing, dampening the production of peptide-MHC complexes on the surface of DCs. As a result, the inhibition of naive CD4(+) T-cell priming was observed. Furthermore, mitochondrial dysfunction in DCs also significantly reduced CD8(+) T cell-dependent type 1 diabetes onset in vivo. These results showed for the first time that CO interferes with T-cell priming by blocking an unknown mitochondria-dependent antigen-processing pathway in mature DC. Interestingly, other immune functions in DCs such as antigen capture, cytokine secretion, costimulation, and cell survival relied on glycolysis, suggesting that oxidative phosphorylation might only play a key role for the maturation of antigen-containing endosomes. In conclusion, CO produced by HO-1 impairs antigen-dependent inflammation by regulating DC immunogenicity by a mitochondria-dependent mechanism. PMID:26461179

  7. Cytokine profiles and phenotype regulation of antigen presenting cells by genotype-I porcine reproductive and respiratory syndrome virus isolates

    PubMed Central

    2011-01-01

    The present study examined the immunological response of antigen presenting cells (APC) to genotype-I isolates of porcine reproductive and respiratory syndrome virus (PRRSV) infection by analysing the cytokine profile induced and evaluating the changes taking place upon infection on immunologically relevant cell markers (MHCI, MHCII, CD80/86, CD14, CD16, CD163, CD172a, SWC9). Several types of APC were infected with 39 PRRSV isolates. The results show that different isolates were able to induce different patterns of IL-10 and TNF-α. The four possible phenotypes based on the ability to induce IL-10 and/or TNF-α were observed, although different cell types seemed to have different capabilities. In addition, isolates inducing different cytokine-release profiles on APC could induce different expression of cell markers. PMID:21314968

  8. Cytokine profiles and phenotype regulation of antigen presenting cells by genotype-I porcine reproductive and respiratory syndrome virus isolates.

    PubMed

    Gimeno, Mariona; Darwich, Laila; Diaz, Ivan; de la Torre, Eugenia; Pujols, Joan; Martín, Marga; Inumaru, Shigeki; Cano, Esmeralda; Domingo, Mariano; Montoya, Maria; Mateu, Enric

    2011-01-01

    The present study examined the immunological response of antigen presenting cells (APC) to genotype-I isolates of porcine reproductive and respiratory syndrome virus (PRRSV) infection by analysing the cytokine profile induced and evaluating the changes taking place upon infection on immunologically relevant cell markers (MHCI, MHCII, CD80/86, CD14, CD16, CD163, CD172a, SWC9). Several types of APC were infected with 39 PRRSV isolates. The results show that different isolates were able to induce different patterns of IL-10 and TNF-α. The four possible phenotypes based on the ability to induce IL-10 and/or TNF-α were observed, although different cell types seemed to have different capabilities. In addition, isolates inducing different cytokine-release profiles on APC could induce different expression of cell markers. PMID:21314968

  9. A functional recombinant single-chain T cell receptor fragment capable of selectively targeting antigen-presenting cells.

    PubMed

    Epel, Malka; Ellenhorn, Joshua D; Diamond, Don J; Reiter, Yoram

    2002-11-01

    Specificity in the immune system is dictated and regulated by specific recognition of peptide/major histocompatibility complexes (MHC) by the T cell receptor (TCR). Such peptide/MHC complexes are a desirable target for novel approaches in immunotherapy because of their highly restricted fine specificity. Recently a potent anti-human p53 CD8(+) cytotoxic T lymphocyte (CTL) response has been developed in HLA-A2 transgenic mice after immunization with peptides corresponding to HLA-A2 motifs from human p53. An alpha/beta T-cell receptor was cloned from such CTL which exhibited a moderately high affinity to the human p53(149-157) peptide. In this report, we investigated the possibility of using a recombinant tumor-specific TCR for antigen-specific elimination of cells that express the specific MHC-peptide complex. To this end, we constructed a functional single-chain Fv fragment from the cloned TCR and fused it to a very potent cytotoxic molecule, a truncated form of Pseudomonas exotoxin A (PE38). The p53 TCR scFv-P38 fusion protein was generated by in vitro refolding from bacterially-expressed inclusion bodies, and was found to be functional by its ability to bind antigen-presenting cells (APC) which express the specific p53-derived peptide. Moreover, we have shown that the p53-specific TCR scFv-PE38 molecule specifically kills APC in a peptide-dependent manner. These results represent the first time that a TCR-derived recombinant single-chain Fv fragment has been used as a targeting moiety to deliver a cytotoxic effector molecule to cells and has been able to mediate the efficient killing of the particular cell population that expresses the specific MHC/peptide complex. Similarly to antibody-based targeting approaches, TCR with tumor cell specificity represent attractive candidates for generating new, very specific targeting moieties for various modes of cancer immunotherapy. PMID:12384808

  10. Human plasmacytoid dendritic cells efficiently capture HIV-1 envelope glycoproteins via CD4 for antigen presentation.

    PubMed

    Sandgren, Kerrie J; Smed-Sörensen, Anna; Forsell, Mattias N; Soldemo, Martina; Adams, William C; Liang, Frank; Perbeck, Leif; Koup, Richard A; Wyatt, Richard T; Karlsson Hedestam, Gunilla B; Loré, Karin

    2013-07-01

    Advances in HIV-1 vaccine clinical trials and preclinical research indicate that the virus envelope glycoproteins (Env) are likely to be an essential component of a prophylactic vaccine. Efficient Ag uptake and presentation by dendritic cells (DCs) is important for strong CD4(+) Th cell responses and the development of effective humoral immune responses. In this study, we examined the capacity of distinct primary human DC subsets to internalize and present recombinant Env to CD4(+) T cells. Consistent with their specific receptor expression, skin DCs bound and internalized Env via C-type lectin receptors, whereas blood DC subsets, including CD1c(+) myeloid DCs, CD123(+) plasmacytoid DCs (PDCs), and CD141(+) DCs exhibited a restricted repertoire of C-type lectin receptors and relied on CD4 for uptake of Env. Despite a generally poor capacity for Ag uptake compared with myeloid DCs, the high expression of CD4 on PDCs allowed them to bind and internalize Env very efficiently. CD4-mediated uptake delivered Env to EEA1(+) endosomes that progressed to Lamp1(+) and MHC class II(+) lysosomes where internalized Env was degraded rapidly. Finally, all three blood DC subsets were able to internalize an Env-CMV pp65 fusion protein via CD4 and stimulate pp65-specific CD4(+) T cells. Thus, in the in vitro systems described in this paper, CD4-mediated uptake of Env is a functional pathway leading to Ag presentation, and this may therefore be a mechanism used by blood DCs, including PDCs, for generating immune responses to Env-based vaccines. PMID:23729440

  11. A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

    SciTech Connect

    Han, Hui; Peng, Ji-Run; Chen, Peng-Cheng; Gong, Lei; Qiao, Shi-Shi; Wang, Wen-Zhen; Cui, Zhu-Qingqing; Yu, Xin; Wei, Yu-Hua; Leng, Xi-Sheng

    2011-08-05

    Highlights: {yields} Adoptive immunotherapy depends on relevant numbers of cytolytic T lymphocytes. {yields} An ideal artificial APCs system was successfully prepared in vivo. {yields} Controlled release of IL-2 leads to much more T-cell expansion. {yields} This system is better than general cellular APCs on T-cell expansion. -- Abstract: Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

  12. PI3Kδ promotes CD4+ T-cell interactions with antigen-presenting cells by increasing LFA-1 binding to ICAM-1

    PubMed Central

    Garçon, Fabien; Okkenhaug, Klaus

    2016-01-01

    Activation of T lymphocytes by peptide/major histocompatibility complex on antigen-presenting cells (APCs) involves dynamic contacts between the two cells, during which T cells undergo marked morphological changes. These interactions are facilitated by integrins. Activation of the T cells increases the binding of the integrin lymphocyte function-associated antigen 1 (LFA-1) expressed by T cells to intercellular adhesion molecule (ICAM)-1 and ICAM-2 expressed by APCs. The signalling pathways that control integrin affinities are incompletely defined. The phosphoinositide 3-kinases (PI3Ks) generate second-messenger signalling molecules that control cell growth, proliferation, differentiation and trafficking. Here we show that in T cells, PI3Kδ attenuates the activation of Rac1, but sustains the activation of Rap1. Consequently, PI3Kδ increases LFA-1-dependent adhesion to form stable conjugates with APCs. Increased Rap1 activity and LFA-1 adhesion were only in part mediated by the downstream kinase Akt, suggesting the involvement of additional phosphatidylinositol(3,4,5)P3-binding proteins. These results establish a link between PI3K activity, cytoskeletal changes and integrin binding and help explain the impaired T-cell-dependent immune responses in PI3Kδ-deficient mice. PMID:26740009

  13. PI3Kδ promotes CD4(+) T-cell interactions with antigen-presenting cells by increasing LFA-1 binding to ICAM-1.

    PubMed

    Garçon, Fabien; Okkenhaug, Klaus

    2016-05-01

    Activation of T lymphocytes by peptide/major histocompatibility complex on antigen-presenting cells (APCs) involves dynamic contacts between the two cells, during which T cells undergo marked morphological changes. These interactions are facilitated by integrins. Activation of the T cells increases the binding of the integrin lymphocyte function-associated antigen 1 (LFA-1) expressed by T cells to intercellular adhesion molecule (ICAM)-1 and ICAM-2 expressed by APCs. The signalling pathways that control integrin affinities are incompletely defined. The phosphoinositide 3-kinases (PI3Ks) generate second-messenger signalling molecules that control cell growth, proliferation, differentiation and trafficking. Here we show that in T cells, PI3Kδ attenuates the activation of Rac1, but sustains the activation of Rap1. Consequently, PI3Kδ increases LFA-1-dependent adhesion to form stable conjugates with APCs. Increased Rap1 activity and LFA-1 adhesion were only in part mediated by the downstream kinase Akt, suggesting the involvement of additional phosphatidylinositol(3,4,5)P3-binding proteins. These results establish a link between PI3K activity, cytoskeletal changes and integrin binding and help explain the impaired T-cell-dependent immune responses in PI3Kδ-deficient mice. PMID:26740009

  14. The role of islet antigen presenting cells and the presentation of insulin in the initiation of autoimmune diabetes in the NOD mouse.

    PubMed

    Unanue, Emil R; Ferris, Stephen T; Carrero, Javier A

    2016-07-01

    We have been examining antigen presentation and the antigen presenting cells (APCs) in the islets of Langerhans of the non-obese diabetic (NOD) mouse. The purpose is to identify the earliest events that initiate autoimmunity in this confined tissue. Islets normally have a population of macrophages that is distinct from those that inhabit the exocrine pancreas. Also found in NOD islets is a minor population of dendritic cells (DCs) that bear the CD103 integrin. We find close interactions between beta cells and the two APCs that result in the initiation of the autoimmunity. Even under non-inflammatory conditions, beta cells transfer insulin-containing vesicles to the APCs of the islet. This reaction requires live cells and intimate contact. The autoimmune process starts in islets with the entrance of CD4(+) T cells and an increase in the CD103(+) DCs. Mice deficient in the Batf3 transcription factor never develop diabetes due to the absence of the CD103/CD8α lineage of DCs. We hypothesize that the 12-20 peptide of the beta chain of insulin is responsible for activation of the initial CD4(+) T-cell response during diabetogenesis. PMID:27319351

  15. Minimum information about tolerogenic antigen-presenting cells (MITAP): a first step towards reproducibility and standardisation of cellular therapies

    PubMed Central

    Spiering, Rachel; Aguillon, Juan C.; Anderson, Amy E.; Appel, Silke; Benitez-Ribas, Daniel; ten Brinke, Anja; Broere, Femke; Cools, Nathalie; Cuturi, Maria Cristina; Diboll, Julie; Geissler, Edward K.; Giannoukakis, Nick; Gregori, Silvia; van Ham, S. Marieke; Lattimer, Staci; Marshall, Lindsay; Harry, Rachel A.; Hutchinson, James A.; Isaacs, John D.; Joosten, Irma; van Kooten, Cees; Lopez Diaz de Cerio, Ascension; Nikolic, Tatjana; Oral, Haluk Barbaros; Sofronic-Milosavljevic, Ljiljana; Ritter, Thomas; Riquelme, Paloma; Thomson, Angus W.; Trucco, Massimo; Vives-Pi, Marta; Martinez-Caceres, Eva M.

    2016-01-01

    Cellular therapies with tolerogenic antigen-presenting cells (tolAPC) show great promise for the treatment of autoimmune diseases and for the prevention of destructive immune responses after transplantation. The methodologies for generating tolAPC vary greatly between different laboratories, making it difficult to compare data from different studies; thus constituting a major hurdle for the development of standardised tolAPC therapeutic products. Here we describe an initiative by members of the tolAPC field to generate a minimum information model for tolAPC (MITAP), providing a reporting framework that will make differences and similarities between tolAPC products transparent. In this way, MITAP constitutes a first but important step towards the production of standardised and reproducible tolAPC for clinical application.

  16. Human sunlight-induced basal-cell-carcinoma-associated dendritic cells are deficient in T cell co-stimulatory molecules and are impaired as antigen-presenting cells.

    PubMed Central

    Nestle, F. O.; Burg, G.; Fäh, J.; Wrone-Smith, T.; Nickoloff, B. J.

    1997-01-01

    Immune surveillance of skin cancer involves the stimulation of effector T cells by tumor-derived antigens and antigen-presenting cells (APCs). An effective APC must not only display processed antigen in the context of MHC molecules but also express co-stimulatory molecules that are required to fully activate T cells. One of the most common cutaneous neoplasms is basal cell carcinoma. To investigate expression of the co-stimulatory molecules CD80 (B7-1) and CD86 (B7-2) on tumor-associated dendritic cells (TADCs), cryosections from basal cell carcinomas were immunostained. In basal cell carcinomas, only 1 to 2% of intratumor and 5 to 10% of peritumor APCs expressed CD80 or CD86. In contrast, biopsies of immunological/inflammatory dermatoses revealed that 38 to 73% of APCs expressed CD80 and CD86. To further evaluate their phenotype and function, TADCs were isolated from tissue samples of basal cell carcinomas; they were non-adherent to plastic, displayed a typical dendritic morphology, and expressed high levels of major histocompatibility class II molecules on their surface. When TADCs were compared with dendritic cells from blood for presentation of superantigens (staphylococcal enterotoxins A and B) to resting autologous T cells, TADCs were consistently weaker stimulators of T cell proliferation than blood dendritic cells. When analyzed by flow cytometry, TADCs expressed high levels of HLA-DR, but only 5 to 10% co-expressed CD80 or CD86. A 3-day culture in granulocyte/macrophage colony-stimulating factor-containing medium partially reconstituted the TADC expression of CD80 and CD86 as well as their immunostimulatory capacity. Thus, in this common skin cancer, although there are prominent collections of HLA-DR-positive APCs in and around tumor cells, the TADCs are deficient in important co-stimulatory molecules as well as being weak stimulators of T cell proliferation. The paucity of co-stimulatory molecule expression and functional activity of TADCs may explain why

  17. Role of p47phox in Antigen-Presenting Cell-Mediated Regulation of Humoral Immunity in Mice

    PubMed Central

    Vasilevsky, Sam; Liu, Qi; Koontz, Sherry M.; Kastenmayer, Robin; Shea, Katherine; Jackson, Sharon H.

    2011-01-01

    Microbial-induced inflammation is important for eliciting humoral immunity. Genetic defects of NADPH oxidase 2–based proteins interrupt phagocyte superoxide generation and are the basis for the human immunodeficiency chronic granulomatous disease (CGD). Hyperinflammation is also a significant clinical manifestation of CGD. Herein, we evaluated humoral immunity in the phagocyte oxidase p47phox-deficient model of CGD and found that UV-inactivated Streptococcus pneumoniae and Listeria monocytogenes (Lm) elicited higher specific antibody (Ab) titers in p47phox-/- mice than wild-type (WT) mice. Both organisms elicited robust and distinct antigen-presenting cell maturation phenotypes, including IL-12 hypersecretion, and higher major histocompatibility complex II and costimulatory protein expression in Lm-stimulated p47phox-/- dendritic cells (DCs) relative to WT DCs. Furthermore, p47phox-/- DCs pulsed with Lm and adoptively transferred into naïve WT mice elicited Ab titers, whereas Lm-pulsed WT DCs did not elicit these titers. The observed robust p47phox-/- mouse humoral response was recapitulated with live Lm and sustained in vivo in p47phox-/- mice. Notably, anti–serum samples from p47phox-/- mice that survived secondary Lm infection were protective in WT and p47phox-/- mice that were rechallenged with secondary lethal Lm infection. These findings demonstrate a novel benefit of NADPH oxidase 2 deficiency (ie, dependent inflammation in antigen-presenting cell–mediated humoral immunity) and that anti-Lm Ab can be protective in an immunodeficient CGD host. PMID:21641399

  18. Antigen-presenting cells but not lymphocytes in the joint may indicate the cause of reactive arthritis.

    PubMed

    Stagg, A J; Hughes, R A; Keat, A C; Elsley, W A; Knight, S C

    1996-11-01

    T cells and antigen-presenting cells (APC) accumulate in the joint in reactive arthritis and there are reports that the T cells are a population selected for responsiveness to the causative agent. In this work, the latter view is questioned by detailed studies of the antigen specificities of the lymphocytes within the joint (SFMC) and peripheral blood (PBMC) of patients with reactive arthritis triggered by infection with Chlamydia trachomatis. Using a hanging-drop microculture system. SFMC displayed enhanced responses not only to antigens from the triggering organism, but also to other antigens, including PPD and tetanus toxoid, to which the patients were likely to have had prior exposure. No evidence was obtained for a dominant cross-reactive T-cell response to epitopes common to these antigen preparations, confirming the polyclonal nature of the infiltrate. In contrast to the broad specificity of the T-cell infiltrate, two experimental approaches indicated that APC within the joint carried chlamydial antigen. The failure of antigen-bearing APC to interact with T cells at this site may underlie the inability to clear microbial antigen from the joint. PMID:8948293

  19. Self-Antigen Presentation by Keratinocytes in the Inflamed Adult Skin Modulates T-Cell Auto-Reactivity.

    PubMed

    Meister, Michael; Tounsi, Amel; Gaffal, Evelyn; Bald, Tobias; Papatriantafyllou, Maria; Ludwig, Julia; Pougialis, Georg; Bestvater, Felix; Klotz, Luisa; Moldenhauer, Gerhard; Tüting, Thomas; Hämmerling, Günter J; Arnold, Bernd; Oelert, Thilo

    2015-08-01

    Keratinocytes have a pivotal role in the regulation of immune responses, but the impact of antigen presentation by these cells is still poorly understood, particularly in a situation where the antigen will be presented only in adult life. Here, we generated a transgenic mouse model in which keratinocytes exclusively present a myelin basic protein (MBP) peptide covalently linked to the major histocompatibility complex class II β-chain, solely under inflammatory conditions. In these mice, inflammation caused by epicutaneous contact sensitizer treatment resulted in keratinocyte-mediated expansion of MBP-specific CD4(+) T cells in the skin. Moreover, repeated contact sensitizer application preceding a systemic MBP immunization reduced the reactivity of the respective CD4(+) T cells and lowered the symptoms of the resulting experimental autoimmune encephalomyelitis. This downregulation was CD4(+) T-cell-mediated and dependent on the presence of the immune modulator Dickkopf-3. Thus, presentation of a neo self-antigen by keratinocytes in the inflamed, adult skin can modulate CD4(+) T-cell auto-aggression at a distal organ. PMID:25835957

  20. Original Encounter with Antigen Determines Antigen-Presenting Cell Imprinting of the Quality of the Immune Response in Mice

    PubMed Central

    Abadie, Valérie; Bonduelle, Olivia; Duffy, Darragh; Parizot, Christophe; Verrier, Bernard; Combadière, Béhazine

    2009-01-01

    Background Obtaining a certain multi-functionality of cellular immunity for the control of infectious diseases is a burning question in immunology and in vaccine design. Early events, including antigen shuttling to secondary lymphoid organs and recruitment of innate immune cells for adaptive immune response, determine host responsiveness to antigens. However, the sequence of these events and their impact on the quality of the immune response remain to be elucidated. Here, we chose to study Modified Vaccinia virus Ankara (MVA) which is now replacing live Smallpox vaccines and is proposed as an attenuated vector for vaccination strategies against infectious diseases. Methodology/Principal findings We analyzed in vivo mechanisms triggered following intradermal (i.d.) and intramuscular (i.m.) Modified Vaccinia virus Ankara (MVA) administration. We demonstrated significant differences in the antigen shuttling to lymphoid organs by macrophages (MΦs), myeloid dendritic cells (DCs), and neutrophils (PMNs). MVA i.d. administration resulted in better antigen distribution and more sustained antigen-presenting cells (APCs) recruitment into draining lymph nodes than with i.m. administration. These APCs, which comprise both DCs and MΦs, were differentially involved in T cell priming and shaped remarkably the quality of cytokine-producing virus-specific T cells according to the entry route of MVA. Conclusions/Significance This study improves our understanding of the mechanisms of antigen delivery and their consequences on the quality of immune responses and provides new insights for vaccine development. PMID:19997562

  1. The receptor for interleukin-17E is induced by Th2 cytokines in antigen-presenting cells.

    PubMed

    Gratchev, A; Kzhyshkowska, J; Duperrier, K; Utikal, J; Velten, F W; Goerdt, S

    2004-09-01

    Interleukin-17E (IL-17E) (IL-25) is a recently identified cytokine capable to induce Th2-associated cytokine production (IL-5 and IL-13) and T helper 2 (Th2)-type pathologies in animal models. The IL-17E-responsive cell population in vivo was described to be a further uncharacterized non-T-, non-B-splenic accessory cell. Despite the identification of IL-17BR as the receptor for IL-17E, the cell population expressing IL-17BR has hitherto not been identified. Here, we show that human monocyte-derived Th2-skewed antigen-presenting cells (APC2) express membrane-bound and soluble forms of IL-17BR on the mRNA and protein level upon stimulation with IL-4, IL-10, IL-13 or transforming growth factor-betain vitro. These results indicate that IL-17BR-expressing APC2s may mediate the development of the IL-17E-mediated immunological reaction patterns observed in vivo. PMID:15320879

  2. Runx1 Regulates Myeloid Precursor Differentiation Into Osteoclasts Without Affecting Differentiation Into Antigen Presenting or Phagocytic Cells in Both Males and Females.

    PubMed

    Paglia, David N; Yang, Xiaochuan; Kalinowski, Judith; Jastrzebski, Sandra; Drissi, Hicham; Lorenzo, Joseph

    2016-08-01

    Runt-related transcription factor 1 (Runx1), a master regulator of hematopoiesis, is expressed in preosteoclasts. Previously we evaluated the bone phenotype of CD11b-Cre Runx1(fl/fl) mice and demonstrated enhanced osteoclasts and decreased bone mass in males. However, an assessment of the effects of Runx1 deletion in female osteoclast precursors was impossible with this model. Moreover, the role of Runx1 in myeloid cell differentiation into other lineages is unknown. Therefore, we generated LysM-Cre Runx1(fl/fl) mice, which delete Runx1 equally (∼80% deletion) in myeloid precursor cells from both sexes and examined the capacity of these cells to differentiate into osteoclasts and phagocytic and antigen-presenting cells. Both female and male LysM-Cre Runx1(fl/fl) mice had decreased trabecular bone mass (72% decrease in bone volume fraction) and increased osteoclast number (2-3 times) (P < .05) without alteration of osteoblast histomorphometric indices. We also demonstrated that loss of Runx1 in pluripotential myeloid precursors with LysM-Cre did not alter the number of myeloid precursor cells in bone marrow or their ability to differentiate into phagocytizing or antigen-presenting cells. This study demonstrates that abrogation of Runx1 in multipotential myeloid precursor cells significantly and specifically enhanced the ability of receptor activator of nuclear factor-κB ligand to stimulate osteoclast formation and fusion in female and male mice without affecting other myeloid cell fates. In turn, increased osteoclast activity in LysM-Cre Runx1(fl/fl) mice likely contributed to a decrease in bone mass. These dramatic effects were not due to increased osteoclast precursors in the deleted mutants and argue that inhibition of Runx1 in multipotential myeloid precursor cells is important for osteoclast formation and function. PMID:27267711

  3. Neonatal Colonisation Expands a Specific Intestinal Antigen-Presenting Cell Subset Prior to CD4 T-Cell Expansion, without Altering T-Cell Repertoire

    PubMed Central

    Mitchard, Louisa; Harley, Ross; Warwick, James; Burt, Rachel; van Diemen, Pauline M.; Stevens, Mark; Bailey, Mick

    2012-01-01

    Interactions between the early-life colonising intestinal microbiota and the developing immune system are critical in determining the nature of immune responses in later life. Studies in neonatal animals in which this interaction can be examined are central to understanding the mechanisms by which the microbiota impacts on immune development and to developing therapies based on manipulation of the microbiome. The inbred piglet model represents a system that is comparable to human neonates and allows for control of the impact of maternal factors. Here we show that colonisation with a defined microbiota produces expansion of mucosal plasma cells and of T-lymphocytes without altering the repertoire of alpha beta T-cells in the intestine. Importantly, this is preceded by microbially-induced expansion of a signal regulatory protein α-positive (SIRPα+) antigen-presenting cell subset, whilst SIRPα−CD11R1+ antigen-presenting cells (APCs) are unaffected by colonisation. The central role of intestinal APCs in the induction and maintenance of mucosal immunity implicates SIRPα+ antigen-presenting cells as orchestrators of early-life mucosal immune development. PMID:22442714

  4. Selection of restriction specificities of virus-specific cytotoxic T cells in the thymus: no evidence for a crucial role of antigen-presenting cells

    SciTech Connect

    Zinkernagel, R.M.

    1982-12-01

    The proposal was tested that (P1 X P2) F1 leads to P1 irradiation bone marrow chimeras expressed predominantly P1-restricted T cells because donor derived stem cells were exposed to recipient derived antigen-presenting cells in the thymus. Because P1 recipient-derived antigen-presenting cells are replaced only slowly after 6-8 wk by (P1 X P2) donor-derived antigen-presenting cells in the thymus and because replenished pools of mature T cells may by then prevent substantial numbers of P2-restricted T cells to be generated, a large portion of thymus cells and mature T cells were eliminated using the following treatments of 12-20-wk-old (P1 X P2) F1 leads to P1 irradiation bone marrow chimeras: (a) cortisone plus antilymphocyte serum, (b) Cytoxan, (c) three doses of sublethal irradiation (300 rad) 2d apart, and (d) lethal irradiation (850 rad) and reconstitution with T cell-depleted (P1 X P2) F1 stem cells. 12-20 wk after this second treatment, (P1 X P2) leads to P1 chimeras were infected with vaccinia-virus. Virus-specific cytotoxic T cell reactivity was expressed by chimeric T cells of (P1 X P(2) F1 origin and was restricted predominantly to P1. Virus-specific cytotoxic T cells, therefore, do not seem to be selected to measurable extent by the immigrating donor-derived antigen-presenting cells in the thymus; their selection depends apparently from the recipient-derived radioresistant thymus cells.

  5. Dual stimulation of antigen presenting cells using carbon nanotube-based vaccine delivery system for cancer immunotherapy.

    PubMed

    Hassan, Hatem A F M; Smyth, Lesley; Wang, Julie T-W; Costa, Pedro M; Ratnasothy, Kulachelvy; Diebold, Sandra S; Lombardi, Giovanna; Al-Jamal, Khuloud T

    2016-10-01

    Although anti-cancer immuno-based combinatorial therapeutic approaches have shown promising results, efficient tumour eradication demands further intensification of anti-tumour immune response. With the emerging field of nanovaccinology, multi-walled carbon nanotubes (MWNTs) have manifested prominent potentials as tumour antigen nanocarriers. Nevertheless, the utilization of MWNTs in co-delivering antigen along with different types of immunoadjuvants to antigen presenting cells (APCs) has not been investigated yet. We hypothesized that harnessing MWNT for concurrent delivery of cytosine-phosphate-guanine oligodeoxynucleotide (CpG) and anti-CD40 Ig (αCD40), as immunoadjuvants, along with the model antigen ovalbumin (OVA) could potentiate immune response induced against OVA-expressing tumour cells. We initially investigated the effective method to co-deliver OVA and CpG using MWNT to the APC. Covalent conjugation of OVA and CpG prior to loading onto MWNTs markedly augmented the CpG-mediated adjuvanticity, as demonstrated by the significantly increased OVA-specific T cell responses in vitro and in C57BL/6 mice. αCD40 was then included as a second immunoadjuvant to further intensify the immune response. Immune response elicited in vitro and in vivo by OVA, CpG and αCD40 was significantly potentiated by their co-incorporation onto the MWNTs. Furthermore, MWNT remarkably improved the ability of co-loaded OVA, CpG and αCD40 in inhibiting the growth of OVA-expressing B16F10 melanoma cells in subcutaneous or lung pseudo-metastatic tumour models. Therefore, this study suggests that the utilization of MWNTs for the co-delivery of tumour-derived antigen, CpG and αCD40 could be a competent approach for efficient tumours eradication. PMID:27475727

  6. Immunobiotic Lactobacillus rhamnosus strains differentially modulate antiviral immune response in porcine intestinal epithelial and antigen presenting cells

    PubMed Central

    2014-01-01

    Background Previous findings suggested that Lactobacillus rhamnosus CRL1505 is able to increase resistance of children to intestinal viral infections. However, the intestinal cells, cytokines and receptors involved in the immunoregulatory effect of this probiotic strain have not been fully characterized. Results We aimed to gain insight into the mechanisms involved in the immunomodulatory effect of the CRL1505 strain and therefore evaluated in vitro the crosstalk between L. rhamnosus CRL1505, porcine intestinal epithelial cells (IECs) and antigen presenting cells (APCs) from swine Peyer’s patches in order to deepen our knowledge about the mechanisms, through which this strain may help preventing viral diarrhoea episodes. L. rhamnosus CRL1505 was able to induce IFN–α and –β in IECs and improve the production of type I IFNs in response to poly(I:C) challenge independently of Toll-like receptor (TLR)-2 or TLR9 signalling. In addition, the CRL1505 strain induced mRNA expression of IL-6 and TNF-α via TLR2 in IECs. Furthermore, the strain significantly increased surface molecules expression and cytokine production in intestinal APCs. The improved Th1 response induced by L. rhamnosus CRL1505 was triggered by TLR2 signalling and included augmented expression of MHC-II and co-stimulatory molecules and expression of IL-1β, IL-6, and IFN-γ in APCs. IL-10 was also significantly up-regulated by CRL1505 in APCs. Conclusions It was recently reviewed the emergence of TLR agonists as new ways to transform antiviral treatments by introducing panviral therapeutics with less adverse effects than IFN therapies. The use of L. rhamnosus CRL1505 as modulator of innate immunity and inductor of antiviral type I IFNs, IFN-γ, and regulatory IL-10 clearly offers the potential to overcome this challenge. PMID:24886142

  7. Pharmacologic IKK/NF-κB inhibition causes antigen presenting cells to undergo TNFα dependent ROS-mediated programmed cell death

    NASA Astrophysics Data System (ADS)

    Tilstra, Jeremy S.; Gaddy, Daniel F.; Zhao, Jing; Davé, Shaival H.; Niedernhofer, Laura J.; Plevy, Scott E.; Robbins, Paul D.

    2014-01-01

    Monocyte-derived antigen presenting cells (APC) are central mediators of the innate and adaptive immune response in inflammatory diseases. As such, APC are appropriate targets for therapeutic intervention to ameliorate certain diseases. APC differentiation, activation and functions are regulated by the NF-κB family of transcription factors. Herein, we examined the effect of NF-κB inhibition, via suppression of the IκB Kinase (IKK) complex, on APC function. Murine bone marrow-derived macrophages and dendritic cells (DC), as well as macrophage and DC lines, underwent rapid programmed cell death (PCD) after treatment with several IKK/NF-κB inhibitors through a TNFα-dependent mechanism. PCD was induced proximally by reactive oxygen species (ROS) formation, which causes a loss of mitochondrial membrane potential and activation of a caspase signaling cascade. NF-κB-inhibition-induced PCD of APC may be a key mechanism through which therapeutic targeting of NF-κB reduces inflammatory pathologies.

  8. Pharmacologic IKK/NF-κB inhibition causes antigen presenting cells to undergo TNFα dependent ROS-mediated programmed cell death

    PubMed Central

    Tilstra, Jeremy S.; Gaddy, Daniel F.; Zhao, Jing; Davé, Shaival H.; Niedernhofer, Laura J.; Plevy, Scott E.; Robbins, Paul D.

    2014-01-01

    Monocyte-derived antigen presenting cells (APC) are central mediators of the innate and adaptive immune response in inflammatory diseases. As such, APC are appropriate targets for therapeutic intervention to ameliorate certain diseases. APC differentiation, activation and functions are regulated by the NF-κB family of transcription factors. Herein, we examined the effect of NF-κB inhibition, via suppression of the IκB Kinase (IKK) complex, on APC function. Murine bone marrow-derived macrophages and dendritic cells (DC), as well as macrophage and DC lines, underwent rapid programmed cell death (PCD) after treatment with several IKK/NF-κB inhibitors through a TNFα-dependent mechanism. PCD was induced proximally by reactive oxygen species (ROS) formation, which causes a loss of mitochondrial membrane potential and activation of a caspase signaling cascade. NF-κB-inhibition-induced PCD of APC may be a key mechanism through which therapeutic targeting of NF-κB reduces inflammatory pathologies. PMID:24406986

  9. Mycobacterium-Specific γ9δ2 T Cells Mediate Both Pathogen-Inhibitory and CD40 Ligand-Dependent Antigen Presentation Effects Important for Tuberculosis Immunity

    PubMed Central

    Spencer, Charles T.; Hamzabegovic, Fahreta; Blazevic, Azra; Xia, Mei

    2015-01-01

    Numerous pathogens, including Mycobacterium tuberculosis, can activate human γ9δ2 T cells to proliferate and express effector mechanisms. γ9δ2 T cells can directly inhibit the growth of intracellular mycobacteria and may also act as antigen-presenting cells (APC). Despite evidence for γδ T cells having the capacity to function as APC, the mechanisms involved and importance of these effects on overall tuberculosis (TB) immunity are unknown. We prepared M. tuberculosis-specific γ9δ2 T cell lines to study their direct protective effects and APC functions for M. tuberculosis-specific αβ T cells. The direct inhibitory effects on intracellular mycobacteria were measured, and the enhancing effects on proliferative and effector responses of αβ T cells assessed. Furthermore, the importance of cell-to-cell contact and soluble products for γ9δ2 T cell effector responses and APC functions were investigated. We demonstrate, in addition to direct inhibitory effects on intracellular mycobacteria, the following: (i) γ9δ2 T cells enhance the expansion of M. tuberculosis-specific αβ T cells and increase the ability of αβ T cells to inhibit intracellular mycobacteria; (ii) although soluble mediators are critical for the direct inhibitory effects of γ9δ2 T cells, their APC functions do not require soluble mediators; (iii) the APC functions of γ9δ2 T cells involve cell-to-cell contact that is dependent on CD40-CD40 ligand (CD40L) interactions; and (iv) fully activated CD4+ αβ T cells and γ9δ2 T cells provide similar immune enhancing/APC functions for M. tuberculosis-specific T cells. These effector and helper effects of γ9δ2 T cells further indicate that these T cells should be considered important new targets for new TB vaccines. PMID:26644385

  10. Artificial antigen-presenting cells expressing HLA class II molecules as an effective tool for amplifying human specific memory CD4(+) T cells.

    PubMed

    Garnier, Anthony; Hamieh, Mohamad; Drouet, Aurélie; Leprince, Jérôme; Vivien, Denis; Frébourg, Thierry; Le Mauff, Brigitte; Latouche, Jean-Baptiste; Toutirais, Olivier

    2016-08-01

    Owing to their multiple immune functions, CD4(+) T cells are of major interest for immunotherapy in chronic viral infections and cancer, as well as for severe autoimmune diseases and transplantation. Therefore, standardized methods allowing rapid generation of a large number of CD4(+) T cells for adoptive immunotherapy are still awaited. We constructed stable artificial antigen-presenting cells (AAPCs) derived from mouse fibroblasts. They were genetically modified to express human leukocyte antigen (HLA)-DR molecules and the human accessory molecules B7.1, Intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-3 (LFA-3). AAPCs expressing HLA-DR1, HLA-DR15 or HLA-DR51 molecules and loaded with peptides derived from influenza hemagglutinin (HA), myelin basic protein (MBP) or factor VIII, respectively, activated specific CD4(+) T-cell clones more effectively than Epstein-Barr virus (EBV)-transformed B cells. We also showed that AAPCs were able to take up and process whole Ag proteins, and present epitopes to specific T cells. In primary cultures, AAPCs loaded with HA peptide allowed generation of specific Th1 lymphocytes from healthy donors as demonstrated by tetramer and intracellular cytokine staining. Although AAPCs were less effective than autologous peripheral blood mononuclear cells (PBMCs) to stimulate CD4(+) T cells in primary culture, AAPCs were more potent to reactivate and expand memory Th1 cells in a strictly Ag-dependent manner. As the availability of autologous APCs is limited, the AAPC system represents a stable and reliable tool to achieve clinically relevant numbers of CD4(+) T cells for adoptive immunotherapy. For fundamental research in immunology, AAPCs are also useful to decipher mechanisms involved in the development of human CD4 T-cell responses. PMID:26924643

  11. ESAT-6 Targeting to DEC205+ Antigen Presenting Cells Induces Specific-T Cell Responses against ESAT-6 and Reduces Pulmonary Infection with Virulent Mycobacterium tuberculosis

    PubMed Central

    Silva-Sánchez, Aarón; Meza-Pérez, Selene; Flores-Langarica, Adriana; Donis-Maturano, Luis; Estrada-García, Iris; Calderón-Amador, Juana; Hernández-Pando, Rogelio; Idoyaga, Juliana; Flores-Romo, Leopoldo

    2015-01-01

    Airways infection with Mycobacterium tuberculosis (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen’s naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (α-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205+ APCs and to assess its in vivo effects on protection associated responses (IFN-γ production, in vivo CTL killing, and pulmonary mycobacterial load). Treatment with α-DEC-ESAT alone induced ESAT-6-specific IFN-γ producing CD4+ T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-γ+ production by specific T cells in the lungs, compared to scarce IFN-γ production in control mice. In vivo CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, α-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205+ APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-γ production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study in vivo the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions. PMID:25915045

  12. CD8+ Effector T Cell Migration to Pancreatic Islet Grafts Is Dependent on Cognate Antigen Presentation by Donor Graft Cells.

    PubMed

    Zhang, Qianqian; Dai, Hehua; Yatim, Karim M; Abou-Daya, Khodor; Williams, Amanda L; Oberbarnscheidt, Martin H; Camirand, Geoffrey; Rudd, Christopher E; Lakkis, Fadi G

    2016-08-15

    Pancreatic islet transplantation is a promising therapy for diabetes, but acute rejection of the islets by host effector T cells has hindered clinical application. In this study, we addressed the mechanisms of CD8(+) effector T cell migration to islet grafts because interrupting this step is key to preventing rejection. We found that effector T cell migration to revascularized islet transplants in mice is dependent on non-self Ag recognition rather than signaling via Gαi-coupled chemokine receptors. Presentation of non-self Ag by donor cells was necessary for migration, whereas Ag presentation by recipient cells was dispensable. We also observed that deficiency of SKAP1, an immune cell adaptor downstream of the TCR and important for integrin activation, prolongs allograft survival but does not reduce effector T cell migration to the graft. Therefore, effector T cell migration to transplanted islets is Ag driven, not chemokine driven, but SKAP1 does not play a critical role in this process. PMID:27357151

  13. Analysis of chronic rejection and obliterative arteriopathy. Possible contributions of donor antigen-presenting cells and lymphatic disruption.

    PubMed Central

    Demetris, A. J.; Murase, N.; Ye, Q.; Galvao, F. H.; Richert, C.; Saad, R.; Pham, S.; Duquesnoy, R. J.; Zeevi, A.; Fung, J. J.; Starzl, T. E.

    1997-01-01

    hypothesis that direct presentation of alloantigen by donor antigen-presenting cells is required for long-term, chronic-rejection-free allograft acceptance. In addition, chronic intermittent lymphatic disruption is implicated as a possible mechanism for the association between chronic interstitial allograft inflammation and the development of obliterative arteriopathy. Images Figure 1 Figure 3 Figure 6 Figure 7 Figure 9 Figure 10 PMID:9033271

  14. Particle-based transcutaneous administration of HIV-1 p24 protein to human skin explants and targeting of epidermal antigen presenting cells.

    PubMed

    Rancan, Fiorenza; Amselgruber, Sarah; Hadam, Sabrina; Munier, Sevérine; Pavot, Vincent; Verrier, Bernard; Hackbarth, Steffen; Combadiere, Behazine; Blume-Peytavi, Ulrike; Vogt, Annika

    2014-02-28

    Transcutaneous immunization is a promising vaccination strategy for the treatment of infectious diseases and cancer. In this study, we investigate the combination of cyanoacrylate skin surface stripping (CSSS) and particle-based antigen delivery to target the HIV-1 p24 protein to skin antigen presenting cells (APC). The CSSS treatment pre-activates skin APC and opens hair follicles, where protein-loaded particles accumulate and allow for sustained delivery of the loaded antigen to perifollicular APC. We found that poly-lactic acid (PLA) and polystyrene (PS) particles targeted the adsorbed HIV-1 p24 protein to the hair follicles. Small amounts of PS and PLA particles were found to translocate to the epidermis and be internalized by skin cells, whereas most of the particles aggregated in the hair follicle canal, where they released the loaded antigen. The p24 protein diffused to the epidermis and dermis and was detected in skin cells, especially in Langerhans cells and dermal dendritic cells. Furthermore, the combination of CSSS and particle-based delivery resulted in activation and maturation of Langerhans cells (HLA-DR, CD80 and CD83). We conclude that particle-based antigen delivery across partially disrupted skin barrier is a feasible and effective approach to needle-free transcutaneous vaccination. PMID:24384300

  15. The role of a human antigen specific T8+ cell subset in antigen presentation, helper function and contrasuppression.

    PubMed Central

    Lehner, T; Avery, J; Jones, T

    1985-01-01

    Regulation of the human immune response was studied by sequential separation of subsets of T cells, followed by assessment of their helper and suppressor functions in a series of reconstitution experiments. T8+ lymphocytes were separated by panning on streptococcal antigen (SA) coated plates into T8+ SA-adherent cells (T8+SA+) and T8+ SA-non-adherent cells (T8+SA-). The helper and suppressor functions of the T8+SA+ and T8+SA- cells, reconstituted with T4+ helper cells were then studied by a direct antibody forming cell assay. T4+ cells will not induce helper activity by 1000 ng SA alone but require the accessory function of monocytes (Mo). However, replacing Mo by T8+SA+ cells will elicit a similar helper activity by T4+ cells and SA as that induced by Mo. In addition to the antigen-specific presentation and induction of helper activity, the T8+SA+ subset displays the properties of antigen-specific contrasuppressor cells. Thus, reconstitution of T4+ cells and T8+SA- (suppressor cells) with T8+SA+ and 1000 ng SA induces helper and no suppressor activity. Substitution of Mo for the T8+SA+ cells converts the helper to a predominantly suppressor-cell function. T8+SA- cells elicit suppression with 1 ng SA in the absence of accessory cells and reconstitution with Mo, T8+SA+ or T4+ cells failed to affect the suppressor activity. Total reconstitution of the four principle subsets of T4+, T8+SA+, T8+SA- cells and Mo elicited similar antigen dose-dependent responses as those of the unseparated mononuclear cells. It seems that all four cell subsets are required for optimal immunoregulation. We suggest that the T8+SA+ can present antigen to T4+ helper cells and induce helper activity, but in addition these cells can prevent the suppressor subset of T8+ cells from inhibiting T4+ helper cells and function as contrasuppressor cells. The mechanism of these functions is not known but HLA class II antigens might play an essential role in antigen binding, presentation and

  16. 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. PMID:27077111

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

    PubMed Central

    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

    2016-01-01

    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. PMID:27077111

  18. Unique Appearance of Proliferating Antigen-Presenting Cells Expressing DC-SIGN (CD209) in the Decidua of Early Human Pregnancy

    PubMed Central

    Kämmerer, Ulrike; Eggert, Andreas O.; Kapp, Michaela; McLellan, Alexander D.; Geijtenbeek, Teunis B. H.; Dietl, Johannes; van Kooyk, Yvette; Kämpgen, Eckhart

    2003-01-01

    Intact human pregnancy can be regarded as an immunological paradox in that the maternal immune system accepts the allogeneic embryo without general immunosuppression. Because dendritic cell (DC) subsets could be involved in peripheral tolerance, the uterine mucosa (decidua) was investigated for DC populations. Here we describe the detailed immunohistochemical and functional characterization of HLA-DR-positive antigen-presenting cells (APCs) in early pregnancy decidua. In contrast to classical macrophages and CD83+ DCs, which were found in comparable numbers in decidua and nonpregnant endometrium, only decidua harbored a significant population of HLA-DR+/DC-SIGN+ APCs further phenotyped as CD14+/CD4+/CD68+/−/CD83−/CD25−. These cells exhibited a remarkable proliferation rate (9.2 to 9.8% of all CD209+ cells) by double staining with Ki67 and proliferating cell nuclear antigen. Unique within the DC-family, the majority of DC-SIGN+ decidual APCs were observed in situ to have intimate contact with CD56+/CD16−/ICAM-3+ decidual natural killer cells, another pregnancy-restricted cell population. In vitro, freshly isolated CD14+/DC-SIGN+ decidual cells efficiently took up antigen, but could not stimulate naive allogeneic T cells at all. Treatment with an inflammatory cytokine cocktail resulted in down-regulation of antigen uptake capacity and evolving capacity to effectively stimulate resting T cells. Fluorescence-activated cell sorting analysis confirmed the maturation of CD14+/DC-SIGN+ decidual cells into CD25+/CD83+ mature DCs. In summary, this is the first identification of a uterine immature DC population expressing DC-SIGN, that appears only in pregnancy-associated tissue, has a high proliferation rate, and a conspicuous association with a natural killer subset. PMID:12598322

  19. Programmed Death-Ligand 1 on Antigen-presenting Cells Facilitates the Induction of Antigen-specific Cytotoxic T Lymphocytes: Application to Adoptive T-Cell Immunotherapy.

