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Sample records for cells retain antigen

  1. 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.

  2. Characterization of a double-CRD-mutated Gal-8 recombinant protein that retains co-stimulatory activity on antigen-specific T-cell response.

    PubMed

    Schroeder, Matías Nicolás; Tribulatti, María Virginia; Carabelli, Julieta; André-Leroux, Gwenaëlle; Caramelo, Julio Javier; Cattaneo, Valentina; Campetella, Oscar

    2016-04-01

    Galectins (Gals) constitute a family of mammalian lectins with affinity for β-galactosides, characterized by the presence of conserved CRDs (carbohydrate-recognition domains). We have found previously that Gal-8, from the tandem-repeat group with two linked CRDs, exerts two separate actions on CD4(+)T-cells: antigen-independent proliferation and, at lower concentration, antigen-specific co-stimulation. Whereas proliferation can be ascribed to the pro-inflammatory role of Gal-8, the co-stimulatory activity of borderline T-cell-specific responses allows the proposal of Gal-8 as an adjuvant in vaccination. To study the relevance of glycan-lectin interaction to these T-cell activities, we generated a double-mutated protein (Gal-8mut) by replacing canonical arginine residues on each CRD, so as to abolish sugar-binding capacity. As expected, Gal-8mut was unable to bind to lactosyl-Sepharose, confirming that lactose recognition was precluded; however, preservation of lectin activity was still evident since Gal-8mut displayed haemoagglutinatory effects and binding capacity to the T-cell surface. To search for glycan affinity, a glycan microarray analysis was conducted which revealed that Gal-8mut lost most low- and intermediate-, but retained high-, affinity interactions, mainly to polylactosamines and blood group antigens. These findings were supported further by molecular modelling. Regarding biological activity, Gal-8mut was unable to induce T-cell proliferation, but efficiently co-stimulated antigen-specific responses, bothin vitroandin vivo.Therefore Gal-8mut represents a useful tool to dissect the specificities of lectin-glycan interactions underlying distinctive Gal-8 activities on T-cell biology. Moreover, given its distinguishing properties, Gal-8mut could be used to enhance borderline immune responses without the non-specific pro-inflammatory activity or other potential adverse effects.

  3. Follicular Dendritic Cells Retain Infectious HIV in Cycling Endosomes.

    PubMed

    Heesters, Balthasar A; Lindqvist, Madelene; Vagefi, Parsia A; Scully, Eileen P; Schildberg, Frank A; Altfeld, Marcus; Walker, Bruce D; Kaufmann, Daniel E; Carroll, Michael C

    2015-12-01

    Despite the success of antiretroviral therapy (ART), it does not cure Human Immunodeficiency Virus (HIV) and discontinuation results in viral rebound. Follicular dendritic cells (FDC) are in direct contact with CD4+ T cells and they retain intact antigen for prolonged periods. We found that human FDC isolated from patients on ART retain infectious HIV within a non-degradative cycling compartment and transmit infectious virus to uninfected CD4 T cells in vitro. Importantly, treatment of the HIV+ FDC with a soluble complement receptor 2 purges the FDC of HIV virions and prevents viral transmission in vitro. Our results provide an explanation for how FDC can retain infectious HIV for extended periods and suggest a therapeutic strategy to achieve cure in HIV-infected humans.

  4. ANTIGENIC STRUCTURE OF CELL SURFACES

    PubMed Central

    Aoki, Tadao; Hämmerling, Ulrich; de Harven, Etienne; Boyse, Edward A.; Old, Lloyd J.

    1969-01-01

    The representation of mouse alloantigens belonging to three systems, H-2, θ and TL, on the surface of cells from thymus, spleen, lymph nodes, and peritoneal cavity, was studied by electron microscopy with ferritin-labeled antibody. As expected from earlier serological data, TL was confined to thymocytes, θ was found on thymocytes and lymphocytes, and H-2 occurred to some extent on all cell types observed. On reticular cells, lymphocytes, plasma cells, and eosinophils, the majority of the cell surface was occupied by H-2; thymocytes had considerably less H-2, and erythrocytes and peritoneal macrophages least of all. In every instance the representation of antigen was discontinuous, the fraction of the cell surface covered being characteristic both of the antigen and of the type of cell. H-2 and θ provide a striking example of this; H-2 is present in far higher amounts on lymphocytes than on thymocytes, whereas the converse is true of θ. Within areas positive for H-2 or θ, protuberances of the surface membrane were often antigen-negative. A better definition of cell surface structure, gained from studies such as this, is necessary for further inquiry into how the cell surface is assembled, and into selective gene action in relation to cellular differentiation. PMID:5347699

  5. The Memory Function of the B Cell Antigen Receptor.

    PubMed

    Wienands, Jürgen; Engels, Niklas

    2016-01-01

    Activated B lymphocytes preserve their antigen experience by differentiating into long-lived pools of antibody-secreting plasma cells or various types of memory B cells (MBCs). The former population constantly produces serum immunoglobulins with sufficient specificity and affinity to thwart infections with recurrent pathogens. By contrast, memory B cell populations retain their antigen receptors on the cell surface and hence need pathogen-induced differentiation steps before they can actively contribute to host defense. The terminal differentiation of MBCs into antibody-secreting plasma cells is hallmarked by the absence of the lag phase characteristic for primary antibody responses. Moreover, secondary antibody responses are predominantly driven by MBCs that bear an antigen receptor of the IgG class on their surface although IgM-positive memory populations exist as well. These fundamental principles of B cell memory were enigmatic for decades. Only recently, we have begun to understand the underlying mechanisms. This review summarizes our current understanding of how different subpopulations of MBCs are generated during primary immune responses and how their functional heterogeneity on antigen recall is controlled by different signaling capabilities of B cell antigen receptor (BCR) isotypes and by the nature of the antigen.

  6. Identification and behavior of label-retaining cells in epithelia

    SciTech Connect

    Bickenbach, J.R.

    1982-01-01

    A subpopulation of stem cells has been demonstrated in several renewing tissues. Such cells have a slow cell cycle and provide differentiating cells during normal turnover and during regeneration of the tissue following damage. The presence of slowly-cycling cells in epithelia from regions of skin and oral mucosa was examined by labeling 10-day-old mice and 5-day-old hamsters with tritiated thymidine (/sup 3/H-TdR) and observing the rate at which label was diluted from the basal cells. Label was rapidly diluted by cell division in most cells but a small percentage of basal cells (label-retaining cells, LRCS) was found to retain label for up to ninety days. Electron microscopic autoradiography and ..beta..-glucuronidase histochemistry with autoradiography were used to distinguish slowly-cycling keratinocytes from Langerhans cells. Such findings of slowly-cycling keratinocytes in epithelia with the ability to proliferate in culture and with a direct relationship to patterns of tissue architecture suggest that LRCs in epithelia correspond to stem cells described in other continuously renewing tissues.

  7. Intestinal Antigen-Presenting Cells

    PubMed Central

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

    2016-01-01

    The microbiota that populate the mammalian intestine are critical for proper host physiology, yet simultaneously pose a potential danger. Intestinal antigen-presenting cells, namely macrophages and dendritic cells (DCs), are integral components of the mucosal innate immune system that maintain co-existence with the microbiota in face of this constant threat. Intestinal macrophages and DCs integrate signals from the microenvironment to orchestrate innate and adaptive immune responses that ultimately lead to durable tolerance of the microbiota. Tolerance is not a default response, however, because macrophages and DCs remain poised to vigorously respond to pathogens that breach the epithelial barrier. In this review, we summarize the salient features of macrophages and DCs in the healthy and inflamed intestine and discuss how signals from the microbiota can influence their function. PMID:25976247

  8. Presentation of lipid antigens to T cells.

    PubMed

    Mori, Lucia; De Libero, Gennaro

    2008-04-15

    T cells specific for lipid antigens participate in regulation of the immune response during infections, tumor immunosurveillance, allergy and autoimmune diseases. T cells recognize lipid antigens as complexes formed with CD1 antigen-presenting molecules, thus resembling recognition of MHC-peptide complexes. The biophysical properties of lipids impose unique mechanisms for their delivery, internalization into antigen-presenting cells, membrane trafficking, processing, and loading of CD1 molecules. Each of these steps is controlled at molecular and celular levels and determines lipid immunogenicity. Lipid antigens may derive from microbes and from the cellular metabolism, thus allowing the immune system to survey a large repertoire of immunogenic molecules. Recognition of lipid antigens facilitates the detection of infectious agents and the initiation of responses involved in immunoregulation and autoimmunity. This review focuses on the presentation mechanisms and specific recognition of self and bacterial lipid antigens and discusses the important open issues.

  9. CCR7 deficient inflammatory Dendritic Cells are retained in the Central Nervous System

    PubMed Central

    Clarkson, Benjamin D.; Walker, Alec; Harris, Melissa G.; Rayasam, Aditya; Hsu, Martin; Sandor, Matyas; Fabry, Zsuzsanna

    2017-01-01

    Dendritic cells (DC) accumulate in the CNS during neuroinflammation, yet, how these cells contribute to CNS antigen drainage is still unknown. We have previously shown that after intracerebral injection, antigen-loaded bone marrow DC migrate to deep cervical lymph nodes where they prime antigen-specific T cells and exacerbate experimental autoimmune encephalomyelitis (EAE) in mice. Here, we report that DC migration from brain parenchyma is dependent upon the chemokine receptor CCR7. During EAE, both wild type and CCR7−/− CD11c-eYFP cells infiltrated into the CNS but cells that lacked CCR7 were retained in brain and spinal cord while wild type DC migrated to cervical lymph nodes. Retention of CCR7-deficient CD11c-eYFP cells in the CNS exacerbated EAE. These data are the first to show that CD11chigh DC use CCR7 for migration out of the CNS, and in the absence of this receptor they remain in the CNS in situ and exacerbate EAE. PMID:28216674

  10. Stem cell programs are retained in human leukemic lymphoblasts.

    PubMed

    Fan, D; Zhou, X; Li, Z; Li, Z-Q; Duan, C; Liu, T; Zhang, F; Huang, Y; Zhang, Y; Gao, F; Guo, Y; Gupta, R; Chen, G; Enver, T; Tang, J; Hong, D

    2015-04-16

    Leukemic lymphoblasts within different immunophenotypic populations possess stem cell properties. However, whether or not the self-renewal program is retained from stem cells or conferred on progenitors by leukemogenic molecules remains unknown. We have addressed the issue in the context of TEL-AML1-associated acute lymphoblastic leukemia (ALL) by profiling a refined program edited from genes essential for self-renewal of hematopoietic stem cells and B-cell development. Bioinformatic analysis shows that ALL populations are loosely clustered and close to the normal population that contains stem and primitive progenitor cells. This finding indicates that immunophenotypes do not reflect maturation stages in ALL and that the self-renewal program may be retained from stem cells. Results of assessing 'first hit' function of TEL-AML1 in different populations of normal cells demonstrate the molecular model. Therefore, the current study shows a leukemogenic scenario of human ALL in which programs of stem cells are sustained in distinct fractions by leukemogenic mutations.

  11. Antigen presentation by Hodgkin's disease cells.

    PubMed

    Fisher, R I; Cossman, J; Diehl, V; Volkman, D J

    1985-11-01

    The L428 tumor cell line is a long-term tissue culture of Reed-Sternberg cells which was derived from the pleural effusion of a patient with Hodgkin's disease. The L428 cells express all known cell surface antigens, cytochemical staining, and cytologic features of freshly explanted Reed-Sternberg cells. In addition to the previously described HLA-DR cell surface antigens, the L428 cells are now demonstrated to express both DS and SB alloantigens. Thus, the L428 cells express all of the known subclasses of the human immune response genes that are located in the major histocompatibility complex. Furthermore, the L428 cells are capable of presenting soluble antigen to T cells in a genetically restricted fashion. T cell lines were established from normal donors previously immunized with tetanus toxoid. The T cells utilized were incapable of tetanus toxoid-induced proliferation unless antigen-presenting cells were added to the cultures. However, T cells from the two normal donors, which like the L428 cells expressed HLA-DR 5, demonstrated significant proliferative responses when cultured with tetanus toxoid and L428 cells. No proliferative response was observed when the L428 cells were used as antigen-presenting cells for a DR (4,-), DR (2,-) or DR (1,7) T cell line. The tetanus toxoid dose-response curve was similar regardless of whether autologous mononuclear leukocytes or L428 cells were used as antigen-presenting cells. The T cell proliferation induced by soluble antigen was also blocked by anti-HLA-DR antibody. Thus, functionally, Hodgkin's disease may be classified as a tumor of antigen-presenting cells.

  12. Optofluidic realization and retaining of cell-cell contact using an abrupt tapered optical fibre

    NASA Astrophysics Data System (ADS)

    Xin, Hongbao; Zhang, Yao; Lei, Hongxiang; Li, Yayi; Zhang, Huixian; Li, Baojun

    2013-06-01

    Studies reveal that there exists much interaction and communication between bacterial cells, with parts of these social behaviors depending on cell-cell contacts. The cell-cell contact has proved to be crucial for determining various biochemical processes. However, for cell culture with relatively low cell concentration, it is difficult to precisely control and retain the contact of a small group of cells. Particularly, the retaining of cell-cell contact is difficult when flows occur in the medium. Here, we report an optofluidic method for realization and retaining of Escherichia coli cell-cell contact in a microfluidic channel using an abrupt tapered optical fibre. The contact process is based on launching a 980-nm wavelength laser into the fibre, E. coli cells were trapped onto the fibre tip one after another, retaining cell-cell contact and forming a highly organized cell chain. The formed chains further show the ability as bio-optical waveguides.

  13. B cell antigen extraction is regulated by physical properties of antigen-presenting cells

    PubMed Central

    2017-01-01

    Antibody production and affinity maturation are driven by B cell extraction and internalization of antigen from immune synapses. However, the extraction mechanism remains poorly understood. Here we develop DNA-based nanosensors to interrogate two previously proposed mechanisms, enzymatic liberation and mechanical force. Using antigens presented by either artificial substrates or live cells, we show that B cells primarily use force-dependent extraction and resort to enzymatic liberation only if mechanical forces fail to retrieve antigen. The use of mechanical forces renders antigen extraction sensitive to the physical properties of the presenting cells. We show that follicular dendritic cells are stiff cells that promote strong B cell pulling forces and stringent affinity discrimination. In contrast, dendritic cells are soft and promote acquisition of low-affinity antigens through low forces. Thus, the mechanical properties of B cell synapses regulate antigen extraction, suggesting that distinct properties of presenting cells support different stages of B cell responses. PMID:27923880

  14. Vertebrate Cells Express Protozoan Antigen after Hybridization

    NASA Astrophysics Data System (ADS)

    Crane, Mark St. J.; Dvorak, James A.

    1980-04-01

    Epimastigotes, the invertebrate host stage of Trypanosoma cruzi, the protozoan parasite causing Chagas' disease in man, were fused with vertebrate cells by using polyethylene glycol. Hybrid cells were selected on the basis of T. cruzi DNA complementation of biochemical deficiencies in the vertebrate cells. Some clones of the hybrid cells expressed T. cruzi-specific antigen. It might be possible to use selected antigens obtained from the hybrids as vaccines for immunodiagnosis or for elucidation of the pathogenesis of Chagas' disease.

  15. Porous electrolyte retainer for molten carbonate fuel cell

    DOEpatents

    Singh, Raj N.; Dusek, Joseph T.

    1983-06-21

    A porous tile for retaining molten electrolyte within a fuel cell is prepared by sintering particles of lithium aluminate into a stable structure. The tile is assembled between two porous metal plates which serve as electrodes with fuels gases such as H.sub.2 and CO opposite to oxidant gases such as O.sub.2 and CO.sub.2. The tile is prepared with a porosity of 55-65% and a pore size distribution selected to permit release of sufficient molten electrolyte to wet but not to flood the adjacent electrodes.

  16. Porous electrolyte retainer for molten carbonate fuel cell. [lithium aluminate

    DOEpatents

    Singh, R.N.; Dusek, J.T.

    1979-12-27

    A porous tile for retaining molten electrolyte within a fuel cell is prepared by sintering particles of lithium aluminate into a stable structure. The tile is assembled between two porous metal plates which serve as electrodes with fuels gases such as H/sub 2/ and CO opposite to oxidant gases such as O/sub 2/ and CO/sub 2/. The tile is prepared with a porosity of 55 to 65% and a pore size distribution selected to permit release of sufficient molten electrolyte to wet but not to flood the adjacent electrodes.

  17. 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

  18. 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.

  19. Mature IgM-expressing plasma cells sense antigen and develop competence for cytokine production upon antigenic challenge

    PubMed Central

    Blanc, Pascal; Moro-Sibilot, Ludovic; Barthly, Lucas; Jagot, Ferdinand; This, Sébastien; de Bernard, Simon; Buffat, Laurent; Dussurgey, Sébastien; Colisson, Renaud; Hobeika, Elias; Fest, Thierry; Taillardet, Morgan; Thaunat, Olivier; Sicard, Antoine; Mondière, Paul; Genestier, Laurent; Nutt, Stephen L.; Defrance, Thierry

    2016-01-01

    Dogma holds that plasma cells, as opposed to B cells, cannot bind antigen because they have switched from expression of membrane-bound immunoglobulins (Ig) that constitute the B-cell receptor (BCR) to production of the secreted form of immunoglobulins. Here we compare the phenotypical and functional attributes of plasma cells generated by the T-cell-dependent and T-cell-independent forms of the hapten NP. We show that the nature of the secreted Ig isotype, rather than the chemical structure of the immunizing antigen, defines two functionally distinct populations of plasma cells. Fully mature IgM-expressing plasma cells resident in the bone marrow retain expression of a functional BCR, whereas their IgG+ counterparts do not. Antigen boost modifies the gene expression profile of IgM+ plasma cells and initiates a cytokine production program, characterized by upregulation of CCL5 and IL-10. Our results demonstrate that IgM-expressing plasma cells can sense antigen and acquire competence for cytokine production upon antigenic challenge. PMID:27924814

  20. Antigen Presentation by MHC-Dressed Cells

    PubMed Central

    Nakayama, Masafumi

    2015-01-01

    Professional antigen-presenting cells (APCs) such as conventional dendritic cells (DCs) process protein antigens to MHC-bound peptides and then present the peptide–MHC complexes to T cells. In addition to this canonical antigen presentation pathway, recent studies have revealed that DCs and non-APCs can acquire MHC class I (MHCI) and/or MHC class II (MHCII) from neighboring cells through a process of cell–cell contact-dependent membrane transfer called trogocytosis. These MHC-dressed cells subsequently activate or regulate T cells via the preformed antigen peptide–MHC complexes without requiring any further processing. In addition to trogocytosis, intercellular transfer of MHCI and MHCII can be mediated by secretion of membrane vesicles such as exosomes from APCs, generating MHC-dressed cells. This review focuses on the physiological role of antigen presentation by MHCI- or MHCII-dressed cells, and also discusses differences and similarities between trogocytosis and exosome-mediated transfer of MHC. PMID:25601867

  1. Asymmetric Cell Division of T Cells Upon Antigen Presentation Utilizes Multiple Conserved Mechanisms

    PubMed Central

    Oliaro, Jane; Van Ham, Vanessa; Sacirbegovic, Faruk; Pasam, Anupama; Bomzon, Ze’ev; Pham, Kim; Ludford-Menting, Mandy J.; Waterhouse, Nigel J.; Bots, Michael; Hawkins, Edwin D.; Watt, Sally V.; Cluse, Leonie A.; Clarke, Chris J.P.; Izon, David J.; Chang, John T.; Thompson, Natalie; Gu, Min; Johnstone, Ricky W.; Smyth, Mark J.; Humbert, Patrick O.; Reiner, Steven L.; Russell, Sarah M.

    2013-01-01

    Asymmetric cell division is a potential means by which cell fate choices during an immune response are orchestrated. Defining the molecular mechanisms that underlie asymmetric division of T cells is paramount for determining the role of this process in the generation of effector and memory T cell subsets. In other cell types, asymmetric cell division is regulated by conserved polarity protein complexes that control the localization of cell fate determinants and spindle orientation during division. We have developed a tractable, in vitro model of naïve CD8+ T cells undergoing initial division while attached to dendritic cells during antigen presentation to investigate whether similar mechanisms might regulate asymmetric division of T cells. Using this system, we show that direct interactions with antigen presenting cells provide the cue for polarization of T cells. Interestingly, the immunological synapse disseminates before division even though the T cells retain contact with the antigen presenting cell. The cue from the antigen presenting cell is translated into polarization of cell fate determinants via the polarity network of the Par3 and Scribble complexes and orientation of the mitotic spindle during division is orchestrated by the Pins/G protein complex. These findings suggest that T cells have selectively adapted a number of evolutionarily conserved mechanisms to generate diversity through asymmetric cell division. PMID:20530266

  2. Podosomes of dendritic cells facilitate antigen sampling

    PubMed Central

    Reinieren-Beeren, Inge; Cambi, Alessandra; Figdor, Carl G.; van den Bogaart, Geert

    2014-01-01

    Summary Dendritic cells sample the environment for antigens and play an important role in establishing the link between innate and acquired immunity. Dendritic cells contain mechanosensitive adhesive structures called podosomes that consist of an actin-rich core surrounded by integrins, adaptor proteins and actin network filaments. They facilitate cell migration via localized degradation of extracellular matrix. Here we show that podosomes of human dendritic cells locate to spots of low physical resistance in the substrate (soft spots) where they can evolve into protrusive structures. Pathogen recognition receptors locate to these protrusive structures where they can trigger localized antigen uptake, processing and presentation to activate T-cells. Our data demonstrate a novel role in antigen sampling for podosomes of dendritic cells. PMID:24424029

  3. Podosomes of dendritic cells facilitate antigen sampling.

    PubMed

    Baranov, Maksim V; Ter Beest, Martin; Reinieren-Beeren, Inge; Cambi, Alessandra; Figdor, Carl G; van den Bogaart, Geert

    2014-03-01

    Dendritic cells sample the environment for antigens and play an important role in establishing the link between innate and acquired immunity. Dendritic cells contain mechanosensitive adhesive structures called podosomes that consist of an actin-rich core surrounded by integrins, adaptor proteins and actin network filaments. They facilitate cell migration via localized degradation of extracellular matrix. Here, we show that podosomes of human dendritic cells locate to spots of low physical resistance in the substrate (soft spots) where they can evolve into protrusive structures. Pathogen recognition receptors locate to these protrusive structures where they can trigger localized antigen uptake, processing and presentation to activate T-cells. Our data demonstrate a novel role in antigen sampling for the podosomes of dendritic cells.

  4. Connecting the Dots: Artificial Antigen Presenting Cell-Mediated Modulation of Natural Killer T Cells

    PubMed Central

    Sun, Wenji; Subrahmanyam, Priyanka B.; East, James E.

    2012-01-01

    Natural killer T (NKT) cells constitute an important subset of T cells that can both directly and indirectly mediate antitumor immunity. However, we and others have reported that cancer patients have a reduction in both NKT cell number and function. NKT cells can be stimulated and expanded with α-GalCer and cytokines and these expanded NKT cells retain their phenotype, remain responsive to antigenic stimulation, and display cytotoxic function against tumor cell lines. These data strongly favor the use of ex vivo expanded NKT cells in adoptive immunotherapy. NKT cell based-immunotherapy has been limited by the use of autologous antigen-presenting cells, which can vary substantially in their quantity and quality. A standardized system that relies on artificial antigen-presenting cells (aAPCs) could produce the stimulating effects of dendritic cell (DC) without the pitfalls of allo- or xenogeneic cells. In this review, we discuss the progress that has been made using CD1d-based aAPC and how this acellular antigen presenting system can be used in the future to enhance our understanding of NKT cell biology and to develop NKT cell-specific adoptive immunotherapeutic strategies. PMID:23050947

  5. Allogeneic substitution for nominal antigen-specific T-cell clone reactivity in schistosomiasis.

    PubMed Central

    Linette, G P; Lammie, P J; Phillips, S M

    1986-01-01

    The present studies have established the nature of a T-cell clone which demonstrates dual reactivity directed against Schistosoma mansoni antigen presented by syngeneic antigen presenting cells and against allogeneic cells. Clone G4, when stimulated by either antigen (SEA) or allogeneic cells (PL/J), exhibits similar functional and phenotypic characteristics. A subclone of G4, G4A.1, which has been maintained in continuous mixed lymphocyte culture for 12 months (in the absence of SEA), retains comparable reactivity with respect to proliferation and ability to produce lymphokines, transfer delayed-type hypersensitivity, and produce in vitro granulomas in response to SEA. Normal antigenic stimulation is highly contingent upon I-Ab compatibility while antibody blocking experiments map allo-reactivity to I-Eu. The failure of B10.PL spleen cells to stimulate G4, however, suggests that alloreactivity may be directed against the recently described Mls X locus. Both allogeneic and nominal antigen induced T-cell activation are blocked by antibody directed against L3T4A, confirming Class II MHC restriction for both types of stimulation. These studies suggest that stimulation of T cells by either alloantigen or nominal antigen elicits qualitatively similar functional profiles, and further suggest the feasibility of producing large numbers of nominal antigen reactive cloned T cells in the absence of nominal antigen under mixed lymphocyte culture conditions. PMID:2420707

  6. Red cell antigens: Structure and function

    PubMed Central

    Pourazar, Abbasali

    2007-01-01

    Landsteiner and his colleagues demonstrated that human beings could be classified into four groups depending on the presence of one (A) or another (B) or both (AB) or none (O) of the antigens on their red cells. The number of the blood group antigens up to 1984 was 410. In the next 20 years, there were 16 systems with 144 antigens and quite a collection of antigens waiting to be assigned to systems, pending the discovery of new information about their relationship to the established systems. The importance of most blood group antigens had been recognized by immunological complications of blood transfusion or pregnancies; their molecular structure and function however remained undefined for many decades. Recent advances in molecular genetics and cellular biochemistry resulted in an abundance of new information in this field of research. In this review, we try to give some examples of advances made in the field of ‘structure and function of the red cell surface molecules.’ PMID:21938229

  7. Th17 cells are refractory to senescence and retain robust antitumor activity after long-term ex vivo expansion

    PubMed Central

    Bowers, Jacob S.; Nelson, Michelle H.; Majchrzak, Kinga; Bailey, Stefanie R.; Rohrer, Baerbel; Kaiser, Andrew D.M.; Atkinson, Carl; Paulos, Chrystal M.

    2017-01-01

    Adoptive immunotherapy for solid tumors relies on infusing large numbers of T cells to mediate successful antitumor responses in patients. While long-term rapid-expansion protocols (REPs) produce sufficient numbers of CD8+ T cells for treatment, they also cause decline in the cell’s therapeutic fitness. In contrast, we discovered that IL-17–producing CD4+ T cells (Th17 cells) do not require REPs to expand 5,000-fold over 3 weeks. Also, unlike Th1 cells, Th17 cells do not exhibit hallmarks of senescence or apoptosis, retaining robust antitumor efficacy in vivo. Three-week-expanded Th17 cells eliminated melanoma as effectively as Th17 cells expanded for 1 week when infused in equal numbers into mice. However, treating mice with large recalcitrant tumors required the infusion of all cells generated after 2 or 3 weeks of expansion, while the cell yield obtained after 1-week expansion was insufficient. Long-term-expanded Th17 cells also protected mice from tumor rechallenge including lung metastasis. Importantly, 2-week-expanded human chimeric antigen receptor–positive (CAR+) Th17 cells also retained their ability to regress human mesothelioma, while CAR+ Th1 cells did not. Our results indicate that tumor-reactive Th17 cells are an effective cell therapy for cancer, remaining uncompromised when expanded for a long duration owing to their resistance to senescence. PMID:28289713

  8. Diverse Endogenous Antigens for Mouse Natural Killer T Cells: Self-Antigens That Are Not Glycosphingolipids

    PubMed Central

    Pei, Bo; Speak, Anneliese O; Shepherd, Dawn; Butters, Terry; Cerundolo, Vincenzo; Platt, Frances M; Kronenberg, Mitchell

    2011-01-01

    Natural killer T cells with an invariant antigen receptor (iNKT cells) represent a highly conserved and unique subset of T lymphocytes having properties of innate and adaptive immune cells. They have been reported to regulate a variety of immune responses, including the response to cancers and the development of autoimmunity. The development and activation of iNKT cells is dependent on self-antigens presented by the CD1d antigen-presenting molecule. It is widely believed that these self-antigens are glycosphingolipids (GSLs), molecules that contain ceramide as the lipid backbone. Here we used a variety of methods to show that mammalian antigens for mouse iNKT cells need not be GSLs, including the use of cell lines deficient in GSL biosynthesis and an inhibitor of GSL biosynthesis. Presentation of these antigens required the expression of CD1d molecules that could traffic to late endosomes, the site where self-antigen is acquired. Extracts of antigen-presenting cells (APCs) contain a self-antigen that could stimulate iNKT cells when added to plates coated with soluble, recombinant CD1d molecules. The antigen(s) in these extracts are resistant to sphingolipid-specific hydrolase digestion, consistent with the results using live APCs. Lyosphosphatidylcholine, a potential self-antigen that activated human iNKT cell lines, did not activate mouse iNKT cell hybridomas. Our data indicate that there may be more than one type of self-antigen for iNKT cells, that the self-antigens comparing mouse and human may not be conserved, and that the search to identify these molecules should not be confined to GSLs. PMID:21191069

  9. Optofluidic realization and retaining of cell–cell contact using an abrupt tapered optical fibre

    PubMed Central

    Xin, Hongbao; Zhang, Yao; Lei, Hongxiang; Li, Yayi; Zhang, Huixian; Li, Baojun

    2013-01-01

    Studies reveal that there exists much interaction and communication between bacterial cells, with parts of these social behaviors depending on cell–cell contacts. The cell–cell contact has proved to be crucial for determining various biochemical processes. However, for cell culture with relatively low cell concentration, it is difficult to precisely control and retain the contact of a small group of cells. Particularly, the retaining of cell–cell contact is difficult when flows occur in the medium. Here, we report an optofluidic method for realization and retaining of Escherichia coli cell–cell contact in a microfluidic channel using an abrupt tapered optical fibre. The contact process is based on launching a 980-nm wavelength laser into the fibre, E. coli cells were trapped onto the fibre tip one after another, retaining cell–cell contact and forming a highly organized cell chain. The formed chains further show the ability as bio-optical waveguides. PMID:23771190

  10. Antigen-Presenting Cells and Antigen Presentation in Tertiary Lymphoid Organs

    PubMed Central

    Hughes, Catherine E.; Benson, Robert A.; Bedaj, Marija; Maffia, Pasquale

    2016-01-01

    Tertiary lymphoid organs (TLOs) form in territorialized niches of peripheral tissues characterized by the presence of antigens; however, little is known about mechanism(s) of antigen handling by ectopic lymphoid structures. In this mini review, we will discuss the role of antigen-presenting cells and mechanisms of antigen presentation in TLOs, summarizing what is currently known about this facet of the formation and function of these tissues as well as identifying questions yet to be addressed. PMID:27872626

  11. Stem/progenitor cells from inflamed human dental pulp retain tissue regeneration potential

    PubMed Central

    Alongi, Dominick J; Yamaza, Takayoshi; Song, Yingjie; Fouad, Ashraf F; Romberg, Elaine E; Shi, Songtao; Tuan, Rocky S; Huang, George T-J

    2011-01-01

    Background Potent stem/progenitor cells have been isolated from normal human dental pulps termed dental pulp stem cells (DPSCs). However, it is unknown whether these cells exist in inflamed pulps (IPs). Aims To determine whether DPSCs can be identified and isolated from IPs; and if they can be successfully cultured, whether they retain tissue regeneration potential in vivo. Materials & methods DPSCs from freshly collected normal pulps (NPs) and IPs were characterized in vitro and their tissue regeneration potential tested using an in vivo study model. Results The immunohistochemical analysis showed that IPs expressed higher levels of mesenchymal stem cell markers STRO-1, CD90, CD105 and CD146 compared with NPs (p < 0.05). Flow cytometry analysis showed that DPSCs from both NPs and IPs expressed moderate to high levels of CD146, stage-specific embryonic antigen-4, CD73 and CD166. Total population doubling of DPSCs-IPs (44.6 ± 2.9) was lower than that of DPSCs-NPs (58.9 ± 2.5) (p < 0.05), and DPSCs-IPs appeared to have a decreased osteo/dentinogenic potential compared with DPSCs-NPs based on the mineral deposition in cultures. Nonetheless, DPSCs-IPs formed pulp/dentin complexes similar to DPSCs-NPs when transplanted into immunocompromised mice. Conclusion DPSCs-IPs can be isolated and their mesenchymal stem cell marker profiles are similar to those from NPs. Although some stem cell properties of DPSCs-IPs were altered, cells from some samples remained potent in tissue regeneration in vivo. PMID:20465527

  12. Normal ovarian surface epithelial label-retaining cells exhibit stem/progenitor cell characteristics.

    PubMed

    Szotek, Paul P; Chang, Henry L; Brennand, Kristen; Fujino, Akihiro; Pieretti-Vanmarcke, Rafael; Lo Celso, Cristina; Dombkowski, David; Preffer, Frederic; Cohen, Kenneth S; Teixeira, Jose; Donahoe, Patricia K

    2008-08-26

    Ovulation induces cyclic rupture and regenerative repair of the ovarian coelomic epithelium. This process of repeated disruption and repair accompanied by complex remodeling typifies a somatic stem/progenitor cell-mediated process. Using BrdU incorporation and doxycycline inducible histone2B-green fluorescent protein pulse-chase techniques, we identify a label-retaining cell population in the coelomic epithelium of the adult mouse ovary as candidate somatic stem/progenitor cells. The identified population exhibits quiescence with asymmetric label retention, functional response to estrous cycling in vivo by proliferation, enhanced growth characteristics by in vitro colony formation, and cytoprotective mechanisms by enrichment for the side population. Together, these characteristics identify the label-retaining cell population as a candidate for the putative somatic stem/progenitor cells of the coelomic epithelium of the mouse ovary.

  13. Normal ovarian surface epithelial label-retaining cells exhibit stem/progenitor cell characteristics

    PubMed Central

    Szotek, Paul P.; Chang, Henry L.; Brennand, Kristen; Fujino, Akihiro; Pieretti-Vanmarcke, Rafael; Lo Celso, Cristina; Dombkowski, David; Preffer, Frederic; Cohen, Kenneth S.; Teixeira, Jose; Donahoe, Patricia K.

    2008-01-01

    Ovulation induces cyclic rupture and regenerative repair of the ovarian coelomic epithelium. This process of repeated disruption and repair accompanied by complex remodeling typifies a somatic stem/progenitor cell-mediated process. Using BrdU incorporation and doxycycline inducible histone2B-green fluorescent protein pulse–chase techniques, we identify a label-retaining cell population in the coelomic epithelium of the adult mouse ovary as candidate somatic stem/progenitor cells. The identified population exhibits quiescence with asymmetric label retention, functional response to estrous cycling in vivo by proliferation, enhanced growth characteristics by in vitro colony formation, and cytoprotective mechanisms by enrichment for the side population. Together, these characteristics identify the label-retaining cell population as a candidate for the putative somatic stem/progenitor cells of the coelomic epithelium of the mouse ovary. PMID:18711140

  14. Shashkov`s method retaining cell-edge unknowns

    SciTech Connect

    Roberts, R.M.

    1996-01-05

    Shashkov`s method for scalar cell-edge and cell-center variables is derived. Dot products for cell-edge vectors are computed for a corner of the cell. Next, the divergence and gradient are discretized. The diffusion equation is solved with cell-edge continuity and boundary conditions. A symmetric positive definite solution matrix is proven.

  15. 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

  16. Activation of Type II Cells into Regenerative Stem Cell Antigen-1+ Cells during Alveolar Repair

    PubMed Central

    Kumar, Varsha Suresh; Zhang, Wei; Rehman, Jalees; Malik, Asrar B.

    2015-01-01

    The alveolar epithelium is composed of two cell types: type I cells comprise 95% of the gas exchange surface area, whereas type II cells secrete surfactant, while retaining the ability to convert into type I cells to induce alveolar repair. Using lineage-tracing analyses in the mouse model of Pseudomonas aeruginosa–induced lung injury, we identified a population of stem cell antigen (Sca)-1–expressing type II cells with progenitor cell properties that mediate alveolar repair. These cells were shown to be distinct from previously reported Sca-1–expressing bronchioalveolar stem cells. Microarray and Wnt reporter studies showed that surfactant protein (Sp)-C+Sca-1+ cells expressed Wnt signaling pathway genes, and inhibiting Wnt/β-catenin signaling prevented the regenerative function of Sp-C+Sca-1+ cells in vitro. Thus, P. aeruginosa–mediated lung injury induces the generation of a Sca-1+ subset of type II cells. The progenitor phenotype of the Sp-C+Sca-1+ cells that mediates alveolar epithelial repair might involve Wnt signaling. PMID:25474582

  17. TL antigen as a transplantation antigen recognized by TL-restricted cytotoxic T cells

    PubMed Central

    1994-01-01

    In contrast to broadly expressed classical class I antigens of the major histocompatibility complex, structurally closely related TL antigens are expressed in a highly restricted fashion. Unlike classical class I antigens, TL antigens are not known to be targets of cytotoxic T cells or to mediate graft rejection. Whereas classical class I antigens function as antigen-presenting molecules to T cell receptors (TCR), the role of TL is yet to be defined. To elucidate the function of TL, we have derived transgenic mice expressing TL in most tissues including skin by introducing a TL gene, T3b of C57BL/6 mouse origin, driven by the H-2Kb promoter. By grafting the skin of transgenic mice, we demonstrate that TL can serve as a transplantation antigen and mediate a TCR-alpha/beta+ CD8+ cytotoxic T cell response. This T cell recognition of TL does not require antigen presentation by H-2 molecules. Furthermore, we show that C57BL/6 F1 mice develop CD8+ T cells that are cytotoxic for C57BL/6 TL+ leukemia cells, providing further support for the concept that aberrantly expressed nonmutated proteins such as TL can be recognized as tumor antigens. PMID:8113675

  18. Antigen-Presenting Cells in the Skin.

    PubMed

    Kashem, Sakeen W; Haniffa, Muzlifah; Kaplan, Daniel H

    2017-02-06

    Professional antigen-presenting cells (APCs) in the skin include dendritic cells, monocytes, and macrophages. They are highly dynamic, with the capacity to enter skin from the peripheral circulation, patrol within tissue, and migrate through lymphatics to draining lymph nodes. Skin APCs are endowed with antigen sensing, processing, and presenting machinery and play key roles in initiating, modulating, and resolving cutaneous inflammation. Skin APCs are a highly heterogeneous population with functionally specialized subsets that are developmentally imprinted and modulated by local tissue microenvironmental and inflammatory cues. This review explores recent advances that have allowed for a more accurate taxonomy of APC subsets found in both mouse and human skin. It also examines the functional specificity of individual APC subsets and their collaboration with other immune cell types that together promote adaptive T cell and regional cutaneous immune responses during homeostasis, inflammation, and disease. Expected final online publication date for the Annual Review of Immunology Volume 35 is April 26, 2017 . Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  19. Immortalization of human myogenic progenitor cell clone retaining multipotentiality

    SciTech Connect

    Hashimoto, Naohiro . E-mail: nao@nils.go.jp; Kiyono, Tohru; Wada, Michiko R.; Shimizu, Shirabe; Yasumoto, Shigeru; Inagawa, Masayo

    2006-10-06

    Human myogenic cells have limited ability to proliferate in culture. Although forced expression of telomerase can immortalize some cell types, telomerase alone delays senescence of human primary cultured myogenic cells, but fails to immortalize them. In contrast, constitutive expression of both telomerase and the E7 gene from human papillomavirus type 16 immortalizes primary human myogenic cells. We have established an immortalized primary human myogenic cell line preserving multipotentiality by ectopic expression of telomerase and E7. The immortalized human myogenic cells exhibit the phenotypic characteristics of their primary parent, including an ability to undergo myogenic, osteogenic, and adipogenic terminal differentiation under appropriate culture conditions. The immortalized cells will be useful for both basic and applied studies aimed at human muscle disorders. Furthermore, immortalization by transduction of telomerase and E7 represents a useful method by which to expand human myogenic cells in vitro without compromising their ability to differentiate.

  20. Detection of cell surface antigens on monolayer cells

    PubMed Central

    Tachibana, Takehiko; Klein, Eva

    1970-01-01

    A small scale modified method of the immune adherence on monolayer cells grown in Falcon plastic `Microtest' plates is described. This method is especially useful in mouse systems as it simplifies the washing procedure which is an indispensable step for removal of the anticomplementary activity present in mouse serum. Histocompatibility antigens and the surface antigen induced by the Moloney virus could be detected with this method. ImagesFIG. 1FIG. 1 PMID:5530203

  1. LOCALIZATION OF ANTIGEN IN TISSUE CELLS

    PubMed Central

    Coons, Albert H.; Leduc, Elizabeth H.; Kaplan, Melvin H.

    1951-01-01

    The fate of three proteins, crystalline hen's egg albumin, crystalline bovine plasma albumin, and human plasma γ-globulin, was traced after intravenous injection into mice. This was done by preparing frozen sections of quick-frozen tissue, allowing what foreign protein might be present in the section to react with homologous antibody labelled with fluorescein, and examining the section under the fluorescence microscope. By this means, which employs the serological specificity of the protein as a natural "marker," all three of these proteins were found in the cells of the reticulo-endothelial system, the connective tissue, the vascular endothelium, the lymphocytes of spleen and lymph node, and the epithelium of the kidney tubules, the liver, and in very small amounts in the adrenal. The central nervous system was not studied. All three persisted longest in the reticulo-endothelial system and the connective tissue, and in the doses employed egg white (10 mg.) was no longer detectable after 1 day, bovine albumin (10 mg.) after 2 days, and human γ-globulin (4 mg.) after 6 days, although in a somewhat higher dose (10 mg.) human γ-globulin persisted longer than 8 days. Egg albumin differed from the others in not being detectable in the cells of the renal glomerulus. It was found that each of the three proteins was present in the nuclei of each cell type enumerated above, often in higher concentration than in the cytoplasm. Further, some of the nuclei not only contained antigen, soon after injection, but were also surrounded by a bright ring associated with the nuclear membrane. By means of photographic records under the fluorescence microscope of sections stained for antigen, and direct observation under the light microscope of the same field subsequently stained with hematoxylin and eosin, it could be determined that the antigen was not adsorbed to chromatin or nucleoli, but was apparently in solution in the nuclear sap. PMID:14803641

  2. Satellite cells from dystrophic muscle retain regenerative capacity.

    PubMed

    Boldrin, Luisa; Zammit, Peter S; Morgan, Jennifer E

    2015-01-01

    Duchenne muscular dystrophy is an inherited disorder that is characterized by progressive skeletal muscle weakness and wasting, with a failure of muscle maintenance/repair mediated by satellite cells (muscle stem cells). The function of skeletal muscle stem cells resident in dystrophic muscle may be perturbed by being in an increasing pathogenic environment, coupled with constant demands for repairing muscle. To investigate the contribution of satellite cell exhaustion to this process, we tested the functionality of satellite cells isolated from the mdx mouse model of Duchenne muscular dystrophy. We found that satellite cells derived from young mdx mice contributed efficiently to muscle regeneration within our in vivo mouse model. To then test the effects of long-term residence in a dystrophic environment, satellite cells were isolated from aged mdx muscle. Surprisingly, they were as functional as those derived from young or aged wild type donors. Removing satellite cells from a dystrophic milieu reveals that their regenerative capacity remains both intact and similar to satellite cells derived from healthy muscle, indicating that the host environment is critical for controlling satellite cell function.

  3. T-cell responses to minor histocompatibility antigens.

    PubMed Central

    Lai, P K; Waterfield, J D; Gascoigne, N R; Sharrock, C E; Mitchison, N A

    1982-01-01

    We have investigated the helper and cytotoxic T-cell response to minor histocompatibility antigens and generated long term antigen-specific cell lines to them. Antigen-specific activity was selected for by regular restimulation with irradiated cells bearing the antigens in the presence of interleukin 2, so that alloreactivity to other cell surface antigens was gradually lost. Helper T cells cultured over several months were active in vivo and in vitro, but the culturing method eventually selected for cytotoxic T cells at the expense of helper T cells, with concomitant changes in the proportions of cells expressing the Lyt phenotypes. Individual long term cultures of cytotoxic T cells specific for minor histocompatibility antigens were restricted by either H2K or D but not both. Helper T cells to minor histocompatibility antigens derived directly from primed F1 mice did not show restriction to the priming parental haplotype. This is consistent with antigen reprocessing by the F1 antigen presenting cells such that populations of helper T cells restricted by both parental H-2 haplotypes were primed. F1 cytotoxic T cells were restricted to the parental H-2 haplotype used for in vitro boosting, irrespective of which H-2 was used for in vivo priming. PMID:6214502

  4. T Helper Cell Tolerance to Ubiquitous Nuclear Antigens

    PubMed Central

    NAKKEN, BRITT; DAVIS, KAREN E.; PAN, ZIJIAN; BACHMANN, MICHAEL; FARRIS, A. DARISE

    2007-01-01

    Systemic autoimmune diseases are characterized by the development of anti-nuclear autoantibodies. In order to understand the immunologic events leading to the development of such antibodies, knowledge of mechanisms of immune tolerance to nuclear antigens is required. By utilizing adoptive T cell transfer strategies with transgenic mouse models expressing nuclear neo-self antigens, T cell tolerance to the lupus-related nuclear antigens human La and nRNP A has been demonstrated. These findings also indicate the existence in normal animals of autoreactive B cells continuously presenting nuclear antigen, suggesting that nuclear antigens are not sequestered from the immune system. Investigations of CD4+ T cell tolerance to non-nuclear antigens have revealed a number of mechanisms that protect the host from autoreactivity, including autoreactive T cell deletion, regulatory T cell development and anergy induction. Recent studies using T cell receptor and neo-self nuclear antigen transgenic mice are revealing the importance of such mechanisms in maintaining tolerance to nuclear antigens. Mechanisms of tolerogenic antigen presentation, identification of tolerogenic antigen source(s), and the pathways leading to loss of tolerance to nuclear antigens in systemic autoimmune disease states are currently being sought. PMID:14629620

  5. Antigen presentation for priming T cells in central system.

    PubMed

    Dasgupta, Shaoni; Dasgupta, Subhajit

    2017-01-01

    Generation of myelin antigen-specific T cells is a major event in neuroimmune responses that causes demyelination. The antigen-priming of T cells and its location is important in chronic and acute inflammation. In autoimmune multiple sclerosis, the effector T cells are considered to generate in periphery. However, the reasons for chronic relapsing-remitting events are obscure. Considering mechanisms, a feasible aim of research is to investigate the role of antigen-primed T cells in lupus cerebritis. Last thirty years of investigations emphasize the relevance of microglia and infiltrated dendritic cells/macrophages as antigen presenting cells in the central nervous system. The recent approach towards circulating B-lymphocytes is an important area in the context. Here, we analyze the existing findings on antigen presentation in the central nervous system. The aim is to visualize signaling events of myelin antigen presentation to T cells and lead to the strategy of future goals on immunotherapy research.

  6. Nanoscale artificial antigen presenting cells for T cell immunotherapy.

    PubMed

    Perica, Karlo; De León Medero, Andrés; Durai, Malarvizhi; Chiu, Yen Ling; Bieler, Joan Glick; Sibener, Leah; Niemöller, Michaela; Assenmacher, Mario; Richter, Anne; Edidin, Michael; Oelke, Mathias; Schneck, Jonathan

    2014-01-01

    Artificial antigen presenting cells (aAPC), which deliver stimulatory signals to cytotoxic lymphocytes, are a powerful tool for both adoptive and active immunotherapy. Thus far, aAPC have been synthesized by coupling T cell activating proteins such as CD3 or MHC-peptide to micron-sized beads. Nanoscale platforms have different trafficking and biophysical interaction properties and may allow development of new immunotherapeutic strategies. We therefore manufactured aAPC based on two types of nanoscale particle platforms: biocompatible iron-dextran paramagnetic particles (50-100 nm in diameter) and avidin-coated quantum dot nanocrystals (~30 nm). Nanoscale aAPC induced antigen-specific T cell proliferation from mouse splenocytes and human peripheral blood T cells. When injected in vivo, both iron-dextran particles and quantum dot nanocrystals enhanced tumor rejection in a subcutaneous mouse melanoma model. This is the first description of nanoscale aAPC that induce antigen-specific T cell proliferation in vitro and lead to effective T cell stimulation and inhibition of tumor growth in vivo. Artifical antigen presenting cells could revolutionize the field of cancer-directed immunotherapy. This team of investigators have manufactured two types of nanoscale particle platform-based aAPCs and demonstrates that both iron-dextran particles and quantum dot nanocrystals enhance tumor rejection in a melanoma model, providing the first description of nanoscale aAPCs that lead to effective T cell stimulation and inhibition of tumor growth. © 2013.

  7. Murine cell-mediated immune response recognizes an enterovirus group-specific antigen(s).

    PubMed Central

    Beck, M A; Tracy, S M

    1989-01-01

    Splenocytes taken from mice inoculated with coxsackievirus B3 (CVB3) (Nancy) developed an in vitro proliferative response against CVB3 antigen. This response could not be detected earlier than 8 days postinoculation but could be detected up to 28 days after exposure to CB3. CVB3-sensitized splenocytes responded not only to the CVB3 antigen but to other enteroviruses as well. This response was found to be enterovirus specific in that no response was detected to a non-enteroviral picornavirus, encephalomyocarditis virus, or to an unrelated influenza virus. The generation of a splenocyte population capable of responding to an enterovirus group antigen(s) was not limited to inoculation of mice with CVB3, as similar responses were generated when mice were inoculated with CVB2. Cell subset depletions revealed that the major cell type responding to the enterovirus group antigen(s) was the CD4+ T cell. Current evidence suggests that the group antigen(s) resides in the structural proteins of the virus, since spleen cells from mice inoculated with a UV-inactivated, highly purified preparation of CVB3 virions also responded in vitro against enteroviral antigens. PMID:2476566

  8. CD169(+) macrophages present lipid antigens to mediate early activation of iNKT cells in lymph nodes.

    PubMed

    Barral, Patricia; Polzella, Paolo; Bruckbauer, Andreas; van Rooijen, Nico; Besra, Gurdyal S; Cerundolo, Vincenzo; Batista, Facundo D

    2010-04-01

    Invariant natural killer T cells (iNKT cells) are involved in the host defense against microbial infection. Although it is known that iNKT cells recognize glycolipids presented by CD1d, how and where they encounter antigen in vivo remains unclear. Here we used multiphoton microscopy to visualize the dynamics and activation of iNKT cells in lymph nodes. After antigen administration, iNKT cells became confined in a CD1d-dependent manner in close proximity to subcapsular sinus CD169(+) macrophages. These macrophages retained, internalized and presented lipid antigen and were required for iNKT cell activation, cytokine production and population expansion. Thus, CD169(+) macrophages can act as true antigen-presenting cells controlling early iNKT cell activation and favoring the fast initiation of immune responses.

  9. A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division.

    PubMed

    Rocheteau, Pierre; Gayraud-Morel, Barbara; Siegl-Cachedenier, Irene; Blasco, Maria A; Tajbakhsh, Shahragim

    2012-01-20

    Satellite cells are adult skeletal muscle stem cells that are quiescent and constitute a poorly defined heterogeneous population. Using transgenic Tg:Pax7-nGFP mice, we show that Pax7-nGFP(Hi) cells are less primed for commitment and have a lower metabolic status and delayed first mitosis compared to Pax7-nGFP(Lo) cells. Pax7-nGFP(Hi) can give rise to Pax7-nGFP(Lo) cells after serial transplantations. Proliferating Pax7-nGFP(Hi) cells exhibit lower metabolic activity, and the majority performs asymmetric DNA segregation during cell division, wherein daughter cells retaining template DNA strands express stem cell markers. Using chromosome orientation-fluorescence in situ hybridization, we demonstrate that all chromatids segregate asymmetrically, whereas Pax7-nGFP(Lo) cells perform random DNA segregation. Therefore, quiescent Pax7-nGFP(Hi) cells represent a reversible dormant stem cell state, and during muscle regeneration, Pax7-nGFP(Hi) cells generate distinct daughter cell fates by asymmetrically segregating template DNA strands to the stem cell. These findings provide major insights into the biology of stem cells that segregate DNA asymmetrically.

  10. Tumor-initiating label-retaining cancer cells in human gastrointestinal cancers undergo asymmetric cell division.

    PubMed

    Xin, Hong-Wu; Hari, Danielle M; Mullinax, John E; Ambe, Chenwi M; Koizumi, Tomotake; Ray, Satyajit; Anderson, Andrew J; Wiegand, Gordon W; Garfield, Susan H; Thorgeirsson, Snorri S; Avital, Itzhak

    2012-04-01

    Label-retaining cells (LRCs) have been proposed to represent adult tissue stem cells. LRCs are hypothesized to result from either slow cycling or asymmetric cell division (ACD). However, the stem cell nature and whether LRC undergo ACD remain controversial. Here, we demonstrate label-retaining cancer cells (LRCCs) in several gastrointestinal (GI) cancers including fresh surgical specimens. Using a novel method for isolation of live LRCC, we demonstrate that a subpopulation of LRCC is actively dividing and exhibits stem cells and pluripotency gene expression profiles. Using real-time confocal microscopic cinematography, we show live LRCC undergoing asymmetric nonrandom chromosomal cosegregation LRC division. Importantly, LRCCs have greater tumor-initiating capacity than non-LRCCs. Based on our data and that cancers develop in tissues that harbor normal-LRC, we propose that LRCC might represent a novel population of GI stem-like cancer cells. LRCC may provide novel mechanistic insights into the biology of cancer and regenerative medicine and present novel targets for cancer treatment.

  11. Tumor-Initiating Label-Retaining Cancer Cells in Human Gastrointestinal Cancers Undergo Asymmetric Cell Division

    PubMed Central

    Xin, Hong-Wu; Hari, Danielle M.; Mullinax, John E.; Ambe, Chenwi M.; Koizumi, Tomotake; Ray, Satyajit; Anderson, Andrew J.; Wiegand, Gordon W.; Garfield, Susan H.; Thorgeirsson, Snorri S.; Avital, Itzhak

    2012-01-01

    Label-retaining cells (LRCs) have been proposed to represent adult tissue stem cells. LRCs are hypothesized to result from either slow cycling or asymmetric cell division (ACD). However, the stem cell nature and whether LRC undergo ACD remain controversial. Here, we demonstrate label-retaining cancer cells (LRCCs) in several gastrointestinal (GI) cancers including fresh surgical specimens. Using a novel method for isolation of live LRCC, we demonstrate that a subpopulation of LRCC is actively dividing and exhibits stem cells and pluripotency gene expression profiles. Using real-time confocal microscopic cinematography, we show live LRCC undergoing asymmetric nonrandom chromosomal cosegregation LRC division. Importantly, LRCCs have greater tumor-initiating capacity than non-LRCCs. Based on our data and that cancers develop in tissues that harbor normal-LRC, we propose that LRCC might represent a novel population of GI stem-like cancer cells. LRCC may provide novel mechanistic insights into the biology of cancer and regenerative medicine and present novel targets for cancer treatment. PMID:22331764

  12. Two genetically identical antigen-presenting cell clones display heterogeneity in antigen processing.

    PubMed Central

    Michalek, M T; Benacerraf, B; Rock, K L

    1989-01-01

    Evidence from various antigen systems suggests that antigen processing can be one factor that determines the repertoire of immunogenic peptides. Thus, processing events may account for some of the disparity between the available and expressed helper T-cell repertoires. In this report, we demonstrate that the immunodominant T-cell determinant in ovalbumin [p323-339; ovalbumin-(323-339) heptadecapeptide] is processed differently by two genetically identical antigen-presenting cell lines, M12 and A20. The ovalbumin-specific T-cell-T-cell hybridomas, DO-11.10 and 3DO-54.8, were used to detect processed antigen. These T-T hybridomas have different fine specificities for the p323-339 determinant. A20 cells presented native ovalbumin well to both T-T hybridomas, whereas M12 cells presented native ovalbumin well to 3DO-54.8 but very inefficiently to DO-11.10. M12 and A20 cells effectively stimulated both T-T hybridomas with the same concentrations of the immunogenic synthetic peptide p323-339. Therefore, M12 cells and DO-11.10 can interact with each other, and both T-T hybridomas have similar sensitivities for the same immunogenic peptide. We conclude that genetically identical antigen-presenting cells can display heterogeneity in the fine processing of an immunodominant T-cell determinant, and synthetic model peptides that represent the minimal stimulatory sequence of a T-cell determinant are not necessarily identical to the structure of in vivo processed antigen. Heterogeneity in antigen processing by individual antigen-presenting cells would serve to increase the repertoire of immunogenic peptides that are presented to T cells. PMID:2470101

  13. CD169+ MACROPHAGES PRESENT LIPID ANTIGENS TO MEDIATE EARLY ACTIVATION OF INVARIANT NKT CELLS IN LYMPH NODES

    PubMed Central

    Barral, Patricia; Polzella, Paolo; Bruckbauer, Andreas; van Rooijen, Nico; Besra, Gurdyal S.; Cerundolo, Vincenzo; Batista, Facundo D.