    PubMed

    Goto, Tatsunori; Nishida, Tetsuya; Takagi, Erina; Miyao, Kotaro; Koyama, Daisuke; Sakemura, Reona; Hanajiri, Ryo; Watanabe, Keisuke; Imahashi, Nobuhiko; Terakura, Seitaro; Murata, Makoto; Kiyoi, Hitoshi

    2016-10-01

    Programmed death-ligand 1 (PD-L1) binds to programmed death-1 (PD-1) on activated T cells and contributes to T-cell exhaustion. PD-L1 expressed on antigen-presenting cells (APCs) could be thought to inhibit the induction of Ag-specific cytotoxic T lymphocytes (CTLs) by transducing negative signal into T cells; however, the roles of PD-L1 on APCs have not yet been well examined. Therefore, we evaluated the roles of PD-L1 on APCs in the induction of Ag-specific CTLs. CD3 T cells isolated from cytomegalovirus (CMV)-seropositive healthy donors were stimulated with mature dendritic cells pulsed with CMV pp65-derived HLA-restricted peptides in the presence of anti-PD-L1 blocking antibody. Unexpectedly, PD-L1 blockade resulted in a less efficient induction of CMV-specific CTLs, suggesting that PD-L1 play a positive role in the induction of Ag-specific CTLs. For further evaluations and application to adoptive immunotherapy, we generated K562-based artificial APCs, which were retrovirally transduced with HLA class I molecules and various combinations of CD80/86 and PD-L1. K562/HLA+CD80/86+PD-L1 cells produced significantly higher induction of CMV-specific CTLs than K562/HLA or K562/HLA+CD80/86 cells without causing excessive differentiation or functional exhaustion of the induced CTLs, whereas PD-L1 itself did not have a stimulatory effect. Furthermore, only K562/HLA+CD80/86+PD-L1 cells pulsed with HLA-A*24:02-restricted Wilms tumor 1 (WT1) peptide clearly expanded WT1-specific CTLs from healthy donors. Our findings presumed that PD-L1 expressed on APCs along with CD80/86 enhanced the induction of Ag-specific CTLs probably depending on fine-tuning excessive stimulation of CD80/86, and that K562/HLA+CD80/86+PD-L1 cells has therapeutic potential as a novel type of artificial APCs for adoptive immunotherapy. PMID:27548033

  20. Privileged Antigen Presentation in Splenic B Cell Follicles Maximizes T Cell Responses in Prime-Boost Vaccination.

    PubMed

    Bridle, Byram W; Nguyen, Andrew; Salem, Omar; Zhang, Liang; Koshy, Sandeep; Clouthier, Derek; Chen, Lan; Pol, Jonathan; Swift, Stephanie L; Bowdish, Dawn M E; Lichty, Brian D; Bramson, Jonathan L; Wan, Yonghong

    2016-06-01

    Effector T cells (TEFF) are a barrier to booster vaccination because they can rapidly kill Ag-bearing APCs before memory T cells are engaged. We report in this study that i.v. delivery of rhabdoviral vectors leads to direct infection of follicular B cells in the spleen, where the earliest evidence of secondary T cell responses was observed. This allows booster immunizations to rapidly expand CD8(+) central memory T cells (TCM) during the acute phase of the primary response that is dominated by TEFF Interestingly, although the ablation of B cells before boosting with rhabdoviral vectors diminishes the expansion of memory T cells, B cells do not present Ags directly. Instead, depletion of CD11c(+) dendritic cells abrogates secondary T cell expansion, suggesting that virus-infected follicular B cells may function as an Ag source for local DCs to subsequently capture and present the Ag. Because TCM are located within B cell follicles in the spleen whereas TEFF cannot traffic through follicular regions, Ag production and presentation by follicular APCs represent a unique mechanism to secure engagement of TCM during an ongoing effector response. Our data offer insights into novel strategies for rapid expansion of CD8(+) T cells using prime-boost vaccines by targeting privileged sites for Ag presentation. PMID:27183620

  1. Dendritic Cell Migration and Antigen Presentation Are Coordinated by the Opposing Functions of the Tetraspanins CD82 and CD37.

    PubMed

    Jones, Eleanor L; Wee, Janet L; Demaria, Maria C; Blakeley, Jessica; Ho, Po Ki; Vega-Ramos, Javier; Villadangos, Jose A; van Spriel, Annemiek B; Hickey, Michael J; Hämmerling, Günther J; Wright, Mark D

    2016-02-01

    This study supports a new concept where the opposing functions of the tetraspanins CD37 and CD82 may coordinate changes in migration and Ag presentation during dendritic cell (DC) activation. We have previously published that CD37 is downregulated upon monocyte-derived DC activation, promotes migration of both skin and bone marrow-derived dendritic cells (BMDCs), and restrains Ag presentation in splenic and BMDCs. In this article, we show that CD82, the closest phylogenetic relative to CD37, appears to have opposing functions. CD82 is upregulated upon activation of BMDCs and monocyte-derived DCs, restrains migration of skin and BMDCs, supports MHC class II maturation, and promotes stable interactions between T cells and splenic DCs or BMDCs. The underlying mechanism involves the rearrangement of the cytoskeleton via a differential activation of small GTPases. Both CD37(-/-) and CD82(-/-) BMDCs lack cellular projections, but where CD37(-/-) BMDCs spread poorly on fibronectin, CD82(-/-) BMDCs are large and spread to a greater extent than wild-type BMDCs. At the molecular level, CD82 is a negative regulator of RhoA, whereas CD37 promotes activation of Rac-1; both tetraspanins negatively regulate Cdc42. Thus, this study identifies a key aspect of DC biology: an unactivated BMDC is CD37(hi)CD82(lo), resulting in a highly motile cell with a limited ability to activate naive T cells. By contrast, a late activated BMDC is CD37(lo)CD82(hi), and thus has modified its migratory, cytoskeletal, and Ag presentation machinery to become a cell superbly adapted to activating naive T cells. PMID:26729805

  2. Evasion and subversion of antigen presentation by Mycobacterium tuberculosis

    PubMed Central

    Baena, Andres; Porcelli, Steven A.

    2009-01-01

    Mycobacterium tuberculosis is one of the most successful of human pathogens, and has acquired the ability to establish latent or progressive infection and persist even in the presence of a fully functioning immune system. The ability of M. tuberculosis to avoid immune-mediated clearance is likely to reflect a highly evolved and coordinated program of immune evasion strategies, including some that interfere with antigen presentation to prevent or alter the quality of T cell responses. Here we review an extensive array of published studies supporting the view that antigen presentation pathways are targeted at many points by pathogenic mycobacteria. These studies reveal the multiple potential mechanisms by which M. tuberculosis may actively inhibit, subvert or otherwise modulate antigen presentation by MHC class I, class II and CD1 molecules. Unraveling the mechanisms by which M. tuberculosis evades or modulates antigen presentation is of critical importance for the development of more effective new vaccines based on live attenuated mycobacterial strains. PMID:19563525

  3. Lead enhances CD4{sup +} T cell proliferation indirectly by targeting antigen presenting cells and modulating antigen-specific interactions

    SciTech Connect

    Farrer, David G.; Hueber, Sara M.; McCabe, Michael J. . E-mail: michael_mccabe@urmc.rochester.edu

    2005-09-01

    Although Pb is a well-known immunotoxicant, its mechanism of action is not well understood. Low levels of Pb ({approx}1 {mu}M) markedly enhance the proliferative T cell response in mixed lymphocyte culture (MLC), a process we have termed allo-enhancement. As Pb allo-enhancement occurs whether alloantigen presenting cells (APC) are derived from C57BL/6 or BALB.B10, the allo-reactive T cells involved are likely to be specific for peptide in the context of the IA{sup b} molecule as the IE molecule is null in H-2{sup b} mice. Analysis of T cell division in MLC with Pb treatment indicated that there was no significant difference between Pb and non-Pb-treated cultures until day 4 when the frequency of proliferating T cells was much greater than in non-treated cultures. Our data suggest that this increased proliferation is not coupled with increased IL-2 levels in the media as these were actually decreased with Pb treatment and that Pb-induced enhancement in the allo-proliferative response is only partially dependent upon IL-2. Pb allo-enhancement is abrogated when stimulating allo-APCs are paraformaldehyde-fixed, and T cell proliferation stimulated by concanavalin A is not enhanced with Pb treatment, suggesting that the APC is the proximate target of Pb in allo-MLC. Pb allo-enhancement does not occur when T cells respond to irradiated allo-B cells, alone; however, it is restored when syngeneic CD11c-enriched cells are added. Of the CD11c-enriched splenocytes, the fraction that is adherent after 24 h, consistent with macrophages, appears to be the cell type targeted by Pb. Using T cells from DO11.10 transgenic mice, we determined that the effect of Pb is centered around specific p:MHC interactions and that enhanced costimulation is an unlikely mechanism for Pb allo-enhancement.

  4. Vanilloid Receptor 1 Agonists, Capsaicin and Resiniferatoxin, Enhance MHC Class I-restricted Viral Antigen Presentation in Virus-infected Dendritic Cells

    PubMed Central

    Lee, Young-Hee; Im, Sun-A; Kim, Ji-Wan

    2016-01-01

    DCs, like the sensory neurons, express vanilloid receptor 1 (VR1). Here we demonstrate that the VR1 agonists, capsaicin (CP) and resiniferatoxin (RTX), enhance antiviral CTL responses by increasing MHC class I-restricted viral antigen presentation in dendritic cells (DCs). Bone marrow-derived DCs (BM-DCs) were infected with a recombinant vaccinia virus (VV) expressing OVA (VV-OVA), and then treated with CP or RTX. Both CP and RTX increased MHC class I-restricted presentation of virus-encoded endogenous OVA in BM-DCs. Oral administration of CP or RTX significantly increased MHC class I-restricted OVA presentation by splenic and lymph node DCs in VV-OVA-infected mice, as assessed by directly measuring OVA peptide SIINFEKL-Kb complexes on the cell surface and by performing functional assays using OVA-specific CD8 T cells. Accordingly, oral administration of CP or RTX elicited potent OVA-specific CTL activity in VV-OVA-infected mice. The results from this study demonstrate that VR1 agonists enhance anti-viral CTL responses, as well as a neuro-immune connection in anti-viral immune responses. PMID:27574502

  5. Vanilloid Receptor 1 Agonists, Capsaicin and Resiniferatoxin, Enhance MHC Class I-restricted Viral Antigen Presentation in Virus-infected Dendritic Cells.

    PubMed

    Lee, Young-Hee; Im, Sun-A; Kim, Ji-Wan; Lee, Chong-Kil

    2016-08-01

    DCs, like the sensory neurons, express vanilloid receptor 1 (VR1). Here we demonstrate that the VR1 agonists, capsaicin (CP) and resiniferatoxin (RTX), enhance antiviral CTL responses by increasing MHC class I-restricted viral antigen presentation in dendritic cells (DCs). Bone marrow-derived DCs (BM-DCs) were infected with a recombinant vaccinia virus (VV) expressing OVA (VV-OVA), and then treated with CP or RTX. Both CP and RTX increased MHC class I-restricted presentation of virus-encoded endogenous OVA in BM-DCs. Oral administration of CP or RTX significantly increased MHC class I-restricted OVA presentation by splenic and lymph node DCs in VV-OVA-infected mice, as assessed by directly measuring OVA peptide SIINFEKL-K(b) complexes on the cell surface and by performing functional assays using OVA-specific CD8 T cells. Accordingly, oral administration of CP or RTX elicited potent OVA-specific CTL activity in VV-OVA-infected mice. The results from this study demonstrate that VR1 agonists enhance anti-viral CTL responses, as well as a neuro-immune connection in anti-viral immune responses. PMID:27574502

  6. Characterization of Yellow Fever Virus Infection of Human and Non-human Primate Antigen Presenting Cells and Their Interaction with CD4+ T Cells.

    PubMed

    Cong, Yu; McArthur, Monica A; Cohen, Melanie; Jahrling, Peter B; Janosko, Krisztina B; Josleyn, Nicole; Kang, Kai; Zhang, Tengfei; Holbrook, Michael R

    2016-05-01

    Humans infected with yellow fever virus (YFV), a mosquito-borne flavivirus, can develop illness ranging from a mild febrile disease to hemorrhagic fever and death. The 17D vaccine strain of YFV was developed in the 1930s, has been used continuously since development and has proven very effective. Genetic differences between vaccine and wild-type viruses are few, yet viral or host mechanisms associated with protection or disease are not fully understood. Over the past 20 years, a number of cases of vaccine-associated disease have been identified following vaccination with 17D; these cases have been correlated with reduced immune status at the time of vaccination. Recently, several studies have evaluated T cell responses to vaccination in both humans and non-human primates, but none have evaluated the response to wild-type virus infection. In the studies described here, monocyte-derived macrophages (MDM) and dendritic cells (MoDC) from both humans and rhesus macaques were evaluated for their ability to support infection with either wild-type Asibi virus or the 17D vaccine strain and the host cytokine and chemokine response characterized. Human MoDC and MDM were also evaluated for their ability to stimulate CD4+ T cells. It was found that MoDC and MDM supported viral replication and that there were differential cytokine responses to infection with either wild-type or vaccine viruses. Additionally, MoDCs infected with live 17D virus were able to stimulate IFN-γ and IL-2 production in CD4+ T cells, while cells infected with Asibi virus were not. These data demonstrate that wild-type and vaccine YFV stimulate different responses in target antigen presenting cells and that wild-type YFV can inhibit MoDC activation of CD4+ T cells, a critical component in development of protective immunity. These data provide initial, but critical insight into regulatory capabilities of wild-type YFV in development of disease. PMID:27191161

  7. Characterization of Yellow Fever Virus Infection of Human and Non-human Primate Antigen Presenting Cells and Their Interaction with CD4+ T Cells

    PubMed Central

    Cong, Yu; McArthur, Monica A.; Cohen, Melanie; Jahrling, Peter B.; Janosko, Krisztina B.; Josleyn, Nicole; Kang, Kai; Zhang, Tengfei; Holbrook, Michael R.

    2016-01-01

    Humans infected with yellow fever virus (YFV), a mosquito-borne flavivirus, can develop illness ranging from a mild febrile disease to hemorrhagic fever and death. The 17D vaccine strain of YFV was developed in the 1930s, has been used continuously since development and has proven very effective. Genetic differences between vaccine and wild-type viruses are few, yet viral or host mechanisms associated with protection or disease are not fully understood. Over the past 20 years, a number of cases of vaccine-associated disease have been identified following vaccination with 17D; these cases have been correlated with reduced immune status at the time of vaccination. Recently, several studies have evaluated T cell responses to vaccination in both humans and non-human primates, but none have evaluated the response to wild-type virus infection. In the studies described here, monocyte-derived macrophages (MDM) and dendritic cells (MoDC) from both humans and rhesus macaques were evaluated for their ability to support infection with either wild-type Asibi virus or the 17D vaccine strain and the host cytokine and chemokine response characterized. Human MoDC and MDM were also evaluated for their ability to stimulate CD4+ T cells. It was found that MoDC and MDM supported viral replication and that there were differential cytokine responses to infection with either wild-type or vaccine viruses. Additionally, MoDCs infected with live 17D virus were able to stimulate IFN-γ and IL-2 production in CD4+ T cells, while cells infected with Asibi virus were not. These data demonstrate that wild-type and vaccine YFV stimulate different responses in target antigen presenting cells and that wild-type YFV can inhibit MoDC activation of CD4+ T cells, a critical component in development of protective immunity. These data provide initial, but critical insight into regulatory capabilities of wild-type YFV in development of disease. PMID:27191161

  8. The NS1 protein of influenza A virus suppresses interferon-regulated activation of antigen-presentation and immune-proteasome pathways

    PubMed Central

    Tisoncik, Jennifer R.; Billharz, Rosalind; Burmakina, Svetlana; Belisle, Sarah E.; Proll, Sean C.; Korth, Marcus J.; García-Sastre, Adolfo

    2011-01-01

    The NS1 protein of influenza virus counters host antiviral defences primarily by antagonizing the type I interferon (IFN) response. Both the N-terminal dsRNA-binding domain and the C-terminal effector domain are required for optimal suppression of host responses during infection. To better understand the regulatory role of the NS1 effector domain, we used an NS1-truncated mutant virus derived from human H1N1 influenza isolate A/Texas/36/91 (Tx/91) and assessed global transcriptional profiles from two independent human lung cell-culture models. Relative to the wild-type Tx/91-induced gene expression, the NS1 mutant virus induced enhanced expression of innate immune genes, specifically NF-κB signalling-pathway genes and IFN-α and -β target genes. We queried an experimentally derived IFN gene set to gauge the proportion of IFN-responsive genes that are suppressed specifically by NS1. We show that the C-terminally truncated NS1 mutant virus is less efficient at suppressing IFN-regulated gene expression associated with activation of antigen-presentation and immune-proteasome pathways. This is the first report integrating genomic analysis from two independent human culture systems, including primary lung cells, using genetically similar H1N1 influenza viruses that differ only in the length of the NS1 protein. PMID:21593271

  9. In vivo anti-melanoma efficacy of allo-restricted CTLs specific for melanoma expanded by artificial antigen-presenting cells.

    PubMed

    Lu, Xiao-ling; Jiang, Xiao-bing; Liu, Ru-en; Zhang, Sheng-min; Liang, Z-h

    2009-04-01

    Cytotoxic CD8(+) T cells are key effectors in the immunotherapy of malignant and viral diseases. However, autologous T cell responses to tumor antigens presented by self-MHC are usually weak and ineffective. Allo-restricted T cells represent a potent source of tumor-specific T cells for adoptive immunotherapy. This study reports in vivo anti-melanoma efficacy of the pTRP2-specific allo-restricted CTLs expanded from the BALB/c splenocytes by multiple stimulations with aAPCs made by coating H-2K(b)-Ig/pTRP2 dimeric complexes, anti-CD28 antibody, 4-1BBL molecules and CD83 molecules to cell-sized latex beads. The induced allo-restricted CTLs exhibited specific lysis against RMA-S cells pulsed with the peptide pTRP2 and H-2K(b+) melanoma cells expressing TRP2, while a murine Lewis lung carcinoma cell line 3LL could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti-H-2K(b) monoclonal antibody Y3. Adoptive transfer of the allo-restricted CTLs specific for malignant melanoma expanded by the aAPCs can mediate effective anti-melanoma response in vivo. These results suggested that the specific allo-restricted CTLs expanded by aAPCs coated with an MHC-Ig/peptide complex, anti-CD28 antibody, 4-1BBL and CD83 could be a potential option of specific immunotherapy for patients with malignant melanoma. PMID:18682943

  10. Delivery of Large Heterologous Polypeptides across the Cytoplasmic Membrane of Antigen-Presenting Cells by the Bordetella RTX Hemolysin Moiety Lacking the Adenylyl Cyclase Domain

    PubMed Central

    Holubova, Jana; Jelinek, Jiri; Tomala, Jakub; Masin, Jiri; Kosova, Martina; Stanek, Ondrej; Bumba, Ladislav; Michalek, Jaroslav; Kovar, Marek; Sebo, Peter

    2012-01-01

    The Bordetella adenylate cyclase toxin-hemolysin (CyaA; also called ACT or AC-Hly) targets CD11b-expressing phagocytes and translocates into their cytosol an adenylyl cyclase (AC) that hijacks cellular signaling by conversion of ATP to cyclic AMP (cAMP). Intriguingly, insertion of large passenger peptides removes the enzymatic activity but not the cell-invasive capacity of the AC domain. This has repeatedly been exploited for delivery of heterologous antigens into the cytosolic pathway of CD11b-expressing dendritic cells by CyaA/AC− toxoids, thus enabling their processing and presentation on major histocompatibility complex (MHC) class I molecules to cytotoxic CD8+ T lymphocytes (CTLs). We produced a set of toxoids with overlapping deletions within the first 371 residues of CyaA and showed that the structure of the AC enzyme does not contain any sequences indispensable for its translocation across target cell membrane. Moreover, replacement of the AC domain (residues 1 to 371) with heterologous polypeptides of 40, 146, or 203 residues yielded CyaAΔAC constructs that delivered passenger CTL epitopes into antigen-presenting cells (APCs) and induced strong antigen-specific CD8+ CTL responses in vivo in mice and ex vivo in human peripheral blood mononuclear cell cultures. This shows that the RTX (repeats in toxin) hemolysin moiety, consisting of residues 374 to 1706 of CyaA, harbors all structural information involved in translocation of the N-terminal AC domain across target cell membranes. These results decipher the extraordinary capacity of the AC domain of CyaA to transport large heterologous cargo polypeptides into the cytosol of CD11b+ target cells and pave the way for the construction of CyaAΔAC-based polyvalent immunotherapeutic T cell vaccines. PMID:22215742

  11. Chronic pulmonary accumulation of iron oxide nanoparticles induced Th1-type immune response stimulating the function of antigen-presenting cells.

    PubMed

    Park, Eun-Jung; Oh, Seung Yun; Lee, Sang Jin; Lee, Kyuhong; Kim, Younghun; Lee, Byoung-Seok; Kim, Jong Sung

    2015-11-01

    Although there is growing evidence that suggests that pulmonary exposure to nanoparticles causes adverse health effects by modulating immune system of the body, available information is very limited. In this study, we investigated immune response following chronic pulmonary accumulation of iron oxide nanoparticles (FeNPs, Fe2O3). FeNPs have a needle-like shape in suspension (101.3±4.2 nm). On day 90 after a single intratracheal instillation (0.5, 1, and 2 mg/kg), the FeNPs remained in the lung and particle-laden macrophages were clearly observed in the BAL fluid of the treated-mice. The number of total cells and proportions of neutrophils and lymphocytes significantly increased at 2 mg/kg dose, and the percentage of apoptotic cells and LDH release increased in a dose-dependent manner. We also found that Th1-polarized inflammatory response was induced in the lung of the treated group accompanying the elevated secretion of chemokines, including GM-CSF, MCP-1, and MIP-1. Additionally, FeNPs enhanced the expression of antigen presentation-related proteins, including CD80, CD86, and MHC class II, on antigen-presenting cells in BAL fluid. Taken together, we suggest that chronic pulmonary accumulation of FeNPs may induce Th1-polarized immune response augmenting the function of antigen-presenting cells in the lung. PMID:26492398

  12. Virus-triggered acquired immunodeficiency by cytotoxic T-cell-dependent destruction of antigen-presenting cells and lymph follicle structure.

    PubMed Central

    Odermatt, B; Eppler, M; Leist, T P; Hengartner, H; Zinkernagel, R M

    1991-01-01

    Virus-induced acquired immune suppression in mice infected with lymphocytic choriomeningitis virus is shown here to be caused by the CD8+-T-cell-dependent elimination of macrophages/antigen-presenting cells. Surprisingly, this is associated with severe destruction of the follicular organization of lymphoid organs, indicating a crucial role for dendritic cells and marginal zone macrophages in maintaining follicular structure. Once established, this immunopathology cannot be readily reversed by the elimination of CD8+ effector cells. Such a T-cell-mediated pathogenesis may play a pivotal role in acquired virus-induced immunosuppression and may represent one strategy by which virus escapes immune surveillance and establishes persistent infections in initially immunocompetent hosts. Images PMID:1910175

  13. Nanoparticle-based targeting of vaccine compounds to skin antigen-presenting cells by hair follicles and their transport in mice.

    PubMed

    Mahe, Brice; Vogt, Annika; Liard, Christelle; Duffy, Darragh; Abadie, Valérie; Bonduelle, Olivia; Boissonnas, Alexandre; Sterry, Wolfram; Verrier, Bernard; Blume-Peytavi, Ulrike; Combadiere, Behazine

    2009-05-01

    Particle-based drug delivery systems target active compounds to the hair follicle and may result in a better penetration and higher efficiency of compound uptake by skin resident cells. As previously proposed, such delivery systems could be important tools for vaccine delivery. In this study, we investigated the penetration of solid fluorescent 40 or 200 nm polystyrene nanoparticles (NPs) as well as virus particles in murine skin to further investigate the efficacy of transcutaneously (TC) applied particulate vaccine delivery route. We demonstrated that 40 and 200 nm NPs and modified vaccinia Ankara (MVA) expressing the green-fluorescent protein penetrated deeply into hair follicles and were internalized by perifollicular antigen-presenting cells (APCs). Fibered-based confocal microscopy analyses allowed visualizing in vivo particle penetration along the follicular duct, diffusion into the surrounding tissue, uptake by APCs and transport to the draining lymph nodes. The application of small particles, such as ovalbumin coding DNA or MVA, induced both humoral and cellular immune responses. Furthermore, TC applied MVA induced protection against vaccinia virus challenge. Our results strengthen the concept of TC targeting of cutaneous APCs by hair follicles and will contribute to the development of advanced vaccination protocols using NPs or viral vectors. PMID:19052565

  14. Cyclosporine inhibits macrophage-mediated antigen presentation

    SciTech Connect

    Ziegler, H.K.; Palay, D.; Wentworth, P.; Cluff, C.

    1986-03-01

    The influence of cyclosporine on antigen-specific, macrophage-dependent T cell activation was analyzed in vitro. Murine T cell activation by antigens derived from Listeria monocytogenes was monitored by the production of interleukin-2. Pretreatment (2 hrs., 37/sup 0/C) of macrophages with cyclosporine resulted in a population of macrophages with a markedly diminished capacity to support the activation of T lymphocytes. When cyclosporine-pretreated macrophages were added to cultures of antigen and untreated T cells, the dose of cyclosporine which produced 50% inhibition was 1.5 ..mu..g/ml. Appropriate control experiments indicated that cyclosporine was indeed inhibiting at the macrophage level. The addition of interleukin-1 or indomethacin to the cultures did not alter the inhibitory effect of cyclosporine. Under conditions which produced >90% inhibition of antigen presentation, macrophage surface Ia expression was not altered, and the uptake and catabolism of radiolabelled antigen was normal. Thus, cyclosporine inhibits antigen presentation by a mechanism which appears unrelated to changes in Il-1 elaboration, prostaglandin production, Ia expression, or antigen uptake and catabolism.

  15. Heparan sulfates targeting increases MHC class I- and MHC class II-restricted antigen presentation and CD8(+) T-cell response.

    PubMed

    Knittel, Delphine; Gadzinski, Adeline; Hua, Stéphane; Denizeau, Jordan; Savatier, Alexandra; de la Rochère, Philippe; Boulain, Jean-Claude; Amigorena, Sebastian; Piaggio, Eliane; Sedlik, Christine; Léonetti, Michel

    2016-06-01

    Heparan sulfates (HS) are carbohydrate moieties of HS proteoglycans (HSPGs). They often represent alternative attachment points for proteins or microorganisms targeting receptors. HSPGs, which are ubiquitously expressed, thereby participate in numerous biological processes. We previously showed that MHC class II-restricted antigen presentation is increased when antigens are coupled to HS ligands, suggesting that HSPGs might contribute to adaptive immune responses. Here, we examined if HSPG targeting influences other aspects of immune responses. We found that coupling of an HS ligand to the antigen increases antigen presentation to CD4(+) and CD8(+) T-cells after antigen targeting to membrane immunoglobulins or to MHC-II molecules. Moreover, this increased stimulating capacity correlates with an enhanced CD8(+) immune response in mice. Last, animals control more effectively the growth of Ova-expressing tumour cells when they are immunized with an Ova construct targeting HSPGs and MHC-II molecules. Our results indicate that ubiquitous molecules can influence both MHC class I- and MHC class II-restricted antigen presentation and behave as co-receptors during T-cell stimulation. Moreover, they suggest that tumour-antigens endowed with the ability to target both HSPGs and MHC-II molecules could be of value to increase CD8(+) immune response and control tumour-growth, opening new perspectives for the design of highly immunogenic protein-based vaccines. PMID:27154391

  16. Inflammatory Proprotein Convertase-Matrix Metalloproteinase Proteolytic Pathway in Antigen-presenting Cells as a Step to Autoimmune Multiple Sclerosis*

    PubMed Central

    Shiryaev, Sergey A.; Remacle, Albert G.; Savinov, Alexei Y.; Chernov, Andrei V.; Cieplak, Piotr; Radichev, Ilian A.; Williams, Roy; Shiryaeva, Tatiana N.; Gawlik, Katarzyna; Postnova, Tatiana I.; Ratnikov, Boris I.; Eroshkin, Alexei M.; Motamedchaboki, Khatereh; Smith, Jeffrey W.; Strongin, Alex Y.

    2009-01-01

    Multiple sclerosis (MS) is a disease of the central nervous system with autoimmune etiology. Susceptibility to MS is linked to viral and bacterial infections. Matrix metalloproteinases (MMPs) play a significant role in the fragmentation of myelin basic protein (MBP) and demyelination. The splice variants of the single MBP gene are expressed in the oligodendrocytes of the central nervous system (classic MBP) and in the immune cells (Golli-MBPs). Our data suggest that persistent inflammation caused by environmental risk factors is a step to MS. We have discovered biochemical evidence suggesting the presence of the inflammatory proteolytic pathway leading to MS. The pathway involves the self-activated furin and PC2 proprotein convertases and membrane type-6 MMP (MT6-MMP/MMP-25) that is activated by furin/PC2. These events are followed by MMP-25 proteolysis of the Golli-MBP isoforms in the immune system cells and stimulation of the specific autoimmune T cell clones. It is likely that the passage of these autoimmune T cell clones through the disrupted blood-brain barrier to the brain and the recognition of neuronal, classic MBP causes inflammation leading to the further up-regulation of the activity of the multiple individual MMPs, the massive cleavage of MBP in the brain, demyelination, and MS. In addition to the cleavage of Golli-MBPs, MMP-25 proteolysis readily inactivates crystallin αB that is a suppressor of MS. These data suggest that MMP-25 plays an important role in MS pathology and that MMP-25, especially because of its restricted cell/tissue expression pattern and cell surface/lipid raft localization, is a promising drug target in MS. PMID:19726693

  17. Inflammatory proprotein convertase-matrix metalloproteinase proteolytic pathway in antigen-presenting cells as a step to autoimmune multiple sclerosis.

    PubMed

    Shiryaev, Sergey A; Remacle, Albert G; Savinov, Alexei Y; Chernov, Andrei V; Cieplak, Piotr; Radichev, Ilian A; Williams, Roy; Shiryaeva, Tatiana N; Gawlik, Katarzyna; Postnova, Tatiana I; Ratnikov, Boris I; Eroshkin, Alexei M; Motamedchaboki, Khatereh; Smith, Jeffrey W; Strongin, Alex Y

    2009-10-30

    Multiple sclerosis (MS) is a disease of the central nervous system with autoimmune etiology. Susceptibility to MS is linked to viral and bacterial infections. Matrix metalloproteinases (MMPs) play a significant role in the fragmentation of myelin basic protein (MBP) and demyelination. The splice variants of the single MBP gene are expressed in the oligodendrocytes of the central nervous system (classic MBP) and in the immune cells (Golli-MBPs). Our data suggest that persistent inflammation caused by environmental risk factors is a step to MS. We have discovered biochemical evidence suggesting the presence of the inflammatory proteolytic pathway leading to MS. The pathway involves the self-activated furin and PC2 proprotein convertases and membrane type-6 MMP (MT6-MMP/MMP-25) that is activated by furin/PC2. These events are followed by MMP-25 proteolysis of the Golli-MBP isoforms in the immune system cells and stimulation of the specific autoimmune T cell clones. It is likely that the passage of these autoimmune T cell clones through the disrupted blood-brain barrier to the brain and the recognition of neuronal, classic MBP causes inflammation leading to the further up-regulation of the activity of the multiple individual MMPs, the massive cleavage of MBP in the brain, demyelination, and MS. In addition to the cleavage of Golli-MBPs, MMP-25 proteolysis readily inactivates crystallin alphaB that is a suppressor of MS. These data suggest that MMP-25 plays an important role in MS pathology and that MMP-25, especially because of its restricted cell/tissue expression pattern and cell surface/lipid raft localization, is a promising drug target in MS. PMID:19726693

  18. Adoptive transfer of pTRP2-specific CTLs expanding by bead-based artificial antigen-presenting cells mediates anti-melanoma response.

    PubMed

    Lu, Xiaoling; Jiang, Xiaobing; Liu, Ruen; Zhao, Hongyang; Liang, Zhihui

    2008-11-18

    Cytotoxic CD8(+) T cells are key effectors in the immunotherapy of malignant and viral diseases. However, the lack of efficient methods for their in vitro priming and expansion has become a bottleneck to the development of vaccines and adoptive transfer strategies. Synthetic artificial antigen-presenting cells (aAPCs) are now emerging as an attractive tool for eliciting and expanding CTL responses. This study reported a novel approach for targeting malignant melanoma with pTRP2-specific cytotoxic T lymphocytes (CTLs) expanded from the C57BL/6 splenocytes by multiple stimulations with aAPCs made by coating H-2K(b)-Ig/pTRP2 dimeric complexes, anti-CD28 antibody, 4-1BBL molecules and CD83 molecules to cell-sized latex beads. The induced CTLs exhibited specific lysis against RMA-S cells pulsed with the peptide pTRP2 and H-2K(b+) melanoma cells expressing TRP2, while a murine Lewis lung carcinoma cell line 3LL could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti-H-2K(b) monoclonal antibody Y3. Adoptive Transfer of CTLs specific for malignant melanoma expanding by the aAPCs can mediate effective anti-melanoma response. These results suggested the bead-based aAPCs coated with an MHC-Ig/peptide complex, anti-CD28 antibody, 4-1BBL and CD83 could provide a useful tool for the reproducible expansion of specific CTLs for adoptive immunotherapy. PMID:18621475

  19. Intracellular transport of MHC class II and associated invariant chain in antigen presenting cells from AP-3-deficient mocha mice.

    PubMed

    Sevilla, L M; Richter, S S; Miller, J

    2001-06-15

    MHC class II-restricted antigen presentation requires trafficking of newly synthesized class II-invariant chain complexes from the trans-Golgi network to endosomal, peptide-loading compartments. This transport is mediated by dileucine-like motifs within the cytosolic tail of the invariant chain. Although these signals have been well characterized, the cytosolic proteins that interact with these dileucine signals and mediate Golgi sorting and endosomal transport have not been identified. Recently, an adaptor complex, AP-3, has been identified that interacts with dileucine motifs and mediates endosomal/lysosomal transport in yeast, Drosophila, and mammals. In this report, we have assessed class II-invariant chain trafficking in a strain of mice (mocha) which lacks expression of AP-3. Our studies demonstrate that the lack of AP-3 does not affect the kinetics of invariant chain degradation, the route of class II-invariant chain transport, or the rate and extent of class II-peptide binding as assessed by the generation of SDS-stable dimers. The possible role of other known or unknown adaptor complexes in class II-invariant chain transport is discussed. PMID:11520080

  20. Microbial Induction of Inflammatory Bowel Disease Associated Gene TL1A (TNFSF15) in Antigen Presenting Cells

    PubMed Central

    Shih, David Q.; Kwan, Lola Y.; Chavez, Valerie; Cohavy, Offer; Gonsky, Rivkah; Chang, Elmer Y.; Chang, Christopher; Elson, Charles O.; Targan, Stephan R.

    2010-01-01

    Summary TL1A is a member of the TNF superfamily and its expression is increased in the mucosa of inflammatory bowel disease (IBD) patients. Neutralizing anti-mouse TL1A Ab attenuates chronic colitis in two T cell driven murine models, suggesting that TL1A is a central modulator of gut mucosal inflammation in IBD. We showed previously that TL1A is induced by immune complexes (IC) via the FcγR signaling pathway. In this study, we report that multiple bacteria, including gram negative organisms (E. coli, E. coli Nissle 1917, S. typhimurium), gram positive organisms (L. monocytogenes, S. epidermidis), partial anaerobes (C. jejuni), and obligate anaerobes (B. thetaiotaomicron, B. breve, Clostridium A4) activate TL1A expression in human APC, including monocytes and monocyte-derived DC. Bacterially induced TL1A mRNA expression correlates with the detection of TL1A protein levels. TL1A induced by bacteria is mediated in part by the TLR signaling pathway and inhibited by downstream blockade of p38 MAPK and NF-κB activation. Microbial induction of TL1A production by human APC potentiated CD4+ T cell effector function by augmenting IFN-γ production. Our findings suggest a role for TL1A in pro-inflammatory APC-T cell interactions and implicate TL1A in host responses to enteric microorganisms. PMID:19839006

  1. Phase I study utilizing a novel antigen-presenting cell-targeted vaccine with Toll-like receptor stimulation to induce immunity to self antigens in cancer patients

    PubMed Central

    Morse, Michael A.; Chapman, Robert; Powderly, John; Blackwell, Kimberly; Keler, Tibor; Green, Jennifer; Riggs, Renee; He, Li-Zhen; Ramakrishna, Venky; Vitale, Laura; Zhao, Biwei; Butler, Stephen A.; Hobeika, Amy; Osada, Takuya; Davis, Thomas; Clay, Timothy; Lyerly, H. Kim

    2011-01-01

    Purpose The use of tumor-derived proteins as cancer vaccines is complicated by tolerance to these self antigens. Tolerance may be broken by immunization with activated, autologous, ex vivo generated and antigen-loaded, antigen-presenting cells (APC); however, targeting tumor antigen directly to APC in vivo would be a less complicated strategy. We wished to test whether targeted delivery of an otherwise poorly immunogenic, soluble antigen to APC through their mannose receptors (MR) would induce clinically relevant immunity. Experimental Design Two phase I studies were performed with CDX-1307, a vaccine composed of human chorionic gonadotropin beta chain (hCG-β) fused to a MR-specific monoclonal antibody, administered either locally (intradermally) or systemically (intravenously) in patients with advanced epithelial malignancies. An initial dose-escalation of single agent CDX-1307 was followed by additional cohorts of CDX-1307 combined with GM-CSF and the Toll-like receptor (TLR)-3 agonist poly-ICLC and TLR7/8 agonist resiquimod to activate the APC. Results CDX-1307 induced consistent humoral and T cell responses to hCG-β when co-administered with TLR agonists. Greater immune responses and clinical benefit, including the longest duration of stable disease, were observed with immunization combined with local TLR agonists. Immune responses were induced equally efficiently in patients with elevated and non-elevated levels of serum hCG-β. Antibodies within the serum of vaccinated participants had tumor suppressive function in vitro. Toxicity consisted chiefly of mild injection site reactions. Conclusions APC targeting and activation induce adaptive immunity against poorly immunogenic self antigens which has implications for enhancing the efficacy of cancer immunotherapy. PMID:21632857

  2. CNS myelin induces regulatory functions of DC-SIGN-expressing, antigen-presenting cells via cognate interaction with MOG.

    PubMed

    García-Vallejo, J J; Ilarregui, J M; Kalay, H; Chamorro, S; Koning, N; Unger, W W; Ambrosini, M; Montserrat, V; Fernandes, R J; Bruijns, S C M; van Weering, J R T; Paauw, N J; O'Toole, T; van Horssen, J; van der Valk, P; Nazmi, K; Bolscher, J G M; Bajramovic, J; Dijkstra, C D; 't Hart, B A; van Kooyk, Y

    2014-06-30

    Myelin oligodendrocyte glycoprotein (MOG), a constituent of central nervous system myelin, is an important autoantigen in the neuroinflammatory disease multiple sclerosis (MS). However, its function remains unknown. Here, we show that, in healthy human myelin, MOG is decorated with fucosylated N-glycans that support recognition by the C-type lectin receptor (CLR) DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) on microglia and DCs. The interaction of MOG with DC-SIGN in the context of simultaneous TLR4 activation resulted in enhanced IL-10 secretion and decreased T cell proliferation in a DC-SIGN-, glycosylation-, and Raf1-dependent manner. Exposure of oligodendrocytes to proinflammatory factors resulted in the down-regulation of fucosyltransferase expression, reflected by altered glycosylation at the MS lesion site. Indeed, removal of fucose on myelin reduced DC-SIGN-dependent homeostatic control, and resulted in inflammasome activation, increased T cell proliferation, and differentiation toward a Th17-prone phenotype. These data demonstrate a new role for myelin glycosylation in the control of immune homeostasis in the healthy human brain through the MOG-DC-SIGN homeostatic regulatory axis, which is comprised by inflammatory insults that affect glycosylation. This phenomenon should be considered as a basis to restore immune tolerance in MS. PMID:24935259

  3. Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using Sleeping Beauty system and artificial antigen presenting cells.

    PubMed

    Singh, Harjeet; Figliola, Matthew J; Dawson, Margaret J; Olivares, Simon; Zhang, Ling; Yang, Ge; Maiti, Sourindra; Manuri, Pallavi; Senyukov, Vladimir; Jena, Bipulendu; Kebriaei, Partow; Champlin, Richard E; Huls, Helen; Cooper, Laurence J N

    2013-01-01

    Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28) that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB) transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC) in the presence of interleukin (IL)-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT). We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼10(10) T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion. PMID:23741305

  4. Manufacture of Clinical-Grade CD19-Specific T Cells Stably Expressing Chimeric Antigen Receptor Using Sleeping Beauty System and Artificial Antigen Presenting Cells

    PubMed Central

    Singh, Harjeet; Figliola, Matthew J.; Dawson, Margaret J.; Olivares, Simon; Zhang, Ling; Yang, Ge; Maiti, Sourindra; Manuri, Pallavi; Senyukov, Vladimir; Jena, Bipulendu; Kebriaei, Partow; Champlin, Richard E.; Huls, Helen; Cooper, Laurence J. N.

    2013-01-01

    Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28) that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB) transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC) in the presence of interleukin (IL)-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT). We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼1010 T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion. PMID:23741305

  5. Th1-biased immune responses induced by DNA-based immunizations are mediated via action on professional antigen-presenting cells to up-regulate IL-12 production

    PubMed Central

    Asakura, Y; Liu, L -J; Shono, N; Hinkula, J; Kjerrström, A; Aoki, I; Okuda, K; Wahren, B; Fukushima, J

    2000-01-01

    The efficacy of DNA-based immunization in conferring protective immunity against certain microbial pathogens including human immunodeficiency virus type 1 (HIV-1) has been described. The potential advantage of DNA-based immunization over the traditional vaccines largely results from its capacity to efficiently induce Th1-biased immune responses against an encoded antigen. We describe how Th1-biased immune responses are induced by DNA-based immunization, using a DNA vaccine construct encoding HIV-1 gp160 cDNA and an eukaryotic expression plasmid carrying murine IFN-γ cDNA. Transfection of an eukaryotic expression plasmid carrying immunostimulatory sequences (ISS) as well as a gene of interest (DNA vaccine) into professional antigen presenting cells (APC) induced transactivation of IL-12 mRNA, which resulted in antigen-specific Th1-biased immune responses against the encoded antigen. Th1-biased immune responses induced by DNA-based immunization were substantially upregulated by a codelivery of an ectopic IFN-γ expression system, and this augmentation was mediated via action on professional antigen presenting cells to upregulate IL-12 production. Taken together, it appears likely that Th1-biased immune responses induced by DNA-based immunization are mediated via action on professional antigen-presenting cells to produce IL-12. Interestingly, the model provided strikingly resembles that previously described in infection with Listeria monocytogenes, an intracellular Gram-positive bacterium that induces strong Th1-biased immune responses. The result suggests that DNA-based immunization mimics certain aspects of natural infection with microbial organisms like attenuated vaccines, which in turn provides a rationale to the question of why DNA-based immunization so efficiently induces protective immunity against these microbial pathogens. PMID:10606974

  6. Rainbow trout CK9, a CCL25-like ancient chemokine that attracts and regulates B cells and macrophages, the main antigen presenting cells in fish

    PubMed Central

    Aquilino, Carolina; Granja, Aitor G.; Castro, Rosario; Wang, Tiehui; Abos, Beatriz; Parra, David; Secombes, Christopher J.; Tafalla, Carolina

    2016-01-01

    CK9 is a rainbow trout (Oncorhynchus mykiss) CC chemokine phylogenetically related to mammalian CCL25. Although CK9 is known to be transcriptionally regulated in response to inflammation particularly in mucosal tissues, its functionality has never been revealed. In the current work, we have demonstrated that CK9 is chemoattractant for antigen presenting cells (APCs) expressing major histocompatibility complex class II (MHC II) on the cell surface. Among these APCs, CK9 has a strong chemotactic capacity for both B cells (IgM+ and IgT+) and macrophages. Along with its chemotactic capacities, CK9 modulated the MHC II turnover of B lymphocytes and up-regulated the phagocytic capacity of both IgM+ cells and macrophages. Although CK9 had no lymphoproliferative effects, it increased the survival of IgT+ lymphocytes. Furthermore, we have established that the chemoattractant capacity of CK9 is strongly increased after pre-incubation of leukocytes with a T-independent antigen, whereas B cell receptor (BCR) cross-linking strongly abrogated their capacity to migrate to CK9, indicating that CK9 preferentially attracts B cells at the steady state or under BCR-independent stimulation. These results point to CK9 being a key regulator of B lymphocyte trafficking in rainbow trout, able to modulate innate functions of teleost B lymphocytes and macrophages. PMID:27003360

  7. Modulation of antigen presentation by autoreactive B cell clones specific for GAD65 from a type I diabetic patient

    PubMed Central

    BANGA, J P; MOORE, J K; DUHINDAN, N; MADEC, A M; VAN ENDERT, P M; ORGIAZZI, J; ENDL, J

    2004-01-01

    We used a GAD65-specific human B–T cell line cognate system in vitro to investigate the modulation of GAD65 presentation by autoantibody, assessed in a proliferation assay. Generally, if the T cell determinant overlaps or resides within the antibody epitope, effects of presentation are blunted while if they are distant can lead to potent presentation. For three different autoreactive B–T cell line cognate pairs, the modulation of GAD65 presentation followed the mode of overlapping or distant epitopes with resultant potent or undetectable presentation. However, other cognate pairs elicited variability in this pattern of presentation. Notably, one B cell line, DPC, whose antibody epitope did not overlap with the T cell determinants, was consistently poor in presenting GAD65. Using the fluorescent dye Alexa Fluor 647 conjugated to GAD65 to study receptor-mediated antigen endocytosis showed that all the antigen-specific B cell clones were efficient in intracellular accumulation of the antigen. Additionally, multicolour immunofluorescence microscopy showed that the internalized GAD65/surface IgG complexes were rapidly targeted to a perinuclear compartment in all GAD-specific B cell clones. This analysis also demonstrated that HLA-DM expression was reduced strongly in DPC compared to the stimulatory B cell clones. Thus the capability of antigen-specific B cells to capture and present antigen to human T cell lines is dependent on the spatial relationship of B and T cell epitopes as well other factors which contribute to the efficiency of presentation. PMID:14678267

  8. Neuroblastoma killing properties of V-delta 2 and V-delta2 negative gamma delta T cells following expansion by artificial antigen presenting cells

    PubMed Central

    Fisher, Jonathan P.H.; Yan, Mengyong; Heuijerjens, Jennifer; Carter, Lisa; Abolhassani, Ayda; Frosch, Jennifer; Wallace, Rebecca; Flutter, Barry; Capsomidis, Anna; Hubank, Mike; Klein, Nigel; Callard, Robin; Gustafsson, Kenth; Anderson, John

    2015-01-01

    Purpose The majority of circulating human γδT lymphocytes are of the Vγ9Vδ2 lineage, and have TCR specificity for non-peptide phosphoantigens. Previous attempts to stimulate and expand these cells have therefore focussed on stimulation using ligands of the Vγ9Vδ2 receptor, whilst relatively little is known about variant blood γδT subsets and their potential role in cancer immunotherapy. Experimental Design To expand the full repertoire of γδT without bias towards specific T cell receptors, we made use of artificial antigen presenting cells loaded with an anti gamma delta T cell receptor antibody that promoted unbiased expansion of the γδT repertoire. Expanded cells from adult blood donors were sorted into 3 populations expressing respectively Vδ2 TCR chains (Vδ2+), Vδ1 chains (Vδ1+) and TCR of other delta chain subtypes (Vδ1negVδ2neg) Results Both freshly isolated and expanded cells showed heterogeneity of differentiation markers, with a less differentiated phenotype in the Vδ1 and Vδ1negVδ2neg populations. Expanded cells were largely of an effector memory phenotype although there were higher numbers of less differentiated cells in the Vδ1+ and Vδ1negVδ2neg populations. Using neuroblastoma tumor cells and the anti-GD2 therapeutic monoclonal antibody ch14.18 as a model system, all three populations showed clinically relevant cytotoxicity. Whilst killing by expanded Vδ2 cells was predominantly antibody dependent and proportionate to upregulated CD16, Vδ1 cells killed by antibody independent mechanisms. Conclusions In conclusion we have demonstrated that polyclonal expanded populations of γδT cells are capable of both antibody dependent and independent effector functions in neuroblastoma. PMID:24893631

  9. Direct recognition by alphabeta cytolytic T cells of Hfe, a MHC class Ib molecule without antigen-presenting function.

    PubMed

    Rohrlich, Pierre S; Fazilleau, Nicolas; Ginhoux, Florent; Firat, Hüseyin; Michel, Frédérique; Cochet, Madeleine; Laham, Nihay; Roth, Marie Paule; Pascolo, Steve; Nato, Faridabano; Coppin, Hélène; Charneau, Pierre; Danos, Olivier; Acuto, Oreste; Ehrlich, Rachel; Kanellopoulos, Jean; Lemonnier, François A

    2005-09-01

    Crystallographic analysis of human Hfe has documented an overall structure similar to classical (class Ia) MHC molecules with a peptide binding groove deprived of ligand. Thus, to address the question of whether alphabeta T cells could recognize MHC molecules independently of bound ligands, we studied human and mouse Hfe interactions with T lymphocytes. We provide formal evidence of direct cytolytic recognition of human Hfe by mouse alphabeta T cell receptors (TCR) in HLA-A*0201 transgenic mice and that this interaction results in ZAP-70 phosphorylation. Furthermore, direct recognition of mouse Hfe molecules by cytotoxic T lymphocytes (CTLs) was demonstrated in DBA/2 Hfe knockout mice. These CTLs express predominantly two T cell antigen receptor alpha variable gene segments (AV6.1 and AV6.6). Interestingly, in wild-type mice we identified a subset of CD8+ T cells positively selected by Hfe that expresses the AV6.1/AV6.6 gene segments. T cell antigen receptor recognition of MHC molecules independently of bound ligand has potential general implications in alloreactivity and identifies in the Hfe case a cognitive link supporting the concept that the immune system could be involved in the control of iron metabolism. PMID:16123136

  10. A minor subset of Batf3-dependent antigen presenting cells in islets of Langerhans is essential for the development of autoimmune diabetes

    PubMed Central

    Ferris, Stephen T.; Carrero, Javier A.; Mohan, James F.; Calderon, Boris; Murphy, Kenneth M.; Unanue, Emil R.

    2014-01-01

    Summary Autoimmune diabetes is characterized by inflammatory infiltration; however the initiating events are poorly understood. We found that the islets of Langerhans in young non-obese diabetic (NOD) mice contained two antigen presenting cell (APC) populations: a major macrophage and a minor CD103+ dendritic cell (DC) population. By four weeks of age, CD4+ T cells entered islets coincident with an increase of CD103+ DCs. In order to examine the role of the CD103+ DCs in diabetes, we examined Batf3-deficient NOD mice that lacked the CD103+ DCs in islets and pancreatic lymph nodes. This led to a lack of autoreactive T cells in islets and, importantly, no incidence of diabetes. Additional examination revealed that presentation of major histocompatibility complex (MHC) class I epitopes in the pancreatic lymph nodes was absent with a partial impairment of MHC class II presentation. Altogether, this study reveals that CD103+ DCs were essential for autoimmune diabetes development. PMID:25367577

  11. Cathepsin B in antigen-presenting cells controls mediators of the Th1 immune response during Leishmania major infection.

    PubMed

    Gonzalez-Leal, Iris J; Röger, Bianca; Schwarz, Angela; Schirmeister, Tanja; Reinheckel, Thomas; Lutz, Manfred B; Moll, Heidrun

    2014-09-01

    Resistance and susceptibility to Leishmania major infection in the murine model is determined by the capacity of the host to mount either a protective Th1 response or a Th2 response associated with disease progression. Previous reports involving the use of cysteine cathepsin inhibitors indicated that cathepsins B (Ctsb) and L (Ctsl) play important roles in Th1/Th2 polarization during L. major infection in both susceptible and resistant mouse strains. Although it was hypothesized that these effects are a consequence of differential patterns of antigen processing, the mechanisms underlying these differences were not further investigated. Given the pivotal roles that dendritic cells and macrophages play during Leishmania infection, we generated bone-marrow derived dendritic cells (BMDC) and macrophages (BMM) from Ctsb-/- and Ctsl-/- mice, and studied the effects of Ctsb and Ctsl deficiency on the survival of L. major in infected cells. Furthermore, the signals used by dendritic cells to instruct Th cell polarization were addressed: the expression of MHC class II and co-stimulatory molecules, and cytokine production. We found that Ctsb-/- BMDC express higher levels of MHC class II molecules than wild-type (WT) and Ctsl-/- BMDC, while there were no significant differences in the expression of co-stimulatory molecules between cathepsin-deficient and WT cells. Moreover, both BMDC and BMM from Ctsb-/- mice significantly up-regulated the levels of interleukin 12 (IL-12) expression, a key Th1-inducing cytokine. These findings indicate that Ctsb-/- BMDC display more pro-Th1 properties than their WT and Ctsl-/- counterparts, and therefore suggest that Ctsb down-regulates the Th1 response to L. major. Moreover, they propose a novel role for Ctsb as a regulator of cytokine expression. PMID:25255101

  12. Transduction of Human Antigen-Presenting Cells with Integrase-Defective Lentiviral Vector Enables Functional Expansion of Primed Antigen-Specific CD8+ T Cells

    PubMed Central

    Bona, Roberta; Michelini, Zuleika; Leone, Pasqualina; Macchia, Iole; Klotman, Mary E.; Salvatore, Mirella

    2010-01-01

    Abstract Nonintegrating lentiviral vectors are being developed as a efficient and safe delivery system for both gene therapy and vaccine purposes. Several reports have demonstrated that a single immunization with integration-defective lentiviral vectors (IDLVs) delivering viral or tumor model antigens in mice was able to elicit broad and long-lasting specific immune responses in the absence of vector integration. At present, no evidence has been reported showing that IDLVs are able to expand preexisting immune responses in the human context. In the present study, we demonstrate that infection of human antigen-presenting cells (APCs), such as monocyte-derived dendritic cells (DCs) and macrophages with IDLVs expressing influenza matrix M1 protein resulted in effective induction of in vitro expansion of M1-primed CD8+ T cells, as evaluated by both pentamer staining and cytokine production. This is the first demonstration that IDLVs represent an efficient delivery system for gene transfer and expression in human APCs, useful for immunotherapeutic applications. PMID:20210625

  13. Half-antibody functionalized lipid-polymer hybrid nanoparticles for targeted drug delivery to carcinoembryonic antigen presenting pancreatic cancer cells.

    PubMed

    Hu, Che-Ming Jack; Kaushal, Sharmeela; Tran Cao, Hop S; Aryal, Santosh; Sartor, Marta; Esener, Sadik; Bouvet, Michael; Zhang, Liangfang

    2010-06-01

    Current chemotherapy regimens against pancreatic cancer are met with little success as poor tumor vascularization significantly limits the delivery of oncological drugs. High-dose targeted drug delivery, through which a drug delivery vehicle releases a large payload upon tumor localization, is thus a promising alternative strategy against this lethal disease. Herein, we synthesize anti-carcinoembryonic antigen (CEA) half-antibody conjugated lipid-polymer hybrid nanoparticles and characterize their ligand conjugation yields, physicochemical properties, and targeting ability against pancreatic cancer cells. Under the same drug loading, the half-antibody targeted nanoparticles show enhanced cancer killing effect compared to the corresponding nontargeted nanoparticles. PMID:20394436

  14. Insulin-like growth factor I messenger RNA and protein are expressed in the human lymph node and distinctly confined to subtypes of macrophages, antigen-presenting cells, lymphocytes and endothelial cells.

    PubMed

    Oberlin, Dominique; Fellbaum, Christian; Eppler, Elisabeth

    2009-11-01

    Insulin-like growth factor I (IGF-I) is a potent hormone that stimulates growth and differentiation and inhibits apoptosis in numerous tissues. Preliminary evidence suggests that IGF-I exerts differentiating, mitogenic and restoring activities in the immune system but the sites of synthesis of local IGF-I are unknown. Identification of these sites would allow the functional role of local IGF-I to be clarified. The presence of IGF-I in non-immune cells suggests that it acts as a trophic factor, while its occurrence in subtypes of lymphocytes or antigen-presenting cells indicates paracrine/autocrine direct regulatory involvement of IGF-I in the human immune response. The present study investigated the location of IGF-I messenger RNA and protein on archival human lymph node samples by in situ hybridization, immunohistochemistry and double immunofluorescence staining using an IGF-I probe and antisera specific for human IGF-I and CD3 (T lymphocytes), CD20 (B lymphocytes), CD68 (macrophages), CD21 (follicular dendritic cells), S100 (interdigitating dendritic cells) and podoplanin (fibroblastic reticular cells). Numerous cells within the B- and T-cell compartments expressed the IGF-I gene, and the majority of these cells were identified as macrophages. Solitary follicular dendritic cells exhibited IGF-I. A few T lymphocytes, and no B lymphocytes, contained IGF-I immunoreactive material. Furthermore, IGF-I immunoreactive cells outside the follicles that did not react with CD3, CD20, S100 or podoplanin markers were identified as high-endothelial venule (HEV) cells. From this we conclude that the main task of IGF-I in human non-tumoral lymph node may be autocrine and paracrine regulation of the differentiation, stimulation and survival of lymphocytes, antigen-presenting cells and macrophages and the differentiation and maintenance of HEV cells. PMID:20067534

  15. Insulin-like growth factor I messenger RNA and protein are expressed in the human lymph node and distinctly confined to subtypes of macrophages, antigen-presenting cells, lymphocytes and endothelial cells

    PubMed Central

    Oberlin, Dominique; Fellbaum, Christian; Eppler, Elisabeth

    2009-01-01

    Insulin-like growth factor I (IGF-I) is a potent hormone that stimulates growth and differentiation and inhibits apoptosis in numerous tissues. Preliminary evidence suggests that IGF-I exerts differentiating, mitogenic and restoring activities in the immune system but the sites of synthesis of local IGF-I are unknown. Identification of these sites would allow the functional role of local IGF-I to be clarified. The presence of IGF-I in non-immune cells suggests that it acts as a trophic factor, while its occurrence in subtypes of lymphocytes or antigen-presenting cells indicates paracrine/autocrine direct regulatory involvement of IGF-I in the human immune response. The present study investigated the location of IGF-I messenger RNA and protein on archival human lymph node samples by in situ hybridization, immunohistochemistry and double immunofluorescence staining using an IGF-I probe and antisera specific for human IGF-I and CD3 (T lymphocytes), CD20 (B lymphocytes), CD68 (macrophages), CD21 (follicular dendritic cells), S100 (interdigitating dendritic cells) and podoplanin (fibroblastic reticular cells). Numerous cells within the B- and T-cell compartments expressed the IGF-I gene, and the majority of these cells were identified as macrophages. Solitary follicular dendritic cells exhibited IGF-I. A few T lymphocytes, and no B lymphocytes, contained IGF-I immunoreactive material. Furthermore, IGF-I immunoreactive cells outside the follicles that did not react with CD3, CD20, S100 or podoplanin markers were identified as high-endothelial venule (HEV) cells. From this we conclude that the main task of IGF-I in human non-tumoral lymph node may be autocrine and paracrine regulation of the differentiation, stimulation and survival of lymphocytes, antigen-presenting cells and macrophages and the differentiation and maintenance of HEV cells. PMID:20067534

  16. Rapid Detection of Dendritic Cell and Monocyte Disorders Using CD4 as a Lineage Marker of the Human Peripheral Blood Antigen-Presenting Cell Compartment

    PubMed Central

    Jardine, Laura; Barge, Dawn; Ames-Draycott, Ashley; Pagan, Sarah; Cookson, Sharon; Spickett, Gavin; Haniffa, Muzlifah; Collin, Matthew; Bigley, Venetia

    2013-01-01

    Dendritic cells (DCs) and monocytes are critical regulators and effectors of innate and adaptive immune responses. Monocyte expansion has been described in many pathological states while monocyte and DC deficiency syndromes are relatively recent additions to the catalog of human primary immunodeficiency disorders. Clinically applicable screening tests to diagnose and monitor these conditions are lacking. Conventional strategies for identifying human DCs and monocytes have been based on the use of a lineage gate to exclude lymphocytes, thus preventing simultaneous detection of DCs, monocytes, and lymphocyte subsets. Here we demonstrate that CD4 is a reliable lineage marker for the human peripheral blood antigen-presenting cell compartment that can be used to identify DCs and monocytes in parallel with lymphocytes. Based on this principle, simple modification of a standard lymphocyte phenotyping assay permits simultaneous enumeration of four lymphocyte and five DC/monocyte populations from a single sample. This approach is applicable to clinical samples and facilitates the diagnosis of DC and monocyte disorders in a wide range of clinical settings, including genetic deficiency, neoplasia, and inflammation. PMID:24416034

  17. Ternary complex consisting of DNA, polycation, and a natural polysaccharide of schizophyllan to induce cellular uptake by antigen presenting cells.

    PubMed

    Takeda, Yoichi; Shimada, Naohiko; Kaneko, Kenji; Shinkai, Seiji; Sakurai, Kazuo

    2007-04-01

    A natural polysaccharide called schizophyllan (SPG) can form a complex with polynucleotides, and the complex has been shown to deliver biofunctional short DNAs such as antisense DNAs and CpG-DNAs. Although it is a novel and efficient method, there is a drawback: attachment of homo-polynucleotide tails [for example, poly(dA) or poly(C)] to the end of DNA is necessary to stabilize the complex, because DNA heterosequences cannot bind to SPG. The aim of this paper is to present an alternative method in which SPG/DNA complexes can be made without using the tails. The basic strategy is as follows: since SPG can form hydrophobic domains in aqueous solutions, hydrophobic objects should be encapsulated by this domain. DNA alone is highly hydrophilic; however, once DNA/polycation complexes are made, they should be included by the SPG hydrophobic domain. The aim of this paper is to prove the formation of the polycation/DNA/SPG ternary complex. Gel electrophoresis showed that presence of SPG influenced the migration pattern of polycation+DNA mixtures. With increasing the SPG ratio, the zeta potential (zeta) of the polycation+DNA+SPG mixture decreased drastically to reach almost zeta = 0 and the particle size distributions were altered due to the ternary complex formation. Confocal laser scanning microscopy revealed that the polycation/DNA/SPG ternary complexes showed high uptake efficiency when the complexes were exposed to macrophage-like cells (J774.A1). IL-12 secretion was enhanced when CpG-DNA was added as the ternary complex. These features can be ascribed to the fact that J774.A1 has a SPG recognition site called Dectin-1 on the cellular surface and the ternary complex can be ingested by this pathway. PMID:17328571

  18. A polysaccharide carrier to effectively deliver native phosphodiester CpG DNA to antigen-presenting cells.

    PubMed

    Shimada, Naohiko; Coban, Cevayir; Takeda, Yoichi; Mizu, Masami; Minari, Jusaku; Anada, Takahisa; Torii, Yuichi; Shinkai, Seiji; Akira, Shizuo; Ishii, Ken J; Sakurai, Kazuo

    2007-01-01

    Oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNAs) activate the vertebrate innate immune system via toll-like receptor 9 (TLR-9). Although CpG DNA is a promising immunotherapeutic agent, its short circulation time in biological fluids due to nuclease is the major drawback. This paper proposes that a natural polysaccharide called schizophyllan (SPG) can be used as an effective CpG DNA carrier because SPG can complex with CpG DNA and the resultant complex shows the nuclease resistance of the bound DNA. In order to increase cellular uptake in vitro, we chemically attached spermine, cholesterol, arginine octamer, or RGD peptide to SPG. The complexes made of the chemically modified SPG and CpG DNA having a phosphorothioate (PS) or phosphodiester (PO) backbone led to increased secretion of cytokines of about 4- to 15-fold, compared with the uncomplexed dose. Furthermore, when PO CpG DNA was complexed with unmodified SPG, the IL-12 level increased by almost 3- to 11-fold compared with the naked dose. The PO CpG DNA/unmodified SPG complex data suggested that unmodified SPG might effectively deliver PO in vivo due to the electrically neutral nature of unmodified SPG. When the complexed CpG DNAs were injected intraperitoneally, a large amount of IL-12 production was observed compared with the uncomplexed material. Both in vivo and vitro assays indicated that the SPG complex may be of use for CpG DNA therapy. PMID:17530815

  19. Origin and Role of a Subset of Tumor-Associated Neutrophils with Antigen-Presenting Cell Features in Early-Stage Human Lung Cancer.

    PubMed

    Singhal, Sunil; Bhojnagarwala, Pratik S; O'Brien, Shaun; Moon, Edmund K; Garfall, Alfred L; Rao, Abhishek S; Quatromoni, Jon G; Stephen, Tom Li; Litzky, Leslie; Deshpande, Charuhas; Feldman, Michael D; Hancock, Wayne W; Conejo-Garcia, Jose R; Albelda, Steven M; Eruslanov, Evgeniy B

    2016-07-11

    Based on studies in mouse tumor models, granulocytes appear to play a tumor-promoting role. However, there are limited data about the phenotype and function of tumor-associated neutrophils (TANs) in humans. Here, we identify a subset of TANs that exhibited characteristics of both neutrophils and antigen-presenting cells (APCs) in early-stage human lung cancer. These APC-like "hybrid neutrophils," which originate from CD11b(+)CD15(hi)CD10(-)CD16(low) immature progenitors, are able to cross-present antigens, as well as trigger and augment anti-tumor T cell responses. Interferon-γ and granulocyte-macrophage colony-stimulating factor are requisite factors in the tumor that, working through the Ikaros transcription factor, synergistically exert their APC-promoting effects on the progenitors. Overall, these data demonstrate the existence of a specialized TAN subset with anti-tumor capabilities in human cancer. PMID:27374224

  20. Mechanisms of antigen presentation to T cells in murine graft-versus-host disease: cross-presentation and the appearance of cross-presentation

    PubMed Central

    Wang, Xiaojian; Li, Hongmei; Matte-Martone, Catherine; Cui, Weiguo; Li, Ning; Tan, Hung Sheng; Roopenian, Derry

    2011-01-01

    Recipient antigen-presenting cells (APCs) initiate GVHD by directly presenting host minor histocompatibility antigens (miHAs) to donor CD8 cells. However, later after transplantation, host APCs are replaced by donor APCs, and if pathogenic CD8 cells continue to require APC stimulation, then donor APCs must cross-present host miHAs. Consistent with this, CD8-mediated GVHD is reduced when donor APCs are MHC class I−. To study cross-presentation, we used hosts that express defined MHC class I Kb-restricted miHAs, crossed to Kb-deficient backgrounds, such that these antigens cannot be directly presented. Cross-priming was surprisingly efficient, whether antigen was restricted to the hematopoietic or nonhematopoietic compartments. Cross-primed CD8 cells were cytolytic and produced IFN-γ. CD8 cells were exclusively primed by donor CD11c+ cells, and optimal cross-priming required that they are stimulated by both type I IFNs and CD40L. In studying which donor APCs acquire host miHAs, we made the surprising discovery that there was a large-scale transfer of transmembrane proteins from irradiated hosts, including MHC class I–peptide complexes, to donor cells, including dendritic cells. Donor dendritic cells that acquired host MHC class I–peptide complexes were potent stimulators of peptide-specific T cells. These studies identify new therapeutic targets for GVHD treatment and a novel mechanism whereby donor APCs prime host-reactive T cells. PMID:21963602

  1. Phenotypic and Functional Properties of Human Steady State CD14+ and CD1a+ Antigen Presenting Cells and Epidermal Langerhans Cells

    PubMed Central

    Fehres, Cynthia. M.; Bruijns, Sven C. M.; Sotthewes, Brigit N.; Kalay, Hakan; Schaffer, Lana; Head, Steven R.; de Gruijl, Tanja D.; Garcia-Vallejo, Juan J.; van Kooyk, Yvette

    2015-01-01

    Cutaneous antigen presenting cells (APCs) are critical for the induction and regulation of skin immune responses. The human skin contains phenotypically and functionally distinct APCs subsets that are present at two separated locations. While CD1ahigh LCs form a dense network in the epidermis, the CD14+ and CD1a+ APCs reside in the dermal compartment. A better understanding of the biology of human skin APC subsets is necessary for the improvement of vaccine strategies that use the skin as administration route. In particular, progress in the characterization of uptake and activatory receptors will certainly improve APC-targeting strategies in vaccination. Here we performed a detailed analysis of the expression and function of glycan-binding and pattern-recognition receptors in skin APC subsets. The results demonstrate that under steady state conditions human CD1a+ dermal dendritic cells (DCs) were phenotypically most mature as measured by the expression of CD83 and CD86, whereas the CD14+ cells showed a higher expression of the CLRs DC-SIGN, mannose receptor and DCIR and had potent antigen uptake capacity. Furthermore, steady state LCs showed superior antigen cross-presentation as compared to the dermal APC subsets. Our results also demonstrate that the TLR3 ligand polyribosinic-polyribocytidylic acid (pI:C) was the most potent stimulator of cytokine production by both LCs and dDCs. These studies warrant further exploration of human CD1a+ dDCs and LCs as target cells for cancer vaccination to induce anti-tumor immune responses. PMID:26605924

  2. Mitomycin C-treated antigen-presenting cells as a tool for control of allograft rejection and autoimmunity: from bench to bedside.

    PubMed

    Terness, Peter; Kleist, Christian; Simon, Helmut; Sandra-Petrescu, Flavius; Ehser, Sandra; Chuang, Jing-Jing; Mohr, Elisabeth; Jiga, Lucian; Greil, Johann; Opelz, Gerhard

    2009-07-01

    Cells have been previously used in experimental models for tolerance induction in organ transplantation and autoimmune diseases. One problem with the therapeutic use of cells is standardization of their preparation. We discuss an immunosuppressive strategy relying on cells irreversibly transformed by a chemotherapeutic drug. Dendritic cells (DCs) of transplant donors pretreated with mitomycin C (MMC) strongly prolonged rat heart allograft survival when injected into recipients before transplantation. Likewise, MMC-DCs loaded with myelin basic protein suppressed autoreactive T cells of MS patients in vitro and prevented experimental autoimmune encephalitis in mice. Comprehensive gene microarray analysis identified genes that possibly make up the suppressive phenotype, comprising glucocorticoid leucine zipper, immunoglobulin-like transcript 3, CD80, CD83, CD86, and apoptotic genes. Based on these findings, a hypothetical model of tolerance induction by MMC-treated DCs is delineated. Finally, we describe the first clinical application of MMC-treated monocyte-enriched donor cells in an attempt to control the rejection of a haploidentical stem cell transplant in a sensitized recipient and discuss the pros and cons of using MMC-treated antigen-presenting cells for tolerance induction. Although many questions remain, MMC-treated cells are a promising clinical tool for controlling allograft rejection and deleterious immune responses in autoimmune diseases. PMID:19393276

  3. Enhancement in number and function of antigen-presenting cells in the lymphatic tissue of rats after in vivo administration of diphenylhydantoin (DPH).

    PubMed Central

    Petrasch, S; Wacker, H H; Zou, P; Bechtold, D; Brittinger, G

    1989-01-01

    We present a monoclonal IgG1 antibody, KiMy1R, which is specific for macrophages and their derivatives in the lymphatic tissue of the rat, and evaluate the distribution of subsets of mononuclear cells in popliteal and para-aortal lymph nodes of Wistar rats after injection of 50 mg DPH into the hindpads. Compared with resting lymphatic tissue and lymph nodes of animals treated with phenobarbital, DPH induced a significant increase (P less than 0.01) of the proportion of phagocytic cells. Furthermore, the soluble antigen alkaline phosphatase was traced after inoculation into the footpads of rats: in locoregional lymph nodes the percentage (mean 12.8/10(3] and the total number (mean 476 x 10(3) cells/lymph node) of cells with membrane-bound or intracytoplasmic alkaline phosphatase, as detected by a monoclonal anti-alkaline phosphatase antibody, were significantly higher (P less than 0.001) in animals pretreated with DPH than in rats pretreated with phenobarbital (mean 2.1/10(3); 31.2 x 10(3) cells/lymph node) and in untreated animals (mean 1.9/10(3); 4.1 x 10(3) cells/lymph node). If verified in humans, the effect of DPH in enhancing the number and function of macrophages and other antigen-presenting cells may exert favourable effects in patients with impairment of the mononuclear phagocytic system. Images Fig. 1 PMID:2805417

  4. Tumour immunogenicity, antigen presentation and immunological barriers in cancer immunotherapy.

    PubMed

    Escors, David

    2014-01-01

    Since the beginning of the 20(th) century, scientists have tried to stimulate the anti-tumour activities of the immune system to fight against cancer. However, the scientific effort devoted on the development of cancer immunotherapy has not been translated into the expected clinical success. On the contrary, classical anti-neoplastic treatments such as surgery, radiotherapy and chemotherapy are the first line of treatment. Nevertheless, there is compelling evidence on the immunogenicity of cancer cells, and the capacity of the immune system to expand cancer-specific effector cytotoxic T cells. However, the effective activation of anti-cancer T cell responses strongly depends on efficient tumour antigen presentation from professional antigen presenting cells such as dendritic cells (DCs). Several strategies have been used to boost DC antigen presenting functions, but at the end cancer immunotherapy is not as effective as would be expected according to preclinical models. In this review we comment on these discrepancies, focusing our attention on the contribution of regulatory T cells and myeloid-derived suppressor cells to the lack of therapeutic success of DC-based cancer immunotherapy. PMID:24634791

  5. Antigen presentation by non-immune B-cell hybridoma clones: presentation of synthetic antigenic sites reveals clones that exhibit no specificity and clones that present only one epitope

    NASA Technical Reports Server (NTRS)

    Cohly, H. H.; Morrison, D. R.; Atassi, M. Z.

    1989-01-01

    Recently, we reported the preparation and antigen-presenting properties of hybridoma B-cell clones obtained after fusing non-secreting, non-antigen presenting Balb/c 653-myeloma cells with non-immune SJL spleen cells. It was found that antigen presentation at the clonal level can be specific or non-specific, depending on the particular B-cell clone. In the present work, one specific and one general presenter B-cell clones were tested for their epitope presentation ability to SJL T-cells that were specific to lysozyme or myoglobin. B-cell clone A1G12, a general presenter which presented both lysozyme and myoglobin to their respective T-cell lines, was found to present all five myoglobin epitopes while clone A1L16, a lysozyme specific presenter presented only one of the three epitopes of lysozyme. The latter reveals a hitherto unknown submolecular specificity (to a given epitope within a protein) for antigen presenting cells at the clonal level. Therefore, the specificity of T-cell recognition does not only derive from the T-cell but may also be dependent on the epitope specificity of the antigen-presenting B-cell.

  6. Differential Impact of PD-1 and/or Interleukin-10 Blockade on HIV-1-Specific CD4 T Cell and Antigen-Presenting Cell Functions

    PubMed Central

    Porichis, Filippos; Hart, Meghan G.; Zupkosky, Jennifer; Barblu, Lucie; Kwon, Douglas S.; McMullen, Ashley; Brennan, Thomas; Ahmed, Rafi; Freeman, Gordon J.; Kavanagh, Daniel G.

    2014-01-01

    that a population of white blood cells called CD4 T cells that targets the virus fails to work properly. At least part of this impairment is under the control of inhibitory mechanisms that can be blocked to improve the function of these CD4 T cells. In this report, we show that blocking one or two of the molecules involved, called PD-1 and IL-10, has different effects on the individual functions of these cells and that one is strongly improved. We investigate how these effects are caused by interactions between CD4 T cells and antigen-presenting cells. These observations can have implications for new therapeutic approaches in HIV infection. PMID:24352453

  7. Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

    PubMed Central

    Huls, M. Helen; Figliola, Matthew J.; Dawson, Margaret J.; Olivares, Simon; Kebriaei, Partow; Shpall, Elizabeth J.; Champlin, Richard E.; Singh, Harjeet; Cooper, Laurence J.N.

    2013-01-01

    The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-γ receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA

  8. Evaluation of cationic solid lipid microparticles as synthetic carriers for the targeted delivery of macromolecules to phagocytic antigen-presenting cells.

    PubMed

    Erni, Corinne; Suard, Catherine; Freitas, Sergio; Dreher, Donatus; Merkle, Hans P; Walter, Elke

    2002-12-01

    Biodegradable microparticles represent a promising carrier system for the efficient delivery of therapeutic macromolecules to phagocytic professional antigen-presenting cells (APC). Solid lipid microparticles (SLM) consisting of a tripalmitin matrix were prepared using a novel micromixer-based solvent extraction process. A positive surface charge was introduced by the incorporation of cationic lipids into the formulation. All obtained SLM were efficiently phagocytosed by primary macrophages in vitro. Complete intracellular degradation was observed already within 24 h, making SLM a suitable carrier for the immediate delivery of therapeutics to APC. Cationic SLM adsorbed plasmid DNA and bovine serum albumin (BSA) used as a model protein, and triggered the cellular internalization of the macromolecules by phagocytic macrophages. Surprisingly, the cationic SLM also triggered the internalization of these molecules by non-phagocytic 293 cells. This was probably due to the detachment of nanocomplexes formed of cationic lipid and DNA or BSA, respectively, from the surface of DNA- or BSA-loaded SLM and their subsequent uptake into the cells. Transfection efficiency of the DNA-loaded SLM was most pronounced in non-phagocytic cells and was not detected in the macrophage cell line or in primary macrophages. Our further studies revealed that cytotoxic effects of cationic SLM were more pronounced in the phagocytic cells, which could be explained by the very rapid uptake and degradation of the cationic SLM in these cells. In conclusion, SLM may provide a new, efficient means for the immediate intracellular delivery of therapeutic macromolecules into APC. Caution is warranted for cationic carriers, which may accentuate cytotoxic effects in the phagocytic cells. PMID:12322988

  9. An experimental subunit vaccine based on Bluetongue virus 4 VP2 protein fused to an antigen-presenting cells single chain antibody elicits cellular and humoral immune responses in cattle, guinea pigs and IFNAR(-/-) mice.

    PubMed

    Legisa, D M; Perez Aguirreburualde, M S; Gonzalez, F N; Marin-Lopez, A; Ruiz, V; Wigdorovitz, A; Martinez-Escribano, J A; Ortego, J; Dus Santos, M J

    2015-05-21

    Bluetongue virus (BTV), the causative agent of bluetongue disease (BT) in domestic and wild ruminants, is worldwide distributed. A total of 27 serotypes have been described so far, and several outbreaks have been reported. Vaccination is critical for controlling the spread of BTV. In the last years, subunit vaccines, viral vector vaccines and reverse genetic-based vaccines have emerged as new alternatives to conventional ones. In this study, we developed an experimental subunit vaccine against BTV4, with the benefit of targeting the recombinant protein to antigen-presenting cells. The VP2 protein from an Argentine BTV4 isolate was expressed alone or fused to the antigen presenting cell homing (APCH) molecule, in the baculovirus insect cell expression system. The immunogenicity of both proteins was evaluated in guinea pigs and cattle. Titers of specific neutralizing antibodies in guinea pigs and cattle immunized with VP2 or APCH-VP2 were high and similar to those induced by a conventional inactivated vaccine. The immunogenicity of recombinant proteins was further studied in the IFNAR(-/-) mouse model where the fusion of VP2 to APCH enhanced the cellular immune response and the neutralizing activity induced by VP2. PMID:25858859

  10. Influence of Cationic Lipid Composition on Gene Silencing Properties of Lipid Nanoparticle Formulations of siRNA in Antigen-Presenting Cells

    PubMed Central

    Basha, Genc; Novobrantseva, Tatiana I; Rosin, Nicole; Tam, Yuen Yi C; Hafez, Ismail M; Wong, Matthew K; Sugo, Tsukasa; Ruda, Vera M; Qin, June; Klebanov, Boris; Ciufolini, Marco; Akinc, Akin; Tam, Ying K; Hope, Michael J; Cullis, Pieter R

    2011-01-01

    Lipid nanoparticles (LNPs) are currently the most effective in vivo delivery systems for silencing target genes in hepatocytes employing small interfering RNA. Antigen-presenting cells (APCs) are also potential targets for LNP siRNA. We examined the uptake, intracellular trafficking, and gene silencing potency in primary bone marrow macrophages (bmMΦ) and dendritic cells of siRNA formulated in LNPs containing four different ionizable cationic lipids namely DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA. LNPs containing DLinKC2-DMA were the most potent formulations as determined by their ability to inhibit the production of GAPDH target protein. Also, LNPs containing DLinKC2-DMA were the most potent intracellular delivery agents as indicated by confocal studies of endosomal versus cytoplamic siRNA location using fluorescently labeled siRNA. DLinK-DMA and DLinKC2-DMA formulations exhibited improved gene silencing potencies relative to DLinDMA but were less toxic. In vivo results showed that LNP siRNA systems containing DLinKC2-DMA are effective agents for silencing GAPDH in APCs in the spleen and peritoneal cavity following systemic administration. Gene silencing in APCs was RNAi mediated and the use of larger LNPs resulted in substantially reduced hepatocyte silencing, while similar efficacy was maintained in APCs. These results are discussed with regard to the potential of LNP siRNA formulations to treat immunologically mediated diseases. PMID:21971424

  11. Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells.

    PubMed

    Pecora, Andrea; Malacari, Darío A; Pérez Aguirreburualde, María S; Bellido, Demian; Escribano, José M; Dus Santos, María J; Wigdorovitz, Andrés

    2015-01-01

    Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5 μg of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle. PMID:25697468

  12. Limited Density of an Antigen Presented by RMA-S Cells Requires B7-1/CD28 Signaling to Enhance T-Cell Immunity at the Effector Phase

    PubMed Central

    Li, Xiao-Lin; Sluijter, Marjolein; Doorduijn, Elien M.; Kale, Shubha P.; McFerrin, Harris; Liu, Yong-Yu; Li, Yan; Mottamal, Madhusoodanan; Yao, Xin; Du, Fengkun; Gu, Baihan; Hoang, Kim; Nguyen, Yen H.; Taylor, Nichelle; Stephens, Chelsea R.; van Hall, Thorbald; Zhang, Qian-Jin

    2014-01-01

    The association of B7-1/CD28 between antigen presenting cells (APCs) and T-cells provides a second signal to proliferate and activate T-cell immunity at the induction phase. Many reports indicate that tumor cells transfected with B7-1 induced augmented antitumor immunity at the induction phase by mimicking APC function; however, the function of B7-1 on antitumor immunity at the effector phase is unknown. Here, we report direct evidence of enhanced T-cell antitumor immunity at the effector phase by the B7-1 molecule. Our experiments in vivo and in vitro indicated that reactivity of antigen-specific monoclonal and polyclonal T-cell effectors against a Lass5 epitope presented by RMA-S cells is increased when the cells expressed B7-1. Use of either anti-B7-1 or anti-CD28 antibodies to block the B7-1/CD28 association reduced reactivity of the T effectors against B7-1 positive RMA-S cells. Transfection of Lass5 cDNA into or pulse of Lass5 peptide onto B7-1 positive RMA-S cells overcomes the requirement of the B7-1/CD28 signal for T effector response. To our knowledge, the data offers, for the first time, strong evidence that supports the requirement of B7-1/CD28 secondary signal at the effector phase of antitumor T-cell immunity being dependent on the density of an antigenic peptide. PMID:25383875

  13. Enhanced immune stimulation by a therapeutic lymphoma tumor antigen vaccine produced in insect cells involves mannose receptor targeting to antigen presenting cells.

    PubMed

    Betting, David J; Mu, Xi Y; Kafi, Kamran; McDonnel, Desmond; Rosas, Francisco; Gold, Daniel P; Timmerman, John M

    2009-01-01

    Therapeutic vaccination of lymphoma patients with tumor-specific immunoglobulin (idiotype, Id) coupled to the carrier protein keyhole limpet hemocyanin (Id-KLH) is undergoing clinical investigation, and methods to improve the immunogenicity of these and other protein tumor antigen vaccines are being sought. Id proteins can be produced via tumor-myeloma hybridomas or recombinant methods in mammalian, bacteria, or insect cells. We now demonstrate that terminal mannose residues, characteristic of recombinant proteins produced in insect cells, yield Id proteins with significantly enhanced immunostimulatory properties compared to Id proteins derived from mammalian cells. Recombinant baculovirus-infected insect cell-derived Id showed higher binding to and activation of human dendritic cells mediated by mannose receptors. In vivo, insect cell-derived Id elicited higher levels of tumor-specific CD8+ cytotoxic T lymphocyte (CTL) and improved eradication of pre-established murine lymphoma. Insect cell and mammalian Id generated similar levels of tumor-specific antibodies, showing no impairment in antibody responses to native tumor antigen despite the glycoslylation differences in the immunogen. Combining insect cell production and maleimide-based KLH conjugation offered the highest levels of anti-tumor immunity. Our data comparing sources of recombinant Id protein tumor antigens used in therapeutic cancer vaccines demonstrate that insect cell-derived antigens can offer several immunologic advantages over proteins derived from mammalian sources. PMID:19000731

  14. Enhanced immune stimulation by a therapeutic lymphoma tumor antigen vaccine produced in insect cells involves mannose receptor targeting to antigen presenting cells

    PubMed Central

    Betting, David J.; Mu, Xi Y.; Kafi, Kamran; McDonnel, Desmond; Rosas, Francisco; Gold, Daniel P.; Timmerman, John M.

    2009-01-01

    Therapeutic vaccination of lymphoma patients with tumor-specific immunoglobulin (idiotype, Id) coupled to the carrier protein keyhole limpet hemocyanin (Id-KLH) is undergoing clinical investigation, and methods to improve the immunogenicity of these and other protein tumor antigen vaccines are being sought. Id proteins can be produced via tumor-myeloma hybridomas or recombinant methods in mammalian, bacteria, or insect cells. We now demonstrate that terminal mannose residues, characteristic of recombinant proteins produced in insect cells, yield Id proteins with significantly enhanced immunostimulatory properties compared to Id proteins derived from mammalian cells. Recombinant baculovirus-infected insect cell-derived Id showed higher binding to and activation of human dendritic cells mediated by mannose receptors. In vivo, insect cell-derived Id elicited higher levels of tumor-specific CD8+ CTL and improved eradication of pre-established murine lymphoma. Insect cell and mammalian Id generated similar levels of tumor-specific antibodies, showing no impairment in antibody responses to native tumor antigen despite the glycoslylation differences in the immunogen. Combining insect cell production and maleimide-based KLH conjugation offered the highest levels of anti-tumor immunity. Our data comparing sources of recombinant Id protein tumor antigens used in therapeutic cancer vaccines demonstrate that insect cell-derived antigens can offer several immunologic advantages over proteins derived from mammalian sources. PMID:19000731

  15. Immunogenic, but Not Steady-State, Antigen Presentation Permits Regulatory T-Cells To Control CD8+ T-Cell Effector Differentiation by IL-2 Modulation

    PubMed Central

    McNally, Alice; McNally, Michael; Galea, Ryan; Thomas, Ranjeny; Steptoe, Raymond J.