    2010-01-01

    Invariant NKT (iNKT) cells are involved in host defence against microbial infections. While it is known that iNKT cells recognize glycolipids presented by CD1d, how and where they encounter antigen in vivo remains unclear. We used multi-photon microscopy to visualize the dynamics and activation of iNKT cells in lymph nodes. Following antigen administration, iNKT cells become confined in a CD1d-dependent manner in close proximity to subcapsular sinus CD169+ macrophages. These macrophages retain, internalize and present lipid antigen, and are required for iNKT cell activation, cytokine production and expansion. Thus, CD169+ macrophages can act as bona fide antigen presenting cells controlling early iNKT cell activation and favouring fast initiation of immune responses. PMID:20228797

  14. Mouse Label-Retaining Cells Are Molecularly And Functionally Distinct From Reserve Intestinal Stem Cells

    PubMed Central

    Li, Ning; Nakauka-Ddamba, Angela; Tobias, John; Jensen, Shane T.; Lengner, Christopher J.

    2016-01-01

    BACKGROUND & AIMS Intestinal homeostasis and regeneration after injury is controlled by 2 different types of cells–slow cycling, injury-resistant reserve intestinal stem cells (ISC) and actively proliferative ISC. Putative reserve ISCs have been identified using a variety of methods, including CreER insertions at Hopx or Bmi1 loci in mice and DNA label retention. Label-retaining cells (LRCs) include dormant stem cells in several tissues; in the intestine, LRCs appear to share some properties with reserve ISCs, which can be marked by reporter alleles. We investigated the relationships between these populations. METHODS Studies were performed in Lgr5–EGFP-IRES-creERT2, Bmi1-CreERT2, Hopx-CreERT2, and TRE-H2BGFP::Hopx-CreERT2::lox-stop-lox-tdTomato mice. Intestinal epithelial cell populations were purified; we compared reporter allele-marked reserve ISCs and several LRC populations (marked by H2B-GFP retention) using histologic, flow cytometry and functional and single-cell gene expression assays. RESULTS LRCs were dynamic and their cellular composition changed with time. Short-term LRCs had properties of secretory progenitor cells undergoing commitment to the Paneth or enteroendocrine lineages while retaining some stem cell activity. Long-term LRCs lost stem cell activity and were a homogenous population of terminally differentiated Paneth cells. Reserve ISCs marked with HopxCreER were primarily quiescent (in G0), with inactive Wnt signaling and robust stem cell activity. In contrast, most LRCs were in G1 arrest and expressed genes that are regulated by the Wnt pathway or are in the secretory lineage. Conclusions LRCs are molecularly and functionally distinct from reporter-marked reserve intestinal stem cells. This information provides an important basis for future studies of relationships among intestinal stem cell populations. PMID:27237597

  15. Langerhans cells and dermal dendritic cells capture protein antigens in the skin: Possible targets for vaccination through the skin

    PubMed Central

    Sparber, Florian; Tripp, Christoph H.; Hermann, Martin; Romani, Nikolaus; Stoitzner, Patrizia

    2010-01-01

    Dendritic cells capture and process antigen and present it to T lymphocytes in the lymphoid organs. Dendritic cells of the skin, including epidermal Langerhans cells, langerin+ and langerinnegative dermal dendritic cells are ideally positioned to take up pathogens that enter the body through the skin or vaccines that are administered into (intradermal) or onto (epicutaneous) the skin. The antigen uptake properties of skin dendritic cells have not thoroughly been studied yet. We therefore investigated the uptake of the fluorochrome-conjugated model antigen ovalbumin (OVA) by skin dendritic cells in the mouse. OVA was readily taken up by immature Langerhans cells both in situ and in cell suspensions. When offered to Langerhans cells in situ either by “bathing” skin explants in OVA-containing culture medium or by intradermal injection they retained the captured OVA for at least 2–3 days when migrating into the culture medium and, importantly, into the draining lymph nodes. Also langerin+ and – to a larger extent – langerinnegative skin dendritic cells took up and transported OVA to the lymph nodes. Interestingly, mature Langerhans cells were still capable of ingesting substantial amounts of OVA, indicating that predominantly receptor-mediated endocytosis is operative in these cells. Unlike macropinocytosis, this pathway of endocytosis is not shut down upon dendritic cell maturation. These observations indicate that in intradermal vaccination schemes, Langerhans cells from the epidermis are prominently involved. They were recently shown to possess the capacity to induce functional cytotoxic T lymphocytes. Furthermore, the potential to markedly enhance antigen uptake and processing by targeting antigen to c-type lectin receptors on Langerhans cells was also recently demonstrated. Our data provide a rationale and an incentive to explore in more detail antigen targeting to Langerhans cells with the aim of harnessing it for immunotherapy. PMID:20599290

  16. Modulation of Ia and photoreactive antigen on antigen-presenting cells: fun with a photoprobe.

    PubMed

    Thomas, D W; Eades, L; Wilson, C; Solvay, M J

    1985-12-01

    To identify the antigen-specific recognition complex containing elements from T cells and antigen-presenting cells (APC), a photoactivatable antigen system was developed which could potentially crosslink the complex during the specific cellular responses. In this paper we describe the development of this system using murine T-cell hybridomas responding to stimulator cells chemically conjugated with N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) and genetically restricted by I-Ad. In initial experiments it was found that several I-Ad-positive B-cell lines were nonstimulatory when coupled with HSAB, but that I-Ad-positive P388D1 macrophage-like cells were efficient stimulators of HSAB-specific T-cell responses. These results suggested that the relevant HSAB coupled surface structure was not likely I-Ad. To substantiate this point, Ia-positive or Ia-negative P388D1 cells were initially coupled with HSAB and the expression of Ia was modulated by the addition and withdrawal of Con A-stimulated spleen cell supernatant fluid through several days of culture. Under these conditions, efficient stimulation was only observed when Ia was expressed, although the HSAB antigen was continuously present. In other experiments it was found that exposure of HSAB-coupled APC to light selectively eliminated their stimulatory capacity for HSAB-specific T hybridomas, suggesting that the light-induced crosslinking by HSAB directly eliminates the antigenic determinant. This antigen system allows a unique opportunity to manipulate the antigen during specific cellular interactions, and to introduce covalent crosslinking of the specific antigen recognition complex that may allow its isolation and characterization.

  17. Detection of Avian Antigen-Specific T Cells Induced by Viral Vaccines.

    PubMed

    Dalgaard, Tina Sørensen; Norup, Liselotte Rothmann; Juul-Madsen, Helle Risdahl

    2016-01-01

    Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen. There is a particular interest in developing robust high-throughput assays as chicken vaccine trials usually comprise many individuals. In many respects, the avian immune system differs from the mammalian, and T cell assessment protocols must be adjusted accordingly to account for, e.g., differences in leukocyte subsets.The carboxyfluorescein succinimidyl ester (CFSE) method described in this chapter has been adapted to chicken cells. In this test, cells of interest are stained with CFSE. The succinimidyl ester group covalently binds to cellular amines forming fluorescent conjugates that are retained in the cells even throughout division. This leads to daughter cells containing half the fluorescence of their parents. When lymphocytes are loaded with CFSE prior to ex vivo stimulation with specific antigen, the measurement of serial halving of its fluorescence by flow cytometry identifies the cells responding to the stimulation. This method has been successfully applied to studies of chicken antigen-specific T cells.

  18. Mouse Ovarian Very Small Embryonic-Like Stem Cells Resist Chemotherapy and Retain Ability to Initiate Oocyte-Specific Differentiation

    PubMed Central

    Sriraman, Kalpana; Anand, Sandhya; Bhutda, Smita

    2015-01-01

    This study was undertaken to investigate stem cells in adult mouse ovary, the effect of chemotherapy on them and their potential to differentiate into germ cells. Very small embryonic-like stem cells (VSELs) that were SCA-1+/Lin−/CD45−, positive for nuclear octamer-binding transforming factor 4 (OCT-4), Nanog, and cell surface stage-specific embryonic antigen 1, were identified in adult mouse ovary. Chemotherapy resulted in complete loss of follicular reserve and cytoplasmic OCT-4 positive progenitors (ovarian germ stem cells) but VSELs survived. In ovarian surface epithelial (OSE) cell cultures from chemoablated ovary, proliferating germ cell clusters and mouse vasa homolog/growth differentiation factor 9-positive oocyte-like structure were observed by day 6, probably arising as a result of differentiation of the surviving VSELs. Follicle-stimulating hormone (FSH) exerted a direct stimulatory action on the OSE and induced stem cells proliferation and differentiation into premeiotic germ cell clusters during intact chemoablated ovaries culture. The FSH analog pregnant mare serum gonadotropin treatment to chemoablated mice increased the percentage of surviving VSELs in ovary. The results of this study provide evidence for the presence of potential VSELs in mouse ovaries and show that they survive chemotherapy, are modulated by FSH, and retain the ability to undergo oocyte-specific differentiation. These results show relevance to women who undergo premature ovarian failure because of oncotherapy. PMID:25779995

  19. Mesenchymal Stem Cells Retain Their Defining Stem Cell Characteristics After Exposure to Ionizing Radiation

    SciTech Connect

    Nicolay, Nils H.; Sommer, Eva; Lopez, Ramon; Wirkner, Ute; Trinh, Thuy; Sisombath, Sonevisay; Debus, Jürgen; Ho, Anthony D.; Saffrich, Rainer; Huber, Peter E.

    2013-12-01

    Purpose: Mesenchymal stem cells (MSCs) have the ability to migrate to lesion sites and undergo differentiation into functional tissues. Although this function may be important for tissue regeneration after radiation therapy, the influence of ionizing radiation (IR) on cellular survival and the functional aspects of differentiation and stem cell characteristics of MSCs have remained largely unknown. Methods and Materials: Radiation sensitivity of human primary MSCs from healthy volunteers and primary human fibroblast cells was examined, and cellular morphology, cell cycle effects, apoptosis, and differentiation potential after exposure to IR were assessed. Stem cell gene expression patterns after exposure to IR were studied using gene arrays. Results: MSCs were not more radiosensitive than human primary fibroblasts, whereas there were considerable differences regarding radiation sensitivity within individual MSCs. Cellular morphology, cytoskeletal architecture, and cell motility were not markedly altered by IR. Even after high radiation doses up to 10 Gy, MSCs maintained their differentiation potential. Compared to primary fibroblast cells, MSCs did not show an increase in irradiation-induced apoptosis. Gene expression analyses revealed an upregulation of various genes involved in DNA damage response and DNA repair, but expression of established MSC surface markers appeared only marginally influenced by IR. Conclusions: These data suggest that human MSCs are not more radiosensitive than differentiated primary fibroblasts. In addition, upon photon irradiation, MSCs were able to retain their defining stem cell characteristics both on a functional level and regarding stem cell marker expression.

  20. Extraction of Cell-Wall Polysaccharide Antigen from Streptococci

    PubMed Central

    Slade, Hutton D.

    1965-01-01

    Slade, Hutton D. (Northwestern University Medical School, Chicago, Ill., and Max-Planck Institut für Immunbiologie, Freiburg, Germany). Extraction of cell-wall polysaccharide antigen from streptococci. J. Bacteriol. 90:667–672. 1965.—The carbohydrate grouping antigens in the cell walls of streptococci belonging to groups A, E, G, L, and T were extracted with 5% trichloroacetic acid at 90 C. The antigens were removed also from dry whole cells by extraction with trichloroacetic acid followed by treatment with phenol-water. Details of the methods are presented. The antigens obtained by use of either of these procedures were suitable for studies on immunological specificity and chemical structure. Quantitative enzymatic and chemical analyses of two group E antigens and one group T preparation showed the presence of l-rhamnose (22 to 44%), d-glucose (7 to 22%), d-galactose (T antigen only, 26%), glucosamine (2 to 16%), and galactosamine (T antigen only, 3%). In addition, analyses of A and G antigen preparations are presented. The protein and phosphate content of the A and E antigens were about 1% each. Quantitative precipitin curves of these antigens are presented. PMID:16562065

  1. Rubber and alumina gaskets retain vacuum seal in high temperature EMF cell

    NASA Technical Reports Server (NTRS)

    Hesson, J. C.

    1966-01-01

    Silicone rubber gasket and an alumina gasket retain a vacuum inside a high temperature EMF cell in which higher and lower density liquid metal electrodes are separated by an intermediate density fused salt electrolyte. This innovation is in use on a sodium bismuth regenerable EMF cell in which the fused salts and metals are at about 500 deg to 600 deg C.

  2. Chimeric Antigen Receptor T-cell Therapies for Multiple Myeloma.

    PubMed

    Mikkilineni, Lekha; Kochenderfer, James N

    2017-09-19

    Multiple myeloma (MM) is a nearly always incurable malignancy of plasma cells, so new approaches to treatment are needed. T-cell therapies are a promising approach for treating MM, with a mechanism of action different than those of standard MM treatments. Chimeric antigen receptors (CARs) are fusion proteins incorporating antigen-recognition domains and T-cell signaling domains. T-cells genetically engineered to express CARs can specifically recognize antigens. Success of CAR T-cells against leukemia and lymphoma has encouraged development of CAR T-cell therapies for MM. Target antigens for CARs must be expressed on malignant cells, but expression on normal cells must be absent or limited. B-cell maturation antigen (BCMA) is expressed by normal and malignant plasma cells. CAR T-cells targeting B-cell maturation antigen have demonstrated significant anti-myeloma activity in early clinical trials. Toxicities in these trials, including cytokine-release syndrome, have been similarto toxicities observed in CAR T-cell trials for leukemia. Targeting postulated CD19(+) myeloma stem cells with anti-CD19 CAR T-cells is a novel approach to MM therapy. MM antigens including CD138, CD38, signaling lymphocyte-activating molecule 7 (SLAMF7), and kappa light chain are under investigation as CAR targets. MM is genetically and phenotypically heterogeneous, so targeting of more than one antigen might often be required for effective treatment of MM with CAR T cells. Integration of CAR T cells with other myeloma therapies is an important area of future research. CAR T cell therapies for MM are at an early stage of development but have great promise to improve MM treatment. Copyright © 2017 American Society of Hematology.

  3. Tumorigenic activity of Merkel cell polyomavirus T antigens expressed in the stratified epithelium of mice

    PubMed Central

    Spurgeon, Megan E.; Cheng, Jingwei; Bronson, Roderick T.; Lambert, Paul F.; DeCaprio, James A.

    2015-01-01

    Merkel cell polyomavirus (MCPyV) is frequently associated with Merkel cell carcinoma (MCC), a highly aggressive neuroendocrine skin cancer. Most MCC tumors contain integrated copies of the viral genome with persistent expression of the MCPyV large T (LT) and small T (ST) antigen. MCPyV isolated from MCC typically contain wild type ST but truncated forms of LT that retain the N-terminus but delete the C-terminus and render LT incapable of supporting virus replication. To determine the oncogenic activity of MCC tumor-derived T antigens in vivo, a conditional, tissue-specific mouse model was developed. Keratin 14-mediated Cre recombinase expression induced expression of MCPyV T antigens in stratified squamous epithelial cells and Merkel cells of the skin epidermis. Mice expressing MCPyV T antigens developed hyperplasia, hyperkeratosis, and acanthosis of the skin with additional abnormalities in whisker pads, footpads and eyes. Nearly half of the mice also developed cutaneous papillomas. Evidence for neoplastic progression within stratified epithelia included increased cellular proliferation, unscheduled DNA synthesis, increased E2F-responsive genes levels, disrupted differentiation, and presence of a DNA damage response. These results indicate that MCPyV T antigens are tumorigenic in vivo, consistent with their suspected etiological role in human cancer. PMID:25596282

  4. Hematopoietic stem cell recipients do not develop post-transplantation immune tolerance to antigens present on minimal residual disease.

    PubMed

    Natzke, Amanda Martinelli; Shaw, Joanne L; McKeller, Morgan R; Emo, Kris Lambert; Mullen, Craig A

    2007-01-01

    The immune environment present after allogeneic hematopoietic stem cell transplantation (HSCT) contributes to the control of leukemia. Our laboratory has demonstrated in a murine model that vaccination of recipients after transplantation with recipient tumor vaccines does not exacerbate graft-versus-host disease but does induce meaningful graft-versus-tumor effects. We previously demonstrated that part of the reason for the lack of graft-versus-host disease from post-transplantation vaccination is due to gradual acquisition of tolerance or unresponsiveness to recipient immunodominant minor histocompatibility antigens that are ubiquitously expressed in the recipient. However, our prior studies have not critically addressed the question of whether a similar process of acquisition of unresponsiveness to or tolerance of antigens present on minimal residual disease also occurs. The present study tested the hypothesis that unresponsiveness to antigens present on minimal residual disease present at the time of HSCT would also occur. The answer to this question would have a significant effect on the potential efficacy of post-transplantation tumor vaccines. In a murine model of major histocompatibility complex matched, minor histocompatibility antigen mismatched HSCT (C3.SW female donors and C57BL/6 female recipients), we tested whether transplant recipients would acquire unresponsiveness to antigens present on small numbers of residual leukemia/lymphoma cells. We employed a male C57BL/6 lymphoid malignancy with an immunoglobulin/c-myc oncogene in these studies using as a model of tumor-restricted antigen the well-characterized male (HY) antigen system present only on the tumor but not present as ubiquitous minor antigens in the recipient. After HSCT, recipients did not mount immune responses to the ubiquitously distributed immunodominant recipient strain H7 minor histocompatibility antigen, but did retain the capacity to mount significant T cell responses to HY antigens

  5. Ceramide Inhibits Antigen Uptake and Presentation by Dendritic Cells

    PubMed Central

    Sallusto, Federica; Nicolò, Chiara; De Maria, Ruggero; Corinti, Silvia; Testi, Roberto

    1996-01-01

    Ceramides are intramembrane diffusible mediators involved in transducing signals originated from a variety of cell surface receptors. Different adaptive and differentiative cellular responses, including apoptotic cell death, use ceramide-mediated pathways as an essential part of the program. Here, we show that human dendritic cells respond to CD40 ligand, as well as to tumor necrosis factor-α and IL-1β, with intracellular ceramide accumulation, as they are induced to differentiate. Dendritic cells down-modulate their capacity to take up soluble antigens in response to exogenously added or endogenously produced ceramides. This is followed by an impairment in presenting soluble antigens to specific T cell clones, while cell viability and the capacity to stimulate allogeneic responses or to present immunogenic peptides is fully preserved. Thus, ceramide-mediated pathways initiated by different cytokines can actively modulate professional antigen-presenting cell function and antigen-specific immune responses. PMID:8976196

  6. Cell-free antigens of Sporothrix brasiliensis: antigenic diversity and application in an immunoblot assay.

    PubMed

    Almeida-Paes, Rodrigo; Bailão, Alexandre Melo; Pizzini, Cláudia Vera; Reis, Rosani Santos; Soares, Célia Maria de Almeida; Peralta, José Mauro; Gutierrez-Galhardo, Maria Clara; Zancopé-Oliveira, Rosely Maria

    2012-11-01

    Sporotrichosis is a subcutaneous mycosis diagnosed by isolation of the fungus in culture. Serological tests for help in diagnosis in general do not use purified or recombinant antigens, because there is a paucity of described immunoreactive proteins, especially for the new described Sporothrix species, such as Sporothrix brasiliensis. This study aims to characterise antigens from S. brasiliensis and verify their application in serodiagnosis of sporotrichosis. An immunoblot assay allied with computer-based analysis was used to identify putative antigenic molecules in a cell-free extracts of both morphological phases of this fungus, and to delineate antigenic polymorphism among seven S. brasiliensis isolates and one S. schenckii Brazilian strain. The mycelial and yeast phase of the fungus originated 14 and 23 reactive bands, respectively, which were variable in intensity. An 85 kDa antigen, verified in the yeast phase of the fungus, was observed in all strains used and the immunodominant protein was identified. This protein, however, cross-react with serum samples from patients infected with other pathogens. The results show that the S. brasiliensis cell-free antigen extract is a single and inexpensive source of antigens, and can be applied on the sporotrichosis serodiagnosis.

  7. Antigen Presenting Properties of a Myeloid Dendritic-Like Cell in Murine Spleen

    PubMed Central

    Hey, Ying-ying; O’Neill, Helen C.

    2016-01-01

    This paper distinguishes a rare subset of myeloid dendritic-like cells found in mouse spleen from conventional (c) dendritic cells (DC) in terms of phenotype, function and gene expression. These cells are tentatively named “L-DC” since they resemble dendritic-like cells produced in longterm cultures of spleen. L-DC can be distinguished on the basis of their unique phenotype as CD11bhiCD11cloMHCII-CD43+Ly6C-Ly6G-Siglec-F- cells. They demonstrate similar ability as cDC to uptake and retain complex antigens like mannan via mannose receptors, but much lower ability to endocytose and retain soluble antigen. While L-DC differ from cDC by their inability to activate CD4+ T cells, they are capable of antigen cross-presentation for activation of CD8+ T cells, although less effectively so than the cDC subsets. In terms of gene expression, CD8- cDC and CD8+ cDC are quite distinct from L-DC. CD8+ cDC are distinguishable from the other two subsets by expression of CD24a, Clec9a, Xcr1 and Tlr11, while CD8- cDC are distinguished by expression of Ccnd1 and H-2Eb2. L-DC are distinct from the two cDC subsets through upregulated expression of Clec4a3, Emr4, Itgam, Csf1r and CD300ld. The L-DC gene profile is quite distinct from that of cDC, confirming a myeloid cell type with distinct antigen presenting properties. PMID:27654936

  8. Antigen Presenting Properties of a Myeloid Dendritic-Like Cell in Murine Spleen.

    PubMed

    Hey, Ying-Ying; O'Neill, Helen C

    This paper distinguishes a rare subset of myeloid dendritic-like cells found in mouse spleen from conventional (c) dendritic cells (DC) in terms of phenotype, function and gene expression. These cells are tentatively named "L-DC" since they resemble dendritic-like cells produced in longterm cultures of spleen. L-DC can be distinguished on the basis of their unique phenotype as CD11bhiCD11cloMHCII-CD43+Ly6C-Ly6G-Siglec-F- cells. They demonstrate similar ability as cDC to uptake and retain complex antigens like mannan via mannose receptors, but much lower ability to endocytose and retain soluble antigen. While L-DC differ from cDC by their inability to activate CD4+ T cells, they are capable of antigen cross-presentation for activation of CD8+ T cells, although less effectively so than the cDC subsets. In terms of gene expression, CD8- cDC and CD8+ cDC are quite distinct from L-DC. CD8+ cDC are distinguishable from the other two subsets by expression of CD24a, Clec9a, Xcr1 and Tlr11, while CD8- cDC are distinguished by expression of Ccnd1 and H-2Eb2. L-DC are distinct from the two cDC subsets through upregulated expression of Clec4a3, Emr4, Itgam, Csf1r and CD300ld. The L-DC gene profile is quite distinct from that of cDC, confirming a myeloid cell type with distinct antigen presenting properties.

  9. Cancer testis antigen expression in testicular germ cell tumorigenesis.

    PubMed

    Bode, Peter K; Thielken, Andrea; Brandt, Simone; Barghorn, André; Lohe, Bernd; Knuth, Alexander; Moch, Holger

    2014-06-01

    Cancer testis antigens are encoded by germ line-associated genes that are present in normal germ cells of testis and ovary but not in differentiated tissues. Their expression in various human cancer types has been interpreted as 're-expression' or as intratumoral progenitor cell signature. Cancer testis antigen expression patterns have not yet been studied in germ cell tumorigenesis with specific emphasis on intratubular germ cell neoplasia unclassified as a precursor lesion for testicular germ cell tumors. Immunohistochemistry was used to study MAGEA3, MAGEA4, MAGEC1, GAGE1 and CTAG1B expression in 325 primary testicular germ cell tumors, including 94 mixed germ cell tumors. Seminomatous and non-seminomatous components were separately arranged and evaluated on tissue microarrays. Spermatogonia in the normal testis were positive, whereas intratubular germ cell neoplasia unclassified was negative for all five CT antigens. Cancer testis antigen expression was only found in 3% (CTAG1B), 10% (GAGE1, MAGEA4), 33% (MAGEA3) and 40% (MAGEC1) of classic seminoma but not in non-seminomatous testicular germ cell tumors. In contrast, all spermatocytic seminomas were positive for cancer testis antigens. These data are consistent with a different cell origin in spermatocytic seminoma compared with classic seminoma and support a progression model with loss of cancer testis antigens in early tumorigenesis of testicular germ cell tumors and later re-expression in a subset of seminomas.

  10. In vitro long-term treatment with MAPK inhibitors induces melanoma cells with resistance plasticity to inhibitors while retaining sensitivity to CD8 T cells

    PubMed Central

    Rowdo, Florencia Paula Madorsky; Barón, Antonela; Von Euw, Erika María; Mordoh, José

    2017-01-01

    The development of BRAF V600 and MEK inhibitors constitutes a breakthrough in the treatment of patients with BRAF-mutated metastatic melanoma. However, although there is an increase in overall survival, these patients generally confront recurrence, and several resistance mechanisms have already been described. In the present study we describe a different resistance mechanism. After several weeks of long-term in vitro treatment of two different V600E BRAF-mutated melanoma cell lines with MARK inhibitors, PLX4032 and/or GDC-0973, the majority of the cells died whereas some remained viable and quiescent (SUR). Markedly, discontinuing treatment of SUR cells with MAPK inhibitors allowed the population to regrow and these cells retained drug sensitivity equal to that of the parental cells. SUR cells had increased expression levels of CD271 and ABCB5 and presented senescence-associated characteristics. Notably, SUR cells were efficiently lysed by cytotoxic T lymphocytes recognizing MART-1 and gp100 melanoma differentiation antigens. We propose quiescent plasticity as a mechanism of resistance to BRAF and MEK inhibitors while retaining sensitivity to immune effectors. PMID:28098866

  11. In vitro long-term treatment with MAPK inhibitors induces melanoma cells with resistance plasticity to inhibitors while retaining sensitivity to CD8 T cells.

    PubMed

    Madorsky Rowdo, Florencia Paula; Barón, Antonela; von Euw, Erika María; Mordoh, José

    2017-03-01

    The development of BRAF V600 and MEK inhibitors constitutes a breakthrough in the treatment of patients with BRAF-mutated metastatic melanoma. However, although there is an increase in overall survival, these patients generally confront recurrence, and several resistance mechanisms have already been described. In the present study we describe a different resistance mechanism. After several weeks of long‑term in vitro treatment of two different V600E BRAF‑mutated melanoma cell lines with MARK inhibitors, PLX4032 and/or GDC-0973, the majority of the cells died whereas some remained viable and quiescent (SUR). Markedly, discontinuing treatment of SUR cells with MAPK inhibitors allowed the population to regrow and these cells retained drug sensitivity equal to that of the parental cells. SUR cells had increased expression levels of CD271 and ABCB5 and presented senescence-associated characteristics. Notably, SUR cells were efficiently lysed by cytotoxic T lymphocytes recognizing MART-1 and gp100 melanoma differentiation antigens. We propose quiescent plasticity as a mechanism of resistance to BRAF and MEK inhibitors while retaining sensitivity to immune effectors.

  12. Antigen specificity of invariant natural killer T-cells.

    PubMed

    Birkholz, Alysia M; Kronenberg, Mitchell

    2015-12-01

    Natural killer T-cells, with an invariant T-cell antigen receptor α-chain (iNKT cells), are unique and conserved subset of lymphocytes capable of altering the immune system through their rapid and potent cytokine responses. They are reactive to lipid antigens presented by the CD1d molecule, an antigen-presenting molecule that is not highly polymorphic. iNKT cell responses frequently involve mixtures of cytokines that work against each other, and therefore attempts are underway to develop synthetic antigens that elicit only strong interferon-gamma (IFNγ) or only strong interleukin-4 responses but not both. Strong IFNγ responses may correlate with tighter binding to CD1d and prolonged stimulation of iNKT cells, and this may be useful for vaccine adjuvants and for stimulating anti-tumor responses. iNKT cells are self-reactive although the structure of the endogenous antigen is controversial. By contrast, bacterial and fungal lipids that engage the T-cell receptor and activate IFNγ from iNKT cells have been identified from both pathogenic and commensal organisms and the responses are in some cases highly protective from pathogens in mice. It is possible that the expanding knowledge of iNKT cell antigens and iNKT cell activation will provide the basis for therapies for patients suffering from infectious and immune diseases and cancer.

  13. The actin cytoskeleton coordinates the signal transduction and antigen processing functions of the B cell antigen receptor

    PubMed Central

    LIU, Chaohong; FALLEN, Margaret K.; MILLER, Heather; UPADHYAYA, Arpita; SONG, Wenxia

    2014-01-01

    The B cell antigen receptor (BCR) is the sensor on the B cell surface that surveys foreign molecules (antigen) in our bodies and activates B cells to generate antibody responses upon encountering cognate antigen. The binding of antigen to the BCR induces signaling cascades in the cytoplasm, which provides the first signal for B cell activation. Subsequently, BCRs internalize and target bound antigen to endosomes, where antigen is processed into T cell recognizable forms. T helper cells generate the second activation signal upon binding to antigen presented by B cells. The optimal activation of B cells requires both signals, thereby depending on the coordination of BCR signaling and antigen transport functions. Antigen binding to the BCR also induces rapid remodeling of the cortical actin network of B cells. While being initiated and controlled by BCR signaling, recent studies reveal that this actin remodeling is critical for both the signaling and antigen processing functions of the BCR, indicating a role for actin in coordinating these two pathways. Here we will review previous and recent studies on actin reorganization during BCR activation and BCR-mediated antigen processing, and discuss how actin remodeling translates BCR signaling into rapid antigen uptake and processing while providing positive and negative feedback to BCR signaling. PMID:24999354

  14. A label-retaining but unipotent cell population resides in biliary compartment of mammalian liver

    PubMed Central

    Viil, Janeli; Klaas, Mariliis; Valter, Kadri; Belitškin, Denis; Ilmjärv, Sten; Jaks, Viljar

    2017-01-01

    Cells with slow proliferation kinetics that retain the nuclear label over long time periods–the label-retaining cells (LRCs)–represent multipotent stem cells in a number of adult tissues. Since the identity of liver LRCs (LLRCs) had remained elusive we utilized a genetic approach to reveal LLRCs in normal non-injured livers and characterized their regenerative properties in vivo and in culture. We found that LLRCs were located in biliary vessels and participated in the regeneration of biliary but not hepatocyte injury. In culture experiments the sorted LLRCs displayed an enhanced self-renewal capacity but a unipotent biliary differentiation potential. Transcriptome analysis revealed a unique set of tumorigenesis- and nervous system-related genes upregulated in LLRCs when compared to non-LRC cholangiocytes. We conclude that the LLRCs established during the normal morphogenesis of the liver do not represent a multipotent primitive somatic stem cell population but act as unipotent biliary progenitor cells. PMID:28084309

  15. Siglecs induce tolerance to cell surface antigens by BIM-dependent deletion of the antigen-reactive B cells

    PubMed Central

    Macauley, Matthew S.; Paulson, James C.

    2014-01-01

    Infusion of blood cells from a donor can induce humoral tolerance in a recipient and increase the probability of successful organ transplant; a clinical method defined as donor-specific transfusion (DST). Despite the clinical success of DST, the immunological mechanism(s) by which blood cells displaying a foreign antigen induce tolerance remain poorly understood. Based on recent findings showing that the B cell siglecs, CD22 and Siglec-G, can promote tolerance to antigens presented on the same surface as their ligands, we speculated that the B cell siglecs are key players in tolerance induced by DST. Using a variety of chemical and genetic approaches, we show that the B cell siglecs mediate tolerance to cell surface antigens by initiating an inhibitory signal that culminates in elimination of the antigen-reactive B cell. CD22 and Siglec-G are recruited to the immunological synapse by sialic acid ligands on the antigen-bearing cells, producing a tolerogenic signal involving Lyn and the pro-apoptotic factor BIM that promotes deletion of the B cell and failure of mice to develop antibodies to the antigen upon subsequent challenge. We speculate that this tolerogenic mechanism is a contributing factor in DST and a mechanism of peripheral B cell tolerance to cell surface autoantigens. PMID:25252961

  16. Cell-mediated immunity to soluble and particulate inhaled antigens

    PubMed Central

    Hill, J. O.; Burrell, R.

    1979-01-01

    In order to determine the influence of an antigen's physical properties on the development of cell-mediated immunity (CMI) in the lung following aerosol immunization, human serum albumin (HSA) was prepared in either a soluble or a particulate form, the latter being coupled to respirable, carboxylated latex beads. Antigen was administered via an aerosol to groups of guinea-pigs, twice weekly for up to 4 weeks. Additional groups of animals served as unexposed and unconjugated latex controls. Lymphoid cells for CMI assays were isolated from the lung by bronchopulmonary lavage and from blood for use in mitogen- and antigen-induced lymphocyte transformation assays, as well as indirect macrophage migration inhibition tests. Particulate HSA-exposed animals yielded the highest numbers of free lung cells containing predominantly macrophages, with up to 33% lymphocytes. These were followed by the latex control, soluble HSA and unexposed control groups, respectively. Only the animals exposed to particulate HSA had evidence of antigen reactivation in the lung cell populations as measured by lymphocyte stimulation assays. In contrast, a response to polyclonal mitogens was found only in animals exposed to antigen in a soluble form. Data from macrophage depletion experiments suggest that the antigenicity of inhaled antigens may be due to the types and numbers of cells responding to the stimulus, and the subsequent role the alveolar macrophage may play in the modulation of cellular immunity. PMID:393444

  17. Carbohydrate-Mediated Targeting of Antigen to Dendritic Cells Leads to Enhanced Presentation of Antigen to T Cells

    PubMed Central

    Adams, Eddie W.; Ratner, Daniel M.; Seeberger, Peter H.; Hacohen, Nir

    2009-01-01

    The unique therapeutic value of dendritic cells (DCs) for the treatment of allergy, autoimmunity and transplant rejection is predicated upon our ability to selectively deliver antigens, drugs or nucleic acids to DCs in vivo. Here we describe a method for delivering whole protein antigens to DCs based on carbohydrate-mediated targeting of DC-expressed lectins. A series of synthetic carbohydrates was chemically-coupled to a model antigen, ovalbumin (OVA), and each conjugate was evaluated for its ability to increase the efficiency of antigen presentation by murine DCs to OVA-specific T cells (CD4+ and CD8+). In vitro data are presented that demonstrate that carbohydrate modification of OVA leads to a 50-fold enhancement of presentation of antigenic peptide to CD4+ T cells. A tenfold enhancement is observed for CD8+ T cells; this indicates that the targeted lectin(s) can mediate cross-presentation of antigens on MHC class I. Our data indicate that the observed enhancements in antigen presentation are unique to OVA that is conjugated to complex oligosaccharides, such as a high-mannose nonasaccharide, but not to monosaccharides. Taken together, our data suggest that a DC targeting strategy that is based upon carbohydrate-lectin interactions is a promising approach for enhancing antigen presentation via class I and class II molecules. PMID:18186095

  18. From the antigen-presenting cell to the antigen-presenting vesicle: the exosomes.

    PubMed

    Schartz, Noël Emile Célestin; Chaput, Nathalie; André, Fabrice; Zitvogel, Laurence

    2002-08-01

    Exosomes are membrane vesicles of 30 to 100 nm in diameter, of endocytic origin, and are produced and secreted in vitro by living cells of diverse origin. In vivo and in vitro experiments suggest, from their particular proteomic composition, that exosomes are involved in the transfer of tumor antigens to antigen presenting cells, and in the stimulation of a specific immune response. In this review, we provide a molecular characterization of exosomes. The hypotheses accounting for exosome biogenesis will be outlined. Finally, we will describe their bioactivities and discuss their potential relevance and clinical implementation for cancer immunotherapy.

  19. 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

  20. Induction of antigen-specific T suppressor cells by soluble Paracoccidioides brasiliensis antigen.

    PubMed Central

    Jimenez-Finkel, B E; Murphy, J W

    1988-01-01

    In naturally acquired paracoccidioidomycosis, patients have depressed in vivo and in vitro cell-mediated immune (CMI) responses to Paracoccidioides brasiliensis antigen. In addition, it has been reported that these patients have significant levels of circulating paracoccidioidal antigen in their sera. The primary purpose of this investigation was to assess the effects of P. brasiliensis antigen on the CMI responses in a mouse model. On the basis of findings with other fungal agents, we predicted that circulating paracoccidioidal antigen may be inducing suppressor cells which modulate the CMI response. In this study, we show (i) that a soluble P. brasiliensis culture filtrate antigen (Pb.Ag) emulsified in complete Freund adjuvant and injected subcutaneously into mice induces reasonably high levels of delayed-type hypersensitivity (DTH) in CBA/J mice; (ii) that Pb.Ag elicits DTH reactions specific for P. brasiliensis when injected into footpads of immunized mice; and (iii) that an intravenous injection of Pb.Ag induces a population of lymph node and spleen cells which, upon adoptive transfer, suppress the afferent limb of the DTH response to paracoccidioidal antigen. The afferent suppressor cells can be detected in spleens as early as 5 days after Pb.Ag treatment, are present in significant numbers by 7 days in both spleens and lymph nodes, and are virtually absent by 14 days. In contrast, at 14 days after antigen injection, efferent suppressor cells were detected in spleens and lymph nodes. The Pb.Ag-induced afferent suppressor cells specifically inhibit the antiparacoccidioidal DTH response. They are nylon wool-nonadherent cells, and their activity is abrogated by anti-Thy-1 and complement treatment, indicating that they are T lymphocytes. The phenotype of these afferent suppressor T cells is L3T4+ Lyt-1+2- I-J+. The Pb.Ag-specific suppressor cells described in this paper are similar to the Ts1 cells in the azobenzenearsonate, 4-hydroxy-3-nitrophenyl acetyl, and

  1. Identification of long-lived proteins retained in cells undergoing repeated asymmetric divisions

    PubMed Central

    Thayer, Nathaniel H.; Leverich, Christina K.; Fitzgibbon, Matthew P.; Nelson, Zara W.; Henderson, Kiersten A.; Gafken, Philip R.; Hsu, Jessica J.; Gottschling, Daniel E.

    2014-01-01

    Long-lived proteins have been implicated in age-associated decline in metazoa, but they have only been identified in extracellular matrices or postmitotic cells. However, the aging process also occurs in dividing cells undergoing repeated asymmetric divisions. It was not clear whether long-lived proteins exist in asymmetrically dividing cells or whether they are involved in aging. Here we identify long-lived proteins in dividing cells during aging using the budding yeast, Saccharomyces cerevisiae. Yeast mother cells undergo a limited number of asymmetric divisions that define replicative lifespan. We used stable-isotope pulse-chase and total proteome mass-spectrometry to identify proteins that were both long-lived and retained in aging mother cells after ∼18 cells divisions. We identified ∼135 proteins that we designate as long-lived asymmetrically retained proteins (LARPS). Surprisingly, the majority of LARPs appeared to be stable fragments of their original full-length protein. However, 15% of LARPs were full-length proteins and we confirmed several candidates to be long-lived and retained in mother cells by time-lapse microscopy. Some LARPs localized to the plasma membrane and remained robustly in the mother cell upon cell division. Other full-length LARPs were assembled into large cytoplasmic structures that had a strong bias to remain in mother cells. We identified age-associated changes to LARPs that include an increase in their levels during aging because of their continued synthesis, which is not balanced by turnover. Additionally, several LARPs were posttranslationally modified during aging. We suggest that LARPs contribute to age-associated phenotypes and likely exist in other organisms. PMID:25228775

  2. Human embryonic stem cells passaged using enzymatic methods retain a normal karyotype and express CD30.

    PubMed

    Thomson, Alison; Wojtacha, Davina; Hewitt, Zoë; Priddle, Helen; Sottile, Virginie; Di Domenico, Alex; Fletcher, Judy; Waterfall, Martin; Corrales, Néstor López; Ansell, Ray; McWhir, Jim

    2008-03-01

    Human embryonic stem cells (hESCs) are thought to be susceptible to chromosomal rearrangements as a consequence of single cell dissociation. Compared in this study are two methods of dissociation that do not generate single cell suspensions (collagenase and EDTA) with an enzymatic procedure using trypsin combined with the calcium-specific chelator EGTA (TEG), that does generate a single cell suspension, over 10 passages. Cells passaged by single cell dissociation using TEG retained a normal karyotype. However, cells passaged using EDTA, without trypsin, acquired an isochromosome p7 in three replicates of one experiment. In all of the TEG, collagenase and EDTA-treated cultures, cells retained consistent telomere length and potentiality, demonstrating that single cell dissociation can be used to maintain karyotypically and phenotypically normal hESCs. However, competitive genomic hybridization revealed that subkaryotypic deletions and amplifications could accumulate over time, reinforcing that present culture regimes remain suboptimal. In all cultures the cell surface marker CD30, reportedly expressed on embryonal carcinoma but not karyoptically normal ESCs, was expressed on hESCs with both normal and abnormal karyotype, but was upregulated on the latter.

  3. Retention of empty MHC class I molecules by tapasin is essential to reconstitute antigen presentation in invertebrate cells.

    PubMed Central

    Schoenhals, G J; Krishna, R M; Grandea, A G; Spies, T; Peterson, P A; Yang, Y; Früh, K

    1999-01-01

    Presentation of antigen-derived peptides by major histocompatibility complex (MHC) class I molecules is dependent on an endoplasmic reticulum (ER) resident glycoprotein, tapasin, which mediates their interaction with the transporter associated with antigen processing (TAP). Independently of TAP, tapasin was required for the presentation of peptides targeted to the ER by signal sequences in MHC class I-transfected insect cells. Tapasin increased MHC class I peptide loading by retaining empty but not peptide-containing MHC class I molecules in the ER. Upon co-expression of TAP, this retention/release function of tapasin was sufficient to reconstitute MHC class I antigen presentation in insect cells, thus defining the minimal non-housekeeping functions required for MHC class I antigen presentation. PMID:9927434

  4. Multimolecular associations of the T-cell antigen receptor.

    PubMed

    Beyers, A D; Spruyt, L L; Williams, A F

    1992-09-01

    T cells are activated when the T-cell receptor for antigen (TCR) interacts with an antigenic peptide bound to a major histocompatibility complex (MHC) molecule on the surface of another cell. It is often assumed that T-cell activation is induced by the crosslinking of TCRs. In this article, Albertus Beyers, Louise Spruyt and Alan Williams argue that this mechanism is not generally applicable. They hypothesize that the key event in T-cell activation is the formation of multimolecular complexes consisting of the TCR and several other polypeptides, including CD4 or CD8, CD2, CD5 and the associated tyrosine kinases p59(fyn) and p56(lck).

  5. Photoaffinity antigens for human γδ T cells1

    PubMed Central

    Sarikonda, Ghanashyam; Wang, Hong; Puan, Kia-Joo; Liu, Xiao-hui; Lee, Hoi K.; Song, Yongcheng; Distefano, Mark D.; Oldfield, Eric; Prestwich, Glenn D.; Morita, Craig T.

    2009-01-01

    Vγ2Vδ2 T cells comprise the major subset of peripheral blood γ δ T cells in humans and expand during infections by recognizing small, nonpeptide prenyl pyrophosphates. These molecules include (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP), a microbial isoprenoid intermediate, and isopentenyl pyrophosphate (IPP), an endogenous isoprenoid intermediate. Recognition of these nonpeptide antigens is mediated by the Vγ2Vδ2 T cell antigen receptor (TCR). Several findings suggest that prenyl pyrophosphates are presented by an antigen presenting molecule: contact between T cells and APCs is required; the antigens do not bind the Vγ2Vδ2 TCR directly; and antigen recognition is abrogated by TCR mutations in CDRs distant from the putative antigen recognition site. Identification of the putative antigen presenting molecule, however, has been hindered by the inability to achieve stable association of nonpeptide prenyl pyrophosphate antigens with the presenting molecule. In this study, we show that photoaffinity analogs of HMBPP, meta/para-benzophenone-(methylene)-prenyl pyrophosphates (m/p-BZ-(C)-C5-OPP), can cross-link to the surface of tumor cell lines and be presented as antigens to γ δ T cells. Mutant tumor cell lines lacking MHC class I, MHC class II, β2-microglobulin, and CD1, as well as tumor cell lines from a variety of tissues and individuals, will all crosslink to and present m-BZ-C5-OPP. Finally, pulsing of BZ-(C)-C5-OPP is inhibited by IPP and an inactive analog, suggesting that they bind to the same molecule. Taken together, these results suggest that nonpeptide antigens are presented by a novel antigen presenting molecule that is widely distributed, non-polymorphic, but not classical MHC class I, MHC class II, or CD1. This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript

  6. Autopresentation of hepatitis B virus envelope antigens by T cells.

    PubMed Central

    Ferrari, C; Pilli, M; Penna, A; Bertoletti, A; Valli, A; Cavalli, A; Pasetti, G; Fiaccadori, F

    1992-01-01

    Processing and presentation by T cells appear to be limited to antigens that can directly interact with the T-cell surface, thereby overcoming the T-cell inefficiency in antigen capture and internalization. Our study provides evidence that the hepatitis B virus (HBV) envelope proteins can also be efficiently processed and presented by CD4+ and CD8+ T cells to other T cells in a human leukocyte antigen class II-restricted fashion. This phenomenon suggests a receptor-mediated interaction between T cells and the HBV envelope and defines a system that can, we hope, be exploited for the identification of the receptor binding site within the HBV envelope and for the characterization of the putative cellular HBV receptor. PMID:1548778

  7. Mature DC from skin and skin-draining LN retain the ability to acquire and efficiently present targeted antigen.

    PubMed

    Henri, Sandrine; Siret, Carole; Machy, Patrick; Kissenpfennig, Adrien; Malissen, Bernard; Leserman, Lee

    2007-05-01

    Skin-draining LN contain several phenotypically distinguishable DC populations, which may be immature or mature. Mature DC are generally considered to have lost the capacity to acquire and present newly encountered Ag. Using antibody-opsonized liposomes as Ag carriers, we show that mature DC purified from skin explants are able to efficiently capture liposomes, process Ag encapsulated within them and activate Ag-specific CD4(+) T cells. Explant DC from mice with Langerhans cells (LC) expressing the primate diphtheria toxin receptor that were exposed to diphtheria toxin in vivo presented Ag as well as explant DC from wild-type mice, indicating that LC are not required and dermal DC are probably responsible for this presentation. We further show that all DC subtypes from LN that capture opsonized Ag are capable of cross-presenting it to CD8(+) T cells. Induction of additional maturation in vivo by LPS or treatment with double-stranded RNA did not alter the Ag presentation capacity of the skin or LN DC subtypes. These results suggest that mature DC present in skin-draining LN may play an important role in the induction of primary and/or secondary immune responses against Ag delivered to the LN that they take up by receptor-mediated endocytosis.

  8. Coproduction of carcinoembryonic antigen and nonspecific cross-reacting antigen by a continuous cell line from a human pancreatic tumor.

    PubMed

    Kuroki, M; Ichiki, S; Kuroki, M; Matsuoka, Y

    1982-08-01

    A simultaneous production of nonspecific cross-reacting antigen (NCA) and carcinoembryonic antigen (CEA) by the same individual cells of an established human pancreatic cell line (QGP-1) was demonstrated by the immunoperoxidase method. Kinetics of cell proliferation and production of CEA and NCA were analyzed, and active synthesis of both antigens was found to be accompanied with the active proliferation of cultured cells. Both antigens in culture medium were purified by immunoadsorption and gel filtration. Immunochemical studies confirmed that CEA and NCA produced by the QGP-1 cells had properties identical to those of authentic CEA derived from metastatic colorectal carcinoma and to those of NCA from normal lungs, respectively.

  9. Semiquantitative measure of immune responses against erythropoietic stem cell antigens

    SciTech Connect

    Harrison, D.E.

    1987-01-01

    A semiquantitative assay was developed and used to measure the effects of immune responses against 16 independent non-H-2 antigenic loci on erythropoietic stem cells. The assay compares repopulation in genetically anemic WBB6F1-W/Wv recipients that have normal immune responses, and in lethally irradiated WBB6F1 +/+ mice whose immune responses are suppressed by the irradiation. The differences in repopulating ability between these two types of recipients measure how immune responses affect erythropoietic stem cells. Stem cell repopulating abilities for the cells with antigens specified by the Thy-1, H-1, H-24, Ly-1, H-37, and H-17 loci were affected slightly, if at all. Repopulating abilities were moderately reduced by responses against antigens specified by H-15, 16, Ea-2, and Ly-2, 3 loci, and against the differences between the B6 and B10 genotypes, although marrow of these types cured W/Wv recipients. A surprising result occurred for the antigen specified by the H-8 locus, in which immune responses strongly reduced repopulating abilities, although this type of marrow cell cured W/Wv recipients. A comparison of these results with skin graft survival times suggests that the antigens specified by the H-17 and H-24 loci are strongly immunogenic on skin but not on marrow stem cells, while those specified by the H-12 and H-8 loci are strongly immunogenic on marrow stem cells but not on skin.

  10. Targeting B-cell maturation antigen in multiple myeloma

    PubMed Central

    Tai, Yu-Tzu; Anderson, Kenneth C

    2015-01-01

    Novel effective immunotherapies are needed for patients with multiple myeloma (MM), since disease recurrence remains a major obstacle. B-cell maturation antigen (BCMA), a cell surface protein universally expressed on malignant plasma cells , has emerged as a very selective antigen to be targeted in novel treatments for MM. We here first review BCMA-related biology, and then highlight the recent clinical development of a novel afucosylated anti-BCMA monoclonal antibody conjugated with monomethyl auristatin F via noncleavable linker (GSK2857916). Chimeric antigen receptor-expressing T cells targeting BCMA may also induce specific and durable anti-MM responses by patients’ own effector cells. Clinical trials testing these two approaches (NCT02064387, NCT02215967) are currently ongoing in relapsed and refractory MM patients. PMID:26370838

  11. Antibody-induced antigenic modulation is antigen dependent: characterization of 22 proteins on a malignant human B cell line

    SciTech Connect

    Pesando, J.M.; Hoffman, P.; Abed, M.

    1986-12-01

    Expression of several of the surface antigens on normal and malignant hematopoietic cells is reduced or is modulated by incubation with specific antibodies. Although antigenic modulation provides a means by which cells can escape antibody-mediated immune destruction, the physiologic significance and frequency of this phenomenon are both poorly understood. To begin to address these issues, the authors identified and characterized surface antigens on the malignant B cell line Laz 221 established from a patient with acute lymphoblastic leukemia (ALL). Indirect immunofluorescence analysis with the use of 26 hematopoietic cell populations and immune precipitation studies with the use of iodinated ALL cells indicate the 163 monoclonal antibodies (MoAb) identify 22 different proteins on this cell line, including at least six previously described surface molecules. Seven of these antigens are expressed by all nucleated cells examined, whereas only the ..mu.. chain of immunoglobulin is B cell specific. Studies that made use of multiple MoAb specific for the same antigen suggest that the capacity for antigenic modulation is an intrinsic property of individual antigens. These studies also suggest that the murine immune response to shared human antigens varies from one immunizing cell population to another. Immunogenicity of individual human antigens in the mouse may be a function of their cell surface environment.

  12. Label-Retaining Cells in the Adult Murine Salivary Glands Possess Characteristics of Adult Progenitor Cells

    PubMed Central

    Chibly, Alejandro M.; Querin, Lauren; Harris, Zoey; Limesand, Kirsten H.

    2014-01-01

    Radiotherapy is the primary treatment for patients with head and neck cancer, which account for roughly 500,000 annual cases worldwide. Dysfunction of the salivary glands and associated conditions like xerostomia and dysphagia are often developed by these patients, greatly diminishing their life quality. Current preventative and palliative care fail to deliver an improvement in the quality of life, thus accentuating the need for regenerative therapies. In this study, a model of label retaining cells (LRCs) in murine salivary glands was developed, in which LRCs demonstrated proliferative potential and possessed markers of putative salivary progenitors. Mice were labeled with 5-Ethynyl-2′-deoxyuridine (EdU) at postnatal day 10 and chased for 8 weeks. Tissue sections from salivary glands obtained at the end of chase demonstrated co-localization between LRCs and the salivary progenitor markers keratin 5 and keratin 14, as well as kit mRNA, indicating that LRCs encompass a heterogeneous population of salivary progenitors. Proliferative potential of LRCs was demonstrated by a sphere assay, in which LRCs were found in primary and secondary spheres and they co-localized with the proliferation marker Ki67 throughout sphere formation. Surprisingly, LRCs were shown to be radio-resistant and evade apoptosis following radiation treatment. The clinical significance of these findings lie in the potential of this model to study the mechanisms that prevent salivary progenitors from maintaining homeostasis upon exposure to radiation, which will in turn facilitate the development of regenerative therapies for salivary gland dysfunction. PMID:25238060

  13. Recognition of antigen-specific B-cell receptors from chronic lymphocytic leukemia patients by synthetic antigen surrogates.

    PubMed

    Sarkar, Mohosin; Liu, Yun; Morimoto, Jumpei; Peng, Haiyong; Aquino, Claudio; Rader, Christoph; Chiorazzi, Nicholas; Kodadek, Thomas

    2014-12-18

    In patients with chronic lymphocytic leukemia (CLL), a single neoplastic antigen-specific B cell accumulates and overgrows other B cells, leading to immune deficiency. CLL is often treated with drugs that ablate all B cells, leading to further weakening of humoral immunity, and a more focused therapeutic strategy capable of targeting only the pathogenic B cells would represent a significant advance. One approach to this would be to develop synthetic surrogates of the CLL antigens allowing differentiation of the CLL cells and healthy B cells in a patient. Here, we describe nonpeptidic molecules capable of targeting antigen-specific B cell receptors with good affinity and selectivity using a combinatorial library screen. We demonstrate that our hit compounds act as synthetic antigen surrogates and recognize CLL cells and not healthy B cells. Additionally, we argue that the technology we developed can be used to identify other classes of antigen surrogates. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. T cells expressing CD19/CD20 bi-specific chimeric antigen receptors prevent antigen escape by malignant B cells

    PubMed Central

    Zah, Eugenia; Lin, Meng-Yin; Silva-Benedict, Anne; Jensen, Michael C.; Chen, Yvonne Y.