    2014-01-01

    Absorption of IL-2 is one proposed mechanism of CD4+CD25+FoxP3+ regulatory T cell (Treg) suppression. Direct in vivo experimental evidence for this has recently been obtained. While modulation of IL-2 bioavailability controls CD8+ T-cell effector differentiation under strongly immunogenic conditions it is not known whether Treg modulate CD8+ T cell responses through this mechanism under steady-state conditions. Here we assess this using a mouse model in which dendritic cells (DC) are manipulated to present cognate antigen to CD8+ T cells either in the steady-state or after activation. Our observations show that Treg exert a check on expansion and effector differentiation of CD8+ T cells under strongly immunogenic conditions associated with TLR ligand activation of DC, and this is mediated by limiting IL-2 availability. In contrast, when DC remain unactivated, depletion of Treg has little apparent effect on effector differentiation or IL-2 homeostasis. We conclude that while modulation of IL-2 homeostasis is an important mechanism through which Treg control CD8+ effector differentiation under immunogenic conditions, this mechanism plays little role in modulating CD8+ T-cell differentiation under steady-state conditions. PMID:24454872

  16. Differential Effects of a Saturated and a Monounsaturated Fatty Acid on MHC Class I Antigen Presentation

    PubMed Central

    Shaikh, S. R.; Mitchell, D.; Carroll, E.; Li, M.; Schneck, J.; Edidin, M.

    2009-01-01

    Lipid overload, associated with metabolic disorders, occurs when fatty acids accumulate in non-adipose tissues. Cells of these tissues use major histocompatibility complex (MHC) class I molecules to present antigen to T cells in order to eliminate pathogens. As obesity is associated with impaired immune responses, we tested the hypothesis that the early stages of lipid overload with saturated fatty acids (SFA) alters MHC class I antigen presentation. Antigen presenting cells (APC) were treated with either the saturated palmitic acid (PA), abundant in the high fat Western diet, or the monounsaturated oleic acid (OA), a component of the Mediterranean diet. PA-treatment lowered APC lysis by activated cytotoxic T lymphocytes and inhibited APC ability to stimulate naïve T cells. Inhibition of immune responses with PA was due to a significant reduction in MHC class I surface expression, inhibition in the rate of APC–T-cell conjugation, and lowering of plasma membrane F-actin levels. OA-treatment had no effect on antigen presentation and upon exposure with PA, prevented the phenotypic effects of PA. OA-treatment conferred protection against changes in antigen presentation by accumulating fatty acids into triglyceride-rich lipid droplets of APC. Our findings establish for the first time a link between the early stages of lipid overload and antigen presentation and suggest that dietary SFA could impair immunity by affecting MHC I-mediated antigen presentation; this could be prevented, paradoxically, by accumulation of triglycerides rich in monounsaturated fatty acids. PMID:18533931

  17. Differential effects of a saturated and a monounsaturated fatty acid on MHC class I antigen presentation.

    PubMed

    Shaikh, S R; Mitchell, D; Carroll, E; Li, M; Schneck, J; Edidin, M

    2008-07-01

    Lipid overload, associated with metabolic disorders, occurs when fatty acids accumulate in non-adipose tissues. Cells of these tissues use major histocompatibility complex (MHC) class I molecules to present antigen to T cells in order to eliminate pathogens. As obesity is associated with impaired immune responses, we tested the hypothesis that the early stages of lipid overload with saturated fatty acids (SFA) alters MHC class I antigen presentation. Antigen presenting cells (APC) were treated with either the saturated palmitic acid (PA), abundant in the high fat Western diet, or the monounsaturated oleic acid (OA), a component of the Mediterranean diet. PA-treatment lowered APC lysis by activated cytotoxic T lymphocytes and inhibited APC ability to stimulate naïve T cells. Inhibition of immune responses with PA was due to a significant reduction in MHC class I surface expression, inhibition in the rate of APC-T-cell conjugation, and lowering of plasma membrane F-actin levels. OA-treatment had no effect on antigen presentation and upon exposure with PA, prevented the phenotypic effects of PA. OA-treatment conferred protection against changes in antigen presentation by accumulating fatty acids into triglyceride-rich lipid droplets of APC. Our findings establish for the first time a link between the early stages of lipid overload and antigen presentation and suggest that dietary SFA could impair immunity by affecting MHC I-mediated antigen presentation; this could be prevented, paradoxically, by accumulation of triglycerides rich in monounsaturated fatty acids. PMID:18533931

  18. 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

  19. IgE-mediated enhancement of CD4+ T cell responses requires antigen presentation by CD8α− conventional dendritic cells

    PubMed Central

    Ding, Zhoujie; Dahlin, Joakim S.; Xu, Hui; Heyman, Birgitta

    2016-01-01

    IgE, forming an immune complex with small proteins, can enhance the specific antibody and CD4+ T cell responses in vivo. The effects require the presence of CD23 (Fcε-receptor II)+ B cells, which capture IgE-complexed antigens (Ag) in the circulation and transport them to splenic B cell follicles. In addition, also CD11c+ cells, which do not express CD23, are required for IgE-mediated enhancement of T cell responses. This suggests that some type of dendritic cell obtains IgE-Ag complexes from B cells and presents antigenic peptides to T cells. To elucidate the nature of this dendritic cell, mice were immunized with ovalbumin (OVA)-specific IgE and OVA, and different populations of CD11c+ cells, obtained from the spleens four hours after immunization, were tested for their ability to present OVA. CD8α− conventional dendritic cells (cDCs) were much more efficient in inducing specific CD4+ T cell proliferation ex vivo than were CD8α+ cDCs or plasmacytoid dendritic cells. Thus, IgE-Ag complexes administered intravenously are rapidly transported to the spleen by recirculating B cells where they are delivered to CD8α− cDCs which induce proliferation of CD4+ T cells. PMID:27306570

  20. IgE-mediated enhancement of CD4(+) T cell responses requires antigen presentation by CD8α(-) conventional dendritic cells.

    PubMed

    Ding, Zhoujie; Dahlin, Joakim S; Xu, Hui; Heyman, Birgitta

    2016-01-01

    IgE, forming an immune complex with small proteins, can enhance the specific antibody and CD4(+) T cell responses in vivo. The effects require the presence of CD23 (Fcε-receptor II)(+) B cells, which capture IgE-complexed antigens (Ag) in the circulation and transport them to splenic B cell follicles. In addition, also CD11c(+) cells, which do not express CD23, are required for IgE-mediated enhancement of T cell responses. This suggests that some type of dendritic cell obtains IgE-Ag complexes from B cells and presents antigenic peptides to T cells. To elucidate the nature of this dendritic cell, mice were immunized with ovalbumin (OVA)-specific IgE and OVA, and different populations of CD11c(+) cells, obtained from the spleens four hours after immunization, were tested for their ability to present OVA. CD8α(-) conventional dendritic cells (cDCs) were much more efficient in inducing specific CD4(+) T cell proliferation ex vivo than were CD8α(+) cDCs or plasmacytoid dendritic cells. Thus, IgE-Ag complexes administered intravenously are rapidly transported to the spleen by recirculating B cells where they are delivered to CD8α(-) cDCs which induce proliferation of CD4(+) T cells. PMID:27306570

  1. Murine Melanoma-Infiltrating Dendritic Cells Are Defective in Antigen Presenting Function Regardless of the Presence of CD4+CD25+ Regulatory T Cells

    PubMed Central

    Ataera, Haley; Hyde, Evelyn; Price, Kylie M.; Stoitzner, Patrizia; Ronchese, Franca

    2011-01-01

    Tumor-infiltrating dendritic cells are often ineffective at presenting tumor-derived antigen in vivo, a defect usually ascribed to the suppressive tumor environment. We investigated the effects of depleting CD4+CD25+ “natural” regulatory T cells (Treg) on the frequency, phenotype and function of total dendritic cell populations in B16.OVA tumors and in tumor-draining lymph nodes. Intraperitoneal injection of the anti-CD25 monoclonal antibody PC61 reduced Treg frequency in blood and tumors, but did not affect the frequency of tumor-infiltrating dendritic cells, or their expression of CD40, CD86 and MHCII. Tumor-infiltrating dendritic cells from PC61-treated or untreated mice induced the proliferation of allogeneic T cells in vitro, but could not induce proliferation of OVA-specific OTI and OTII T cells unless specific peptide antigen was added in culture. Some proliferation of naïve, OVA-specific OTI T cells, but not OTII T cells, was observed in the tumor-draining LN of mice carrying B16.OVA tumors, however, this was not improved by PC61 treatment. Experiments using RAG1−/− hosts adoptively transferred with OTI and CD25-depleted OTII cells also failed to show improved OTI and OTII T cell proliferation in vivo compared to C57BL/6 hosts. We conclude that the defective presentation of B16.OVA tumor antigen by tumor-infiltrating dendritic cells and in the tumor-draining lymph node is not due to the presence of “natural” CD4+CD25+ Treg. PMID:21390236

  2. Calcitonin Gene-Related Peptide-Exposed Endothelial Cells Bias Antigen Presentation to CD4+ T Cells toward a Th17 Response.

    PubMed

    Ding, Wanhong; Stohl, Lori L; Xu, Linghui; Zhou, Xi K; Manni, Michela; Wagner, John A; Granstein, Richard D

    2016-03-01

    Calcitonin gene-related peptide (CGRP) is a neuropeptide with well-established immunomodulatory functions. CGRP-containing nerves innervate dermal blood vessels and lymph nodes. We examined whether CGRP regulates the outcome of Ag presentation by Langerhans cells (LCs) to T cells through actions on microvascular endothelial cells (ECs). Exposure of primary murine dermal microvascular ECs (pDMECs) to CGRP followed by coculture with LCs, responsive CD4(+) T cells and Ag resulted in increased production of IL-6 and IL-17A accompanied by inhibition of IFN-γ, IL-4, and IL-22 compared with wells containing pDMECs treated with medium alone. Physical contact between ECs and LCs or T cells was not required for this effect and, except for IL-4, we demonstrated that IL-6 production by CGRP-treated pDMECs was involved in these effects. CD4(+) cells expressing cytoplasmic IL-17A were increased, whereas cells expressing cytoplasmic IFN-γ or IL-4 were decreased by the presence of CGRP-treated pDMECs. In addition, the level of retinoic acid receptor-related orphan receptor γt mRNA was significantly increased, whereas T-bet and GATA3 expression was inhibited. Immunization at the site of intradermally administered CGRP led to a similar bias in CD4(+) T cells from draining lymph node cells toward IL-17A and away from IFN-γ. Actions of nerve-derived CGRP on ECs may have important regulatory effects on the outcome of Ag presentation with consequences for the expression of inflammatory skin disorders involving Th17 cells. PMID:26829986

  3. Selectively Reduced Intracellular Proliferation of Salmonella enterica Serovar Typhimurium within APCs Limits Antigen Presentation and Development of a Rapid CD8 T Cell Response1

    PubMed Central

    Albaghdadi, Homam; Robinson, Nirmal; Finlay, Brett; Krishnan, Lakshmi; Sad, Subash

    2014-01-01

    Ag presentation to CD8+ T cells commences immediately after infection, which facilitates their rapid expansion and control of pathogen. This paradigm is not followed during infection with virulent Salmonella enterica serovar Typhimurium (ST), an intracellular bacterium that causes mortality in susceptible C57BL/6J mice within 7 days and a chronic infection in resistant mice (129 × 1SvJ). Infection of mice with OVA-expressing ST results in the development of a CD8+ T cell response that is detectable only after the second week of infection despite the early detectable bacterial burden. The mechanism behind the delayed CD8+ T cell activation was evaluated, and it was found that dendritic cells/macrophages or mice infected with ST-OVA failed to present Ag to OVA-specific CD8+ T cells. Lack of early Ag presentation was not rescued when mice or dendritic cells/macrophages were infected with an attenuated aroA mutant of ST or with mutants having defective Salmonella pathogenicity island I/II genes. Although extracellular ST proliferated extensively, the replication of ST was highly muted once inside macrophages. This muted intracellular proliferation of ST resulted in the generation of poor levels of intracellular Ag and peptide-MHC complex on the surface of dendritic cells. Additional experiments revealed that ST did not actively inhibit Ag presentation, rather it inhibited the uptake of another intracellular pathogen, Listeria monocytogenes, thereby causing inhibition of Ag presentation against L. monocytogenes. Taken together, this study reveals a dichotomy in the proliferation of ST and indicates that selectively reduced intra-cellular proliferation of virulent pathogens may be an important mechanism of immune evasion. PMID:19692639

  4. Exposure to T helper 2 cytokines in vivo before encounter with antigen selects for T helper subsets via alterations in antigen-presenting cell function.

    PubMed

    Cua, D J; Coffman, R L; Stohlman, S A

    1996-10-01

    The Th1 subset of CD4+ T cells mediate both delayed-type hypersensitivity (DTH) responses and experimental allergic encephalomyelitis (EAE). Th1 cells are induced by immunization of young adult female and older (> or = 10 wk of age) male SJL mice. By contrast, young adult (< or = 8 wk of age) male mice are characterized by the inability of immunization to induce either a DTH response or EAE, demonstrating a clear sex and age dependence to these Th1-mediated responses in SJL mice. T cell activation in age-matched female and male SJL mice was compared to understand the mechanism(s) of these differential responses. Here, we report that immunization of DTH responder female mice primes for Ag-specific secretion of IFN-gamma but not IL-4 and IL-10. In contrast, immunization of DTH nonresponder male mice primes Ag-specific T cells that secrete IL-4 and IL-10, but not IFN-gamma. Depletion of either IL-4 or IL-10 recovers DTH responsiveness in young adult male mice, demonstrating expansion of Th1 cells in these mice when Th2 cytokines are suppressed. The age- and sex-dependent inability to prime Th1 cells in young male mice is due to the functional absence of a macrophage APC population defined by co-expression of Mac-1 and Mac-3. To determine whether Th2 cytokines directly affect the APC's ability to support the priming of Th1 cells, Mac-3+ APC isolated from naive young male donors, which had been depleted of either IL-4 or IL-10, were transferred into DTH nonresponder males. Induction of DTH responses in these recipients demonstrates that in vivo suppression of Th2 cytokines enables the male-derived Mac-3+ APC to support priming of Th1 responses. These data indicate that, in addition to their regulatory roles in controlling preferential T cell subset expansion, exposure of APC to cytokines in vivo before the initial encounter with Ag may regulate induction of CD4+ T cell subsets. PMID:8816386

  5. Production of CXC and CC chemokines by human antigen-presenting cells in response to Lassa virus or closely related immunogenic viruses, and in cynomolgus monkeys with lassa fever.

    PubMed

    Pannetier, Delphine; Reynard, Stéphanie; Russier, Marion; Carnec, Xavier; Baize, Sylvain

    2014-01-01

    The pathogenesis of Lassa fever (LF), a hemorrhagic fever endemic to West Africa, remains unclear. We previously compared Lassa virus (LASV) with its genetically close, but nonpathogenic homolog Mopeia virus (MOPV) and demonstrated that the strong activation of antigen-presenting cells (APC), including type I IFN production, observed in response to MOPV probably plays a crucial role in controlling infection. We show here that human macrophages (MP) produce large amounts of CC and CXC chemokines in response to MOPV infection, whereas dendritic cells (DC) release only moderate amounts of CXC chemokines. However, in the presence of autologous T cells, DCs produced CC and CXC chemokines. Chemokines were produced in response to type I IFN synthesis, as the levels of both mediators were strongly correlated and the neutralization of type I IFN resulted in an inhibition of chemokine production. By contrast, LASV induced only low levels of CXCL-10 and CXCL-11 production. These differences in chemokine production may profoundly affect the generation of virus-specific T-cell responses and may therefore contribute to the difference of pathogenicity between these two viruses. In addition, a recombinant LASV (rLASV) harboring the NP-D389A/G392A mutations, which abolish the inhibition of type I IFN response by nucleoprotein (NP), induced the massive synthesis of CC and CXC chemokines in both DC and MP, confirming the crucial role of arenavirus NP in immunosuppression and pathogenicity. Finally, we confirmed, using PBMC samples and lymph nodes obtained from LASV-infected cynomolgus monkeys, that LF was associated with high levels of CXC chemokine mRNA synthesis, suggesting that the very early synthesis of these mediators may be correlated with a favourable outcome. PMID:24421914

  6. Production of CXC and CC Chemokines by Human Antigen-Presenting Cells in Response to Lassa Virus or Closely Related Immunogenic Viruses, and in Cynomolgus Monkeys with Lassa Fever

    PubMed Central

    Russier, Marion; Carnec, Xavier; Baize, Sylvain

    2014-01-01

    The pathogenesis of Lassa fever (LF), a hemorrhagic fever endemic to West Africa, remains unclear. We previously compared Lassa virus (LASV) with its genetically close, but nonpathogenic homolog Mopeia virus (MOPV) and demonstrated that the strong activation of antigen-presenting cells (APC), including type I IFN production, observed in response to MOPV probably plays a crucial role in controlling infection. We show here that human macrophages (MP) produce large amounts of CC and CXC chemokines in response to MOPV infection, whereas dendritic cells (DC) release only moderate amounts of CXC chemokines. However, in the presence of autologous T cells, DCs produced CC and CXC chemokines. Chemokines were produced in response to type I IFN synthesis, as the levels of both mediators were strongly correlated and the neutralization of type I IFN resulted in an inhibition of chemokine production. By contrast, LASV induced only low levels of CXCL-10 and CXCL-11 production. These differences in chemokine production may profoundly affect the generation of virus-specific T-cell responses and may therefore contribute to the difference of pathogenicity between these two viruses. In addition, a recombinant LASV (rLASV) harboring the NP-D389A/G392A mutations, which abolish the inhibition of type I IFN response by nucleoprotein (NP), induced the massive synthesis of CC and CXC chemokines in both DC and MP, confirming the crucial role of arenavirus NP in immunosuppression and pathogenicity. Finally, we confirmed, using PBMC samples and lymph nodes obtained from LASV-infected cynomolgus monkeys, that LF was associated with high levels of CXC chemokine mRNA synthesis, suggesting that the very early synthesis of these mediators may be correlated with a favourable outcome. PMID:24421914

  7. Bone marrow transplantation alters lung antigen presenting cells to promote TH17 response and the development of pneumonitis and fibrosis following gammaherpesvirus infection

    PubMed Central

    Zhou, Xiaofeng; Loomis-King, Hillary; Gurczynski, Stephen J.; Wilke, Carol A.; Konopka, Kristine E.; Ptaschinski, Catherine; Coomes, Stephanie M; Iwakura, Yoichiro; van Dyk, Linda F.; Lukacs, Nicholas W.; Moore, Bethany B.

    2015-01-01

    Hematopoietic stem cell transplantation (HSCT) efficacy is limited by numerous pulmonary complications. We developed a model of syngeneic bone marrow transplant (BMT) followed by infection with murine gamma herpesvirus (γHV-68) that results in pneumonitis and fibrosis and mimics human “non-infectious” HSCT complications. BMT mice experience increased early lytic replication, but establish viral latency by 21 days post infection (dpi). CD4 T cells in BMT mice are skewed towards IL-17A rather than IFN-γ production. Transplantation of bone marrow from Il-17a−/− donors or treatment with anti-IL-17A neutralization antibodies at late stages attenuates pneumonitis and fibrosis in infected BMT mice, suggesting that hematopoietic-derived IL-17A is essential for development of pathology. IL-17A directly influences activation and extracellular matrix production by lung mesenchymal cells. Lung CD11c+ cells of BMT mice secrete more TGF-β1, and pro-TH17 mRNAs for IL-23 and IL-6, and less TH1-promoting cytokine mRNA for IFN-γ but slightly more IL-12 mRNA in response to viral infection. Adoptive transfer of non-BMT lung CD11c-enriched cells restores robust TH1 response and suppresses aberrant TH17 response in BMT mice to improve lung pathology. Our data suggest “non-infectious” HSCT lung complications may reflect preceding viral infections and demonstrate that IL-17A neutralization may offer therapeutic advantage even after disease onset. PMID:26376362

  8. Parkinson's Disease-Related Proteins PINK1 and Parkin Repress Mitochondrial Antigen Presentation.

    PubMed

    Matheoud, Diana; Sugiura, Ayumu; Bellemare-Pelletier, Angélique; Laplante, Annie; Rondeau, Christiane; Chemali, Magali; Fazel, Ali; Bergeron, John J; Trudeau, Louis-Eric; Burelle, Yan; Gagnon, Etienne; McBride, Heidi M; Desjardins, Michel

    2016-07-14

    Antigen presentation is essential for establishing immune tolerance and for immune responses against infectious disease and cancer. Although antigen presentation can be mediated by autophagy, here we demonstrate a pathway for mitochondrial antigen presentation (MitAP) that relies on the generation and trafficking of mitochondrial-derived vesicles (MDVs) rather than on autophagy/mitophagy. We find that PINK1 and Parkin, two mitochondrial proteins linked to Parkinson's disease (PD), actively inhibit MDV formation and MitAP. In absence of PINK1 or Parkin, inflammatory conditions trigger MitAP in immune cells, both in vitro and in vivo. MitAP and the formation of MDVs require Rab9 and Sorting nexin 9, whose recruitment to mitochondria is inhibited by Parkin. The identification of PINK1 and Parkin as suppressors of an immune-response-eliciting pathway provoked by inflammation suggests new insights into PD pathology. PMID:27345367

  9. Major histocompatibility complex class I-intercellular adhesion molecule-1 association on the surface of target cells: implications for antigen presentation to cytotoxic T lymphocytes.

    PubMed

    Lebedeva, Tatiana; Anikeeva, Nadja; Kalams, Spyros A; Walker, Bruce D; Gaidarov, Ibragim; Keen, James H; Sykulev, Yuri

    2004-12-01

    Polarization and segregation of the T-cell receptor (TCR) and integrins upon productive cytotoxic T-lymphocyte (CTL) target cell encounters are well documented. Much less is known about the redistribution of major histocompatibility complex class I (MHC-I) and intercellular adhesion molecule-1 (ICAM-1) proteins on target cells interacting with CTLs. Here we show that human leucocyte antigen-A2 (HLA-A2) MHC-I and ICAM-1 are physically associated and recovered from both the raft fraction and the fraction of soluble membranes of target cells. Conjugation of target cells with surrogate CTLs, i.e. polystyrene beads loaded with antibodies specific for HLA-A2 and ICAM-1, induced the accumulation of membrane rafts, and beads loaded with ICAM-1-specific antibodies caused the selective recruitment of HLA-A2 MHC-I at the contact area of the target cells. Disruption of raft integrity on target cells led to a release of HLA-A2 and ICAM-1 from the raft fraction, abatement of HLA-A2 polarization, and diminished the ability of target cells bearing viral peptides to induce a Ca(2+) flux in virus-specific CTLs. These data suggest that productive engagement of ICAM-1 on target cells facilitates the polarization of MHC-I at the CTL-target cell interface, augmenting presentation of cognate peptide-MHC (pMHC) complexes to CTLs. We propose that ICAM-1-MHC-I association on the cell membrane is a mechanism that enhances the linkage between antigen recognition and early immunological synapse formation. PMID:15554924

  10. The Central Role of Antigen Presentation in Islets of Langerhans in Autoimmune Diabetes

    PubMed Central

    Calderon, Boris; Carrero, Javier A.; Unanue, Emil R.

    2014-01-01

    The islets of Langerhans normally contain resident antigen presenting cells (APCs), which in normal conditions are mostly represented by macrophages, with a few dendritic cells (DC). We present here the features of these islet APCs, making the point that they have a supportive function in islet homeostasis. Islet APCs express high levels of major histocompatibility complexes (MHC) molecules on their surfaces and are highly active in antigen presentation in the autoimmune diabetes of the NOD mouse: they do this by presenting peptides derived from molecules of the β-cells. These APCs also are instrumental in the localization of diabetogenic T cells into islets. The islet APC present exogenous peptides derived from secretory granules of the beta cell, giving rise to unique peptide-MHC complexes (pMHC) that activate those non-conventional T cells that bypass thymus selection. PMID:24556398

  11. The central role of antigen presentation in islets of Langerhans in autoimmune diabetes.

    PubMed

    Calderon, Boris; Carrero, Javier A; Unanue, Emil R

    2014-02-01

    The islets of Langerhans normally contain resident antigen presenting cells (APCs), which in normal conditions are mostly represented by macrophages, with a few dendritic cells (DC). We present here the features of these islet APCs, making the point that they have a supportive function in islet homeostasis. Islet APCs express high levels of major histocompatibility complexes (MHC) molecules on their surfaces and are highly active in antigen presentation in the autoimmune diabetes of the NOD mouse: they do this by presenting peptides derived from molecules of the β-cells. These APCs also are instrumental in the localization of diabetogenic T cells into islets. The islet APC present exogenous peptides derived from secretory granules of the β-cell, giving rise to unique peptide-MHC complexes (pMHC) that activate those non-conventional T cells that bypass thymus selection. PMID:24556398

  12. Phenotypic modulation and cytokine profiles of antigen presenting cells by European subtype 1 and 3 porcine reproductive and respiratory syndrome virus strains in vitro and in vivo.

    PubMed

    Weesendorp, Eefke; Stockhofe-Zurwieden, Norbert; Popma-De Graaf, Ditta J; Fijten, Helmi; Rebel, Johanna M J

    2013-12-27

    Porcine reproductive and respiratory syndrome virus (PRRSV) causes continuous problems in the pig industry, due to high costs of outbreaks and reduced welfare of diseased pigs. The severity of infection is, partly, dependent on the virus strain. Recently isolated Eastern-European subtype 3 strains are more pathogenic than the widespread subtype 1 strains. There is, however, almost no information available about the mechanisms involved in the pathogenicity of these subtype 3 strains. The objective of the present study was to characterize the in vitro and in vivo response of two European subtype 1 strains, Belgium A and Lelystad-Ter Huurne (LV), and a virulent subtype 3 strain, Lena, in bone marrow-derived dendritic cells (BM-DC) (in vitro) and alveolar macrophages (in vitro and in vivo). It was shown that infection with the Lena strain resulted in a higher apoptosis of cells in vitro and a higher level of infectivity in vitro and in vivo than the other virus strains. Furthermore, infection with Lena resulted in a small downregulation of the immunologically relevant cell surface molecules SLA-I, SLA-II and CD80/86 in vitro, and SLA-II in vivo. In spite of these differences, in vitro cytokine responses did not differ significantly between strains, except for the absence of IL-10 production by Lena in BM-DC. The higher infectivity, apoptosis and downregulation of the cell surface molecules, may have contributed to the increased pathogenicity of Lena, and have dampened specific immune responses. This could explain the delayed and decreased adaptive immune responses observed after infections with this strain. PMID:24120935

  13. MiR-381-3p Regulates the Antigen-Presenting Capability of Dendritic Cells and Represses Antituberculosis Cellular Immune Responses by Targeting CD1c.

    PubMed

    Wen, Qian; Zhou, Chaoying; Xiong, Wenjing; Su, Jing; He, Jianchun; Zhang, Shimeng; Du, Xialin; Liu, Sudong; Wang, Juanjuan; Ma, Li

    2016-07-15

    Tuberculosis is still the widest spread infectious disease in the world, and more in-depth studies are needed on the interaction between the pathogen and the host. Due to the highest lipid components in Mycobacterium tuberculosis, the CD1 family that specifically presents antigenic lipids plays important roles in the antituberculosis immunity, especially CD1c, which functions as the intracellular Ag inspector at the full intracellular range. However, downregulation of the CD1c mRNA level has been observed in M. tuberculosis-infected cells, which is consistent with the regulatory mechanism of miRNA on gene expression. In this study, through combinatory analysis of previous miRNA transcriptomic assays and bioinformatic predictions by web-based algorithms, miR-381-3p was predicted to bind the 3'-untranslated region of CD1c gene. In vivo expression of miR-381-3p in dendritic cells (DCs) of TB patients is higher than in DCs of healthy individuals, inversely related to CD1c. Suppression of CD1c expression in bacillus Calmette-Guérin (BCG)-infected DCs was accompanied with upregulation of miR-381-3p, whereas inhibition of miR-381-3p could reverse suppression of CD1c expression and promote T cell responses against BCG infection. Further study indicated that miR-381-3p is also one of the mediators of the immune suppressor IL-10. Collectively, these results demonstrated the mechanism that suppression of CD1c by BCG infection is mediated by miR-381-3p. This finding may provide a novel approach to boost immune responses to M. tuberculosis. PMID:27296666

  14. The Neck Region of the C-type Lectin DC-SIGN Regulates Its Surface Spatiotemporal Organization and Virus-binding Capacity on Antigen-presenting Cells*

    PubMed Central

    Manzo, Carlo; Torreno-Pina, Juan A.; Joosten, Ben; Reinieren-Beeren, Inge; Gualda, Emilio J.; Loza-Alvarez, Pablo; Figdor, Carl G.; Garcia-Parajo, Maria F.; Cambi, Alessandra

    2012-01-01

    The C-type lectin DC-SIGN expressed on dendritic cells (DCs) facilitates capture and internalization of a plethora of different pathogens. Although it is known that DC-SIGN organizes in nanoclusters at the surface of DCs, the molecular mechanisms responsible for this well defined nanopatterning and role in viral binding remain enigmatic. By combining biochemical and advanced biophysical techniques, including optical superresolution and single particle tracking, we demonstrate that DC-SIGN intrinsic nanoclustering strictly depends on its molecular structure. DC-SIGN nanoclusters exhibited free, Brownian diffusion on the cell membrane. Truncation of the extracellular neck region, known to abrogate tetramerization, significantly reduced nanoclustering and concomitantly increased lateral diffusion. Importantly, DC-SIGN nanocluster dissolution exclusively compromised binding to nanoscale size pathogens. Monte Carlo simulations revealed that heterogeneity on nanocluster density and spatial distribution confers broader binding capabilities to DC-SIGN. As such, our results underscore a direct relationship between spatial nanopatterning, driven by intermolecular interactions between the neck regions, and receptor diffusion to provide DC-SIGN with the exquisite ability to dock pathogens at the virus length scale. Insight into how virus receptors are organized prior to virus binding and how they assemble into functional platforms for virus docking is helpful to develop novel strategies to prevent virus entry and infection. PMID:23019323

  15. Umbilical cord blood regulatory T-cell expansion and functional effects of tumor necrosis factor receptor family members OX40 and 4-1BB expressed on artificial antigen-presenting cells

    PubMed Central

    Harker-Murray, Paul; Porter, Stephen B.; Merkel, Sarah C.; Londer, Aryel; Taylor, Dawn K.; Bina, Megan; Panoskaltsis-Mortari, Angela; Rubinstein, Pablo; Van Rooijen, Nico; Golovina, Tatiana N.; Suhoski, Megan M.; Miller, Jeffrey S.; Wagner, John E.; June, Carl H.; Riley, James L.

    2008-01-01

    Previously, we showed that human umbilical cord blood (UCB) regulatory T cells (Tregs) could be expanded approximately 100-fold using anti-CD3/28 monoclonal antibody (mAb)–coated beads to provide T-cell receptor and costimulatory signals. Because Treg numbers from a single UCB unit are limited, we explored the use of cell-based artificial antigen-presenting cells (aAPCs) preloaded with anti-CD3/28 mAbs to achieve higher levels of Treg expansion. Compared with beads, aAPCs had similar expansion properties while significantly increasing transforming growth factor β (TGF-β) secretion and the potency of Treg suppressor function. aAPCs modified to coexpress OX40L or 4-1BBL expanded UCB Tregs to a significantly greater extent than bead- or nonmodified aAPC cultures, reaching mean expansion levels exceeding 1250-fold. Despite the high expansion and in contrast to studies using other Treg sources, neither OX40 nor 4-1BB signaling of UCB Tregs reduced in vitro suppression. UCB Tregs expanded with 4-1BBL expressing aAPCs had decreased levels of proapoptotic bim. UCB Tregs expanded with nonmodified or modified aAPCs versus beads resulted in higher survival associated with increased Treg persistence in a xeno-geneic graft-versus-host disease lethality model. These data offer a novel approach for UCB Treg expansion using aAPCs, including those coexpressing OX40L or 4-1BBL. PMID:18645038

  16. Cathepsin G: roles in antigen presentation and beyond

    PubMed Central

    Burster, Timo; Macmillan, Henriette; Hou, Tieying; Boehm, Bernhard O.; Mellins, Elizabeth D.

    2014-01-01

    Summary Contributions from multiple cathepsins within endosomal antigen processing compartments are necessary to process antigenic proteins into antigenic peptides. Cysteine and aspartyl cathepsins have been known to digest antigenic proteins. A role for the serine protease, Cathepsin G (CatG), in this process has been described only recently, although CatG has long been known to be a granule-associated proteolytic enzyme of neutrophils. In line with a role for this enzyme in antigen presentation, CatG is found in endocytic compartments of a variety of antigen presenting cells. CatG is found in primary human monocytes, B cells, myeloid dendritic cells 1 (mDC1), mDC2, plasmacytoid DC (pDC), and murine microglia, but is not expressed in B cell lines or monocyte-derived DC. Purified CatG can be internalized into endocytic compartments in CatG non-expressing cells, widening the range of cells where this enzyme may play a role in antigen processing. Functional assays have implicated CatG as a critical enzyme in processing of several antigens and autoantigens. In this review, historical and recent data on CatG expression, distribution, function and involvement in disease will be summarized and discussed, with a focus on its role in antigen presentation and immune-related events. PMID:19910052

  17. Specific killing of cytotoxic T cells and antigen-presenting cells by CD4+ cytotoxic T cell clones. A novel potentially immunoregulatory T-T cell interaction in man.

    PubMed

    Ottenhoff, T H; Mutis, T

    1990-06-01

    Mycobacterial antigens not only stimulate Th cells that produce macrophage-activating factors, but also CD4+ and CD8+ CTL that lyse human macrophages. The mycobacterial recombinant 65-kD hsp was previously found to be an important target antigen for polyclonal CD4+ CTL. Because of the major role of 65-kD hsp in the immune response to mycobacterial as well as autoantigens, we have studied CTL activity to this protein at the clonal level. HLA-DR or HLA-DQ restricted, CD4+CD8- T cell clones that recognize different peptides of the M. leprae 65-kD hsp strongly lysed EBV-BLCL pulsed with specific but not irrelevant peptide. No bystander lysis of B cells, T cells, or tumor cells was seen. Target cell lysis could not be triggered by PMA + Ca2+ ionophore alone and depended on active metabolism. Interestingly, these CD4+ CTL also strongly lysed themselves and other HLA-class II compatible CD4+ (TCR-alpha/beta or -gamma/delta) or CD8+ CTL clones in the presence of peptide, suggesting that CTL are not actively protected from CTL-mediated lysis. Cold target competition experiments suggested that EBV-BLCL targets were more efficiently recognized than CD4+ CTL targets. These results demonstrate that hsp65 peptide-specific HLA class II-restricted CD4+ T cell clones display strong peptide-dependent cytolytic activity towards both APCs, and, unexpectedly, CD4+ and CD8+ CTL clones, including themselves. Since, in contrast to murine T cells human T cells express class II, CTL-mediated T cell killing may represent a novel immunoregulatory pathway in man. PMID:1972178

  18. Mice completely lacking immunoproteasomes display major alterations in antigen presentation

    PubMed Central

    Kincaid, Eleanor Z; Che, Jenny W; York, Ian; Escobar, Hernando; Reyes-Vargas, Eduardo; Delgado, Julio C.; Welsh, Raymond M; Karow, Margaret L.; Murphy, Andrew J.; Valenzuela, David M.; Yancopoulos, George D.; Rock, Kenneth L

    2011-01-01

    The importance of immunoproteasomes to antigen presentation has been unclear because animals totally lacking immunoproteasomes have not been previously developed. Here we show that dendritic cells from mice lacking the three immunoproteasome catalytic subunits display defects in presenting multiple major histocompatability (MHC) class I epitopes. During viral infection in vivo, the presentation of a majority of MHC class I epitopes is markedly reduced in immunoproteasome-deficient animals, while presentation of MHC class II peptides is unaffected. By mass spectrometry the repertoire of MHC class I-presented peptides is ~50% different and these differences are sufficient to stimulate robust transplant rejection of wild type cells in mutant mice. These results indicate that immunoproteasomes play a much more important role in antigen presentation than previously thought. PMID:22197977

  19. MHC structure and function – antigen presentation. Part 1

    PubMed Central

    Goldberg, Anna Carla; Rizzo, Luiz Vicente

    2015-01-01

    The setting for the occurrence of an immune response is that of the need to cope with a vast array of different antigens from both pathogenic and non-pathogenic sources. When the first barriers against infection and innate defense fail, adaptive immune response enters the stage for recognition of the antigens by means of extremely variable molecules, namely immunoglobulins and T-cell receptors. The latter recognize the antigen exposed on cell surfaces, in the form of peptides presented by the HLA molecule. The first part of this review details the central role played by these molecules, establishing the close connection existing between their structure and their antigen presenting function. PMID:25807245

  20. The molecular basis of antigen presentation.

    PubMed

    Germain, R N; Sant, A J; Braunstein, N S; Ronchese, F

    1988-01-01

    We have used a multifactorial approach to investigate the relationship between the structure of class II major histocompatibility complex (MHC)-encoded cell surface molecules (Ia) and the recognition of Ia-bound peptide antigens by clonally distributed alpha beta heterodimeric T cell receptors (TCR). Four distinct parameters of Ia structure-function--1) control of Ia assembly and transport to the surface membrane; 2) serological reactivity with panels of monoclonal antibodies; 3) ability to present peptide antigens to T cells for functional activation; and 4) differential peptide binding independent of the TCR--have been measured. This has allowed assignment of allelically polymorphic subregions or individual residues to locations in a model class II molecular structure and attribution to these subregions and residues of specific functions such as control of alpha beta chain interaction and protein folding, antigenic peptide binding, or TCR binding. The results of these analyses have provided an internally consistent picture of the class II molecule that fits well with a hypothetical model for Ia derived by analogy from the recently solved HLA class I crystal structure. They have also given us the first clear definition of specific peptide binding residues within the general peptide binding region of Ia and have revealed an unexpected asymmetry in the structure-function relationships of the putative alpha and beta chain helical regions. Overall, the results of these studies indicate the critical importance of multi-parameter analysis in creating useful molecular models using non-chemical techniques. They also suggest that hypotheses about TCR-Ia interaction may have to take into account a significant asymmetry in the function of the two major polymorphic regions of histocompatibility molecules. PMID:3269359

  1. MHC Class II Auto-Antigen Presentation is Unconventional

    PubMed Central

    Sadegh-Nasseri, Scheherazade; Kim, AeRyon

    2015-01-01

    Antigen presentation is highly critical in adoptive immunity. Only by interacting with antigens presented by major histocompatibility complex class II molecules, helper T cells can be stimulated to fight infections or diseases. The degradation of a full protein into small peptide fragments bound to class II molecules is a dynamic, lengthy process consisting of many steps and chaperons. Deregulation in any step of antigen processing could lead to the development of self-reactive T cells or defective immune response to pathogens. Indeed, human leukocyte antigens class II genes are the predominant contributors to susceptibility to autoimmune diseases. Conventional antigen-processing calls for internalization of extracellular antigens followed by processing and epitope selection within antigen-processing subcellular compartments, enriched with all necessary accessory molecules, processing enzymes, and proper pH and denaturing conditions. However, recent data examining the temporal relationship between antigen uptakes, processing, and epitope selection revealed unexpected characteristics for auto-antigenic epitopes, which were not shared with antigenic epitopes from pathogens. This review provides a discussion of the relevance of these findings to the mechanisms of autoimmunity. PMID:26257739

  2. MHC structure and function − antigen presentation. Part 2

    PubMed Central

    Goldberg, Anna Carla; Rizzo, Luiz Vicente

    2015-01-01

    The second part of this review deals with the molecules and processes involved in the processing and presentation of the antigenic fragments to the T-cell receptor. Though the nature of the antigens presented varies, the most significant class of antigens is proteins, processed within the cell to be then recognized in the form of peptides, a mechanism that confers an extraordinary degree of precision to this mode of immune response. The efficiency and accuracy of this system is also the result of the myriad of mechanisms involved in the processing of proteins and production of peptides, in addition to the capture and recycling of alternative sources aiming to generate further diversity in the presentation to T-cells. PMID:25807243

  3. Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation

    PubMed Central

    Ryu, Jae Kyu; Petersen, Mark A.; Murray, Sara G.; Baeten, Kim M.; Meyer-Franke, Anke; Chan, Justin P.; Vagena, Eirini; Bedard, Catherine; Machado, Michael R.; Coronado, Pamela E. Rios; Prod'homme, Thomas; Charo, Israel F.; Lassmann, Hans; Degen, Jay L.; Zamvil, Scott S.; Akassoglou, Katerina

    2015-01-01

    Autoimmunity and macrophage recruitment into the central nervous system (CNS) are critical determinants of neuroinflammatory diseases. However, the mechanisms that drive immunological responses targeted to the CNS remain largely unknown. Here we show that fibrinogen, a central blood coagulation protein deposited in the CNS after blood–brain barrier disruption, induces encephalitogenic adaptive immune responses and peripheral macrophage recruitment into the CNS leading to demyelination. Fibrinogen stimulates a unique transcriptional signature in CD11b+ antigen-presenting cells inducing the recruitment and local CNS activation of myelin antigen-specific Th1 cells. Fibrinogen depletion reduces Th1 cells in the multiple sclerosis model, experimental autoimmune encephalomyelitis. Major histocompatibility complex (MHC) II-dependent antigen presentation, CXCL10- and CCL2-mediated recruitment of T cells and macrophages, respectively, are required for fibrinogen-induced encephalomyelitis. Inhibition of the fibrinogen receptor CD11b/CD18 protects from all immune and neuropathologic effects. Our results show that the final product of the coagulation cascade is a key determinant of CNS autoimmunity. PMID:26353940

  4. Regulation of antigen presentation by Mycobacterium tuberculosis: a role for Toll-like receptors

    PubMed Central

    Harding, Clifford V.; Boom, W. Henry

    2011-01-01

    Mycobacterium tuberculosis survives in antigen-presenting cells (APCs) such as macrophages and dendritic cells. APCs present antigens in association with major histocompatibility complex (MHC) class II molecules to stimulate CD4+ T cells, and this process is essential to contain M. tuberculosis infection. Immune evasion allows M. tuberculosis to establish persistent or latent infection in macrophages and results in Toll-like receptor 2 (TLR2)-dependent inhibition of MHC class II transactivator expression, MHC class II molecule expression and antigen presentation. This reduction of antigen presentation might reflect a general mechanism of negative-feedback regulation that prevents excessive T cell-mediated inflammation and that M. tuberculosis has subverted to create a niche for survival in infected macrophages and evasion of recognition by CD4+ T cells. PMID:20234378

  5. Characterization of cutaneous antigen presentation in partially inbred miniature swine.

    PubMed

    Grabbe, S; Fishbein, J M; Sachs, D H; Flotte, T J; Granstein, R D

    1994-12-01

    MHC class I and II-defined, partially inbred miniature swine have recently become available as a large animal model in transplantation immunology. To investigate cutaneous immunocompetence in this model, cutaneous antigen presenting cell (APC) function was assessed. For morphologic analysis, punch biopsies were examined by electron microscopy. By this technique, epidermal Langerhans cells bearing typical Birbeck granules could be detected. For functional studies, epidermal cell (EC) suspensions were prepared from split thickness skin specimens. Using FACS analysis, freshly prepared epidermal cell suspensions contained 1.8-4.7% MHC class II-positive cells. These EC potently stimulated allogeneic nylon wool-enriched peripheral blood T cells in the primary mixed EC-lymphocyte reaction. For in vivo assessment of cutaneous APC function, EC suspensions enriched for or depleted of class II-positive EC were generated by panning of class II-positive EC using mouse anti-MHC class II antibodies and anti-mouse IgG-coated petri dishes. EC were then coupled to the hapten trinitrophenol (TNP) and injected s.c. into autologous or MHC-mismatched pigs twice at a one week interval. One week later, pigs were challenged by s.c.-injection of 0.5-1 x 10(7) TNP-coupled or uncoupled EC. Autologous unseparated EC as well as EC enriched for MHC class II-positive cells were able to sensitize naive animals against TNP, whereas neither TNP-coupled EC depleted of class II-positive APC, MHC-mismatched EC coupled to TNP, nor uncoupled EC induced immunity to TNP. Our data indicate that inbred miniature swine possess competent cutaneous APC which are able to induce cutaneous APC which are able to induce cutaneous immunity in a matter similar to Langerhans cells in murine or human skin. PMID:7749572

  6. Metformin Suppresses MHC-Restricted Antigen Presentation by Inhibiting Co-Stimulatory Factors and MHC Molecules in APCs

    PubMed Central

    Shin, Seulmee; Hyun, Bobae; Lee, Aeri; Kong, Hyunseok; Han, Shinha; Lee, Chong-Kil; Ha, Nam-Joo; Kim, Kyungjae

    2013-01-01

    Metformin is widely used for T2D therapy but its cellular mechanism of action is undefined. Recent studies on the mechanism of metformin in T2D have demonstrated involvement of the immune system. Current immunotherapies focus on the potential of immunomodulatory strategies for the treatment of T2D. In this study, we examined the effects of metformin on the antigen-presenting function of antigen-presenting cells (APCs). Metformin decreased both MHC class I and class II-restricted presentation of OVA and suppressed the expression of both MHC molecules and co-stimulatory factors such as CD54, CD80, and CD86 in DCs, but did not affect the phagocytic activity toward exogenous OVA. The class II-restricted OVA presentation-regulating activity of metformin was also confirmed using mice that had been injected with metformin followed by soluble OVA. These results provide an understanding of the mechanisms of the T cell response-regulating activity of metformin through the inhibition of MHC-restricted antigen presentation in relation to its actions on APCs. PMID:24009856

  7. Boosting the MHC Class II-Restricted Tumor Antigen Presentation to CD4+ T Helper Cells: A Critical Issue for Triggering Protective Immunity and Re-Orienting the Tumor Microenvironment Toward an Anti-Tumor State

    PubMed Central

    Accolla, Roberto S.; Lombardo, Letizia; Abdallah, Rawan; Raval, Goutham; Forlani, Greta; Tosi, Giovanna

    2014-01-01

    Although the existence of an immune response against tumor cells is well documented, the fact that tumors take off in cancer patients indicates that neoplastic cells can circumvent this response. Over the years many investigators have described strategies to rescue the anti-tumor immune response with the aim of creating specific and long-lasting protection against the disease. When exported to human clinical settings, these strategies have revealed in most cases a very limited, if any, positive outcome. We believe that the failure is mostly due to the inadequate triggering of the CD4+ T helper (TH) cell arm of the adaptive immunity, as TH cells are necessary to trigger all the immune effector mechanisms required to eliminate tumor cells. In this review, we focus on novel strategies that by stimulating MHC class II-restricted activation of TH cells generate a specific and persistent adaptive immunity against the tumor. This point is of critical importance for both preventive and therapeutic anti-tumor vaccination protocols, because adaptive immunity with its capacity to produce specific, long-lasting protection and memory responses is indeed the final goal of vaccination. We will discuss data from our as well as other laboratories which strongly suggest that triggering a specific and persistent anti-tumor CD4+ TH cell response stably modify not only the tumor microenvironment but also tumor-dependent extratumor microenvironments by eliminating and/or reducing the blood-derived tumor infiltrating cells that may have a pro-tumor growth function such as regulatory CD4+/CD25+ T cells and myeloid-derived-suppressor cells. Within this frame, therefore, we believe that the establishment of a pro-tumor environment is not the cause but simply the consequence of the tumor strategy to primarily counteract components of the adaptive cellular immunity, particularly TH lymphocytes. PMID:24600588

  8. Enhanced Direct Major Histocompatibility Complex Class I Self-Antigen Presentation Induced by Chlamydia Infection

    PubMed Central

    Cram, Erik D.; Simmons, Ryan S.; Palmer, Amy L.; Hildebrand, William H.; Rockey, Daniel D.