    2016-01-01

    The adoptive transfer of T cells expressing anti-CD19 chimeric antigen receptors (CARs) has shown remarkable curative potential against advanced B-cell malignancies, but multiple trials have also reported patient relapses due to the emergence of CD19-negative leukemic cells. Here, we report the design and optimization of single-chain, bi-specific CARs that trigger robust cytotoxicity against target cells expressing either CD19 or CD20, two clinically validated targets for B-cell malignancies. We determined the structural parameters required for efficient dual-antigen recognition, and we demonstrate that optimized bi-specific CARs can control both wild-type B-cell lymphoma and CD19− mutants with equal efficiency in vivo. To our knowledge, this is the first bi-specific CAR capable of preventing antigen escape by performing true OR-gate signal computation on a clinically relevant pair of tumor-associated antigens. The CD19-OR-CD20 CAR is fully compatible with existing T-cell manufacturing procedures and implementable by current clinical protocols. These results present an effective solution to the challenge of antigen escape in CD19 CAR T-cell therapy, and they highlight the utility of structure-based rational design in the development of receptors with higher-level complexity. PMID:27059623

  15. Breaking the In Vitro Alveolar Type II Cell Proliferation Barrier while Retaining Ion Transport Properties

    PubMed Central

    Dang, Hong; Cheluvaraju, Chaitra; Jones, Lisa C.; Liu, Xuefeng; O’Neal, Wanda K.; Randell, Scott H.; Schlegel, Richard; Boucher, Richard C.

    2014-01-01

    Alveolar type (AT)I and ATII cells are central to maintaining normal alveolar fluid homeostasis. When disrupted, they contribute to the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome. Research on ATII cells has been limited by the inability to propagate primary cells in vitro to study their specific functional properties. Moreover, primary ATII cells in vitro quickly transdifferentiate into nonproliferative “ATI-like” cells under traditional culture conditions. Recent studies have demonstrated that normal and tumor cells grown in culture with a combination of fibroblast (feeder cells) and a pharmacological Rho kinase inhibitor (Y-27632) exhibit indefinite cell proliferation that resembled a “conditionally reprogrammed cell” phenotype. Using this coculture system, we found that primary human ATII cells (1) proliferated at an exponential rate, (2) established epithelial colonies expressing ATII-specific and “ATI-like” mRNA and proteins after serial passage, (3) up-regulated genes important in cell proliferation and migration, and (4) on removal of feeder cells and Rho kinase inhibitor under air–liquid interface conditions, exhibited bioelectric and volume transport characteristics similar to freshly cultured ATII cells. Collectively, our results demonstrate that this novel coculture technique breaks the in vitro ATII cell proliferation barrier while retaining cell-specific functional properties. This work will allow for a significant increase in studies designed to elucidate ATII cell function with the goal of accelerating the development of novel therapies for alveolar diseases. PMID:24191670

  16. HLA class II antigen presentation by prostate cancer cells.

    PubMed

    Younger, A R; Amria, S; Jeffrey, W A; Mahdy, A E M; Goldstein, O G; Norris, J S; Haque, A

    2008-01-01

    Prostate cancer is the second most commonly diagnosed cancer in men. Recent evidence suggests that reduced expression of target protein antigens and human leukocyte antigen (HLA) molecules is the predominant immune escape mechanism of malignant prostate tumor cells. The purpose of this study was to investigate the prospect of antigen specific immunotherapy against prostate cancer via the HLA class II pathway of immune recognition. Here, we show for the first time that prostate cancer cells express HLA class II proteins that are recognized by CD4+ T cells. Prostate tumor cells transduced with class II molecules efficiently presented tumor-associated antigens/peptides to CD4+ T cells. This data suggests that malignant prostate tumors can be targeted via the HLA class II pathway, and that class II-positive tumors could be employed for direct antigen presentation, and CD4+ T-cell mediated tumor immunotherapy.Prostate Cancer and Prostatic Diseases (2008) 11, 334-341; doi:10.1038/sj.pcan.4501021; published online 16 October 2007.

  17. Regulator T cells: specific for antigen and/or antigen receptors?

    PubMed

    Rubin, B; de Durana, Y Diaz; Li, N; Sercarz, E E

    2003-05-01

    Adaptive immune responses are regulated by many different molecular and cellular effectors. Regulator T cells are coming to their rights again, and these T cells seem to have ordinary alpha/beta T-cell receptors (TCRs) and to develop in the thymus. Autoimmune responses are tightly regulated by such regulatory T cells, a phenomenon which is beneficial to the host in autoimmune situations. However, the regulation of autoimmune responses to tumour cells is harmful to the host, as this regulation delays the defence against the outgrowth of neoplastic cells. In the present review, we discuss whether regulatory T cells are specific for antigen and/or for antigen receptors. Our interest in these phenomena comes from the findings that T cells produce many more TCR-alpha and TCR-beta chains than are necessary for surface membrane expression of TCR-alphabeta heterodimers with CD3 complexes. Excess TCR chains are degraded by the proteasomes, and TCR peptides thus become available to the assembly pathway of major histocompatibility complex class I molecules. Consequently, do T cells express two different identification markers on the cell membrane, the TCR-alphabeta clonotype for recognition by B-cell receptors and clonotypic TCR-alphabeta peptides for recognition by T cells?

  18. Synovial fluid antigen-presenting cells unmask peripheral blood T cell responses to bacterial antigens in inflammatory arthritis.

    PubMed Central

    Life, P F; Viner, N J; Bacon, P A; Gaston, J S

    1990-01-01

    We and others have previously shown that synovial fluid (SF) mononuclear cells (MC) from patients with both reactive arthritis and other inflammatory arthritides proliferate in vitro in response to bacteria clinically associated with the triggering of reactive arthritis. In all cases, such SFMC responses are greater than the corresponding peripheral blood (PB) MC responses, often markedly so, and the mechanism for this is unclear. We have investigated this phenomenon by comparing the relative abilities of irradiated non-T cells derived from PB and SF to support autologous T cell responses to ReA-associated bacteria. Seven patients whose SFMC had been shown previously to respond to bacteria were studied. We demonstrate antigen-specific responses of PB T cells to bacteria in the presence of SF non-T cells which are in marked contrast to the minimal responses of either unfractionated PBMC or PB T cells reconstituted with PB non-T cells. We also show that PB, but not SF T cells respond strongly to autologous SF non-T cells in the absence of antigen or mitogen. These findings demonstrate that SF antigen-presenting cells (APC) are potent activators of PB T cells. We conclude that the contrasting responses of SFMC and PBMC to bacterial antigens may be accounted for at least in part by an enhanced ability of SF APC to support T cell proliferative responses. PMID:2311298

  19. Cell-to-cell transfer of M. tuberculosis antigens optimizes CD4 T cell priming.

    PubMed

    Srivastava, Smita; Ernst, Joel D

    2014-06-11

    During Mycobacterium tuberculosis and other respiratory infections, optimal T cell activation requires pathogen transport from the lung to a local draining lymph node (LN). However, the infected inflammatory monocyte-derived dendritic cells (DCs) that transport M. tuberculosis to the local lymph node are relatively inefficient at activating CD4 T cells, possibly due to bacterial inhibition of antigen presentation. We found that infected migratory DCs release M. tuberculosis antigens as soluble, unprocessed proteins for uptake and presentation by uninfected resident lymph node DCs. This transfer of bacterial proteins from migratory to local DCs results in optimal priming of antigen-specific CD4 T cells, which are essential in controlling tuberculosis. Additionally, this mechanism does not involve transfer of the whole bacterium and is distinct from apoptosis or exosome shedding. These findings reveal a mechanism that bypasses pathogen inhibition of antigen presentation by infected cells and generates CD4 T cell responses that control the infection.

  20. T Cells as Antigen Carriers for Anti-tumor Vaccination.

    PubMed

    Traversari, Catia; Russo, Vincenzo

    2016-01-01

    The exploitation of the physiologic processing and presenting machinery of dendritic cells (DCs) by in vivo loading of tumor-associated antigens may improve the immunogenic potential and clinical efficacy of DC-based cancer vaccines. The approach developed by our group was based on the clinical observation that some patients treated with the infusion of donor lymphocytes transduced to express the HSV-TK suicide gene for relapse of hematologic malignancies, after allogeneic hematopoietic stem cell transplantation, developed a T cell-mediated immune response specifically directed against the HSV-TK gene product.We demonstrated that lymphocytes genetically modified to express HSV-TK as well as self/tumor antigens, acting as antigen carriers, efficiently target DCs in vivo in tumor-bearing mice. The infusion of TRP-2-transduced lymphocytes induced the establishment of protective immunity and long-term memory in tumor-bearing mice by cross-presentation of the antigen mediated by the CD11c(+)CD8a(+) DCs subset. A similar approach was applied in a clinical setting. Ten patients affected by MAGE-3(+) metastatic melanoma were treated with autologous lymphocytes retrovirally transduced to express the MAGE-3 tumor antigen. In three patients, the treatment led to the increase of MAGE-3 specific CD8+ and CD4+ effectors and the development of long-term memory, which ultimately correlated with a favorable clinical outcome. Transduced lymphocytes represent an efficient way for in vivo loading of tumor-associated antigens of DCs.

  1. Characterization of antigen association with accessory cells: specific removal of processed antigens from the cell surface by phospholipases.

    PubMed Central

    Falo, L D; Haber, S I; Herrmann, S; Benacerraf, B; Rock, K L

    1987-01-01

    To characterize the basis for the cell surface association of processed antigen with the antigen-presenting cell (APC) we analyzed its sensitivity to enzymatic digestion. Antigen-exposed APC that are treated with phospholipase and then immediately fixed lose their ability to stimulate antigen-plus-Ia-specific T-T hybridomas. This effect is seen with highly purified phospholipase A2 and phospholipase C. In addition it is observed with three distinct antigens--ovalbumin, bovine insulin, and poly(LGlu56LLys35LPhe9) [(GluLysPhe)n]. The effect of phospholipases is highly specific. Identically treated APC are equivalent to controls in their ability to stimulate alloreactive hybridomas specific for precisely the same Ia molecule that is corecognized by antigen-plus-Ia-specific hybrids. Furthermore, the antigen-presenting function of enzyme-treated, fixed APC can be reconstituted by the addition of exogenous in vitro processed or "processing independent" antigens. In parallel studies 125I-labeled avidin was shown to specifically bind to APC that were previously exposed and allowed to process biotin-insulin. Biotin-insulin-exposed APC that are pretreated with phospholipase bind significantly less 125I-labeled avidin than do untreated, exposed APC. Identical enzyme treatment does not reduce the binding of avidin to a biotinylated antibody already bound to class II major histocompatibility complex molecules of APC. At least some of the biotin-insulin surface sites are immunologically relevant, because the presentation of processed biotin-insulin by fixed APC is blocked by avidin. This effect is specific. Avidin binding to biotin-insulin-exposed APC does not inhibit allospecific stimulation nor the presentation of unconjugated insulin. These studies demonstrate that phospholipase effectively removes processed cell surface antigen. PMID:3467371

  2. Label-retaining stromal cells in mouse endometrium awaken for expansion and repair after parturition.

    PubMed

    Cao, Mingzhu; Chan, Rachel W S; Yeung, William S B

    2015-03-15

    Human and mouse endometrium undergo dramatic cellular reorganization during pregnancy and postpartum. Somatic stem cells maintain homeostasis of the tissue by providing a cell reservoir for regeneration. We hypothesized that endometrial cells with quiescent properties (stem/progenitor cells) were involved in the regeneration of the endometrial tissue. Given that stem cells divide infrequently, they can retain the DNA synthesis label [bromodeoxyuridine (BrdU)] after a prolonged chase period. In this study, prepubertal mice were pulsed with BrdU and after a 6-week chase a small population of label-retaining stromal cells (LRSC) was located primarily beneath the luminal epithelium, adjacent to blood vessels, and near the endometrial-myometrial junction. Marker analyses suggested that they were of mesenchymal origin expressing CD44(+), CD90(+), CD140b(+), CD146(+), and Sca-1(+). During pregnancy, nonproliferating LRSC predominately resided at the interimplantation/placental loci of the gestational endometrium. Immediately after parturition, a significant portion of the LRSC underwent proliferation (BrdU(+)/Ki-67(+)) and expressed total and active β-catenin. The β-catenin expression in the LRSC was transiently elevated at postpartum day (PPD) 1. The proliferation of LRSC resulted in a significant decline in the proportion of LRSC in the postpartum uterus. The LRSC returned to dormancy at PPD7, and the percentage of LRSC remained stable thereafter until 11 weeks. This study demonstrated that LRSC can respond efficiently to physiological stimuli upon initiation of uterine involution and return to its quiescent state after postpartum repair.

  3. Bovine CD49 positive-cell subpopulation remarkably increases in mammary epithelial cells that retain a stem-like phenotype.

    PubMed

    Cravero, Diego; Martignani, Eugenio; Miretti, Silvia; Accornero, Paolo; Baratta, Mario

    2015-10-01

    We previously proved that adult stem cells reside in the bovine mammary gland and possess an intrinsic potential to generate a functional mammary outgrowth. The aim of this study was to investigate on the immunophenotyping features retained by mammary stem-like cells detected in long term culture. Flow cytometry analysis showed different subpopulations of mammary epithelial cells emerging according to the timing of cell culture. CD49f(+)-cells significantly increased during the culture (p<0.01) and a similar trend was observed, even if less regular, for CD29(+) and ALDH1 positive cell populations. No difference during the culture was observed for CD24 positive cells but after 35 days of culture a subset of cells, CD49f positive, still retained regenerative capabilities in in vivo xenotransplants. These cells were able to form organized pseudo-alveoli when transplanted in immunodeficient mice. These results prove the presence of a multipotent cell subpopulation that retain a strong epithelial induction, confirmed in in vivo xenotransplants with a presumable in vitro expansion of the primitive population of adult mammary stem cells.

  4. Activated Brain Endothelial Cells Cross-Present Malaria Antigen.

    PubMed

    Howland, Shanshan W; Poh, Chek Meng; Rénia, Laurent

    2015-06-01

    In the murine model of cerebral malaria caused by P. berghei ANKA (PbA), parasite-specific CD8+ T cells directly induce pathology and have long been hypothesized to kill brain endothelial cells that have internalized PbA antigen. We previously reported that brain microvessel fragments from infected mice cross-present PbA epitopes, using reporter cells transduced with epitope-specific T cell receptors. Here, we confirm that endothelial cells are the population responsible for cross-presentation in vivo, not pericytes or microglia. PbA antigen cross-presentation by primary brain endothelial cells in vitro confers susceptibility to killing by CD8+ T cells from infected mice. IFNγ stimulation is required for brain endothelial cross-presentation in vivo and in vitro, which occurs by a proteasome- and TAP-dependent mechanism. Parasite strains that do not induce cerebral malaria were phagocytosed and cross-presented less efficiently than PbA in vitro. The main source of antigen appears to be free merozoites, which were avidly phagocytosed. A human brain endothelial cell line also phagocytosed P. falciparum merozoites. Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention.

  5. T Cells Redirected to a Minor Histocompatibility Antigen Instruct Intratumoral TNFα Expression and Empower Adoptive Cell Therapy for Solid Tumors.

    PubMed

    Manzo, Teresa; Sturmheit, Tabea; Basso, Veronica; Petrozziello, Elisabetta; Hess Michelini, Rodrigo; Riba, Michela; Freschi, Massimo; Elia, Angela R; Grioni, Matteo; Curnis, Flavio; Protti, Maria Pia; Schumacher, Ton N; Debets, Reno; Swartz, Melody A; Corti, Angelo; Bellone, Matteo; Mondino, Anna

    2017-02-01

    Donor-derived allogeneic T cells evoke potent graft versus tumor (GVT) effects likely due to the simultaneous recognition of tumor-specific and host-restricted minor histocompatibility (H) antigens. Here we investigated whether such effects could be reproduced in autologous settings by TCR gene-engineered lymphocytes. We report that T cells redirected either to a broadly expressed Y-encoded minor H antigen or to a tumor-associated antigen, although poorly effective if individually transferred, when simultaneously administered enabled acute autochthonous tumor debulking and resulted in durable clinical remission. Y-redirected T cells proved hyporesponsive in peripheral lymphoid organs, whereas they retained effector function at the tumor site, where in synergy with tumor-redirected lymphocytes, they instructed TNFα expression, endothelial cell activation, and intratumoral T-cell infiltration. While neutralizing TNFα hindered GVT effects by the combined T-cell infusion, a single injection of picogram amounts of NGR-TNF, a tumor vessel-targeted TNFα derivative currently in phase III clinical trials, substituted for Y-redirected cells and enabled tumor debulking by tumor-redirected lymphocytes. Together, our results provide new mechanistic insights into allogeneic GVT, validate the importance of targeting the tumor and its associated stroma, and prove the potency of a novel combined approach suitable for immediate clinical implementation. Cancer Res; 77(3); 658-71. ©2016 AACR.

  6. Proliferation and migration of label-retaining cells of the kidney papilla.

    PubMed

    Oliver, Juan A; Klinakis, Apostolos; Cheema, Faisal H; Friedlander, Jonathan; Sampogna, Rosemary V; Martens, Timothy P; Liu, Charles; Efstratiadis, Argiris; Al-Awqati, Qais

    2009-11-01

    The kidney papilla contains a population of cells with several characteristics of adult stem cells, including the retention of proliferation markers during long chase periods (i.e., they are label-retaining cells [LRCs]). To determine whether the papillary LRCs generate new cells in the normal adult kidney, we examined cell proliferation throughout the kidney and found that the upper papilla is a site of enhanced cell cycling. Using genetically modified mice that conditionally expressed green fluorescence protein fused to histone 2B, we observed that the LRCs of the papilla proliferated only in its upper part, where they associate with "chains" of cycling cells. The papillary LRCs decreased in number with age, suggesting that the cells migrated to the upper papilla before entering the cell cycle. To test this directly, we marked papillary cells with vital dyes in vivo and found that some cells in the kidney papilla, including LRCs, migrated toward other parts of the kidney. Acute kidney injury enhanced both cell migration and proliferation. These results suggest that during normal homeostasis, LRCs of the kidney papilla (or their immediate progeny) migrate to the upper papilla and form a compartment of rapidly proliferating cells, which may play a role in repair after ischemic injury.

  7. Induction of the autoantigen proliferating cell nuclear antigen in T lymphocytes by a mycobacterial antigen.

    PubMed Central

    Haftel, H M; Chang, Y; Hinderer, R; Hanash, S M; Holoshitz, J

    1994-01-01

    Mycobacteria have been implicated in the pathogenesis of autoimmunity. To determine the potential effect of mycobacterial antigens on peripheral blood mononuclear cells (PBMC), we analyzed PBMC incubated with the acetone-precipitable fraction of Mycobacterium tuberculosis (APMT) for changes in cellular protein expression. Two-dimensional gel analysis showed induction of a 36-kD polypeptide identified as proliferating cell nuclear antigen (PCNA), a known autoantigen, after incubation with AP-MT. PCNA plays a role in cell proliferation and is expressed as a late growth regulated factor. However, its synthesis in response to AP-MT was induced as an early event. The early induction of PCNA was regulated at a posttranscriptional level and was restricted to T cells. Treatment of PBMC with known T cell mitogens, namely PHA, anti-CD3 antibodies, and staphylococcal superantigens failed to induce an early PCNA increase. The distinct characteristics of the AP-MT effect on PCNA expression suggest a separate mechanism of induction in response to AP-MT, compared with the late increase observed in response to mitogens. The induction of PCNA in response to mycobacterial antigens may represent a pathogenically relevant mechanism in autoimmunity. Images PMID:7929811

  8. Predicted complementarity determining regions of the T cell antigen receptor determine antigen specificity.

    PubMed Central

    Katayama, C D; Eidelman, F J; Duncan, A; Hooshmand, F; Hedrick, S M

    1995-01-01

    The antigen receptor on T cells (TCR) has been predicted to have a structure similar to a membrane-anchored form of an immunoglobulin F(ab) fragment. Virtually all of the conserved amino acids that are important for inter- and intramolecular interactions in the VH-VL pair are also conserved in the TCR V alpha and V beta chains. A molecular model of the TCR has been constructed by homology and we have used the information from this, as well as the earlier structural predictions of others, to study the basis for specificity. Specifically, regions of a TCR cloned from an antigen-specific T cell were stitched into the corresponding framework of a second TCR. Results indicate that the substitution of amino acid sequences corresponding to the complementarity determining regions (CDRs) of immunoglobulin can convey the specificity for antigen and major histocompatibility complex molecules. These data are consistent with a role, but not an exclusive role, for CDR3 in antigen peptide recognition. Images PMID:7534228

  9. MAIT cells are depleted early but retain functional cytokine expression in HIV infection.

    PubMed

    Fernandez, Caroline S; Amarasena, Thakshila; Kelleher, Anthony D; Rossjohn, Jamie; McCluskey, James; Godfrey, Dale I; Kent, Stephen J

    2015-02-01

    Mucosal-associated invariant T (MAIT) cells home to mucosal sites and exert antimicrobial activity against bacteria and other microorganisms. HIV infection leads to early depletion of gut T cells and translocation of bacterial products. There are reports that MAIT cells, defined by coexpression of Vα7.2 and CD161, are depleted during HIV infection and residual MAIT cells are functionally impaired. However, one study suggested that MAIT cells might remain after HIV infection but evade detection through CD161 downregulation. Thus, the impact of HIV infection on MAIT cells is unclear. We studied longitudinal blood samples from 31 HIV-infected subjects for MAIT cell numbers, phenotype and function using both standard Vα7.2/CD161 surface markers and an MR1 tetramer. We found that MAIT cells were depleted early during HIV infection, and although there was a concomitant rise in Vα7.2(+)CD161(-) cells, these were MR1 tetramer negative, indicating that these are unlikely to be altered MAIT cells. Antigen-mediated activation of residual MAIT cells showed that they remained functional out to 2 years following HIV infection. Although MAIT cells are depleted in HIV infection, residual and functionally active MAIT cells persist and may still be able to assist in controlling bacterial translocation during HIV infection.

  10. Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy

    PubMed Central

    Dai, Hanren; Wang, Yao; Lu, Xuechun

    2016-01-01

    The genetic modification and characterization of T-cells with chimeric antigen receptors (CARs) allow functionally distinct T-cell subsets to recognize specific tumor cells. The incorporation of costimulatory molecules or cytokines can enable engineered T-cells to eliminate tumor cells. CARs are generated by fusing the antigen-binding region of a monoclonal antibody (mAb) or other ligand to membrane-spanning and intracellular-signaling domains. They have recently shown clinical benefit in patients treated with CD19-directed autologous T-cells. Recent successes suggest that the modification of T-cells with CARs could be a powerful approach for developing safe and effective cancer therapeutics. Here, we briefly review early studies, consider strategies to improve the therapeutic potential and safety, and discuss the challenges and future prospects for CAR T-cells in cancer therapy. PMID:26819347

  11. Chimaeric antigen receptor T-cell therapy for tumour immunotherapy

    PubMed Central

    Sha, Huan-huan; Wang, Dan-dan; Yan, Da-li; Hu, Yong; Yang, Su-jin; Liu, Si-wen

    2017-01-01

    Chimaeric antigen receptor (CAR) T-cell therapies, as one of the cancer immunotherapies, have heralded a new era of treating cancer. The accumulating data, especially about CAR-modified T cells against CD19 support that CAR T-cell therapy is a highly effective immune therapy for B-cell malignancies. Apart from CD19, there have been many trials of CAR T cells directed other tumour specific or associated antigens (TSAs/TAAs) in haematologic malignancies and solid tumours. This review will briefly summarize basic CAR structure, parts of reported TSAs/TAAs, results of the clinical trials of CAR T-cell therapies as well as two life-threatening side effects. Experiments in vivo or in vitro, ongoing clinical trials and the outlook for CAR T-cell therapies also be included. Our future efforts will focus on identification of more viable cancer targets and more strategies to make CAR T-cell therapy safer. PMID:28053197

  12. Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy.

    PubMed

    Dai, Hanren; Wang, Yao; Lu, Xuechun; Han, Weidong

    2016-07-01

    The genetic modification and characterization of T-cells with chimeric antigen receptors (CARs) allow functionally distinct T-cell subsets to recognize specific tumor cells. The incorporation of costimulatory molecules or cytokines can enable engineered T-cells to eliminate tumor cells. CARs are generated by fusing the antigen-binding region of a monoclonal antibody (mAb) or other ligand to membrane-spanning and intracellular-signaling domains. They have recently shown clinical benefit in patients treated with CD19-directed autologous T-cells. Recent successes suggest that the modification of T-cells with CARs could be a powerful approach for developing safe and effective cancer therapeutics. Here, we briefly review early studies, consider strategies to improve the therapeutic potential and safety, and discuss the challenges and future prospects for CAR T-cells in cancer therapy.

  13. How Chimeric Antigen Receptor Design Affects Adoptive T Cell Therapy.

    PubMed

    Gacerez, Albert T; Arellano, Benjamine; Sentman, Charles L

    2016-12-01

    Chimeric antigen receptor (CAR) T cells have been developed to treat tumors and have shown great success against B cell malignancies. Exploiting modular designs and swappable domains, CARs can target an array of cell surface antigens and, upon receptor-ligand interactions, direct signaling cascades, thereby driving T cell effector functions. CARs have been designed using receptors, ligands, or scFv binding domains. Different regions of a CAR have each been found to play a role in determining the overall efficacy of CAR T cells. Therefore, this review provides an overview of CAR construction and common designs. Each CAR region is discussed in the context of its importance to a CAR's function. Additionally, the review explores how various engineering strategies have been applied to CAR T cells in order to regulate CAR T cell function and activity. J. Cell. Physiol. 231: 2590-2598, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  14. Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins

    SciTech Connect

    Storrie, B.; Sachdeva, M.; Viers, V.S.

    1984-02-01

    We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t 1/2 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000 X g under conditions in which greater than 80% of the lysosomal marker enzyme beta-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37 degrees C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts.

  15. Turning tumour cells into antigen presenting cells: The next step to improve cancer immunotherapy?

    PubMed

    de Charette, Marie; Marabelle, Aurélien; Houot, Roch

    2016-11-01

    Downregulation/loss of the antigen presentation is a major immune escape mechanism in cancer. It allows tumour cells to become 'invisible' and avoid immune attack by antitumour T cells. In tumour harbouring properties of professional antigen presenting cells (i.e. tumour B cells in lymphoma), downregulation/loss of the antigen presentation may also prevent direct priming of naïve T cells by tumour cells. Here, we review treatments that may induce/restore antigen presentation by the tumour cells. These treatments may increase the generation of antitumour T cells and/or their capacity to recognise and eliminate tumour cells. By forcing tumour cells to present their antigens, these treatments may sensitise patients to T cell-based immunotherapies, including checkpoint inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Long-term label retaining cells localize to distinct regions within the female reproductive epithelium.

    PubMed

    Patterson, Amanda L; Pru, James K

    2013-09-01

    The uterus is an extremely plastic organ that undergoes cyclical remodeling including endometrial regeneration during the menstrual cycle. Endometrial remodeling and regeneration also occur during pregnancy and following parturition, particularly in hemochorial implanting species. The mechanisms of endometrial regeneration are not well understood. Endometrial stem/progenitor cells are proposed to contribute to endometrial regeneration in both humans and mice. BrdU label retention has been used to identify potential stem/progenitor cells in mouse endometrium. However, methods are not available to isolate BrdU label-retaining cells (LRC) for functional analyses. Therefore, we employed a transgenic mouse model to identify H2B-GFP LRCs throughout the female reproductive tract with particular interest on the endometrium. We hypothesized that the female reproductive tract contains a population of long-term LRCs that persist even following pregnancy and endometrial regeneration. Endometrial cells were labeled (pulsed) either transplacentally/translactationally or peripubertally. When mice were pulsed transplacentally/translactationally, the label was not retained in the uterus. However, LRCs were concentrated to the distal oviduct and endocervical transition zone (TZ) following natural (i.e., pregnancy/parturition induced) and mechanically induced endometrial regeneration. LRCs in the distal oviduct and endocervical TZ expressed stem cell markers and did not express ERα or PGR, implying the undifferentiated phenotype of these cells. Oviduct and endocervical TZ LRCs did not proliferate during endometrial re-epithelialization, suggesting that they do not contribute to the endometrium in a stem/progenitor cell capacity. In contrast, when mice were pulsed peripubertally long-term LRCs were identified in the endometrial glandular compartment in mice as far out as 9 months post-pulse. These findings suggest that epithelial tissue of the female reproductive tract contains 3

  17. Epithelial Label-Retaining Cells Are Absent during Tooth Cycling in Salmo salar and Polypterus senegalus

    PubMed Central

    Vandenplas, Sam; Willems, Maxime; Witten, P. Eckhard; Hansen, Tom; Fjelldal, Per Gunnar; Huysseune, Ann

    2016-01-01

    The Atlantic salmon (Salmo salar) and African bichir (Polypterus senegalus) are both actinopterygian fish species that continuously replace their teeth without the involvement of a successional dental lamina. Instead, they share the presence of a middle dental epithelium: an epithelial tier enclosed by inner and outer dental epithelium. It has been hypothesized that this tier could functionally substitute for a successional dental lamina and might be a potential niche to house epithelial stem cells involved in tooth cycling. Therefore, in this study we performed a BrdU pulse chase experiment on both species to (1) determine the localization and extent of proliferating cells in the dental epithelial layers, (2) describe cell dynamics and (3) investigate if label-retaining cells are present, suggestive for the putative presence of stem cells. Cells proliferate in the middle dental epithelium, outer dental epithelium and cervical loop at the lingual side of the dental organ to form a new tooth germ. Using long chase times, both in S. salar (eight weeks) and P. senegalus (eight weeks and twelve weeks), we could not reveal the presence of label-retaining cells in the dental organ. Immunostaining of P. senegalus dental organs for the transcription factor Sox2, often used as a stem cell marker, labelled cells in the zone of outer dental epithelium which grades into the oral epithelium (ODE transition zone) and the inner dental epithelium of a successor only. The location of Sox2 distribution does not provide evidence for epithelial stem cells in the dental organ and, more specifically, in the middle dental epithelium. Comparison of S. salar and P. senegalus reveals shared traits in tooth cycling and thus advances our understanding of the developmental mechanism that ensures lifelong replacement. PMID:27049953

  18. Expression of a multi-epitope DPT fusion protein in transplastomic tobacco plants retains both antigenicity and immunogenicity of all three components of the functional oligomer.

    PubMed

    Soria-Guerra, Ruth Elena; Alpuche-Solís, Angel G; Rosales-Mendoza, Sergio; Moreno-Fierros, Leticia; Bendik, Elise M; Martínez-González, Luzmila; Korban, Schuyler S

    2009-05-01

    Expression of genes in plant chloroplasts provides an opportunity for enhanced production of target proteins. We report the introduction and expression of a fusion DPT protein containing immunoprotective exotoxin epitopes of Corynebacterium diphtheriae, Bordetella pertussis, and Clostridium tetani in tobacco chloroplasts. Using biolistic-mediated transformation, a plant-optimized synthetic DPT gene was successfully transferred to tobacco plastomes. Putative transplastomic T0 plants were identified by PCR, and Southern blot analysis confirmed homoplasmy in T1 progeny. ELISA assays demonstrated that the DPT protein retained antigenicity of the three components of the fusion protein. The highest level of expression in these transplastomic plants reached 0.8% of total soluble protein. To assess whether the functional recombinant protein expressed in tobacco plants would induce specific antibodies in test animals, a mice feeding experiment was conducted. For mice orally immunized with freeze-dried transplastomic leaves, production of IgG and IgA antibodies specific to each toxin were detected in serum and mucosal tissues.

  19. Antigen presentation by monocytes and monocyte-derived cells.

    PubMed

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

    2008-02-01

    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 before 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.

  20. Expression of blood group antigens on red cell microvesicles.

    PubMed

    Oreskovic, R T; Dumaswala, U J; Greenwalt, T J

    1992-01-01

    The purpose of this study was to determine whether epitopes of the A, B, D, Fya, M, N, S, s, and K blood group antigens are present on microvesicle membranes shed by red cells during storage. Vesicles were isolated from outdated units of blood having and lacking the specified antigens. Diluted antisera were absorbed with fixed quantities of vesicles from red cells with the test antigen and red cells lacking that antigen (controls). The adsorbed and unadsorbed antisera were titrated and scored by using panel cells from persons known to be heterozygous for all the non-AB antigens. The mean titration scores following adsorption with the vesicles from A, B, D, M+N-, M-N+, S+s-, S-s+, and Fy(a+b-) units were appreciably lower than the control scores (0, 0, 3, 2, 2, 0, 4, and 4 vs. 19, 23, 34, 13, 12, 16, 18, and 29, respectively), which indicated the presence of these epitopes on the membrane of shed vesicles. The results following adsorption with K:1,2 vesicles were equivocal.

  1. Specificity for the tumor-associated self-antigen WT1 drives the development of fully functional memory T cells in the absence of vaccination.

    PubMed

    Pospori, Constandina; Xue, Shao-An; Holler, Angelika; Voisine, Cecile; Perro, Mario; King, Judith; Fallah-Arani, Farnaz; Flutter, Barry; Chakraverty, Ronjon; Stauss, Hans J; Morris, Emma C

    2011-06-23

    Recently, vaccines against the Wilms Tumor antigen 1 (WT1) have been tested in cancer patients. However, it is currently not known whether physiologic levels of WT1 expression in stem and progenitor cells of normal tissue result in the deletion or tolerance induction of WT1-specific T cells. Here, we used an human leukocyte antigen-transgenic murine model to study the fate of human leukocyte antigen class-I restricted, WT1-specific T cells in the thymus and in the periphery. Thymocytes expressing a WT1-specific T-cell receptor derived from high avidity human CD8 T cells were positively selected into the single-positive CD8 population. In the periphery, T cells specific for the WT1 antigen differentiated into CD44-high memory phenotype cells, whereas T cells specific for a non-self-viral antigen retained a CD44(low) naive phenotype. Only the WT1-specific T cells, but not the virus-specific T cells, displayed rapid antigen-specific effector function without prior vaccination. Despite long-term persistence of WT1-specific memory T cells, the animals did not develop autoimmunity, and the function of hematopoietic stem and progenitor cells was unimpaired. This is the first demonstration that specificity for a tumor-associated self-antigen may drive differentiation of functionally competent memory T cells.

  2. Multinucleated Giant Cancer Cells Produced in Response to Ionizing Radiation Retain Viability and Replicate Their Genome

    PubMed Central

    Mirzayans, Razmik; Andrais, Bonnie; Scott, April; Wang, Ying W.; Kumar, Piyush; Murray, David

    2017-01-01

    Loss of wild-type p53 function is widely accepted to be permissive for the development of multinucleated giant cells. However, whether therapy-induced multinucleation is associated with cancer cell death or survival remains controversial. Herein, we demonstrate that exposure of p53-deficient or p21WAF1 (p21)-deficient solid tumor-derived cell lines to ionizing radiation (between 2 and 8 Gy) results in the development of multinucleated giant cells that remain adherent to the culture dish for long times post-irradiation. Somewhat surprisingly, single-cell observations revealed that virtually all multinucleated giant cells that remain adherent for the duration of the experiments (up to three weeks post-irradiation) retain viability and metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and the majority (>60%) exhibit DNA synthesis. We further report that treatment of multinucleated giant cells with pharmacological activators of apoptosis (e.g., sodium salicylate) triggers their demise. Our observations reinforce the notion that radiation-induced multinucleation may reflect a survival mechanism for p53/p21-deficient cancer cells. With respect to evaluating radiosensitivity, our observations underscore the importance of single-cell experimental approaches (e.g., single-cell MTT) as the creation of viable multinucleated giant cells complicates the interpretation of the experimental data obtained by commonly-used multi-well plate colorimetric assays. PMID:28208747

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

    PubMed

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

    2013-02-21

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

  4. Regulatory effect of monocytes on T cell proliferative responses to oral microbial antigens.

    PubMed Central

    Stashenko, P

    1982-01-01

    Mononuclear cell preparations isolated by Ficoll-Hypaque centrifugation from human peripheral blood were found to vary considerably in the number of monocytes they contained (mean, 20.3%; range, 13 to 33%). The regulatory role of monocytes in T cell proliferative responses to sonic extracts of a panel of oral microorganisms was therefore investigated. T cells were fractionated by anti-immunoglobulin chromatography and depleted of monocytes by treatment with a monoclonal anti-human Ia-like (DR locus antigen) antibody and complement. Purified populations of monocytes were obtained by extensive adherence procedures. The resultant cell populations were greater than 95% pure, as judged by indirect immunofluorescence on a fluorescence-activated cell sorter. Monocyte-depleted T cells failed to respond by proliferation to the nonoral antigen tetanus toxoid, as well as to any oral microorganism, but retained responsiveness to phytohemagglutinin. Readdition of monocytes in final concentrations of from 5 to 15% resulted in the restoration of maximal T cell proliferation. Monocytes in greater numbers suppressed T cell responses to all sonic extracts tested. PMID:6984019

  5. Bone marrow long label-retaining cells reside in the sinusoidal hypoxic niche

    SciTech Connect

    Kubota, Yoshiaki; Takubo, Keiyo; Suda, Toshio

    2008-02-08

    In response to changing signals, quiescent hematopoietic stem cells (HSCs) can be induced to an activated cycling state and provide multi-lineage hematopoietic cells to the whole body via blood vessels. However, the precise localization of quiescent HSCs in bone marrow microenvironment is not fully characterized. Here, we performed whole-mount immunostaining of bone marrow and found that BrdU label-retaining cells (LRCs) definitively reside in the sinusoidal hypoxic zone distant from the 'vascular niche'. Although LRCs expressed very low level of a well-known HSC marker, c-kit in normal circumstances, myeloablation by 5-FU treatment caused LRCs to abundantly express c-kit and proliferate actively. These results demonstrate that bone marrow LRCs reside in the sinusoidal hypoxic niche, and function as a regenerative cell pool of HSCs.

  6. Drug antigenicity, immunogenicity and co-stimulatory signalling: evidence for formation of a functional antigen through immune cell metabolism1

    PubMed Central

    Elsheikh, Ayman; Lavergne, Sidonie N.; Castrejon, J. Luis; Farrell, John; Wang, Haiyi; Sathish, Jean; Pichler, Werner J.; Park, B. Kevin; Naisbitt, Dean J.

    2011-01-01

    Recognition of drugs by immune cells is usually explained by the hapten model, which states that endogenous metabolites bind irreversibly to protein to stimulate immune cells. Synthetic metabolites interact directly with protein generating antigenic determinants for T-cells; however, experimental evidence relating intracellular metabolism in immune cells and the generation of physiologically relevant antigens to functional immune responses is lacking. The aim of this study was to develop an integrated approach using both animal and human experimental systems to characterize sulfamethoxazole (SMX) metabolism-derived antigenic protein adduct formation in immune cells and define the relationship between adduct formation, cell death, co-stimulatory signalling and stimulation of a T-cell response. Formation of SMX-derived adducts in antigen presenting cells was dose- and time-dependent, detectable at non-toxic concentrations and dependent on drug metabolizing enzyme activity. Adduct formation above a threshold induced necrotic cell death, dendritic cell co-stimulatory molecule expression and cytokine secretion. Antigen presenting cells cultured with SMX for 16h, the time needed for drug metabolism, stimulated T-cells from sensitized mice and lymphocytes and T-cell clones from allergic patients. Enzyme inhibition decreased SMX-derived protein adduct formation and the T-cell response. Dendritic cells cultured with SMX and adoptively transferred to recipient mice initiated an immune response; however, T-cells were stimulated with adducts derived from SMX metabolism in antigen presenting cells, not the parent drug. This study shows that antigen presenting cells metabolize SMX; subsequent protein binding generates a functional T-cell antigen. Adduct formation above a threshold stimulates cell death, which provides a maturation signal for dendritic cells. PMID:20980635

  7. SDF-1 activates papillary label-retaining cells during kidney repair from injury.

    PubMed

    Oliver, Juan A; Maarouf, Omar; Cheema, Faisal H; Liu, Charles; Zhang, Qing-Yin; Kraus, Carl; Zeeshan Afzal, M; Firdous, Mamoona; Klinakis, Apostolos; Efstratiadis, Argiris; Al-Awqati, Qais

    2012-06-01

    The adult kidney contains a population of low-cycling cells that resides in the papilla. These cells retain for long periods S-phase markers given as a short pulse early in life; i.e., they are label-retaining cells (LRC). In previous studies in adult rat and mice, we found that shortly after acute kidney injury many of the quiescent papillary LRC started proliferating (Oliver JA, Klinakis A, Cheema FH, Friedlander J, Sampogna RV, Martens TP, Liu C, Efstratiadis A, Al-Awqati Q. J Am Soc Nephrol 20: 2315-2327, 2009; Oliver JA, Maarouf O, Cheema FH, Martens TP, Al-Awqati Q. J Clin Invest 114: 795-804, 2004) and, with cell-tracking experiments, we found upward migration of some papillary cells including LRC (Oliver JA, Klinakis A, Cheema FH, Friedlander J, Sampogna RV, Martens TP, Liu C, Efstratiadis A, Al-Awqati Q. J Am Soc Nephrol 20: 2315-2327, 2009). To identify molecular cues involved in the activation (i.e., proliferation and/or migration) of the papillary LRC that follows injury, we isolated these cells from the H2B-GFP mice and found that they migrated and proliferated in response to the cytokine stromal cell-derived factor-1 (SDF-1). Moreover, in a papillary organ culture assay, the cell growth out of the upper papilla was dependent on the interaction of SDF-1 with its receptor Cxcr4. Interestingly, location of these two proteins in the kidney revealed a complementary location, with SDF-1 being preferentially expressed in the medulla and Cxcr4 more abundant in the papilla. Blockade of Cxcr4 in vivo prevented mobilization of papillary LRC after transient kidney ischemic injury and worsened its functional consequences. The data indicate that the SDF-1/Cxcr4 axis is a critical regulator of papillary LRC activation following transient kidney injury and during organ repair.

  8. SDF-1 activates papillary label-retaining cells during kidney repair from injury

    PubMed Central

    Maarouf, Omar; Cheema, Faisal H.; Liu, Charles; Zhang, Qing-Yin; Kraus, Carl; Zeeshan Afzal, M.; Firdous, Mamoona; Klinakis, Apostolos; Efstratiadis, Argiris; Al-Awqati, Qais

    2012-01-01

    The adult kidney contains a population of low-cycling cells that resides in the papilla. These cells retain for long periods S-phase markers given as a short pulse early in life; i.e., they are label-retaining cells (LRC). In previous studies in adult rat and mice, we found that shortly after acute kidney injury many of the quiescent papillary LRC started proliferating (Oliver JA, Klinakis A, Cheema FH, Friedlander J, Sampogna RV, Martens TP, Liu C, Efstratiadis A, Al-Awqati Q. J Am Soc Nephrol 20: 2315–2327, 2009; Oliver JA, Maarouf O, Cheema FH, Martens TP, Al-Awqati Q. J Clin Invest 114: 795–804, 2004) and, with cell-tracking experiments, we found upward migration of some papillary cells including LRC (Oliver JA, Klinakis A, Cheema FH, Friedlander J, Sampogna RV, Martens TP, Liu C, Efstratiadis A, Al-Awqati Q. J Am Soc Nephrol 20: 2315–2327, 2009). To identify molecular cues involved in the activation (i.e., proliferation and/or migration) of the papillary LRC that follows injury, we isolated these cells from the H2B-GFP mice and found that they migrated and proliferated in response to the cytokine stromal cell-derived factor-1 (SDF-1). Moreover, in a papillary organ culture assay, the cell growth out of the upper papilla was dependent on the interaction of SDF-1 with its receptor Cxcr4. Interestingly, location of these two proteins in the kidney revealed a complementary location, with SDF-1 being preferentially expressed in the medulla and Cxcr4 more abundant in the papilla. Blockade of Cxcr4 in vivo prevented mobilization of papillary LRC after transient kidney ischemic injury and worsened its functional consequences. The data indicate that the SDF-1/Cxcr4 axis is a critical regulator of papillary LRC activation following transient kidney injury and during organ repair. PMID:22461304

  9. In Vitro Generation of Antigen-Specific T Cells from Induced Pluripotent Stem Cells of Antigen-Specific T Cell Origin.

    PubMed

    Kaneko, Shin

    2016-01-01

    Induced pluripotent stem (iPS) cells derived from T lymphocyte (T-iPS cells) preserve the T cell receptor (TCR) α and β gene rearrangements identical to the original T cell clone. Re-differentiated CD8 single positive αβ T cells from the T-iPS cells exhibited antigen-specific cytotoxicity, improved proliferative response, and elongation of telomere indicating rejuvenation of antigen specific T cell immunity in vitro. To regenerate antigen specific cytotoxic T lymphocytes (CTL), first, we have optimized a method for reprogramming-resistant CD8 T cell clones into T-iPS cells by using sendaiviral vectors. Second, we have optimized stepwise differentiation methods for inducing hematopoietic progenitor cells, T cell progenitors, and functionally matured CD8 single positive CTL. These protocols provide useful in vitro tools and models both for research of antigen-specific T cell immunotherapy and for research of normal and pathological thymopoiesis.

  10. Regulation of proliferating cell nuclear antigen ubiquitination in mammalian cells

    PubMed Central

    Niimi, Atsuko; Brown, Stephanie; Sabbioneda, Simone; Kannouche, Patricia L.; Scott, Andrew; Yasui, Akira; Green, Catherine M.; Lehmann, Alan R.

    2008-01-01

    After exposure to DNA-damaging agents that block the progress of the replication fork, monoubiquitination of proliferating cell nuclear antigen (PCNA) mediates the switch from replicative to translesion synthesis DNA polymerases. We show that in human cells, PCNA is monoubiquitinated in response to methyl methanesulfonate and mitomycin C, as well as UV light, albeit with different kinetics, but not in response to bleomycin or camptothecin. Cyclobutane pyrimidine dimers are responsible for most of the PCNA ubiquitination events after UV-irradiation. Failure to ubiquitinate PCNA results in substantial sensitivity to UV and methyl methanesulfonate, but not to camptothecin or bleomycin. PCNA ubiquitination depends on Replication Protein A (RPA), but is independent of ATR-mediated checkpoint activation. After UV-irradiation, there is a temporal correlation between the disappearance of the deubiquitinating enzyme USP1 and the presence of PCNA ubiquitination, but this correlation was not found after chemical mutagen treatment. By using cells expressing photolyases, we are able to remove the UV lesions, and we show that PCNA ubiquitination persists for many hours after the damage has been removed. We present a model of translesion synthesis behind the replication fork to explain the persistence of ubiquitinated PCNA. PMID:18845679

  11. Divergent members of a single autoreactive B cell clone retain specificity for apoptotic blebs.

    PubMed

    Neeli, Indira; Richardson, Mekel M; Khan, Salar N; Nicolo, Danielle; Monestier, Marc; Radic, Marko Z

    2007-03-01

    Specificity for double-stranded DNA can arise due to somatic mutations within one of the branches of an autoreactive B cell clone. However, it is not known whether a different autospecificity predates anti-dsDNA and whether separate offshoots of an expanding B cell clone retain or evolve alternative specificities. We compared 3H9, an anti-dsDNA IgG, to 4H8 and 1A11, antibodies produced by hybridomas representing an alternative branch of the 3H9 B cell clone. All three IgG bound chromatin in ELISA and apoptotic cells in confocal microscopy, yet only 3H9 bound dsDNA, as measured by plasmon resonance. Moreover, we demonstrate that despite the unique specificity of 3H9 for dsDNA, all three clone members exhibited indistinguishable binding to chromatin. The binding to chromatin and apoptotic cells was unaffected by N-linked glycosylation in L chain CDR1, a modification that results from a replacement of serine 26 with asparagine in 4H8 and 1A11. These data provide the first evidence that specificity for nucleosome epitopes on apoptotic cells provides the initial positive stimulus for somatic variants that comprise a B cell clone, including those that subsequently acquire specificity for dsDNA. Conversely, selection of autoreactive B cells for binding to apoptotic cells leads to clonal expansion, antibody diversification, and the development of linked sets of anti-nuclear autoantibodies.

  12. Label-Retaining Stromal Cells in Mouse Endometrium Awaken for Expansion and Repair After Parturition

    PubMed Central

    Cao, Mingzhu; Yeung, William S.B.

    2015-01-01

    Human and mouse endometrium undergo dramatic cellular reorganization during pregnancy and postpartum. Somatic stem cells maintain homeostasis of the tissue by providing a cell reservoir for regeneration. We hypothesized that endometrial cells with quiescent properties (stem/progenitor cells) were involved in the regeneration of the endometrial tissue. Given that stem cells divide infrequently, they can retain the DNA synthesis label [bromodeoxyuridine (BrdU)] after a prolonged chase period. In this study, prepubertal mice were pulsed with BrdU and after a 6-week chase a small population of label-retaining stromal cells (LRSC) was located primarily beneath the luminal epithelium, adjacent to blood vessels, and near the endometrial–myometrial junction. Marker analyses suggested that they were of mesenchymal origin expressing CD44+, CD90+, CD140b+, CD146+, and Sca-1+. During pregnancy, nonproliferating LRSC predominately resided at the interimplantation/placental loci of the gestational endometrium. Immediately after parturition, a significant portion of the LRSC underwent proliferation (BrdU+/Ki-67+) and expressed total and active β-catenin. The β-catenin expression in the LRSC was transiently elevated at postpartum day (PPD) 1. The proliferation of LRSC resulted in a significant decline in the proportion of LRSC in the postpartum uterus. The LRSC returned to dormancy at PPD7, and the percentage of LRSC remained stable thereafter until 11 weeks. This study demonstrated that LRSC can respond efficiently to physiological stimuli upon initiation of uterine involution and return to its quiescent state after postpartum repair. PMID:25386902

  13. Genomic Typing of Red Cell Antigens

    DTIC Science & Technology

    2011-09-01

    Antigen‐Matched  Red  Cells   for  Sickle   Cell   Anemia  Patients  Using  Molecular Typing to Augment Testing: Meghan Delaney, Prashant Gaur, Askale...H, Constans J, Quilici JC, Lefevre‐Witier P, Sevin J, Stevens M: Study of red blood  cell  and serum enzymes in  five  Pyrenean communities and in a...Antigen‐Matched Red  Cells  for  Sickle   Cell  Anemia Patients  Using Molecular Typing to Augment Testing: AABB (poster) 2009.  Background: Patients with  sickle

  14. Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

    PubMed Central

    Schütz, Christian; Fleck, Martin; Schneck, Jonathan P.; Oelke, Mathias

    2014-01-01

    Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion. PMID:25145915

  15. B Cells Loaded with Synthetic Particulate Antigens: A Versatile Platform To Generate Antigen-Specific Helper T Cells for Cell Therapy.

    PubMed

    Sicard, Antoine; Koenig, Alice; Graff-Dubois, Stéphanie; Dussurgey, Sébastien; Rouers, Angéline; Dubois, Valérie; Blanc, Pascal; Chartoire, Dimitri; Errazuriz-Cerda, Elisabeth; Paidassi, Helena; Taillardet, Morgan; Morelon, Emmanuel; Moris, Arnaud; Defrance, Thierry; Thaunat, Olivier

    2016-01-13

    Adoptive cell therapy represents a promising approach for several chronic diseases. This study describes an innovative strategy for biofunctionalization of nanoparticles, allowing the generation of synthetic particulate antigens (SPAg). SPAg activate polyclonal B cells and vectorize noncognate proteins into their endosomes, generating highly efficient stimulators for ex vivo expansion of antigen-specific CD4+ T cells. This method also allows harnessing the ability of B cells to polarize CD4+ T cells into effectors or regulators.

  16. Telomerase-immortalized non-malignant human prostate epithelial cells retain the properties of multipotent stem cells

    SciTech Connect

    Li Hongzhen; Zhou Jianjun; Miki, Jun; Furusato, Bungo; Gu Yongpeng; Srivastava, Shiv; McLeod, David G.; Vogel, Jonathan C.; Rhim, Johng S.

    2008-01-01

    Understanding prostate stem cells may provide insight into the origin of prostate cancer. Primary cells have been cultured from human prostate tissue but they usually survive only 15-20 population doublings before undergoing senescence. We report here that RC-170N/h/clone 7 cells, a clonal cell line from hTERT-immortalized primary non-malignant tissue-derived human prostate epithelial cell line (RC170N/h), retain multipotent stem cell properties. The RC-170N/h/clone 7 cells expressed a human embryonic stem cell marker, Oct-4, and potential prostate epithelial stem cell markers, CD133, integrin {alpha}2{beta}1{sup hi} and CD44. The RC-170N/h/clone 7 cells proliferated in KGM and Dulbecco's Modified Eagle Medium with 10% fetal bovine serum and 5 {mu}g/ml insulin (DMEM + 10% FBS + Ins.) medium, and differentiated into epithelial stem cells that expressed epithelial cell markers, including CK5/14, CD44, p63 and cytokeratin 18 (CK18); as well as the mesenchymal cell markers, vimentin, desmin; the neuron and neuroendocrine cell marker, chromogranin A. Furthermore the RC170 N/h/clone 7 cells differentiated into multi tissues when transplanted into the sub-renal capsule and subcutaneously of NOD-SCID mice. The results indicate that RC170N/h/clone 7 cells retain the properties of multipotent stem cells and will be useful as a novel cell model for studying the mechanisms of human prostate stem cell differentiation and transformation.

  17. Cell surface expression of hepatitis B surface and core antigens in transfected rat fibroblast cell lines.

    PubMed

    Gholson, C F; Siddiqui, A; Vierling, J M

    1990-04-01

    Hepatocellular necrosis during hepatitis B virus infection is hypothesized to result from host immune responses against either hepatitis B surface antigen or hepatitis B core antigen expressed on the surface membrane of infected hepatocytes. To study the capacity of hepatitis B deoxyribonucleic acid to induce membrane expression of either hepatitis B surface antigen or hepatitis B core antigen in vitro, we assessed transfected rat fibroblast cell lines by indirect immunofluorescence. Rat fibroblasts were transfected with plasmid vectors containing the natural promoters, native enhancer, and uninterrupted sequences of either the Pre S/S gene or core gene. Resulting cell lines produced hepatitis B surface antigen and hepatitis B core antigen/hepatitis B e antigen, respectively. Immunofluorescence microscopy or flow cytometry showed that hepatitis B surface antigen and hepatitis B core antigen were expressed in a granular pattern in the surface membrane of transfected cells. We conclude that surface membrane expression of both hepatitis B surface antigen and hepatitis B core antigen is an intrinsic consequence of expression of either the Pre S/S or core gene.

  18. Chimeric antigen receptor T-cell therapies for lymphoma.

    PubMed

    Brudno, Jennifer N; Kochenderfer, James N

    2017-08-31

    New therapies are needed for patients with Hodgkin or non-Hodgkin lymphomas that are resistant to standard therapies. Indeed, unresponsiveness to standard chemotherapy and relapse after autologous stem-cell transplantation are indicators of an especially poor prognosis. Chimeric antigen receptor (CAR) T cells are emerging as a novel treatment modality for these patients. Clinical trial data have demonstrated the potent activity of anti-CD19 CAR T cells against multiple subtypes of B-cell lymphoma, including diffuse large-B-cell lymphoma (DLBCL), follicular lymphoma, mantle-cell lymphoma, and marginal-zone lymphoma. Importantly, anti-CD19 CAR T cells have impressive activity against chemotherapy-refractory lymphoma, inducing durable complete remissions lasting >2 years in some patients with refractory DLBCL. CAR-T-cell therapies are, however, associated with potentially fatal toxicities, including cytokine-release syndrome and neurological toxicities. CAR T cells with novel target antigens, including CD20, CD22, and κ-light chain for B-cell lymphomas, and CD30 for Hodgkin and T-cell lymphomas, are currently being investigated in clinical trials. Centrally manufactured CAR T cells are also being tested in industry-sponsored multicentre clinical trials, and will probably soon become a standard therapy. Herein, we review the clinical efficacy and toxicity of CAR-T-cell therapies for lymphoma, and discuss their limitations and future directions with regard to toxicity management, CAR designs and CAR-T-cell phenotypes, conditioning regimens, and combination therapies.

  19. Twist1-positive epithelial cells retain adhesive and proliferative capacity throughout dissemination

    PubMed Central

    Shamir, Eliah R.; Coutinho, Kester; Georgess, Dan; Auer, Manfred

    2016-01-01

    ABSTRACT Dissemination is the process by which cells detach and migrate away from a multicellular tissue. The epithelial-to-mesenchymal transition (EMT) conceptualizes dissemination in a stepwise fashion, with downregulation of E-cadherin leading to loss of intercellular junctions, induction of motility, and then escape from the epithelium. This gain of migratory activity is proposed to be mutually exclusive with proliferation. We previously developed a dissemination assay based on inducible expression of the transcription factor Twist1 and here utilize it to characterize the timing and dynamics of intercellular adhesion, proliferation and migration during dissemination. Surprisingly, Twist1+ epithelium displayed extensive intercellular junctions, and Twist1– luminal epithelial cells could still adhere to disseminating Twist1+ cells. Although proteolysis and proliferation were both observed throughout dissemination, neither was absolutely required. Finally, Twist1+ cells exhibited a hybrid migration mode; their morphology and nuclear deformation were characteristic of amoeboid cells, whereas their dynamic protrusive activity, pericellular proteolysis and migration speeds were more typical of mesenchymal cells. Our data reveal that epithelial cells can disseminate while retaining competence to adhere and proliferate. PMID:27402962

  20. Stem cells expanded from the human embryonic hindbrain stably retain regional specification and high neurogenic potency.

    PubMed

    Tailor, Jignesh; Kittappa, Raja; Leto, Ketty; Gates, Monte; Borel, Melodie; Paulsen, Ole; Spitzer, Sonia; Karadottir, Ragnhildur Thora; Rossi, Ferdinando; Falk, Anna; Smith, Austin

    2013-07-24

    Stem cell lines that faithfully maintain the regional identity and developmental potency of progenitors in the human brain would create new opportunities in developmental neurobiology and provide a resource for generating specialized human neurons. However, to date, neural progenitor cultures derived from the human brain have either been short-lived or exhibit restricted, predominantly glial, differentiation capacity. Pluripotent stem cells are an alternative source, but to ascertain definitively the identity and fidelity of cell types generated solely in vitro is problematic. Here, we show that hindbrain neuroepithelial stem (hbNES) cells can be derived and massively expanded from early human embryos (week 5-7, Carnegie stage 15-17). These cell lines are propagated in adherent culture in the presence of EGF and FGF2 and retain progenitor characteristics, including SOX1 expression, formation of rosette-like structures, and high neurogenic capacity. They generate GABAergic, glutamatergic and, at lower frequency, serotonergic neurons. Importantly, hbNES cells stably maintain hindbrain specification and generate upper rhombic lip derivatives on exposure to bone morphogenetic protein (BMP). When grafted into neonatal rat brain, they show potential for integration into cerebellar development and produce cerebellar granule-like cells, albeit at low frequency. hbNES cells offer a new system to study human cerebellar specification and development and to model diseases of the hindbrain. They also provide a benchmark for the production of similar long-term neuroepithelial-like stem cells (lt-NES) from pluripotent cell lines. To our knowledge, hbNES cells are the first demonstration of highly expandable neuroepithelial stem cells derived from the human embryo without genetic immortalization.

  1. Decreased neutralizing antigenicity in IBV S1 protein expressed from mammalian cells.

    PubMed

    Andoh, Kiyohiko; Suenaga, Kiyotaka; Sakaguchi, Masashi; Yamazaki, Kenichi; Honda, Takashi

    2015-10-02

    We evaluated the antigenicity of recombinant infectious bronchitis virus (IBV) S1 protein expressed in mammalian cells. Recombinant S1 was expressed as a secreted protein fused with a trimerization motif peptide, then purified using Ni Sepharose. The purified protein was analyzed by Western blotting, mixed with oil adjuvant, and administered to 29-day-old specific-pathogen-free chickens. Six weeks after immunization, anti-IBV neutralizing titer and anti-S1 ELISA titer were determined; immunized chickens then were inoculated with IBV via the trachea and ciliary activity was observed. Results showed that the recombinant S1 protein was highly glycosylated, and the neutralizing antigenicity of recombinant S1 protein was lower than that of inactivated virus. However, anti-S1 ELISA indicated that the recombinant S1 protein induced antibodies against S1. These results suggest that the recombinant S1 may retain non-neutralizing epitopes but have unnatural glycosylation pattern and conformation, resulting in lacking neutralizing conformational epitopes. In conclusion, the neutralizing antigenicity of recombinant S1 protein expressed from mammalian cells was decreased, and was not sufficient to induce neutralizing antibodies. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Hepatitis B surface antigen (HBsAg) expression in plant cell culture: Kinetics of antigen accumulation in batch culture and its intracellular form.

    PubMed

    Smith, Mark L; Mason, Hugh S; Shuler, Michael L

    2002-12-30

    The production of edible vaccines in transgenic plants and plant cell culture may be improved through a better understanding of antigen processing and assembly. The hepatitis B surface antigen (HBsAg) was chosen for study because it undergoes substantial and complex post-translational modifications, which are necessary for its immunogenicity. This antigen was expressed in soybean (Glycine max L. Merr. cv Williams 82) and tobacco NT1 (Nicotiana tabacum L.) cell suspension cultures, and HBsAg production in batch culture was characterized. The plant-derived antigen consisted predominantly of disulfide cross-linked HBsAg protein (p24(s)) dimers, which were all membrane associated. Similar to yeast, the plant-expressed HBsAg was retained intracellularly. The maximal HBsAg titers were obtained with soybean suspension cultures (20-22 mg/L) with titers in tobacco cultures being approximately 10-fold lower. For soybean cells, electron microscopy and immunolocalization demonstrated that all the HBsAg was localized to the endoplasmic reticulum (ER) and provoked dilation and proliferation of the ER network. Sucrose gradient analysis of crude extracts showed that HBsAg had a complex size distribution uncharacteristic of the antigen's normal structure of uniform 22-nm virus-like particles. The extent of authentic epitope formation was assessed by comparing total p24(s) synthesized to that reactive by polyclonal and monoclonal immunoassays. Depending on culture age, between 40% and 100% of total p24(s) was polyclonal antibody reactive whereas between 6% and 37% was recognized by a commercial monoclonal antibody assay. Possible strategies to increase HBsAg production and improve post-translational processing are discussed.

  3. Photoaffinity antigens for human gammadelta T cells.

    PubMed

    Sarikonda, Ghanashyam; Wang, Hong; Puan, Kia-Joo; Liu, Xiao-hui; Lee, Hoi K; Song, Yongcheng; Distefano, Mark D; Oldfield, Eric; Prestwich, Glenn D; Morita, Craig T

    2008-12-01

    Vgamma2Vdelta2 T cells comprise the major subset of peripheral blood gammadelta T cells in humans and expand during infections by recognizing small nonpeptide prenyl pyrophosphates. These molecules include (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP), a microbial isoprenoid intermediate, and isopentenyl pyrophosphate, an endogenous isoprenoid intermediate. Recognition of these nonpeptide Ags is mediated by the Vgamma2Vdelta2 T cell Ag receptor. Several findings suggest that prenyl pyrophosphates are presented by an Ag-presenting molecule: contact between T cells and APC is required, the Ags do not bind the Vgamma2Vdelta2 TCR directly, and Ag recognition is abrogated by TCR mutations in CDRs distant from the putative Ag recognition site. Identification of the putative Ag-presenting molecule, however, has been hindered by the inability to achieve stable association of nonpeptide prenyl pyrophosphate Ags with the presenting molecule. In this study, we show that photoaffinity analogues of HMBPP, meta/para-benzophenone-(methylene)-prenyl pyrophosphates (m/p-BZ-(C)-C(5)-OPP), can crosslink to the surface of tumor cell lines and be presented as Ags to gammadelta T cells. Mutant tumor cell lines lacking MHC class I, MHC class II, beta(2)-microglobulin, and CD1, as well as tumor cell lines from a variety of tissues and individuals, will all crosslink to and present m-BZ-C(5)-OPP. Finally, pulsing of BZ-(C)-C(5)-OPP is inhibited by isopentenyl pyrophosphate and an inactive analog, suggesting that they bind to the same molecule. Taken together, these results suggest that nonpeptide Ags are presented by a novel-Ag-presenting molecule that is widely distributed and nonpolymorphic, but not classical MHC class I, MHC class II, or CD1.

  4. Orthotropic Laminated Open-cell Frameworks Retaining Strong Auxeticity under Large Uniaxial Loading

    NASA Astrophysics Data System (ADS)

    Tanaka, Hiro; Suga, Kaito; Iwata, Naoki; Shibutani, Yoji

    2017-01-01

    Anisotropic materials form inside living tissue and are widely applied in engineered structures, where sophisticated structural and functional design principles are essential to employing these materials. This paper presents a candidate laminated open-cell framework, which is an anisotropic material that shows remarkable mechanical performance. Using additive manufacturing, artificial frameworks are fabricated by lamination of in-plane orthotropic microstructures made of elbowed beam and column members; this fabricated structure features orthogonal anisotropy in three-dimensional space. Uniaxial loading tests reveal strong auxeticity (high negative Poisson’s ratios) in the out-of-plane direction, which is retained reproducibly up to the nonlinear elastic region, and is equal under tensile and compressive loading. Finite element simulations support the observed auxetic behaviors for a unit cell in the periodic framework, which preserve the theoretical elastic properties of an orthogonal solid. These findings open the possibility of conceptual materials design based on geometry.

  5. Orthotropic Laminated Open-cell Frameworks Retaining Strong Auxeticity under Large Uniaxial Loading.

    PubMed

    Tanaka, Hiro; Suga, Kaito; Iwata, Naoki; Shibutani, Yoji

    2017-01-04

    Anisotropic materials form inside living tissue and are widely applied in engineered structures, where sophisticated structural and functional design principles are essential to employing these materials. This paper presents a candidate laminated open-cell framework, which is an anisotropic material that shows remarkable mechanical performance. Using additive manufacturing, artificial frameworks are fabricated by lamination of in-plane orthotropic microstructures made of elbowed beam and column members; this fabricated structure features orthogonal anisotropy in three-dimensional space. Uniaxial loading tests reveal strong auxeticity (high negative Poisson's ratios) in the out-of-plane direction, which is retained reproducibly up to the nonlinear elastic region, and is equal under tensile and compressive loading. Finite element simulations support the observed auxetic behaviors for a unit cell in the periodic framework, which preserve the theoretical elastic properties of an orthogonal solid. These findings open the possibility of conceptual materials design based on geometry.

  6. Orthotropic Laminated Open-cell Frameworks Retaining Strong Auxeticity under Large Uniaxial Loading

    PubMed Central

    Tanaka, Hiro; Suga, Kaito; Iwata, Naoki; Shibutani, Yoji

    2017-01-01

    Anisotropic materials form inside living tissue and are widely applied in engineered structures, where sophisticated structural and functional design principles are essential to employing these materials. This paper presents a candidate laminated open-cell framework, which is an anisotropic material that shows remarkable mechanical performance. Using additive manufacturing, artificial frameworks are fabricated by lamination of in-plane orthotropic microstructures made of elbowed beam and column members; this fabricated structure features orthogonal anisotropy in three-dimensional space. Uniaxial loading tests reveal strong auxeticity (high negative Poisson’s ratios) in the out-of-plane direction, which is retained reproducibly up to the nonlinear elastic region, and is equal under tensile and compressive loading. Finite element simulations support the observed auxetic behaviors for a unit cell in the periodic framework, which preserve the theoretical elastic properties of an orthogonal solid. These findings open the possibility of conceptual materials design based on geometry. PMID:28051133

  7. Transformation of human T-cell clones by Herpesvirus saimiri: intact antigen recognition by autonomously growing myelin basic protein-specific T cells.

    PubMed Central

    Weber, F; Meinl, E; Drexler, K; Czlonkowska, A; Huber, S; Fickenscher, H; Müller-Fleckenstein, I; Fleckenstein, B; Wekerle, H; Hohlfeld, R

    1993-01-01

    Herpesvirus saimiri has recently been shown to immortalize human T cells. It was unknown, however, whether Herpesvirus saimiri transformation affects T-cell receptor (TCR) expression and signal transduction. In the present study, we have transformed CD4+ human T-cell clones specific for human myelin basic protein. The transformed T cells were grown in interleukin 2 and divided in the absence of antigen and antigen-presenting cells. They retained the membrane phenotype of activated T cells and secreted the cytokines interferon gamma and lymphotoxin, but interleukin 4 was not detected. Further, the transformed T cells continued to express the original TCR as demonstrated by TCR variable-region-V beta-specific monoclonal antibodies and TCR sequencing. Antigen-specific recognition and signal transduction by the TCR were demonstrated by myelin-basic-protein-induced HLA-DR-restricted secretion of interferon gamma and lymphotoxin and by myelin-basic-protein-specific proliferation. Antigen specificity and reactivity have been maintained for > 1 year after transformation. Transformation with Herpesvirus saimiri now allows the production of virtually unlimited numbers of (auto)antigen-specific T cells expressing functional TCR and a stable membrane phenotype. This technology will facilitate studies of the pathogenesis of putative autoimmune diseases, such as multiple sclerosis, and may be of help in TCR-targeted immunotherapy. PMID:7504291

  8. Bovine mammary epithelial cells retain stem-like phenotype in long-term cultures.

    PubMed

    Cravero, Diego; Diego, Cravero; Martignani, Eugenio; Eugenio, Martignani; Miretti, Silvia; Silvia, Miretti; Macchi, Elisabetta; Elisabetta, Macchi; Accornero, Paolo; Paolo, Accornero; Baratta, Mario; Mario, Baratta

    2014-10-01

    The detection and characterization of bovine mammary stem cells may give a better understanding of the cyclic characteristic of mammary gland development. In turn, this could potentially offer techniques to manipulate lactation yield and for regenerative medicine. We previously demonstrated that adult stem cells reside in the bovine mammary gland and possess an intrinsic regenerative potential. In vitro maintenance and expansion of this primitive population is a challenging task that could make easier the study of adult mammary stem cells. The aim of this study is to investigate this possibility. Different subpopulations of mammary epithelial cells emerge when they are cultured in two defined culture conditions. Specific cell differentiation markers as cytokeratin 18 (CK18) and cytokeratin 14 (CK14) were expressed with significant differences according to culture conditions. Vimentin, a well-known fibroblast marker was observed to increase significantly (P < 0.5) only after day 20. In both conditions, after prolonged culture (25 days) a subset of cells still retained regenerative capabilities. These cells were able to form organized pseudo-alveoli when transplanted in immunodeficient mice as shown by the expression of cytokeratin 14 (CK14), cytokeratin 18 (CK18), p63 (a mammary basal cell layer marker) and Epithelial Cell Adhesion Molecule (EpCAM). We also were able to observe the presence of milk proteins signal in these regenerated structures, which is a specific marker of functional mammary alveoli. Progenitor content was also analyzed in vitro through Colony-Forming Cell (CFC) assays with no substantial differences among culture conditions and time points. These results demonstrate that long-term culture of a multipotent cell subpopulation with intrinsic regenerative potential is possible.

  9. The activation of the adaptive immune system: cross-talk between antigen-presenting cells, T cells and B cells.

    PubMed

    den Haan, Joke M M; Arens, Ramon; van Zelm, Menno C

    2014-12-01

    The adaptive immune system consists of T and B cells that express clonally distributed antigen receptors. To achieve functional adaptive immune responses, antigen-specific T cell populations are stimulated by professional antigen-presenting cells like dendritic cells (DCs), which provide crucial stimulatory signals for efficient expansion and development of effector functions. Antigen-specific B cells receive costimulatory signals from helper T cells to stimulate affinity maturation and isotype switching. Here we elaborate on the interactions between DCs, T cells and B cells, and on the important signals for efficient induction of adaptive immune responses. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Chimeric antigen receptor T-cell therapy for ALL.

    PubMed

    Maude, Shannon L; Shpall, Elizabeth J; Grupp, Stephan A

    2014-12-05

    Relapsed and refractory leukemias pose substantial challenges in both children and adults, with very little progress being made in more than a decade. Targeted immunotherapy using chimeric antigen receptor (CAR)-modified T cells has emerged as a potent therapy with an innovative mechanism. Dramatic clinical responses with complete remission rates as high as 90% have been reported using CAR-modified T cells directed against the B-cell-specific antigen CD19 in patients with relapsed/refractory acute lymphoblastic leukemia. Supraphysiologic T-cell proliferation, a hallmark of this therapy, contributes to both efficacy and the most notable toxicity, cytokine release syndrome, posing a unique challenge for toxicity management. Further studies are necessary to identify additional targets, standardize approaches to cytokine release syndrome management, and determine the durability of remissions. © 2014 by The American Society of Hematology. All rights reserved.

  11. Computational prediction of B cell epitopes from antigen sequences.

    PubMed

    Gao, Jianzhao; Kurgan, Lukasz

    2014-01-01

    Computational identification of B-cell epitopes from antigen chains is a difficult and actively pursued research topic. Efforts towards the development of method for the prediction of linear epitopes span over the last three decades, while only recently several predictors of conformational epitopes were released. We review a comprehensive set of 13 recent approaches that predict linear and 4 methods that predict conformational B-cell epitopes from the antigen sequences. We introduce several databases of B-cell epitopes, since the availability of the corresponding data is at the heart of the development and validation of computational predictors. We also offer practical insights concerning the use and availability of these B-cell epitope predictors, and motivate and discuss feature research in this area.

  12. Label-retaining liver cancer cells are relatively resistant to sorafenib.

    PubMed

    Xin, Hong-Wu; Ambe, Chenwi M; Hari, Danielle M; Wiegand, Gordon W; Miller, Tyler C; Chen, Jin-Qiu; Anderson, Andrew J; Ray, Satyajit; Mullinax, John E; Koizumi, Tomotake; Langan, Russell C; Burka, Douglas; Herrmann, Michelle A; Goldsmith, Paul K; Stojadinovic, Alexander; Rudloff, Udo; Thorgeirsson, Snorri S; Avital, Itzhak

    2013-12-01

    The standard therapy for advanced hepatocellular carcinoma (HCC) is sorafenib, with most patients experiencing disease progression within 6 months. Label-retaining cancer cells (LRCC) represent a novel subpopulation of cancer stem cells (CSC). The objective was to test whether LRCC are resistant to sorafenib. We tested human HCC derived LRCC and non-LRCC before and after treatment with sorafenib. LRCC derived from human HCC are relatively resistant to sorafenib. The proportion of LRCC in HCC cell lines is increased after sorafenib while the general population of cancer cells undergoes growth suppression. We show that LRCC demonstrate improved viability and toxicity profiles, and reduced apoptosis, over non-LRCC. We show that after treatment with sorafenib, LRCC upregulate the CSC marker aldehyde dehydrogenase 1 family, wingless-type MMTV-integration-site family, cell survival and proliferation genes, and downregulate apoptosis, cell cycle arrest, cell adhesion and stem cells differentiation genes. This phenomenon was accompanied by non-uniform activation of specific isoforms of the sorafenib target proteins extracellular-signal-regulated kinases and v-akt-murine-thymoma-viral-oncogene homologue (AKT) in LRCC but not in non-LRCC. A molecular pathway map for sorafenib treated LRCC is proposed. Our results suggest that HCC derived LRCC are relatively resistant to sorafenib. Since LRCC can generate tumours with as few as 10 cells, our data suggest a potential role for these cells in disease recurrence. Further investigation of this phenomenon might provide novel insights into cancer biology, cancer recurrence and drug resistance with important implications for the development of novel cancer therapies based on targeting LRCC.

  13. Expanded Human Blood-Derived γδT Cells Display Potent Antigen-Presentation Functions

    PubMed Central

    Khan, Mohd Wajid A.; Curbishley, Stuart M.; Chen, Hung-Chang; Thomas, Andrew D.; Pircher, Hanspeter; Mavilio, Domenico; Steven, Neil M.; Eberl, Matthias; Moser, Bernhard

    2014-01-01

    Cell-based immunotherapy strategies target tumors directly (via cytolytic effector cells) or aim at mobilizing endogenous anti-tumor immunity. The latter approach includes dendritic cells (DC) most frequently in the form of in vitro cultured peripheral blood monocytes-derived DC. Human blood γδT cells are selective for a single class of non-peptide agonists (“phosphoantigens”) and develop into potent antigen-presenting cells (APC), termed γδT-APC within 1–3 days of in vitro culture. Availability of large numbers of γδT-APC would be advantageous for use as a novel cellular vaccine. We here report optimal γδT cell expansion (>107 cells/ml blood) when peripheral blood mononuclear cells (PBMC) from healthy individuals and melanoma patients were stimulated with zoledronate and then cultured for 14 days in the presence of IL-2 and IL-15, yielding γδT cell cultures of variable purity (77 ± 21 and 56 ± 26%, respectively). They resembled effector memory αβT (TEM) cells and retained full functionality as assessed by in vitro tumor cell killing as well as secretion of pro-inflammatory cytokines (IFNγ, TNFα) and cell proliferation in response to stimulation with phosphoantigens. Importantly, day 14 γδT cells expressed numerous APC-related cell surface markers and, in agreement, displayed potent in vitro APC functions. Day 14 γδT cells from PBMC of patients with cancer were equally effective as their counterparts derived from blood of healthy individuals and triggered potent CD8+ αβT cell responses following processing and cross-presentation of simple (influenza M1) and complex (tuberculin purified protein derivative) protein antigens. Of note, and in clear contrast to peripheral blood γδT cells, the ability of day 14 γδT cells to trigger antigen-specific αβT cell responses did not depend on re-stimulation. We conclude that day 14 γδT cell cultures provide a convenient source of autologous APC for use in immunotherapy of patients

  14. Existence of a squamous cell carcinoma antigen-immunoglobulin complex causes a deviation between squamous cell carcinoma antigen concentrations determined using two different immunoassays: first report of squamous cell carcinoma antigen coupling with immunoglobulin A.

    PubMed

    Mori, Eriko; Kurano, Makoto; Tobita, Akiko; Shimosaka, Hironori; Yatomi, Yutaka

    2017-01-01

    Background Squamous cell carcinoma antigen is used as a tumour marker and is routinely measured in clinical laboratories. We validated two different immunoassays and found three cases in which the squamous cell carcinoma antigen concentrations deviated greatly between the two immunoassays. Here, we aimed to elucidate the mechanisms responsible for these deviations. Methods The squamous cell carcinoma antigen concentrations were determined using the ARCHITECT SCC (CLIA method) and the ST AIA-PACK SCC (FEIA method). We performed polyethylene glycol precipitation and size exclusion chromatography to assess the molecular weight and spike recovery and absorption tests to examine the presence of an autoantibody. Results Both methods exhibited good performances for the measurement of squamous cell carcinoma antigen, although a correlation test showed large differences in the squamous cell carcinoma antigen concentrations measured using the two methods in three cases. The results of polyethylene glycol treatment and size exclusion chromatography indicated the existence of a large molecular weight squamous cell carcinoma antigen in these three cases. The spike recovery tests suggested the possible presence of an autoantibody against squamous cell carcinoma antigen. Moreover, the absorption test revealed that large squamous cell carcinoma antigen complexes were formed by the association of squamous cell carcinoma antigen with IgG in two cases and with both IgG and IgA in one case. Conclusions This study describes the existence of large molecular weight squamous cell carcinoma antigen that has complexed with immunoglobulin in the serum samples. The reason for the deviations between the two immunoassays might be due to differences of their reactivities against the squamous cell carcinoma antigen immune complexes with their autoantibody. To our knowledge, this is the first report to describe the coupling of squamous cell carcinoma antigen with IgA.

  15. M. tuberculosis T Cell Epitope Analysis Reveals Paucity of Antigenic Variation and Identifies Rare Variable TB Antigens.

    PubMed

    Coscolla, Mireia; Copin, Richard; Sutherland, Jayne; Gehre, Florian; de Jong, Bouke; Owolabi, Olumuiya; Mbayo, Georgetta; Giardina, Federica; Ernst, Joel D; Gagneux, Sebastien

    2015-11-11

    Pathogens that evade adaptive immunity typically exhibit antigenic variation. By contrast, it appears that although the chronic human tuberculosis (TB)-causing pathogen Mycobacterium tuberculosis needs to counter host T cell responses, its T cell epitopes are hyperconserved. Here we present an extensive analysis of the T cell epitopes of M. tuberculosis. We combined population genomics with experimental immunology to determine the number and identity of T cell epitope sequence variants in 216 phylogenetically diverse strains of M. tuberculosis. Antigen conservation is indeed a hallmark of M. tuberculosis. However, our analysis revealed a set of seven variable antigens that were immunogenic in subjects with active TB. These findings suggest that M. tuberculosis uses mechanisms other than antigenic variation to evade T cells. T cell epitopes that exhibit sequence variation may not be subject to the same evasion mechanisms, and hence vaccines that include such variable epitopes may be more efficacious.

  16. Two distinct antigen systems in human B lymphocytes: identification of cell surface and intracellular antigens using monoclonal antibodies.

    PubMed Central

    Ishii, Y; Takami, T; Yuasa, H; Takei, T; Kikuchi, K

    1984-01-01

    Two distinct antigen systems (L26 and L27) specifically expressed in human B lymphocytes were identified using TB2-2B3 (2B3) and T3-5B3 (5B3) monoclonal antibodies, respectively. Whereas L26 antigen defined by 2B3 were rarely expressed on the surface of B cells but abundant in the cytoplasm, 127 antigens detected by 5B3 was clearly expressed on the cell surface. These two antigens appeared to be restricted in their expression to B cells, as they were found in most B cells but not other cell types including thymocytes, T cells, monocytes and granulocytes. Functional studies demonstrated that L27 was more easily lost from B cells after activation with pokeweed mitogen than was L26. Likewise, plasma cell myeloma, as well as normal plasma cells, was devoid of both L26 and L27, whereas immunoblastic sarcoma of B cell type expressed L26 but not L27. These two antigens co-existed in the same B cell lines including Epstein-Barr virus transformed B cell lines, B cell type acute lymphatic leukaemia (B-ALL) cell line, Burkitt's lymphoma cell lines and myeloma cell lines, but pre-B and common ALL cell lines were entirely negative for both L26 and L27. Immunoprecipitation studies showed that L26 consisted of at least two polypeptide chains with molecular weights of 30K and 33K daltons, which were clearly distinct from HLA-DR antigens. The antigen L27 is presently under study. Images Fig. 2 Fig. 3 PMID:6332692

  17. Dry-Coated Live Viral Vector Vaccines Delivered by Nanopatch Microprojections Retain Long-Term Thermostability and Induce Transgene-Specific T Cell Responses in Mice

    PubMed Central

    Pearson, Frances E.; McNeilly, Celia L.; Crichton, Michael L.; Primiero, Clare A.; Yukiko, Sally R.; Fernando, Germain J. P.; Chen, Xianfeng; Gilbert, Sarah C.; Hill, Adrian V. S.; Kendall, Mark A. F.

    2013-01-01

    The disadvantages of needle-based immunisation motivate the development of simple, low cost, needle-free alternatives. Vaccine delivery to cutaneous environments rich in specialised antigen-presenting cells using microprojection patches has practical and immunological advantages over conventional needle delivery. Additionally, stable coating of vaccine onto microprojections removes logistical obstacles presented by the strict requirement for cold-chain storage and distribution of liquid vaccine, or lyophilised vaccine plus diluent. These attributes make these technologies particularly suitable for delivery of vaccines against diseases such as malaria, which exerts its worst effects in countries with poorly-resourced healthcare systems. Live viral vectors including adenoviruses and poxviruses encoding exogenous antigens have shown significant clinical promise as vaccines, due to their ability to generate high numbers of antigen-specific T cells. Here, the simian adenovirus serotype 63 and the poxvirus modified vaccinia Ankara – two vectors under evaluation for the delivery of malaria antigens to humans – were formulated for coating onto Nanopatch microprojections and applied to murine skin. Co-formulation with the stabilising disaccharides trehalose and sucrose protected virions during the dry-coating process. Transgene-specific CD8+ T cell responses following Nanopatch delivery of both vectors were similar to intradermal injection controls after a single immunisation (despite a much lower delivered dose), though MVA boosting of pre-primed responses with Nanopatch was found to be less effective than the ID route. Importantly, disaccharide-stabilised ChAd63 could be stored for 10 weeks at 37°C with less than 1 log10 loss of viability, and retained single-dose immunogenicity after storage. These data support the further development of microprojection patches for the deployment of live vaccines in hot climates. PMID:23874462

  18. Dry-coated live viral vector vaccines delivered by nanopatch microprojections retain long-term thermostability and induce transgene-specific T cell responses in mice.

    PubMed

    Pearson, Frances E; McNeilly, Celia L; Crichton, Michael L; Primiero, Clare A; Yukiko, Sally R; Fernando, Germain J P; Chen, Xianfeng; Gilbert, Sarah C; Hill, Adrian V S; Kendall, Mark A F

    2013-01-01

    The disadvantages of needle-based immunisation motivate the development of simple, low cost, needle-free alternatives. Vaccine delivery to cutaneous environments rich in specialised antigen-presenting cells using microprojection patches has practical and immunological advantages over conventional needle delivery. Additionally, stable coating of vaccine onto microprojections removes logistical obstacles presented by the strict requirement for cold-chain storage and distribution of liquid vaccine, or lyophilised vaccine plus diluent. These attributes make these technologies particularly suitable for delivery of vaccines against diseases such as malaria, which exerts its worst effects in countries with poorly-resourced healthcare systems. Live viral vectors including adenoviruses and poxviruses encoding exogenous antigens have shown significant clinical promise as vaccines, due to their ability to generate high numbers of antigen-specific T cells. Here, the simian adenovirus serotype 63 and the poxvirus modified vaccinia Ankara--two vectors under evaluation for the delivery of malaria antigens to humans--were formulated for coating onto Nanopatch microprojections and applied to murine skin. Co-formulation with the stabilising disaccharides trehalose and sucrose protected virions during the dry-coating process. Transgene-specific CD8(+) T cell responses following Nanopatch delivery of both vectors were similar to intradermal injection controls after a single immunisation (despite a much lower delivered dose), though MVA boosting of pre-primed responses with Nanopatch was found to be less effective than the ID route. Importantly, disaccharide-stabilised ChAd63 could be stored for 10 weeks at 37°C with less than 1 log10 loss of viability, and retained single-dose immunogenicity after storage. These data support the further development of microprojection patches for the deployment of live vaccines in hot climates.

  19. Immunochemical properties of antigen-specific monkey T-cell suppressor factor induced with a Streptococcus mutans antigen.

    PubMed Central

    Lamb, J R; Zanders, E D; Kontiainen, S; Lehner, T

    1980-01-01

    Antigen-specific suppressor factor could be released from monkey suppressor T cells induced in vitro with a protein antigen isolated from the carcinogenic bacterium Streptococcus mutans. The suppressor activity was due to the factor itself and not to carryover of free antigen. Characterization of the monkey factor revealed it to have a molecular weight of ca. 70,000, and to contain a constant region and determinants encoded by the major histocompatibility complex. The presence of immunoglobulin determinants could not be demonstrated. However, by virtue of its adsorption to specific antigen, an antigen-combining site was shown to be present. The possible regulatory role of streptococcal antigen-specific suppressor factor in protection against dental caries is discussed. PMID:6164645

  20. Toxicities of chimeric antigen receptor T cells: recognition and management

    PubMed Central

    Brudno, Jennifer N.

    2016-01-01

    Chimeric antigen receptor (CAR) T cells can produce durable remissions in hematologic malignancies that are not responsive to standard therapies. Yet the use of CAR T cells is limited by potentially severe toxicities. Early case reports of unexpected organ damage and deaths following CAR T-cell therapy first highlighted the possible dangers of this new treatment. CAR T cells can potentially damage normal tissues by specifically targeting a tumor-associated antigen that is also expressed on those tissues. Cytokine release syndrome (CRS), a systemic inflammatory response caused by cytokines released by infused CAR T cells can lead to widespread reversible organ dysfunction. CRS is the most common type of toxicity caused by CAR T cells. Neurologic toxicity due to CAR T cells might in some cases have a different pathophysiology than CRS and requires different management. Aggressive supportive care is necessary for all patients experiencing CAR T-cell toxicities, with early intervention for hypotension and treatment of concurrent infections being essential. Interleukin-6 receptor blockade with tocilizumab remains the mainstay pharmacologic therapy for CRS, though indications for administration vary among centers. Corticosteroids should be reserved for neurologic toxicities and CRS not responsive to tocilizumab. Pharmacologic management is complicated by the risk of immunosuppressive therapy abrogating the antimalignancy activity of the CAR T cells. This review describes the toxicities caused by CAR T cells and reviews the published approaches used to manage toxicities. We present guidelines for treating patients experiencing CRS and other adverse events following CAR T-cell therapy. PMID:27207799

  1. Microplate cell-retaining methodology for high-content analysis of individual non-adherent unanchored cells in a population.

    PubMed

    Deutsch, Assaf; Zurgil, Naomi; Hurevich, Ihar; Shafran, Yana; Afrimzon, Elena; Lebovich, Pnina; Deutsch, Mordechai

    2006-12-01

    A high throughput Microtiter plate Cell Retainer (MCR) has been developed to enable, for the first time, high-content, time-dependent analysis of the same single non-adherent and non-anchored cells in a large cell population, while bio-manipulating the cells. The identity of each cell in the investigated population is secured, even during bio-manipulation, by cell retention in a specially designed concave microlens, acting as a picoliter well (PW). The MCR technique combines micro-optical features and microtiter plate methodology. The array of PWs serves as the bottom of a microtiter plate, fitted with a unique flow damper element. The latter enables rapid fluid exchange without dislodging the cells from their original PWs, thus maintaining the cells' identity. Loading cell suspensions and reagents into the MCR is performed by simple pouring, followed by gravitational sedimentation and settling of cells into the PWs. Cell viability and cell division within the MCR were shown to be similar to those obtained under similar conditions in a standard microtiter plate. The efficiency of single cell occupancy in the MCR exceeded 90%. No cell dislodging was observed when comparing images before and after bio-manipulations (rinsing, staining, etc.). The MCR permits the performance of kinetic measurements on an individual cell basis. Data acquisition is governed by software, controlling microscope performance, stage position and image acquisition and analysis. The PW's unique micro-optical features enable rapid, simultaneous signal analysis of each individual cell, bypassing lengthy image analysis.

  2. Impact of antigen-presenting cells on cytokine profiles of human Th clones established after stimulation with Mycobacterium tuberculosis antigens.

    PubMed Central

    Conradt, P; Kaufmann, S H

    1995-01-01

    Human T cells reactive with mycobacterial antigens are generally considered to correlate with a Th1 cytokine profile. Our data show that, in addition, Th0 and Th2 clones develop in bulk culture with appropriate antigen-presenting cells before cloning. CD4+ blasts activated by mycobacterial antigens were cloned, and their mRNA patterns for the interleukins (IL) IL-2, IL-4, IL-5, IL-6, and IL-10 and gamma interferon were characterized by reverse-transcribed PCR. Nonadherent, nonrosetting, enriched peripheral blood mononuclear cells promoted development of Th0; after further depletion of monocytes and natural killer cells, Th2 clones were also found. Epstein-Barr virus-transformed B cells, with specificity for the stimulating antigen, increased the proportion of Th2 clones. PMID:7729923

  3. Serine Proteases Enhance Immunogenic Antigen Presentation on Lung Cancer Cells.

    PubMed

    Peters, Haley L; Tripathi, Satyendra C; Kerros, Celine; Katayama, Hiroyuki; Garber, Haven R; St John, Lisa S; Federico, Lorenzo; Meraz, Ismail M; Roth, Jack A; Sepesi, Boris; Majidi, Mourad; Ruisaard, Kathryn; Clise-Dwyer, Karen; Roszik, Jason; Gibbons, Don L; Heymach, John V; Swisher, Stephen G; Bernatchez, Chantale; Alatrash, Gheath; Hanash, Samir; Molldrem, Jeffrey J

    2017-03-02

    Immunotherapies targeting immune checkpoints have proven efficacious in reducing the burden of lung cancer in patients; however, the antigenic targets of these reinvigorated T cells remain poorly defined. Lung cancer tumors contain tumor-associated macrophages (TAM) and neutrophils, which release the serine proteases neutrophil elastase (NE) and proteinase 3 (P3) into the tumor microenvironment. NE and P3 shape the antitumor adaptive immune response in breast cancer and melanoma. In this report, we demonstrate that lung cancer cells cross-presented the tumor-associated antigen PR1, derived from NE and P3. Additionally, NE and P3 enhanced the expression of human leukocyte antigen (HLA) class I molecules on lung cancer cells and induced unique, endogenous peptides in the immunopeptidome, as detected with mass spectrometry sequencing. Lung cancer patient tissues with high intratumoral TAMs were enriched for MHC class I genes and T-cell markers, and patients with high TAM and cytotoxic T lymphocyte (CTL) infiltration had improved overall survival. We confirmed the immunogenicity of unique, endogenous peptides with cytotoxicity assays against lung cancer cell lines, using CTLs from healthy donors that had been expanded against select peptides. Finally, CTLs specific for serine proteases-induced endogenous peptides were detected in lung cancer patients using peptide/HLA-A2 tetramers and were elevated in tumor-infiltrating lymphocytes. Thus, serine proteases in the tumor microenvironment of lung cancers promote the presentation of HLA class I immunogenic peptides that are expressed by lung cancer cells, thereby increasing the antigen repertoire that can be targeted in lung cancer. Cancer Immunol Res; 5(4); 1-11. ©2017 AACR.

  4. Properties of B cells and Thy-1-antigen-expressing cells infiltrating rat renal allografts

    SciTech Connect

    Leszczynski, D.; Halttunen, J.; Tiisala, S.; Ustinov, J.; Renkonen, R.; Haeyry, P. )

    1990-10-01

    We have examined (1) the frequency of B cells secreting antibodies against donor major histocompatibility complex (MHC) antigens and (2) the properties of Thy-1-antigen-expressing leukocytes in rats rejecting renal allografts. Our results show that B cells secreting antibodies are present in the inflammatory cell population at the frequency of 1:850. Among them only 1 out of 2-150 is engaged in production of antibodies directed to the graft MHC antigens, depending on the method of assay. This suggests that despite the observed significant production of nonspecific immunoglobulin in situ, only a minority of the B-cell population is specifically committed to the graft MHC antigens. This finding is concordant with the described previously low frequencies of the T cells specifically directed toward the graft MHC antigen. The role of the immunologically noncommitted cells in graft rejection is unknown. We have found that a substantial part (up to 60%) of inflammatory cells invading a rat kidney allograft express the Thy-1 antigen. This suggests that they might be immature (progenitor ) cells and, therefore, unable to respond to the graft antigens. Progenitor-like properties of these cells have been confirmed by their ability to reconstitute lethally irradiated syngeneic rat. Finally, these immature cells are of lymphoid, not of myeloid, linkage, because they do not proliferate in the presence of GM-CSF.

  5. Nuclear localization of Merkel cell polyomavirus large T antigen in Merkel cell carcinoma

    SciTech Connect

    Nakamura, Tomoyuki; Sato, Yuko; Watanabe, Daisuke; Ito, Hideki; Shimonohara, Nozomi; Tsuji, Takahiro; Nakajima, Noriko; Suzuki, Yoshio; Matsuo, Koma; Nakagawa, Hidemi; Sata, Tetsutaro; Katano, Harutaka

    2010-03-15

    To clarify whether mutations in the large T gene encoded by Merkel cell polyomavirus affect the expression and function of large T antigen in Merkel cell carcinoma cases, we investigated the expression of large T antigen in vitro and in vivo. Immunohistochemistry using a rabbit polyclonal antibody revealed that large T antigen was expressed in the nuclei of Merkel cell carcinoma cells with Merkel cell polyomavirus infection. Deletion mutant analyses identified an Arg-Lys-Arg-Lys sequence (amino acids 277-280) as a nuclear localization signal in large T antigen. Sequence analyses revealed that there were no mutations in the nuclear localization signal in any of the eleven Merkel cell polyomavirus strains examined. Furthermore, stop codons were not observed in the upstream of the nuclear localization signal in any of the Merkel cell carcinoma cases examined. These data suggest that the nuclear localization signal is highly conserved and functional in Merkel cell carcinoma cases.

  6. How B cells capture, process and present antigens: a crucial role for cell polarity.

    PubMed

    Yuseff, Maria-Isabel; Pierobon, Paolo; Reversat, Anne; Lennon-Duménil, Ana-Maria

    2013-07-01

    B cells are key components of the adaptive immune response. Their differentiation into either specific memory B cells or antibody-secreting plasma cells is a consequence of activation steps that involve the processing and presentation of antigens. The engagement of B cell receptors by surface-tethered antigens leads to the formation of an immunological synapse that coordinates cell signalling events and that promotes antigen uptake for presentation on MHC class II molecules. In this Review, we discuss membrane trafficking and the associated molecular mechanisms that are involved in antigen extraction and processing at the B cell synapse, and we highlight how B cells use cell polarity to coordinate the complex events that ultimately lead to efficient humoral responses.

  7. Sockeye Salmon Retain Immunoglobulin-Secreting Plasma Cells Throughout Their Spawning Journey And Post-spawning

    PubMed Central

    Schouten, Jonathan; Clister, Terri; Bruce, Amber; Epp, Lidia; Zwollo, Patty

    2013-01-01

    Antibody-producing plasma cells are a major source of protective immunity in vertebrates, including salmon. During the spawning phase, salmon undergo drastic, hormonally driven changes in their physiology, including elevated levels of cortisol, which are known to suppress the immune system. As adult fish need to survive their long journey to the spawning grounds, we hypothesized that humoral immunity, in the form of IgM-secreting plasma cells, remains functional until post-spawning. This was investigated by measuring changes in membrane and secreted immunoglobulin heavy chain mu and Pax5 transcripts in spleen and kidney from migrating sockeye salmon, using real-time qPCR. As an additional measurement, the abundance of developing B, mature B, and plasma cells was determined in spawning fish, using flow cytometry. Immune tissue samples were collected from fish from the Kenai River drainage and Main Bay, Prince William Sound. Our results reveal that spawning fish express high levels of secreted heavy chain mu transcripts in their spleen and anterior kidney throughout the spawning journey. Furthermore, we show that IgM-secreting PCs (HCmu++/Pax5−) remain abundant in anterior kidney and spleen of post-spawning sockeye salmon, with a concomitant loss in developing B cells (HCmu−/Pax5+). This suggests that successful spawners retain their PCs throughout the spawning journey and post-spawning. PMID:23434463

  8. Targeted delivery of antigen processing inhibitors to antigen presenting cells via mannose receptors

    PubMed Central

    Raiber, Eun-Ang; Tulone, Calogero; Zhang”, Yanjing; Martinez-Pomares, Luisa; Steed, Emily; Sponaas, Anna M.; Langhorne, Jean; Noursadeghi, Mahdad

    2014-01-01

    Improved chemical inhibitors are required to dissect the role of specific antigen processing enzymes and to complement genetic models. In this study we explore the in vitro and in vivo properties of a novel class of targeted inhibitor of aspartic proteinases, in which pepstatin is coupled to mannosylated albumin (MPC6), creating an inhibitor with improved solubility and the potential for selective cell tropism. Using these compounds, we have demonstrated that MPC6 is taken up via mannose receptor facilitated endocytosis, leading to a slow but continuous accumulation of inhibitor within large endocytic vesicles within dendritic cells, and a parallel inhibition of intracellular aspartic proteinase activity. Inhibition of intracellular proteinase activity is associated with reduction in antigen processing activity, but this is epitope specific, preferentially inhibiting processing of T cell epitopes buried within compact proteinase-resistant protein domains. Unexpectedly, we have also demonstrated, using quenched fluorescent substrates, that little or no cleavage of the disulfide linker takes place within dendritic cells, but this does not appear to affect the activity of MPC6 as an inhibitor of cathepsins D and E in vitro and in vivo. Finally, we have shown that MPC6 selectively targets dendritic cells and macrophages in spleen in vivo. Access to non-lymphoid tissues is very limited in the steady state, but is strongly enhanced at local sites of inflammation. The strategy adopted for MPC6 synthesis may therefore represent a more general way to deliver chemical inhibitors to cells of the innate immune system, especially at sites of inflammation. PMID:20349916

  9. Chimeric Antigen Receptor T Cell Therapy in Hematology.

    PubMed

    Ataca, Pınar; Arslan, Önder

    2015-12-01

    It is well demonstrated that the immune system can control and eliminate cancer cells. Immune-mediated elimination of tumor cells has been discovered and is the basis of both cancer vaccines and cellular therapies including hematopoietic stem cell transplantation. Adoptive T cell transfer has been improved to be more specific and potent and to cause less off-target toxicity. Currently, there are two forms of engineered T cells being tested in clinical trials: T cell receptor (TCR) and chimeric antigen receptor (CAR) modified T cells. On 1 July 2014, the United States Food and Drug Administration granted 'breakthrough therapy' designation to anti-CD19 CAR T cell therapy. Many studies were conducted to evaluate the benefits of this exciting and potent new treatment modality. This review summarizes the history of adoptive immunotherapy, adoptive immunotherapy using CARs, the CAR manufacturing process, preclinical and clinical studies, and the effectiveness and drawbacks of this strategy.

  10. BrdU-label-retaining cells in rat eccrine sweat glands over time.

    PubMed

    Li, Haihong; Zhang, Mingjun; Li, Xuexue; Chen, Lu; Zhang, Bingna; Tang, Shijie; Fu, Xiaobing

    2016-03-01

    Cell proliferation and turnover are fueled by stem cells. In a previous study, we demonstrated that rat eccrine sweat glands contained abundant bromodeoxyuridine (BrdU)-label-retaining cells (LRCs). However, morphological observations showed that eccrine sweat glands usually show little or no signs of homeostatic change. In this study, we account for why the homeostatic change is rare in eccrine sweat glands based on cytokinetic changes in BrdU-LRC turnover, and also determine the BrdU-labeled cell type. Thirty-six newborn SD rats, were injected intraperitoneally with 50mg/kg BrdU twice daily at a 2h interval for 4 consecutive days. After a chase period of 4, 6, 8, 12, 24 and 32 weeks, rats were euthanized, and the hind footpads were removed and processed for BrdU immunostaining, and BrdU/α-SMA and BrdU/K14 double-immunostaining. BrdU-LRCs were observed in the ducts, secretory coils and mesenchymal cells at all survival time points. The percentage of BrdU(+) cells in rat eccrine sweat glands averaged 4.2±1.2% after 4 weeks of chase, increased slightly by the 6th week, averaging 4.4±0.9%, and peaked at 8 weeks, averaging 5.3±1.0%. Subsequently, the average percentage of BrdU(+) cells declined to 3.2±0.8% by the 32nd week. There was no difference in the percentage of BrdU-LRCs among the different survival time points except that a significant difference in the percentage of BrdU-LRCs detected at 24 weeks versus 8 weeks, and 32 weeks versus 8 weeks, was observed. We concluded that the BrdU-LRCs turnover is slow in eccrine sweat glands.

  11. CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

    PubMed Central

    Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Nuñez-Prado, Natalia; Diaz, Rosa M; Vile, Richard; Alvarez-Vallina, Luis

    2013-01-01

    A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells. PMID:23695536

  12. Expression of cell surface antigens on mast cells: mast cell phenotyping.

    PubMed

    Hauswirth, Alexander W; Florian, Stefan; Schernthaner, Gerit-Holger; Krauth, Maria-Theresa; Sonneck, Karoline; Sperr, Wolfgang R; Valent, Peter

    2006-01-01

    During the past few decades, a number of functionally important cell surface antigens have been detected on human mast cells (MCs). These antigens include the stem cell factor receptor (SCFR/CD117), the high-affinity immunoglobulin E receptor, adhesion molecules, and activation-linked membrane determinants. Several of these antigens (CD2, CD25, CD35, CD88, CD203c) appear to be upregulated on MCs in patients with systemic mastocytosis and therefore are used as diagnostic markers. Quantitative measurement of these markers on MCs is thus of diagnostic value and is usually performed by multicolor-based flow cytometry techniques utilizing a PE- or APC-labeled antibody against CD117 for MCs detection. This chapter gives an overview about the methods of staining of MC in various tissues with special reference to novel diagnostic markers applied in patients with suspected systemic mastocytosis.

  13. Different cytokine and stimulation conditions influence the expansion and immune phenotype of third-generation chimeric antigen receptor T cells specific for tumor antigen GD2.

    PubMed

    Gargett, Tessa; Brown, Michael P

    2015-04-01

    Chimeric antigen receptor (CAR) T cells are a novel immunotherapy for cancer. To achieve anti-tumor efficacy, these cells must survive, expand, and persist after infusion into patients, functions that are reportedly best achieved by cells with a stem or central-memory rather than effector-memory phenotype. We have developed third-generation CAR T cells specific for the tumor-associated antigen GD2 for use in a phase I clinical trial. We investigated the optimal cell culture conditions for CAR T-cell production, and here we describe the relative effects of 3 activation and cytokine conditions on CAR T-cell expansion, effector function and phenotype. Peripheral blood mononuclear cells were activated by anti-CD3 and anti-CD28 or anti-CD3 and Retronectin. Activated cells were transduced with the CAR-encoding retroviral vector and expanded in either interleukin (IL)-2 or IL-7 and IL-15. Immune phenotype and expansion were tracked throughout the culture, and transduction efficiency, and subsequent GD2-specific effector functions were evaluated by flow cytometry and cytotoxic T lymphocytes assay. CD3/Retronectin stimulation with IL-2 resulted in poorer activation, expansion and Th1 cytokine secretion of CAR T cells than CD3/CD28 stimulation with either IL-2 or IL-7 and IL-15. However, CAR T cells cultured in CD3/CD28/IL7/IL-15 and CD3/Retronectin/IL-2 had superior cytotoxic T lymphocyte activity and a more stem-like phenotype. The combination of CD3 and CD28 with IL-7 and IL-15 gave the best balance of CAR T-cell expansion and potent GD2-specific effector functions while retaining a stem/memory phenotype, and these growth conditions will therefore be used to manufacture CAR T cells for our phase I clinical trial. Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.

  14. The T cell antigen receptor expressed by Vα14i NKT cells has a unique mode of glycosphingolipid antigen recognition

    PubMed Central

    Sidobre, Stéphane; Hammond, Kirsten J. L.; Bénazet-Sidobre, Lise; Maltsev, Sergei D.; Richardson, Stewart K.; Ndonye, Rachel M.; Howell, Amy R.; Sakai, Teruyuki; Besra, Gurdyal S.; Porcelli, Steven A.; Kronenberg, Mitchell

    2004-01-01

    Natural killer (NK) T cells with an invariant Vα14 rearrangement (Vα14i) are the largest population of lipid antigen-specific T lymphocytes identified in animals. They react to the glycolipid α-galactosyl ceramide (α-GalCer) presented by CD1d, and they may have important regulatory functions. It was previously shown that the Vα14i T cell antigen receptor (TCR) has a high affinity for the α-GalCer/CD1d complex, driven by a long half-life (t1/2). Although this result could have reflected the unique attributes of α-GalCer, using several related glycolipid compounds, we show here that the threshold for full activation of Vα14i NKT cells by these glycosphingolipids requires a relatively high-affinity TCR interaction with a long t1/2. Furthermore, our data are consistent with the view that the mechanism of recognition of these compounds presented by CD1d to the Vα14i NKT cell TCR is likely to fit a lock-and-key model. Overall, these findings emphasize the distinct properties of glycosphingolipid antigen recognition by Vα14i NKT cells. PMID:15304644

  15. Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines

    PubMed Central

    Lee Szeto, Gregory; Van Egeren, Debra; Worku, Hermoon; Sharei, Armon; Alejandro, Brian; Park, Clara; Frew, Kirubel; Brefo, Mavis; Mao, Shirley; Heimann, Megan; Langer, Robert; Jensen, Klavs; Irvine, Darrell J

    2015-01-01

    B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to “professional” APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale “cell squeezing” process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8+T-cells, and not CD4+T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8+T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8+T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8+T-cells, and decoupling of antigen uptake from B-cell activation. PMID:25999171

  16. Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines

    NASA Astrophysics Data System (ADS)

    Lee Szeto, Gregory; van Egeren, Debra; Worku, Hermoon; Sharei, Armon; Alejandro, Brian; Park, Clara; Frew, Kirubel; Brefo, Mavis; Mao, Shirley; Heimann, Megan; Langer, Robert; Jensen, Klavs; Irvine, Darrell J.

    2015-05-01

    B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to “professional” APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale “cell squeezing” process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8+T-cells, and not CD4+T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8+T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8+T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8+T-cells, and decoupling of antigen uptake from B-cell activation.

  17. T-cell intracellular antigens in health and disease.

    PubMed

    Sánchez-Jiménez, Carmen; Izquierdo, José M

    2015-01-01

    T-cell intracellular antigen 1 (TIA1) and TIA1-related/like protein (TIAR/TIAL1) are 2 proteins discovered in 1991 as components of cytotoxic T lymphocyte granules. They act in the nucleus as regulators of transcription and pre-mRNA splicing. In the cytoplasm, TIA1 and TIAR regulate and/or modulate the location, stability and/or translation of mRNAs. As knowledge of the different genes regulated by these proteins and the cellular/biological programs in which they are involved increases, it is evident that these antigens are key players in human physiology and pathology. This review will discuss the latest developments in the field, with physiopathological relevance, that point to novel roles for these regulators in the molecular and cell biology of higher eukaryotes.