    2015-01-01

    The direct major histocompatibility complex (MHC) class I antigen presentation pathway ensures intracellular peptides are displayed at the cellular surface for recognition of infected or transformed cells by CD8+ cytotoxic T lymphocytes. Chlamydia spp. are obligate intracellular bacteria and, as such, should be targeted by CD8+ T cells. It is likely that Chlamydia spp. have evolved mechanisms to avoid the CD8+ killer T cell responses by interfering with MHC class I antigen presentation. Using a model system of self-peptide presentation which allows for posttranslational control of the model protein's stability, we tested the ability of various Chlamydia species to alter direct MHC class I antigen presentation. Infection of the JY lymphoblastoid cell line limited the accumulation of a model host protein and increased presentation of the model-protein-derived peptides. Enhanced self-peptide presentation was detected only when presentation was restricted to defective ribosomal products, or DRiPs, and total MHC class I levels remained unaltered. Skewed antigen presentation was dependent on a bacterial synthesized component, as evidenced by reversal of the observed phenotype upon preventing bacterial transcription, translation, and the inhibition of bacterial lipooligosaccharide synthesis. These data suggest that Chlamydia spp. have evolved to alter the host antigen presentation machinery to favor presentation of defective and rapidly degraded forms of self-antigen, possibly as a mechanism to diminish the presentation of peptides derived from bacterial proteins. PMID:26597986

  9. Endogenous Antigen Presentation of MHC Class II Epitopes through Non-Autophagic Pathways

    PubMed Central

    Leung, Carol S. K.

    2015-01-01

    Antigenic peptides presented by major histocompatibility complex (MHC) class II molecules are generally derived from exogenous proteins acquired by antigen presenting cells. However, in some circumstances, MHC class II molecules can present intracellular proteins expressed within the antigen-presenting cells. There are several described pathways by which endogenous antigens are degraded and gain access to MHC class II molecules. These include autophagy and other non-autophagic pathways; the latter category includes the MHC class I-like pathways, heat shock protein 90-mediated pathways, and internalization from the plasma membrane. This review will summarize and discuss the non-autophagic pathways. PMID:26441969

  10. Inhibition by chloroquine of the class II major histocompatibility complex-restricted presentation of endogenous antigens varies according to the cellular origin of the antigen-presenting cells, the nature of the T-cell epitope, and the responding T cell.

    PubMed Central

    Lombard-Platlet, S; Bertolino, P; Deng, H; Gerlier, D; Rabourdin-Combe, C

    1993-01-01

    Chloroquine treatment of antigen-presenting cells (APC) was explored as a tool to investigate the processing pathway for major histocompatibility complex (MHC) class II-restricted presentation of the endogenous secreted hen egg lysozyme (HEL) and transmembrane measles virus haemagglutinin (HA). A 72-hr pretreatment of the APC with 25 microM chloroquine blocked the presentation of the HEL(52-61) T-cell epitope generated from endogenous HEL to the I-Ak-restricted 3A9 T-cell hybridoma by MHC class II-transfected L cells expressing the invariant chain (Ii). The presentation of exogenously added HEL peptides was not affected. Under the same conditions, no inhibition of the presentation of HEL(106-116) to the I-Ed-restricted G28 high-avidity T-cell hybridoma, nor of HA when synthesized by L cells, was observed. When B-lymphoid APC were used, inhibition was observed in every case with a low number of B APC pretreated for 48 hr with chloroquine prior to the T-cell stimulation test. Moreover, addition of chloroquine to untreated B APC during the T-cell stimulation assay was sufficient to inhibit completely the presentation of HEL(106-116) to the B10.D24.42 low avidity T-cell hybridoma. Altogether these studies suggest that an apparent resistance of endogenous Ag presentation to chloroquine inhibition may not necessarily indicate the existence of a non-endosomal pathway but may be due to the nature of the T-cell epitope, to the use of 'non-professional' APC such as L cells, to the use of T cells of high avidity, and to high amounts of pre-existing MHC class II-peptide complexes expressed by the APC. We demonstrate here that, at least in conventional APC such as B cells, class II-restricted presentation of both endogenous secreted HEL and transmembrane HA involves an endosomal pathway. PMID:7508420

  11. Human and Simian Immunodeficiency Virus-Mediated Upregulation of the Apoptotic Factor TRAIL Occurs in Antigen-Presenting Cells from AIDS-Susceptible but Not from AIDS-Resistant Species▿ †

    PubMed Central

    Kim, Nayoung; Dabrowska, Alicja; Jenner, Richard G.; Aldovini, Anna

    2007-01-01

    Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections lead to AIDS in humans and rhesus macaques (RM), while they are asymptomatic in species naturally infected with SIV, such as chimpanzees, sooty mangabeys (SM), and African green monkeys (AGM). Differential CD4+ T-cell apoptosis may be responsible for these species-specific differences in susceptibility to disease. To identify factors that influence the different apoptotic responses of these species, we analyzed virus-infected human and nonhuman primate peripheral blood mononuclear cells (PBMC). We found that the apoptotic factor TRAIL was present at higher levels in human and RM PBMC cultures and was mediating, at least in part, CD4+ T-cell apoptosis in these cultures. The species-specific increase in TRAIL and death receptor expression observed with cultures also occurred in vivo in SIV-infected RM but not in SIV-infected SM. In human and RM myeloid immature dendritic cells and macrophages, the virus-induced expression of TRAIL and other interferon-inducible genes, which did not occur in the same cells from chimpanzee, SM, and AGM, was Tat dependent. Our results link the differential induction of TRAIL in human and nonhuman primate cells to species-specific differences in disease susceptibility. PMID:17494085

  12. Human and simian immunodeficiency virus-mediated upregulation of the apoptotic factor TRAIL occurs in antigen-presenting cells from AIDS-susceptible but not from AIDS-resistant species.

    PubMed

    Kim, Nayoung; Dabrowska, Alicja; Jenner, Richard G; Aldovini, Anna

    2007-07-01

    Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections lead to AIDS in humans and rhesus macaques (RM), while they are asymptomatic in species naturally infected with SIV, such as chimpanzees, sooty mangabeys (SM), and African green monkeys (AGM). Differential CD4(+) T-cell apoptosis may be responsible for these species-specific differences in susceptibility to disease. To identify factors that influence the different apoptotic responses of these species, we analyzed virus-infected human and nonhuman primate peripheral blood mononuclear cells (PBMC). We found that the apoptotic factor TRAIL was present at higher levels in human and RM PBMC cultures and was mediating, at least in part, CD4(+) T-cell apoptosis in these cultures. The species-specific increase in TRAIL and death receptor expression observed with cultures also occurred in vivo in SIV-infected RM but not in SIV-infected SM. In human and RM myeloid immature dendritic cells and macrophages, the virus-induced expression of TRAIL and other interferon-inducible genes, which did not occur in the same cells from chimpanzee, SM, and AGM, was Tat dependent. Our results link the differential induction of TRAIL in human and nonhuman primate cells to species-specific differences in disease susceptibility. PMID:17494085

  13. Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting: Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4{sup +} T cells compared

    SciTech Connect

    Ford, A.L.; Goodsall, A.L.; Sedgwick, J.D.

    1995-05-01

    Ramified microglia in the adult central nervous system (CNS) are the principal glial element up-regulating MHC class I and II expression in response to inflammatory events or neuronal damage. A proportion of these cells also express MHC class II constitutively in the normal CNS. The role of microglia as APCs for CD4{sup +} cells extravasating into the CNS remains undefined. In this study, using irradiation bone marrow chimeras in CD45-congenic rats, the phenotype CD45{sup low}CD11b/c{sup +} is shown to identify microglial cells specifically within the CNS. Highly purified populations of microglia and nonmicroglial but CNS-associated macrophages (CD45{sup high}CD11b/c{sup +}) have been obtained directly from the adult CNS, by using flow cytometric sorting. Morphologically, freshly isolated microglia vs other CNS macrophages are quite distinct. Of the two populations recovered from the normal CNS, it is the minority CD45{sup high}CD11 b/c{sup +} transitional macrophage population, and not microglia, that is the effective APC for experimental autoimmune encephalomyelitis-inducing CD4{sup +} myelin basic protein (MBP)-reactive T cells. CD45{sup high}CD11b/c{sup +} CNS macrophages also stimulate MBP-reactive T cells without addition of MBP to culture suggesting presentation of endogenous Ag. This is the first study in which microglia vs other CNS macrophages have been analyzed for APC ability directly from the CNS, with substantial cross-contamination between the two populations eliminated. The heterogeneity of these populations in terms of APC function is clearly demonstrated. Evidence is still lacking that adult CNS microglia have the capacity to interact with and stimulate CD4{sup +} T cells to proliferate or secrete IL-2. 60 refs., 6 figs., 1 tab.

  14. Suppression of autophagy and antigen presentation by Mycobacterium tuberculosis PE_PGRS47.

    PubMed

    Saini, Neeraj K; Baena, Andres; Ng, Tony W; Venkataswamy, Manjunatha M; Kennedy, Steven C; Kunnath-Velayudhan, Shajo; Carreño, Leandro J; Xu, Jiayong; Chan, John; Larsen, Michelle H; Jacobs, William R; Porcelli, Steven A

    2016-01-01

    Suppression of major histocompatibility complex (MHC) class II antigen presentation is believed to be among the major mechanisms used by Mycobacterium tuberculosis to escape protective host immune responses. Through a genome-wide screen for the genetic loci of M. tuberculosis that inhibit MHC class II-restricted antigen presentation by mycobacteria-infected dendritic cells, we identified the PE_PGRS47 protein as one of the responsible factors. Targeted disruption of the PE_PGRS47 (Rv2741) gene led to attenuated growth of M. tuberculosis in vitro and in vivo, and a PE_PGRS47 mutant showed enhanced MHC class II-restricted antigen presentation during in vivo infection of mice. Analysis of the effects of deletion or over-expression of PE_PGRS47 implicated this protein in the inhibition of autophagy in infected host phagocytes. Our findings identify PE_PGRS47 as a functionally relevant, non-redundant bacterial factor in the modulation of innate and adaptive immunity by M. tuberculosis, suggesting strategies for improving antigen presentation and the generation of protective immunity during vaccination or infection. PMID:27562263

  15. Immune privilege in the anterior chamber of the eye: alloantigens and tumour-specific antigens presented into the anterior chamber simultaneously induce suppression and activation of delayed hypersensitivity to the respective antigens.

    PubMed Central

    Benson, J L; Niederkorn, J Y

    1992-01-01

    Allografts placed into the anterior chamber of the eye enjoy prolonged and sometimes permanent survival while similar grafts are promptly rejected if transplanted to non-privileged sites. The immunological privilege of the anterior chamber is due, in large part, to the systemic suppression of delayed-type hypersensitivity (DTH) that is induced by anterior chamber presentation of alloantigens and is termed anterior chamber-associated immune deviation (ACAID). Although many categories of antigens elicit ACAID, strong tumour-specific transplantation antigens (TSTA) do not induce ACAID and instead, provoke potent DTH responses following anterior chamber presentation. The present study sought to determine if the anterior chamber were simultaneously confronted with these two categories of antigens, which systemic immune response would prevail: DTH or suppression of DTH? The results show that inoculation of minor histocompatibility alloantigens and TSTA into the anterior chamber induced both afferent and efferent suppression of specific anti-alloantigen DTH responses, yet simultaneously induced normal anti-TSTA DTH responses. Both responses (i.e. DTH and suppression) were transferable with lymphoid cells. PMID:1427974

  16. Exploring genome-wide datasets of MHC class II antigen presentation.

    PubMed

    Wijdeven, Ruud H; Bakker, Jeroen M; Paul, Petra; Neefjes, Jacques

    2013-09-01

    MHC class II molecules (MHCII) are critical for presenting antigens to CD4(+) T-cells. They control ignition of CD4(+) T cells and are as such involved in most auto-immune diseases. To define proteins and pathways controlling MHCII antigen presentation and expression, we performed a genome-wide flow cytometry based RNAi screen. Hits were subsequently classified by two screens that monitored the intracellular distribution and transcription of MHCII. This multi-dimensional approach allowed subclassification of hits into functional groups as a first step to defining new pathways controlling MHCII antigen presentation. The datasets from this screen are used as a template for several follow-up studies. This overview focuses on how data from genome-wide screens can be used for target-lead finding, data mining, systems biology and systematic cell biology. PMID:23137594

  17. Antigen Presentation, Autoantigens, and Immune Regulation in Multiple Sclerosis and Other Autoimmune Diseases

    PubMed Central

    Riedhammer, Christine; Weissert, Robert

    2015-01-01

    Antigen presentation is in the center of the immune system, both in host defense against pathogens, but also when the system is unbalanced and autoimmune diseases like multiple sclerosis (MS) develop. It is not just by chance that a major histocompatibility complex gene is the major genetic susceptibility locus in MS; a feature that MS shares with other autoimmune diseases. The exact etiology of the disease, however, has not been fully understood yet. T cells are regarded as the major players in the disease, but most probably a complex interplay of altered central and peripheral tolerance mechanisms, T-cell and B-cell functions, characteristics of putative autoantigens, and a possible interference of environmental factors like microorganisms are at work. In this review, new data on all these different aspects of antigen presentation and their role in MS will be discussed, probable autoantigens will be summarized, and comparisons to other autoimmune diseases will be drawn. PMID:26136751

  18. Comparative Analysis of Gingival Tissue Antigen Presentation Pathways in Aging and Periodontitis

    PubMed Central

    Gonzalez, O.A.; Novak, M.J.; Kirakodu, S.; Orraca, L.; Chen, K.C.; Strom-berg, A.; Gonzalez-Martinez, J.; Ebersole, J. L.

    2014-01-01

    Aim Gingival tissues of periodontitis lesions contribute to local elevations in mediators, including both specific T cell and antibody immune responses to oral bacterial antigens. Thus, antigen processing and presentation activities must exist in these tissues to link antigen-presenting cells with adaptive immunity. We hypothesized that alterations in the transcriptome of antigen processing and presentation genes occur in aging gingival tissues and that periodontitis enhances these differences reflecting tissues less capable of immune resistance to oral pathogens. Materials and Methods Rhesus monkeys (n=34) from 3–23 years of age were examined. A buccal gingival sample from healthy or periodontitis sites were obtained, total RNA isolated, and microarray analysis was used to describe the transcriptome. Results The results demonstrated increased transcription of genes related to the MHC class II and negative regulation of NK cells with aging in healthy gingival tissues. In contrast, both adult and aging periodontitis tissues showed decreased transcription of genes for MHC class II antigens, coincident with up-regulation of MHC class I-associated genes. Conclusion These transcriptional changes suggest a response of healthy aging tissues through the class II pathway (i.e., endocytosed antigens) and altered responses in periodontitis that could reflect host-associated self-antigens or targeting cytosolic intra-cellular microbial pathogens. PMID:24304139

  19. Parasite Manipulation of the Invariant Chain and the Peptide Editor H2-DM Affects Major Histocompatibility Complex Class II Antigen Presentation during Toxoplasma gondii Infection

    PubMed Central

    Leroux, Louis-Philippe; Nishi, Manami; El-Hage, Sandy; Fox, Barbara A.; Bzik, David J.

    2015-01-01

    Toxoplasma gondii is an obligate intracellular protozoan parasite. This apicomplexan is the causative agent of toxoplasmosis, a leading cause of central nervous system disease in AIDS. It has long been known that T. gondii interferes with major histocompatibility complex class II (MHC-II) antigen presentation to attenuate CD4+ T cell responses and establish persisting infections. Transcriptional downregulation of MHC-II genes by T. gondii was previously established, but the precise mechanisms inhibiting MHC-II function are currently unknown. Here, we show that, in addition to transcriptional regulation of MHC-II, the parasite modulates the expression of key components of the MHC-II antigen presentation pathway, namely, the MHC-II-associated invariant chain (Ii or CD74) and the peptide editor H2-DM, in professional antigen-presenting cells (pAPCs). Genetic deletion of CD74 restored the ability of infected dendritic cells to present a parasite antigen in the context of MHC-II in vitro. CD74 mRNA and protein levels were, surprisingly, elevated in infected cells, whereas MHC-II and H2-DM expression was inhibited. CD74 accumulated mainly in the endoplasmic reticulum (ER), and this phenotype required live parasites, but not active replication. Finally, we compared the impacts of genetic deletion of CD74 and H2-DM genes on parasite dissemination toward lymphoid organs in mice, as well as activation of CD4+ T cells and interferon gamma (IFN-γ) levels during acute infection. Cyst burdens and survival during the chronic phase of infection were also evaluated in wild-type and knockout mice. These results highlight the fact that the infection is influenced by multiple levels of parasite manipulation of the MHC-II antigen presentation pathway. PMID:26195549

  20. Parasite Manipulation of the Invariant Chain and the Peptide Editor H2-DM Affects Major Histocompatibility Complex Class II Antigen Presentation during Toxoplasma gondii Infection.

    PubMed

    Leroux, Louis-Philippe; Nishi, Manami; El-Hage, Sandy; Fox, Barbara A; Bzik, David J; Dzierszinski, Florence S

    2015-10-01

    Toxoplasma gondii is an obligate intracellular protozoan parasite. This apicomplexan is the causative agent of toxoplasmosis, a leading cause of central nervous system disease in AIDS. It has long been known that T. gondii interferes with major histocompatibility complex class II (MHC-II) antigen presentation to attenuate CD4(+) T cell responses and establish persisting infections. Transcriptional downregulation of MHC-II genes by T. gondii was previously established, but the precise mechanisms inhibiting MHC-II function are currently unknown. Here, we show that, in addition to transcriptional regulation of MHC-II, the parasite modulates the expression of key components of the MHC-II antigen presentation pathway, namely, the MHC-II-associated invariant chain (Ii or CD74) and the peptide editor H2-DM, in professional antigen-presenting cells (pAPCs). Genetic deletion of CD74 restored the ability of infected dendritic cells to present a parasite antigen in the context of MHC-II in vitro. CD74 mRNA and protein levels were, surprisingly, elevated in infected cells, whereas MHC-II and H2-DM expression was inhibited. CD74 accumulated mainly in the endoplasmic reticulum (ER), and this phenotype required live parasites, but not active replication. Finally, we compared the impacts of genetic deletion of CD74 and H2-DM genes on parasite dissemination toward lymphoid organs in mice, as well as activation of CD4(+) T cells and interferon gamma (IFN-γ) levels during acute infection. Cyst burdens and survival during the chronic phase of infection were also evaluated in wild-type and knockout mice. These results highlight the fact that the infection is influenced by multiple levels of parasite manipulation of the MHC-II antigen presentation pathway. PMID:26195549

  1. Vaccinia Virus A35R Inhibits MHC Class II Antigen Presentation

    PubMed Central

    Rehm, Kristina E.; Connor, Ramsey F.; Jones, Gwendolyn J.B.; Yimbu, Kenneth; Roper, Rachel L.

    2009-01-01

    The Vaccinia virus gene A35R (Copenhagen designation) is highly conserved in mammalian-tropic poxviruses and is an important virulence factor, but its function was unknown. We show herein that A35 does not affect viral infectivity, apoptosis induction, or replication; however, we found that A35 significantly inhibited MHC class II-restricted antigen presentation, immune priming of T lymphocytes, and subsequent chemokine and cytokine synthesis. A35 localized to endosomes and reduced the amount of a model antigenic peptide displayed in the cleft of class II MHC. In addition, A35 decreased VV specific T cell responses in vivo. Thus, this is the first report identifying a function for the A35 protein in virulence as well as the first report identifying a VV gene that inhibits peptide antigen presentation. PMID:19954808

  2. Towards a systems understanding of MHC class I and MHC class II antigen presentation.

    PubMed

    Neefjes, Jacques; Jongsma, Marlieke L M; Paul, Petra; Bakke, Oddmund

    2011-12-01

    The molecular details of antigen processing and presentation by MHC class I and class II molecules have been studied extensively for almost three decades. Although the basic principles of these processes were laid out approximately 10 years ago, the recent years have revealed many details and provided new insights into their control and specificity. MHC molecules use various biochemical reactions to achieve successful presentation of antigenic fragments to the immune system. Here we present a timely evaluation of the biology of antigen presentation and a survey of issues that are considered unresolved. The continuing flow of new details into our understanding of the biology of MHC class I and class II antigen presentation builds a system involving several cell biological processes, which is discussed in this Review. PMID:22076556

  3. Inhibition of MHC class I-restricted antigen presentation by gamma 2-herpesviruses.

    PubMed

    Stevenson, P G; Efstathiou, S; Doherty, P C; Lehner, P J

    2000-07-18

    The gamma-herpesviruses, in contrast to the alpha- and beta-herpesviruses, are not known to inhibit antigen presentation to CD8(+) cytotoxic T lymphocytes (CTLs) during lytic cycle replication. However, murine gamma-herpesvirus 68 causes a chronic lytic infection in CD4(+) T cell-deficient mice despite the persistence of a substantial CTL response, suggesting that CTL evasion occurs. Here we show that, distinct from host protein synthesis shutoff, gamma-herpesvirus 68 down-regulates surface MHC class I expression on lytically infected fibroblasts and inhibits their recognition by antigen-specific CTLs. The viral K3 gene, encoding a zinc-finger-containing protein, dramatically reduced the half-life of nascent class I molecules and the level of surface MHC class I expression and was by itself sufficient to block antigen presentation. The homologous K3 and K5 genes of the related Kaposi's sarcoma-associated virus also inhibited antigen presentation and decreased cell surface expression of HLA class I antigens. Thus it appears that an immune evasion strategy shared by at least two gamma-herpesviruses allows continued lytic infection in the face of strong CTL immunity. PMID:10890918

  4. Effect of oestradiol and pathogen-associated molecular patterns on class II-mediated antigen presentation and immunomodulatory molecule expression in the mouse female reproductive tract

    PubMed Central

    Ochiel, Daniel O; Rossoll, Richard M; Schaefer, Todd M; Wira, Charles R

    2012-01-01

    Cells of the female reproductive tract (FRT) can present antigen to naive and memory T cells. However, the effects of oestrogen, known to modulate immune responses, on antigen presentation in the FRT remain undefined. In the present study, DO11.10 T-cell antigen receptor transgenic mice specific for the class II MHC-restricted ovalbumin (OVA) 323–339 peptide were used to study the effects of oestradiol and pathogen-associated molecular patterns on antigen presentation in the FRT. We report here that oestradiol inhibited antigen presentation of OVA by uterine epithelial cells, uterine stromal cells and vaginal cells to OVA-specific memory T cells. When ovariectomized animals were treated with oestradiol for 1 or 3 days, antigen presentation was decreased by 20–80%. In contrast, incubation with PAMP increased antigen presentation by epithelial cells (Pam3Cys), stromal cells (peptidoglycan, Pam3Cys) and vaginal cells (Pam3Cys). In contrast, CpG inhibited both stromal and vaginal cell antigen presentation. Analysis of mRNA expression by reverse transcription PCR indicated that oestradiol inhibited CD40, CD80 and class II in the uterus and CD40, CD86 and class II in the vagina. Expression in isolated uterine and vaginal cells paralleled that seen in whole tissues. In contrast, oestradiol increased polymeric immunoglobulin receptor mRNA expression in the uterus and decreased it in the vagina. These results indicate that antigen-presenting cells in the uterus and vagina are responsive to oestradiol, which inhibits antigen presentation and co-stimulatory molecule expression. Further, these findings suggest that antigen-presenting cells in the uterus and vagina respond to selected Toll-like receptor agonists with altered antigen presentation. PMID:22043860

  5. Antigen presentation events during the initiation of autoimmune diabetes in the NOD mouse.

    PubMed

    Ferris, Stephen T; Carrero, Javier A; Unanue, Emil R

    2016-07-01

    This is a brief summary of our studies of NOD autoimmune diabetes examining the events during the initial stage of the process. Our focus has been on antigen presentation events and the antigen presenting cells (APC) inside islets. Islets of non-diabetic mice contain resident macrophages that are developmentally distinct from those in the inter-acinar stroma. The autoimmune process starts with the entrance of CD4+ T cells together with a burst of a subset of dendritic cells (DC) bearing CD103. The CD103+ DC develop under the influence of the Batf3 transcription factor. Batf3 deficient mice do not develop diabetes and their islets are uninfiltrated throughout life. Thus, the CD103+ DC are necessary for the progression of autoimmune diabetes. The major CD4+ T cell response in NOD are the T cells directed to insulin. In particular, the non-conventional 12-20 segment of the insulin B chain is presented by the class II MHC molecule I-A(g7) and elicits pathogenic CD4+ T cells. We discuss that the diabetic process requires the CD103+ DC, the CD4+ T cells to insulin peptides, and NOD specific I-Ag(7) MHC-II allele. Finally, our initial studies indicate that beta cells transfer insulin containing vesicles to the local APC in a contact-dependent reaction. Live images of beta cells interactions with the APC and electron micrographs of islet APCs also show the transfer of granules. PMID:27021276

  6. TLR7 and TLR9 ligands regulate antigen presentation by macrophages.

    PubMed

    Celhar, Teja; Pereira-Lopes, Selma; Thornhill, Susannah I; Lee, Hui Yin; Dhillon, Manprit K; Poidinger, Michael; Connolly, John E; Lim, Lina H K; Biswas, Subhra K; Fairhurst, Anna-Marie

    2016-05-01

    The toll-like receptors (TLRs) are important innate receptors recognizing potentially pathogenic material. However, they also play a significant role in the development of Alzheimer's disease, cancer, autoimmunity and the susceptibility to viral infections. Macrophages are essential for an effective immune response to foreign material and the resolution of inflammation. In these studies, we examined the impact of different TLR ligands on macrophage cell function. We demonstrate that stimulation of all TLRs tested increases the phagocytosis of apoptotic cells by macrophages. TLR7 and TLR9 ligation decreased the levels of the surface co-expression molecules CD86 and MHCII, which was associated with a concomitant reduction in antigen presentation and proliferation of T cells. This down-regulation in macrophage function was not due to an increase in cell death. In fact, exposure to TLR7 or TLR9 ligands promoted cell viability for up to 9 days, in contrast to TLR3 or TLR4. Additionally, macrophages exposed to TLR7/TLR9 ligands had a significantly lower ratio of Il-12/Il-10 mRNA expression compared with those treated with the TLR4 ligand, LPS. Taken together, these data demonstrate that TLR7/TLR9 ligands push the macrophage into a phagocytic long-lived cell, with a decreased capacity of antigen presentation and reminiscent of the M2 polarized state. PMID:26567289

  7. The intracellular pathway for the presentation of vitamin B-related antigens by the antigen-presenting molecule MR1.

    PubMed

    McWilliam, Hamish E G; Eckle, Sidonia B G; Theodossis, Alex; Liu, Ligong; Chen, Zhenjun; Wubben, Jacinta M; Fairlie, David P; Strugnell, Richard A; Mintern, Justine D; McCluskey, James; Rossjohn, Jamie; Villadangos, Jose A

    2016-05-01

    The antigen-presenting molecule MR1 presents vitamin B-related antigens (VitB antigens) to mucosal-associated invariant T (MAIT) cells through an uncharacterized pathway. We show that MR1, unlike other antigen-presenting molecules, does not constitutively present self-ligands. In the steady state it accumulates in a ligand-receptive conformation within the endoplasmic reticulum. VitB antigens reach this location and form a Schiff base with MR1, triggering a 'molecular switch' that allows MR1-VitB antigen complexes to traffic to the plasma membrane. These complexes are endocytosed with kinetics independent of the affinity of the MR1-ligand interaction and are degraded intracellularly, although some MR1 molecules acquire new ligands during passage through endosomes and recycle back to the surface. MR1 antigen presentation is characterized by a rapid 'off-on-off' mechanism that is strictly dependent on antigen availability. PMID:27043408

  8. Antigen presentation by hapten-specific B lymphocytes. II. Specificity and properties of antigen-presenting B lymphocytes, and function of immunoglobulin receptors

    SciTech Connect

    Abbas, A.K.; Haber, S.; Rock, K.L.

    1985-09-01

    Studies were designed to examine the ability of hapten-binding murine B lymphocytes to present hapten-protein conjugates to protein antigen-specific, Ia-restricted T cell hybridomas. BALB/c B cells specific for TNP or FITC presented hapten-modified proteins (TNP-G1 phi, TNP-OVA, or FITC-OVA) to the relevant T cell hybridomas at concentrations below 0.1 microgram/ml. Effective presentation of the same antigens by B lymphocyte-depleted splenocytes, and of unmodified proteins by either hapten-binding B cells or Ig spleen cells, required about 10(3)-to 10(4)-fold higher concentrations of antigen. The use of two different haptens and two carrier proteins showed that this extremely efficient presentation of antigen was highly specific, with hapten specificity being a property of the B cells and carrier specificity of the responding T cells. The presentation of hapten-proteins by hapten-binding B lymphocytes was radiosensitive and was not affected by the depletion of plastic-adherent cells, suggesting that conventional APCs (macrophages or dendritic cells) are not required in this phenomenon. Antigen-pulsing and antibody-blocking experiments showed that this hapten-specific antigen presentation required initial binding of antigen to surface Ig receptors. Moreover, linked recognition of hapten and carrier determinants was required, but these recognition events could be temporally separated. Finally, an antigen-processing step was found to be necessary, and this step was disrupted by ionizing radiation. These data suggest a role for B cell surface Ig in providing a specific high-affinity receptor to allow efficient uptake or focusing of antigen for its subsequent processing and presentation to T lymphocytes.

  9. Wheeling and Dealing With Antigen Presentation in Tuberculosis.

    PubMed

    Hudrisier, Denis; Neyrolles, Olivier

    2016-03-01

    In tuberculosis, antigens are transferred from infected to uninfected dendritic cells. Does this favor T lymphocyte response and anti-mycobacterial host defense? In a recent report published in Cell Host & Microbe, Ernst and colleagues show that Mycobacterium tuberculosis seems to have hijacked this mechanism for its own benefit. PMID:26794467

  10. Uptake of HLA Alloantigens via CD89 and CD206 Does Not Enhance Antigen Presentation by Indirect Allorecognition

    PubMed Central

    Breman, Eytan; Ruben, Jurjen M.; Franken, Kees L.; Heemskerk, Mirjam H. M.; Roelen, Dave L.; Claas, Frans H.

    2016-01-01

    In organ transplantation, alloantigens are taken up by antigen presenting cells and presented via the indirect pathway to T-cells which in turn can induce allograft rejection. Monitoring of these T-cells is of major importance; however no reliable assay is available to routinely monitor indirect allorecognition. Recently we showed that HLA monomers can be successfully used to monitor indirect allorecognition. Targeting antigens to endocytic receptors on antigen presenting cells may further enhance the presentation of antigens via HLA class II and improve the efficiency of this assay. In the current study we explored targeting of HLA monomers to either CD89 expressing monocytes or mannose receptor expressing dendritic cells. Monomer-antibody complexes were generated using biotin-labeled monomers and avidin labeling of the antibodies. We demonstrate that targeting the complexes to these receptors resulted in a dose-dependent HLA class II mediated presentation to a T-cell clone. The immune-complexes were efficiently taken up and presented to T-cells. However, the level of T-cell reactivity was similar to that when only exogenous antigen was added. We conclude that HLA-A2 monomers targeted for presentation through CD89 on monocytes or mannose receptor on dendritic cells lead to proper antigen presentation but do not enhance indirect allorecognition via HLA-DR. PMID:27413760

  11. Immunology by numbers: quantitation of antigen presentation completes the quantitative milieu of systems immunology!

    PubMed

    Purcell, Anthony W; Croft, Nathan P; Tscharke, David C

    2016-06-01

    We review approaches to quantitate antigen presentation using a variety of biological and biochemical readouts and highlight the emerging role of mass spectrometry (MS) in defining and quantifying MHC-bound peptides presented at the cell surface. The combination of high mass accuracy in the determination of the molecular weight of the intact peptide of interest and its signature pattern of fragmentation during tandem MS provide an unambiguous and definitive identification. This is in contrast to the potential receptor cross-reactivity towards closely related peptides and variable dose responsiveness seen in biological readouts. In addition, we gaze into the not too distant future where big data approaches in MS can be accommodated to quantify whole immunopeptidomes both in vitro and in vivo. PMID:27060633

  12. Organic extract of diesel exhaust particles stimulates expression of Ia and costimulatory molecules associated with antigen presentation in rat peripheral blood monocytes but not in alveolar macrophages

    SciTech Connect

    Koike, Eiko . E-mail: ekoike@nies.go.jp; Kobayashi, Takahiro

    2005-12-15

    We hypothesized that diesel exhaust particles (DEP) induce the activation of antigen-presenting cells (APC) in lung. The present study was designed to clarify the following about DEP: (1) whether it affects the expression of Ia and B7 molecules in alveolar macrophages (AM) as a mature cell or in peripheral blood monocytes (PBM) as an immature cell (2) if it affects the antigen-presenting (AP) activity of PBM (3) what component of DEP is responsible for the effects, and (4) whether the effect of DEP is related to oxidative stress. DEP was extracted with methylene chloride. Cells were exposed to whole DEP, organic extract, or residual particles for 24 h. Cell-surface molecules were measured by flow cytometry. AP activity was assessed by antigen-specific T cell proliferation. Whole DEP or organic extract significantly increased the expression of Ia and B7 molecules on PBM but not on AM. No significant effect of residual particles was observed. A low concentration of organic extract also increased the AP activity of PBM. When the induction of an antioxidative enzyme was assessed, heme oxygenase-1 protein was found to be significantly increased by exposure to whole DEP, and the organic extract was more effective than the residual particles. Furthermore, the organic extract-induced expression of Ia antigen on PBM was reduced by the addition of an antioxidative agent. These results suggest that DEP may act on immature APC and enhance their AP activity and that the action contributing to oxidative stress may be mediated by organic compounds of DEP.

  13. Pseudomonas aeruginosa Cif Protein Enhances the Ubiquitination and Proteasomal Degradation of the Transporter Associated with Antigen Processing (TAP) and Reduces Major Histocompatibility Complex (MHC) Class I Antigen Presentation*

    PubMed Central

    Bomberger, Jennifer M.; Ely, Kenneth H.; Bangia, Naveen; Ye, Siying; Green, Kathy A.; Green, William R.; Enelow, Richard I.; Stanton, Bruce A.

    2014-01-01

    Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8+ T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation. PMID:24247241

  14. Effects of messenger RNA structure and other translational control mechanisms on major histocompatibility complex-I mediated antigen presentation

    PubMed Central

    Murat, Pierre; Tellam, Judy

    2015-01-01

    Effective T-cell surveillance of antigen-presenting cells is dependent on the expression of an array of antigenic peptides bound to major histocompatibility complex (MHC) class I (MHC-I) or class II (MHC-II) molecules. Pathogens co-evolving with their hosts exploit crucial translational regulatory mechanisms in order to evade host immune recognition and thereby sustain their infection. Evasion strategies that downregulate viral protein synthesis and thereby restrict antigen presentation to cytotoxic T-cells through the endogenous MHC-I pathway have been implicated in the pathogenesis of viral-associated malignancies. An understanding of the mechanisms by which messenger RNA (mRNA) structure modulates both viral mRNA translation and the antigen processing machinery to escape immune surveillance, will stimulate the development of alternative therapeutic strategies focused on RNA-directed drugs designed to enhance immune responses against infected cells. In this review, we discuss regulatory aspects of the MHC-I pathway and summarize current knowledge of the role attributed by mRNA structure and other translational regulatory mechanisms in immune evasion. In particular we highlight the impact of recently identified G-quadruplex structures within virally encoded transcripts as unique regulatory signals for translational control and antigen presentation. WIREs RNA 2015, 6:157–171. doi: 10.1002/wrna.1262 PMID:25264139

  15. Role of antigen presentation in the production of pro-inflammatory cytokines in obese adipose tissue.

    PubMed

    Majdoubi, Abdelilah; Kishta, Osama A; Thibodeau, Jacques

    2016-06-01

    Type II diabetes regroups different physiological anomalies that ultimately lead to low-grade chronic inflammation, insulin resistance and loss of pancreatic β-cells. Obesity is one of the best examples of such a condition that can develop into Metabolic Syndrome, causing serious health problems of great socio-economic consequences. The pathological outcome of obesity has a genetic basis and depends on the delicate balance between pro- and anti-inflammatory effectors of the immune system. The causal link between obesity and inflammation is well established. While innate immunity plays a key role in the development of a pro-inflammatory state in obese adipose tissues, it has now become clear that adaptive immune cells are also involved and participate in the cascade of events that lead to metabolic perturbations. The efficacy of some immunotherapeutic protocols in reducing the symptoms of obesity-driven metabolic syndrome in mice implicated all arms of the immune response. Recently, the production of pathogenic immunoglobulins and pro-inflammatory cytokines by B and T lymphocytes suggested an auto-immune basis for the establishment of a non-healthy obese state. Understanding the cellular landscape of obese adipose tissues and how immune cells sustain chronic inflammation holds the key to the development of targeted therapies. In this review, we emphasize the role of antigen-presenting cells and MHC molecules in obese adipose tissue and the general contribution of the adaptive arm of the immune system in inflammation-induced insulin resistance. PMID:26854212

  16. No Major Role for Insulin-Degrading Enzyme in Antigen Presentation by MHC Molecules

    PubMed Central

    Hsu, Hsiang-Ting; Burgevin, Anne; Guénette, Suzanne; Moser, Anna; van Endert, Peter

    2014-01-01

    Antigen presentation by MHC class I molecules requires degradation of epitope source proteins in the cytosol. Although the preeminent role of the proteasome is clearly established, evidence suggesting a significant role for proteasome-independent generation of class I ligands has been reported repeatedly. However, an enzyme responsible for such a role has not been identified. Recently insulin-degrading enzyme (IDE) was shown to produce an antigenic peptide derived from the tumor antigen MAGE-A3 in an entirely proteasome-independent manner, raising the question of the global impact of IDE in MHC class I antigen processing. Here we report that IDE knockdown in human cell lines, or knockout in two different mouse strains, has no effect on cell surface expression of various MHC class I molecules, including allomorphs such as HLA-A3 and HLA-B27 suggested to be loaded in an at least a partly proteasome-independent manner. Moreover, reduced or absent IDE expression does not affect presentation of five epitopes including epitopes derived from beta amyloid and proinsulin, two preferred IDE substrates. Thus, IDE does not play a major role in MHC class I antigen processing, confirming the dominant and almost exclusive role of the proteasome in cytosolic production of MHC class I ligands. PMID:24516642

  17. Resistance induced by drug abbreviated Schistosoma mansoni infections: treatment with the drug Ro11-3128 leads to enhanced antigen presentation.

    PubMed Central

    Smith, D A; Bickle, Q D; Kaye, P M

    1994-01-01

    Treatment of mice with the benzodiazepine derivative Ro11-3128 1-2 days post-infection with Schistosoma mansoni leads to arrest of virtually all schistosomula at the skin stage, and results in the development of protective immunity to challenge infection. A characteristic feature of Ro11-3128 treatment in vitro is the formation of exudates and membranous blebs at the schistosomular surface; other drugs tested, such as Ro15-5458 and oxamniquine which are also effective against the skin stages but relatively ineffective in inducing protection, do not induce this reaction. Here, we have examined whether such in vitro treatment causes enhanced presentation of schistosomular antigens by host antigen-presenting cells (APC) using an in vitro assay with activated peritoneal adherent cells as APC and T cells from S. mansoni antigen-sensitized mice. We have shown that viable mechanically transformed schistosomula (MS) can be processed and presented with similar kinetics to soluble antigen. However, in vitro drug treatment leads to enhanced presentation of MS. Experiments in which membranous blebs and antigen released by Ro11-3128-treated parasites during in vitro culture were separated from the remaining intact schistosomula, demonstrated significant stimulatory activity in the soluble and particulate-released antigen fractions. Filtration, antigen transfer experiments and SDS-PAGE analysis of the released material further suggested that most of the activity resided in the particulate fraction. Thus, quantitative and qualitative changes to antigen presentation by Ro11-3128 treatment early after infection may underlie the immunoprotective efficacy of Ro11-3128-abbreviated infections. Images Figure 3 Figure 9 PMID:7959877

  18. Antigen presentation of detergent free glutamate decarboxylase (GAD65) is affected by human serum albumin as carrier protein

    PubMed Central

    Steed, Jordan; Gilliam, Lisa K.; Harris, Robert A.; Lernmark, Åke; Hampe, Christiane S.

    2008-01-01

    1. Summary The smaller isoform of glutamate decarboxylase (GAD65) is a major autoantigen in type 1 diabetes (TID). Its hydrophobic character requires detergent to keep the protein in solution, which complicates studies of antigen processing and presentation. In this study an attempt was made to replace detergent with human serum albumin (HSA) for in vitro antigen presentation. Different preparations of recombinant human GAD65 complexed with HSA were incubated with Priess B cells (HLA DRB1*0401) and antigen presentation was tested with HLA DRB1*0401-restricted and epitope-specific T33.1 (GAD65 epitope 274-286) and T35 (GAD65 epitope 115-127) T cell hybridomas. Specific epitope recognition by T33.1 (274-286) and T35 (115-127) cells varied between the different GAD65/HSA preparations, and a reverse pattern of antigen presentation were detected by the two hybridoma. The HSA-specific T-cell hybridoma 17.9 response to the different GAD65/HSA preparations followed the same pattern as that observed for the T33.1 cells. The content of immunoreactive GAD65 measured with four GAD65 antibodies indicated that the lowest GAD65 concentration resulted in the highest 274-286, but the lowest 115-127 presentation. This suggests that HSA-GAD65 complexes qualitatively affect the epitope specificity of GAD65 presentation. HSA may enhance the 274-286 epitope presentation, while suppressing the 115-127 epitope. PMID:18353353

  19. Effective Inhibition of Kb- and Db-Restricted Antigen Presentation in Primary Macrophages by Murine Cytomegalovirus

    PubMed Central

    LoPiccolo, Diane M.; Gold, Marielle C.; Kavanagh, Daniel G.; Wagner, Markus; Koszinowski, Ulrich H.; Hill, Ann B.

    2003-01-01

    Macrophages play an important role in murine cytomegalovirus (MCMV) infection in vivo, both in disseminating infection and in harboring latent virus. MCMV encodes three immune evasion genes (m4, m6, and m152) that interfere with the ability of cytotoxic T cells (CTL) to detect virus-infected fibroblasts, but the efficacy of immune evasion in macrophages has been controversial. Here we show that MCMV immune evasion genes function in H-2b primary bone marrow macrophages (BMMφ) in the same way that they do in fibroblasts. Metabolic labeling experiments showed that class I is retained in the endoplasmic reticulum by MCMV infection and associates with m4/gp34 to a similar extent in fibroblasts and BMMφ. We tested a series of Kb- and Db-restricted CTL clones specific for MCMV early genes against a panel of MCMV wild-type virus and mutants lacking m152, m4, or m6. MCMV immune evasion genes effectively inhibited antigen presentation. m152 appeared sufficient to abolish Db-restricted presentation in infected macrophages, as has been previously observed in infected fibroblasts. However, for inhibition of recognition of infected macrophages by Kb-restricted CTL, m4, m6, and m152 were all required. The contribution of m4 to inhibition of recognition appeared much more important in macrophages than in fibroblasts. Thus, MCMV immune evasion genes function effectively in primary macrophages to prevent CTL recognition of early antigens and show the same pattern of major histocompatibility complex class I allele discrimination as is seen in fibroblasts. Furthermore, for inhibition of Kb-restricted presentation, a strong synergistic effect was noted among m152, m4, and m6. PMID:12477835

  20. Rheumatoid arthritis vaccine therapies: perspectives and lessons from therapeutic ligand epitope antigen presentation system vaccines for models of rheumatoid arthritis

    PubMed Central

    Rosenthal, Kenneth S.; Mikecz, Katalin; Steiner, Harold L.; Glant, Tibor T.; Finnegan, Alison; Carambula, Roy E.; Zimmerman, Daniel H.

    2016-01-01

    The current status of therapeutic vaccines for autoimmune diseases is reviewed with rheumatoid arthritis as the focus. Therapeutic vaccines for autoimmune diseases must regulate or subdue responses to common self-antigens. Ideally, such a vaccine would initiate an antigen-specific modulation of the T-cell immune response that drives the inflammatory disease. Appropriate animal models and types of T helper cells and signature cytokine responses that drive autoimmune disease are also discussed. Interpretation of these animal models must be done cautiously because the means of initiation, autoantigens, and even the signature cytokine and T helper cell (Th1 or Th17) responses that are involved in the disease may differ significantly from those in humans. We describe ligand epitope antigen presentation system vaccine modulation of T-cell autoimmune responses as a strategy for the design of therapeutic vaccines for rheumatoid arthritis, which may also be effective in other autoimmune conditions. PMID:25787143

  1. Salt bridge residues between I-Ak dimer of dimers alpha-chains modulate antigen presentation.

    PubMed

    Yadati, S; Nydam, T; Demian, D; Wade, T K; Gabriel, J L; Barisas, B G; Wade, W F

    1999-03-15

    Class II dimers of dimers are predicted to have functional significance in antigen presentation. The putative contact amino acids of the I-Ak class II dimer of dimers have been identified by molecular modeling based on the DR1 crystal structure (Nydam et al., Int. Immunol. 10, 1237,1998). We have previously reported the role in antigen presentation of dimer of dimers contact amino acids located in the C-terminal domains of the alpha- and beta-chains of class II. Our calculations show that residues Ealpha89 and Ralpha145 in the alpha2-domain form an inter alpha-chain salt bridge between pairs of alphabeta-heterodimers. Other residues, Qalpha92 and Nalpha115, may be involved in close association in that part of the alpha-chain. We investigated the role of these amino acids on class II expression and antigen presentation. Class II composed of an Ealpha89K substituted alpha-chain paired with a wt beta-chain exhibited inhibited antigen presentation and expression of alpha-chain serologic epitopes. In contrast, mutation of Ralpha145E had less affect on antigen presentation and did not affect I-Ak serologic epitopes. Interchanging charges of the salt bridge residues by expressing both Ralpha145E and Ealpha89K on the same chain obviated the large negative effect of the Ealpha89K mutation on antigen presentation but not on the serologic epitopes. Our results are similar for those reported for mutation of DR3's inter-chain salt bridge with the exception that double mutants did not moderate the DR3 defect. Interestingly, the amino acids differences between I-A and DR change the location of the inter-chain salt bridges. In DR1 these residues are located at positions Ealpha88 and Kalpha111; in I-Ak these residues are located at position Ealpha89 and Ralpha145. Inter alpha-chain salt bridges are thus maintained in various class II molecules by amino acids located in different parts of the alpha2-domain. This conservation of structure suggests that considerable functional

  2. Effect of substrate mechanical properties on T cell activation

    NASA Astrophysics Data System (ADS)

    Hui, King; Upadhyaya, Arpita

    2013-03-01

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

  3. The Critical Role of Antigen-Presentation-Induced Cytokine Crosstalk in the Central Nervous System in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Sosa, Rebecca A.

    2011-01-01

    Multiple sclerosis (MS) is a debilitating disease of the central nervous system (CNS) that has been extensively studied using the animal model experimental autoimmune encephalomyelitis (EAE). It is believed that CD4+ T lymphocytes play an important role in the pathogenesis of this disease by mediating the demyelination of neuronal axons via secretion of proinflammatory cytokines resulting in the clinical manifestations. Although a great deal of information has been gained in the last several decades about the cells involved in the inflammatory and disease mediating process, important questions have remained unanswered. It has long been held that initial neuroantigen presentation and T cell activation events occur in the immune periphery and then translocate to the CNS. However, an increasing body of evidence suggests that antigen (Ag) presentation might initiate within the CNS itself. Importantly, it has remained unresolved which antigen presenting cells (APCs) in the CNS are the first to acquire and present neuroantigens during EAE/MS to T cells, and what the conditions are under which this takes place, ie, whether this occurs in the healthy CNS or only during inflammatory conditions and what the related cytokine microenvironment is comprised of. In particular, the central role of interferon-γ as a primary mediator of CNS pathology during EAE has been challenged by the emergence of Th17 cells producing interleukin-17. This review describes our current understanding of potential APCs in the CNS and the contribution of these and other CNS-resident cells to disease pathology. Additionally, we discuss the question of where Ag presentation is initiated and under what conditions neuroantigens are made available to APCs with special emphasis on which cytokines may be important in this process. PMID:21919736

  4. Class-switched anti-insulin antibodies originate from unconventional antigen presentation in multiple lymphoid sites.

    PubMed

    Wan, Xiaoxiao; Thomas, James W; Unanue, Emil R

    2016-05-30

    Autoantibodies to insulin are a harbinger of autoimmunity in type 1 diabetes in humans and in non-obese diabetic mice. To understand the genesis of these autoantibodies, we investigated the interactions of insulin-specific T and B lymphocytes using T cell and B cell receptor transgenic mice. We found spontaneous anti-insulin germinal center (GC) formation throughout lymphoid tissues with GC B cells binding insulin. Moreover, because of the nature of the insulin epitope recognized by the T cells, it was evident that GC B cells presented a broader repertoire of insulin epitopes. Such broader recognition was reproduced by activating naive B cells ex vivo with a combination of CD40 ligand and interleukin 4. Thus, insulin immunoreactivity extends beyond the pancreatic lymph node-islets of Langerhans axis and indicates that circulating insulin, despite its very low levels, can have an influence on diabetogenesis. PMID:27139492

  5. Kinetic discrimination in T-cell activation.

    PubMed Central

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

    1996-01-01

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

  6. Immunoglobulin-like Transcript 4 (ILT4) Inhibits Lipid Antigen Presentation through Direct CD1d Interaction1

    PubMed Central

    Li, Demin; Wang, Lili; Yu, Li; Freundt, Eric C.; Jin, Boquan; Screaton, Gavin R.; Xu, Xiao-Ning

    2008-01-01

    Natural killer T (NKT) cells recognize lipid antigens presented by CD1d molecules and play an important role in the regulation of innate and adaptive immune responses. Here we report the identification of a membrane-associated protein, immunoglobulin-like transcript 4 (ILT4), as a novel human CD1d receptor that inhibits CD1d-mediated immune responses. We found that native CD1d tetramer generated by mammalian cells was able to specifically bind human monocytes in the peripheral blood, and this binding was at least partly mediated by monocyte-expressed ILT4. The interaction between ILT4 and CD1d involves the two N-terminal domains of ILT4 and the antigen-binding groove of CD1d (α1/α2 domain). This interaction has been identified on the cell surface as well as in the endosomal and lysosomal compartments. Functional analysis showed that ILT4 could block the loading of lipid antigens such as α-GalCer, and consequently inhibited NKT recognition. The interaction between ILT4 and CD1d may provide new insights into the regulation of NKT-mediated immunity. PMID:19124746

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

    PubMed Central

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

    2016-01-01

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

  8. First identification of regulatory B cell subsets expressing IL-10 in teleost fish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    IL-10 is an immunoregulatory cytokine with a potent anti-inflammatory activity, thus inhibiting the production of proinflammatory cytokines as well as processes of antigen presentation. IL-10 is produced by variety of cells, including antigen presentation cells (i.e., monocytes, macrophages and den...

  9. Susceptibility to measles virus-induced encephalitis in mice correlates with impaired antigen presentation to cytotoxic T lymphocytes.

    PubMed Central

    Niewiesk, S; Brinckmann, U; Bankamp, B; Sirak, S; Liebert, U G; ter Meulen, V

    1993-01-01

    In measles virus (MV) infection in humans, meningitis and encephalitis are important complications. However, little is known of the pathogenesis of MV encephalitis, in particular about the role of the immune response. We have examined the role of cytotoxic T lymphocytes (CTL) in a mouse model of MV-induced encephalitis. We report here that the resistance of inbred strains of mice to MV-induced encephalitis correlated with the major histocompatibility complex (MHC) haplotype and that only resistant mouse strains mounted an effective CTL response to MV. Mice with low susceptibility to MV infection, such as the BALB/c strain (H-2d), generated CTL, whereas the highly susceptible strains, C3H (H-2k) and C57BL/6 (H-2b), revealed very poor CTL responses. MV-induced CTL were usually CD8+, and the generation of these cells was independent of the route of inoculation or the time postinfection. CD4+ T cells were generally only weakly lytic. The nucleocapsid protein was the major target antigen for CTL in BALB/c mice, although in some experiments the hemagglutinin was also recognized. CTL from C3H and C57BL/6 mice did not lyse MV-infected target cells. However, targets infected with vaccinia virus recombinants expressing the nucleocapsid protein or hemagglutinin were lysed, but levels of cytotoxicity were still low. Experiments using target cells transfected with single MHC class I genes suggested inefficient antigen presentation of MV proteins by the MHC molecules of the H-2k and H-2b haplotypes. PMID:8093223

  10. Association of Polymorphisms in HLA Antigen Presentation-Related Genes with the Outcomes of HCV Infection

    PubMed Central

    Lu, Xiaomei; Xu, Yin; Wang, Jie; Zhang, Yun; Yu, Rongbin; Su, Jing

    2015-01-01

    Antigen-presentation genes play a vital role in the pathogenesis of HCV infection. However, the relationship of variants of these genes with spontaneous outcomes of HCV infection has not been fully investigated. To explore novel loci in the Chinese population, 34 tagging-SNPs in 9 candidate genes were genotyped for their associations with the outcomes of HCV infection. The distributions of different genotypes and haplotypes were compared among 773 HCV-negative controls, 246 subjects with HCV natural clearance, and 218 HCV persistent carriers recruited from hemodialysis patients and intravenous drug users. Our study implicated that TAP2, HLA-DOA, HLA-DOB, and tapasin loci were novel candidate regions for susceptibility to HCV infection and viral clearance in the Chinese population. Logistic regression analyses showed that TAP2 rs1800454 A (OR = 1.48, P = 0.002) and HLA-DOB rs2071469 G (OR = 1.23, P = 0.048) were significantly associated with increased susceptibility to establishment of HCV infection. However, high-risk behavior exposure and age were stronger predictors of HCV infection. Mutation of tapasin rs9277972 T (OR = 1.57, P =0.043) increased the risk of HCV chronicity, and HLA-DOA rs3128935 C (OR = 0.62, P = 0.019) increased the chance of viral resolution. With regards to the effect of rs3128925, interactions were found with high-risk behavior (P = 0.013) and age (P = 0.035). The risk effect of rs3128925 T for persistent HCV infection was higher in injecting drug users (vs. dialysis patients) and in subjects ≥ 40 years old (vs. < 40 years old). PMID:25874709

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

    PubMed Central

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

    2012-01-01

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

  12. The ESAT-6 protein of Mycobacterium tuberculosis interacts with beta-2-microglobulin (β2M) affecting antigen presentation function of macrophage.

    PubMed

    Sreejit, Gopalkrishna; Ahmed, Asma; Parveen, Nazia; Jha, Vishwanath; Valluri, Vijaya Lakshmi; Ghosh, Sudip; Mukhopadhyay, Sangita

    2014-10-01

    ESAT-6, an abundantly secreted protein of Mycobacterium tuberculosis (M. tuberculosis) is an important virulence factor, inactivation of which leads to reduced virulence of M. tuberculosis. ESAT-6 alone, or in complex with its chaperone CFP-10 (ESAT-6:CFP-10), is known to modulate host immune responses; however, the detailed mechanisms are not well understood. The structure of ESAT-6 or ESAT-6:CFP-10 complex does not suggest presence of enzymatic or DNA-binding activities. Therefore, we hypothesized that the crucial role played by ESAT-6 in the virulence of mycobacteria could be due to its interaction with some host cellular factors. Using a yeast two-hybrid screening, we identified that ESAT-6 interacts with the host protein beta-2-microglobulin (β2M), which was further confirmed by other assays, like GST pull down, co-immunoprecipitation and surface plasmon resonance. The C-terminal six amino acid residues (90-95) of ESAT-6 were found to be essential for this interaction. ESAT-6, in complex with CFP-10, also interacts with β2M. We found that ESAT-6/ESAT-6:CFP-10 can enter into the endoplasmic reticulum where it sequesters β2M to inhibit cell surface expression of MHC-I-β2M complexes, resulting in downregulation of class I-mediated antigen presentation. Interestingly, the ESAT-6:β2M complex could be detected in pleural biopsies of individuals suffering from pleural tuberculosis. Our data highlight a novel mechanism by which M. tuberculosis may undermine the host adaptive immune responses to establish a successful infection. Identification of such novel interactions may help us in designing small molecule inhibitors as well as effective vaccine design against tuberculosis. PMID:25356553

  13. The ESAT-6 Protein of Mycobacterium tuberculosis Interacts with Beta-2-Microglobulin (β2M) Affecting Antigen Presentation Function of Macrophage

    PubMed Central

    Parveen, Nazia; Jha, Vishwanath; Valluri, Vijaya Lakshmi; Ghosh, Sudip; Mukhopadhyay, Sangita

    2014-01-01

    ESAT-6, an abundantly secreted protein of Mycobacterium tuberculosis (M. tuberculosis) is an important virulence factor, inactivation of which leads to reduced virulence of M. tuberculosis. ESAT-6 alone, or in complex with its chaperone CFP-10 (ESAT-6:CFP-10), is known to modulate host immune responses; however, the detailed mechanisms are not well understood. The structure of ESAT-6 or ESAT-6:CFP-10 complex does not suggest presence of enzymatic or DNA-binding activities. Therefore, we hypothesized that the crucial role played by ESAT-6 in the virulence of mycobacteria could be due to its interaction with some host cellular factors. Using a yeast two-hybrid screening, we identified that ESAT-6 interacts with the host protein beta-2-microglobulin (β2M), which was further confirmed by other assays, like GST pull down, co-immunoprecipitation and surface plasmon resonance. The C-terminal six amino acid residues (90–95) of ESAT-6 were found to be essential for this interaction. ESAT-6, in complex with CFP-10, also interacts with β2M. We found that ESAT-6/ESAT-6:CFP-10 can enter into the endoplasmic reticulum where it sequesters β2M to inhibit cell surface expression of MHC-I-β2M complexes, resulting in downregulation of class I-mediated antigen presentation. Interestingly, the ESAT-6:β2M complex could be detected in pleural biopsies of individuals suffering from pleural tuberculosis. Our data highlight a novel mechanism by which M. tuberculosis may undermine the host adaptive immune responses to establish a successful infection. Identification of such novel interactions may help us in designing small molecule inhibitors as well as effective vaccine design against tuberculosis. PMID:25356553

  14. Balancing Selection Maintains a Form of ERAP2 that Undergoes Nonsense-Mediated Decay and Affects Antigen Presentation

    PubMed Central

    Kretzschmar, Warren W.; Cannons, Jennifer L.; Lee-Lin, Shih-Queen; Hurle, Belen; Schwartzberg, Pamela L.; Bustamante, Carlos D.; Nielsen, Rasmus; Clark, Andrew G.; Green, Eric D.

    2010-01-01

    A remarkable characteristic of the human major histocompatibility complex (MHC) is its extreme genetic diversity, which is maintained by balancing selection. In fact, the MHC complex remains one of the best-known examples of natural selection in humans, with well-established genetic signatures and biological mechanisms for the action of selection. Here, we present genetic and functional evidence that another gene with a fundamental role in MHC class I presentation, endoplasmic reticulum aminopeptidase 2 (ERAP2), has also evolved under balancing selection and contains a variant that affects antigen presentation. Specifically, genetic analyses of six human populations revealed strong and consistent signatures of balancing selection affecting ERAP2. This selection maintains two highly differentiated haplotypes (Haplotype A and Haplotype B), with frequencies 0.44 and 0.56, respectively. We found that ERAP2 expressed from Haplotype B undergoes differential splicing and encodes a truncated protein, leading to nonsense-mediated decay of the mRNA. To investigate the consequences of ERAP2 deficiency on MHC presentation, we correlated surface MHC class I expression with ERAP2 genotypes in primary lymphocytes. Haplotype B homozygotes had lower levels of MHC class I expressed on the surface of B cells, suggesting that naturally occurring ERAP2 deficiency affects MHC presentation and immune response. Interestingly, an ERAP2 paralog, endoplasmic reticulum aminopeptidase 1 (ERAP1), also shows genetic signatures of balancing selection. Together, our findings link the genetic signatures of selection with an effect on splicing and a cellular phenotype. Although the precise selective pressure that maintains polymorphism is unknown, the demonstrated differences between the ERAP2 splice forms provide important insights into the potential mechanism for the action of selection. PMID:20976248

  15. MHC class II antigen presentation pathway in murine tumours: tumour evasion from immunosurveillance?

    PubMed Central

    Walter, W; Lingnau, K; Schmitt, E; Loos, M; Maeurer, M J

    2000-01-01

    Qualitative differences in the MHC class II antigen processing and presentation pathway may be instrumental in shaping the CD4+ T cell response directed against tumour cells. Efficient loading of many MHC class II alleles with peptides requires the assistance of H2-M, a heterodimeric MHC class II-like molecule. In contrast to the HLA-DM region in humans, the β-chain locus is duplicated in mouse, with the H2-Mb1 (Mb1β-chain distal to H2-Mb2 (Mb2) and the H2-Ma (Ma) α-chain gene). Here, we show that murine MHC class II and H2-M genes are coordinately regulated in murine tumour cell lines by T helper cell 1 (IFN-γ) and T helper cell 2 (IL-4 or IL-10) cytokines in the presence of the MHC class II-specific transactivator CIITA as determined by mRNA expression and Western blot analysis. Furthermore, Mαβ1 and Mαβ2 heterodimers are differentially expressed in murine tumour cell lines of different histology. Both H2-M isoforms promote equally processing and presentation of native protein antigens to H2-Ad- and H2-Ed-restricted CD4+ T cells. Murine tumour cell lines could be divided into three groups: constitutive MHC class II and CIITA expression; inducible MHC class II and CIITA expression upon IFN-γ-treatment; and lack of constitutive and IFN-γ-inducible MHC class II and CIITA expression. These differences may impact on CD4+ T cell recognition of cancer cells in murine tumour models. © 2000 Cancer Research Campaign PMID:11027433

  16. Skin tumor immunity: Site does matter for antigen presentation by DCs.

    PubMed

    Waithman, Jason; Gebhardt, Thomas; Bedoui, Sammy

    2016-03-01

    The immune system has the ability to specifically identify and eliminate tumors, but the underlying mechanisms responsible for this phenomenon are not fully understood. A study published in this issue of the European Journal of Immunology now provides new insights into this important problem. Joncker et al. [Eur. J. Immunol. 2016. 46: 609-618] show that the timely mobilization of tumor antigen-bearing dendritic cells (DCs) from the periphery to the lymph nodes is critical for effective antitumor T-cell immunity, and that DCs present tumor antigens much more efficiently when encountered in the skin rather than in the subcutaneous tissues. PMID:26842676

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

    PubMed Central

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

    2010-01-01

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

  18. N-3 polyunsaturated fatty acids modulate B cell activity in pre-clinical models: Implications for the immune response to infections.

    PubMed

    Whelan, Jarrett; Gowdy, Kymberly M; Shaikh, Saame Raza

    2016-08-15

    B cell antigen presentation, cytokine production, and antibody production are targets of pharmacological intervention in inflammatory and infectious diseases. Here we review recent pre-clinical evidence demonstrating that pharmacologically relevant levels of n-3 polyunsaturated fatty acids (PUFA) derived from marine fish oils influence key aspects of B cell function through multiple mechanisms. N-3 PUFAs modestly diminish B cell mediated stimulation of classically defined naïve CD4(+) Th1 cells through the major histocompatibility complex (MHC) class II pathway. This is consistent with existing data showing that n-3 PUFAs suppress the activation of Th1/Th17 cells through direct effects on helper T cells and indirect effects on antigen presenting cells. Mechanistically, n-3 PUFAs lower antigen presentation and T cell signaling by disrupting the formation of lipid microdomains within the immunological synapse. We then review data to show that n-3 PUFAs boost B cell activation and antibody production in the absence and presence of antigen stimulation. This has potential benefits for several clinical populations such as the aged and obese that have poor humoral immunity. The mode of action by which n-3 PUFA boost B cell activation and antibody production remains unclear, but may involve Th2 cytokines, enhanced production of specialized proresolving lipid mediators, and targeting of protein lateral organization in lipid microdomains. Finally, we highlight evidence to show that different n-3 PUFAs are not biologically equivalent, which has implications for the development of future interventions to target B cell activity. PMID:26022530

  19. Emerging roles for antigen presentation in establishing host-microbiome symbiosis.

    PubMed

    Bessman, Nicholas J; Sonnenberg, Gregory F

    2016-07-01

    Trillions of beneficial bacteria inhabit the intestinal tract of healthy mammals from birth. Accordingly, mammalian hosts have evolved a series of complementary and redundant pathways to limit pathologic immune responses against these bacteria, while simultaneously protecting against enteric pathogen invasion. These pathways can be generically responsive to the presence of any commensal bacteria and innate in nature, as for IL-22-related pathways. Alternatively, specific bacterial antigens can drive a distinct set of adaptive immune cell responses, including IgA affinity maturation and secretion, and a recently described pathway of intestinal selection whereby MHCII(+) ILC3 deletes commensal bacteria-reactive CD4 T cells. These pathways can either promote or inhibit colonization by specific subsets of commensal bacteria, and cooperatively maintain intestinal homeostasis. In this review, we will highlight recent developments in understanding how these diverse pathways complement each other to cooperatively shape the symbiotic relationship between commensal bacteria and mammalian hosts. PMID:27319348

  20. A KALA-modified lipid nanoparticle containing CpG-free plasmid DNA as a potential DNA vaccine carrier for antigen presentation and as an immune-stimulative adjuvant

    PubMed Central

    Miura, Naoya; Shaheen, Sharif M.; Akita, Hidetaka; Nakamura, Takashi; Harashima, Hideyoshi

    2015-01-01

    Technologies that delivery antigen-encoded plasmid DNA (pDNA) to antigen presenting cell and their immune-activation are required for the success of DNA vaccines. Here we report on an artificial nanoparticle that can achieve these; a multifunctional envelope-type nanodevice modified with KALA, a peptide that forms α-helical structure at physiological pH (KALA-MEND). KALA modification and the removal of the CpG-motifs from the pDNA synergistically boosted transfection efficacy. In parallel, transfection with the KALA-MEND enhances the production of multiple cytokines and chemokines and co-stimulatory molecules via the Toll-like receptor 9-independent manner. Endosome-fusogenic lipid envelops and a long length of pDNA are essential for this immune stimulation. Furthermore, cytoplasmic dsDNA sensors that are related to the STING/TBK1 pathway and inflammasome are involved in IFN-β and IL-1β production, respectively. Consequently, the robust induction of antigen-specific cytotoxic T-lymphoma activity and the resulting prophylactic and therapeutic anti-tumor effect was observed in mice that had been immunized with bone marrow-derived dendritic cells ex vivo transfected with antigen-encoding pDNA. Collectively, the KALA-MEND possesses dual functions; gene transfection system and immune-stimulative adjuvant, those are both necessary for the successful DNA vaccine. PMID:25605799

  1. The saga of MHC-bound peptides: a renaissance for antigen presentation?

    PubMed Central

    Teyton, Luc

    2007-01-01

    In this issue of the JCI, two separate studies on MHC-bound peptides reopen the debate on the utility of peptides for the purposes of vaccination and treatment of autoimmune diseases. In the first study, by Wahlström et al., peptides bound to HLA-DR17 from bronchoalveolar lavage cells of sarcoidosis patients were analyzed in order to identify target antigens of the autoimmune response (see the related article beginning on page 3576). In the second study, by Le Gall et al., the modulation of epitope immunodominance and the processing and presentation of HIV peptides for MHC class I recognition were shown to be dependent on flanking residues that were N terminal to the natural epitopes (see the related article beginning on page 3563). Both studies highlight the tremendous therapeutic potential of MHC-bound peptides. They also emphasize that technical issues are still plaguing this field and hindering our understanding of MHC presentation in vivo. PMID:17975658

  2. Solar cell activation system

    SciTech Connect

    Apelian, L.

    1983-07-05

    A system for activating solar cells involves the use of phosphorescent paint, the light from which is amplified by a thin magnifying lens and used to activate solar cells. In a typical system, a member painted with phosphorescent paint is mounted adjacent a thin magnifying lens which focuses the light on a predetermined array of sensitive cells such as selenium, cadmium or silicon, mounted on a plastic board. A one-sided mirror is mounted adjacent the cells to reflect the light back onto said cells for purposes of further intensification. The cells may be coupled to rechargeable batteries or used to directly power a small radio or watch.

  3. Cytotoxic activity of interferon alpha induced dendritic cells as a biomarker of glioblastoma

    NASA Astrophysics Data System (ADS)

    Mishinov, S. V.; Stupak, V. V.; Tyrinova, T. V.; Leplina, O. Yu.; Ostanin, A. A.; Chernykh, E. R.

    2016-08-01

    Dendritic cells (DCs) are the most potent antigen presenting cells that can play direct role in anti-tumor immune response as killer cells. DC tumoricidal activity can be stimulated greatly by type I IFN (IFNα and IFNβ). In the present study, we examined cytostatic and cytotoxic activity of monocyte-derived IFNα-induced DCs generated from patients with brain glioma and evaluated the potential use of these parameters in diagnostics of high-grade gliomas. Herein, we demonstrated that patient DCs do not possess the ability to inhibit the growth of tumor HEp-2 cell line but low-grade and high-grade glioma patients do not differ significantly in DC cytostatic activity. However, glioma patient DCs are characterized by reduced cytotoxic activity against HEp-2 cells. The impairment of DC cytotoxic function is observed mainly in glioblastoma patients. The cytotoxic activity of DCs against HEp-2 cells below 9% is an informative marker for glioblastomas.

  4. Antigen-presenting genes and genomic copy number variations in the Tasmanian devil MHC

    PubMed Central

    2012-01-01

    Background The Tasmanian devil (Sarcophilus harrisii) is currently under threat of extinction due to an unusual fatal contagious cancer called Devil Facial Tumour Disease (DFTD). DFTD is caused by a clonal tumour cell line that is transmitted between unrelated individuals as an allograft without triggering immune rejection due to low levels of Major Histocompatibility Complex (MHC) diversity in Tasmanian devils. Results Here we report the characterization of the genomic regions encompassing MHC Class I and Class II genes in the Tasmanian devil. Four genomic regions approximately 960 kb in length were assembled and annotated using BAC contigs and physically mapped to devil Chromosome 4q. 34 genes and pseudogenes were identified, including five Class I and four Class II loci. Interestingly, when two haplotypes from two individuals were compared, three genomic copy number variants with sizes ranging from 1.6 to 17 kb were observed within the classical Class I gene region. One deletion is particularly important as it turns a Class Ia gene into a pseudogene in one of the haplotypes. This deletion explains the previously observed variation in the Class I allelic number between individuals. The frequency of this deletion is highest in the northwestern devil population and lowest in southeastern areas. Conclusions The third sequenced marsupial MHC provides insights into the evolution of this dynamic genomic region among the diverse marsupial species. The two sequenced devil MHC haplotypes revealed three copy number variations that are likely to significantly affect immune response and suggest that future work should focus on the role of copy number variations in disease susceptibility in this species. PMID:22404855

  5. Human MAIT-cell responses to Escherichia coli: activation, cytokine production, proliferation, and cytotoxicity

    PubMed Central

    Dias, Joana; Sobkowiak, Michał J.; Sandberg, Johan K.; Leeansyah, Edwin

    2016-01-01

    Mucosa-associated invariant T cells are a large and relatively recently described innate-like antimicrobial T-cell subset in humans. These cells recognize riboflavin metabolites from a range of microbes presented by evolutionarily conserved major histocompatibility complex, class I-related molecules. Given the innate-like characteristics of mucosa-associated invariant T cells and the novel type of antigens they recognize, new methodology must be developed and existing methods refined to allow comprehensive studies of their role in human immune defense against microbial infection. In this study, we established protocols to examine a range of mucosa-associated invariant T-cell functions as they respond to antigen produced by Escherichia coli. These improved and dose- and time-optimized experimental protocols allow detailed studies of MR1-dependent mucosa-associated invariant T-cell responses to Escherichia coli pulsed antigen-presenting cells, as assessed by expression of activation markers and cytokines, by proliferation, and by induction of apoptosis and death in major histocompatibility complex, class I-related–expressing target cells. The novel and optimized protocols establish a framework of methods and open new possibilities to study mucosa-associated invariant T-cell immunobiology, using Escherichia coli as a model antigen. Furthermore, we propose that these robust experimental systems can also be adapted to study mucosa-associated invariant T-cell responses to other microbes and types of antigen-presenting cells. PMID:27034405

  6. B Cell Infection and Activation by Rabies Virus-Based Vaccines

    PubMed Central

    Lytle, Andrew G.; Norton, James E.; Dorfmeier, Corin L.; Shen, Shixue

    2013-01-01

    Replication-deficient rabies viruses (RABV) are promising rabies postexposure vaccines due to their prompt and potent stimulation of protective virus neutralizing antibody titers, which are produced in mice by both T-dependent and T-independent mechanisms. To promote such early and robust B cell stimulation, we hypothesized that live RABV-based vaccines directly infect B cells, thereby activating a large pool of antigen-presenting cells (APCs) capable of providing early priming and costimulation to CD4+ T cells. In this report, we show that live RABV-based vaccine vectors efficiently infect naive primary murine and human B cells ex vivo. Infection of B cells resulted in the significant upregulation of early markers of B cell activation and antigen presentation, including CD69, major histocompatibility complex class II (MHC-II), and CD40 in murine B cells or HLA-DR and CD40 in human B cells compared to mock-infected cells or cells treated with an inactivated RABV-based vaccine. Furthermore, primary B cells infected with a live RABV expressing ovalbumin were able to prime and stimulate naive CD4+ OT-II T cells to proliferate and to secrete interleukin-2 (IL-2), demonstrating a functional consequence of B cell infection and activation by live RABV-based vaccine vectors. We propose that this direct B cell stimulation by live RABV-based vaccines is a potential mechanism underlying their induction of early protective T cell-dependent B cell responses, and that designing live RABV-based vaccines to infect and activate B cells represents a promising strategy to develop a single-dose postexposure rabies vaccine where the generation of early protective antibody titers is critical. PMID:23760241

  7. B cell infection and activation by rabies virus-based vaccines.

    PubMed

    Lytle, Andrew G; Norton, James E; Dorfmeier, Corin L; Shen, Shixue; McGettigan, James P

    2013-08-01

    Replication-deficient rabies viruses (RABV) are promising rabies postexposure vaccines due to their prompt and potent stimulation of protective virus neutralizing antibody titers, which are produced in mice by both T-dependent and T-independent mechanisms. To promote such early and robust B cell stimulation, we hypothesized that live RABV-based vaccines directly infect B cells, thereby activating a large pool of antigen-presenting cells (APCs) capable of providing early priming and costimulation to CD4(+) T cells. In this report, we show that live RABV-based vaccine vectors efficiently infect naive primary murine and human B cells ex vivo. Infection of B cells resulted in the significant upregulation of early markers of B cell activation and antigen presentation, including CD69, major histocompatibility complex class II (MHC-II), and CD40 in murine B cells or HLA-DR and CD40 in human B cells compared to mock-infected cells or cells treated with an inactivated RABV-based vaccine. Furthermore, primary B cells infected with a live RABV expressing ovalbumin were able to prime and stimulate naive CD4(+) OT-II T cells to proliferate and to secrete interleukin-2 (IL-2), demonstrating a functional consequence of B cell infection and activation by live RABV-based vaccine vectors. We propose that this direct B cell stimulation by live RABV-based vaccines is a potential mechanism underlying their induction of early protective T cell-dependent B cell responses, and that designing live RABV-based vaccines to infect and activate B cells represents a promising strategy to develop a single-dose postexposure rabies vaccine where the generation of early protective antibody titers is critical. PMID:23760241

  8. 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. PMID:26561366

  9. Neuropilin-1 expression characterizes T follicular helper (Tfh) cells activated during B cell differentiation in human secondary lymphoid organs.

    PubMed

    Renand, Amédée; Milpied, Pierre; Rossignol, Julien; Bruneau, Julie; Lemonnier, François; Dussiot, Michael; Coulon, Séverine; Hermine, Olivier

    2013-01-01

    T follicular helper (Tfh) cells play an essential role in the development of antigen-specific B cell immunity. Tfh cells regulate the differentiation and survival of activated B cells outside and inside germinal centers (GC) of secondary lymphoid organs. They act through cognate contacts with antigen-presenting B cells, but there is no current marker to specifically identify those Tfh cells which productively interact with B cells. Here we show that neuropilin 1 (Nrp1), a cell surface receptor, is selectively expressed by a subset of Tfh cells in human secondary lymphoid organs. Nrp1 expression on Tfh cells correlates with B cell differentiation in vivo and in vitro, is transient, and can be induced upon co-culture with autologous memory B cells in a cell contact-dependent manner. Comparative analysis of ex vivo Nrp1(+) and Nrp1(-) Tfh cells reveals gene expression modulation during activation. Finally, Nrp1 is expressed by malignant Tfh-like cells in a severe case of angioimmunoblastic T-cell lymphoma (AITL) associated with elevated terminal B cell differentiation. Thus, Nrp1 is a specific marker of Tfh cells cognate activation in humans, which may prove useful as a prognostic factor and a therapeutic target in neoplastic diseases associated with Tfh cells activity. PMID:24386482

  10. Immune activation by combination human lymphokine-activated killer and dendritic cell therapy

    PubMed Central

    West, E J; Scott, K J; Jennings, V A; Melcher, A A

    2011-01-01

    Background: Optimal cellular immunotherapy for cancer should ideally harness both the innate and adaptive arms of the immune response. Lymphokine-activated killer cells (LAKs) can trigger early innate killing of tumour targets, whereas long-term adaptive-specific tumour control requires priming of CD8+ cytotoxic lymphocytes (CTLs) following acquisition of tumour-associated antigens (TAAs) by antigen-presenting cells such as dendritic cells (DCs). As DCs stimulate both innate and adaptive effectors, combination cell therapy using LAKs and DCs has the potential to maximise anti-tumour immune priming. Methods: Reciprocal activation between human clinical grade LAKs and DCs on co-culture, and its immune consequences, was monitored by cell phenotype, cytokine release and priming of both innate and adaptive cytotoxicity against melanoma targets. Results: Co-culture of DCs and LAKs led to phenotypic activation of natural killer (NK) cells within the LAK population, which was associated with increased production of inflammatory cytokines and enhanced innate cytotoxicity against tumour cell targets. The LAKs reciprocally matured DCs, and the combination of LAKs and DCs, on addition of melanoma cells, supported priming of specific anti-tumour CTLs better than DCs alone. Conclusion: Clinical-grade LAKs/DCs represents a practical, effective combination cell immunotherapy for stimulation of both innate and adaptive anti-tumour immunity in cancer patients. PMID:21847125

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

    NASA Astrophysics Data System (ADS)

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

    2002-10-01

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

  12. Functional Anatomy of T Cell Activation and Synapse Formation

    PubMed Central

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

    2010-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1998-08-01

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

  14. Mechanisms of Innate Lymphoid Cell and Natural Killer T Cell Activation during Mucosal Inflammation

    PubMed Central

    Altmayer, Nora

    2014-01-01

    Mucosal surfaces in the airways and the gastrointestinal tract are critical for the interactions of the host with its environment. Due to their abundance at mucosal tissue sites and their powerful immunomodulatory capacities, the role of innate lymphoid cells (ILCs) and natural killer T (NKT) cells in the maintenance of mucosal tolerance has recently moved into the focus of attention. While NKT cells as well as ILCs utilize distinct transcription factors for their development and lineage diversification, both cell populations can be further divided into three polarized subpopulations reflecting the distinction into Th1, Th2, and Th17 cells in the adaptive immune system. While bystander activation through cytokines mediates the induction of ILC and NKT cell responses, NKT cells become activated also through the engagement of their canonical T cell receptors (TCRs) by (glyco)lipid antigens (cognate recognition) presented by the atypical MHC I like molecule CD1d on antigen presenting cells (APCs). As both innate lymphocyte populations influence inflammatory responses due to the explosive release of copious amounts of different cytokines, they might represent interesting targets for clinical intervention. Thus, we will provide an outlook on pathways that might be interesting to evaluate in this context. PMID:24987710

  15. Immunostimulatory Activity of the Cytokine-Based Biologic, IRX-2, on Human Papillomavirus-Exposed Langerhans Cells

    PubMed Central

    Da Silva, Diane M.; Woodham, Andrew W.; Naylor, Paul H.; Egan, James E.; Berinstein, Neil L.

    2016-01-01

    Langerhans cells (LCs) are the antigen-presenting cells of the epithelial layer and are responsible for initiating immune responses against skin and mucosa-invading viruses. Human papillomavirus (HPV)-mediated suppression of LC function is a crucial mechanism of HPV immune evasion, which can lead to persistent infection and development of several human cancers, including cervical, anal, and head and neck cancers. The cell-derived cytokine-based biologic, IRX-2, consists of multiple well-defined cytokines and is broadly active on various immune cell subsets. In this study, we investigated primary human LC activation after exposure to HPV16, followed by treatment with IRX-2 in vitro, and evaluated their subsequent ability to induce HPV16-specific T cells. In contrast to its activity on dendritic cells, HPV16 alone is not sufficient to induce phenotypic and functional activation of LCs. However, IRX-2 induces a significant upregulation of antigen presentation and costimulatory molecules, T helper 1 (Th1)-associated cytokine release, and chemokine-directed migration of LCs pre-exposed to HPV16. Furthermore, LCs treated with IRX-2 after HPV16 exposure induced CD8+ T-cell responses against specific HLA-A*0201-binding HPV16 T-cell epitopes. The present study suggests that IRX-2 is an attractive immunomodulator for assisting the immune response in eradication of HPV-infected cells, thereby potentially preventing HPV-induced cancers. PMID:26653678

  16. Immunostimulatory Activity of the Cytokine-Based Biologic, IRX-2, on Human Papillomavirus-Exposed Langerhans Cells.

    PubMed

    Da Silva, Diane M; Woodham, Andrew W; Naylor, Paul H; Egan, James E; Berinstein, Neil L; Kast, W Martin

    2016-05-01

    Langerhans cells (LCs) are the antigen-presenting cells of the epithelial layer and are responsible for initiating immune responses against skin and mucosa-invading viruses. Human papillomavirus (HPV)-mediated suppression of LC function is a crucial mechanism of HPV immune evasion, which can lead to persistent infection and development of several human cancers, including cervical, anal, and head and neck cancers. The cell-derived cytokine-based biologic, IRX-2, consists of multiple well-defined cytokines and is broadly active on various immune cell subsets. In this study, we investigated primary human LC activation after exposure to HPV16, followed by treatment with IRX-2 in vitro, and evaluated their subsequent ability to induce HPV16-specific T cells. In contrast to its activity on dendritic cells, HPV16 alone is not sufficient to induce phenotypic and functional activation of LCs. However, IRX-2 induces a significant upregulation of antigen presentation and costimulatory molecules, T helper 1 (Th1)-associated cytokine release, and chemokine-directed migration of LCs pre-exposed to HPV16. Furthermore, LCs treated with IRX-2 after HPV16 exposure induced CD8(+) T-cell responses against specific HLA-A*0201-binding HPV16 T-cell epitopes. The present study suggests that IRX-2 is an attractive immunomodulator for assisting the immune response in eradication of HPV-infected cells, thereby potentially preventing HPV-induced cancers. PMID:26653678

  17. Antigen uptake and expression of antigen presentation-related immune genes in flounder (Paralichthys olivaceus) after vaccination with an inactivated Edwardsiella tarda immersion vaccine, following hyperosmotic treatment.

    PubMed

    Gao, Yingli; Tang, Xiaoqian; Sheng, Xiuzhen; Xing, Jing; Zhan, Wenbin

    2016-08-01

    Antigen uptake is a critical process for activation of the immune system, and therefore the ability to enhance antigen uptake is a primary consideration in the development of an immersion vaccination of fish. In the present work, flounders (Paralichthys olivaceus) were immersed in three hyperosmotic solutions with 40, 50 and 60‰ salinities, then transferred into seawater of normal salinity (i.e. 30‰) containing formalin-inactivated Edwardsiella tarda for 30 min. The antigen uptake in vaccinated flounder was determined using an absolute quantitative PCR (qPCR). The results showed significantly higher antigen uptake in the tissues of flounders immersed in solutions with 50‰ and 60‰ salinity compared to the control group directly immersed in vaccine (DI) (P < 0.05), and the highest amount of antigen was detected in flounders immersed in the 50‰ salinity solution, whereas there was no significant difference in antigen uptake between the 40‰ salinity group and the DI group (P > 0.05). A rapid and significant increase in antigen uptake was detected in the mucosal-associated tissues including the gill, skin and intestine (P < 0.05) compared with the spleen, kidney and liver. Antigen uptake in the gill and skin both peaked at 30 min post immersion, which was significantly higher than the levels of uptake measured in the other tissues (P < 0.05), and then quickly declined. In contrast, antigen uptake in the spleen, kidney and liver gradually increased 3 h post immersion (hpi). The expression profiles of four antigen presentation-related immune genes (MHC Iα, MHC IIα, CD4-1 and CD8α) were investigated after immersion. These four genes showed a significantly stronger response in the immersed flounders exposed to 50‰ salinity compared with the DI group (P < 0.05). In the mucosal-associated tissues, the expression of MHC Iα and CD8α genes peaked at 24 hpi, while the expression of MHC IIα and CD4-1 genes showed up-regulation in the gill and skin

  18. Human Leukocyte Antigen-Presented Macrophage Migration Inhibitory Factor Is a Surface Biomarker and Potential Therapeutic Target for Ovarian Cancer.