  18. T-cell intracellular antigens in health and disease

    PubMed Central

    Sánchez-Jiménez, Carmen; Izquierdo, José M

    2015-01-01

    T-cell intracellular antigen 1 (TIA1) and TIA1-related/like protein (TIAR/TIAL1) are 2 proteins discovered in 1991 as components of cytotoxic T lymphocyte granules. They act in the nucleus as regulators of transcription and pre-mRNA splicing. In the cytoplasm, TIA1 and TIAR regulate and/or modulate the location, stability and/or translation of mRNAs. As knowledge of the different genes regulated by these proteins and the cellular/biological programs in which they are involved increases, it is evident that these antigens are key players in human physiology and pathology. This review will discuss the latest developments in the field, with physiopathological relevance, that point to novel roles for these regulators in the molecular and cell biology of higher eukaryotes. PMID:26036275

  19. Macrophages retain hematopoietic stem cells in the spleen via VCAM-1

    PubMed Central

    Hoyer, Friedrich Felix; Grigoryeva, Lubov S.; Sager, Hendrik B.; Leuschner, Florian; Courties, Gabriel; Borodovsky, Anna; Novobrantseva, Tatiana; Ruda, Vera M.; Fitzgerald, Kevin; Iwamoto, Yoshiko; Wojtkiewicz, Gregory; Sun, Yuan; Da Silva, Nicolas; Libby, Peter; Anderson, Daniel G.; Swirski, Filip K.; Weissleder, Ralph

    2015-01-01

    Splenic myelopoiesis provides a steady flow of leukocytes to inflamed tissues, and leukocytosis correlates with cardiovascular mortality. Yet regulation of hematopoietic stem cell (HSC) activity in the spleen is incompletely understood. Here, we show that red pulp vascular cell adhesion molecule 1 (VCAM-1)+ macrophages are essential to extramedullary myelopoiesis because these macrophages use the adhesion molecule VCAM-1 to retain HSCs in the spleen. Nanoparticle-enabled in vivo RNAi silencing of the receptor for macrophage colony stimulation factor (M-CSFR) blocked splenic macrophage maturation, reduced splenic VCAM-1 expression and compromised splenic HSC retention. Both, depleting macrophages in CD169 iDTR mice or silencing VCAM-1 in macrophages released HSCs from the spleen. When we silenced either VCAM-1 or M-CSFR in mice with myocardial infarction or in ApoE−/− mice with atherosclerosis, nanoparticle-enabled in vivo RNAi mitigated blood leukocytosis, limited inflammation in the ischemic heart, and reduced myeloid cell numbers in atherosclerotic plaques. PMID:25800955

  20. Regulatory T-cell vaccination independent of auto-antigen.

    PubMed

    Pascual, David W; Yang, Xinghong; Holderness, Kathryn; Jun, SangMu; Maddaloni, Massimo; Kochetkova, Irina

    2014-03-14

    To date, efforts to treat autoimmune diseases have primarily focused on the disease symptoms rather than on the cause of the disease. In large part, this is attributed to not knowing the responsible auto-antigens (auto-Ags) for driving the self-reactivity coupled with the poor success of treating autoimmune diseases using oral tolerance methods. Nonetheless, if tolerogenic approaches or methods that stimulate regulatory T (Treg) cells can be devised, these could subdue autoimmune diseases. To forward such efforts, our approach with colonization factor antigen I (CFA/I) fimbriae is to establish bystander immunity to ultimately drive the development of auto-Ag-specific Treg cells. Using an attenuated Salmonella vaccine expressing CFA/I fimbriae, fimbriae-specific Treg cells were induced without compromising the vaccine's capacity to protect against travelers' diarrhea or salmonellosis. By adapting the vaccine's anti-inflammatory properties, it was found that it could also dampen experimental inflammatory diseases resembling multiple sclerosis (MS) and rheumatoid arthritis. Because of this bystander effect, disease-specific Treg cells are eventually induced to resolve disease. Interestingly, this same vaccine could elicit the required Treg cell subset for each disease. For MS-like disease, conventional CD25(+) Treg cells are stimulated, but for arthritis CD39(+) Treg cells are induced instead. This review article will examine the potential of treating autoimmune diseases without having previous knowledge of the auto-Ag using an innocuous antigen to stimulate Treg cells via the production of transforming growth factor-β and interleukin-10.

  1. Human skeletal muscle-derived stem cells retain stem cell properties after expansion in myosphere culture

    SciTech Connect

    Wei, Yan; Li, Yuan; Chen, Chao; Stoelzel, Katharina; Kaufmann, Andreas M.

    2011-04-15

    Human skeletal muscle contains an accessible adult stem-cell compartment in which differentiated myofibers are maintained and replaced by a self-renewing stem cell pool. Previously, studies using mouse models have established a critical role for resident stem cells in skeletal muscle, but little is known about this paradigm in human muscle. Here, we report the reproducible isolation of a population of cells from human skeletal muscle that is able to proliferate for extended periods of time as floating clusters of rounded cells, termed 'myospheres' or myosphere-derived progenitor cells (MDPCs). The phenotypic characteristics and functional properties of these cells were determined using reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry and immunocytochemistry. Our results showed that these cells are clonogenic, express skeletal progenitor cell markers Pax7, ALDH1, Myod, and Desmin and the stem cell markers Nanog, Sox2, and Oct3/4 significantly elevated over controls. They could be maintained proliferatively active in vitro for more than 20 weeks and passaged at least 18 times, despite an average donor-age of 63 years. Individual clones (4.2%) derived from single cells were successfully expanded showing clonogenic potential and sustained proliferation of a subpopulation in the myospheres. Myosphere-derived cells were capable of spontaneous differentiation into myotubes in differentiation media and into other mesodermal cell lineages in induction media. We demonstrate here that direct culture and expansion of stem cells from human skeletal muscle is straightforward and reproducible with the appropriate technique. These cells may provide a viable resource of adult stem cells for future therapies of disease affecting skeletal muscle or mesenchymal lineage derived cell types.

  2. Tryptase precursors are preferentially and spontaneously released, whereas mature tryptase is retained by HMC-1 cells, Mono-Mac-6 cells, and human skin-derived mast cells.

    PubMed

    Schwartz, Lawrence B; Min, Hae-Ki; Ren, Shunlin; Xia, Han-Zhang; Hu, Jiang; Zhao, Wei; Moxley, George; Fukuoka, Yoshihiro

    2003-06-01

    Tryptase (alpha and beta) levels in serum are used to assess mast cell involvement in human disease. Using cultured cells, the current study examines the hypothesis that protryptase(s) are spontaneously secreted by mast cells at rest, whereas mature tryptase(s) are stored in secretory granules until their release by activated cells. HMC-1 cells have only beta-tryptase genes and the corresponding mRNA. Mono-Mac-6 cells have both alpha- and beta-tryptase genes but preferentially express alpha-tryptase. Mono-Mac-6 cells spontaneously secrete most of their tryptase, which consists of alpha-protryptase, whereas mature tryptase is retained inside these cells. HMC-1 cells also spontaneously secrete most of their tryptase, identified as beta-protryptase, and retain mature tryptase. Skin-derived mast cells retain most of their tryptase, which is mature, and spontaneously secrete protryptase(s). Total tryptase levels in plasma are detectable but no different in healthy subjects with and without the gene for alpha-tryptase, consistent with pro forms of both alpha- and beta-tryptase being spontaneously secreted. Thus, protryptase(s) are spontaneously secreted by resting mast cells, whereas mature tryptase is retained by mast cells until they are activated to degranulate.

  3. Germinal center B cells recognize antigen through a specialized immune synapse architecture

    PubMed Central

    Nowosad, Carla R.; Spillane, Katelyn M.; Tolar, Pavel

    2016-01-01

    B cell activation is regulated by B cell antigen receptor (BCR) signaling and antigen internalization in immune synapses. Using large-scale imaging across B cell subsets, we show that in contrast to naive and memory B cells, which gathered antigen towards the synapse center before internalization, germinal center (GC) B cells extracted antigen by a distinct pathway using small peripheral clusters. Both naive and GC B cell synapses required proximal BCR signaling, but GC cells signaled less through the protein kinase C-β (PKC-β)–NF-κB pathway and produced stronger tugging forces on the BCR, thereby more stringently regulating antigen binding. Consequently, GC B cells extracted antigen with better affinity discrimination than naive B cells, suggesting that specialized biomechanical patterns in B cell synapses regulate T-cell dependent selection of high-affinity B cells in GCs. PMID:27183103

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

    PubMed Central

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

    2015-01-01

    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

  5. Nonselective expression of simian virus 40 large tumor antigen fragments in mouse cells.

    PubMed Central

    Reddy, V B; Tevethia, S S; Tevethia, M J; Weissman, S M

    1982-01-01

    To understand the role of various functional domains of simian virus 40 early tumor antigens, we have cloned and introduced into mouse cells portions of early simian virus 40 DNA. Two types of truncated large tumor antigen (33 and 12.3 kilodaltons), as well as small tumor antigen, were identified by immunoprecipitation. Both truncated large tumor antigens have been found to be overproduced with respect to the small tumor antigen, although the 12.3-kilodalton truncated large tumor antigen was more stable than the 33-kilodalton one. Nonviral 53-kilodalton protein was not found associated with either truncated large tumor antigen in immunoprecipitations. Images PMID:6281793

  6. The growth of B cell receptor microcluster is a universal response of B cells encountering antigens with different motion features.

    PubMed

    Wan, Zhengpeng; Liu, Wanli

    2012-07-01

    B lymphocyte cell senses and acquires foreign antigens through clonal distributed B cell receptors (BCRs) expressed on the surface of plasma membrane. The presentation formats of antigens are quite diverse. Based on their Brownian diffusion mobility, there are three forms: free mobile soluble antigens, lateral mobile membrane bound antigens, and fixed immobile antigens. Here, using high resolution high speed live cell imaging approaches, we provide evidence that BCR microclusters are formed on the surface of B cells shortly after B cell's encountering of antigens with each format of motion features. Through high speed live cell imaging, we determine that these BCR microclusters show dynamic growth feature and by doing so function as the basic platforms for B cells to acquire the antigens. We propose that the formation and dynamic growth of BCR microcluster is a universal mechanism for B cell to response to antigens with diverse motion features.

  7. Polyoma small T antigen triggers cell death via mitotic catastrophe

    PubMed Central

    Fernando, Arun T Pores; Andrabi, Shaida; Cizmecioglu, Onur; Zhu, Cailei; Livingston, David M.; Higgins, Jonathan M.G; Schaffhausen, Brian S; Roberts, Thomas M

    2014-01-01

    Polyoma small T antigen (PyST), an early gene product of the polyoma virus, has been shown to cause cell death in a number of mammalian cells in a protein phosphatase 2A (PP2A)-dependent manner. In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis. Live-cell and immunofluorescence studies showed that the majority of the PyST-expressing cells were arrested in prometaphase with almost no cells progressing beyond metaphase. These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, resulting in the activation of the Spindle Assembly Checkpoint (SAC). Prolonged mitotic arrest then led to cell death via mitotic catastrophe. Cell cycle inhibitors that block cells in G1/S prevented PyST-induced death. PyST-induced cell death that occurs during M is not dependent on p53 status. These data suggested, and our results confirmed that, PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors. PMID:24998850

  8. Cell Wall Anchoring of the Campylobacter Antigens to Lactococcus lactis

    PubMed Central

    Kobierecka, Patrycja A.; Olech, Barbara; Książek, Monika; Derlatka, Katarzyna; Adamska, Iwona; Majewski, Paweł M.; Jagusztyn-Krynicka, Elżbieta K.; Wyszyńska, Agnieszka K.

    2016-01-01

    Campylobacter jejuni is the most frequent cause of human food-borne gastroenteritis and chicken meat is the main source of infection. Recent studies showed that broiler chicken immunization against Campylobacter should be the most efficient way to lower the number of human infections by this pathogen. Induction of the mucosal immune system after oral antigen administration should provide protective immunity to chickens. In this work we tested the usefulness of Lactococcus lactis, the most extensively studied lactic acid bacterium, as a delivery vector for Campylobacter antigens. First we constructed hybrid protein – CjaA antigen presenting CjaD peptide epitopes on its surface. We showed that specific rabbit anti-rCjaAD serum reacted strongly with both CjaA and CjaD produced by a wild type C. jejuni strain. Next, rCjaAD and CjaA were fused to the C-terminus of the L. lactis YndF containing the LPTXG motif. The genes expressing these proteins were transcribed under control of the L. lactis Usp45 promoter and their products contain the Usp45 signal sequences. This strategy ensures a cell surface location of both analyzed proteins, which was confirmed by immunofluorescence assay. In order to evaluate the impact of antigen location on vaccine prototype efficacy, a L. lactis strain producing cytoplasm-located rCjaAD was also generated. Animal experiments showed a decrease of Campylobacter cecal load in vaccinated birds as compared with the control group and showed that the L. lactis harboring the surface-exposed rCjaAD antigen afforded greater protection than the L. lactis producing cytoplasm-located rCjaAD. To the best of our knowledge, this is the first attempt to employ Lactic Acid Bacteria (LAB) strains as a mucosal delivery vehicle for chicken immunization. Although the observed reduction of chicken colonization by Campylobacter resulting from vaccination was rather moderate, the experiments showed that LAB strains can be considered as an alternative vector to

  9. Healthy Human T-Cell Responses to Aspergillus fumigatus Antigens

    PubMed Central

    Chaudhary, Neelkamal; Staab, Janet F.; Marr, Kieren A.

    2010-01-01

    Background Aspergillus fumigatus is associated with both invasive and allergic pulmonary diseases, in different hosts. The organism is inhaled as a spore, which, if not cleared from the airway, germinates into hyphal morphotypes that are responsible for tissue invasion and resultant inflammation. Hyphae secrete multiple products that function as antigens, evoking both a protective (TH1–TH17) and destructive allergic (TH2) immunity. How Aspergillus allergens (Asp f proteins) participate in the development of allergic sensitization is unknown. Methodology/Principal Findings To determine whether Asp f proteins are strictly associated with TH2 responses, or represent soluble hyphal products recognized by healthy hosts, human T cell responses to crude and recombinant products were characterized by ELISPOT. While responses (number of spots producing IFN-γ, IL-4 or IL-17) to crude hyphal antigen preparations were weak, responses to recombinant Asp f proteins were higher. Recombinant allergens stimulated cells to produce IFN-γ more so than IL-4 or IL-17. Volunteers exhibited a diverse CD4+ and CD8+ T cell antigen recognition profile, with prominent CD4 TH1-responses to Asp f3 (a putative peroxismal membrane protein), Asp f9/16 (cell wall glucanase), Asp f11 (cyclophilin type peptidyl-prolyl isomerase) and Asp f22 (enolase). Strong IFN-γ responses were reproduced in most subjects tested over 6 month intervals. Conclusions Products secreted after conidial germination into hyphae are differentially recognized by protective T cells in healthy, non-atopic individuals. Defining the specificity of the human T cell repertoire, and identifying factors that govern early responses may allow for development of novel diagnostics and therapeutics for both invasive and allergic Aspergillus diseases. PMID:20174463

  10. Chimeric antigen receptor-modified T cells strike back

    PubMed Central

    Frigault, Matthew J.

    2016-01-01

    Chimeric antigen receptors (CARs) are engineered molecules designed to endow a polyclonal T-cell population with the ability to recognize tumor-associated surface antigens. In their simplest form, CARs comprise a targeting moiety in the form of a single-chain variable fragment from an antibody connected to various intracellular signaling domains allowing for T-cell activation. This powerful approach combines the specificity of an antibody with the cytotoxic ability of a T cell. There has been much excitement since early phase trials of CAR-T cells targeting CD19 expressed on B-cell malignancies demonstrated remarkable efficacy in inducing long-term, stable remissions in otherwise relapsed/refractory disease. Despite these successes, we have just begun to understand the intricacies of CAR biology with efforts underway to utilize this platform in the treatment of other, previously refractory malignancies. Challenges currently include identification of viable cancer targets, management strategies for potentially severe and irreversible toxicities and overcoming the immunosuppressive nature of the tumor microenvironment. This review will focus on basic CAR structure and function, previous success and new approaches aimed at the broader application of CAR-T-cell therapy. PMID:27021308

  11. Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells.

    PubMed

    Frigault, Matthew J; Lee, Jihyun; Basil, Maria Ciocca; Carpenito, Carmine; Motohashi, Shinichiro; Scholler, John; Kawalekar, Omkar U; Guedan, Sonia; McGettigan, Shannon E; Posey, Avery D; Ang, Sonny; Cooper, Laurence J N; Platt, Jesse M; Johnson, F Brad; Paulos, Chrystal M; Zhao, Yangbing; Kalos, Michael; Milone, Michael C; June, Carl H

    2015-04-01

    This study compared second-generation chimeric antigen receptors (CAR) encoding signaling domains composed of CD28, ICOS, and 4-1BB (TNFRSF9). Here, we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T cells with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to 3 months following a single stimulation through the T-cell receptor (TCR). Sustained numeric expansion was independent of cognate antigen and did not require the addition of exogenous cytokines or feeder cells after a single stimulation of the TCR and CD28. Results from gene array and functional assays linked sustained cytokine secretion and expression of T-bet (TBX21), EOMES, and GATA-3 to the effect. Sustained expression of the endogenous IL2 locus has not been reported in primary T cells. Sustained proliferation was dependent on CAR structure and high expression, the latter of which was necessary but not sufficient. The mechanism involves constitutive signaling through NF-κB, AKT, ERK, and NFAT. The propagated CAR T cells retained a diverse TCR repertoire, and cellular transformation was not observed. The CARs with a constitutive growth phenotype displayed inferior antitumor effects and engraftment in vivo. Therefore, the design of CARs that have a nonconstitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or nonconstitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials. ©2015 American Association for Cancer Research.

  12. Immunoavtoradiographic demonstration of virus antigens in infected cells

    SciTech Connect

    Tarasishin, L.A.; Zhovnovataya, V.L.

    1986-02-01

    This paper describes a simple method of determining virus antigens, which consists essentially of the demonstration of immune complexes, formed by treatment of acetone-fixed infected cells with specific immune serum, by means of labeled protein A of S. aureus. Transplantable human HeLa cells, type 1 human adenovirus (AD 1), normal rabbit serum, and specific immune sera, obtained by immunization of rabbits with purified Ad 1 or with hexone Ad 1, and a commerical preparation of protein A of S. aureus, labeled with I 125 by the chloramine method, were used.

  13. Optimized T-cell receptor-mimic chimeric antigen receptor T cells directed toward the intracellular Wilms Tumor 1 antigen

    PubMed Central

    Rafiq, S; Purdon, TJ; Daniyan, AF; Koneru, M; Dao, T; Liu, C; Scheinberg, DA; Brentjens, RJ

    2017-01-01

    CD19-directed chimeric antigen receptor (CAR) T cells are clinically effective in a limited set of leukemia patients. However, CAR T-cell therapy thus far has been largely restricted to targeting extracellular tumor-associated antigens (TAA). Herein, we report a T-cell receptor-mimic (TCRm) CAR, termed WT1-28z, that is reactive to a peptide portion of the intracellular onco-protein Wilms Tumor 1(WT1), as it is expressed on the surface of the tumor cell in the context of HLA-A*02:01. T cells modified to express WT1-28z specifically targeted and lysed HLA-A*02:01+ WT1+ tumors and enhanced survival of mice engrafted with HLA-A*02:01+, WT1+ leukemia or ovarian tumors. This in vivo functional validation of TCRm CAR T cells provides the proof-of-concept necessary to expand the range of TAA that can be effectively targeted for immunotherapy to include attractive intracellular targets, and may hold great potential to expand on the success of CAR T-cell therapy. PMID:27924074

  14. Human Intestinal M Cells Display the Sialyl Lewis A Antigen

    PubMed Central

    Giannasca, Paul J.; Giannasca, Karen T.; Leichtner, Alan M.; Neutra, Marian R.

    1999-01-01

    The biochemical features that distinguish human M cells from other intestinal epithelial cell types are important for understanding microbial pathogenesis and for targeting vaccines to the mucosal immune system. We applied a large panel of carbohydrate-specific monoclonal antibodies and lectins to Peyer’s patch and cecum biopsy specimens from three normal individuals and a patient with inflammatory bowel disease. The results show that human M-cell glycosylation patterns are distinct from those of other species examined and that human M cells preferentially display the sialyl Lewis A antigen. This carbohydrate epitope is also present in a small subpopulation of enterocytes in the follicle-associated epithelium and in goblet cell mucins. PMID:9916113

  15. Autografting with CD34+ peripheral blood stem cells: retained engraftment capability and reduced tumour cell content.

    PubMed

    Voso, M T; Hohaus, S; Moos, M; Pförsich, M; Cremer, F W; Schlenk, R F; Martin, S; Hegenbart, U; Goldschmidt, H; Haas, R

    1999-02-01

    The efficacy of an immunomagnetic purging method and the Isolex 300 devices were assessed for selecting CD34+ cells from leukapheresis products of 29 patients with non-Hodgkin's lymphoma (NHL), 39 with multiple myeloma and 34 with breast cancer. The mean purity of the CD34+ cell population was 93.6% and the mean recovery was 67.7%. Following enzymatic cleavage by chymopapain the expression of Thy-1 and Leu-8 was significantly reduced without affecting haematological recovery. The population of selected CD34+ cells of 4/8 patients with follicular lymphoma became PCR-negative. A 2.5 log reduction of tumour cells could be achieved in four patients with multiple myeloma as shown by a quantitative PCR assay. There were no tumour cells detectable in any of the 19 CD34+ cell preparations of patients with breast cancer. In 64 patients who received 94 cycles of high-dose therapy, a mean number of 4.7x 10(6) CD34+ cells/kg were autografted. The time needed for platelet reconstitution was different when a comparison was made with 156 patients, who had received unmanipulated leukapheresis products (10 v 12 d, P = 0.006). No significant differences with regard to neutrophil recovery were noted. Five patients had a graft failure. Two of them died (on day 78 and 88 following PBSCT), and three patients were rescued with unmanipulated back-up transplants. In conclusion, the immunomagnetic selection of CD34+ cells provides autografts with reduced tumour cell content and an engraftment ability similar to that of unmanipulated autografts.

  16. Chimeric antigen receptor engineered stem cells: a novel HIV therapy.

    PubMed

    Zhen, Anjie; Carrillo, Mayra A; Kitchen, Scott G

    2017-03-01

    Despite the success of combination antiretroviral therapy (cART) for suppressing HIV and improving patients' quality of life, HIV persists in cART-treated patients and remains an incurable disease. Financial burdens and health consequences of lifelong cART treatment call for novel HIV therapies that result in a permanent cure. Cellular immunity is central in controlling HIV replication. However, HIV adopts numerous strategies to evade immune surveillance. Engineered immunity via genetic manipulation could offer a functional cure by generating cells that have enhanced antiviral activity and are resistant to HIV infection. Recently, encouraging reports from several human clinical trials using an anti-CD19 chimeric antigen receptor (CAR) modified T-cell therapy for treating B-cell malignancies have provided valuable insights and generated remarkable enthusiasm in engineered T-cell therapy. In this review, we discuss the development of HIV-specific chimeric antigen receptors and the use of stem cell based therapies to generate lifelong anti-HIV immunity.

  17. The Role of Antigen Presenting Cells in Multiple Sclerosis

    PubMed Central

    Chastain, Emily M. L.; Duncan, D'Anne S.; Rodgers, Jane M.; Miller, Stephen D.

    2010-01-01

    Multiple Sclerosis (MS) is a debilitating T cell-mediated autoimmune disease of the central nervous system (CNS). Animal models of MS, such as experimental autoimmune encephalomyelitis (EAE) and Theiler's murine encephalomyelitis virus-Induced demyelinating disease (TMEV-IDD) have given light to cellular mechanisms involved in the initiation and progression of this organ-specific autoimmune disease. Within the CNS, antigen presenting cells (APC) such as microglia and astrocytes participate as first line defenders against infections or inflammation. However, during chronic inflammation they can participate in perpetuating the self-destructive environment by secretion of inflammatory factors and/or presentation of myelin epitopes to autoreactive T cells. Dendritic cells (DC) are also participants in the presentation of antigen to T cells, even within the CNS. While the APCs alone are not solely responsible for mediating the destruction to the myelin sheath, they are critical players in perpetuating the inflammatory milieu. This review will highlight relevant studies which have provided insight to the roles played by microglia, DCs and astrocytes in the context of CNS autoimmunity. PMID:20637861

  18. Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells

    NASA Astrophysics Data System (ADS)

    Herce, Henry D.; Schumacher, Dominik; Schneider, Anselm F. L.; Ludwig, Anne K.; Mann, Florian A.; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M. Cristina; Hackenberger, Christian P. R.

    2017-08-01

    Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

  19. Analysis of the limitations of hepatitis B surface antigen expression in soybean cell suspension cultures.

    PubMed

    Ganapathi, T R; Sunil Kumar, G B; Srinivas, L; Revathi, C J; Bapat, V A

    2007-09-01

    Soybean cell suspension cultures were transformed using Agrobacterium tumefaciens harboring pHBS/pHER constructs to express hepatitis B surface antigen (HBsAg). The transformed colonies were selected and analyzed for the expression of HBsAg by PCR, reverse transcription (RT) PCR, Western blot and ELISA analysis. The maximum expression of 700 ng/g F.W. was noted in pHER transformed cells. The highest expressing colonies were used to initiate the cell suspension cultures and the expression of HBsAg was estimated periodically. The expression levels were reduced drastically in cell suspension cultures compared to the colonies maintained on semi-solid medium. Various parameters were studied to maximize the cell growth and to retain the expression levels. The supplementation of culture medium with a protease inhibitor, leupeptin hemisulfate could restore up to 50% of HBsAg expression in cell suspension cultures. This is the first report to investigate the possible cause and solution to the loss of recombinant protein expression levels in plant cell suspension cultures.

  20. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte.

    PubMed

    Claycomb, W C; Lanson, N A; Stallworth, B S; Egeland, D B; Delcarpio, J B; Bahinski, A; Izzo, N J

    1998-03-17

    We have derived a cardiac muscle cell line, designated HL-1, from the AT-1 mouse atrial cardiomyocyte tumor lineage. HL-1 cells can be serially passaged, yet they maintain the ability to contract and retain differentiated cardiac morphological, biochemical, and electrophysiological properties. Ultrastructural characteristics typical of embryonic atrial cardiac muscle cells were found consistently in the cultured HL-1 cells. Reverse transcriptase-PCR-based analyses confirmed a pattern of gene expression similar to that of adult atrial myocytes, including expression of alpha-cardiac myosin heavy chain, alpha-cardiac actin, and connexin43. They also express the gene for atrial natriuretic factor. Immunohistochemical staining of the HL-1 cells indicated that the distribution of the cardiac-specific markers desmin, sarcomeric myosin, and atrial natriuretic factor was similar to that of cultured atrial cardiomyocytes. A delayed rectifier potassium current (IKr) was the most prominent outward current in HL-1 cells. The activating currents displayed inward rectification and deactivating current tails were voltage-dependent, saturated at >+20 mV, and were highly sensitive to dofetilide (IC50 of 46.9 nM). Specific binding of [3H]dofetilide was saturable and fit a one-site binding isotherm with a Kd of 140 +/- 60 nM and a Bmax of 118 fmol per 10(5) cells. HL-1 cells represent a cardiac myocyte cell line that can be repeatedly passaged and yet maintain a cardiac-specific phenotype.

  1. Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells

    PubMed Central

    Frigault, Matthew J; Lee, Jihyun; Basil, Maria Ciocca; Carpenito, Carmine; Motohashi, Shinichiro; Scholler, John; Kawalekar, Omkar U.; Guedan, Sonia; McGettigan, Shannon E.; Posey, Avery D.; Ang, Sonny; Cooper, Laurence J. N.; Platt, Jesse M.; Johnson, F. Brad; Paulos, Chrystal M; Zhao, Yangbing; Kalos, Michael; Milone, Michael C.; June, Carl H.

    2015-01-01

    This study compared second generation chimeric antigen receptors encoding signaling domains composed of CD28, ICOS and 4-1BB. Here we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T-cell with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to three months following a single stimulation through the TCR. Sustained numeric expansion was independent of cognate antigen and did not require the addition of exogenous cytokines or feeder cells after a single stimulation of the TCR and CD28. Results from gene array and functional assays linked sustained cytokine secretion and expression of T-bet, EOMES and GATA-3 to the effect. Sustained expression of the endogenous IL2 locus has not been reported in primary T cells. Sustained proliferation was dependent on CAR structure and high expression, the latter of which was necessary but not sufficient. The mechanism involves constitutive signaling through NF-kB, Akt, Erk and NFAT. The propagated CAR T cells retained a diverse TCR repertoire and cellular transformation was not observed. The CARs with a constitutive growth phenotype displayed inferior antitumor effects and engraftment in vivo. Therefore the design of CARs that have a non-constitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or non-constitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials. PMID:25600436

  2. HIV-1--Infected T Cells Show a Selective Signaling Defect after Perturbation of CD3/Antigen Receptor

    NASA Astrophysics Data System (ADS)

    Linette, Gerald P.; Hartzman, Robert J.; Ledbetter, Jeffrey A.; June, Carl H.

    1988-07-01

    The binding of antigen or monoclonal antibody to the T cell receptor for antigen or the closely associated CD3 complex causes increases in the concentration of intracellular ionized calcium and subsequent cell proliferation. By measuring second messenger production in primary cultures of human immunodeficiency virus (HIV-1)--infected T cells stimulated with monoclonal antibodies specific for either CD3 or CD2, a specific impairment of membrane signaling was revealed. The HIV-1--infected T cells were unable to mobilize Ca2+ after stimulation with anti-CD3, whereas CD2-induced calcium mobilization remained intact. Furthermore, the HIV-1--infected cells proliferated poorly after CD3 stimulation, although the cells retained normal DNA synthesis in response to interleukin-2 stimulation. These results show that the signals initiated by CD2 and CD3 can be regulated independently within the same T cell; uncoupling of signal transduction after antigen-specific stimulation provides a biochemical mechanism to explain, in part, the profound immunodeficiency of patients with HIV-1 infection.

  3. Elutriated lymphocytes for manufacturing chimeric antigen receptor T cells.

    PubMed

    Stroncek, David F; Lee, Daniel W; Ren, Jiaqiang; Sabatino, Marianna; Highfill, Steven; Khuu, Hanh; Shah, Nirali N; Kaplan, Rosandra N; Fry, Terry J; Mackall, Crystal L

    2017-03-16

    Clinical trials of Chimeric Antigen Receptor (CAR) T cells manufactured from autologous peripheral blood mononuclear cell (PBMC) concentrates for the treatment of hematologic malignancies have been promising, but CAR T cell yields have been variable. This variability is due in part to the contamination of the PBMC concentrates with monocytes and granulocytes. Counter-flow elutriation allows for the closed system separation of lymphocytes from monocytes and granulocytes. We investigated the use of PBMC concentrates enriched for lymphocytes using elutriation for manufacturing 8 CD19- and 5 GD2-CAR T cell products. When compared to PBMC concentrates, lymphocyte-enriched elutriation fractions contained greater proportions of CD3+ and CD56+ cells and reduced proportions of CD14+ and CD15+ cells. All 13 CAR T cell products manufactured using the elutriated lymphocytes yielded sufficient quantities of transduced CAR T cells to meet clinical dose criteria. The GD2-CAR T cell products contained significantly more T cells and transduced T cells than the CD19-CAR T cell products. A comparison of the yields of CAR T cells produced from elutriated lymphocytes with the yields of CAR T cells previous produced from cells isolated from PBMC concentrates by anti-CD3/CD28 bead selection or by anti-CD3/CD28 bead selection plus plastic adherence found that greater quantities of GD2-CAR T cells were produced from elutriated lymphocytes, but not CD19-CAR T cells. Enrichment of PBMC concentrates for lymphocytes using elutriation increased the quantity of GD2-CAR T cells produced. These results provide further evidence that CAR T cell expansion is inhibited by monocytes and granulocytes.

  4. T cells expressing an anti–B-cell maturation antigen chimeric antigen receptor cause remissions of multiple myeloma

    PubMed Central

    Ali, Syed Abbas; Shi, Victoria; Maric, Irina; Wang, Michael; Stroncek, David F.; Rose, Jeremy J.; Brudno, Jennifer N.; Stetler-Stevenson, Maryalice; Feldman, Steven A.; Hansen, Brenna G.; Fellowes, Vicki S.; Hakim, Frances T.; Gress, Ronald E.

    2016-01-01

    Therapies with novel mechanisms of action are needed for multiple myeloma (MM). B-cell maturation antigen (BCMA) is expressed in most cases of MM. We conducted the first-in-humans clinical trial of chimeric antigen receptor (CAR) T cells targeting BCMA. T cells expressing the CAR used in this work (CAR-BCMA) specifically recognized BCMA-expressing cells. Twelve patients received CAR-BCMA T cells in this dose-escalation trial. Among the 6 patients treated on the lowest 2 dose levels, limited antimyeloma activity and mild toxicity occurred. On the third dose level, 1 patient obtained a very good partial remission. Two patients were treated on the fourth dose level of 9 × 106 CAR+ T cells/kg body weight. Before treatment, the first patient on the fourth dose level had chemotherapy-resistant MM, making up 90% of bone marrow cells. After treatment, bone marrow plasma cells became undetectable by flow cytometry, and the patient’s MM entered a stringent complete remission that lasted for 17 weeks before relapse. The second patient on the fourth dose level had chemotherapy-resistant MM making up 80% of bone marrow cells before treatment. Twenty-eight weeks after this patient received CAR-BCMA T cells, bone marrow plasma cells were undetectable by flow cytometry, and the serum monoclonal protein had decreased by >95%. This patient is in an ongoing very good partial remission. Both patients treated on the fourth dose level had toxicity consistent with cytokine-release syndrome including fever, hypotension, and dyspnea. Both patients had prolonged cytopenias. Our findings demonstrate antimyeloma activity of CAR-BCMA T cells. This trial was registered to www.clinicaltrials.gov as #NCT02215967. PMID:27412889

  5. Adult T-cell leukemia: antigen in an ATL cell line and detection of antibodies to the antigen in human sera.

    PubMed Central

    Hinuma, Y; Nagata, K; Hanaoka, M; Nakai, M; Matsumoto, T; Kinoshita, K I; Shirakawa, S; Miyoshi, I

    1981-01-01

    Indirect immunofluorescence of certain human sera demonstrated an antigen(s) in the cytoplasm of 1--5% of the cells of a T-cell line, MT-1, from a patient with adult T-cell leukemia (ATL), which is endemic in southwestern Japan. The antigen was not detected in other human lymphoid cell lines, including six T-cell lines, seven B-cell lines, and four non-T non-B cell lines. The antigen did not show cross antigenicity with that of herpesviruses, including Epstein--Barr virus, herpes simplex virus, cytomegalovirus, varicella-zoster virus, herpesvirus saimiri, and Marek disease virus. The proportion of antigen-bearing cells was increased by a factor of approximately 5 on culture in the presence of 5-iodo-2'-deoxyuridine. Antibodies against the antigen in MT-1 cells were found in all 44 patients with ATL examined and in 32 of 40 patients with malignant T-cell lymphomas (most of them had diseases similar to ATL except that leukemic cells were not found in the peripheral blood). The antibodies were also detected in 26% of the healthy adults examined from ATL-endemic areas but in only a few of those examined from ATL-non-endemic areas. On electron microscopy, extracellular type C virus particles were detected in pelleted MT-1 cells cultured in the presence of 5-iodo-2'-deoxyuridine. Images PMID:7031654

  6. Do FY antigens act as minor histocompatibility antigens in the graft-versus-host disease paradigm after human leukocyte antigen-identical sibling hematopoietic stem cell transplantation?

    PubMed

    Sellami, Mohamed Hichem; Chaabane, Manel; Kaabi, Houda; Torjemane, Lamia; Ladeb, Saloua; Ben Othmane, Tarek; Hmida, Slama

    2012-03-01

    FY antigens are candidate minor histocompatibility antigens relevant to renal allograft rejection, but no data have been reported about their role in graft-versus-host disease (GVHD) incidence after human leukocyte antigen (HLA)-identical siblings hematopoietic stem cell transplantation (HSCT). The aim of this study was to examine the effect of donor/recipient disparity at FY antigens on the incidence of GVHD in Tunisian patients receiving an HLA-identical HSCT. This work enrolled 105 Tunisian pairs of recipients and their HLA-identical sibling donors of HSCs. FY genotyping was performed with the polymerase chain reaction-sequence-specific primer method and donor/recipient disparity for these antigens was analyzed at two levels: incompatibility and nonidentity. The case-control analyses showed no significant correlation between FY disparity and the incidence of either acute or chronic GVHD. Sample size calculation showed that 572 cases and 1716 controls would be necessary to be able to detect a significant association with 80% power and two-sided type I error level of 5% (α=0.05). The lack of association in the studied cohort may be explained by the low immunogenicity of FY antigens in HSCT context, compared with other antigens such as HA-1 and CD31.

  7. Nuclear EGFR characterize still controlled proliferation retained in better differentiated clear cell RCC.

    PubMed

    Ahel, J; Dordevic, G; Markic, D; Mozetic, V; Spanjol, J; Grahovac, B; Stifter, S

    2015-08-01

    Renal cell carcinoma (RCC) is the most common solid kidney tumor representing 2-3% of all cancers, with the highest frequency occurring in Western countries. There was a worldwide and European annual increase in incidence of approximately 2% although incidence has been stabilized in last few years. One third of the patients already have metastases in the time of the diagnosis with poor prognosis because RCC are radio and chemoresistant. The prognostic value of EGFR over-expression in RCC is a controversial issue that could be explained by different histological types of study tumors and non-standardized criteria for evaluation of expression. Recent evidences points to a new mode of EGFR signaling pathway in which activated EGFR undergoes nuclear translocalization and then, as transcription factor, mediates gene expression and other cellular events required for highly proliferating activities. According to our observations, the membranous expression of EGFR associates with high nuclear grade and poor differentiated tumors. On the other hand, nuclear EGFR expression was high in low nuclear graded and well differentiated tumors with good prognosis. We hypothesize that this mode of EGFR signaling characterizes still controlled proliferation retained in well differentiated RCC with Furhman nuclear grade I or II.

  8. Antigen exposure shapes the ratio between antigen-specific Tregs and conventional T cells in human peripheral blood

    PubMed Central

    Su, Laura F.; del Alcazar, Daniel; Stelekati, Erietta; Wherry, E. John; Davis, Mark M.

    2016-01-01

    The T-cell receptor (TCR) is required for maturation and function of regulatory T cells (Tregs), but the ligand specificities of Tregs outside the context of transgenic TCRs are largely unknown. Using peptide–MHC tetramers, we isolated rare specific Foxp3+ cells directly ex vivo from adult peripheral blood and defined their frequency and phenotype. We find that a proportion of circulating Tregs recognize foreign antigens and the frequency of these cells are similar to that of self-reactive Tregs in the absence of cognate infection. In contrast, the frequencies of Tregs that recognize some common microbial antigens are significantly reduced in the blood of most adults. Exposure to peripheral antigens likely has a major influence on the balance between Tregs and conventional T-cell subsets because a larger proportion of flu-specific T cells has a regulatory cell phenotype in the cord blood. Consistent with this finding, we show that lymphocytic choriomeningitis virus infection can directly modulate the ratio of virus-specific effectors and Tregs in mice. The resulting change in the balance within an antigen-specific T-cell population further correlates with the magnitude of effector response and the chronicity of infection. Taken together, our data highlight the importance of antigen specificity in the functional dynamics of the T-cell repertoire. Each specific population of CD4+ T cells in human peripheral blood contains a subset of Tregs at birth, but the balance between regulatory and effector subsets changes in response to peripheral antigen exposure and this could impact the robustness of antipathogen immunity. PMID:27681619

  9. Does an alloimmune response to strong immunogenic red blood cell antigens enhance a response to weaker antigens?

    PubMed

    Schonewille, Henk; Brand, Anneke

    2008-05-01

    It has been suggested that an immune response against the high immunogenic D antigen also augments the immune response to less immunogenic red blood cell (RBC) antigens. Based on the high antibody frequency, E and K antigens can also be regarded as strong immunogens. The question is whether the immunization against E and K antigens also enhances the formation of other antibody specificities. This question is in particular relevant for patients who are currently transfused with RH- and/or K-matched RBCs. A retrospective multicenter study analyzed FY, JK, and MNS antibodies alone and in combination with anti-E and/or anti-K. Analysis was performed for primary and subsequent antibody responses. In the cohort analyzed, 5016 patients possessed 5981 antibodies. Antibodies directed to multiple blood group systems were present in 606 of the 779 (78%) patients with multiple antibodies. In 88 of 1270 (6.9%) patients, FY, JK, and/or MNS antibodies appeared simultaneous with anti-E and/or anti-K during a primary antibody response after transfusion. Patients formed antibodies to antigens in the FY, JK, and MNS systems equally often as first antibodies followed by anti-E or anti-K than as second antibodies after anti-E or anti-K were already present. Patients with anti-E and/or anti-K or with antibodies to antigens in the FY, JK, and/or MNS systems equally often formed additional antibodies during a second antibody response. An immune response against allogeneic RBC antigens defines good responders who readily form antibodies against other antigens. No support was found that the response against strong RBC antigens also enhances the formation against weaker antigens.

  10. Y. enterocolitica translocated Yops impair stimulation of T-cells by antigen presenting cells.

    PubMed

    Kramer, Uwe; Wiedig, Carolin A

    2005-09-15

    As T helper cells play a crucial role in the defense of the mouse immune system against Yersinia enterocolitica, an effective subversion strategy for the pathogen would be the inhibition of T-cell activation. In this study, we investigated whether Y. enterocolitica impairs this process on the level of antigen presentation. For this purpose, we used T-cells to measure the antigen presentation capacity of dendritic cells after they had been incubated with different types of Yersinia mutants. We could show that Y. enterocolitica impairs the processing of antigens by dendritic cells, that this effect is dependent on factors translocated by the pathogenicity-plasmid-encoded type III secretion system and that the most important factor appears to be YopP. The YopP effect is partly mediated by the killing of APCs, but in addition to this there appears to be an alternative way of action that results in the inhibition of antigen processing. The YopP effect is not mediated by soluble factors. In contrast to antigen processing, antigen presentation was only weakly affected by pathogenicity plasmid encoded factors in dendritic cells, but obviously in A20.J B-cells.

  11. Stable isotope labeling of oligosaccharide cell surface antigens

    SciTech Connect

    Unkefer, C.J.; Silks, L.A. III; Martinez, R.A.

    1998-12-31

    The overall goal of this Laboratory Directed Research and Development (LDRD) project was to develop new methods for synthesis of {sup 13}C-labeled oligosaccharides that are required for nuclear magnetic resonance (NMR) studies of their solution conformation. Oligosaccharides are components of the cell`s outer surface and are involved in important processes such as cell-cell recognition and adhesion. Recently, Danishefsky and coworkers at Slone-Kettering Cancer Center developed a method for the solid-phase chemical synthesis of oligosaccharides. The specific goal of this LDRD project was to prepare uniform {sup 13}C-labeled aldohexose precursors required for the solid-phase synthesis of the Lewis blood-group antigenic determinants. We report the synthesis of {sup 13}C-labeled D-glucal, D-galactal and Fucosyl precursors. We have been collaborating with the Danishefsky group on the synthesis of the Lewis oligosaccharides and the NMR analysis of their solution conformation.

  12. Prostate stem cell antigen: a Jekyll and Hyde molecule?

    PubMed

    Saeki, Norihisa; Gu, Jian; Yoshida, Teruhiko; Wu, Xifeng

    2010-07-15

    Prostate stem cell antigen (PSCA) is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein. Although PSCA is thought to be involved in intracellular signaling, much remains unknown about its physiological function and regulatory mechanism in normal and cancer cells. It is up-regulated in several major cancers including prostate, bladder, and pancreatic cancers. The expression of PSCA is positively correlated with advanced clinical stage and metastasis in prostate cancers and is also associated with malignant progression of premalignant prostate lesions. Therefore, PSCA has been proposed as a biomarker of diagnosis and prognosis, as well as a target of therapy for these cancers. In addition, PSCA has also shown clinical potential in immunotherapy as a prostate-specific antigen, which, when presented by dendritic cells, may elicit strong tumor-specific immunity. In contrast, PSCA is down-regulated in esophageal and gastric cancer and may have a tumor-suppressing function in the gastric epithelium. Recent exciting findings that genetic variations of PSCA conferred increased risks of gastric cancer and bladder cancer have opened up a new avenue of research about the pathological function of PSCA. PSCA seems to be a Jekyll and Hyde molecule that plays differential roles, tumor promoting or suppressing, depending on the cellular context. Copyright 2010 AACR.

  13. Prostate stem cell antigen (PSCA): a Jekyll and Hyde molecule?

    PubMed Central

    Saeki, Norihisa; Gu, Jian; Yoshida, Teruhiko; Wu, Xifeng

    2010-01-01

    Prostate stem cell antigen (PSCA) is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein. Although PSCA is thought to be involved in intracellular signaling, much remain unknown regarding its physiological function and regulatory mechanism in normal and cancer cells. It is up-regulated in several major cancers including prostate, bladder and pancreatic cancers. The expression of PSCA is positively correlated with advanced clinical stage and metastasis in prostate cancers and is also associated with malignant progression of pre-malignant prostate lesions. Therefore, PSCA has been proposed as a biomarker of diagnosis and prognosis, as well as a target of therapy for these cancers. In addition, PSCA has also shown clinical potential in immunotherapy as a prostate specific antigen which, when presented by dendritic cells, may elicit strong tumor specific immunity. In contrast, PSCA is down-regulated in esophageal and gastric cancer and may have tumor-suppressing function in the gastric epithelium. Recent exciting findings that genetic variations of PSCA conferred increased risks of gastric cancer and bladder cancer have opened up a new avenue of research regarding the pathological function of PSCA. PSCA appears to be a Jekyll and Hyde molecule that plays differential roles, tumor promoting or suppressing, depending on the cellular context. PMID:20501618

  14. Induction of B cell immune tolerance by antigen-modified CTL

    PubMed Central

    Nguyen, Phuong; Geiger, Terrence L.

    2010-01-01

    Background Third party-specific CTL, or veto CTL, are being assessed as a cellular therapeutic for the induction of T cell tolerance during transplantation. Conceptually, veto cell expressed antigens may induce B cell immune responses, and this may have deleterious consequences. Whether veto cells induce immunity, tolerance, or are ignored by B lymphocytes has, however, not been addressed. Methods CTL were retrovirally transduced with a model cell surface antigen to generate veto CTL. The impact of CTL-specific antigen expression on the activation and tolerization of antigen specific B cells was assessed in vitro and, using adoptive transfer models, in vivo. Results In vitro, CTL-expressed antigen induced an abortive proliferative response in specific B lymphocytes, whereby an initial proliferative burst was followed by cell death. In vivo, the administration of veto CTL also induced B cell tolerance. Specific immunoglobulin was not detected after subsequent immunization with a veto cell-expressed antigen. Modeling of this effect with antigen-specific B cell receptor (BCR) transgenic B lymphocytes demonstrated that antigen-specific B cells were eliminated by the veto CTL; cell division was accompanied by the exhaustion and depletion of responding cells. Veto-induced B cell tolerance could be wholly abrogated by treatment with the toll-like receptor ligand LPS, implying that this tolerance resulted from the absence of adequate supplemental signals during antigenic stimulation. Conclusions Veto CTL are effective promoters of B cell tolerance. Further assessment of their therapeutic potential in this regard is warranted. PMID:20065917

  15. Paired Expression Analysis of Tumor Cell Surface Antigens

    PubMed Central

    Orentas, Rimas J.; Sindiri, Sivasish; Duris, Christine; Wen, Xinyu; He, Jianbin; Wei, Jun S.; Jarzembowski, Jason; Khan, Javed

    2017-01-01

    Adoptive immunotherapy with antibody-based therapy or with T cells transduced to express chimeric antigen receptors (CARs) is useful to the extent that the cell surface membrane protein being targeted is not expressed on normal tissues. The most successful CAR-based (anti-CD19) or antibody-based therapy (anti-CD20) in hematologic malignancies has the side effect of eliminating the normal B cell compartment. Targeting solid tumors may not provide a similar expendable marker. Beyond antibody to Her2/NEU and EGFR, very few antibody-based and no CAR-based therapies have seen broad clinical application for solid tumors. To expand the way in which the surfaceome of solid tumors can be analyzed, we created an algorithm that defines the pairwise relative overexpression of surface antigens. This enables the development of specific immunotherapies that require the expression of two discrete antigens on the surface of the tumor target. This dyad analysis was facilitated by employing the Hotelling’s T-squared test (Hotelling–Lawley multivariate analysis of variance) for two independent variables in comparison to a third constant entity (i.e., gene expression levels in normal tissues). We also present a unique consensus scoring mechanism for identifying transcripts that encode cell surface proteins. The unique application of our bioinformatics processing pipeline and statistical tools allowed us to compare the expression of two membrane protein targets as a pair, and to propose a new strategy based on implementing immunotherapies that require both antigens to be expressed on the tumor cell surface to trigger therapeutic effector mechanisms. Specifically, we found that, for MYCN amplified neuroblastoma, pairwise expression of ACVR2B or anaplastic lymphoma kinase (ALK) with GFRA3, GFRA2, Cadherin 24, or with one another provided the strongest hits. For MYCN, non-amplified stage 4 neuroblastoma, neurotrophic tyrosine kinase 1, or ALK paired with GFRA2, GFRA3, SSK1, GPR173, or

  16. Gravity sedimentation of granulocytapheresis concentrates with hydroxyethyl starch efficiently removes red blood cells and retains neutrophils.

    PubMed

    Bryant, Barbara J; Yau, Yu Ying; Byrne, Phyllis J; Stroncek, David F; Leitman, Susan F

    2010-06-01

    Transfusion of granulocytapheresis concentrates can be limited by the volume of incompatible donor red blood cells (RBCs) in the component. Efficient reduction of RBCs in granulocyte units would result in safe transfusion of RBC-incompatible units. Granulocyte concentrates were collected by continuous-flow apheresis from granulocyte-colony-stimulating factor (G-CSF) and dexamethasone-stimulated volunteer donors, with 6% hydroxyethyl starch (HES) added continuously during apheresis as a RBC sedimenting agent to enhance granulocyte collection efficiency. After collection, the component was placed in a plasma extractor for 4 hours. A sharp line of demarcation between the starch-sedimented RBCs and the granulocyte-rich supernatant developed, and the supernatant was transferred to a sterilely docked transfer pack. RBC reduction and white blood cell recovery were determined. Gravity sedimentation was performed on 165 granulocyte concentrates. Mean sedimentation time was 267 minutes (range, 150-440 min). RBC depletion was 92% (range, 71%-99%) with mean residual RBC content of 3.2 +/- 1.4 mL. Twelve percent of components contained less than 2 mL of RBCs. Mean granulocyte and platelet (PLT) recoveries were 80 and 81%, respectively. There were no transfusion reactions or signs of hemolysis after transfusion of 66 RBC-incompatible granulocyte concentrates (RBC volume, 1.6-8.2 mL). The remaining concentrates were used for topical or intrapleural applications. RBCs were significantly reduced and granulocytes and PLTs effectively retained in G-CSF/steroid-mobilized granulocyte components collected with HES and processed by gravity sedimentation. This procedure allows safe transfusion of RBC-incompatible sedimented granulocyte units and may be used to expand the pool of available granulocyte donors for specific recipients.

  17. Chimeric Antigen Receptor T Cells for Sustained Remissions in Leukemia

    PubMed Central

    Maude, Shannon L.; Frey, Noelle; Shaw, Pamela A.; Aplenc, Richard; Barrett, David M.; Bunin, Nancy J.; Chew, Anne; Gonzalez, Vanessa E.; Zheng, Zhaohui; Lacey, Simon F.; Mahnke, Yolanda D.; Melenhorst, Jan J.; Rheingold, Susan R.; Shen, Angela; Teachey, David T.; Levine, Bruce L.; June, Carl H.; Porter, David L.; Grupp, Stephan A.

    2014-01-01

    BACKGROUND Relapsed acute lymphoblastic leukemia (ALL) is difficult to treat despite the availability of aggressive therapies. Chimeric antigen receptor–modified T cells targeting CD19 may overcome many limitations of conventional therapies and induce remission in patients with refractory disease. METHODS We infused autologous T cells transduced with a CD19-directed chimeric antigen receptor (CTL019) lentiviral vector in patients with relapsed or refractory ALL at doses of 0.76×106 to 20.6×106 CTL019 cells per kilogram of body weight. Patients were monitored for a response, toxic effects, and the expansion and persistence of circulating CTL019 T cells. RESULTS A total of 30 children and adults received CTL019. Complete remission was achieved in 27 patients (90%), including 2 patients with blinatumomab-refractory disease and 15 who had undergone stem-cell transplantation. CTL019 cells proliferated in vivo and were detectable in the blood, bone marrow, and cerebrospinal fluid of patients who had a response. Sustained remission was achieved with a 6-month event-free survival rate of 67% (95% confidence interval [CI], 51 to 88) and an overall survival rate of 78% (95% CI, 65 to 95). At 6 months, the probability that a patient would have persistence of CTL019 was 68% (95% CI, 50 to 92) and the probability that a patient would have relapse-free B-cell aplasia was 73% (95% CI, 57 to 94). All the patients had the cytokine-release syndrome. Severe cytokine-release syndrome, which developed in 27% of the patients, was associated with a higher disease burden before infusion and was effectively treated with the anti–interleukin-6 receptor antibody tocilizumab. CONCLUSIONS Chimeric antigen receptor–modified T-cell therapy against CD19 was effective in treating relapsed and refractory ALL. CTL019 was associated with a high remission rate, even among patients for whom stem-cell transplantation had failed, and durable remissions up to 24 months were observed. (Funded by

  18. Chimeric antigen receptor T cells for sustained remissions in leukemia.

    PubMed

    Maude, Shannon L; Frey, Noelle; Shaw, Pamela A; Aplenc, Richard; Barrett, David M; Bunin, Nancy J; Chew, Anne; Gonzalez, Vanessa E; Zheng, Zhaohui; Lacey, Simon F; Mahnke, Yolanda D; Melenhorst, Jan J; Rheingold, Susan R; Shen, Angela; Teachey, David T; Levine, Bruce L; June, Carl H; Porter, David L; Grupp, Stephan A

    2014-10-16

    Relapsed acute lymphoblastic leukemia (ALL) is difficult to treat despite the availability of aggressive therapies. Chimeric antigen receptor-modified T cells targeting CD19 may overcome many limitations of conventional therapies and induce remission in patients with refractory disease. We infused autologous T cells transduced with a CD19-directed chimeric antigen receptor (CTL019) lentiviral vector in patients with relapsed or refractory ALL at doses of 0.76×10(6) to 20.6×10(6) CTL019 cells per kilogram of body weight. Patients were monitored for a response, toxic effects, and the expansion and persistence of circulating CTL019 T cells. A total of 30 children and adults received CTL019. Complete remission was achieved in 27 patients (90%), including 2 patients with blinatumomab-refractory disease and 15 who had undergone stem-cell transplantation. CTL019 cells proliferated in vivo and were detectable in the blood, bone marrow, and cerebrospinal fluid of patients who had a response. Sustained remission was achieved with a 6-month event-free survival rate of 67% (95% confidence interval [CI], 51 to 88) and an overall survival rate of 78% (95% CI, 65 to 95). At 6 months, the probability that a patient would have persistence of CTL019 was 68% (95% CI, 50 to 92) and the probability that a patient would have relapse-free B-cell aplasia was 73% (95% CI, 57 to 94). All the patients had the cytokine-release syndrome. Severe cytokine-release syndrome, which developed in 27% of the patients, was associated with a higher disease burden before infusion and was effectively treated with the anti-interleukin-6 receptor antibody tocilizumab. Chimeric antigen receptor-modified T-cell therapy against CD19 was effective in treating relapsed and refractory ALL. CTL019 was associated with a high remission rate, even among patients for whom stem-cell transplantation had failed, and durable remissions up to 24 months were observed. (Funded by Novartis and others; CART19 Clinical

  19. Antigen-B Cell Receptor Complexes Associate with Intracellular major histocompatibility complex (MHC) Class II Molecules*

    PubMed Central

    Barroso, Margarida; Tucker, Heidi; Drake, Lisa; Nichol, Kathleen; Drake, James R.