    PubMed

    Patterson, Andrea M; Kaabinejadian, Saghar; McMurtrey, Curtis P; Bardet, Wilfried; Jackson, Ken W; Zuna, Rosemary E; Husain, Sanam; Adams, Gregory P; MacDonald, Glen; Dillon, Rachelle L; Ames, Harold; Buchli, Rico; Hawkins, Oriana E; Weidanz, Jon A; Hildebrand, William H

    2016-02-01

    T cells recognize cancer cells via HLA/peptide complexes, and when disease overtakes these immune mechanisms, immunotherapy can exogenously target these same HLA/peptide surface markers. We previously identified an HLA-A2-presented peptide derived from macrophage migration inhibitory factor (MIF) and generated antibody RL21A against this HLA-A2/MIF complex. The objective of the current study was to assess the potential for targeting the HLA-A2/MIF complex in ovarian cancer. First, MIF peptide FLSELTQQL was eluted from the HLA-A2 of the human cancerous ovarian cell lines SKOV3, A2780, OV90, and FHIOSE118hi and detected by mass spectrometry. By flow cytometry, RL21A was shown to specifically stain these four cell lines in the context of HLA-A2. Next, partially matched HLA-A*02:01+ ovarian cancer (n = 27) and normal fallopian tube (n = 24) tissues were stained with RL21A by immunohistochemistry to assess differential HLA-A2/MIF complex expression. Ovarian tumor tissues revealed significantly increased RL21A staining compared with normal fallopian tube epithelium (P < 0.0001), with minimal staining of normal stroma and blood vessels (P < 0.0001 and P < 0.001 compared with tumor cells) suggesting a therapeutic window. We then demonstrated the anticancer activity of toxin-bound RL21A via the dose-dependent killing of ovarian cancer cells. In summary, MIF-derived peptide FLSELTQQL is HLA-A2-presented and recognized by RL21A on ovarian cancer cell lines and patient tumor tissues, and targeting of this HLA-A2/MIF complex with toxin-bound RL21A can induce ovarian cancer cell death. These results suggest that the HLA-A2/MIF complex should be further explored as a cell-surface target for ovarian cancer immunotherapy. PMID:26719579

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

    PubMed

    Benvenuti, Federica

    2016-01-01

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

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

    PubMed Central

    Benvenuti, Federica

    2016-01-01

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

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

    PubMed Central

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

    2009-01-01

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

  2. Phosphatidylinositol 4-Phosphate 5-Kinases in the Regulation of T Cell Activation.

    PubMed

    Porciello, Nicla; Kunkl, Martina; Viola, Antonella; Tuosto, Loretta

    2016-01-01

    Phosphatidylinositol 4,5-biphosphate kinases (PIP5Ks) are critical regulators of T cell activation being the main enzymes involved in the synthesis of phosphatidylinositol 4,5-biphosphate (PIP2). PIP2 is indeed a pivotal regulator of the actin cytoskeleton, thus controlling T cell polarization and migration, stable adhesion to antigen-presenting cells, spatial organization of the immunological synapse, and co-stimulation. Moreover, PIP2 also serves as a precursor for the second messengers inositol triphosphate, diacylglycerol, and phosphatidylinositol 3,4,5-triphosphate, which are essential for the activation of signaling pathways regulating cytokine production, cell cycle progression, survival, metabolism, and differentiation. Here, we discuss the impact of PIP5Ks on several T lymphocyte functions with a specific focus on the role of CD28 co-stimulation in PIP5K compartimentalization and activation. PMID:27242793

  3. Phosphatidylinositol 4-Phosphate 5-Kinases in the Regulation of T Cell Activation

    PubMed Central

    Porciello, Nicla; Kunkl, Martina; Viola, Antonella; Tuosto, Loretta

    2016-01-01

    Phosphatidylinositol 4,5-biphosphate kinases (PIP5Ks) are critical regulators of T cell activation being the main enzymes involved in the synthesis of phosphatidylinositol 4,5-biphosphate (PIP2). PIP2 is indeed a pivotal regulator of the actin cytoskeleton, thus controlling T cell polarization and migration, stable adhesion to antigen-presenting cells, spatial organization of the immunological synapse, and co-stimulation. Moreover, PIP2 also serves as a precursor for the second messengers inositol triphosphate, diacylglycerol, and phosphatidylinositol 3,4,5-triphosphate, which are essential for the activation of signaling pathways regulating cytokine production, cell cycle progression, survival, metabolism, and differentiation. Here, we discuss the impact of PIP5Ks on several T lymphocyte functions with a specific focus on the role of CD28 co-stimulation in PIP5K compartimentalization and activation. PMID:27242793

  4. White button mushroom enhances maturation of bone marrow derived dendritic cells and their antigen presenting function in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mushrooms have been shown to enhance immune response, which contributes to their anti-tumor property. White button mushrooms (Agaricus bisporus) (WBM) constitute 90 percent of the total mushrooms consumed in the United States; however, the health benefit of this strain in general is not well studied...

  5. Auto-presentation of Staphylococcal enterotoxin A by mouse CD4+ T cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The currently accepted model for superantigen (SAg )induced T cell activation suggests that SAg, without being processed, cross links both MHC class II, from Antigen Presenting Cells (APC), and V-beta, from T-cell receptor (TCR), initiating nonspecific T-cell activation. This T-cell proliferation in...

  6. Regulation of protein synthesis and autophagy in activated dendritic cells: implications for antigen processing and presentation.

    PubMed

    Argüello, Rafael J; Reverendo, Marisa; Gatti, Evelina; Pierre, Philippe

    2016-07-01

    Antigenic peptides presented in the context of major histocompatibility complex (MHC) molecules originate from the degradation of both self and non-self proteins. T cells can therefore recognize at the surface of surveyed cells, the self-peptidome produced by the cell itself (mostly inducing tolerance) or immunogenic peptides derived from exogenous origins. The initiation of adaptive immune responses by dendritic cells (DCs), through the antigenic priming of naïve T cells, is associated to microbial pattern recognition receptors engagement. Activation of DCs by microbial product or inflammatory cytokines initiates multiple processes that maximize DC capacity to present exogenous antigens and stimulate T cells by affecting major metabolic and membrane traffic pathways. These include the modulation of protein synthesis, the regulation of MHC and co-stimulatory molecules transport, as well as the regulation of autophagy, that, all together promote exogenous antigen presentation while limiting the display of self-antigens by MHC molecules. PMID:27319340

  7. Hsp90-peptide complexes stimulate antigen presentation through the class II pathway after binding scavenger receptor SREC-I

    PubMed Central

    Murshid, Ayesha; Gong, Jianlin; Calderwood, Stuart K

    2016-01-01

    Molecular chaperones such as heat shock protein 90 (Hsp90) have been shown to form complexes with tumor antigens and can be used to prepare anticancer vaccines largely due to this property. Earlier studies had suggested that, mice immunized with a molecular chaperone based vaccine derived from tumors became immune to further vaccination and that both CD8+ and CD4+ T cells were activated by the chaperone vaccine in a manner dependent on scavenger receptor SREC-I. Here we have investigated mechanisms whereby SREC-I might facilitate uptake of Hsp90 conjugated peptides by APC into the MHC class II pathway for presentation to CD4+ T cells. Our studies showed that antigenic peptides associated with Hsp90 were taken up into the Class II pathway by a mechanism dependent on SREC-I binding and internalization and presented to CD4+ T cells. In addition our studies showed that SREC-I could associate with MHC class II molecules on the cell surface and in intracellular endosomes, suggesting a mechanism involving facilitated uptake of peptides into the MHC class II pathway. These studies in addition to our earlier findings showed SREC-I to play a primary role in chaperone-associated antigen uptake both through cross priming of MHC class I molecules and entry into the class II pathway. PMID:25155057

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Burroughs, Nigel J; van der Merwe, P Anton

    2007-04-01

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

  10. Microparticulate β-glucan vaccine conjugates phagocytized by dendritic cells activate both naïve CD4 and CD8 T cells in vitro.

    PubMed

    Berner, Vanessa K; duPre, Sally A; Redelman, Doug; Hunter, Kenneth W

    2015-01-01

    Microparticulate β-glucan (MG) conjugated to vaccine antigen has been shown to serve as an effective adjuvant in vivo. To further study antigen presentation by MG:vaccine conjugates, bone marrow-derived dendritic cells (BMDC) were treated with MG conjugated to ovalbumin (OVA), then interacted with splenocytes from DO11.10 transgenic mice expressing an OVA peptide-specific T cell receptor. BMDC treated with MG:OVA induced significantly higher numbers of activated (CD25+CD69+) OVA-specific CD4+ T cells than BMDC treated with OVA alone. BMDC treated with MG:OVA upregulated CD86 and CD40 expression as well as MG alone, indicating that conjugation of OVA does not alter the immunostimulatory capacity of MG. Activation of CD8+ OVA-specific OT-1 cells showed that MG:OVA is also capable of enhancing cross-presentation by BMDC to CD8+ cytotoxic T cells. These results show that MG acts as an adjuvant to enhance antigen presentation by dendritic cells to naïve, antigen-specific CD4 and CD8 T cells. PMID:26549577

  11. Metabolic maintenance of cell asymmetry following division in activated T lymphocytes.

    PubMed

    Verbist, Katherine C; Guy, Cliff S; Milasta, Sandra; Liedmann, Swantje; Kamiński, Marcin M; Wang, Ruoning; Green, Douglas R

    2016-04-21

    Asymmetric cell division, the partitioning of cellular components in response to polarizing cues during mitosis, has roles in differentiation and development. It is important for the self-renewal of fertilized zygotes in Caenorhabditis elegans and neuroblasts in Drosophila, and in the development of mammalian nervous and digestive systems. T lymphocytes, upon activation by antigen-presenting cells (APCs), can undergo asymmetric cell division, wherein the daughter cell proximal to the APC is more likely to differentiate into an effector-like T cell and the distal daughter is more likely to differentiate into a memory-like T cell. Upon activation and before cell division, expression of the transcription factor c-Myc drives metabolic reprogramming, necessary for the subsequent proliferative burst. Here we find that during the first division of an activated T cell in mice, c-Myc can sort asymmetrically. Asymmetric distribution of amino acid transporters, amino acid content, and activity of mammalian target of rapamycin complex 1 (mTORC1) is correlated with c-Myc expression, and both amino acids and mTORC1 activity sustain the differences in c-Myc expression in one daughter cell compared to the other. Asymmetric c-Myc levels in daughter T cells affect proliferation, metabolism, and differentiation, and these effects are altered by experimental manipulation of mTORC1 activity or c-Myc expression. Therefore, metabolic signalling pathways cooperate with transcription programs to maintain differential cell fates following asymmetric T-cell division. PMID:27064903

  12. 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. PMID:21674065

  13. The crystal structure of a TL/CD8{alpha}{alpha} complex at 2.1 {angstrom} resolution : implications for modulation of T cell activation and memory.

    SciTech Connect

    Liu, Y.; Xiong, Y.; Naidenko, O. V.; Liu, J.-H.; Zhang, R.; Joachimiak, A.; Kronenberg, M.; Cheroutre, H.; Reinherz, E. L.; Wang, J.-H.; Biosciences Division; Dana-Farber Cancer Inst.; Harvard Medical School; La Jolla Inst. of Allergy and Immunology

    2003-02-01

    TL is a nonclassical MHC class I molecule that modulates T cell activation through relatively high-affinity interaction with CD8{alpha}{alpha}. To investigate how the TL/CD8{alpha}{alpha} interaction influences TCR signaling, we characterized the structure of the TL/CD8{alpha}{alpha} complex using X-ray crystallography. Unlike antigen-presenting molecules, the TL antigen-binding groove is occluded by specific conformational changes. This feature eliminates antigen presentation, severely hampers direct TCR recognition, and prevents TL from participating in the TCR activation complex. At the same time, the TL/CD8{alpha}{alpha} interaction is strengthened through subtle structure changes in the TL {alpha}3 domain. Thus, TL functions to sequester and redirect CD8{alpha}{alpha} away from the TCR, modifying lck-dependent signaling.

  14. In vitro T-cell activation of monocyte-derived macrophages by soluble messengers or cell-to-cell contact in bovine tuberculosis

    PubMed Central

    Liébana, E; Aranaz, A; Welsh, M; Neill, S D; Pollock, J M

    2000-01-01

    The macrophage plays a dual role in tuberculosis, promoting not only protection against mycobacteria, but also survival of the pathogen. Macrophages inhibit multiplication of mycobacteria but also act in concert with lymphocytes through presentation of antigens to T cells. Studies in animal and human infections have suggested a correlation of in vitro growth rates of mycobacteria with in vivo virulence, using uracil uptake to assess mycobacterial metabolism. This study found that blood-derived, non-activated bovine macrophages were capable of controlling Mycobacterium bovis bacillus Calmette–Guérin growth for up to 96 hr, but were permissive to intracellular growth of virulent M. bovis. The present investigation compared the in vitro modulation of these macrophage activities by cytokine-rich T-cell supernatants or cell-to-cell contact. On the one hand, treatment of cultured monocytes with mitogen-produced T-cell supernatants promoted morphological changes suggestive of an activation status, enhanced the antigen presentation capabilities of monocytes and up-regulated major histocompatibility complex class II expression. However, this activation was not associated with enhanced anti-M. bovis activity. On the other hand, incubation of infected monocytes with T-cell populations resulted in proportionally increased inhibition of M. bovis uracil uptake. This inhibition was also seen using cells from uninfected animals and indicated the necessity for cell-to-cell contact to promote antimycobacterial capability. PMID:10886395

  15. Distinct Migration and Contact Dynamics of Resting and IL-2-Activated Human Natural Killer Cells

    PubMed Central

    Olofsson, Per E.; Forslund, Elin; Vanherberghen, Bruno; Chechet, Ksenia; Mickelin, Oscar; Ahlin, Alexander Rivera; Everhorn, Tobias; Önfelt, Björn

    2013-01-01

    Natural killer (NK) cells serve as one of the first lines of defense against viral infections and transformed cells. NK cell cytotoxicity is not dependent on antigen presentation by target cells, but is dependent on integration of activating and inhibitory signals triggered by receptor–ligand interactions formed at a tight intercellular contact between the NK and target cell, i.e., the immune synapse. We have studied the single-cell migration behavior and target-cell contact dynamics of resting and interleukin (IL)-2-activated human peripheral blood NK cells. Small populations of NK cells and target cells were confined in microwells and imaged by fluorescence microscopy for >8 h. Only the IL-2-activated population of NK cells showed efficient cytotoxicity against the human embryonic kidney 293T target cells. We found that although the average migration speeds were comparable, activated NK cells showed significantly more dynamic migration behavior, with more frequent transitions between periods of low and high motility. Resting NK cells formed fewer and weaker contacts with target cells, which manifested as shorter conjugation times and in many cases a complete lack of post-conjugation attachment to target cells. Activated NK cells were approximately twice as big as the resting cells, displayed a more migratory phenotype, and were more likely to employ “motile scanning” of the target-cell surface during conjugation. Taken together, our experiments quantify, at the single-cell level, how activation by IL-2 leads to altered NK cell cytotoxicity, migration behavior, and contact dynamics. PMID:24639676

  16. Mechanism of NK cell activation induced by coculture with dendritic cells derived from peripheral blood monocytes

    PubMed Central

    Amakata, Y; Fujiyama, Y; Andoh, A; Hodohara, K; Bamba, T

    2001-01-01

    Dendritic cells (DCs) have been regarded as one of the effective antigen-presenting cells, but the relationship between DCs and lymphocytes, in particular natural killer (NK) cells, remains unclear. In this study, we evaluated how DCs interact with both lymphocytes and NK cells using a coculture system. The number of lymphocytes increased significantly when cocultured with DCs (1·8-fold increase). In particular, the proliferation of NK cells was prominent. Furthermore, the coculture of DCs with lymphocytes induced a marked increase in IL-12 and IFN-γ secretion. When contact between the DCs and lymphocytes was prevented, the secretion of both IL-12 and IFN-γ was markedly reduced. IFN-γ production was completely blocked by an anti-IL-12 antibody, indicating that IFN-γ secretion was dependent on IL-12 secretion. The stimulating effect of the DCs on the proliferation of the lymphocytes was partially suppressed by anti-IL-12 antibodies, and was completely attenuated when cellular contact was prevented. Furthermore, the NK cell proliferation induced by coculture with DCs was significantly blocked by the inhibition of the interaction of either CD40–CD40L or CD28–B7 molecule. The coculture with DCs enhanced NK activity by 40%, and this was partially suppressed by anti-IL-12 antibodies and was completely blocked by the inhibition of cell-to-cell contact. These results indicate that the activation of NK cells by DCs is partially mediated by IL-12 secretion, and that direct contact between DCs and NK cells play a major role in this response. PMID:11422197

  17. T helper 1 immunity requires complement-driven NLRP3 inflammasome activity in CD4⁺ T cells.

    PubMed

    Arbore, Giuseppina; West, Erin E; Spolski, Rosanne; Robertson, Avril A B; Klos, Andreas; Rheinheimer, Claudia; Dutow, Pavel; Woodruff, Trent M; Yu, Zu Xi; O'Neill, Luke A; Coll, Rebecca C; Sher, Alan; Leonard, Warren J; Köhl, Jörg; Monk, Pete; Cooper, Matthew A; Arno, Matthew; Afzali, Behdad; Lachmann, Helen J; Cope, Andrew P; Mayer-Barber, Katrin D; Kemper, Claudia

    2016-06-17

    The NLRP3 inflammasome controls interleukin-1β maturation in antigen-presenting cells, but a direct role for NLRP3 in human adaptive immune cells has not been described. We found that the NLRP3 inflammasome assembles in human CD4(+) T cells and initiates caspase-1-dependent interleukin-1β secretion, thereby promoting interferon-γ production and T helper 1 (T(H)1) differentiation in an autocrine fashion. NLRP3 assembly requires intracellular C5 activation and stimulation of C5a receptor 1 (C5aR1), which is negatively regulated by surface-expressed C5aR2. Aberrant NLRP3 activity in T cells affects inflammatory responses in human autoinflammatory disease and in mouse models of inflammation and infection. Our results demonstrate that NLRP3 inflammasome activity is not confined to "innate immune cells" but is an integral component of normal adaptive T(H)1 responses. PMID:27313051

  18. IL-36 promotes myeloid cell infiltration, activation and inflammatory activity in skin

    PubMed Central

    Foster, Alexander M.; Baliwag, Jaymie; Chen, Cynthia S.; Guzman, Andrew M.; Stoll, Stefan W.; Gudjonsson, Johann E.; Ward, Nicole L.; Johnston, Andrew

    2014-01-01

    The IL-1 family members IL-36α (IL-1F6), IL-36β (IL-1F8) and IL-36γ (IL-1F9) and the receptor antagonist IL-36Ra (IL-1F5) constitute a novel signaling system that is poorly understood. We now show that these cytokines have profound effects on the skin immune system. Treatment of human keratinocytes with IL-36 cytokines significantly increased the expression of CXCL1, CXCL8, CCL3, CCL5, and CCL20, potent chemotactic agents for activated leukocytes, and IL-36α injected intradermally resulted in chemokine expression, leukocyte infiltration and acanthosis of mouse skin. Blood monocytes, myeloid dendritic cells (DC) and monocyte-derived DC (MO-DC) expressed IL-36R and responded to IL-36. In contrast, no direct effects of IL-36 on resting or activated human CD4+ or CD8+ T cells, or blood neutrophils, could be demonstrated. Monocytes expressed IL-1A, IL-1B and IL-6 mRNA and IL-1β and IL-6 protein and mDC upregulated surface expression of CD83, CD86 and HLADR and secretion of IL-1β and IL-6 after treatment with IL-36. Furthermore, IL-36α-treated MO-DC enhanced allogeneic CD4+ T cell proliferation, demonstrating that IL-36 can stimulate the maturation and function of DC and drive T cell proliferation. These data indicate that IL-36 cytokines actively propagate skin inflammation via the activation of keratinocytes, antigen presenting cells and, indirectly, T cells. PMID:24829417

  19. Mode of dendritic cell activation: the decisive hand in Th2/Th17 cell differentiation. Implications in asthma severity?

    PubMed

    Vroman, Heleen; van den Blink, Bernt; Kool, Mirjam

    2015-02-01

    Asthma is a heterogeneous chronic inflammatory disease of the airways, with reversible airflow limitations and airway remodeling. The classification of asthma phenotypes was initially based on different combinations of clinical symptoms, but they are now unfolding to link biology to phenotype. As such, patients can suffer from a predominant eosinophilic, neutrophilic or even mixed eosinophilic/neutrophilic inflammatory response. In adult asthma patients, eosinophilic inflammation is usually seen in mild-to-moderate disease and neutrophilic inflammation in more severe disease. The underlying T cell response is predominated by T helper (Th) 2, Th17, or a mixed Th2/Th17 cell immune response. Dendritic cells (DCs) are "professional" antigen presenting cells (APCs), since their principal function is to present antigens and induce a primary immune response in resting naive T cells. DCs also drive the differentiation into distinctive Th subsets. The expression of co-stimulatory molecules and cytokines by DCs and surrounding cells determines the outcome of Th cell differentiation. The nature of DC activation will determine the expression of specific co-stimulatory molecules and cytokines, specifically needed for induction of the different Th cell programs. Thus DC activation is crucial for the subsequent effector Th immune responses. In this review, we will discuss underlying mechanisms that initiate DC activation in favor of Th2 differentiation versus Th1/Th17 and Th17 differentiation in the development of mild versus moderate to severe asthma. PMID:25245013

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

    PubMed Central

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

    2013-01-01

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

  1. HLA-B27-restricted antigen presentation in the absence of tapasin reveals polymorphism in mechanisms of HLA class I peptide loading.

    PubMed

    Peh, C A; Burrows, S R; Barnden, M; Khanna, R; Cresswell, P; Moss, D J; McCluskey, J

    1998-05-01

    Tapasin is a resident ER protein believed to be critical for antigen presentation by HLA class I molecules. We demonstrate that allelic variation in MHC class I molecules influences their dependence on tapasin for peptide loading and antigen presentation. HLA-B*2705 molecules achieve high levels of surface expression and present specific viral peptides in the absence of tapasin. In contrast, HLA-B*4402 molecules are highly dependent upon human tapasin for these functions, while HLA-B8 molecules are intermediate in this regard. Significantly, HLA-B*2705 like HLA-B*4402, requires tapasin to associate efficiently with TAP (transporters associated with antigen processing). The unusual ability of HLA-B*2705 to form peptide complexes without associating with TAP or tapasin confers flexibility in the repertoire of peptides presented by this molecule. We speculate that these properties might contribute to the role of HLA-B27 in conferring susceptibility to inflammatory spondyloarthropathies. PMID:9620674

  2. Fluorescence activated cell sorting.

    NASA Technical Reports Server (NTRS)

    Bonner, W. A.; Hulett, H. R.; Sweet, R. G.; Herzenberg, L. A.

    1972-01-01

    An instrument has been developed for sorting biological cells. The cells are rendered differentially fluorescent and incorporated into a small liquid stream illuminated by a laser beam. The cells pass sequentially through the beam, and fluorescent light from the cells gives rise to electrical signals. The stream is broken into a series of uniform size drops downstream of the laser. The cell signals are used to give appropriate electrostatic charges to drops containing the cells. The drops then pass between two charged plates and are deflected to appropriate containers. The system has proved capable of providing fractions containing large numbers of viable cells highly enriched in a particular functional type.

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

    PubMed Central

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

    2014-01-01

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

  4. Peptide microarrays for the profiling of cytotoxic T-lymphocyte activity using minimum numbers of cells

    PubMed Central

    Hoff, Antje; Bagû, Ana-Cristina; André, Thomas; Roth, Günter; Wiesmüller, Karl-Heinz; Gückel, Brigitte

    2010-01-01

    The identification of epitopes that elicit cytotoxic T-lymphocyte activity is a prerequisite for the development of cancer-specific immunotherapies. However, especially the parallel characterization of several epitopes is limited by the availability of T cells. Microarrays have enabled an unprecedented miniaturization and parallelization in biological assays. Here, we developed peptide microarrays for the detection of CTL activity. MHC class I-binding peptide epitopes were pipetted onto polymer-coated glass slides. Target cells, loaded with the cell-impermeant dye calcein, were incubated on these arrays, followed by incubation with antigen-expanded CTLs. Cytotoxic activity was detected by release of calcein and detachment of target cells. With only 200,000 cells per microarray, CTLs could be detected at a frequency of 0.5% corresponding to 1,000 antigen-specific T cells. Target cells and CTLs only settled on peptide spots enabling a clear separation of individual epitopes. Even though no physical boundaries were present between the individual spots, peptide loading only occurred locally and cytolytic activity was confined to the spots carrying the specific epitope. The peptide microarrays provide a robust platform that implements the whole process from antigen presentation to the detection of CTL activity in a miniaturized format. The method surpasses all established methods in the minimum numbers of cells required. With antigen uptake occurring on the microarray, further applications are foreseen in the testing of antigen precursors that require uptake and processing prior to presentation. PMID:20512327

  5. Expression of essential B cell genes and immunoglobulin isotypes suggests active development and gene recombination during equine gestation.

    PubMed

    Tallmadge, Rebecca L; McLaughlin, Kristin; Secor, Erica; Ruano, Diana; Matychak, Mary Beth; Flaminio, M Julia B F

    2009-09-01

    Many features of the equine immune system develop during fetal life, yet the naïve or immature immune state of the neonate renders the foal uniquely susceptible to particular pathogens. RT-PCR and immunohistochemical experiments investigated the progressive expression of developmental B cell markers and immunoglobulins in lymphoid tissues from equine fetus, pre-suckle neonate, foal, and adult horses. Serum IgM, IgG isotype, and IgA concentrations were also quantified in pre-suckle foals and adult horses. The expression of essential B cell genes suggests active development and gene recombination during equine gestation, including immunoglobulin isotype switching. The corresponding production of IgM and IgG proteins is detectable in a limited scale at birth. Although the equine neonate humoral response seems competent, B cell activation factors derived from antigen presenting cells and T cells may control critical developmental regulation and immunoglobulin production during the initial months of life. PMID:19442687

  6. The activation of Epimedium polysaccharide-propolis flavone liposome on Kupffer cells.

    PubMed

    Fan, Yunpeng; Ren, Meimei; Hou, Weifeng; Guo, Chao; Tong, Dewen; Ma, Lin; Zhang, Weimin; He, Mengmeng; Song, Xiaoping

    2015-11-20

    Epimedium polysaccharide-propolis flavone liposome (EPL), a potent immunological pharmaceutical preparation, was investigated for the immunomodulatory activity on Kupffer cells (KCs) in vitro. The results showed that EPL could significantly induce the secretion of chemokines (RANTES and MCP-1), promote the production of nitric oxide and induced nitric oxide synthase, improve the pinocytic and phagocytic activity of KCs, promote the mRNA expression of TNF-α and IL-1β, and enhance the expression of costimulatory molecules (CD11b and CD68) in KCs compared with Epimedium polysaccharide-propolis flavone (EP) at 30-7.5μg/mL. In addition, the abilities of KCs on stimulating lymphocytes proliferation and antigen presenting were significantly enhanced after stimulated with EPL compared with EP. These results suggested that EPL could activate KCs and possessed the stronger immunomodulatory effect, which provided the theoretical basis for further studying the mechanism of EPL on improving the immune response. PMID:26344320

  7. Type I interferons produced by dendritic cells promote their phenotypic and functional activation.

    PubMed

    Montoya, Maria; Schiavoni, Giovanna; Mattei, Fabrizio; Gresser, Ion; Belardelli, Filippo; Borrow, Persephone; Tough, David F

    2002-05-01

    Resting dendritic cells (DCs) are resident in most tissues and can be activated by environmental stimuli to mature into potent antigen-presenting cells. One important stimulus for DC activation is infection; DCs can be triggered through receptors that recognize microbial components directly or by contact with infection-induced cytokines. We show here that murine DCs undergo phenotypic maturation upon exposure to type I interferons (type I IFNs) in vivo or in vitro. Moreover, DCs either derived from bone marrow cells in vitro or isolated from the spleens of normal animals express IFN-alpha and IFN-beta, suggesting that type I IFNs can act in an autocrine manner to activate DCs. Consistent with this idea, the ability to respond to type I IFN was required for the generation of fully activated DCs from bone marrow precursors, as DCs derived from the bone marrow of mice lacking a functional receptor for type I IFN had reduced expression of costimulatory and adhesion molecules and a diminished ability to stimulate naive T-cell proliferation compared with DCs derived from control bone marrow. Furthermore, the addition of neutralizing anti-IFN-alpha/beta antibody to purified splenic DCs in vitro partially blocked the "spontaneous" activation of these cells, inhibiting the up-regulation of costimulatory molecules, secretion of IFN-gamma, and T-cell stimulatory activity. These results show that DCs both secrete and respond to type I IFN, identifying type I interferons as autocrine DC activators. PMID:11964292

  8. Early Activation of Teleost B Cells in Response to Rhabdovirus Infection

    PubMed Central

    Abós, Beatriz; Castro, Rosario; González Granja, Aitor; Havixbeck, Jeffrey J.; Barreda, Daniel R.

    2014-01-01

    ABSTRACT To date, the response of teleost B cells to specific pathogens has been only scarcely addressed. In this work, we have demonstrated that viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, has the capacity to infect rainbow trout spleen IgM-positive (IgM+) cells, although the infection is not productive. Consequently, we have studied the effects of VHSV on IgM+ cell functionality, comparing these effects to those elicited by a Toll-like receptor 3 (TLR3) ligand, poly(I·C). We found that poly(I·C) and VHSV significantly upregulated TLR3 and type I interferon (IFN) transcription in spleen and blood IgM+ cells. Further effects included the upregulated transcription of the CK5B chemokine. The significant inhibition of some of these effects in the presence of bafilomycin A1 (BAF), an inhibitor of endosomal acidification, suggests the involvement of an intracellular TLR in these responses. In the case of VHSV, these transcriptional effects were dependent on viral entry into B cells and the initiation of viral transcription. VHSV also provoked the activation of NF-κB and the upregulation of major histocompatibility complex class II (MHC-II) cell surface expression on IgM+ cells, which, along with the increased transcription of the costimulatory molecules CD80/86 and CD83, pointed to VHSV-induced IgM+ cell activation toward an antigen-presenting profile. Finally, despite the moderate effects of VHSV on IgM+ cell proliferation, a consistent effect on IgM+ cell survival was detected. IMPORTANCE Innate immune responses to pathogens established through their recognition by pattern recognition receptors (PRRs) have been traditionally ascribed to innate cells. However, recent evidence in mammals has revealed that innate pathogen recognition by B lymphocytes is a crucial factor in shaping the type of immune response that is mounted. In teleosts, these immediate effects of viral encounter on B lymphocytes have not been addressed to date. In our study, we

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

    PubMed

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

    2016-03-01

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

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

    PubMed

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

    2016-07-25

    The immunological synapse (IS) formed at the interface between T cells and antigen-presenting cells represents a hallmark of initiation of acquired immunity. T cell activation is initiated at T cell receptor (TCR) microclusters (MCs), in which TCRs and signaling molecules assemble at the interface before IS formation. We found that each TCR-MC was transiently bordered by a ring structure made of integrin and focal adhesion molecules in the early phase of activation, which is similar in structure to the IS in microscale. The micro-adhesion ring is composed of LFA-1, focal adhesion molecules paxillin and Pyk2, and myosin II (MyoII) and is supported by F-actin core and MyoII activity through LFA-1 outside-in signals. The formation of the micro-adhesion ring was transient but especially sustained upon weak TCR stimulation to recruit linker for activation of T cells (LAT) and SLP76. Perturbation of the micro-adhesion ring induced impairment of TCR-MC development and resulted in impaired cellular signaling and cell functions. Thus, the synapse-like structure composed of the core TCR-MC and surrounding micro-adhesion ring is a critical structure for initial T cell activation through integrin outside-in signals. PMID:27354546

  11. Identification of putative cathepsin S in mangrove red snapper Lutjanus argentimaculatus and its role in antigen presentation.

    PubMed

    Zhou, Jin; Li, Lei; Cai, Zhong-Hua

    2012-05-01

    Cathepsin S (CTSS) is a key enzyme employed in the histocompatibility complex (MHC) class II-restricted antigens, which are presented by processing class II-associated invariant chains and loaded antigen peptides into class II molecules. To date, little is known about the character and function of CTSS in fish. In the present study, we screened and identified a CTSS cDNA sequence from the mangrove red snapper head kidney cDNA library. The full-length CTSS cDNA contained 1339-bp nucleotide acids encoding 337 amino acids. The sequence shared high identity and similarity with other known cathepsins, especially CTSS (about 56-78% and 79-89%, respectively). Like other cathepsins, the deduced peptide consisted of regions with N-terminal signal peptides, propeptides, and mature peptides. A typical ERWNIN motif in L-like cathepsins and three conservative catalytic activity sites forming a catalytic triad active center were respectively identified in the pro-peptide and mature peptide regions of CTSS. Phylogenetic analysis revealed that mangrove red snapper CTSS was located in the CTSS clade belonging to the L-like cathepsin group, and evolved from the same ancestry. To further characterize the biological activity of the putative CTSS of mangrove snapper, CTSS was expressed in Escherichia coli M15 strains. Like other mammalian CTSS, the recombinant CTSS (rCTSS) had autocatalytic activation properties, can remove pro-peptides, and can release active mature peptides. Active CTSS had the ability to catalyze Z-Phe-Arg-AMC substrates in acidic conditions (pH 5.0) and weak alkaline environments (pH 7.5); this activity could be blocked by the cysteine protease inhibitor E-64. Active CTSS can process recombinant Ii chains (invariant chains) in a stepwise manner in vitro. The results indicate that mangrove red snapper CTSS is a lysosomal cysteine protease family member with a key role in antigen processing in fish. PMID:22210546

  12. Enhancing dendritic cell activation and HIV vaccine effectiveness through nanoparticle vaccination.

    PubMed

    Glass, Joshua J; Kent, Stephen J; De Rose, Robert

    2016-06-01

    Novel vaccination approaches are needed to prevent and control human immunodeficiency virus (HIV) infection. A growing body of literature demonstrates the potential of nanotechnology to modulate the human immune system and generate targeted, controlled immune responses. In this Review, we summarize important advances in how 'nanovaccinology' can be used to develop safe and effective vaccines for HIV. We highlight the central role of dendritic cells in the immune response to vaccination and describe how nanotechnology can be used to enhance delivery to and activation of these important antigen-presenting cells. Strategies employed to improve biodistribution are discussed, including improved lymph node delivery and mucosal penetration concepts, before detailing methods to enhance the humoral and/or cellular immune response to vaccines. We conclude with a commentary on the current state of nanovaccinology. PMID:26783186

  13. IgE epitope proximity determines immune complex shape and effector cell activation capacity

    PubMed Central

    Gieras, Anna; Linhart, Birgit; Roux, Kenneth H.; Dutta, Moumita; Khodoun, Marat; Zafred, Domen; Cabauatan, Clarissa R.; Lupinek, Christian; Weber, Milena; Focke-Tejkl, Margarete; Keller, Walter; Finkelman, Fred D.; Valenta, Rudolf

    2016-01-01

    Background IgE-allergen complexes induce mast cell and basophil activation and thus immediate allergic inflammation. They are also important for IgE-facilitated allergen presentation to T cells by antigen-presenting cells. Objective To investigate whether the proximity of IgE binding sites on an allergen affects immune complex shape and subsequent effector cell activation in vitro and in vivo. Methods We constructed artificial allergens by grafting IgE epitopes in different numbers and proximity onto a scaffold protein. The shape of immune complexes formed between artificial allergens and the corresponding IgE was studied by negative-stain electron microscopy. Allergenic activity was determined using basophil activation assays. Mice were primed with IgE, followed by injection of artificial allergens to evaluate their in vivo allergenic activity. Severity of systemic anaphylaxis was measured by changes in body temperature. Results We could demonstrate simultaneous binding of 4 IgE antibodies in close vicinity to each other. The proximity of IgE binding sites on allergens influenced the shape of the resulting immune complexes and the magnitude of effector cell activation and in vivo inflammation. Conclusions Our results demonstrate that the proximity of IgE epitopes on an allergen affects its allergenic activity. We thus identified a novel mechanism by which IgE-allergen complexes regulate allergic inflammation. This mechanism should be important for allergy and other immune complex–mediated diseases. PMID:26684291

  14. Determination of the Absolute Number of Cytokine mRNA Molecules within Individual Activated Human T Cells

    NASA Technical Reports Server (NTRS)

    Karr, Laurel J.; Marshall, Gwen; Hockett, Richard D.; Bucy, R. Pat; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    A primary function of activated T cells is the expression and subsequent secretion of cytokines, which orchestrate the differentiation of other lymphocytes, modulate antigen presenting cell activity, and alter vascular endothelium to mediate an immune response. Since many features of immune regulation probably result from modest alterations of endogenous rates of multiple interacting processes, quantitative analysis of the frequency and specific activity of individual T cells is critically important. Using a coordinated set of quantitative methods, the absolute number of molecules of several key cytokine mRNA species in individual T cells has been determined. The frequency of human blood T cells activated in vitro by mitogens and recall protein antigens was determined by intracellular cytokine protein staining, in situ hybridization for cytokine mRNA, and by limiting dilution analysis for cytokine mRNA+ cells. The absolute number of mRNA molecules was simultaneously determined in both homogenates of the entire population of cells and in individual cells obtained by limiting dilution, using a quantitative, competitive RT-PCR assay. The absolute numbers of mRNA molecules in a population of cells divided by the frequency of individual positive cells, yielded essentially the same number of mRNA molecules per cell as direct analysis of individual cells by limiting dilution analysis. Mean numbers of mRNA per positive cell from both mitogen and antigen activated T cells, using these stimulation conditions, were 6000 for IL-2, 6300 for IFN-gamma, and 1600 for IL-4.

  15. Target-Cell Contact Activates a Highly Selective Capacitative Calcium Entry Pathway in Cytotoxic T Lymphocytes

    PubMed Central

    Zweifach, Adam

    2000-01-01

    Calcium influx is critical for T cell activation. Evidence has been presented that T cell receptor–stimulated calcium influx in helper T lymphocytes occurs via channels activated as a consequence of depletion of intracellular calcium stores, a mechanism known as capacitative Ca2+ entry (CCE). However, two key questions have not been addressed. First, the mechanism of calcium influx in cytotoxic T cells has not been examined. While the T cell receptor–mediated early signals in helper and cytotoxic T cells are similar, the physiology of the cells is strikingly different, raising the possibility that the mechanism of calcium influx is also different. Second, contact of T cells with antigen-presenting cells or targets involves a host of intercellular interactions in addition to those between antigen–MHC and the T cell receptor. The possibility that calcium influx pathways in addition to those activated via the T cell receptor may be activated by contact with relevant cells has not been addressed. We have used imaging techniques to show that target-cell–stimulated calcium influx in CTLs occurs primarily through CCE. We investigated the permeability of the CTL influx pathway for divalent cations, and compared it to the permeability of CCE in Jurkat human leukemic T cells. CCE in CTLs shows a similar ability to discriminate between calcium, barium, and strontium as CCE in Jurkat human leukemic T lymphocytes, where CCE is likely to mediated by Ca2+ release–activated Ca2+ current (CRAC) channels, suggesting that CRAC channels also underlie CCE in CTLs. These results are the first determination of the mechanism of calcium influx in cytotoxic T cells and the first demonstration that cell contact–mediated calcium signals in T cells occur via depletion-activated channels. PMID:10662784

  16. Alterations in cellular metabolism modulate CD1d-mediated NKT-cell responses.

    PubMed

    Webb, Tonya J; Carey, Gregory B; East, James E; Sun, Wenji; Bollino, Dominique R; Kimball, Amy S; Brutkiewicz, Randy R

    2016-08-01

    Natural killer T (NKT) cells play a critical role in the host's innate immune response. CD1d-mediated presentation of glycolipid antigens to NKT cells has been established; however, the mechanisms by which NKT cells recognize infected or cancerous cells remain unclear. 5(')-AMP activated protein kinase (AMPK) is a master regulator of lipogenic pathways. We hypothesized that activation of AMPK during infection and malignancy could alter the repertoire of antigens presented by CD1d and serve as a danger signal to NKT cells. In this study, we examined the effect of alterations in metabolism on CD1d-mediated antigen presentation to NKT cells and found that an infection with lymphocytic choriomeningitis virus rapidly increased CD1d-mediated antigen presentation. Hypoxia inducible factors (HIF) enhance T-cell effector functions during infection, therefore antigen presenting cells pretreated with pharmacological agents that inhibit glycolysis, induce HIF and activate AMPK were assessed for their ability to induce NKT-cell responses. Pretreatment with 2-deoxyglucose, cobalt chloride, AICAR and metformin significantly enhanced CD1d-mediated NKT-cell activation. In addition, NKT cells preferentially respond to malignant B cells and B-cell lymphomas express HIF-1α. These data suggest that targeting cellular metabolism may serve as a novel means of inducing innate immune responses. PMID:27297969

  17. Functional analysis of Human Papillomavirus Virus-Like Particle activated Langerhans cells in vitro

    PubMed Central

    Yan, Lisa; Woodham, Andrew W.; Da Silva, Diane M.; Kast, W. Martin

    2016-01-01

    Langerhans cells (LC) are antigen presenting cells responsible for initiating an immune response against human papillomaviruses (HPV) entering the epithelial layer in vivo as they are the first immune cell that HPV comes into contact with. LC become activated in response to foreign antigens, which causes internal signaling resulting in the increased expression of co-stimulatory molecules and the secretion of inflammatory cytokines. Functionally activated LC are then capable of migrating to the lymph nodes where they interact with antigen specific T cells and initiate an adaptive T cell response in vivo. However, HPV has evolved in a manner that suppresses LC function, and thus the induction of antigen specific T cells is hindered. While many methods exist to monitor the activity of LC in vitro, the migration and induction of cytotoxic T-cells is ultimately indicative of a functional immune response. Here, methods in analyzing functional migration and induction of antigen specific T cells after stimulation of LC with HPV virus-like particles in vitro are described. PMID:25348318

  18. T cell immunity using transgenic B lymphocytes

    NASA Astrophysics Data System (ADS)

    Gerloni, Mara; Rizzi, Marta; Castiglioni, Paola; Zanetti, Maurizio

    2004-03-01

    Adaptive immunity exists in all vertebrates and plays a defense role against microbial pathogens and tumors. T cell responses begin when precursor T cells recognize antigen on specialized antigen-presenting cells and differentiate into effector cells. Currently, dendritic cells are considered the only cells capable of stimulating T lymphocytes. Here, we show that mature naïve B lymphocytes can be genetically programmed by using nonviral DNA and turned into powerful antigen-presenting cells with a dual capacity of synthesis and presentation of antigen to T cells in vivo. A single i.v. injection of transgenic lymphocytes activates T cell responses reproducibly and specifically even at very low cell doses (102). We also demonstrate that T cell priming can occur in the absence of dendritic cells and results in immunological memory with protective effector functions. These findings disclose aspects in the regulation of adaptive immunity and indicate possibilities for vaccination against viruses and cancer in humans.

  19. Application of Microneedles to Skin Induces Activation of Epidermal Langerhans Cells and Dermal Dendritic Cells in Mice.