    2015-01-01

    Antigen processing and MHC class II-restricted antigen presentation by antigen-presenting cells such as dendritic cells and B cells allows the activation of naïve CD4+ T cells and cognate interactions between B cells and effector CD4+ T cells, respectively. B cells are unique among class II-restricted antigen-presenting cells in that they have a clonally restricted antigen-specific receptor, the B cell receptor (BCR), which allows the cell to recognize and respond to trace amounts of foreign antigen present in a sea of self-antigens. Moreover, engagement of peptide-class II complexes formed via BCR-mediated processing of cognate antigen has been shown to result in a unique pattern of B cell activation. Using a combined biochemical and imaging/FRET approach, we establish that internalized antigen-BCR complexes associate with intracellular class II molecules. We demonstrate that the M1-paired MHC class II conformer, shown previously to be critical for CD4 T cell activation, is incorporated selectively into these complexes and loaded selectively with peptide derived from BCR-internalized cognate antigen. These results demonstrate that, in B cells, internalized antigen-BCR complexes associate with intracellular MHC class II molecules, potentially defining a site of class II peptide acquisition, and reveal a selective role for the M1-paired class II conformer in the presentation of cognate antigen. These findings provide key insights into the molecular mechanisms used by B cells to control the source of peptides charged onto class II molecules, allowing the immune system to mount an antibody response focused on BCR-reactive cognate antigen. PMID:26400081

  20. Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells

    PubMed Central

    Chabaud, Mélanie; Heuzé, Mélina L.; Bretou, Marine; Vargas, Pablo; Maiuri, Paolo; Solanes, Paola; Maurin, Mathieu; Terriac, Emmanuel; Le Berre, Maël; Lankar, Danielle; Piolot, Tristan; Adelstein, Robert S.; Zhang, Yingfan; Sixt, Michael; Jacobelli, Jordan; Bénichou, Olivier; Voituriez, Raphaël; Piel, Matthieu; Lennon-Duménil, Ana-Maria

    2015-01-01

    The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space. PMID:26109323

  1. CNS Schwann cells display oligodendrocyte precursor-like potassium channel activation and antigenic expression in vitro.

    PubMed

    Kegler, Kristel; Imbschweiler, Ilka; Ulrich, Reiner; Kovermann, Peter; Fahlke, Christoph; Deschl, Ulrich; Kalkuhl, Arno; Baumgärnter, Wolfgang; Wewetzer, Konstantin

    2014-06-01

    Central nervous system (CNS) injury triggers production of myelinating Schwann cells from endogenous oligodendrocyte precursors (OLPs). These CNS Schwann cells may be attractive candidates for novel therapeutic strategies aiming to promote endogenous CNS repair. However, CNS Schwann cells have been so far mainly characterized in situ regarding morphology and marker expression, and it has remained enigmatic whether they display functional properties distinct from peripheral nervous system (PNS) Schwann cells. Potassium channels (K+) have been implicated in progenitor and glial cell proliferation after injury and may, therefore, represent a suitable pharmacological target. In the present study, we focused on the function and expression of voltage-gated K+ channels Kv(1-12) and accessory β-subunits in purified adult canine CNS and PNS Schwann cell cultures using electrophysiology and microarray analysis and characterized their antigenic phenotype. We show here that K+ channels differed significantly in both cell types. While CNS Schwann cells displayed prominent K D-mediated K+ currents, PNS Schwann cells elicited K(D-) and K(A-type) K+ currents. Inhibition of K+ currents by TEA and Ba2+ was more effective in CNS Schwann cells. These functional differences were not paralleled by differential mRNA expression of Kv(1-12) and accessory β-subunits. However, O4/A2B5 and GFAP expressions were significantly higher and lower, respectively, in CNS than in PNS Schwann cells. Taken together, this is the first evidence that CNS Schwann cells display specific properties not shared by their peripheral counterpart. Both Kv currents and increased O4/A2B5 expression were reminiscent of OLPs suggesting that CNS Schwann cells retain OLP features during maturation.

  2. Inhibitory effects of thymus-independent type 2 antigens on MHC class II-restricted antigen presentation: comparative analysis of carbohydrate structures and the antigen presenting cell.

    PubMed

    González-Fernández, M; Carrasco-Marín, E; Alvarez-Domínguez, C; Outschoorn, I M; Leyva-Cobián, F

    1997-02-25

    The role of thymus-independent type 2 (TI-2) antigens (polysaccharides) on the MHC-II-restricted processing of protein antigens was studied in vitro. In general, antigen presentation is inhibited when both peritoneal and splenic macrophages (M phi) as well as Küpffer cells (KC) are preincubated with acidic polysaccharides or branched dextrans. However, the inhibitory effect of neutral polysaccharides was minimal when KC were used as antigen presenting cells (APC). Morphological evaluation of the uptake of fluoresceinated polysaccharides clearly correlates with this selective and differential interference. Polysaccharides do not block MHC-I-restricted antigen presentation. Some chemical characteristics shared by different saccharides seem to be specially related to their potential inhibitory abilities: (i) those where two anomeric carbon atoms of two interlinked sugars and (ii) those containing several sulfate groups per disaccharide repeating unit. No polysaccharide being inhibitory in M phi abrogated antigen processing in other APC: lipopolysaccharide-activated B cells, B lymphoma cells, or dendritic cells (DC). Using radiolabeled polysaccharides it was observed that DC and B cells incorporated less radioactivity as a function of time than M phi. Morphological evaluation of these different APC incubated for extended periods of time with inhibitory concentrations of polysaccharides revealed intense cytoplasmic vacuolization in M phi but not in B cells or DC. The large majority of M phi lysosomes containing polysaccharides fail to fuse with incoming endocytic vesicles and delivery of fluid-phase tracers was reduced, suggesting that indigestible carbohydrates reduced the fusion of these loaded lysosomes with endosomes containing recently internalized tracers. It is suggested that the main causes of this antigen presentation blockade are (i) the chemical characteristics of certain carbohydrates and whether the specific enzymatic machinery for their intracellular

  3. Functionalized Magnetic Nanoparticles for the Detection and Quantitative Analysis of Cell Surface Antigen

    PubMed Central

    Shahbazi-Gahrouei, Daryoush; Abdolahi, Mohammad; Zarkesh-Esfahani, Sayyed Hamid; Laurent, Sophie; Sermeus, Corine; Gruettner, Cordula

    2013-01-01

    Cell surface antigens as biomarkers offer tremendous potential for early diagnosis, prognosis, and therapeutic response in a variety of diseases such as cancers. In this research, a simple, rapid, accurate, inexpensive, and easily available in vitro assay based on magnetic nanoparticles and magnetic cell separation principle was applied to identify and quantitatively analyze the cell surface antigen expression in the case of prostate cancer cells. Comparing the capability of the assay with flow cytometry as a gold standard method showed similar results. The results showed that the antigen-specific magnetic cell separation with antibody-coated magnetic nanoparticles has high potential for quantitative cell surface antigen detection and analysis. PMID:23484112

  4. T-cell intracellular antigens function as tumor suppressor genes.

    PubMed

    Sánchez-Jiménez, C; Ludeña, M D; Izquierdo, J M

    2015-03-05

    Knockdown of T-cell intracellular antigens TIA1 and TIAR in transformed cells triggers cell proliferation and tumor growth. Using a tetracycline-inducible system, we report here that an increased expression of TIA1 or TIAR in 293 cells results in reduced rates of cell proliferation. Ectopic expression of these proteins abolish endogenous TIA1 and TIAR levels via the regulation of splicing of their pre-mRNAs, and partially represses global translation in a phospho-eukaryotic initiation factor 2 alpha-dependent manner. This is accompanied by cell cycle arrest at G1/S and cell death through caspase-dependent apoptosis and autophagy. Genome-wide profiling illustrates a selective upregulation of p53 signaling pathway-related genes. Nude mice injected with doxycycline-inducible cells expressing TIA1 or TIAR retard, or even inhibit, growth of xenotumors. Remarkably, low expressions of TIA1 and TIAR correlate with poor prognosis in patients with lung squamous cell carcinoma. These findings strongly support the concept that TIA proteins act as tumor suppressor genes.

  5. Chimeric Antigen Receptor T Cells in Hematologic Malignancies.

    PubMed

    Shank, Brandon R; Do, Bryan; Sevin, Adrienne; Chen, Sheree E; Neelapu, Sattva S; Horowitz, Sandra B

    2017-03-01

    Patients with B-cell hematologic malignancies who progress through first- or second-line chemotherapy have a poor prognosis. Early clinical trials with autologous anti-CD19 chimeric antigen receptor (CAR) T cells have demonstrated promising results for patients who have relapsed or refractory disease. Lymphodepleting conditioning regimens, including cyclophosphamide, fludarabine, pentostatin, bendamustine, interleukin-2, and total body irradiation, are often administered before the infusion of CAR T cells, allowing for greater T-cell expansion. The major toxicity associated with CAR T-cell infusions is cytokine release syndrome (CRS), a potentially life-threatening systemic inflammatory disorder. The quick onset and progression of CRS require rapid detection and intervention to reduce treatment-related mortality. Management with tocilizumab can help ameliorate the symptoms of severe CRS, allowing steroids, which diminish the expansion and persistence of CAR T cells, to be reserved for tocilizumab-refractory patients. Other toxicities of CAR T-cell therapy include neutropenia and/or febrile neutropenia, infection, tumor lysis syndrome, neurotoxicity and nausea/vomiting. A review of patients' medications is imperative to eliminate medications that may contribute to treatment-related toxicities. Studies are ongoing to help optimize patient selection, preparation, safety, and management of individuals receiving CAR T cells. Long-term follow-up will help establish the place of CAR T cells in therapy.

  6. The T cell response to secreted antigens of Mycobacterium tuberculosis.

    PubMed

    Andersen, P

    1994-10-01

    Recent information from several laboratories points to proteins secreted from live Mycobacterium tuberculosis as being involved in protective immunity. We have studied protein release from M. tuberculosis during growth and have defined 3 different groups of proteins: excreted proteins, secreted proteins of the outer cell wall and cytoplasmic proteins released at late culture timepoints. These findings have lead to the definition of a short-term culture filtrate (ST-CF) enriched in excreted/secreted proteins and with a minimal content of autolytic products. ST-CF was tested as antigen in experimental vaccines against tuberculosis. A vaccine based on the adjuvant dimethyldioctadecylammonium chloride (DDA) was constructed and demonstrated to induce a potent cell mediated immune response of the Th-1 type. The vaccine was tested in parallel with a BCG standard vaccine and both vaccines induced a highly significant protection of the same magnitude. Molecules within the Ag85 complex and a 6-kDA secreted protein were mapped as the major antigenic targets for long-lived T cells involved in protective immunity against M. tuberculosis.

  7. TAP2-defective RMA-S cells present Sendai virus antigen to cytotoxic T lymphocytes.

    PubMed

    Zhou, X; Glas, R; Momburg, F; Hämmerling, G J; Jondal, M; Ljunggren, H G

    1993-08-01

    The murine antigen-processing-defective mutant cell line RMA-S is leaky in the presentation of certain endogenously synthesized minor histocompatibility and viral antigens to major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL). The viral antigens include influenza virus nucleoprotein, vesicular stomatitis virus (VSV) nucleocapsid and Rauscher murine leukemia virus (MuLV) antigen. Here we demonstrate Sendai virus antigen presentation by the HAM2 (murine TAP2, transporter associated with antigen presentation type 2)-defective RMA-S cell line and compare antigen presentation after restoration of the defect by murine TAP1/2 gene transfection. Kinetic studies revealed that RMA-S cells required 2-3 h longer incubation and approximately 10 times higher doses of Sendai virus to reach the same level of killing as the RMA parental line. After transfection of RMA-S cells with the murine TAP1/2 gene, Sendai virus antigen presentation was restored to levels of the RMA wild-type line with regard to time of virus infection and dose of virus needed for sensitizing target cells. The presentation of Sendai virus antigen in RMA-S cells was sensitive to brefeldin A (BFA), suggesting that the presentation was mediated via the endogenous pathway. Our findings confirmed leakiness of antigen presentation in RMA-S cells and extended it to Sendai virus. The results underscored the role for intact expression of the TAP 1/2 molecules for efficient MHC class I-mediated antigen presentation.

  8. Collecting Duct-Derived Cells Display Mesenchymal Stem Cell Properties and Retain Selective In Vitro and In Vivo Epithelial Capacity

    PubMed Central

    Li, Joan; Ariunbold, Usukhbayar; Suhaimi, Norseha; Sunn, Nana; Guo, Jinjin; McMahon, Jill A.; McMahon, Andrew P.

    2015-01-01

    We previously described a mesenchymal stem cell (MSC)-like population within the adult mouse kidney that displays long-term colony-forming efficiency, clonogenicity, immunosuppression, and panmesodermal potential. Although phenotypically similar to bone marrow (BM)-MSCs, kidney MSC–like cells display a distinct expression profile. FACS sorting from Hoxb7/enhanced green fluorescent protein (GFP) mice identified the collecting duct as a source of kidney MSC–like cells, with these cells undergoing an epithelial-to-mesenchymal transition to form clonogenic, long-term, self-renewing MSC-like cells. Notably, after extensive passage, kidney MSC–like cells selectively integrated into the aquaporin 2–positive medullary collecting duct when microinjected into the kidneys of neonatal mice. No epithelial integration was observed after injection of BM-MSCs. Indeed, kidney MSC–like cells retained a capacity to form epithelial structures in vitro and in vivo, and conditioned media from these cells supported epithelial repair in vitro. To investigate the origin of kidney MSC–like cells, we further examined Hoxb7+ fractions within the kidney across postnatal development, identifying a neonatal interstitial GFPlo (Hoxb7lo) population displaying an expression profile intermediate between epithelium and interstitium. Temporal analyses with Wnt4GCE/+:R26tdTomato/+ mice revealed evidence for the intercalation of a Wnt4-expressing interstitial population into the neonatal collecting duct, suggesting that such intercalation may represent a normal developmental mechanism giving rise to a distinct collecting duct subpopulation. These results extend previous observations of papillary stem cell activity and collecting duct plasticity and imply a role for such cells in collecting duct formation and, possibly, repair. PMID:24904087

  9. Collecting duct-derived cells display mesenchymal stem cell properties and retain selective in vitro and in vivo epithelial capacity.

    PubMed

    Li, Joan; Ariunbold, Usukhbayar; Suhaimi, Norseha; Sunn, Nana; Guo, Jinjin; McMahon, Jill A; McMahon, Andrew P; Little, Melissa

    2015-01-01

    We previously described a mesenchymal stem cell (MSC)-like population within the adult mouse kidney that displays long-term colony-forming efficiency, clonogenicity, immunosuppression, and panmesodermal potential. Although phenotypically similar to bone marrow (BM)-MSCs, kidney MSC-like cells display a distinct expression profile. FACS sorting from Hoxb7/enhanced green fluorescent protein (GFP) mice identified the collecting duct as a source of kidney MSC-like cells, with these cells undergoing an epithelial-to-mesenchymal transition to form clonogenic, long-term, self-renewing MSC-like cells. Notably, after extensive passage, kidney MSC-like cells selectively integrated into the aquaporin 2-positive medullary collecting duct when microinjected into the kidneys of neonatal mice. No epithelial integration was observed after injection of BM-MSCs. Indeed, kidney MSC-like cells retained a capacity to form epithelial structures in vitro and in vivo, and conditioned media from these cells supported epithelial repair in vitro. To investigate the origin of kidney MSC-like cells, we further examined Hoxb7(+) fractions within the kidney across postnatal development, identifying a neonatal interstitial GFP(lo) (Hoxb7(lo)) population displaying an expression profile intermediate between epithelium and interstitium. Temporal analyses with Wnt4(GCE/+):R26(tdTomato/+) mice revealed evidence for the intercalation of a Wnt4-expressing interstitial population into the neonatal collecting duct, suggesting that such intercalation may represent a normal developmental mechanism giving rise to a distinct collecting duct subpopulation. These results extend previous observations of papillary stem cell activity and collecting duct plasticity and imply a role for such cells in collecting duct formation and, possibly, repair.

  10. Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

    PubMed Central

    Streng-Ouwehand, Ingeborg; Ho, Nataschja I; Litjens, Manja; Kalay, Hakan; Boks, Martine Annemarie; Cornelissen, Lenneke AM; Kaur Singh, Satwinder; Saeland, Eirikur; Garcia-Vallejo, Juan J; Ossendorp, Ferry A; Unger, Wendy WJ; van Kooyk, Yvette

    2016-01-01

    Antigen uptake by dendritic cells and intracellular routing of antigens to specific compartments is regulated by C-type lectin receptors that recognize glycan structures. We show that the modification of Ovalbumin (OVA) with the glycan-structure LewisX (LeX) re-directs OVA to the C-type lectin receptor MGL1. LeX-modification of OVA favored Th1 skewing of CD4+ T cells and enhanced cross-priming of CD8+ T cells. While cross-presentation of native OVA requires high antigen dose and TLR stimuli, LeX modification reduces the required amount 100-fold and obviates its dependence on TLR signaling. The OVA-LeX-induced enhancement of T cell cross-priming is MGL1-dependent as shown by reduced CD8+ effector T cell frequencies in MGL1-deficient mice. Moreover, MGL1-mediated cross-presentation of OVA-LeX neither required TAP-transporters nor Cathepsin-S and was still observed after prolonged intracellular storage of antigen in Rab11+LAMP1+ compartments. We conclude that controlled neo-glycosylation of antigens can crucially influence intracellular routing of antigens, the nature and strength of immune responses and should be considered for optimizing current vaccination strategies. DOI: http://dx.doi.org/10.7554/eLife.11765.001 PMID:26999763

  11. T-cell recognition of a cross-reactive antigen(s) in erythrocyte stages of Plasmodium falciparum and Plasmodium yoelii: inhibition of parasitemia by this antigen(s).

    PubMed Central

    Lucas, B; Engels, A; Camus, D; Haque, A

    1993-01-01

    In the current study, we investigated the presence of a cross-reactive antigen(s) in the erythrocyte stage from Plasmodium yoelii (265 BY strain) and Plasmodium falciparum through recognition by T cells primed in vivo with antigens from each of these parasites. BALB/c mice are naturally resistant to P. falciparum but are susceptible to P. yoelii infection. Mice that had recovered from P. yoelii primary infection became resistant to a second infection. A higher in vitro proliferative response to a soluble blood stage preparation of P. falciparum was observed in splenic cells from immune animals than in those from mice with a patent P. yoelii infection. The antigen-induced proliferative response was enhanced when animals were exposed to a secondary infection. Animals exposed to a challenge infection were treated with anti-CD4 or anti-CD8 monoclonal antibodies to deplete the corresponding subset of T cells. There was a marked diminution in P. falciparum antigen-induced proliferative response in the total splenic cell populations from CD8-depleted but not from CD4-depleted mice. In CD8-depleted and nondepleted animals, the antigen-induced proliferation in the total cell populations was markedly lower than in the T-cell-rich populations, indicating inhibitory activities of B cells and/or macrophages. There was no such difference in the stimulation between total and T-enriched cell populations from CD4-depleted animals. Flow cytometry analysis demonstrated the presence of an almost equal percentage of CD8+ (59.6%) and CD4+ (64%) T cells in the spleen preparations following in vivo depletion of CD4- and CD8-bearing T cells, respectively. When cultured with P. yoelii blood stage antigen, splenocytes from animals immunized with P. falciparum antigen displayed a significant proliferative response which was markedly diminished by treatment with anti-Thy-1.2 antibody plus complement. Animals immunized with P. falciparum antigen and then challenged with P. yoelii blood stage

  12. B cell receptor revision diminishes the autoreactive B cell response after antigen activation in mice

    PubMed Central

    Wang, Ying-Hua; Diamond, Betty

    2008-01-01

    Autoreactive B cells are regulated in the BM during development through mechanisms, including editing of the B cell receptor (BCR), clonal deletion, and anergy. Peripheral B cell tolerance is also important for protection from autoimmune damage, although the mechanisms are less well defined. Here we demonstrated, using a mouse model of SLE-like serology, that during an autoimmune response, RAG was reinduced in antigen-activated early memory or preplasma B cells. Expression of RAG was specific to antigen-reactive B cells, required the function of the IL-7 receptor (IL-7R), and contributed to maintenance of humoral tolerance. We also showed that soluble antigen could diminish a non-autoreactive antibody response through induction of BCR revision. These data suggest that tolerance induction operates in B cells at a postactivation checkpoint and that BCR revision helps regulate autoreactivity generated during an ongoing immune response. PMID:18636122

  13. Antigen negative red blood cell inventory of Indian blood donors.

    PubMed

    Kulkarni, Swati; Vasantha, K; Ghosh, Kanjaksha

    2016-08-01

    Screening the donor population for clinically important antigens and creating a database of phenotyped donors will eliminate the tedious task of large scale screening for antigen negative units. The aim of the present study is to identify donors lacking common antigens and a combination of common antigens to establish an antigen negative inventory. Blood samples of 1221 regular blood donors were phenotyped for the clinically important common antigens of the Rh, Duffy, Kell, Kidd and MNS blood group systems using standard tube technique. Out of 1221 total donors tested, we observed that 261 donors lacked a combination of clinically important common antigens (C, D, e, Fya, Jka, s). After excluding the RhD negative donors in this study 15.56% lacked a combination of two or three common antigens. Of all donors, 3.2% lacked Fya and Jka antigens, 1.96% Fya and s, 1.88% Jka and s antigens and 0.57% lacked three common antigens. An antigen negative inventory of donors who lack a single common antigen or a combination of common antigens was prepared from regular donors which will prove useful for efficient management of transfusion therapy in patients with multiple antibodies against common antigens. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Proliferating cell nuclear antigen/cyclin in cultured human keratinocytes.

    PubMed

    Okada, N; Miyagawa, S; Steinberg, M L; Yoshikawa, K

    1990-09-01

    Expression of proliferating cell nuclear antigen (PCNA)/cyclin in cultured human keratinocytes was studied using an antibody from an SLE patient as the reagent. By indirect immunofluorescence staining, SV40-transformed human keratinocytes expressed PCNA/cyclin in 40-45% of the cells as a nulcear granular fluorescence. After synchronization of these cells, their nuclear distribution pattern during the S phase was sequential and showed a clear correlation with DNA synthesis. Primary cultured keratinocytes grown in high Ca+ medium expressed PCNA/cyclin in 10-15% of the cells with a similar staining pattern. These positively stained cells were confined to the basal and immediate suprabasal layers of the stratified culture sheet. The keratinocytes disaggregated by trypsin were separated according to cell size through a screen of Nitex monofilament cloth. The cells smaller than 15 microns in diameter synthesized abundant PCNA/cyclin, while the larger cells expressed very low levels. These results indicate that the expression of PCNA/cyclin correlates with DNA synthesis in cultured keratinocytes, but is not associated with their differentiation process.

  15. Non-integrating lentiviral vectors based on the minimal S/MAR sequence retain transgene expression in dividing cells.

    PubMed

    Xu, Zhen; Chen, Feng; Zhang, Lingling; Lu, Jing; Xu, Peng; Liu, Guang; Xie, Xuemin; Mu, Wenli; Wang, Yajun; Liu, Depei

    2016-10-01

    Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral (NIL) vectors are among the most promising candidates for gene transfer tools, because they exhibit high transfer efficiency in both dividing and non-dividing cells and do not present a risk of insertional mutagenesis. However, non-integrating lentiviral vectors cannot introduce stable exogenous gene expression to dividing cells, thereby limiting their application. Here, we report the design of a non-integrating lentiviral vector that contains the minimal scaffold/matrix attachment region (S/MAR) sequence (SNIL), and this SNIL vector is able to retain episomal transgene expression in dividing cells. Using SNIL vectors, we detected the expression of the eGFP gene for 61 days in SNIL-transduced stable CHO cells, either with selection or not. In the NIL group without the S/MAR sequence, however, the transduced cells died under selection for the transient expression of NIL vectors. Furthermore, Southern blot assays demonstrated that the SNIL vectors were retained extrachromosomally in the CHO cells. In conclusion, the minimal S/MAR sequence retained the non-integrating lentiviral vectors in dividing cells, which indicates that SNIL vectors have the potential for use as a gene transfer tool.

  16. Aβ and Inflammatory Stimulus Activate Diverse Signaling Pathways in Monocytic Cells: Implications in Retaining Phagocytosis in Aβ-Laden Environment

    PubMed Central

    Savchenko, Ekaterina; Malm, Tarja; Konttinen, Henna; Hämäläinen, Riikka H.; Guerrero-Toro, Cindy; Wojciechowski, Sara; Giniatullin, Rashid; Koistinaho, Jari; Magga, Johanna

    2016-01-01

    Background: Accumulation of amyloid β (Aβ) is one of the main hallmarks of Alzheimer’s disease (AD). The enhancement of Aβ clearance may provide therapeutic means to restrict AD pathology. The cellular responses to different forms of Aβ in monocytic cells are poorly known. We aimed to study whether different forms of Aβ induce inflammatory responses in monocytic phagocytes and how Aβ may affect monocytic cell survival and function to retain phagocytosis in Aβ-laden environment. Methods: Monocytic cells were differentiated from bone marrow hematopoietic stem cells (HSC) in the presence of macrophage-colony stimulating factor. Monocytic cells were stimulated with synthetic Aβ42 and intracellular calcium responses were recorded with calcium imaging. The formation of reactive oxygen species (ROS), secretion of cytokines and cell viability were also assessed. Finally, monocytic cells were introduced to native Aβ deposits ex vivo and the cellular responses in terms of cell viability, pro-inflammatory activation and phagocytosis were determined. The ability of monocytic cells to phagocytose Aβ plaques was determined after intrahippocampal transplantation in vivo. Results: Freshly solubilized Aβ induced calcium oscillations, which persisted after removal of the stimulus. After few hours of aggregation, Aβ was not able to induce oscillations in monocytic cells. Instead, lipopolysaccharide (LPS) induced calcium responses divergent from Aβ-induced response. Furthermore, while LPS induced massive production of pro-inflammatory cytokines, neither synthetic Aβ species nor native Aβ deposits were able to induce pro-inflammatory activation of monocytic cells, contrary to primary microglia. Finally, monocytic cells retained their viability in the presence of Aβ and exhibited phagocytic activity towards native fibrillar Aβ deposits and congophilic Aβ plaques. Conclusion: Monocytic cells carry diverse cellular responses to Aβ and inflammatory stimulus LPS. Even

  17. Aβ and Inflammatory Stimulus Activate Diverse Signaling Pathways in Monocytic Cells: Implications in Retaining Phagocytosis in Aβ-Laden Environment.

    PubMed

    Savchenko, Ekaterina; Malm, Tarja; Konttinen, Henna; Hämäläinen, Riikka H; Guerrero-Toro, Cindy; Wojciechowski, Sara; Giniatullin, Rashid; Koistinaho, Jari; Magga, Johanna

    2016-01-01

    Background: Accumulation of amyloid β (Aβ) is one of the main hallmarks of Alzheimer's disease (AD). The enhancement of Aβ clearance may provide therapeutic means to restrict AD pathology. The cellular responses to different forms of Aβ in monocytic cells are poorly known. We aimed to study whether different forms of Aβ induce inflammatory responses in monocytic phagocytes and how Aβ may affect monocytic cell survival and function to retain phagocytosis in Aβ-laden environment. Methods: Monocytic cells were differentiated from bone marrow hematopoietic stem cells (HSC) in the presence of macrophage-colony stimulating factor. Monocytic cells were stimulated with synthetic Aβ42 and intracellular calcium responses were recorded with calcium imaging. The formation of reactive oxygen species (ROS), secretion of cytokines and cell viability were also assessed. Finally, monocytic cells were introduced to native Aβ deposits ex vivo and the cellular responses in terms of cell viability, pro-inflammatory activation and phagocytosis were determined. The ability of monocytic cells to phagocytose Aβ plaques was determined after intrahippocampal transplantation in vivo. Results: Freshly solubilized Aβ induced calcium oscillations, which persisted after removal of the stimulus. After few hours of aggregation, Aβ was not able to induce oscillations in monocytic cells. Instead, lipopolysaccharide (LPS) induced calcium responses divergent from Aβ-induced response. Furthermore, while LPS induced massive production of pro-inflammatory cytokines, neither synthetic Aβ species nor native Aβ deposits were able to induce pro-inflammatory activation of monocytic cells, contrary to primary microglia. Finally, monocytic cells retained their viability in the presence of Aβ and exhibited phagocytic activity towards native fibrillar Aβ deposits and congophilic Aβ plaques. Conclusion: Monocytic cells carry diverse cellular responses to Aβ and inflammatory stimulus LPS. Even

  18. Human cell-mediated immune responses to chlamydial antigens.

    PubMed

    Hanna, L; Schmidt, L; Sharp, M; Stites, D P; Jawetz, E

    1979-02-01

    A reproducible method was developed to determine the ability of chlamydial antigens to stimulate lymphocytes from volunteers. In tests repeated 4 to 14 times, the cells from a given volunteer gave a relatively narrow range of responses, but there were great differences in the mean response of different volunteers. In the entire group of 52 volunteers, lymphocyte stimulation was significantly associated with the presence of antibody, but in a given individual results of one test did not aid in predicting the results of the other. A majority of persons with either antichlamydial antibody or elevated lymphocyte stimulation, or both, did not have a history of signs or symptoms within a spectrum of chlamydial diseases. This may reflect the great frequency of asymptomatic infection with these organisms. The lymphocytes of some individuals were stimulated to a significantly greater degree by antigens of one chlamydial species (Chlamydia trachomatis or C. psittaci) than by the other. These and other cell-mediated reactions in human chlamydial infections, and their possible medical significance, are under continued study.

  19. [Antigen-binding clone cells in hematopoietic spleen colonies].

    PubMed

    Khazanova, I V; Van'ko, L V; Malaĭtsev, V V; Khamitova, N S; Zhuravel', G M

    1976-05-01

    The antigen-binding cell clones and the Ig-positive cells were found and quantitatively assessed in the primary hemopoietic splenic colonies. The data ogtained were analyzed assuming that the ratio of clone of specialized B-cells should reflect the quantitative ration in the corresponding V-genes in the given lymphocyte populations at definite stages of its development. The colonies differed from one another markedly by the curves of inhibition by DNP-lysine of rosette-formation with DNP-erythrocytes, i.e. by the avidity of B-cells of the given specificity. The colonies differed significantly by the ratio of the volumes of the two clone groups (cell with anti-DNP and anti-BE Ig-receptors) between themselves and the combination of the Ig-positive cells. These quantitaive regularities were incompatible with the view that each B-cell had any conceivable set of V-genes, i.e. with the germ-line hypothesis of the antibody and receptor diversity.

  20. A cytokine-independent approach to identify antigen-specific human germinal center Tfh cells and rare antigen-specific CD4+ T cells in blood

    PubMed Central

    Dan, Jennifer M.; Arlehamn, Cecilia S. Lindestam; Weiskopf, Daniela; Antunes, Ricardo da Silva; Havenar-Daughton, Colin; Reiss, Samantha; Brigger, Matthew; Bothwell, Marcella; Sette, Alessandro; Crotty, Shane

    2016-01-01

    Detection of antigen-specific CD4+ T cells is central to the study of many human infectious diseases, vaccines, and autoimmune diseases. However, such cells are generally rare and heterogeneous in their cytokine profiles. Identification of antigen-specific germinal center (GC) T follicular helper (Tfh) cells by cytokine production has been particularly problematic. The function of a GC Tfh cell is to selectively help adjacent GC B cells via cognate interaction; thus, GC Tfh cells may be ‘stingy’ cytokine producers, fundamentally different than Th1 or Th17 cells in the quantities of cytokines produced. Conventional identification of antigen-specific cells by intracellular cytokine staining (ICS) relies on the ability of the CD4+ T cell to generate substantial amounts of cytokine. To address this problem, we have developed a cytokine-independent activation induced marker (AIM) methodology to identify antigen-specific GC Tfh cells in human lymphoid tissue. Whereas Group A Streptococcus (Strep)-specific GC Tfh cells produced minimal detectable cytokines by ICS, the AIM method identified 85-fold more antigen-specific GC Tfh cells. Intriguingly, these GC Tfh cells consistently expressed programmed death ligand 1 (PD-L1) upon activation. AIM also detected non-Tfh cells in lymphoid tissue. As such, we applied AIM for identification of rare antigen-specific CD4+ T cells in human peripheral blood. Dengue-, tuberculosis-, and pertussis-vaccine-specific CD4+ T cells were readily detectable by AIM. In sum, cytokine assays missed 98% of antigen-specific human GC Tfh cells, reflecting the biology of these cells, which could instead be sensitively identified by co-expression of TCR-dependent activation markers. PMID:27342848

  1. Antibody specificity and antigen characterization of rat monoclonal antibodies against Streptococcus mutans cell wall-associated protein antigens.

    PubMed Central

    Ackermans, F; Klein, J P; Cormont, F; Bazin, H; Ogier, J A; Frank, R M; Vreven, J

    1985-01-01

    Monoclonal antibodies to Streptococcus mutans OMZ175 (serotype f) cell wall-associated antigens (wall-extracted antigens [WEA]) were derived from the fusion of Lou C plasmocytoma rat cells (IR 983 F) and spleen cells from Wistar R inbred rats immunized with WEA. Four cell lines producing monoclonal antibodies directed against a component of S. mutans WEA have been established. All four monoclonal antibodies reacted only with two antigens of WEA from S. mutans OMZ175 by Western blotting and immunoprecipitation techniques, enzyme-linked immunosorbent assay (ELISA), and competitive ELISA. Western blot analysis of WEA showed that the four monoclonal antibodies recognized two related cell wall-associated proteins with apparent molecular weights of 125,000 and 76,000. Immunoprecipitation of whole cells with the monoclonal antibodies confirmed the surface localization of the two antigens. The ELISA and competitive ELISA were used to analyze the distribution of the epitopes on seven S. mutans serotypes. All S. mutans serotypes were found to express the recognized epitopes; however, different reactivity patterns could be distinguished among the various strains tested, and the four monoclonal antibodies reacted only weakly with S. mutans serotypes d and g. Images PMID:2410364

  2. Label retaining cells (LRCs) with myoepithelial characteristic from the proximal acinar region define stem cells in the sweat gland.

    PubMed

    Leung, Yvonne; Kandyba, Eve; Chen, Yi-Bu; Ruffins, Seth; Kobielak, Krzysztof

    2013-01-01

    Slow cycling is a common feature shared among several stem cells (SCs) identified in adult tissues including hair follicle and cornea. Recently, existence of unipotent SCs in basal and lumenal layers of sweat gland (SG) has been described and label retaining cells (LRCs) have also been localized in SGs; however, whether these LRCs possess SCs characteristic has not been investigated further. Here, we used a H2BGFP LRCs system for in vivo detection of infrequently dividing cells. This system allowed us to specifically localize and isolate SCs with label-retention and myoepithelial characteristics restricted to the SG proximal acinar region. Using an alternative genetic approach, we demonstrated that SG LRCs expressed keratin 15 (K15) in the acinar region and lineage tracing determined that K15 labeled cells contributed long term to the SG structure but not to epidermal homeostasis. Surprisingly, wound healing experiments did not activate proximal acinar SG cells to participate in epidermal healing. Instead, predominantly non-LRCs in the SG duct actively divided, whereas the majority of SG LRCs remained quiescent. However, when we further challenged the system under more favorable isolated wound healing conditions, we were able to trigger normally quiescent acinar LRCs to trans-differentiate into the epidermis and adopt its long term fate. In addition, dissociated SG cells were able to regenerate SGs and, surprisingly, hair follicles demonstrating their in vivo plasticity. By determining the gene expression profile of isolated SG LRCs and non-LRCs in vivo, we identified several Bone Morphogenetic Protein (BMP) pathway genes to be up-regulated and confirmed a functional requirement for BMP receptor 1A (BMPR1A)-mediated signaling in SG formation. Our data highlight the existence of SG stem cells (SGSCs) and their primary importance in SG homeostasis. It also emphasizes SGSCs as an alternative source of cells in wound healing and their plasticity for regenerating

  3. Label Retaining Cells (LRCs) with Myoepithelial Characteristic from the Proximal Acinar Region Define Stem Cells in the Sweat Gland

    PubMed Central

    Leung, Yvonne; Kandyba, Eve; Chen, Yi-Bu; Ruffins, Seth; Kobielak, Krzysztof

    2013-01-01

    Slow cycling is a common feature shared among several stem cells (SCs) identified in adult tissues including hair follicle and cornea. Recently, existence of unipotent SCs in basal and lumenal layers of sweat gland (SG) has been described and label retaining cells (LRCs) have also been localized in SGs; however, whether these LRCs possess SCs characteristic has not been investigated further. Here, we used a H2BGFP LRCs system for in vivo detection of infrequently dividing cells. This system allowed us to specifically localize and isolate SCs with label-retention and myoepithelial characteristics restricted to the SG proximal acinar region. Using an alternative genetic approach, we demonstrated that SG LRCs expressed keratin 15 (K15) in the acinar region and lineage tracing determined that K15 labeled cells contributed long term to the SG structure but not to epidermal homeostasis. Surprisingly, wound healing experiments did not activate proximal acinar SG cells to participate in epidermal healing. Instead, predominantly non-LRCs in the SG duct actively divided, whereas the majority of SG LRCs remained quiescent. However, when we further challenged the system under more favorable isolated wound healing conditions, we were able to trigger normally quiescent acinar LRCs to trans-differentiate into the epidermis and adopt its long term fate. In addition, dissociated SG cells were able to regenerate SGs and, surprisingly, hair follicles demonstrating their in vivo plasticity. By determining the gene expression profile of isolated SG LRCs and non-LRCs in vivo, we identified several Bone Morphogenetic Protein (BMP) pathway genes to be up-regulated and confirmed a functional requirement for BMP receptor 1A (BMPR1A)-mediated signaling in SG formation. Our data highlight the existence of SG stem cells (SGSCs) and their primary importance in SG homeostasis. It also emphasizes SGSCs as an alternative source of cells in wound healing and their plasticity for regenerating

  4. 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

  5. T cell antigen discovery using soluble vaccinia proteome reveals recognition of antigens with both virion and non-virion association*

    PubMed Central

    Davies, D. Huw; Chun, Sookhee; Hermanson, Gary; Tucker, Jo Anne; Jain, Aarti; Nakajima, Rie; Pablo, Jozelyn; Felgner, Philip L.; Liang, Xiaowu

    2014-01-01

    Vaccinia virus (VACV) is a useful model system for understanding the immune response to a complex pathogen. Proteome-wide antibody profiling studies reveal the humoral response to be strongly biased towards virion associated antigens, and several membrane proteins induce antibody-mediated protection against VACV challenge in mice. Some studies have indicated the CD4 response is also skewed toward proteins with virion association, whereas the CD8 response is more biased toward proteins with early expression. In this study, we have leveraged a VACV-WR plasmid expression library, produced previously for proteome microarrays for antibody profiling, to make a solubilized full VACV-WR proteome for T cell antigen profiling. Splenocytes from VACV-WR-infected mice were assayed without prior expansion against the soluble proteome in assays for Th1 and Th2 signature cytokines. The response to infection was polarized toward a Th1 response, with the distribution of reactive T cell antigens comprising both early and late VACV proteins. Interestingly, the proportions of different functional subsets were similar to that present in the whole proteome. In contrast, the targets of antibodies from the same mice were enriched for membrane and other virion components, as described previously. We conclude that a ‘non-biasing’ approach to T cell antigen discovery reveals a T cell antigen profile in VACV that is broader and less skewed to virion-association than the antibody profile. The T cell antigen mapping method developed here should be applicable to other organisms where expressible ‘ORFeome’ libraries are also available, and is readily scalable for larger pathogens. PMID:25024392

  6. Targeting antigens through blood dendritic cell antigen 2 (BDCA2) on plasmacytoid dendritic cells promotes immunologic tolerance1

    PubMed Central

    Draves, Kevin E.; Chen, ChangHung; Hayden-Ledbetter, Martha S.; Shlomchik, Mark J.; Kaplan, Daniel H.; Clark, Edward A.

    2014-01-01

    The C-type lectin receptor blood dendritic cell antigen 2 (BDCA2) is expressed exclusively on human plasmacytoid dendritic cells (pDCs) and plays a role in Ag capture, internalization and presentation to T cells. We used transgenic mice that express human BDCA2 and anti-BDCA2 mAbs to deliver Ags directly to BDCA2 on pDCs in vivo. Targeting Ag to pDCs in this manner resulted in significant suppression of Ag-specific CD4+ T cell and Ab responses upon secondary exposure to Ag in the presence of adjuvant. Suppression of Ab responses required both a decrease in effector CD4+ T cells and preservation of Foxp3+ regulatory T cells (Tregs). Reduction in Treg cell numbers following Ag delivery to BDCA2 restored both CD4+ T cell activation and Ab responses, demonstrating that Tregs were required for the observed tolerance. Our results demonstrate that Ag delivery to pDCs through BDCA2 is an effective method to induce immunological tolerance, which may be useful for treating autoimmune diseases or to inhibit unwanted Ab responses. PMID:24829416

  7. Mast cells and dendritic cells form synapses that facilitate antigen transfer for T cell activation.

    PubMed

    Carroll-Portillo, Amanda; Cannon, Judy L; te Riet, Joost; Holmes, Anna; Kawakami, Yuko; Kawakami, Toshiaki; Cambi, Alessandra; Lidke, Diane S

    2015-08-31

    Mast cells (MCs) produce soluble mediators such as histamine and prostaglandins that are known to influence dendritic cell (DC) function by stimulating maturation and antigen processing. Whether direct cell-cell interactions are important in modulating MC/DC function is unclear. In this paper, we show that direct contact between MCs and DCs occurs and plays an important role in modulating the immune response. Activation of MCs through FcεRI cross-linking triggers the formation of stable cell-cell interactions with immature DCs that are reminiscent of the immunological synapse. Direct cellular contact differentially regulates the secreted cytokine profile, indicating that MC modulation of DC populations is influenced by the nature of their interaction. Synapse formation requires integrin engagement and facilitates the transfer of internalized MC-specific antigen from MCs to DCs. The transferred material is ultimately processed and presented by DCs and can activate T cells. The physiological outcomes of the MC-DC synapse suggest a new role for intercellular crosstalk in defining the immune response.

  8. Chimeric antigen receptors and bispecific antibodies to retarget T cells in pediatric oncology

    PubMed Central

    Suzuki, Maya; Curran, Kevin J.; Cheung, Nai-Kong V.

    2016-01-01

    Cancer immunotherapy using antigen-specific T cells has broad therapeutic potential. Chimeric antigen receptors and bispecific antibodies can redirect T cells to kill tumors without human leukocyte antigens (HLA) restriction. Key determinants of clinical potential include the choice of target antigen, antibody specificity, antibody affinity, tumor accessibility, T cell persistence, and tumor immune evasion. For pediatric cancers, additional constraints include their propensity for bulky metastatic disease and the concern for late toxicities from treatment. Nonetheless, the recent preclinical and clinical developments of these T cell based therapies are highly encouraging. PMID:25832831

  9. Lymphocyte imprinting with melanoma antigens acquired by trogocytosis facilitates identification of tumor-reactive T cells

    PubMed Central

    Eisenberg, Galit; Uzana, Ronny; Pato, Aviad; Frankenburg, Shoshana; Merims, Sharon; Yefenof, Eitan; Ferrone, Soldano; Peretz, Tamar; Machlenkin, Arthur; Lotem, Michal

    2013-01-01

    Trogocytosis is a contact-dependent inter-cellular transfer of membrane fragments and associated molecules from antigen presenting cells to effector lymphocytes. We previously demonstrated that trogocytosis also occurs between tumor target and cognate melanoma antigen-specific cytotoxic T cells (CTL). Here we show that, following trogocytosis, immune effector cells acquire molecular components of the tumor, including surface antigens, which are detectable by specific monoclonal antibodies. We demonstrate that CD8+ and CD4+ T cells from melanoma patients’ PBMC and tumor infiltrating lymphocytes (TIL) capture melanoma antigens, enabling identification of trogocytosing lymphocytes by staining with antigen-specific antibodies. This finding circumvents the necessity of tumor pre-labeling, which in the past was mandatory to detect membrane-capturing T cells. Through the detection of melanoma antigens on TIL, we sorted trogocytosing T cells and verified their preferential reactivity and cytotoxicity. Furthermore, tumor-antigen imprinted T cells were detected at low frequency in fresh TIL cultures shortly after extraction from the tumor. Thus, T cell imprinting by tumor antigens may allow the enrichment of melanoma antigen-specific T cells for research and potentially even for the adoptive immunotherapy of patients with cancer. PMID:23626012

  10. Identification of a potent microbial lipid antigen for diverse Natural Killer T cells1

    PubMed Central

    Wolf, Benjamin J.; Tatituri, Raju V. V.; Almeida, Catarina F.; Le Nours, Jérôme; Bhowruth, Veemal; Johnson, Darryl; Uldrich, Adam P.; Hsu, Fong-Fu; Brigl, Manfred; Besra, Gurdyal S.; Rossjohn, Jamie; Godfrey, Dale I.; Brenner, Michael B.

    2016-01-01

    Invariant Natural Killer T (iNKT) cells are a well-characterized CD1d-restricted T cell subset. The availability of potent antigens and tetramers for iNKT cells has allowed this population to be extensively studied and has revealed their central roles in infection, autoimmunity, and tumor immunity. In contrast, diverse Natural Killer T (dNKT) cells are poorly understood because the lipid antigens they recognize are largely unknown. We sought to identify dNKT cell lipid antigen(s) by interrogating a panel of dNKT mouse cell hybridomas with lipid extracts from the pathogen Listeria monocytogenes. We identified Listeria phosphatidylglycerol (PG) as a microbial antigen that was significantly more potent than a previously characterized dNKT cell antigen, mammalian PG. Further, while mammalian PG loaded CD1d tetramers did not stain dNKT cells, the Listeria-derived PG loaded tetramers did. The structure of Listeria PG was distinct from mammalian PG since it contained shorter, fully-saturated anteiso fatty acid lipid tails. CD1d binding lipid displacement studies revealed that the microbial PG antigen binds significantly better to CD1d than counterparts with the same headgroup. These data reveal a highly-potent microbial lipid antigen for a subset of dNKT cells and provide an explanation for its increased antigen potency compared to the mammalian counterpart. PMID:26254340

  11. Low dose antigen promotes induction of FOXP3 in human CD4+ T cells

    PubMed Central

    Long, S. Alice; Rieck, Mary; Tatum, Megan; Bollyky, Paul L.; Wu, Rebecca P.; Muller, Isabelle; Ho, Jhon-Chun; Shilling, Heather G.; Buckner, Jane H.

    2011-01-01

    Low antigen dose promotes induction and persistence of Treg in mice, yet few studies have addressed the role of antigen dose in the induction of adaptive CD4+FOXP3+ Treg in humans. To this end, we examined the level of FOXP3 expression in human CD4+CD25− T cells upon activation with autologous antigen presenting cells and varying doses of peptide. Antigen specific T cells expressing FOXP3 were identified by flow cytometry using MHC Class II tetramer (Tmr). We found an inverse relationship between antigen dose and the frequency of FOXP3+ cells for both foreign and self antigen specific T cells. Through studies of FOXP3 locus demethylation and helios expression, we determined that variation in the frequency of Tmr+FOXP3+ T cells was not due to expansion of natural Treg, but instead, we found that induction, proliferation and persistence of FOXP3+ cells was similar in high and low dose cultures whereas proliferation of FOXP3− T cells was favored in high antigen dose cultures. The frequency of FOXP3+ cells positively correlated with suppressive function, indicative of adaptive Treg generation. The frequency of FOXP3+ cells were maintained with IL-2, but not upon re-stimulation with antigen. Together, these data suggest that low antigen dose favors the transient generation of human antigen specific adaptive Treg over the proliferation of antigen specific FOXP3- effector T cells. These adaptive Treg could function to reduce ongoing inflammatory responses and promote low dose tolerance in humans, especially when antigen exposure and tolerance is transient. PMID:21865550

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

    PubMed

    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.

  13. Dendritic cell preactivation impairs MHC class II presentation of vaccines and endogenous viral antigens

    PubMed Central

    Young, Louise J.; Wilson, Nicholas S.; Schnorrer, Petra; Mount, Adele; Lundie, Rachel J.; La Gruta, Nicole L.; Crabb, Brendan S.; Belz, Gabrielle T.; Heath, William R.; Villadangos, Jose A.

    2007-01-01

    When dendritic cells (DCs) encounter signals associated with infection or inflammation, they become activated and undergo maturation. Mature DCs are very efficient at presenting antigens captured in association with their activating signal but fail to present subsequently encountered antigens, at least in vitro. Such impairment of MHC class II (MHC II) antigen presentation has generally been thought to be a consequence of down-regulation of endocytosis, so it might be expected that antigens synthesized by the DCs themselves (for instance, viral antigens) would still be presented by mature DCs. Here, we show that DCs matured in vivo could still capture and process soluble antigens, but were unable to present peptides derived from these antigens. Furthermore, presentation of viral antigens synthesized by the DCs themselves was also severely impaired. Indeed, i.v. injection of pathogen mimics, which caused systemic DC activation in vivo, impaired the induction of CD4 T cell responses against subsequently encountered protein antigens. This immunosuppressed state could be reversed by adoptive transfer of DCs loaded exogenously with antigens, demonstrating that impairment of CD4 T cell responses was due to lack of antigen presentation rather than to overt suppression of T cell activation. The biochemical mechanism underlying this phenomenon was the down-regulation of MHC II–peptide complex formation that accompanied DC maturation. These observations have important implications for the design of prophylactic and therapeutic DC vaccines and contribute to the understanding of the mechanisms causing immunosuppression during systemic blood infections. PMID:17978177

  14. Chemotactic Migration of T Cells towards Dendritic Cells Promotes the Detection of Rare Antigens

    PubMed Central

    Vroomans, Renske M. A.; Marée, Athanasius F. M.; de Boer, Rob J.; Beltman, Joost B.

    2012-01-01

    In many immunological processes chemoattraction is thought to play a role in guiding cells to their sites of action. However, based on in vivo two-photon microscopy experiments in the absence of cognate antigen, T cell migration in lymph nodes (LNs) has been roughly described as a random walk. Although it has been shown that dendritic cells (DCs) carrying cognate antigen in some circumstances attract T cells chemotactically, it is currently still unclear whether chemoattraction of T cells towards DCs helps or hampers scanning. Chemoattraction towards DCs could on the one hand help T cells to rapidly find DCs. On the other hand, it could be deleterious if DCs become shielded by a multitude of attracted yet non-specific T cells. Results from a recent simulation study suggested that the deleterious effect dominates. We re-addressed the question whether T cell chemoattraction towards DCs is expected to promote or hamper the detection of rare antigens using the Cellular Potts Model, a formalism that allows for dynamic, flexible cellular shapes and cell migration. Our simulations show that chemoattraction of T cells enhances the DC scanning efficiency, leading to an increased probability that rare antigen-specific T cells find DCs carrying cognate antigen. Desensitization of T cells after contact with a DC further improves the scanning efficiency, yielding an almost threefold enhancement compared to random migration. Moreover, the chemotaxis-driven migration still roughly appears as a random walk, hence fine-tuned analysis of cell tracks will be required to detect chemotaxis within microscopy data. PMID:23166480

  15. Chemotactic migration of T cells towards dendritic cells promotes the detection of rare antigens.

    PubMed

    Vroomans, Renske M A; Marée, Athanasius F M; de Boer, Rob J; Beltman, Joost B

    2012-01-01

    In many immunological processes chemoattraction is thought to play a role in guiding cells to their sites of action. However, based on in vivo two-photon microscopy experiments in the absence of cognate antigen, T cell migration in lymph nodes (LNs) has been roughly described as a random walk. Although it has been shown that dendritic cells (DCs) carrying cognate antigen in some circumstances attract T cells chemotactically, it is currently still unclear whether chemoattraction of T cells towards DCs helps or hampers scanning. Chemoattraction towards DCs could on the one hand help T cells to rapidly find DCs. On the other hand, it could be deleterious if DCs become shielded by a multitude of attracted yet non-specific T cells. Results from a recent simulation study suggested that the deleterious effect dominates. We re-addressed the question whether T cell chemoattraction towards DCs is expected to promote or hamper the detection of rare antigens using the Cellular Potts Model, a formalism that allows for dynamic, flexible cellular shapes and cell migration. Our simulations show that chemoattraction of T cells enhances the DC scanning efficiency, leading to an increased probability that rare antigen-specific T cells find DCs carrying cognate antigen. Desensitization of T cells after contact with a DC further improves the scanning efficiency, yielding an almost threefold enhancement compared to random migration. Moreover, the chemotaxis-driven migration still roughly appears as a random walk, hence fine-tuned analysis of cell tracks will be required to detect chemotaxis within microscopy data.

  16. Viral Sequestration of Antigen Subverts Cross Presentation to CD8+ T Cells

    PubMed Central

    Tewalt, Eric F.; Grant, Jean M.; Granger, Erica L.; Palmer, Douglas C.; Heuss, Neal D.; Gregerson, Dale S.; Restifo, Nicholas P.; Norbury, Christopher C.

    2009-01-01

    Virus-specific CD8+ T cells (TCD8+) are initially triggered by peptide-MHC Class I complexes on the surface of professional antigen presenting cells (pAPC). Peptide-MHC complexes are produced by two spatially distinct pathways during virus infection. Endogenous antigens synthesized within virus-infected pAPC are presented via the direct-presentation pathway. Many viruses have developed strategies to subvert direct presentation. When direct presentation is blocked, the cross-presentation pathway, in which antigen is transferred from virus-infected cells to uninfected pAPC, is thought to compensate and allow the generation of effector TCD8+. Direct presentation of vaccinia virus (VACV) antigens driven by late promoters does not occur, as an abortive infection of pAPC prevents production of these late antigens. This lack of direct presentation results in a greatly diminished or ablated TCD8+ response to late antigens. We demonstrate that late poxvirus antigens do not enter the cross-presentation pathway, even when identical antigens driven by early promoters access this pathway efficiently. The mechanism mediating this novel means of viral modulation of antigen presentation involves the sequestration of late antigens within virus factories. Early antigens and cellular antigens are cross-presented from virus-infected cells, as are late antigens that are targeted to compartments outside of the virus factories. This virus-mediated blockade specifically targets the cross-presentation pathway, since late antigen that is not cross-presented efficiently enters the MHC Class II presentation pathway. These data are the first to describe an evasion mechanism employed by pathogens to prevent entry into the cross-presentation pathway. In the absence of direct presentation, this evasion mechanism leads to a complete ablation of the TCD8+ response and a potential replicative advantage for the virus. Such mechanisms of viral modulation of antigen presentation must also be taken into

  17. The cell proliferation antigen Ki-67 organises heterochromatin

    PubMed Central

    Sobecki, Michal; Mrouj, Karim; Camasses, Alain; Parisis, Nikolaos; Nicolas, Emilien; Llères, David; Gerbe, François; Prieto, Susana; Krasinska, Liliana; David, Alexandre; Eguren, Manuel; Birling, Marie-Christine; Urbach, Serge; Hem, Sonia; Déjardin, Jérôme; Malumbres, Marcos; Jay, Philippe; Dulic, Vjekoslav; Lafontaine, Denis LJ; Feil, Robert; Fisher, Daniel

    2016-01-01

    Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression. DOI: http://dx.doi.org/10.7554/eLife.13722.001 PMID:26949251

  18. The cell proliferation antigen Ki-67 organises heterochromatin.

    PubMed

    Sobecki, Michal; Mrouj, Karim; Camasses, Alain; Parisis, Nikolaos; Nicolas, Emilien; Llères, David; Gerbe, François; Prieto, Susana; Krasinska, Liliana; David, Alexandre; Eguren, Manuel; Birling, Marie-Christine; Urbach, Serge; Hem, Sonia; Déjardin, Jérôme; Malumbres, Marcos; Jay, Philippe; Dulic, Vjekoslav; Lafontaine, Denis Lj; Feil, Robert; Fisher, Daniel

    2016-03-07

    Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression.