    PubMed

    Takeuchi, Asuka; Nomoto, Yusuke; Watanabe, Mai; Kimura, Soichiro; Morimoto, Yasunori; Ueda, Hideo

    2016-08-01

    An adequate immune response to percutaneous vaccine application is generated by delivery of sufficient amounts of antigen to skin and by administration of toxin adjuvants or invasive skin abrasion that leads to an adjuvant effect. Microneedles penetrate the stratum corneum, the outermost layer of the skin, and enable direct delivery of vaccines from the surface into the skin, where immunocompetent dendritic cells are densely distributed. However, whether the application of microneedles to the skin activates antigen-presenting cells (APCs) has not been demonstrated. Here we aimed to demonstrate that microneedles may act as a potent physical adjuvant for successful transcutaneous immunization (TCI). We prepared samples of isolated epidermal and dermal cells and analyzed the expression of major histocompatibility complex (MHC) class II and costimulatory molecules on Langerhans or dermal dendritic cells in the prepared samples using flow cytometry. The expression of MHC class II and costimulatory molecules demonstrated an upward trend in APCs in the skin after the application of 500- and 300-µm microneedles. In addition, in the epidermal cells, application of microneedles induced more effective activation of Langerhans cells than did an invasive tape-stripping (positive control). In conclusion, the use of microneedles is likely to have a positive effect not only as an antigen delivery system but also as a physical technique inducing an adjuvant-like effect for TCI. PMID:27251665

  20. Activation of spleen cells by ArtinM may account for its immunomodulatory properties.

    PubMed

    Silva, Thiago Aparecido da; Souza, Maria Aparecida de; Cecílio, Nerry Tatiana; Roque-Barreira, Maria Cristina

    2014-09-01

    ArtinM is a D-mannose-binding lectin extracted from Artocarpus heterophyllus that promotes interleukin-12 production by macrophages and dendritic cells. This property is considered responsible for T helper 1 immunity induced in vivo after ArtinM administration. In this study, we investigated the effect of native (jArtinM) and recombinant (rArtinM) forms of lectin on murine spleen cells and isolated T lymphocytes. We found that ArtinM binds to the surface of spleen cells. This interaction, which was blocked by D-mannose, induced cell activation, as manifested by increased mitochondrial activity, interleukin-2 production, and cell proliferation. We verified that a 30-times higher concentration of rArtinM was required to trigger optimal activation of spleen cells compared with that needed with jArtinM, although these proteins have identical sugar recognition properties and use the same signaling molecules to trigger cell activation. Because the distinction between native and recombinant is restricted to their tertiary structure (tetrameric and monomeric, respectively), we postulated that the multi-valence of jArtinM accounts for its superiority in promoting clustering of cell surface glycoreceptors and activation. The jArtinM and rArtinM activation effect exerted on spleen cells was reproduced on purified CD4(+) T cells. Our results suggest that ArtinM interaction with T cells leads to responses that may act in concert with the interleukin-12 produced by antigen-presenting cells to modulate immunity toward the T helper 1 axis. Further studies are necessary to dissect ArtinM/T-cell interactions to more fully understand the immunomodulation induced by carbohydrate recognition. PMID:24842046

  1. Single molecule analysis of B cell receptor motion during signaling activation

    NASA Astrophysics Data System (ADS)

    Rey Suarez, Ivan; Koo, Peter; Mochrie, Simon; Song, Wenxia; Upadhyaya, Arpita

    B cells are an essential part of the adaptive immune system. They patrol the body looking for signs of infection in the form of antigen on the surface of antigen presenting cells. The binding of the B cell receptor (BCR) to antigen induces signaling cascades that lead to B cell activation and eventual production of high affinity antibodies. During activation, BCR organize into signaling microclusters, which are platforms for signal amplification. The physical processes underlying receptor movement and aggregation are not well understood. Here we study the dynamics of single BCRs on activated murine primary B cells using TIRF imaging and single particle tracking. The tracks obtained are analyzed using perturbation expectation-maximization (pEM) a systems-level analysis that allows the identification of different short-time diffusive states from a set of single particle tracks. We identified five different diffusive states on wild type cells, which correspond to different molecular states of the BCR. By using actin polymerization inhibitors and mutant cells lacking important actin regulators we were able to identify the BCR molecule configuration associated with each diffusive state.

  2. NK Cells Respond to Haptens by the Activation of Calcium Permeable Plasma Membrane Channels

    PubMed Central

    Grandclément, Camille; Pick, Horst; Vogel, Horst; Held, Werner

    2016-01-01

    Natural Killer (NK) cells mediate innate immunity to infected and transformed cells. Yet, NK cells can also mount hapten-specific recall responses thereby contributing to contact hypersensitivity (CHS). However, since NK cells lack antigen receptors that are used by the adaptive immune system to recognize haptens, it is not clear if NK cells respond directly to haptens and, if so, what mediates these responses. Here we show that among four haptens the two that are known to induce NK cell-dependent CHS trigger the rapid influx of extracellular Ca2+ into NK cells and lymphocyte cell lines. Thus lymphocytes can respond to haptens independent of antigen presentation and antigen receptors. We identify the Ca2+-permeable cation channel TRPC3 as a component of the lymphocyte response to one of these haptens. These data suggest that the response to the second hapten is based on a distinct mechanism, consistent with the capacity of NK cells to discriminate haptens. These findings raise the possibility that antigen-receptor independent activation of immune cells contributes to CHS. PMID:26963818

  3. Dendritic cells treated with resveratrol during differentiation from monocytes gain substantial tolerogenic properties upon activation

    PubMed Central

    Švajger, Urban; Obermajer, Nataša; Jeras, Matjaž

    2010-01-01

    Resveratrol is a polyphenol that acts on multiple molecular targets important for cell differentiation and activation. Dendritic cells (DCs) are a functionally diverse cell type and represent the most potent antigen-presenting cells of the immune system. In this study, we investigated resveratrol-induced effects on DCs during their differentiation and maturation. Our results show that resveratrol induces DC-associated tolerance, particularly when applied during DC differentiation. Costimulatory molecules CD40, CD80 and CD86 were down-regulated, as was the expression of major histocompatibility complex (MHC) class II molecules. Surface expression of inhibitory immunoglobulin-like transcript 3 (ILT3) and ILT4 molecules was induced, while human leucocyte antigen (HLA)-G expression was not affected. Resveratrol-treated DCs lost the ability to produce interleukin (IL)-12p70 after activation, but had an increased ability to produce IL-10. Such DCs were poor stimulators of allogeneic T cells and had lowered ability to induce CD4+ T-cell migration. Furthermore, treated cells were able to generate allogeneic IL-10-secreting T cells, but were not competent in inducing FoxP3 expression These tolerogenic effects are probably associated with the effect of resveratrol on multiple molecular targets through which it interferes with DC differentiation and nuclear factor (NF)-κB translocation. Our data provide new insights into the molecular and functional mechanisms of the tolerogenic effects that resveratrol exerts on DCs. PMID:20002210

  4. G1-4A, a polysaccharide from Tinospora cordifolia induces peroxynitrite dependent killer dendritic cell (KDC) activity against tumor cells.

    PubMed

    Pandey, Vipul K; Amin, Prayag J; Shankar, Bhavani S

    2014-12-01

    Dendritic cells (DC) play a central role in the development of an adaptive immune response against tumor. In addition to its role in antigen presentation, DC also possesses cytotoxic activity against tumor cells. We have earlier shown phenotypic and functional maturation of bone marrow derived dendritic cells (BMDC) by G1-4A, an arabinogalactan derived from Tinospora cordifolia. In this study, we have investigated the killer phenotype of BMDC matured in the presence of G1-4A, [mBMDC (G1-4A)] on tumor cells. We have observed several fold increase in killing of tumor cells by mBMDC (G1-4A). The tumoricidal activity was not specific to syngeneic tumors cells but could kill xenogenic tumors also. Nitric oxide released by mBMDC (G1-4A) generates peroxynitrite in tumor cells and is responsible for killing of target cells. This killing was completely abrogated by inducible nitric oxide synthase (iNOS) inhibitor 1400W and NADPH oxidase inhibitor apocyanin. The killed target cells are phagocytosed by BMDC which further activate syngeneic cytotoxic T cells. These results thus show that G1-4A treated mBMDC acquire killer phenotype along with maturation which plays an important role in activation of cytotoxic T cells. PMID:25278461

  5. Antitumor Efficacy of Radiation plus Immunotherapy Depends upon Dendritic Cell Activation of Effector CD8+ T Cells.

    PubMed

    Dovedi, Simon J; Lipowska-Bhalla, Grazyna; Beers, Stephen A; Cheadle, Eleanor J; Mu, Lijun; Glennie, Martin J; Illidge, Timothy M; Honeychurch, Jamie

    2016-07-01

    Tumor cells dying after cytotoxic therapy are a potential source of antigen for T-cell priming. Antigen-presenting cells (APC) can cross-present MHC I-restricted peptides after the uptake of dying cells. Depending on the nature of the surrounding environmental signals, APCs then orchestrate a spectrum of responses ranging from immune activation to inhibition. Previously, we had demonstrated that combining radiation with either agonistic monoclonal antibody (mAb) to CD40 or a systemically administered TLR7 agonist could enhance CD8 T-cell-dependent protection against syngeneic murine lymphoma models. However, it remains unknown how individual APC populations affect this antitumor immune response. Using APC depletion models, we now show that dendritic cells (DC), but not macrophages or B cells, were responsible for the generation of long-term immunologic protection following combination therapy with radiotherapy and either agonistic CD40 mAb or systemic TLR7 agonist therapy. Novel immunotherapeutic approaches that augment antigen uptake and presentation by DCs may further enhance the generation of therapeutic antitumor immune responses, leading to improved outcomes after radiotherapy. Cancer Immunol Res; 4(7); 621-30. ©2016 AACR. PMID:27241845

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

    PubMed

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

    2015-01-01

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

  7. Acute GVHD in patients receiving IL-15/4-1BBL activated NK cells following T-cell-depleted stem cell transplantation.

    PubMed

    Shah, Nirali N; Baird, Kristin; Delbrook, Cynthia P; Fleisher, Thomas A; Kohler, Mark E; Rampertaap, Shakuntala; Lemberg, Kimberly; Hurley, Carolyn K; Kleiner, David E; Merchant, Melinda S; Pittaluga, Stefania; Sabatino, Marianna; Stroncek, David F; Wayne, Alan S; Zhang, Hua; Fry, Terry J; Mackall, Crystal L

    2015-01-29

    Natural killer (NK) cells can enhance engraftment and mediate graft-versus-leukemia following allogeneic hematopoietic stem cell transplantation (HSCT), but the potency of graft-versus-leukemia mediated by naturally reconstituting NK cells following HSCT is limited. Preclinical studies demonstrate that activation of NK cells using interleukin-15 (IL-15) plus 4-1BBL upregulates activating receptor expression and augments killing capacity. In an effort to amplify the beneficial effects of NK cells post-HSCT, we conducted a first-in-human trial of adoptive transfer of donor-derived IL-15/4-1BBL-activated NK cells (aNK-DLI) following HLA-matched, T-cell-depleted (1-2 × 10(4) T cells/kg) nonmyeloablative peripheral blood stem cell transplantation in children and young adults with ultra-high-risk solid tumors. aNK-DLI were CD3(+)-depleted, CD56(+)-selected lymphocytes, cultured for 9 to 11 days with recombinant human IL-15 plus 4-1BBL(+)IL-15Rα(+) artificial antigen-presenting cells. aNK-DLI demonstrated potent killing capacity and displayed high levels of activating receptor expression. Five of 9 transplant recipients experienced acute graft-versus-host disease (GVHD) following aNK-DLI, with grade 4 GVHD observed in 3 subjects. GVHD was more common in matched unrelated donor vs matched sibling donor recipients and was associated with higher donor CD3 chimerism. Given that the T-cell dose was below the threshold required for GVHD in this setting, we conclude that aNK-DLI contributed to the acute GVHD observed, likely by augmenting underlying T-cell alloreactivity. This trial was registered at www.clinicaltrials.gov as #NCT01287104. PMID:25452614

  8. Proteolysis, proteasomes and antigen presentation

    NASA Technical Reports Server (NTRS)

    Goldberg, A. L.; Rock, K. L.

    1992-01-01

    Proteins presented to the immune system must first be cleaved to small peptides by intracellular proteinases. Proteasomes are proteolytic complexes that degrade cytosolic and nuclear proteins. These particles have been implicated in ATP-ubiquitin-dependent proteolysis and in the processing of intracellular antigens for cytolytic immune responses.

  9. Adaptive (T and B cells) immunity and control by dendritic cells in atherosclerosis.

    PubMed

    Ait-Oufella, Hafid; Sage, Andrew P; Mallat, Ziad; Tedgui, Alain

    2014-05-01

    Chronic inflammation in response to lipoprotein accumulation in the arterial wall is central in the development of atherosclerosis. Both innate and adaptive immunity are involved in this process. Adaptive immune responses develop against an array of potential antigens presented to effector T lymphocytes by antigen-presenting cells, especially dendritic cells. Functional analysis of the role of different T-cell subsets identified the Th1 responses as proatherogenic, whereas regulatory T-cell responses exert antiatherogenic activities. The effect of Th2 and Th17 responses is still debated. Atherosclerosis is also associated with B-cell activation. Recent evidence established that conventional B-2 cells promote atherosclerosis. In contrast, innate B-1 B cells offer protection through secretion of natural IgM antibodies. This review discusses the recent development in our understanding of the role of T- and B-cell subsets in atherosclerosis and addresses the role of dendritic cell subpopulations in the control of adaptive immunity. PMID:24812352

  10. Effects of methoxypoly (Ethylene glycol) mediated immunocamouflage on leukocyte surface marker detection, cell conjugation, activation and alloproliferation.

    PubMed

    Kyluik-Price, Dana L; Scott, Mark D

    2016-01-01

    Tissue rejection occurs subsequent to the recognition of foreign antigens via receptor-ligand contacts between APC (antigen presenting cells) and T cells, resulting in initialization of signaling cascades and T cell proliferation. Bioengineering of donor cells by the covalent attachment of methoxypolyethylene glycol (mPEG) to membrane proteins (PEGylation) provides a novel means to attenuate these interactions consequent to mPEG-induced charge and steric camouflage. While previous studies demonstrated that polymer-mediated immunocamouflage decreased immune recognition both in vitro and in vivo, these studies monitored late events in immune recognition and activation such as T cell proliferation. Consequently little information has been provided concerning the early cellular events governing this response. Therefore, the effect of PEGylation was assessed by examining initial cell-cell interactions, changes to activation pathways, and apoptosis to understand the role that each may play in the decreased proliferative response observed in modified cells during the course of a mixed lymphocyte reaction (MLR). The mPEG-modified T cells resulted in significant immunocamouflage of lymphocyte surface proteins and decreased interactions with APC. Furthermore, mPEG-MLR exhibited decreased NFκB pathway activation, while exhibiting no significant differences in degree of cell death compared to the control MLR. These results suggest that PEGylation may prevent the direct recognition of foreign alloantigens by decreasing the stability and duration of initial cell-cell interactions. PMID:26457834

  11. Tetherin/BST-2 promotes dendritic cell activation and function during acute retrovirus infection

    PubMed Central

    Li, Sam X.; Barrett, Bradley S.; Guo, Kejun; Kassiotis, George; Hasenkrug, Kim J.; Dittmer, Ulf; Gibbert, Kathrin; Santiago, Mario L.

    2016-01-01

    Tetherin/BST-2 is a host restriction factor that inhibits retrovirus release from infected cells in vitro by tethering nascent virions to the plasma membrane. However, contradictory data exists on whether Tetherin inhibits acute retrovirus infection in vivo. Previously, we reported that Tetherin-mediated inhibition of Friend retrovirus (FV) replication at 2 weeks post-infection correlated with stronger natural killer, CD4+ T and CD8+ T cell responses. Here, we further investigated the role of Tetherin in counteracting retrovirus replication in vivo. FV infection levels were similar between wild-type (WT) and Tetherin KO mice at 3 to 7 days post-infection despite removal of a potent restriction factor, Apobec3/Rfv3. However, during this phase of acute infection, Tetherin enhanced myeloid dendritic cell (DC) function. DCs from infected, but not uninfected, WT mice expressed significantly higher MHC class II and the co-stimulatory molecule CD80 compared to Tetherin KO DCs. Tetherin-associated DC activation during acute FV infection correlated with stronger NK cell responses. Furthermore, Tetherin+ DCs from FV-infected mice more strongly stimulated FV-specific CD4+ T cells ex vivo compared to Tetherin KO DCs. The results link the antiretroviral and immunomodulatory activity of Tetherin in vivo to improved DC activation and MHC class II antigen presentation. PMID:26846717

  12. Presentation of the candidate rheumatoid arthritis autoantigen aggrecan by antigen-specific B cells induces enhanced CD4(+) T helper type 1 subset differentiation.

    PubMed

    Wilson, Caroline L; Hine, Dominic W; Pradipta, Ariel; Pearson, Jeffrey P; van Eden, Willem; Robinson, John H; Knight, Andrew M

    2012-04-01

    Effective immune responses require antigen uptake by antigen-presenting cells (APC), followed by controlled endocytic proteolysis resulting in the generation of antigen-derived peptide fragments that associate with intracellular MHC class II molecules. The resultant peptide-MHC class II complexes then move to the APC surface where they activate CD4(+) T cells. Dendritic cells (DC), macrophages and B cells act as efficient APC. In many settings, including the T helper type 1 (Th1) -dependent, proteoglycan-induced arthritis model of rheumatoid arthritis, accumulating evidence demonstrates that antigen presentation by B cells is required for optimal CD4(+) T cell activation. The reasons behind this however, remain unclear. In this study we have compared the activation of CD4(+) T cells specific for the proteoglycan aggrecan following antigen presentation by DC, macrophages and B cells. We show that aggrecan-specific B cells are equally efficient APC as DC and macrophages and use similar intracellular antigen-processing pathways. Importantly, we also show that antigen presentation by aggrecan-specific B cells to TCR transgenic CD4(+) T cells results in enhanced CD4(+) T cell interferon-γ production and Th1 effector sub-set differentiation compared with that seen with DC. We conclude that preferential CD4(+) Th1 differentiation may define the requirement for B cell APC function in both proteoglycan-induced arthritis and rheumatoid arthritis. PMID:22182481

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

    PubMed

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

    2016-08-01

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

  14. Regulation of Cathepsin G Reduces the Activation of Proinsulin-Reactive T Cells from Type 1 Diabetes Patients

    PubMed Central

    Zou, Fang; Schäfer, Nadja; Palesch, David; Brücken, Ruth; Beck, Alexander; Sienczyk, Marcin; Kalbacher, Hubert; Sun, ZiLin; Boehm, Bernhard O.; Burster, Timo

    2011-01-01

    Autoantigenic peptides resulting from self-proteins such as proinsulin are important players in the development of type 1 diabetes mellitus (T1D). Self-proteins can be processed by cathepsins (Cats) within endocytic compartments and loaded to major histocompatibility complex (MHC) class II molecules for CD4+ T cell inspection. However, the processing and presentation of proinsulin by antigen-presenting cells (APC) in humans is only partially understood. Here we demonstrate that the processing of proinsulin by B cell or myeloid dendritic cell (mDC1)-derived lysosomal cathepsins resulted in several proinsulin-derived intermediates. These intermediates were similar to those obtained using purified CatG and, to a lesser extent, CatD, S, and V in vitro. Some of these intermediates polarized T cell activation in peripheral blood mononuclear cells (PBMC) from T1D patients indicative for naturally processed T cell epitopes. Furthermore, CatG activity was found to be elevated in PBMC from T1D patients and abrogation of CatG activity resulted in functional inhibition of proinsulin-reactive T cells. Our data suggested the notion that CatG plays a critical role in proinsulin processing and is important in the activation process of diabetogenic T cells. PMID:21850236

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

    PubMed Central

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

    2012-01-01

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

  16. Multitarget magnetic activated cell sorter

    PubMed Central

    Adams, Jonathan D.; Kim, Unyoung; Soh, H. Tom

    2008-01-01

    Magnetic selection allows high-throughput sorting of target cells based on surface markers, and it is extensively used in biotechnology for a wide range of applications from in vitro diagnostics to cell-based therapies. However, existing methods can only perform separation based on a single parameter (i.e., the presence or absence of magnetization), and therefore, the simultaneous sorting of multiple targets at high levels of purity, recovery, and throughput remains a challenge. In this work, we present an alternative system, the multitarget magnetic activated cell sorter (MT-MACS), which makes use of microfluidics technology to achieve simultaneous spatially-addressable sorting of multiple target cell types in a continuous-flow manner. We used the MT-MACS device to purify 2 types of target cells, which had been labeled via target-specific affinity reagents with 2 different magnetic tags with distinct saturation magnetization and size. The device was engineered so that the combined effects of the hydrodynamic force produced from the laminar flow and the magnetophoretic force produced from patterned ferromagnetic structures within the microchannel result in the selective purification of the differentially labeled target cells into multiple independent outlets. We demonstrate here the capability to simultaneously sort multiple magnetic tags with >90% purity and >5,000-fold enrichment and multiple bacterial cell types with >90% purity and >500-fold enrichment at a throughput of 109 cells per hour. PMID:19015523

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

    PubMed Central

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

    2014-01-01

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

  18. Hyperthermia stimulates plasminogen activator inhibitor type 1 expression in human umbilical vein endothelial cells in vitro.

    PubMed Central

    Wojta, J.; Holzer, M.; Hufnagl, P.; Christ, G.; Hoover, R. L.; Binder, B. R.

    1991-01-01

    The effect of exposure to hyperthermia on the fibrinolytic potential of human umbilical vein endothelial cells (HUVEC) in culture was studied. HUVEC responded to exposure to 42 degrees C with a time-dependent increase in plasminogen activator inhibitor type 1 (PAI-1) activity and antigen accompanied by a four- to fivefold increase in PAI-1 specific m-RNA and a decrease in tissue-type plasminogen activator (t-PA) antigen. The effect of 8 hours exposure to hyperthermia on PAI-1 activity and antigen could not be reversed by reexposure of the cells to 37 degrees C for 24 hours as evidenced by continuously increased amounts of PAI-1 released into the conditioned media. t-PA release, however, decreased during the 24-hour period at 37 degrees C after exposure to hyperthermia. No difference in PAI-1 antigen present in the extracellular matrix of heat treated HUVEC as compared to HUVEC kept at 37 degrees C could be found. Our data supports the idea that hyperthermia is one stress factor that influences the fibrinolytic potential of endothelial cells. Images Figure 6 PMID:1928306

  19. 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. PMID:11592060

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

    PubMed Central

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Sackmann, Erich

    2011-06-01

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

  2. Suppression of Adaptive Immune Cell Activation Does Not Alter Innate Immune Adipose Inflammation or Insulin Resistance in Obesity

    PubMed Central

    Subramanian, Manikandan; Ozcan, Lale; Ghorpade, Devram Sampat; Ferrante, Anthony W.; Tabas, Ira

    2015-01-01

    Obesity-induced inflammation in visceral adipose tissue (VAT) is a major contributor to insulin resistance and type 2 diabetes. Whereas innate immune cells, notably macrophages, contribute to visceral adipose tissue (VAT) inflammation and insulin resistance, the role of adaptive immunity is less well defined. To address this critical gap, we used a model in which endogenous activation of T cells was suppressed in obese mice by blocking MyD88-mediated maturation of CD11c+ antigen-presenting cells. VAT CD11c+ cells from Cd11cCre+Myd88fl/fl vs. control Myd88fl/fl mice were defective in activating T cells in vitro, and VAT T and B cell activation was markedly reduced in Cd11cCre+Myd88fl/fl obese mice. However, neither macrophage-mediated VAT inflammation nor systemic inflammation were altered in Cd11cCre+Myd88fl/fl mice, thereby enabling a focused analysis on adaptive immunity. Unexpectedly, fasting blood glucose, plasma insulin, and the glucose response to glucose and insulin were completely unaltered in Cd11cCre+Myd88fl/fl vs. control obese mice. Thus, CD11c+ cells activate VAT T and B cells in obese mice, but suppression of this process does not have a discernible effect on macrophage-mediated VAT inflammation or systemic glucose homeostasis. PMID:26317499

  3. In vivo sensitized and in vitro activated B cells mediate tumor regression in cancer adoptive immunotherapy1

    PubMed Central

    Li, Qiao; Song, Hongbin; Teitz-Tennenbaum, Seagal; Donald, Elizabeth J.; Li, Mu; Chang, Alfred E.

    2011-01-01

    Adoptive cellular immunotherapy utilizing tumor-reactive T cells has proven to be a promising strategy for cancer treatment. However, we hypothesize that successful treatment strategies will have to appropriately stimulate not only cellular immunity, but also humoral immunity. We previously reported that B cells in tumor-draining lymph nodes (TDLN) may function as antigen-presenting cells. In this study, we identified TDLN B cells as effector cells in an adoptive immunotherapy model. In vivo primed and in vitro activated TDLN B cells alone mediated effective (p<0.05) tumor regression after adoptive transfer into two histologically distinct murine pulmonary metastatic tumor models. Prior lymphodepletion of the host with either chemotherapy or whole-body irradiation augmented the therapeutic efficacy of the adoptively transferred TDLN B cells in the treatment of subcutaneous tumors as well as metastatic pulmonary tumors. Furthermore, B cell plus T cell transfers resulted in substantially more efficient antitumor responses than B cells or T cells alone (p<0.05). Activated TDLN B cells conferred strong humoral responses to tumor. This was evident by the production of IgM, IgG and IgG2b, which bound specifically to tumor cells and led to specific tumor cell lysis in the presence of complement. Collectively, these data indicate that in vivo primed and in vitro activated B cells can be employed as effector cells for cancer therapy. The synergistic antitumor efficacy of co-transferred activated B effector cells and T effector cells represents a novel approach for cancer adoptive immunotherapy. PMID:19667089

  4. Exposure to Δ9-Tetrahydrocannabinol Impairs the Differentiation of Human Monocyte-derived Dendritic Cells and their Capacity for T cell Activation

    PubMed Central

    Castaneda, Julie T.; Kiertscher, Sylvia M.

    2015-01-01

    The capacity for human monocytes to differentiate into antigen-presenting dendritic cells (DC) can be influenced by a number of immune modulating signals. Monocytes express intracellular cannabinoid type 1 (CB1) and 2 (CB2) receptors and we demonstrate that exposure to Δ9-tetrahydrocannabinol (THC) inhibits the forskolin-induced generation of cyclic adenosine monophosphate in a CB2-specific manner. In order to examine the potential impact of cannabinoids on the generation of monocyte-derived DC, monocytes were cultured in vitro with differentiation medium alone [containing granulocyte/macrophage-colony stimulating factor (GM-CSF) and Interleukin-4 (IL-4)] or in combination with THC. The presence of THC (0.25–1.0 μg/ml) altered key features of DC differentiation, producing a concentration-dependent decrease in surface expression of CD11c, HLA-DR and costimulatory molecules (CD40 and CD86), less effective antigen uptake, and signs of functional skewing with decreased production of IL-12 but normal levels of IL-10. When examined in a mixed leukocyte reaction, DC that had been generated in the presence of THC were poor T cell activators as evidenced by their inability to generate effector/memory T cells or to stimulate robust IFN-γ responses. Some of these effects were partially restored by exposure to exogenous IL-7 and bacterial superantigen (S. aureus Cowans strain). These studies demonstrate that human monocytes express functional cannabinoid receptors and suggest that exposure to THC can alter their differentiation into functional antigen presenting cells; an effect that may be counter-balanced by the presence of other immunoregulatory factors. The impact of cannabinoids on adaptive immune responses in individuals with frequent drug exposure remains to be determined. PMID:25614186

  5. The phosphoinositide 3-kinase signaling pathway in normal and malignant B cells: activation mechanisms, regulation and impact on cellular functions.

    PubMed

    Pauls, Samantha D; Lafarge, Sandrine T; Landego, Ivan; Zhang, Tingting; Marshall, Aaron J

    2012-01-01

    The phosphoinositide 3-kinase (PI3K) pathway is a central signal transduction axis controlling normal B cell homeostasis and activation in humoral immunity. The p110δ PI3K catalytic subunit has emerged as a critical mediator of multiple B cell functions. The activity of this pathway is regulated at multiple levels, with inositol phosphatases PTEN and SHIP both playing critical roles. When deregulated, the PI3K pathway can contribute to B cell malignancies and autoantibody production. This review summarizes current knowledge on key mechanisms that activate and regulate the PI3K pathway and influence normal B cell functional responses including the development of B cell subsets, antigen presentation, immunoglobulin isotype switch, germinal center responses, and maintenance of B cell anergy. We also discuss PI3K pathway alterations reported in select B cell malignancies and highlight studies indicating the functional significance of this pathway in malignant B cell survival and growth within tissue microenvironments. Finally, we comment on early clinical trial results, which support PI3K inhibition as a promising treatment of chronic lymphocytic leukemia. PMID:22908014

  6. The phosphoinositide 3-kinase signaling pathway in normal and malignant B cells: activation mechanisms, regulation and impact on cellular functions

    PubMed Central

    Pauls, Samantha D.; Lafarge, Sandrine T.; Landego, Ivan; Zhang, Tingting; Marshall, Aaron J.

    2012-01-01

    The phosphoinositide 3-kinase (PI3K) pathway is a central signal transduction axis controlling normal B cell homeostasis and activation in humoral immunity. The p110δ PI3K catalytic subunit has emerged as a critical mediator of multiple B cell functions. The activity of this pathway is regulated at multiple levels, with inositol phosphatases PTEN and SHIP both playing critical roles. When deregulated, the PI3K pathway can contribute to B cell malignancies and autoantibody production. This review summarizes current knowledge on key mechanisms that activate and regulate the PI3K pathway and influence normal B cell functional responses including the development of B cell subsets, antigen presentation, immunoglobulin isotype switch, germinal center responses, and maintenance of B cell anergy. We also discuss PI3K pathway alterations reported in select B cell malignancies and highlight studies indicating the functional significance of this pathway in malignant B cell survival and growth within tissue microenvironments. Finally, we comment on early clinical trial results, which support PI3K inhibition as a promising treatment of chronic lymphocytic leukemia. PMID:22908014

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

    PubMed Central

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

    1998-01-01

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

  8. A cell-based microarray to investigate combinatorial effects of microparticle-encapsulated adjuvants on dendritic cell activation

    PubMed Central

    Acharya, Abhinav P.; Carstens, Matthew R.; Lewis, Jamal S.; Dolgova, Natalia; Xia, C. Q.; Clare-Salzler, Michael J.

    2016-01-01

    Experimental vaccine adjuvants are being designed to target specific toll-like receptors (TLRs) alone or in combination, expressed by antigen presenting cells, notably dendritic cells (DCs). There is a need for high-content screening (HCS) platforms to explore how DC activation is affected by adjuvant combinations. Presented is a cell-based microarray approach, “immunoarray”, exposing DCs to a large number of adjuvant combinations. Microparticles encapsulating TLR ligands are printed onto arrays in a range of doses for each ligand, in all possible dose combinations. Dendritic cells are then co-localized with physisorbed microparticles on the immunoarray, adherent to isolated islands surrounded by a non-fouling background, and DC activation is quantified. Delivery of individual TLR ligands was capable of eliciting high levels of specific DC activation markers. For example, either TLR9 ligand, CpG, or TLR3 ligand, poly I:C, was capable of inducing among the highest 10% expression levels of CD86. In contrast, MHC-II expression in response to TLR4 agonist MPLA was among the highest, whereas either MPLA or poly I:C, was capable of producing among the highest levels of CCR7 expression, as well as inflammatory cytokine IL-12. However, in order to produce robust responses across all activation markers, adjuvant combinations were required, and combinations were more represented among the high responders. The immunoarray also enables investigation of interactions between adjuvants, and each TLR ligand suggested antagonism to other ligands, for various markers. Altogether, this work demonstrates feasibility of the immunoarray platform to screen microparticle-encapsulated adjuvant combinations for the development of improved and personalized vaccines. PMID:26985393

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

    PubMed Central

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

    2014-01-01

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

  10. CD62L+ NKT cells have prolonged persistence and antitumor activity in vivo

    PubMed Central

    Tian, Gengwen; Courtney, Amy N.; Jena, Bipulendu; Heczey, Andras; Liu, Daofeng; Marinova, Ekaterina; Guo, Linjie; Xu, Xin; Torikai, Hiroki; Mo, Qianxing; Dotti, Gianpietro; Cooper, Laurence J.; Metelitsa, Leonid S.

    2016-01-01

    Vα24-invariant natural killer T cells (NKTs) localize to tumors and have inherent antitumor properties, making them attractive chimeric antigen receptor (CAR) carriers for redirected cancer immunotherapy. However, clinical application of CAR-NKTs has been impeded, as mechanisms responsible for NKT expansion and the in vivo persistence of these cells are unknown. Here, we demonstrated that antigen-induced expansion of primary NKTs in vitro associates with the accumulation of a CD62L+ subset and exhaustion of CD62L– cells. Only CD62L+ NKTs survived and proliferated in response to secondary stimulation. When transferred to immune-deficient NSG mice, CD62L+ NKTs persisted 5 times longer than CD62L– NKTs. Moreover, CD62L+ cells transduced with a CD19-specific CAR achieved sustained tumor regression in a B cell lymphoma model. Proliferating CD62L+ cells downregulated or maintained CD62L expression when activated via T cell receptor alone or in combination with costimulatory receptors. We generated HLAnull K562 cell clones that were engineered to express CD1d and costimulatory ligands. Clone B-8-2 (HLAnullCD1dmedCD86high4-1BBLmedOX40Lhigh) induced the highest rates of NKT expansion and CD62L expression. B-8-2–expanded CAR-NKTs exhibited prolonged in vivo persistence and superior therapeutic activities in models of lymphoma and neuroblastoma. Therefore, we have identified CD62L as a marker of a distinct NKT subset endowed with high proliferative potential and have developed artificial antigen-presenting cells that generate CD62L-enriched NKTs for effective cancer immunotherapy. PMID:27183388

  11. CD62L+ NKT cells have prolonged persistence and antitumor activity in vivo.

    PubMed

    Tian, Gengwen; Courtney, Amy N; Jena, Bipulendu; Heczey, Andras; Liu, Daofeng; Marinova, Ekaterina; Guo, Linjie; Xu, Xin; Torikai, Hiroki; Mo, Qianxing; Dotti, Gianpietro; Cooper, Laurence J; Metelitsa, Leonid S

    2016-06-01

    Vα24-invariant natural killer T cells (NKTs) localize to tumors and have inherent antitumor properties, making them attractive chimeric antigen receptor (CAR) carriers for redirected cancer immunotherapy. However, clinical application of CAR-NKTs has been impeded, as mechanisms responsible for NKT expansion and the in vivo persistence of these cells are unknown. Here, we demonstrated that antigen-induced expansion of primary NKTs in vitro associates with the accumulation of a CD62L+ subset and exhaustion of CD62L- cells. Only CD62L+ NKTs survived and proliferated in response to secondary stimulation. When transferred to immune-deficient NSG mice, CD62L+ NKTs persisted 5 times longer than CD62L- NKTs. Moreover, CD62L+ cells transduced with a CD19-specific CAR achieved sustained tumor regression in a B cell lymphoma model. Proliferating CD62L+ cells downregulated or maintained CD62L expression when activated via T cell receptor alone or in combination with costimulatory receptors. We generated HLAnull K562 cell clones that were engineered to express CD1d and costimulatory ligands. Clone B-8-2 (HLAnullCD1dmedCD86high4-1BBLmedOX40Lhigh) induced the highest rates of NKT expansion and CD62L expression. B-8-2-expanded CAR-NKTs exhibited prolonged in vivo persistence and superior therapeutic activities in models of lymphoma and neuroblastoma. Therefore, we have identified CD62L as a marker of a distinct NKT subset endowed with high proliferative potential and have developed artificial antigen-presenting cells that generate CD62L-enriched NKTs for effective cancer immunotherapy. PMID:27183388

  12. Altered interleukin-12 responsiveness in Th1 and Th2 cells is associated with the differential activation of STAT5 and STAT1.

    PubMed

    Gollob, J A; Murphy, E A; Mahajan, S; Schnipper, C P; Ritz, J; Frank, D A

    1998-02-15

    T-cell activation in response to interleukin-12 (IL-12) is mediated through signaling events that include the tyrosine phosphorylation of STAT4. IL-12 responsiveness and the ability of IL-12 to activate STAT4 is different in T cells induced to differentiate into a Th1 or Th2 phenotype. In this report, we show that STAT5, STAT1alpha, and STAT1beta, in addition to STAT4, are tyrosine phosphorylated in response to IL-12 in phytohemagglutinin (PHA)-activated human T cells. To understand how the activation of these STATs contributes to T-cell IL-12 responsiveness, we analyzed the IL-12-induced activation of STAT5 and STAT1 in T cells stimulated to undergo Th1 or Th2 differentiation. The IL-12-induced tyrosine phosphorylation of STAT5 and STAT1, but not STAT4, is augmented in T cells activated into Th1 cells with PHA + interferon-gamma (IFN-gamma) compared with T cells activated with PHA alone. STAT5 DNA binding induced by IL-12 is also augmented in T cells activated with PHA + IFN-gamma compared with T cells activated with PHA alone, whereas STAT4 DNA binding is not increased. In contrast, the IL-12-induced activation of these STATs is inhibited in T cells activated into Th2 cells with PHA + IL-4. The enhancement of IL-12 signaling by IFN-gamma is not a direct effect of IFN-gamma on T cells, but rather is mediated by IL-12 that is produced by antigen-presenting cells in response to IFN-gamma. This positive autoregulatory effect of IL-12 on the activation of select STATs correlates with an increase in T-cell IFN-gamma production in response to IL-12. These findings suggest that the activation of STAT5 and STAT1 may augment select STAT4-dependent functional responses to IL-12 in Th1 cells. PMID:9454765

  13. Human SOCS1 Controls Immunostimulatory Activity of Monocyte-derived Dendritic Cells

    PubMed Central

    Hong, Bangxing; Ren, Wenhong; Song, Xiao-Tong; Evel-Kabler, Kevin; Chen, Si-Yi; Huang, Xue F

    2009-01-01

    Dendritic cell (DC)-based tumor vaccines have only achieved limited clinical efficacy, underscoring the limitation of stimulatory strategies to elicit effective cytotoxic T lymphocyte (CTL) responses against self tumor-associated antigens. Here we investigate the role of human suppressor of cytokine signaling (SOCS) 1, a feedback inhibitor of the JAK/STAT signaling pathway, in regulating antigen presentation by human DCs. We find that human SOCS1-silenced DCs have an enhanced stimulatory ability to prime self antigen-specific CTLs in vitro and in an SCID-hu mouse model. Human CTLs activated by SOCS1-silenced DCs, but not wild-type DCs, have an active lytic activity to natural antigen-expressing tumor cells. We further find that the capacity of human DCs to prime CTLs is likely controlled by SOCS1 restricted production and signaling of proinflammatory cytokines such as IL-12. These results indicate a critical role of human SOCS1 in negatively regulating the immunostimulatory capacity of DCs and imply a translational potential of this alternative, SOCS1 silencing strategy to develop effective DC vaccines. PMID:19789342

  14. Inhibition of human dendritic cell activation by hydroethanolic but not lipophilic extracts of turmeric (Curcuma longa).

    PubMed

    Krasovsky, Joseph; Chang, David H; Deng, Gary; Yeung, Simon; Lee, Mavis; Leung, Ping Chung; Cunningham-Rundles, Susanna; Cassileth, Barrie; Dhodapkar, Madhav V

    2009-03-01

    Turmeric has been extensively utilized in Indian and Chinese medicine for its immune-modulatory properties. Dendritic cells (DCs) are antigen-presenting cells specialized to initiate and regulate immunity. The ability of DCs to initiate immunity is linked to their activation status. The effects of turmeric on human DCs have not been studied. Here we show that hydroethanolic (HEE) but not lipophilic "supercritical" extraction (SCE) of turmeric inhibits the activation of human DCs in response to inflammatory cytokines. Treatment of DCs with HEE also inhibits the ability of DCs to stimulate the mixed lymphocyte reaction (MLR). Importantly, the lipophilic fraction does not synergize with the hydroethanolic fraction for the ability of inhibiting DC maturation. Rather, culturing of DCs with the combination of HEE and SCE leads to partial abrogation of the effects of HEE on the MLR initiated by DCs. These data provide a mechanism for the anti-inflammatory properties of turmeric. However, they suggest that these extracts are not synergistic and may contain components with mutually antagonistic effects on human DCs. Harnessing the immune effects of turmeric may benefit from specifically targeting the active fractions. PMID:19034830

  15. Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells.

    PubMed

    Ibiza, Sales; Pérez-Rodríguez, Andrea; Ortega, Angel; Martínez-Ruiz, Antonio; Barreiro, Olga; García-Domínguez, Carlota A; Víctor, Víctor M; Esplugues, Juan V; Rojas, José M; Sánchez-Madrid, Francisco; Serrador, Juan M

    2008-07-29

    Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S-nitrosylation on Cys(118), suggesting that upon TCR engagement, eNOS-derived NO directly activates N-Ras on the Golgi. Moreover, wild-type but not C118S N-Ras increased TCR-dependent apoptosis, suggesting that S-nitrosylation of Cys(118) contributes to activation-induced T cell death. Our data define a signaling mechanism for the regulation of the Ras/ERK pathway based on the eNOS-dependent differential activation of N-Ras and K-Ras at specific cell compartments. PMID:18641128

  16. Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells

    PubMed Central

    Ibiza, Sales; Pérez-Rodríguez, Andrea; Ortega, Ángel; Martínez-Ruiz, Antonio; Barreiro, Olga; García-Domínguez, Carlota A.; Víctor, Víctor M.; Esplugues, Juan V.; Rojas, José M.; Sánchez-Madrid, Francisco; Serrador, Juan M.

    2008-01-01

    Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S-nitrosylation on Cys118, suggesting that upon TCR engagement, eNOS-derived NO directly activates N-Ras on the Golgi. Moreover, wild-type but not C118S N-Ras increased TCR-dependent apoptosis, suggesting that S-nitrosylation of Cys118 contributes to activation-induced T cell death. Our data define a signaling mechanism for the regulation of the Ras/ERK pathway based on the eNOS-dependent differential activation of N-Ras and K-Ras at specific cell compartments. PMID:18641128

  17. A Septin Requirement Differentiates Autonomous- and Contact-Facilitated T Cell Proliferation

    PubMed Central

    Mujal, Adriana M.; Gilden, Julia K.; Gérard, Audrey; Kinoshita, Makoto; Krummel, Matthew F.

    2015-01-01

    T cell proliferation is initiated by T cell antigen receptor (TCR) triggering and/or by soluble growth factors. In characterizing T cells lacking the septin cytoskeleton, we found that successful cell division has discrete septin-dependent and -independent pathways. Septin-deficient T cells failed cytokinesis when prompted by pharmacological activation or cytokines. In contrast, cell division was independent of septins when cell-cell contacts, such as those from antigen-presenting cells, provided a niche. This septin-independent pathway was mediated by phosphatidylinositol-3-OH kinase activation through a combination of integrins and co-stimulatory signals. We could differentiate cytokine- versus antigen-driven expansion in vivo and thus demonstrate that targeting septins has strong potential to moderate detrimental bystander or homeostatic cytokine-driven proliferation without influencing expansion driven by conventional antigen-presentation. PMID:26692174

  18. Production of lentiviral vectors with enhanced efficiency to target dendritic cells by attenuating mannosidase activity of mammalian cells

    PubMed Central

    2011-01-01

    Background Dendritic cells (DCs) are antigen-presenting immune cells that interact with T cells and have been widely studied for vaccine applications. To achieve this, DCs can be manipulated by lentiviral vectors (LVs) to express antigens to stimulate the desired antigen-specific T cell response, which gives this approach great potential to fight diseases such as cancers, HIV, and autoimmune diseases. Previously we showed that LVs enveloped with an engineered Sindbis virus glycoprotein (SVGmu) could target DCs through a specific interaction with DC-SIGN, a surface molecule predominantly expressed by DCs. We hypothesized that SVGmu interacts with DC-SIGN in a mannose-dependent manner, and that an increase in high-mannose structures on the glycoprotein surface could result in higher targeting efficiencies of LVs towards DCs. It is known that 1-deoxymannojirimycin (DMJ) can inhibit mannosidase, which is an enzyme that removes high-mannose structures during the glycosylation process. Thus, we investigated the possibility of generating LVs with enhanced capability to modify DCs by supplying DMJ during vector production. Results Through western blot analysis and binding tests, we were able to infer that binding of SVGmu to DC-SIGN is directly related to amount of high-mannose structures on SVGmu. We also found that the titer for the LV (FUGW/SVGmu) produced with DMJ against 293T.DCSIGN, a human cell line expressing the human DC-SIGN atnibody, was over four times higher than that of vector produced without DMJ. In addition, transduction of a human DC cell line, MUTZ-3, yielded a higher transduction efficiency for the LV produced with DMJ. Conclusion We conclude that LVs produced under conditions with inhibited mannosidase activity can effectively modify cells displaying the DC-specific marker DC-SIGN. This study offers evidence to support the utilization of DMJ in producing LVs that are enhanced carriers for the development of DC-directed vaccines. PMID:21276219

  19. Toll-like receptor activation enhances cell-mediated immunity induced by an antibody vaccine targeting human dendritic cells

    PubMed Central

    Ramakrishna, Venky; Vasilakos, John P; Tario, Joseph D; Berger, Marc A; Wallace, Paul K; Keler, Tibor

    2007-01-01

    Previously, we have successfully targeted the mannose receptor (MR) expressed on monocyte-derived dendritic cells (DCs) using a fully human MR-specific antibody, B11, as a vehicle to deliver whole protein tumor antigens such as the human chorionic gonadotropin hormone (hCGβ). Since MRs play a role in bridging innate immunity with adaptive immunity we have explored several toll-like receptor (TLR)-specific ligands that may synergize with MR targeting and be applicable as adjuvants in the clinic. We demonstrate that antigen-specific helper and cytolytic T cells from both healthy donors and cancer patients were effectively primed with B11-hCGβ-treated autologous DCs when a combination of one or several TLR ligands is used. Specifically, concomitant signaling of DCs via TLR3 with dsRNA (poly I:C) and DC TLR 7/8 with Resiquimod (R-848), respectively, elicited efficient antigen presentation-mediated by MR-targeting. We demonstrate that MR and TLRs contribute towards maturation and activation of DCs by a mechanism that may be driven by a combination of adjuvant and antibody vaccines that specifically deliver antigenic targets to DCs. PMID:17254349

  20. Tracking and treating activated T cells

    PubMed Central

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

    2014-01-01

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