  19. Future directions in chimeric antigen receptor T cell therapy.

    PubMed

    Maude, Shannon L

    2017-02-01

    The impact of immunotherapy has grown exponentially in the past 5 years. Principle illustrations are encouraging results with engineered T cells expressing a chimeric antigen receptor (CAR). This experimental therapy is developing simultaneously in pediatric and adult clinical trials, making this field particularly relevant and exciting for pediatric oncologists. CAR-modified T cells targeting CD19 have produced dramatic antitumor responses in patients with relapsed/refractory B cell acute lymphoblastic leukemia. Clinical trials from several institutions, in both children and adults, using distinct CAR T cell products have demonstrated similar high complete remission rates of 61-93%, with durable remissions observed. Although the development of CARs for other malignancies has lagged behind, research into novel approaches to overcome inherent challenges is promising. Clinical trials of CAR-modified T cells have produced unprecedented results and are anticipated to have a broader impact as this approach expands into other indications, including other cancers and frontline therapy. The potential for long-term disease control, if fully realized, will have a transformative impact on the field.

  20. T follicular helper cells differentiate from Th2 cells in response to helminth antigens

    PubMed Central

    Zaretsky, Arielle Glatman; Taylor, Justin J.; King, Irah L.; Marshall, Fraser A.; Mohrs, Markus

    2009-01-01

    The relationship of T follicular helper (TFH) cells to other T helper (Th) subsets is controversial. We find that after helminth infection, or immunization with helminth antigens, reactive lymphoid organs of 4get IL-4/GFP reporter mice contain populations of IL-4/GFP-expressing CD4+ T cells that display the TFH markers CXCR5, PD-1, and ICOS. These TFH cells express the canonical TFH markers BCL6 and IL-21, but also GATA3, the master regulator of Th2 cell differentiation. Consistent with a relationship between Th2 and TFH cells, IL-4 protein production, reported by expression of huCD2 in IL-4 dual reporter (4get/KN2) mice, was a robust marker of TFH cells in LNs responding to helminth antigens. Moreover, the majority of huCD2/IL-4–producing Th cells were found within B cell follicles, consistent with their definition as TFH cells. TFH cell development after immunization failed to occur in mice lacking B cells or CD154. The relationship of TFH cells to the Th2 lineage was confirmed when TFH cells were found to develop from CXCR5− PD-1− IL-4/GFP+ CD4+ T cells after their transfer into naive mice and antigen challenge in vivo. PMID:19380637

  1. Precision Tumor Recognition by T Cells With Combinatorial Antigen Sensing Circuits

    PubMed Central

    Roybal, Kole T.; Rupp, Levi J.; Morsut, Leonardo; Walker, Whitney J.; McNally, Krista A.; Park, Jason S.; Lim, Wendell A.

    2016-01-01

    SUMMARY T cells can be re-directed to kill cancer cells using chimeric antigen receptors (CARs) or T cell receptors (TCRs). This approach, however, is constrained by the rarity of tumor-specific single antigens. Targeting antigens also found on bystander tissues can cause life-threatening adverse effects. A powerful way to enhance ON-target activity of therapeutic T cells is to engineer them to require combinatorial antigens. Here we engineer a combinatorially activated T cell circuit in which a synthetic Notch receptor for one antigen induces the expression of a CAR for a second antigen. These dual receptor AND-gate T cells are only armed and activated in the presence of dual antigen tumor cells. These T cells show precise therapeutic discrimination in vivo – sparing single antigen “bystander” tumors while efficiently clearing combinatorial antigen “disease” tumors. This type of precision dual receptor circuit opens the door to immune recognition of a wider range of tumors. PMID:26830879

  2. Immunophenotypic and antigen receptor gene rearrangement analysis in T cell neoplasia.

    PubMed Central

    Knowles, D. M.

    1989-01-01

    The author reviews the immunophenotypic profiles displayed by the major clinicopathologic categories of T cell neoplasia, the immunophenotypic criteria useful in the immunodiagnosis of T cell neoplasia, and the contributions made by antigen receptor gene rearrangement analysis to the understanding of T cell neoplasia. Neoplasms belonging to distinct clinicopathologic categories of T cell neoplasia often exhibit characteristic immunophenotypic profiles. Approximately 80% of lymphoblastic lymphomas and 20% of acute lymphoblastic leukemias express phenotypes consistent with prethymic and intrathymic stages of T cell differentiation, including intranuclear terminal deoxynucleotidyl transferase. Cutaneous T cell lymphomas of mycosis fungoides type usually express pan-T cell antigens CD2, CD5, and CD3, often lack the pan-T cell antigen CD7, and usually express the mature, peripheral helper subset phenotype, CD4+ CD8-. Cutaneous T cell lymphomas of nonmycosis fungoides type and peripheral T cell lymphomas often lack one or more pan-T cell antigens and, in addition, occasionally express the anomalous CD4+ CD8+ or CD4- CD8- phenotypes. T gamma-lymphoproliferative disease is divisable into two broad categories: those cases that are CD3 antigen positive and exhibit clonal T cell receptor beta chain (TCR-beta) gene rearrangements and those cases that are CD3 antigen negative and exhibit the TCR-beta gene germline configuration. Human T cell lymphotropic virus-I (HTLV-I) associated Japanese, Carribean, and sporadic adult T cell leukemia/lymphomas usually express pan-T cell antigens, the CD4+ CD8- phenotype, and various T cell-associated activation antigens, including the interleukin-2 receptor (CD25). Immunophenotypic criteria useful in the immunodiagnosis of T cell neoplasia include, in increasing order of utility, T cell predominance, T cell subset antigen restriction, anomalous T cell subset antigen expression, and deletion of one or more pan-T cell antigens. Only in

  3. The Other Function: Class II-Restricted Antigen Presentation by B Cells

    PubMed Central

    Adler, Lital N.; Jiang, Wei; Bhamidipati, Kartik; Millican, Matthew; Macaubas, Claudia; Hung, Shu-chen; Mellins, Elizabeth D.

    2017-01-01

    Mature B lymphocytes (B cells) recognize antigens using their B cell receptor (BCR) and are activated to become antibody-producing cells. In addition, and integral to the development of a high-affinity antibodies, B cells utilize the specialized major histocompatibility complex class II (MHCII) antigen presentation pathway to process BCR-bound and internalized protein antigens and present selected peptides in complex with MHCII to CD4+ T cells. This interaction influences the fate of both types of lymphocytes and shapes immune outcomes. Specific, effective, and optimally timed antigen presentation by B cells requires well-controlled intracellular machinery, often regulated by the combined effects of several molecular events. Here, we delineate and summarize these events in four steps along the antigen presentation pathway: (1) antigen capture and uptake by B cells; (2) intersection of internalized antigen/BCRs complexes with MHCII in peptide-loading compartments; (3) generation and regulation of MHCII/peptide complexes; and (4) exocytic transport for presentation of MHCII/peptide complexes at the surface of B cells. Finally, we discuss modulation of the MHCII presentation pathway across B cell development and maturation to effector cells, with an emphasis on the shaping of the MHCII/peptide repertoire by two key antigen presentation regulators in B cells: HLA-DM and HLA-DO. PMID:28386257

  4. Regulation of NK-cell function by mucins via antigen-presenting cells.

    PubMed

    Laskarin, G; Redzovic, A; Medancic, S Srsen; Rukavina, D

    2010-12-01

    Decidual antigen-presenting cells including dendritic cells (DCs) and CD14(+) macrophages, as mediators of the first encounter with fetal antigens, appear to be critically involved in the initiation of primary immune response by regulating innate- and adaptive immunity. Interleukin-15, produced by them, permits the proliferation and differentiation of CD3(-)CD16(-)CD94(+)NKG2A(+)CD56(+bright) decidual NK cells that identify trophoblast cells. These cells are able to kill them after Th1 cytokine overstimulation and by increasing the release of preformed cytotoxic mediators. Thus, the local microenvironment is a potent modulator of antigen-presenting cell functions. Tumor associated glycoprotein-72 (TAG-72) and mucine 1 (MUC-1) are glycoproteins secreted by uterine epithelial cells. Our hypothesis is that TAG-72 and MUC-1 are the natural ligands for carbohydrate recognition domains (CRDs) of endocytic mannose receptor (MR or CD206) and DC-specific ICAM non-integrin (DC-SIGN or CD209) expressed on decidual CD14(+) macrophages and CD1a(+) DCs. They might be able to condition antigen-presenting cells to produce distinct profiles of cyto/chemokines with consequential reduction in NK-cell numbers and cytotoxic potential leading to insufficient control over trophoblast growth. This hypothesis could explain the disappearance of MUC-1 beneath the attached embryo during the process of successful implantation when tight regulation of trophoblast invasion is needed. As IL-15 is the earliest and the most important factor in NK-cell proliferation, differentiation, and maturation, we expected primarily an increase of IL-15 expression in antigen-presenting cells concomitant with the disappearance of mucins and the enhancement in NK cells numbers and of cytotoxic potential after their close contact with early pregnancy decidual antigen-presenting cells. If our hypothesis is correct, it would contribute to the understanding of the role of mucins in the redirection of immune response

  5. Multiple antigen-specific processing pathways for activating naive CD8+ T cells in vivo.

    PubMed

    Norbury, C C; Princiotta, M F; Bacik, I; Brutkiewicz, R R; Wood, P; Elliott, T; Bennink, J R; Yewdell, J W

    2001-04-01

    Current knowledge of the processing of viral Ags into MHC class I-associated ligands is based almost completely on in vitro studies using nonprofessional APCs (pAPCs). This is two steps removed from real immune responses to pathogens and vaccines, in which pAPCs activate naive CD8(+) T cells in vivo. Rational vaccine design requires answers to numerous questions surrounding the function of pAPCs in vivo, including their abilities to process and present peptides derived from endogenous and exogenous viral Ags. In the present study, we characterize the in vivo dependence of Ag presentation on the expression of TAP by testing the immunogenicity of model Ags synthesized by recombinant vaccinia viruses in TAP1(-/-) mice. We show that the efficiency of TAP-independent presentation in vitro correlates with TAP-independent activation of naive T cells in vivo and provide the first in vivo evidence for proteolytic processing of antigenic peptides in the secretory pathway. There was, however, a clear exception to this correlation; although the presentation of the minimal SIINFEKL determinant from chicken egg OVA in vitro was strictly TAP dependent, it was presented in a TAP-independent manner in vivo. In vivo presentation of the same peptide from a fusion protein retained its TAP dependence. These results show that determinant-specific processing pathways exist in vivo for the generation of antiviral T cell responses. We present additional findings that point to cross-priming as the likely mechanism for these protein-specific differences.

  6. Antigen affinity discrimination is an intrinsic function of the B cell receptor

    PubMed Central

    Liu, Wanli; Meckel, Tobias; Tolar, Pavel; Sohn, Hae Won

    2010-01-01

    Antibody affinity maturation, a hallmark of adaptive immune responses, results from the selection of B cells expressing somatically hypermutated B cell receptors (BCRs) with increased affinity for antigens. Despite the central role of affinity maturation in antibody responses, the molecular mechanisms by which the increased affinity of a B cell for antigen is translated into a selective advantage for that B cell in immune responses is incompletely understood. We use high resolution live-cell imaging to provide evidence that the earliest BCR-intrinsic events that follow within seconds of BCR–antigen binding are highly sensitive to the affinity of the BCR for antigen. High affinity BCRs readily form oligomers and the resulting microclusters grow rapidly, resulting in enhanced recruitment of Syk kinase and calcium fluxes. Thus, B cells are able to read the affinity of antigen by BCR-intrinsic mechanisms during the earliest phases of BCR clustering, leading to the initiation of B cell responses. PMID:20404102

  7. Augmenting antitumor T-cell responses to mimotope vaccination by boosting with native tumor antigens.

    PubMed

    Buhrman, Jonathan D; Jordan, Kimberly R; U'ren, Lance; Sprague, Jonathan; Kemmler, Charles B; Slansky, Jill E

    2013-01-01

    Vaccination with antigens expressed by tumors is one strategy for stimulating enhanced T-cell responses against tumors. However, these peptide vaccines rarely result in efficient expansion of tumor-specific T cells or responses that protect against tumor growth. Mimotopes, or peptide mimics of tumor antigens, elicit increased numbers of T cells that crossreact with the native tumor antigen, resulting in potent antitumor responses. Unfortunately, mimotopes may also elicit cells that do not crossreact or have low affinity for tumor antigen. We previously showed that one such mimotope of the dominant MHC class I tumor antigen of a mouse colon carcinoma cell line stimulates a tumor-specific T-cell clone and elicits antigen-specific cells in vivo, yet protects poorly against tumor growth. We hypothesized that boosting the mimotope vaccine with the native tumor antigen would focus the T-cell response elicited by the mimotope toward high affinity, tumor-specific T cells. We show that priming T cells with the mimotope, followed by a native tumor-antigen boost, improves tumor immunity compared with T cells elicited by the same prime with a mimotope boost. Our data suggest that the improved tumor immunity results from the expansion of mimotope-elicited tumor-specific T cells that have increased avidity for the tumor antigen. The enhanced T cells are phenotypically distinct and enriched for T-cell receptors previously correlated with improved antitumor immunity. These results suggest that incorporation of native antigen into clinical mimotope vaccine regimens may improve the efficacy of antitumor T-cell responses.

  8. Specific antigen targeting to surface IgE and IgG on mouse bone marrow-derived mast cells enhances efficiency of antigen presentation.

    PubMed Central

    Tkaczyk, C; Viguier, M; Boutin, Y; Frandji, P; David, B; Hébert, J; Mécheri, S

    1998-01-01

    The discovery that bone marrow-derived mast cells can express major histocompatibility complex class II molecules and act as antigen-presenting cells prompted us to evaluate this function when antigen is internalized through fluid-phase endocytosis or via specific uptake by using IgG and IgE antibodies. This study was performed using a specific T-cell hybridoma developed against Lol p 1, the major allergen of grass pollen Lolium perenne. Expression of Fc gamma R and Fc epsilon RI by mast cells led us to investigate the influence of IgG- and IgE-targeted antigen on the antigen-presenting function of mast cells. Internalization of Lol p 1 through different specific IgG monoclonal antibodies (mAb) resulted in the activation of Lol p 1-specific T-cell hybridoma at concentrations about 100-fold less than that required for T-cell stimulation by uncomplexed antigen. IgE-complexed Lol p 1, which facilitates trapping of antigen by mast cells, induced an accelerated and more efficient antigen-presenting capacity of mast cells than that obtained with uncomplexed antigen. However, aggregation of anti-dinitrophenyl (DNP) IgE mAb by the irrelevant antigen DNP-human serum albumin did not substantially increase the capacity of mast cells to present Lol p 1 to T cells. This suggests that the mere aggregation of Fc epsilon RI is not sufficient for enhanced antigen presentation mediated by IgE. Tissue distribution and strategic location of mast cells at the mucosal barriers and their capacity to process the antigen through efficient fluid-phase pinocytosis as well as IgG- and IgE-dependent targeting of antigens provide mast cells with a prominent role in immune surveillance. Images Figure 1 PMID:9767412

  9. Herpesvirus saimiri transformed T cells and peripheral blood mononuclear cells restimulate identical antigen-specific human T cell clones.

    PubMed

    Daubenberger, C A; Nickel, B; Hübner, B; Siegler, U; Meinl, E; Pluschke, G

    2001-08-01

    Panels of human antigen-specific T cell clones (TCC) have been established by limiting dilution using Herpesvirus saimiri (HVS) subtype C transformed T cells as antigen presenting cells (APC). They showed antigen-specific proliferation when peripheral blood mononuclear cells (PBMC), HVS-transformed T cells and Epstein Barr Virus transformed lymphoblastoid B cell lines (EBV-LCL) were used as APC. All T cell clones were CD4+ and HLA class II restricted. For a detailed analysis, two panels of T cell clones specific for an epitope located in the N-terminus of the Merozoite Surface Protein 1 (MSP-1) of Plasmodium falciparum were established from the same founder T cell line using either PBMC or HVS-transformed T cells as APC. TCR analysis of the two panels of TCC demonstrated that the same founder cells could be propagated in both culture systems. Furthermore, no difference in the cytokine expression pattern or antigen processing and co-stimulatory requirements was observed between TCC established on PBMC or HVS-transformed T cells. Based on the finding that HVS-transformed T cells can replace PBMC as APC for isolation and propagation of antigen-specific TCC, a protocol was developed and successfully executed, which allows to establish and maintain vaccine-specific T cell clones from 20 ml of blood. This method might be particularly significant in clinical trials of immune intervention strategies.

  10. Induction of Neonatal Tolerance to Mls^a Antigens by CD8^+ T Cells

    NASA Astrophysics Data System (ADS)

    Webb, Susan R.; Sprent, Jonathan

    1990-06-01

    Antigen-specific tolerance of T cells to minor lymphocyte stimulatory (Mis) antigens can be induced in mice by neonatal injection of foreign lymphohematopoietic cells. Although immune responses to Mls^a antigens are controlled by B cells, CD8^+ T cells were the most effective cell type for induction of Mls^a tolerance. Tolerance was evident in both thymus and lymph nodes and could be induced by as few as 2 x 10^4 CD8^+ T cells; these cells were 50 to 100 times as potent as CD4^+ cells or B cells in causing functional tolerance and deletion of Vβ6^+ T cells. Thus, intrathymic contact with antigens expressed on CD8^+ T cells may play an important role in controlling the normal development of tolerance.

  11. Expression of basement membrane antigens in spindle cell melanoma.

    PubMed

    Prieto, V G; Woodruff, J M

    1998-07-01

    Spindle cell melanoma (SCM) is an uncommon form of melanoma that may be confused histologically with other tumors, including malignant peripheral nerve sheath tumors (MPNST). Tumors with neural differentiation and melanocytic nevi may both show basement membrane immunohistochemically and at the ultrastructural level. However, most ultrastructural studies of melanoma have failed to demonstrate well formed basement membrane around tumor cells. The presence of basement membrane has been used by some authors as evidence favoring MPNST, as opposed to SCM. To evaluate this distinction immunohistochemically, 22 primary and metastatic cutaneous melanomas having a spindle cell component (SCM) were studied using monoclonal antibodies against laminin and Type IV collagen. S100 protein and HMB45 antigen expression were also studied. All but one of the SCM were reactive for S100 protein in at least 25% of the cells. Thirteen of 20 tumors (65%) were focally reactive with HMB45. Laminin was expressed in 42% of the tumors (only membranous pattern in 3; cytoplasmic and membranous in 5). Seventeen tumors (77%) expressed type IV collagen (only membranous pattern in 7; cytoplasmic and membranous pattern in 10). Laminin and type IV collagen, known components of basement membrane, are often found in SCM. Therefore, their detection cannot be used to distinguish SCM from MPNST.

  12. Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens.

    PubMed Central

    Pfeifer, A M; Cole, K E; Smoot, D T; Weston, A; Groopman, J D; Shields, P G; Vignaud, J M; Juillerat, M; Lipsky, M M; Trump, B F

    1993-01-01

    Normal human liver tissue and cultured human hepatocytes are valuable models to study xenobiotic metabolism and toxicity, but they only have a limited in vitro life-span and are not readily available. This report describes the establishment of replicative cultures of human adult liver epithelial cells in serum-free medium. The longevity of three of these cultures, derived from different donors, was extended by introduction of the simian virus 40 large T antigen gene. Two cell lines, THLE-2 and -3, established with a recombinant simian virus 40 large T antigen virus have undergone > 100 population doublings, are nontumorigenic when injected into athymic nude mice, have near-diploid karyotypes, and do not express alpha-fetoprotein. The cells express cytokeratin 18 and albumin in early passage, whereas higher-passage cells in logarithmic-phase growth also express cytokeratin 19. THLE-2 and -3 cells metabolize benzo[a]pyrene, N-nitrosodimethylamine, and aflatoxin B1 to their ultimate carcinogenic metabolites that adduct DNA, which indicates functional cytochrome P450 pathways. Other enzymes involved in metabolism of chemical carcinogens, such as epoxide hydrolase, NADPH cytochrome P450 reductase, superoxide dismutase, catalase, glutathione S-transferases, and glutathione peroxidase are also retained by THLE cells. Thus, these immortalized human liver cells constitute an in vitro model for pharmacotoxicological studies and for the investigation of etiology and pathogenesis of human hepatocellular carcinoma. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7685115

  13. Langerhans cells utilize CD1a and langerin to efficiently present nonpeptide antigens to T cells

    PubMed Central

    Hunger, Robert E.; Sieling, Peter A.; Ochoa, Maria Teresa; Sugaya, Makoto; Burdick, Anne E.; Rea, Thomas H.; Brennan, Patrick J.; Belisle, John T.; Blauvelt, Andrew; Porcelli, Steven A.; Modlin, Robert L.

    2004-01-01

    Langerhans cells (LCs) constitute a subset of DCs that initiate immune responses in skin. Using leprosy as a model, we investigated whether expression of CD1a and langerin, an LC-specific C-type lectin, imparts a specific functional role to LCs. LC-like DCs and freshly isolated epidermal LCs presented nonpeptide antigens of Mycobacterium leprae to T cell clones derived from a leprosy patient in a CD1a-restricted and langerin-dependent manner. LC-like DCs were more efficient at CD1a-restricted antigen presentation than monocyte-derived DCs. LCs in leprosy lesions coexpress CD1a and langerin, placing LCs in position to efficiently present a subset of antigens to T cells as part of the host response to human infectious disease. PMID:14991068

  14. MSI-H colorectal cancers preferentially retain and expand intraepithelial lymphocytes rather than peripherally derived CD8+ T cells.

    PubMed

    Baker, Kristi; Foulkes, William D; Jass, Jeremy R

    2009-01-01

    The healthy colorectal mucosa contains many resident intraepithelial lymphocytes (IELs) consisting of partially activated yet hyporesponsive CD8(+) T cells. A predominant feature of colorectal cancers (CRCs) characterized by high levels of microsatellite instability (MSI-H) is heavy infiltration by an intraepithelial population of tumor infiltrating lymphocytes (iTILs). While it has been assumed that these iTILs originate from tumor infiltration by peripheral CD8(+) effector T cells, their origin remains unknown. In light of the phenotypic and functional differences exhibited by IELs and peripheral T cells, elucidation of the precursor population of iTILs in MSI-H CRCs could clarify the role played by these lymphocytes in tumor progression. The aim of the present study was to investigate whether MSI-H CRCs interact differently with IEL- versus peripherally-derived CD8(+) T cells. Using a Transwell assay system to mimic basolateral infiltration of tumor cells by lymphocytes, T cell migration, retention, proliferation and phenotypic alterations were investigated. Results indicate that MSI-H CRCs preferentially retain and expand IEL-derived cells to a greater degree than their microsatellite stable (MSS) counterparts. While MSI-H CRCs also retained more peripherally derived T cells, this number was considerably less than that from the IEL population. While interaction of IELs with either CRC type led to baseline lymphocyte activation, MSS CRCs induced upregulation of additional activation markers on retained IELs compared to MSI-H CRCs. These results suggest that the abundant iTILs present in MSI-H CRCs result from expansion of the preexisting mucosal IEL population and imply a limited prognostic role for iTILs in MSI-H CRC.

  15. The effects of Fasciola hepatica tegumental antigens on mast cell function.

    PubMed

    Vukman, Krisztina V; Adams, Paul N; Dowling, David; Metz, Martin; Maurer, Marcus; O'Neill, Sandra M

    2013-06-01

    Fasciola hepatica infection is associated with T helper 2/T regulatory immune responses and increased mast cell numbers. The aim of this study was to examine the interaction between F. hepatica tegumental coat antigen and mast cells in vivo and in vitro. Firstly, BALB/C, C57BL/6 or STAT6(-/-) mice were infected with F. hepatica metacercarie or mice were treated with F. hepatica tegumental coat antigen and then mast cells numbers in the peritoneal cavity and/or the liver were quantified. Also, the proliferation, chemotaxis, degranulation and cytokine secretion of mast cells from bone marrow or from peritoneal exudate cells stimulated with F. hepatica tegumental coat antigen were measured. Finally, we tested whether F. hepatica tegumental coat antigen inhibits degranulation of mast cells in vivo in a passive cutaneous and systemic anaphylaxis mouse model. Mast cell numbers increased in the peritoneal cavity and liver of F. hepatica infected mice, and this was mimicked by injection of F. hepatica tegumental coat antigen in a STAT6(-/-) independent manner. The increase in mast cell number was not the result of F. hepatica tegumental coat antigen-induced proliferation; rather F. hepatica tegumental coat antigen indirectly induces mast cell migration by dendritic cell-derived chemokines. Fasciola hepatica tegumental coat antigen interactions with mast cells do not drive T helper 2 or T regulatory immune responses. These studies on mast cell and F. hepatica tegumental coat antigen interaction may help us to understand the function of mast cells in immunity against F. hepatica and the immunomodulatory effect of F. hepatica tegumental coat antigen on these cells.

  16. Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen

    SciTech Connect

    Noda, T.; Satake, M.; Robins, T.; Ito, Y.

    1986-10-01

    The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of PSI2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10/sup 6/ cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture.

  17. Antigenic modulation of metastatic breast and ovary carcinoma cells by intracavitary injection of IFN-alpha.

    PubMed Central

    Giacomini, P.; Mottolese, M.; Fraioli, R.; Benevolo, M.; Venturo, I.; Natali, P. G.

    1992-01-01

    Antigenic modulation of major histocompatibility and tumour associated antigens was observed in neoplastic cells obtained from patients with pleural and abdominal effusions of breast and ovary carcinomas following a single intracavitary dose of 18 x 10(6) U recombinant IFN-alpha. This regimen resulted in antigenic modulation in seven out of 11 tested cases, suggesting a potential, although limited, responsiveness of at least a fraction of breast and ovary carcinoma cells to in situ biomodification with IFN-alpha. PMID:1503908

  18. Phospholipase treatment of accessory cells that have been exposed to antigen selectively inhibits antigen-specific Ia-restricted, but not allospecific, stimulation of T lymphocytes.

    PubMed Central

    Falo, L D; Benacerraf, B; Rock, K L

    1986-01-01

    The corecognition of antigen and class II major histocompatibility complex (MHC) molecules (Ia molecules) by the T-cell receptor is a cell surface event. Before antigen is recognized, it must be taken up, processed, and displayed on the surface of an Ia-bearing accessory cell (antigen-presenting cell, APC). The exact nature of antigen processing and the subsequent associations of antigen with the APC plasma membrane, Ia molecules, and/or the T-cell receptor are not well defined. To further analyze these events, we have characterized the processing and presentation of the soluble polypeptide antigen bovine insulin. We found that this antigen requires APC-dependent processing, as evidenced by the inability of metabolically inactivated APCs to present native antigen to antigen plus Ia-specific T-T hybridomas. The ability of the same APCs to present antigen after uptake and processing showed that this antigen subsequently becomes stably associated with the APC plasma membrane. To characterize the basis for this association, we analyzed its sensitivity to enzymatic digestion. APCs exposed to antigen, treated with phospholipase A2, and then immediately fixed lost the ability to stimulate bovine insulin plus I-Ad-specific hybridomas. In contrast, the ability of these same APCs to stimulate I-Ad allospecific hybridomas was unaffected. This effect of phospholipase is not mimicked by the broadly active protease Pronase, nor is there evidence for contaminating proteases in the phospholipase preparation. These results suggest that one consequence of antigen processing may be an antigen-lipid association that contributes to the anchoring of antigen to the APC membrane. The implications of this model are discussed. PMID:3529095

  19. Cell adhesion markers are expressed by a stable human endothelial cell line transformed by the SV40 large T antigen under vimentin promoter control.

    PubMed

    Vicart, P; Testut, P; Schwartz, B; Llorens-Cortes, C; Perdomo, J J; Paulin, D

    1993-10-01

    Markers of endothelium have been studied in a new endothelial cell line derived from human umbilical cord vein cells by microinjection of a recombinant gene that includes a deletion mutant of the human vimentin gene regulatory region controlling the large T and small t antigen coding region of the SV40 virus. In culture, this immortalized venous endothelial cell line (IVEC) demonstrated morphological characteristics of endothelium; uptake of acetylated low density lipoprotein and presence of the Factor VIII-related antigen. Treatment of IVEC cells with Interleukin-1 beta (IL-1 beta) at 10 U.ml-1 activates the expression of cell adhesion molecules such as endothelial leucocyte adhesion molecule (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), as observed in primary culture. Prostacyclin secretion was induced in the IVEC cells by 100 nM PMA treatment and thrombin at 0.5 U/ml. Angiotensin converting enzyme (ACE) activity detected in IVEC cells was present but lower than ACE activity in primary endothelial cells and was completely blocked by enalaprilat (1 microM), a specific ACE inhibitor. The presence of ACE mRNA was also demonstrated in IVEC cells by RT-PCR amplification. Our data demonstrate that endothelial cells immortalized by use of this recombinant gene retain the morphological organization and numerous differentiated properties of endothelium.

  20. Red blood cells as innovative antigen carrier to induce specific immune tolerance.

    PubMed

    Cremel, Magali; Guérin, Nathalie; Horand, Françoise; Banz, Alice; Godfrin, Yann

    2013-02-25

    The route of administration, the dose of antigen as well as the type of antigen-presenting cells (APCs) targeted are important factors to induce immune tolerance. Despite encouraging results obtained in animal models, intravenous injection of soluble antigen is unsuccessful in human clinical trials on autoimmune disease due to inefficient antigen delivery. To improve antigen delivery, we used mouse red blood cells (RBCs) as antigen vehicles to specifically target APCs which are responsible for removal of senescent RBCs after phagocytosis. In this study, we demonstrated that antigen-delivery by RBCs induced a strong decrease in the humoral response compared with the ovalbumin (OVA) free form in mice. In addition, OVA-loaded RBC treated with [bis(sulphosuccinimidyl)] suberate (BS3), a chemical compound known to enhance RBC phagocytosis, induced an inhibition of antigen-specific T cell responses and an increase in the percentage of regulatory T cells. The state of tolerance induced is long lasting, antigen-specific and sufficiently robust to withstand immunization with antigen mixed with cholera toxin adjuvant. This RBC strategy, which does not abolish the immune system, constitutes an attractive approach for induction of tolerance compared to systemic immunosuppressant therapies already in use.

  1. Survival of human lymphoma cells requires B-cell receptor engagement by self-antigens

    PubMed Central

    Young, Ryan M.; Wu, Tianyi; Schmitz, Roland; Dawood, Moez; Xiao, Wenming; Phelan, James D.; Xu, Weihong; Menard, Laurence; Meffre, Eric; Chan, Wing-Chung C.; Jaffe, Elaine S.; Gascoyne, Randy D.; Campo, Elías; Rosenwald, Andreas; Ott, German; Delabie, Jan; Rimsza, Lisa M.; Staudt, Louis M.

    2015-01-01

    The activated B-cell–like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) relies on chronic active B-cell receptor (BCR) signaling. BCR pathway inhibitors induce remissions in a subset of ABC DLBCL patients. BCR microclusters on the surface of ABC cells resemble those generated following antigen engagement of normal B cells. We speculated that binding of lymphoma BCRs to self-antigens initiates and maintains chronic active BCR signaling in ABC DLBCL. To assess whether antigenic engagement of the BCR is required for the ongoing survival of ABC cells, we developed isogenic ABC cells that differed solely with respect to the IgH V region of their BCRs. In competitive assays with wild-type cells, substitution of a heterologous V region impaired the survival of three ABC lines. The viability of one VH4-34+ ABC line and the ability of its BCR to bind to its own cell surface depended on V region residues that mediate the intrinsic autoreactivity of VH4-34 to self-glycoproteins. The BCR of another ABC line reacted with self-antigens in apoptotic debris, and the survival of a third ABC line was sustained by reactivity of its BCR to an idiotypic epitope in its own V region. Hence, a diverse set of self-antigens is responsible for maintaining the malignant survival of ABC DLBCL cells. IgH V regions used by the BCRs of ABC DLBCL biopsy samples varied in their ability to sustain survival of these ABC lines, suggesting a screening procedure to identify patients who might benefit from BCR pathway inhibition. PMID:26483459

  2. Identification of a Highly Antigenic Linear B Cell Epitope within Plasmodium vivax Apical Membrane Antigen 1 (AMA-1)

    PubMed Central

    Bueno, Lilian Lacerda; Lobo, Francisco Pereira; Morais, Cristiane Guimarães; Mourão, Luíza Carvalho; de Ávila, Ricardo Andrez Machado; Soares, Irene Silva; Fontes, Cor Jesus; Lacerda, Marcus Vinícius; Olórtegui, Carlos Chavez; Bartholomeu, Daniella Castanheira; Fujiwara, Ricardo Toshio; Braga, Érika Martins

    2011-01-01

    Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/disordered regions (IURs). The B cell epitope comprising the amino acid sequence 290–307 of PvAMA-1 (SASDQPTQYEEEMTDYQK), with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P = 0.0001 for both), respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies. PMID:21713006

  3. Microbioassay system for antiallergic drug screening using suspension cells retaining in a poly(dimethylsiloxane) microfluidic device.

    PubMed

    Tokuyama, Takahito; Fujii, Shin-Ichiro; Sato, Kiichi; Abo, Mitsuru; Okubo, Akira

    2005-05-15

    This article describes an antiallergic drug-screening system by the detection of histamine released from mast cells (suspension cells) on a multilayer microchip. In this study, the elastmeric material, poly(dimethylsiloxane) (PDMS), was employed to fabricate microchannels and microchambers. The microchip consists of two sections: a histamine-releasing one, which has a cell chamber, and a histamine-derivatizing one. Both were laminated to one microchip. Rat peritoneal mast cells were retained in the cell chamber (1.2 microL) with a filtering system using a cellulose nitrate membrane. This filtering system could easily retain suspension cells without cell damage. Mast cells were viable for a sufficient time to conduct the assay on the cell chamber. The cells were stimulated with a chemical release compound 48/80 (C48/80), and then histamine flowed into the lower layer, where it was derivatized to the fluorescent molecules with o-phthalaldehyde and its fluorescence was detected on the microchip. This flow system could detect the time course of the histamine release, and this microchip system required only 20 min for the assay. By this integrated system, 51 pmol of histamine released from 500 cells was detected, and the number of cells required for the assay was reduced to 1% compared with conventional bulk systems. By comparing the released histamine levels with and without drugs, their effect could be evaluated. The inhibition ratio of C48/80 induced-histamine release using an antiallergic drug, disodium cromoglicate (DSCG), was related to the concentration of DSCG. This flow system was applicable for antiallergy drug screening by rapid measurement of the inhibition of histamine release from a very small amount of mast cells.

  4. Neutrophils and monocytes transport tumor cell antigens from the peritoneal cavity to secondary lymphoid tissues

    SciTech Connect

    Terasawa, Masao; Nagata, Kisaburo; Kobayashi, Yoshiro

    2008-12-12

    Antigen-transporting cells take up pathogens, and then migrate from sites of inflammation to secondary lymphoid tissues to induce an immune response. Among antigen-transporting cells, dendritic cells (DCs) are believed to be the most potent and professional antigen-presenting cells that can stimulate naive T cells. However, the cells that transport antigens, tumor cell antigens in particular, have not been clearly identified. In this study we have analyzed what types of cells transport tumor cell antigens to secondary lymphoid tissues. We show that neutrophils, monocytes and macrophages but not DCs engulf X-irradiated P388 leukemic cells after their injection into the peritoneal cavity, and that neutrophils and monocytes but not macrophages migrate to the parathymic lymph nodes (pLN), the blood, and then the spleen. The monocytes in the pLN comprise Gr-1{sup -} and Gr-1{sup +} ones, and some of these cells express CD11c. Overall, this study demonstrates that neutrophils and monocytes transport tumor cell antigens from the peritoneal cavity to secondary lymphoid tissues.

  5. Usage of Murine T-cell Hybridoma Cells as Responder Cells Reveals Interference of Helicobacter Pylori with Human Dendritic Cell-mediated Antigen Presentation

    PubMed Central

    Fehlings, Michael; Drobbe, Lea; Beigier-Bompadre, Macarena; Viveros, Pablo Renner; Moos, Verena; Schneider, Thomas; Meyer, Thomas F.; Aebischer, Toni; Ignatius, Ralf

    2016-01-01

    Direct effects of Helicobacter pylori (H. pylori) on human CD4+ T-cells hamper disentangling a possible bacterial-mediated interference with major histocompatibility complex class II (MHC-II)-dependent antigen presentation to these cells. To overcome this limitation, we employed a previously described assay, which enables assessing human antigen-processing cell function by using murine T-cell hybridoma cells restricted by human leukocyte antigen (HLA) alleles. HLA-DR1+ monocyte-derived dendritic cells were exposed to H. pylori and pulsed with the antigen 85B from Mycobacterium tuberculosis (M. tuberculosis). Interleukin-2 (IL-2) secretion by AG85Baa97-112-specific hybridoma cells was then evaluated as an integral reporter of cognate antigen presentation. This methodology enabled revealing of interference of H. pylori with the antigen-presenting capacity of human dendritic cells. PMID:27980859

  6. Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells

    PubMed Central

    Mittelbrunn, María; Gutiérrez-Vázquez, Cristina; Villarroya-Beltri, Carolina; González, Susana; Sánchez-Cabo, Fátima; González, Manuel Ángel; Bernad, Antonio; Sánchez-Madrid, Francisco

    2011-01-01

    The immune synapse is an exquisitely evolved means of communication between T cells and antigen-presenting cells (APCs) during antigen recognition. Recent evidence points to the transfer of RNA via exosomes as a novel mode of intercellular communication. Here we show that exosomes of T, B and dendritic immune cells contain microRNA (miRNA) repertoires that differ from those of their parent cells. We investigate whether miRNAs are exchanged during cognate immune interactions, and demonstrate the existence of antigen-driven unidirectional transfer of miRNAs from the T cell to the APC, mediated by the delivery of CD63+ exosomes on immune synapse formation. Inhibition of exosome production by targeting neutral sphingomyelinase-2 impairs transfer of miRNAs to APCs. Moreover, miRNAs transferred during immune synapsis are able to modulate gene expression in recipient cells. Thus, our results support a mechanism of cellular communication involving antigen-dependent, unidirectional intercellular transfer of miRNAs by exosomes during immune synapsis. PMID:21505438

  7. Killer artificial antigen-presenting cells: a novel strategy to delete specific T cells.

    PubMed

    Schütz, Christian; Fleck, Martin; Mackensen, Andreas; Zoso, Alessia; Halbritter, Dagmar; Schneck, Jonathan P; Oelke, Mathias

    2008-04-01

    Several cell-based immunotherapy strategies have been developed to specifically modulate T cell-mediated immune responses. These methods frequently rely on the utilization of tolerogenic cell-based antigen-presenting cells (APCs). However, APCs are highly sensitive to cytotoxic T-cell responses, thus limiting their therapeutic capacity. Here, we describe a novel bead-based approach to modulate T-cell responses in an antigen-specific fashion. We have generated killer artificial APCs (kappaaAPCs) by coupling an apoptosis-inducing alpha-Fas (CD95) IgM mAb together with HLA-A2 Ig molecules onto beads. These kappaaAPCs deplete targeted antigen-specific T cells in a Fas/Fas ligand (FasL)-dependent fashion. T-cell depletion in cocultures is rapidly initiated (30 minutes), dependent on the amount of kappaaAPCs and independent of activation-induced cell death (AICD). kappaaAPCs represent a novel technology that can control T cell-mediated immune responses, and therefore has potential for use in treatment of autoimmune diseases and allograft rejection.

  8. Rational design of nanoparticles towards targeting antigen-presenting cells and improved T cell priming.

    PubMed

    Zupančič, Eva; Curato, Caterina; Paisana, Maria; Rodrigues, Catarina; Porat, Ziv; Viana, Ana S; Afonso, Carlos A M; Pinto, João; Gaspar, Rogério; Moreira, João N; Satchi-Fainaro, Ronit; Jung, Steffen; Florindo, Helena F

    2017-07-28

    Vaccination is a promising strategy to trigger and boost immune responses against cancer or infectious disease. We have designed, synthesized and characterized aliphatic-polyester (poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) to investigate how the nature of protein association (adsorbed versus entrapped) and polymer/surfactant concentrations impact on the generation and modulation of antigen-specific immune responses. The ability of the NP formulations to target dendritic cells (DC), be internalized and activate the T cells was characterized and optimized in vitro and in vivo using markers of DC activation and co-stimulatory molecules. Ovalbumin (OVA) was used as a model antigen in combination with the engraftment of CD4(+) and CD8(+) T cells, carrying a transgenic OVA-responding T cell receptor (TCR), to trace and characterize the activation of antigen-specific CD4(+) and CD8(+) lymph node T cells upon NP vaccination. Accordingly, the phenotype and frequency of immune cell stimulation induced by the NP loaded with OVA, isolated or in combination with synthetic unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotide (ODN) motifs, were characterized. DC-NP interactions increased with incubation time, presenting internalization values between 50 and 60% and 30-40%, in vitro and in vivo, respectively. Interestingly, animal immunization with antigen-adsorbed NP up-regulated major histocompatibility complex (MHC) class II (MHCII), while NP entrapping the antigen up-regulated MHCI, suggesting a more efficient cross-presentation. On the other hand, rather surprisingly, the surfactant used in the NP formulation had a major impact on the activation of antigen presenting cells (APC). In fact, DC collected from lymph nodes of animals immunized with NP prepared using poly(vinil alcohol) (PVA), as a surfactant, expressed significantly higher levels of CD86, MHCI and MHCII. In addition, those NP prepared with PVA and co-entrapping OVA and the toll

  9. Surface-Engineering of Red Blood Cells as Artificial Antigen Presenting Cells Promising for Cancer Immunotherapy.

    PubMed

    Sun, Xiaoqi; Han, Xiao; Xu, Ligeng; Gao, Min; Xu, Jun; Yang, Rong; Liu, Zhuang

    2017-09-01

    The development of artificial antigen presenting cells (aAPCs) to mimic the functions of APCs such as dendritic cells (DCs) to stimulate T cells and induce antitumor immune responses has attracted substantial interests in cancer immunotherapy. In this work, a unique red blood cell (RBC)-based aAPC system is designed by engineering antigen peptide-loaded major histocompatibility complex-I and CD28 activation antibody on RBC surface, which are further tethered with interleukin-2 (IL2) as a proliferation and differentiation signal. Such RBC-based aAPC-IL2 (R-aAPC-IL2) can not only provide a flexible cell surface with appropriate biophysical parameters, but also mimic the cytokine paracrine delivery. Similar to the functions of matured DCs, the R-aAPC-IL2 cells can facilitate the proliferation of antigen-specific CD8+ T cells and increase the secretion of inflammatory cytokines. As a proof-of-concept, we treated splenocytes from C57 mice with R-aAPC-IL2 and discovered those splenocytes induced significant cancer-cell-specific lysis, implying that the R-aAPC-IL2 were able to re-educate T cells and induce adoptive immune response. This work thus presents a novel RBC-based aAPC system which can mimic the functions of antigen presenting DCs to activate T cells, promising for applications in adoptive T cell transfer or even in direct activation of circulating T cells for cancer immunotherapy. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Generation of mouse pluripotent stem cell-derived proliferating myeloid cells as an unlimited source of functional antigen-presenting cells.

    PubMed

    Zhang, Rong; Liu, Tian-Yi; Senju, Satoru; Haruta, Miwa; Hirosawa, Narumi; Suzuki, Motoharu; Tatsumi, Minako; Ueda, Norihiro; Maki, Hiroyuki; Nakatsuka, Ryusuke; Matsuoka, Yoshikazu; Sasaki, Yutaka; Tsuzuki, Shinobu; Nakanishi, Hayao; Araki, Ryoko; Abe, Masumi; Akatsuka, Yoshiki; Sakamoto, Yasushi; Sonoda, Yoshiaki; Nishimura, Yasuharu; Kuzushima, Kiyotaka; Uemura, Yasushi

    2015-06-01

    The use of dendritic cells (DC) to prime tumor-associated antigen-specific T-cell responses provides a promising approach to cancer immunotherapy. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can differentiate into functional DCs, thus providing an unlimited source of DCs. However, the previously established methods of generating practical volumes of DCs from pluripotent stem cells (PSC) require a large number of PSCs at the start of the differentiation culture. In this study, we generated mouse proliferating myeloid cells (pMC) as a source of antigen-presenting cells (APC) using lentivirus-mediated transduction of the c-Myc gene into mouse PSC-derived myeloid cells. The pMCs could propagate almost indefinitely in a cytokine-dependent manner, while retaining their potential to differentiate into functional APCs. After treatment with IL4 plus GM-CSF, the pMCs showed impaired proliferation and differentiated into immature DC-like cells (pMC-DC) expressing low levels of major histocompatibility complex (MHC)-I, MHC-II, CD40, CD80, and CD86. In addition, exposure to maturation stimuli induced the production of TNFα and IL12p70, and enhanced the expression of MHC-II, CD40, and CD86, which is thus suggestive of typical DC maturation. Similar to bone marrow-derived DCs, they stimulated a primary mixed lymphocyte reaction. Furthermore, the in vivo transfer of pMC-DCs pulsed with H-2K(b)-restricted OVA257-264 peptide primed OVA-specific cytotoxic T cells and elicited protection in mice against challenge with OVA-expressing melanoma. Overall, myeloid cells exhibiting cytokine-dependent proliferation and DC-like differentiation may be used to address issues associated with the preparation of DCs. ©2015 American Association for Cancer Research.

  11. Ubiquitinated Proteins Isolated From Tumor Cells Are Efficient Substrates for Antigen Cross-Presentation.

    PubMed

    Yu, Guangjie; Moudgil, Tarsem; Cui, Zhihua; Mou, Yongbin; Wang, Lixin; Fox, Bernard A; Hu, Hong-Ming

    2017-03-31

    We have previously shown that inhibition of the proteasome causes defective ribosomal products to be shunted into autophagosomes and subsequently released from tumor cells as defective ribosomal products in Blebs (DRibbles). These DRibbles serve as an excellent source of antigens for cross-priming of tumor-specific T cells. Here, we examine the role of ubiquitinated proteins (Ub-proteins) in this pathway. Using purified Ub-proteins from tumor cells that express endogenous tumor-associated antigen or exogenous viral antigen, we tested the ability of these proteins to stimulate antigen-specific T-cell responses, by activation of monocyte-derived dendritic cells generated from human peripheral blood mononuclear cells. Compared with total cell lysates, we found that purified Ub-proteins from both a gp100-specific melanoma cell line and from a lung cancer cell line expressing cytomegalovirus pp65 antigen produced a significantly higher level of IFN-γ in gp100- or pp65-specific T cells, respectively. In addition, Ub-proteins from an allogeneic tumor cell line could be used to stimulate tumor-infiltrating lymphocytes isolated and expanded from non-small cell lung cancer patients. These results establish that Ub-proteins provide a relevant source of antigens for cross-priming of antitumor immune responses in a variety of settings, including endogenous melanoma and exogenous viral antigen presentation, as well as antigen-specific tumor-infiltrating lymphocytes. Thus, ubiquitin can be used as an affinity tag to enrich for unknown tumor-specific antigens from tumor cell lysates to stimulate tumor-specific T cells ex vivo or to be used as vaccines to target short-lived proteins.

  12. 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

    2016-06-02

    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.

  13. 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.

  14. Molecular characterization of a fully human chimeric T-cell antigen receptor for tumor-associated antigen EpCAM.

    PubMed

    Shirasu, Naoto; Yamada, Hiromi; Shibaguchi, Hirotomo; Kuroki, Motomu; Kuroki, Masahide

    2012-01-01

    The transduction of T cells to express chimeric T-cell antigen receptor (CAR) is an attractive strategy for adaptive immunotherapy for cancer, because the CAR can redirect the recognition specificity of T cells to tumor-associated antigens (TAAs) on the surface of target cells, thereby avoiding the limitations of HLA restriction. However, there are considerable problems with the clinical application of CAR, mostly due to its xenogeneic components, which could be immunogenic in humans. Moreover, while extensive studies on the CARs have been performed, the detailed molecular mechanisms underlying the activation of CAR-grafted T cells remain unclear. In order to eliminate potential immunogenicity and investigate the molecular basis of the CAR-mediated T-cell activation, we constructed a novel CAR (CAR57-28ζ) specific for one of the most important TAAs, epithelial cell adhesion molecule (EpCAM), using only human-derived genes. We revealed that in Jurkat T cells, lentivirally expressed CAR57-28ζ can transmit the T-cell-activating signals sufficient to induce IL-2 production upon EpCAM stimulation. An immunofluorescent analysis clearly showed that the CAR57-28ζ induces the formation of signaling clusters containing endogenous CD3ζ at the CAR/EpCAM interaction interface. These results suggest that this CAR gene may be safely and effectively applied for adaptive T-cell immunotherapy.

  15. Molecular Characterization of a Fully Human Chimeric T-Cell Antigen Receptor for Tumor-Associated Antigen EpCAM

    PubMed Central

    Shirasu, Naoto; Yamada, Hiromi; Shibaguchi, Hirotomo; Kuroki, Motomu; Kuroki, Masahide

    2012-01-01

    The transduction of T cells to express chimeric T-cell antigen receptor (CAR) is an attractive strategy for adaptive immunotherapy for cancer, because the CAR can redirect the recognition specificity of T cells to tumor-associated antigens (TAAs) on the surface of target cells, thereby avoiding the limitations of HLA restriction. However, there are considerable problems with the clinical application of CAR, mostly due to its xenogeneic components, which could be immunogenic in humans. Moreover, while extensive studies on the CARs have been performed, the detailed molecular mechanisms underlying the activation of CAR-grafted T cells remain unclear. In order to eliminate potential immunogenicity and investigate the molecular basis of the CAR-mediated T-cell activation, we constructed a novel CAR (CAR57-28ζ) specific for one of the most important TAAs, epithelial cell adhesion molecule (EpCAM), using only human-derived genes. We revealed that in Jurkat T cells, lentivirally expressed CAR57-28ζ can transmit the T-cell-activating signals sufficient to induce IL-2 production upon EpCAM stimulation. An immunofluorescent analysis clearly showed that the CAR57-28ζ induces the formation of signaling clusters containing endogenous CD3ζ at the CAR/EpCAM interaction interface. These results suggest that this CAR gene may be safely and effectively applied for adaptive T-cell immunotherapy. PMID:22547929

  16. Identification of novel Mycobacterium tuberculosis CD4 T-cell antigens via high throughput proteome screening

    PubMed Central

    Nayak, Kaustuv; Jing, Lichen; Russell, Ronnie M.; Davies, D. Huw; Hermanson, Gary; Molina, Douglas M.; Liang, Xiaowu; Sherman, David R.; Kwok, William W.; Yang, Junbao; Kenneth, John; Ahamed, Syed F.; Chandele, Anmol; Kaja, Murali-Krishna; Koelle, David M.

    2015-01-01

    Elicitation of CD4 IFN-gamma T cell responses to Mycobacterium tuberculosis (MTB) is a rational vaccine strategy to prevent clinical tuberculosis. Diagnosis of MTB infection is based on T-cell immune memory to MTB antigens. The MTB proteome contains over four thousand open reading frames (ORFs). We conducted a pilot antigen identification study using 164 MTB proteins and MTB-specific T-cells expanded in vitro from 12 persons with latent MTB infection. Enrichment of MTB-reactive T-cells from PBMC used cell sorting or an alternate system compatible with limited resources. MTB proteins were used as single antigens or combinatorial matrices in proliferation and cytokine secretion readouts. Overall, our study found that 44 MTB proteins were antigenic, including 27 not previously characterized as CD4 T-cell antigens. Antigen truncation, peptide, NTM homology, and HLA class II tetramer studies confirmed malate synthase G (encoded by gene Rv1837) as a CD4 T-cell antigen. This simple, scalable system has potential utility for the identification of candidate MTB vaccine and biomarker antigens. PMID:25857935

  17. T cell tolerance and activation to a transgene-encoded tumor antigen.

    PubMed

    Antoniou, A; McCormick, D; Scott, D; Yeoman, H; Chandler, P; Mellor, A; Dyson, J

    1996-05-01

    Much has been learned in recent years concerning the nature of tumor antigens recognized by T cells. To apply this knowledge clinically, the nature of the host response to individual and multiple tumor antigens has to be characterized. This will help to define the efficacy of immune surveillance and the immune status of the host following exposure to tumor antigens expressed on pre-neoplastic tissue. To approach these questions, we have developed a transgenic mouse which expresses the tumor-specific antigen P91A. The single amino acid substitution in P91A results in the expression of a new MHC class I (H-2Ld)-binding peptide. In transgenic tissue, the H-2Ld/P91A complex is expressed in isolation from other tumor-associated antigens, allowing definition of the immune response to a single defined tumor antigen, a situation closely analogous to events during tumorigenesis. We show that CD8+ T cell immune surveillance of P91A is ineffective without the introduction of a helper determinant operating through stimulation of CD4+ T cells. Recognition of the isolated P91A tumor antigen on normal tissue by CD8+ T cells is a tolerogenic process. Induction of T cell tolerance suggests tumor antigen-T cell interactions occurring during tumorigenesis may elicit T cell tolerance and hence confound some immunotherapeutic approaches.

  18. Censoring of self-reactive B cells by follicular dendritic cell-displayed self-antigen.

    PubMed

    Yau, Irene W; Cato, Matthew H; Jellusova, Julia; Hurtado de Mendoza, Tatiana; Brink, Robert; Rickert, Robert C

    2013-08-01

    In the secondary lymphoid organs, intimate contact with follicular dendritic cells (FDCs) is required for B cell retention and Ag-driven selection during the germinal center response. However, selection of self-reactive B cells by Ag on FDCs has not been addressed. To this end, we generated a mouse model to conditionally express a membrane-bound self-antigen on FDCs and to monitor the fate of developing self-reactive B cells. In this article, we show that self-antigen displayed on FDCs mediates effective elimination of self-reactive B cells at the transitional stage. Notwithstanding, some self-reactive B cells persist beyond this checkpoint, showing evidence of Ag experience and intact proximal BCR signaling, but they are short-lived and unable to elicit T cell help. These results implicate FDCs as an important component of peripheral B cell tolerance that prevents the emergence of naive B cells capable of responding to sequestered self-antigens.

  19. Gamma delta T cells recognize a microbial encoded B Cell antigen to initiate a rapid antigen-specific Interleukin-17 response

    USDA-ARS?s Scientific Manuscript database

    Gamma delta T cells contribute uniquely to host immune defense, but the way in which they do so remains an enigma. Here we show that an algae protein, phycoerythrin (PE) is recognized by gamma delta T cells from mice, bovine and humans and binds directly to specific gamma delta T cell antigen recept...

  20. 20-kDa protein associated with the murine T-cell antigen receptor is phosphorylated in response to activation by antigen or concanavalin A

    SciTech Connect

    Samelson, L.E.; Harford, J.; Schwartz, R.H.; Klausner, R.D.

    1985-04-01

    Antigen or concanavalin A activation of a murine T-cell hybrid specific for pigeon cytochrome resulted in phosphorylation of a 20-kDa protein that was specifically coprecipitated by a monoclonal antibody binding the T-cell antigen receptor. There was no evidence for phosphorylation of the antigen receptor itself. The phosphorylation of the 20-kDa polypeptide was dependent on the concentration of antigen or lectin used to activate the T-cell hybrid and reached a maximum 40 min after the addition of antigen. The 20-kDa protein was also radioiodinated with a hydrophobic photoactivatable labeling reagent. The amount of iodinated 20-kDa protein immunoprecipitable with the anti-receptor antibody did not increase with T-cell activation, indicating that the phosphorylation occurred on a molecule that was constitutively associated with the antigen receptor. Concanavalin A also induced phosphorylation of a 20-kDa polypeptide in a second antigen-specific major histocompatibility complex-restricted T-cell hybrid. Again, the phosphorylated polypeptide was precipitated only by a monoclonal antibody specific for the antigen receptor on this hybrid. Thus, the antigen or concanavalin A-induced activation of T-cell hybrids results in the rapid phosphorylation of a 20-kDa protein that is associated with the T-cell receptor.

  1. Germ tube-specific antigens of Candida albicans cell walls

    SciTech Connect

    Sundstrom, P.R.

    1986-01-01

    Studies were performed to characterize the surface differences between blastospores and germ tubes of the pathogenic, dimorphic yeast, Candida albicans, and to identify components of yeast cells responsible for these differences. Investigation of surfaces differences of the two growth forms was facilitated by the production of rabbit antiserum prepared against Formalin-treated yeast possessing germ tubes. To prepare antiserum specific for germ tubes, this serum was adsorbed with stationary phase blastospores. Whereas the unadsorbed antiserum reacted with both blastospore and germ tube forms by immunofluorescence and Enzyme-Linked Immunosorbent Assay, the adsorbed antiserum did not react with blastospores but detected germ tube-specific antigens in hyphal forms. The differences between blastospores and germ tubes of Candida albicans, were further studied by comparing enzymatic digests of cell walls of both growth forms in radiolabeled organisms. Organisms were labeled either on the surface with /sup 125/I, or metabolically with (/sup 35/S) methionine or (/sup 3/H) mannose. Three-surface-located components (as shown by antibody adsorption and elution experiments) were precipitated from Zymolase digests. All three components were mannoproteins as shown by their ability to bind Concanavalin A, and to be labeled in protein labeling procedures, and two of these (200,000 and 155,000 molecular weight) were germ tube specific, as shown by their ability to be precipitated by germ tube-specific antiserum. Monoclonal antibodies were prepared to C. albicans, using blastospores bearing germ tubes as immunogen.

  2. Stem Cell Antigen-1 in Skeletal Muscle Function

    PubMed Central

    Bernstein, Harold S.; Samad, Tahmina; Cholsiripunlert, Sompob; Khalifian, Saami; Gong, Wenhui; Ritner, Carissa; Aurigui, Julian; Ling, Vivian; Wilschut, Karlijn J.; Bennett, Stephen; Hoffman, Julien; Oishi, Peter

    2013-01-01

    Stem cell antigen-1 (Sca-1) is a member of the Ly-6 multigene family encoding highly homologous, glycosyl-phosphatidylinositol-anchored membrane proteins. Sca-1 is expressed on muscle-derived stem cells and myogenic precursors recruited to sites of muscle injury. We previously reported that inhibition of Sca-1 expression stimulated myoblast proliferation in vitro and regulated the tempo of muscle repair in vivo. Despite its function in myoblast expansion during muscle repair, a role for Sca-1 in normal, post-natal muscle has not been thoroughly investigated. We systematically compared Sca-1-/- (KO) and Sca-1+/+ (WT) mice and hindlimb muscles to elucidate the tissue, contractile, and functional effects of Sca-1 in young and aging animals. Comparison of muscle volume, fibrosis, myofiber cross-sectional area, and Pax7+ myoblast number showed little differences between ages or genotypes. Exercise protocols, however, demonstrated decreased stamina in KO versus WT mice, with young KO mice achieving results similar to aging WT animals. In addition, KO mice did not improve with practice, while WT animals demonstrated conditioning over time. Surprisingly, myomechanical analysis of isolated muscles showed that KO young muscle generated more force and experienced less fatigue. However, KO muscle also demonstrated incomplete relaxation with fatigue. These findings suggest that Sca-1 is necessary for muscle conditioning with exercise, and that deficient conditioning in Sca-1 KO animals becomes more pronounced with age. PMID:24042315

  3. Expression of T cell antigen receptor during differentiation

    SciTech Connect

    Allison, J.P.; Lanier, L.L.; Guyden, J.; Richie, E.R.

    1986-03-01

    The authors have used flow cytometry with monoclonal antibodies, radioimmuneprecipitation with a rabbit antiserum to common epitopes of the TCR, and Northern and Southern blot analysis with cloned TCR genes to study antigen receptor (TCR) expression by normal murine and human thymocytes and by primary murine thymomas. L3T4-,Lyt2- murine thymomas corresponding to the earliest stage of thymic differentiation, were found to have rearranged TCR beta genes, and to express low levels of beta transcript, but lacked alpha gene transcript and failed to express TCR on the cell surface. L3T4+,Lyt2+ thymomas were variable, but the majority were found to contain significant levels of both alpha and beta transcripts and to express TCR at the cell surface. Similarly, alpha and beta transcripts and TCR protein were detected in sorted L3T4+,Lyt2+ murine thymocytes. Using three color fluorescence, the authors determined that app. 70% of human T4+T8+ thymocytes also expressed T3, a component of the TCR complex. These data indicate that in mouse and man expression of TCR occurs in the immature, or cortical, thymic population.

  4. Human T cell activation. III. Induction of an early activation antigen, EA 1 by TPA, mitogens and antigens

    SciTech Connect

    Hara, T.; Jung, L.K.L.; FU, S.M.

    1986-03-01

    With human T cells activated for 12 hours by 12-o-tetradecanoyl phorbol-13-acetate (TPA) as immunogen, an IgG/sub 2a/ monoclonal antibody, mAb Ea 1, has been generated to a 60KD phosphorylated protein with 32KD and 28KD subunits. The antigen, Ea 1, is readily detected on 60% of isolated thymocytes by indirect immunofluorescence. A low level of Ea 1 expression is detectable on 2-6% of blood lymphocytes. Isolated T cells have been induced to express Ea 1 by TPA, mitogens and anitgens. TPA activated T cells express Ea 1 as early as 1 hour after activation. By 4 hours, greater than 95% of the T cells stain with mAb Ea 1. About 50% of the PHA or Con A activated T cells express Ea 1 with a similar kinetics. Ea 1 expression proceeds that of IL-2 receptor in these activation processes. T cells activated by soluble antigens (tetanus toxoid and PPD) and alloantigens in MLR also express Ea 1 after a long incubation. About 20% of the T cells stain for Ea 1 at day 6. Ea 1 expression is not limited to activated T cells. B cells activated by TPA or anti-IgM Ab plus B cell growth factor express Ea 1. The kinetics of Ea 1 expression is slower and the staining is less intense. Repeated attempts to detect Ea 1 on resting and activated monocytes and granulocytes have not been successful. Ea 1 expression is due to de novo synthesis for its induction is blocked by cycloheximide and actinomycin D. Ea 1 is the earliest activation antigen detectable to-date.

  5. Human immune response to Vibrio cholerae O1 whole cells and isolated outer membrane antigens.

    PubMed Central

    Richardson, K; Kaper, J B; Levine, M M

    1989-01-01

    The serum immunoglobulin G (IgG) and mucosal secretory IgA (SIgA) response of human volunteers challenged with Vibrio cholerae O1 was analyzed for reactivity to V. cholerae O1 antigens by the immunoblot technique. Components of both in vitro- and in vivo (rabbit ligated ileal loop)-grown V. cholerae O1 were separated by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. Postchallenge serum IgG reacted uniquely with 15 antigens and with greater intensity than did prechallenge serum with at least 16 antigens. Serum IgG and SIgA reacted with antigens present in preparations from the homologous challenge strain of V. cholerae as well as antigens from strains of heterologous biotype or serotype. These heterologous antigens may represent antigens responsible for protection to rechallenge with a heterologous strain of V. cholerae. All the antigens detected by postchallenge jejunal fluid SIgA had an apparent molecular size of less than 25 kilodaltons. Serum IgG and jejunal fluid SIgA also reacted with antigens unique to in vivo-grown cells and several antigens in outer membrane preparations, suggesting that studies of protective immunity and V. cholerae O1 pathogenesis should include examination of both in vitro- and in vivo-grown V. cholerae O1 cellular antigens. Images PMID:2912896

  6. Definition of target antigens for naturally occurring CD4(+) CD25(+) regulatory T cells.

    PubMed

    Nishikawa, Hiroyoshi; Kato, Takuma; Tawara, Isao; Saito, Kanako; Ikeda, Hiroaki; Kuribayashi, Kagemasa; Allen, Paul M; Schreiber, Robert D; Sakaguchi, Shimon; Old, Lloyd J; Shiku, Hiroshi

    2005-03-07

    The antigenic targets recognized by naturally occurring CD4(+) CD25(+) regulatory T cells (T reg cells) have been elusive. We have serologically defined a series of broadly expressed self-antigens derived from chemically induced mouse sarcomas by serological identification of antigens by recombinant expression cloning (SEREX). CD4(+) CD25(+) T cells from mice immunized with SEREX-defined self-antigens had strong suppressive activity on peptide-specific proliferation of CD4(+) CD25(-) T cells and CD8(+) T cells. The suppressive effect was observed without in vitro T cell stimulation. Foxp3 expression in these CD4(+) CD25(+) T cells from immunized mice was 5-10 times greater than CD4(+) CD25(+) T cells derived from naive mice. The suppressive effect required cellular contact and was blocked by anti-glucocorticoid-induced tumor necrosis factor receptor family-related gene antibody. In vitro suppressive activity essentially disappeared 8 wk after the last immunization. However, it was regained by in vitro restimulation with cognate self-antigen protein but not with control protein. We propose that SEREX-defined self-antigens such as those used in this study represent self-antigens that elicit naturally occurring CD4(+) CD25(+) T reg cells.

  7. Enhanced antigen-presenting capacity of cultured Langerhans' cells is associated with markedly increased expression of Ia antigen

    SciTech Connect

    Shimada, S.; Caughman, S.W.; Sharrow, S.O.; Stephany, D.; Katz, S.I.

    1987-10-15

    Recent studies indicate that when epidermal Langerhans' cells (LC) are cultured for 2 to 3 days they, in comparison to freshly prepared LC, exhibit markedly enhanced ability to stimulate T cell proliferative responses in oxidative mitogenesis and in the mixed epidermal-leukocyte reaction. In this study, we determined whether cultured LC enhance antigen-specific T cell responses, and whether such enhanced stimulatory capacity correlates with the level of Ia antigen expressed on LC. We used C3H/He (Iak) epidermal cells as stimulators and, as responder cells, both the trinitrophenyl-specific clones D8 and SE4, which were assayed for (/sup 3/H)dThd incorporation, and the pigeon cytochrome c specific hybridoma 2C2, which was assayed for interleukin 2 production. Cultured LC induced 10 to 100 times greater proliferation or interleukin 2 production by responder cells than did freshly prepared LC. The intensity of I-Ak and I-Ek, expressed on cultured LC as assessed by immunofluorescence and flow cytometry, was found to be 10 to 36 times greater on a per cell basis than that on freshly prepared LC. Depletion of LC from fresh epidermal cell suspensions by anti-Iak and complement or treatment with 50 mJ/cm/sup 2/ medium range ultraviolet light or cycloheximide before culture abrogated both the increase in Ia expression and antigen-specific clonal proliferation. The results suggest that when LC are removed from their usual epidermal milieu, they express increased amounts of Ia and become more potent stimulators of T cell responses.

  8. Serum B-cell maturation antigen: a novel biomarker to predict outcomes for multiple myeloma patients.

    PubMed

    Ghermezi, Michael; Li, Mingjie; Vardanyan, Suzie; Harutyunyan, Nika Manik; Gottlieb, Jillian; Berenson, Ariana; Spektor, Tanya M; Andreu-Vieyra, Claudia; Petraki, Sophia; Sanchez, Eric; Udd, Kyle; Wang, Cathy S; Swift, Regina A; Chen, Haiming; Berenson, James R

    2017-04-01

    B-cell maturation antigen is expressed on plasma cells. In this study, we have identified serum B-cell maturation antigen as a novel biomarker that can monitor and predict outcomes for multiple myeloma patients. Compared to healthy donors, patients with multiple myeloma showed elevated serum B-cell maturation antigen levels (P<0.0001). Serum B-cell maturation antigen levels correlated with the proportion of plasma cells in bone marrow biopsies (Spearman's rho = 0.710; P<0.001), clinical status (complete response vs partial response, P=0.0374; complete response vs progressive disease, P<0.0001), and tracked with changes in M-protein levels. Among patients with non-secretory disease, serum B-cell maturation antigen levels correlated with bone marrow plasma cell levels and findings from positron emission tomography scans. Kaplan-Meier analysis demonstrated that serum B-cell maturation antigen levels above the median levels were predictive of a shorter progression-free survival (P=0.0006) and overall survival (P=0.0108) among multiple myeloma patients (n=243). Specifically, patients with serum B-cell maturation antigen levels above the median level at the time of starting front-line (P=0.0043) or a new salvage therapy (P=0.0044) were found to have shorter progression-free survival. Importantly, serum B-cell maturation antigen levels did not show any dependence on renal function and maintained independent significance when tested against other known prognostic markers for multiple myeloma such as age, serum β2 microglobulin, hemoglobin, and bone disease. These data identify serum B-cell maturation antigen as a new biomarker to manage multiple myeloma patients. Copyright© Ferrata Storti Foundation.

  9. Splenic B cells and antigen-specific B cells process anti-Ig in a similar manner

    SciTech Connect

    Myers, C.D.; Vitetta, E.S.

    1989-06-01

    B lymphocytes can process and present antigen to T cells. However, the fate of native antigen after its binding to specific B cells, i.e., the intracellular events involved in the processing and recycling of the antigenic fragments to the cell surface for antigen presentation, are not well understood. In the present study, we demonstrate that murine B cells degrade anti-Ig molecules bound to their surface and release acid soluble fragments into the supernatant. We also demonstrate that the kinetics of this process are identical for anti-mu, anti-delta, and anti-light chain antibodies, indicating that both surface IgM and surface IgD are equally effective in binding antigen and directing its processing. We also describe the effects of azide, chloroquine, and irradiation on this process. To extend these studies to the processing of specifically bound antigen, we demonstrate that highly purified trinitrophenyl antigen-binding cells degrade anti-Ig molecules with the same kinetics as unpurified splenic B cells. Thus, this purified population provides a suitable model system for the analysis of antigen degradation by antigen-specific cells.

  10. Cyclic re-entry of germinal center B cells in dealing with switching antigen

    NASA Astrophysics Data System (ADS)

    He, Chaoyang

    1999-08-01

    Germinal center spatial compartmentalization may help immune cells to optimize their mutation schedule so that affinity maturation through somatic hypermutation achieves higher efficiency. Some pathogens can alter their antigen expression (surface glycoprotein) by evolution of antigen or antigen switching or drifting to counteract the immune defense. We examine the switching antigen situation by introducing a prey-predator model in the string space representation of B cells and antigen, using Pontryagin's maximum principle to seek out the optimal mutation schedule, The optimal mutation schedule is still phase like. We conclude that re-entry of germinal center B cells is still crucial to affinity maturation. We further speculate a model of diffusing B cells coupling with pair correlation function may provide the underlying mechanism for the phasic like mutation schedule.

  11. Localization of Label-Retaining Cells in Murine Vocal Fold Epithelium

    ERIC Educational Resources Information Center

    Leydon, Ciara; Bartlett, Rebecca S.; Roenneburg, Drew A.; Thibeault, Susan L.

    2011-01-01

    Purpose: Epithelial homeostasis is critical for vocal fold health, yet little is known about the cells that support epithelial self-renewal. As a known characteristic of stem cells is that they are slow-cycling in vivo, the purpose of this prospective controlled study was to identify and quantify slow-cycling cells or putative stem cells in murine…

  12. Localization of Label-Retaining Cells in Murine Vocal Fold Epithelium

    ERIC Educational Resources Information Center

    Leydon, Ciara; Bartlett, Rebecca S.; Roenneburg, Drew A.; Thibeault, Susan L.

    2011-01-01

    Purpose: Epithelial homeostasis is critical for vocal fold health, yet little is known about the cells that support epithelial self-renewal. As a known characteristic of stem cells is that they are slow-cycling in vivo, the purpose of this prospective controlled study was to identify and quantify slow-cycling cells or putative stem cells in murine…

  13. The adult CNS retains the potential to direct region-specific differentiation of a transplanted neuronal precursor cell line.

    PubMed

    Shihabuddin, L S; Hertz, J A; Holets, V R; Whittemore, S R

    1995-10-01

    The chronic survival and differentiation of the conditionally immortalized neuronal cell line, RN33B, was examined following transplantation into the adult and neonatal rat hippocampus and cerebral cortex. In clonal culture, differentiated RN33B cells express p75NTR and trkB mRNA and protein, and respond to brain-derived neurotrophic factor treatment by inducing c-fos mRNA. Transplanted cells, identified using immunohistochemistry to detect beta-galactosidase expression, were seen in most animals up to 24 weeks posttransplantation (the latest time point examined). Stably integrated cells with various morphologies consistent with their transplantation site were observed. In the cerebral cortex, many RN33B cells differentiated with morphologies similar to pyramidal neurons and stellate cells. In the hippocampal formation, many RN33B cells assumed morphologies similar to pyramidal neurons characteristic of CA1 and CA3 regions, granular cell layer neurons of the dentate gyrus, and polymorphic neurons of the hilar region. Identical morphologies were observed in both adult and neonatal hosts, although a greater percentage of beta-galactosidase immunoreactive cells had differentiated in the neonatal brains. These results suggest that RN33B cells have the developmental plasticity to respond to local microenvironmental signals and that the adult brain retains the capacity to direct the differentiation of neuronal precursor cells in a direction that is consistent with that of endogenous neurons.

  14. Immunohistochemical detection of major histocompatibility complex antigens and quantitative analysis of tumour-infiltrating mononuclear cells in renal cell cancer.

    PubMed Central

    Tomita, Y.; Nishiyama, T.; Fujiwara, M.; Sato, S.

    1990-01-01

    In order to investigate the anti-tumour immune responsiveness of patients with renal cell cancer (RCC), we examined 30 such patients for the degree of expression of major histocompatibility complex (MHC) class I and class II antigens on RCC and the populations of tumour-infiltrating mononuclear cells (TIM). Normal renal tubular cells expressed class I but not class II antigens. Most of the tumour cells expressed class I antigens in 25 (83%) cases, but the proportion of such cells was reduced in five cases, three of which were of granular cell type histologically. Class II antigens were detected in all specimens with class I positivity. Various numbers of TIM were detected in 25 cases, being composed mainly of T cells and a smaller number of macrophages. Examination for the phenotype of T cells showed that CD8-positive cells were the dominant population. B cells were not detected. Quantitative analysis revealed that the numbers of TIM were significantly lower in cases showing class I reduction than in those with normal class I expression. Therefore, it was clear that class I antigens were preserved in RCC cells in most cases. Furthermore, a higher rate of reduction of class I antigens was observed in cases of granular cell type, which has been reported to have a worse prognosis than the clear cell type. The present data suggest that degree of the expression of MHC class I antigen on RCC might influence the host immune responsiveness against it. Images Figure 1 Figure 2 Figure 3 PMID:2206942

  15. Squamous cell carcinoma antigen in human liver carcinogenesis.

    PubMed

    Guido, M; Roskams, T; Pontisso, P; Fassan, M; Thung, S N; Giacomelli, L; Sergio, A; Farinati, F; Cillo, U; Rugge, M

    2008-04-01

    Squamous cell carcinoma antigen (SCCA) is a serine protease inhibitor that can be overexpressed in hepatocellular carcinoma (HCC) at both molecular and protein level, but no data are available on its expression in pre-malignant stages. To assess SCCA expression by immunohistochemistry in HCC and its nodular precursors in cirrhotic livers. 55 nodules from 42 explanted livers were evaluated: 7 large regenerative nodules (LRNs), 7 low-grade dysplastic nodules (LG-DNs), 10 high-grade DNs (HG-DNs), and 31 HCC. SCCA expression was semiquantitatively scored on a four-tiered scale. SCCA hepatocyte immunostaining was always restricted to the cytoplasm, mainly exhibiting a granular pattern. Stain intensity varied, ranging from weak to very strong. Within the nodules, positive cells were unevenly distributed, either scattered or in irregular clusters. The prevalence of SCCA expression was 29% in LRNs, 100% in DNs and 93% in HCC. A significant difference emerged in both prevalence and score for LRNs versus LG-DNs (p<0.039), HG-DNs (p = 0.001), and HCC (p = 0.000). A barely significant difference (p = 0.49) was observed between LG-DNs and HG-DNs, while no difference in SCCA expression was detected between HG-DNs and HCC. Cirrhotic tissue adjacent to the nodules was positive in 96% of cases, with a significant difference in the score (p = 0.000) between hepatocytes adjacent to HCC and those surrounding LRNs. This study provides the first evidence that aberrant SCCA expression is an early event in liver cell carcinomatous transformation.

  16. Trivalent CAR T-cells Overcome Interpatient Antigenic Variability in Glioblastoma.

    PubMed

    Bielamowicz, Kevin; Fousek, Kristen; Byrd, Tiara T; Samaha, Hebatalla; Mukherjee, Malini; Aware, Nikita; Wu, Meng-Fen; Orange, Jordan S; Sumazin, Pavel; Man, Tsz-Kwong; Joseph, Sujith K; Hegde, Meenakshi; Ahmed, Nabil

    2017-09-16

    Glioblastoma (GBM) is the most common primary malignant brain cancer, and is currently incurable. Chimeric Antigen Receptor (CAR) T-cells have shown promise in GBM treatment. While we have shown that combinatorial targeting of two glioma antigens offsets antigen escape and enhances T-cell effector functions, the inter-patient variability in surface antigen expression between patients hinders the clinical impact of targeting two antigen pairs. This study addresses targeting 3 antigens using a single CAR T-cell product for broader application. We analyzed the surface expression of 3 targetable glioma antigens (HER2, IL13Rα2 and EphA2) in 15 primary GBM samples. Accordingly, we created a trivalent T-cell product armed with three CAR molecules specific for these validated targets encoded by a single universal (U) tricistronic transgene (UCAR T-cells). Our data showed that co-targeting HER2, IL13Rα2 and EphA2 could overcome inter-patient variability by a tendency to capture near 100% of tumor cells in most tumors tested in this cohort. UCAR T-cells made from GBM patients' blood uniformly expressed all three CAR molecules with distinct antigen specificity. UCAR T-cells mediated robust immune synapses with tumor targets forming more polarized microtubule organizing centers (MTOC) and exhibited improved cytotoxicity and cytokine release over best monospecific and bispecific CAR T-cells per patient tumor profile. Lastly, low doses of UCAR T-cells controlled established autologous GBM patient derived xenografts (PDX) and improved survival of treated animals. UCAR T-cells can overcome antigenic heterogeneity in GBM and lead to improved treatment outcomes.

  17. Detecting Antigen-Specific T Cell Responses: From Bulk Populations to Single Cells

    PubMed Central

    Phetsouphanh, Chansavath; Zaunders, John James; Kelleher, Anthony Dominic

    2015-01-01

    A new generation of sensitive T cell-based assays facilitates the direct quantitation and characterization of antigen-specific T cell responses. Single-cell analyses have focused on measuring the quality and breadth of a response. Accumulating data from these studies demonstrate that there is considerable, previously-unrecognized, heterogeneity. Standard assays, such as the ICS, are often insufficient for characterization of rare subsets of cells. Enhanced flow cytometry with imaging capabilities enables the determination of cell morphology, as well as the spatial localization of the protein molecules within a single cell. Advances in both microfluidics and digital PCR have improved the efficiency of single-cell sorting and allowed multiplexed gene detection at the single-cell level. Delving further into the transcriptome of single-cells using RNA-seq is likely to reveal the fine-specificity of cellular events such as alternative splicing (i.e., splice variants) and allele-specific expression, and will also define the roles of new genes. Finally, detailed analysis of clonally related antigen-specific T cells using single-cell TCR RNA-seq will provide information on pathways of differentiation of memory T cells. With these state of the art technologies the transcriptomics and genomics of Ag-specific T cells can be more definitively elucidated. PMID:26274954

  18. Detecting Antigen-Specific T Cell Responses: From Bulk Populations to Single Cells.

    PubMed

    Phetsouphanh, Chansavath; Zaunders, John James; Kelleher, Anthony Dominic

    2015-08-12

    A new generation of sensitive T cell-based assays facilitates the direct quantitation and characterization of antigen-specific T cell responses. Single-cell analyses have focused on measuring the quality and breadth of a response. Accumulating data from these studies demonstrate that there is considerable, previously-unrecognized, heterogeneity. Standard assays, such as the ICS, are often insufficient for characterization of rare subsets of cells. Enhanced flow cytometry with imaging capabilities enables the determination of cell morphology, as well as the spatial localization of the protein molecules within a single cell. Advances in both microfluidics and digital PCR have improved the efficiency of single-cell sorting and allowed multiplexed gene detection at the single-cell level. Delving further into the transcriptome of single-cells using RNA-seq is likely to reveal the fine-specificity of cellular events such as alternative splicing (i.e., splice variants) and allele-specific expression, and will also define the roles of new genes. Finally, detailed analysis of clonally related antigen-specific T cells using single-cell TCR RNA-seq will provide information on pathways of differentiation of memory T cells. With these state of the art technologies the transcriptomics and genomics of Ag-specific T cells can be more definitively elucidated.

  19. Kinetics of Tumor Destruction by Chimeric Antigen Receptor-modified T Cells

    PubMed Central

    Anurathapan, Usanarat; Chan, Robert C; Hindi, Hakeem F; Mucharla, Roopa; Bajgain, Pradip; Hayes, Brendan C; Fisher, William E; Heslop, Helen E; Rooney, Cliona M; Brenner, Malcolm K; Leen, Ann M; Vera, Juan F

    2014-01-01

    The use of chimeric antigen receptor (CAR)–modified T cells as a therapy for hematologic malignancies and solid tumors is becoming more widespread. However, the infusion of a T-cell product targeting a single tumor-associated antigen may lead to target antigen modulation under this selective pressure, with subsequent tumor immune escape. With the purpose of preventing this phenomenon, we have studied the impact of simultaneously targeting two distinct antigens present on tumor cells: namely mucin 1 and prostate stem cell antigen, both of which are expressed in a variety of solid tumors, including pancreatic and prostate cancer. When used individually, CAR T cells directed against either tumor antigen were able to kill target-expressing cancer cells, but tumor heterogeneity led to immune escape. As a combination therapy, we demonstrate superior antitumor effects using both CARs simultaneously, but this was nevertheless insufficient to achieve a complete response. To understand the mechanism of escape, we studied the kinetics of T-cell killing and found that the magnitude of tumor destruction depended not only on the presence of target antigens but also on the intensity of expression—a feature that could be altered by administering epigenetic modulators that upregulated target expression and enhanced CAR T-cell potency. PMID:24213558

  20. Potent antigen-specific immune response induced by infusion of spleen cells coupled with succinimidyl-4-(N-maleimidomethyl cyclohexane)-1-carboxylate (SMCC) conjugated antigens.

    PubMed

    Guo, Yixian; Werbel, Tyler; Wan, Suigui; Wu, Haitao; Li, Yaohua; Clare-Salzler, Michael; Xia, Chang-Qing

    2016-02-01

    In the present study, we report our recently developed new approach to inducing antigen-specific immune response. We use two nucleophilic substitution "click" chemistry processes to successfully couple protein antigens or peptides to mouse spleen cells or T cells by a heterobifunctional crosslinker, succinimidyl-4-(N-maleimidomethyl cyclohexane)-1-carboxylate (SMCC) or sulfo-SMCC. SMCC and its water-soluble analog sulfo-SMCC contain N-hydroxysuccinimide (NHS) ester and maleimide groups, which allow stable covalent conjugation of amine- and sulfhydryl-containing molecules in trans. Protein coupling to cells relies on the free sulfhydryls (thiols) on cell surfaces and the free amines on protein antigens. Although the amount of protein coupled to cells is limited due to the limited number of cell surface thiols, the injection of spleen cells coupled with antigenic proteins, such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA), induces a potent antigen-specific immune response in vivo, which is even stronger than that induced by the injection of a large dose of protein plus adjuvants. In addition, short peptides coupled to purified splenic T cells also potently elicit peptide-specific T cell proliferation in vivo after injection. Further studies show that antigen-coupled spleen cell treatment leads to augmented IFN-γ-producing T cells. Our study provides a unique antigen delivery method that efficiently distributes antigen to the entire immune system, subsequently eliciting a potent antigen-specific immune response with enhanced IFN-γ production. The findings in the present study suggest that this antigen-cell coupling strategy could be employed in immunotherapy for cancers, infectious diseases as well as immune-mediated disorders.

  1. Tumor escape mechanisms: Potential role of soluble HLA antigens and NK cells activating ligands

    PubMed Central

    Campoli, Michael; Ferrone, Soldano

    2009-01-01

    The crucial role played by HLA antigens and natural killer (NK) cell activating ligands in the interactions of malignant cells with components of the host's immune system has stimulated interest in the characterization of their expression by malignant cells. Convincing evidence generated by the immunohistochemical staining of surgically removed malignant lesions with monoclonal antibodies (mAb) recognizing HLA antigens and NK cell activating ligands indicates that the surface expression of these molecules is frequently altered on malignant cells. These changes appear to have clinical significance, since in some types of malignant disease they are associated with the histopathological characteristics of the lesions as well as with disease free interval and survival. These associations have been suggested to reflect the effect of HLA antigen and NK cell activating ligand abnormalities on the interactions of tumor cells with antigen-specific cytotoxic T lymphocytes (CTL) and with NK cells. Nevertheless, there are examples in which disease progresses in the face of appropriate HLA antigen and/or NK cell activating ligand as well as tumor antigen expression by malignant cells and of functional antigen-specific CTL in the investigated patient. In such scenarios, it is likely that the tumor microenvironment is unfavorable for CTL and NK cell activity and contributes to tumor immune escape. Many distinct escape mechanisms have been shown to protect malignant cells from immune recognition and destruction in the tumor microenvironment. In this paper, following the description of the structural and functional characteristics of soluble HLA antigens and NK cell activating ligands, we will review changes in their serum level in malignant disease and discuss their potential role in the escape mechanisms utilized by tumor cells to avoid recognition and destruction. PMID:18700879

  2. Angiomotin promotes renal epithelial and carcinoma cell proliferation by retaining the nuclear YAP.

    PubMed

    Lv, Meng; Li, Shuting; Luo, Changqin; Zhang, Xiaoman; Shen, Yanwei; Sui, Yan Xia; Wang, Fan; Wang, Xin; Yang, Jiao; Liu, Peijun; Yang, Jin

    2016-03-15

    Renal cell carcinoma (RCC) is one of the common tumors in the urinary system without effective therapies. Angiomotin (Amot) can interact with Yes-associated protein (YAP) to either stimulate or inhibit YAP activity, playing a potential role in cell proliferation. However, the role of Amot in regulating the proliferation of renal epithelial and RCC cells is unknown. Here, we show that Amot is expressed predominantly in the nucleus of RCC cells and tissues, and in the cytoplasm and nucleus of renal epithelial cells and paracancerous tissues. Furthermore, Amot silencing inhibited proliferation of HK-2 and 786-O cells while Amot upregulation promoted proliferation of ACHN cells. Interestingly, the location of Amot and YAP in RCC clinical samples and cells was similar. Amot interacted with YAP in HK-2 and 786-O cells, particularly in the nucleus. Moreover, Amot silencing mitigated the levels of nuclear YAP in HK-2 and 786-O cells and reduced YAP-related CTGF and Cyr61 expression in 786-O cells. Amot upregulation slightly increased the nuclear YAP and YAP-related gene expression in ACHN cells. Finally, enhanced YAP expression restored proliferation of Amot-silencing 786-O cells. Together, these data indicate that Amot is crucial for the maintenance of nuclear YAP to promote renal epithelial and RCC proliferation.

  3. Prostate Stem Cell Antigen DNA Vaccination Breaks Tolerance to Self-antigen and Inhibits Prostate Cancer Growth

    PubMed Central

    Ahmad, Sarfraz; Casey, Garrett; Sweeney, Paul; Tangney, Mark; O'Sullivan, Gerald C

    2009-01-01

    Prostate stem cell antigen (PSCA) is a cell surface antigen expressed in normal human prostate and over expressed in prostate cancer. Elevated levels of PSCA protein in prostate cancer correlate with increased tumor stage/grade, with androgen independence and have higher expression in bone metastases. In this study, the PSCA gene was isolated from the transgenic adenocarcinoma mouse prostate cell line (TRAMPC1), and a vaccine plasmid construct was generated. This plasmid PSCA (pmPSCA) was delivered by intramuscular electroporation (EP) and induced effective antitumor immune responses against subcutaneous TRAMPC1 tumors in male C57 BL/6 mice. The pmPSCA vaccination inhibited tumor growth, resulting in cure or prolongation in survival. Similarly, the vaccine inhibited metastases in PSCA expressing B16 F10 tumors. There was activation of Th-1 type immunity against PSCA, indicating the breaking of tolerance to a self-antigen. This immunity was tumor specific and was transferable by adoptive transfer of splenocytes. The mice remained healthy and there was no evidence of collateral autoimmune responses in normal tissues. EP-assisted delivery of the pmPSCA evoked strong specific responses and could, in neoadjuvant or adjuvant settings, provide a safe and effective immune control of prostate cancer, given that there is significant homology between human and mouse PSCA. PMID:19337234

  4. Intestinal antigen-presenting cells in mucosal immune homeostasis: crosstalk between dendritic cells, macrophages and B-cells.

    PubMed

    Mann, Elizabeth R; Li, Xuhang

    2014-08-07

    The intestinal immune system maintains a delicate balance between immunogenicity against invading pathogens and tolerance of the commensal microbiota. Inflammatory bowel disease (IBD) involves a breakdown in tolerance towards the microbiota. Dendritic cells (DC), macrophages (MΦ) and B-cells are known as professional antigen-presenting cells (APC) due to their specialization in presenting processed antigen to T-cells, and in turn shaping types of T-cell responses generated. Intestinal DC are migratory cells, unique in their ability to generate primary T-cell responses in mesenteric lymph nodes or Peyer's patches, whilst MΦ and B-cells contribute to polarization and differentiation of secondary T-cell responses in the gut lamina propria. The antigen-sampling function of gut DC and MΦ enables them to sample bacterial antigens from the gut lumen to determine types of T-cell responses generated. The primary function of intestinal B-cells involves their secretion of large amounts of immunoglobulin A, which in turn contributes to epithelial barrier function and limits immune responses towards to microbiota. Here, we review the role of all three types of APC in intestinal immunity, both in the steady state and in inflammation, and how these cells interact with one another, as well as with the intestinal microenvironment, to shape mucosal immune responses. We describe mechanisms of maintaining intestinal immune tolerance in the steady state but also inappropriate responses of APC to components of the gut microbiota that contribute to pathology in IBD.

  5. Intestinal antigen-presenting cells in mucosal immune homeostasis: Crosstalk between dendritic cells, macrophages and B-cells

    PubMed Central

    Mann, Elizabeth R; Li, Xuhang

    2014-01-01

    The intestinal immune system maintains a delicate balance between immunogenicity against invading pathogens and tolerance of the commensal microbiota. Inflammatory bowel disease (IBD) involves a breakdown in tolerance towards the microbiota. Dendritic cells (DC), macrophages (MΦ) and B-cells are known as professional antigen-presenting cells (APC) due to their specialization in presenting processed antigen to T-cells, and in turn shaping types of T-cell responses generated. Intestinal DC are migratory cells, unique in their ability to generate primary T-cell responses in mesenteric lymph nodes or Peyer’s patches, whilst MΦ and B-cells contribute to polarization and differentiation of secondary T-cell responses in the gut lamina propria. The antigen-sampling function of gut DC and MΦ enables them to sample bacterial antigens from the gut lumen to determine types of T-cell responses generated. The primary function of intestinal B-cells involves their secretion of large amounts of immunoglobulin A, which in turn contributes to epithelial barrier function and limits immune responses towards to microbiota. Here, we review the role of all three types of APC in intestinal immunity, both in the steady state and in inflammation, and how these cells interact with one another, as well as with the intestinal microenvironment, to shape mucosal immune responses. We describe mechanisms of maintaining intestinal immune tolerance in the steady state but also inappropriate responses of APC to components of the gut microbiota that contribute to pathology in IBD. PMID:25110405

  6. A small protein inhibits proliferating cell nuclear antigen by breaking the DNA clamp

    PubMed Central

    Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo; Robinson, Howard; Sallman, Zahur F.; Marino, John P.; Kelman, Zvi

    2016-01-01

    Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain a canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP. PMID:27141962

  7. A small protein inhibits proliferating cell nuclear antigen by breaking the DNA clamp

    SciTech Connect

    Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo; Robinson, Howard; Sallman, Zahur F.; Marino, John P.; Kelman, Zvi

    2016-05-03

    Here, proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain a canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.

  8. A small protein inhibits proliferating cell nuclear antigen by breaking the DNA clamp

    DOE PAGES

    Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo; ...

    2016-05-03

    Here, proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain amore » canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.« less

  9. A small protein inhibits proliferating cell nuclear antigen by breaking the DNA clamp

    SciTech Connect

    Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo; Robinson, Howard; Sallman, Zahur F.; Marino, John P.; Kelman, Zvi

    2016-05-03

    Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain a canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.

  10. A small protein inhibits proliferating cell nuclear antigen by breaking the DNA clamp

    SciTech Connect

    Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo; Robinson, Howard; Sallman, Zahur F.; Marino, John P.; Kelman, Zvi

    2016-05-03

    Here, proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain a canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.

  11. External antigen uptake by Langerhans cells with reorganization of epidermal tight junction barriers.

    PubMed

    Kubo, Akiharu; Nagao, Keisuke; Yokouchi, Mariko; Sasaki, Hiroyuki; Amagai, Masayuki

    2009-12-21

    Outermost barriers are critical for terrestrial animals to avoid desiccation and to protect their bodies from foreign insults. Mammalian skin consists of two sets of barriers: stratum corneum (SC) and tight junctions (TJs). How acquisition of external antigens (Ags) by epidermal Langerhans cells (LCs) occur despite these barriers has remained unknown. We show that activation-induced LCs elongate their dendrites to penetrate keratinocyte (KC) TJs and survey the extra-TJ environment located outside of the TJ barrier, just beneath the SC. Penetrated dendrites uptake Ags from the tip where Ags colocalize with langerin/Birbeck granules. TJs at KC-KC contacts allow penetration of LC dendrites by dynamically forming new claudin-dependent bicellular- and tricellulin-dependent tricellular TJs at LC-KC contacts, thereby maintaining TJ integrity during Ag uptake. Thus, covertly under keratinized SC barriers, LCs and KCs demonstrate remarkable cooperation that enables LCs to gain access to external Ags that have violated the SC barrier while concomitantly retaining TJ barriers to protect intra-TJ environment.

  12. Epstein-Barr virus nuclear antigen 2 specifically induces expression of the B-cell activation antigen CD23

    SciTech Connect

    Wang, F.; Gregory, C.D.; Rowe, M.; Rickinson, A.B.; Wang, D.; Birkenbach, M.; Kikutani, H.; Kishimoto, T.; Kieff, E.

    1987-05-01

    Epstein-Barr virus (EBV) infection of EBV-negative Burkitt lymphoma (BL) cells includes some changes similar to those seen in normal B lymphocytes that have been growth transformed by EBV. The role of individual EBV genes in this process was evaluated by introducing each of the viral genes that are normally expressed in EBV growth-transformed and latently infected lymphoblasts into an EBV-negative BL cell line, using recombinant retrovirus-mediated transfer. Clones of cells were derived that stably express the EBV nuclear antigen 1 (EBNA-1), EBNA-2, EBNA-3, EBNA-leader protein, or EBV latent membrane protein (LMP). These were compared with control clones infected with the retrovirus vector. All 10 clones converted to EBNA-2 expression differed from control clones or clones expressing other EBV proteins by growth in tight clumps and by markedly increased expression of one particular surface marker of B-cell activation, CD23. Other activation antigens were unaffected by EBNA-2 expression, as were markers already expressed on the parent BL cell line. The results indicate that EBNA-2 is a specific direct or indirect trans-activator of CD23. This establishes a link between an EBV gene and cell gene expression. Since CD23 has been implicated in the transduction of B-cell growth signals, its specific induction by EBNA-2 could be important in EBV induction of B-lymphocyte transformation.

  13. Rapid desensitization induces internalization of antigen-specific IgE on mouse mast cells

    PubMed Central

    Oka, Tatsuya; Rios, Eon J.; Tsai, Mindy; Kalesnikoff, Janet; Galli, Stephen J.

    2013-01-01

    Background Rapid desensitization transiently prevents severe allergic reactions, allowing administration of life-saving therapies in previously sensitized patients. However, the mechanisms underlying successful rapid desensitization are not fully understood. Objectives We sought to investigate whether the mast cell (MC) is an important target of rapid desensitization in mice sensitized to exhibit IgE-dependent passive systemic anaphylaxis in vivo and to investigate the antigen specificity and underlying mechanisms of rapid desensitization in our mouse model. Methods C57BL/6 mice (in vivo) or primary isolated C57BL/6 mouse peritoneal mast cells (PMCs; in vitro) were passively sensitized with antigen-specific anti–2,4-dinitrophenyl IgE, anti-ovalbumin IgE, or both. MCs were exposed over a short period of time to increasing amounts of antigen (2,4-dinitrophenyl–human serum albumin or ovalbumin) in the presence of extracellular calcium in vitro or by means of intravenous administration to sensitized mice in vivo before challenging the mice with or exposing the PMCs to optimal amounts of specific or irrelevant antigen. Results Rapidly exposing mice or PMCs to progressively increasing amounts of specific antigen inhibited the development of antigen-induced hypothermia in sensitized mice in vivo and inhibited antigen-induced PMC degranulation and prostaglandin D2 synthesis in vitro. Such MC hyporesponsiveness was induced antigen-specifically and was associated with a significant reduction in antigen-specific IgE levels on MC surfaces. Conclusions Rapidly exposing MCs to progressively increasing amounts of antigen can both enhance the internalization of antigen-specific IgE on the MC surface and also desensitize these cells in an antigen-specific manner in vivo and in vitro. PMID:23810240

  14. Rapid desensitization induces internalization of antigen-specific IgE on mouse mast cells.

    PubMed

    Oka, Tatsuya; Rios, Eon J; Tsai, Mindy; Kalesnikoff, Janet; Galli, Stephen J

    2013-10-01

    Rapid desensitization transiently prevents severe allergic reactions, allowing administration of life-saving therapies in previously sensitized patients. However, the mechanisms underlying successful rapid desensitization are not fully understood. We sought to investigate whether the mast cell (MC) is an important target of rapid desensitization in mice sensitized to exhibit IgE-dependent passive systemic anaphylaxis in vivo and to investigate the antigen specificity and underlying mechanisms of rapid desensitization in our mouse model. C57BL/6 mice (in vivo) or primary isolated C57BL/6 mouse peritoneal mast cells (PMCs; in vitro) were passively sensitized with antigen-specific anti-2,4-dinitrophenyl IgE, anti-ovalbumin IgE, or both. MCs were exposed over a short period of time to increasing amounts of antigen (2,4-dinitrophenyl-human serum albumin or ovalbumin) in the presence of extracellular calcium in vitro or by means of intravenous administration to sensitized mice in vivo before challenging the mice with or exposing the PMCs to optimal amounts of specific or irrelevant antigen. Rapidly exposing mice or PMCs to progressively increasing amounts of specific antigen inhibited the development of antigen-induced hypothermia in sensitized mice in vivo and inhibited antigen-induced PMC degranulation and prostaglandin D2 synthesis in vitro. Such MC hyporesponsiveness was induced antigen-specifically and was associated with a significant reduction in antigen-specific IgE levels on MC surfaces. Rapidly exposing MCs to progressively increasing amounts of antigen can both enhance the internalization of antigen-specific IgE on the MC surface and also desensitize these cells in an antigen-specific manner in vivo and in vitro. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  15. Stabilization of Transfected Cells Expressing Low-Incidence Blood Group Antigens: Novel Methods Facilitating Their Use as Reagent-Cells

    PubMed Central

    González, Cecilia; Esteban, Rosa; Canals, Carme; Muñiz-Díaz, Eduardo; Nogués, Núria

    2016-01-01

    Background The identification of erythrocyte antibodies in the serum of patients rely on panels of human red blood cells (RBCs), which coexpress many antigens and are not easily available for low-incidence blood group phenotypes. These problems have been addressed by generating cell lines expressing unique blood group antigens, which may be used as an alternative to human RBCs. However, the use of cell lines implies several drawbacks, like the requirement of cell culture facilities and the high cost of cryopreservation. The application of cell stabilization methods could facilitate their use as reagent cells in clinical laboratories. Methods We generated stably-transfected cells expressing low-incidence blood group antigens (Dia and Lua). High-expresser clones were used to assess the effect of TransFix® treatment and lyophilization as cell preservation methods. Cells were kept at 4°C and cell morphology, membrane permeability and antigenic properties were evaluated at several time-points after treatment. Results TransFix® addition to cell suspensions allows cell stabilization and proper antigen detection for at least 120 days, despite an increase in membrane permeability and a reduction in antigen expression levels. Lyophilized cells showed minor morphological changes and antigen expression levels were rather conserved at days 1, 15 and 120, indicating a high stability of the freeze-dried product. These stabilized cells have been proved to react specifically with human sera containing alloantibodies. Conclusions Both stabilization methods allow long-term preservation of the transfected cells antigenic properties and may facilitate their distribution and use as reagent-cells expressing low-incidence antigens, overcoming the limited availability of such rare RBCs. PMID:27603310

  16. Stabilization of Transfected Cells Expressing Low-Incidence Blood Group Antigens: Novel Methods Facilitating Their Use as Reagent-Cells.

    PubMed

    González, Cecilia; Esteban, Rosa; Canals, Carme; Muñiz-Díaz, Eduardo; Nogués, Núria

    2016-01-01

    The identification of erythrocyte antibodies in the serum of patients rely on panels of human red blood cells (RBCs), which coexpress many antigens and are not easily available for low-incidence blood group phenotypes. These problems have been addressed by generating cell lines expressing unique blood group antigens, which may be used as an alternative to human RBCs. However, the use of cell lines implies several drawbacks, like the requirement of cell culture facilities and the high cost of cryopreservation. The application of cell stabilization methods could facilitate their use as reagent cells in clinical laboratories. We generated stably-transfected cells expressing low-incidence blood group antigens (Dia and Lua). High-expresser clones were used to assess the effect of TransFix® treatment and lyophilization as cell preservation methods. Cells were kept at 4°C and cell morphology, membrane permeability and antigenic properties were evaluated at several time-points after treatment. TransFix® addition to cell suspensions allows cell stabilization and proper antigen detection for at least 120 days, despite an increase in membrane permeability and a reduction in antigen expression levels. Lyophilized cells showed minor morphological changes and antigen expression levels were rather conserved at days 1, 15 and 120, indicating a high stability of the freeze-dried product. These stabilized cells have been proved to react specifically with human sera containing alloantibodies. Both stabilization methods allow long-term preservation of the transfected cells antigenic properties and may facilitate their distribution and use as reagent-cells expressing low-incidence antigens, overcoming the limited availability of such rare RBCs.

  17. Intestinal label-retaining cells are secretory precursors expressing Lgr5.

    PubMed

    Buczacki, Simon J A; Zecchini, Heather Ireland; Nicholson, Anna M; Russell, Roslin; Vermeulen, Louis; Kemp, Richard; Winton, Douglas J

    2013-03-07

    The rapid cell turnover of the intestinal epithelium is achieved from small numbers of stem cells located in the base of glandular crypts. These stem cells have been variously described as rapidly cycling or quiescent. A functional arrangement of stem cells that reconciles both of these behaviours has so far been difficult to obtain. Alternative explanations for quiescent cells have been that they act as a parallel or reserve population that replace rapidly cycling stem cells periodically or after injury; their exact nature remains unknown. Here we show mouse intestinal quiescent cells to be precursors that are committed to mature into differentiated secretory cells of the Paneth and enteroendocrine lineage. However, crucially we find that after intestinal injury they are capable of extensive proliferation and can give rise to clones comprising the main epithelial cell types. Thus, quiescent cells can be recalled to the stem-cell state. These findings establish quiescent cells as an effective clonogenic reserve and provide a motivation for investigating their role in pathologies such as colorectal cancers and intestinal inflammation.

  18. Characterization of an antigen associated with the Marek's disease lymphoblastoid cell line MSB-1.

    PubMed

    Ross, L J

    1982-06-01

    A Marek's disease lymphoblastoid cell line (MSB-1) has been analysed by immunoprecipitation for expression of tumour-associated antigen, Marek's disease virus (MDV)-specific antigens and antigens specific to avian leukosis-sarcoma viruses. Rabbit antisera raised against two independently derived cell lines after extensive absorption with normal chick cells reacted with a polypeptide of mol. wt. 40 000 (40K) in extracts of MSB-1 cells. The 40K polypeptide was not present in myeloblasts or in chick embryo fibroblasts (CEF) infected with MDV and did not react with antiserum raised against normal chicken thymus antigens. The possibility that the 40K polypeptide is a tumour-associated antigen is discussed. Seven MDV-specific antigens were noted in infected CEF (mol. wt. 110K, 100K, 80K, 70K, 50K, 35K and 32K) but none of these was detected in MSB-1 cells. The avian leukosis-sarcoma group-specific antigen P27gag and its precursor Pr76gag were not found in MSB-1 cells, confirming that expression of mature gag protein is not required for transformation by MDV. However, two polypeptides of unknown origin and function (mol. wt. 180K and 110K) were precipitated from MSB-1 cells with a rabbit anti-Rous sarcoma (Schmidt-Rupin, subgroup D) antiserum.

  19. MHC Class II tetramers and the pursuit of antigen-specific T cells: define, deviate, delete.

    PubMed

    Mallone, Roberto; Nepom, Gerald T

    2004-03-01

    Selective expansion and activation of a very small number of antigen-specific CD4(+) T cells is a remarkable and essential property of the adaptive immune response. Antigen-specific T cells were until recently identified only indirectly by functional assays, such as antigen-induced cytokine secretion and proliferation. The advent of MHC Class II tetramers has added a pivotal tool to our research armamentarium, allowing the definition of allo- and autoimmune responses in deeper detail. Rare antigen-specific CD4(+) cells can now be selectively identified, isolated and characterized. The same tetramer reagents also provide a new mean of stimulating T cells, more closely reproducing the MHC-peptide/TCR interaction. This property allows the use of tetramers to direct T cells toward the more desirable outcome, that is, activation (in malignancies and infectious diseases) or Th2/T regulatory cell deviation, anergy and deletion (in autoimmune diseases). These experimental approaches hold promise for diagnostic, prognostic and therapeutic applications.

  20. RNAi screen for kinases and phosphatases that play a role in antigen presentation by dendritic cells.

    PubMed

    Moita, Catarina F; Chora, Ângelo; Hacohen, Nir; Moita, Luis F

    2012-07-01

    Effective CD8(+) T-cell responses against tumor or microbial antigens that are not directly expressed in antigen-presenting cells (APCs) depend on the cross-presentation of these antigens on MHC class I in APCs. To identify signaling molecules that regulate cross-presentation, we used lentiviral-based RNA interference to test the roles of hundreds of kinases and phosphatases in this process. Our study uncovered eight previously unknown genes, consisting of one positive and seven negative regulators of antigen cross-presentation. Depletion of Acvr1c, a type I receptor for TGF-β family of signaling molecules, led to an increase in CD80 and CD86 co-stimulator surface expression and secreted IL-12 in mouse bone marrow-derived DCs, as well as antigen-specific T-cell proliferation.

  1. Cutting Edge: Murine NK Cells Degranulate and Retain Cytotoxic Function without Store-Operated Calcium Entry.

    PubMed

    Freund-Brown, Jacquelyn; Choa, Ruth; Singh, Brenal K; Robertson, Tanner Ford; Ferry, Gabrielle M; Viver, Eric; Bassiri, Hamid; Burkhardt, Janis K; Kambayashi, Taku

    2017-08-09

    Sustained Ca(2+) signaling, known as store-operated calcium entry (SOCE), occurs downstream of immunoreceptor engagement and is critical for cytotoxic lymphocyte signaling and effector function. CD8(+) T cells require sustained Ca(2+) signaling for inflammatory cytokine production and the killing of target cells; however, much less is known about its role in NK cells. In this study, we use mice deficient in stromal interacting molecules 1 and 2, which are required for SOCE, to examine the contribution of sustained Ca(2+) signaling to murine NK cell function. Surprisingly, we found that, although SOCE is required for NK cell IFN-γ production in an NFAT-dependent manner, NK cell degranulation/cytotoxicity and tumor rejection in vivo remained intact in the absence of sustained Ca(2+) signaling. Our data suggest that mouse NK cells use different signaling mechanisms for cytotoxicity compared with other cytotoxic lymphocytes. Copyright © 2017 by The American Association of Immunologists, Inc.

  2. HIV Nef expression favors the relative preservation of CD4+ T regulatory cells that retain some important suppressive functions.

    PubMed

    Chrobak, Pavel; Afkhami, Soheila; Priceputu, Elena; Poudrier, Johanne; Meunier, Clémence; Hanna, Zaher; Sparwasser, Tim; Jolicoeur, Paul

    2014-02-15

    HIV-1 infection causes depletion and/or dysfunction of distinct CD4(+) T cell subsets and may affect these differently. Using the CD4C/HIV-1(Nef) transgenic (Tg) mice as a model, we report that HIV-1 Nef causes depletion of total CD4(+) T cells, but preserves and relatively enriches CD4(+) regulatory T cells (Treg). We found that Nef-mediated CD4(+) Treg enrichment is the direct result of Nef expression in CD4(+) T cells, occurs independently of Nef-induced lymphopenia, and most likely results from multiple mechanisms: lower apoptosis, enhanced cell division, and increased generation from precursors. Interestingly, Tg Treg relative enrichment could be reversed by enhancing Lck activity. Most importantly, we show that, in contrast to Tg helper CD4(+) T cells that have lost their function, Nef-expressing CD4(+) Treg retain their regulatory function in vitro and also in vivo, under some settings. In particular, we found that Treg prevent expansion of Tg B and non-Treg T cells in vivo. Our study reveals that Nef affects distinct CD4(+) T cell subsets differently and uncovers the high proliferative potential of B and non-Treg T cells in this mouse model.

  3. A DEPRESSION OF CELL-MEDIATED IMMUNITY TO MEASLES ANTIGEN IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS

    PubMed Central

    Utermohlen, Virginia; Winfield, John B.; Zabriskie, John B.; Kunkel, Henry G.

    1974-01-01

    Using the direct migration inhibition test, response to measles antigen in patients with systemic lupus erythematosus (SLE) was found to be decreased when compared with that of normal subjects. No alteration was observed in similar experiments using parainfluenza type 1 and rubella antigens. The specific decrease in measles antigen effect showed no obvious correlation with activity of SLE or with the presence of lymphocytotoxic antibodies. Whether the specificity of the decrease in reactivity is due to some particular relationship between the measles virus or antigen and SLE, or to the possibility that measles reactivity is a more sensitive indicator of a generalized defect of cell-mediated immunity, remains unclear. PMID:4361242

  4. Dendritic Cells in the Periphery Control Antigen-Specific Natural and Induced Regulatory T Cells

    PubMed Central

    Yamazaki, Sayuri; Morita, Akimichi

    2013-01-01

    Dendritic cells (DCs) are specialized antigen-presenting cells that regulate both immunity and tolerance. DCs in the periphery play a key role in expanding naturally occurring Foxp3+ CD25+ CD4+ regulatory T cells (Natural T-regs) and inducing Foxp3 expression (Induced T-regs) in Foxp3− CD4+ T cells. DCs are phenotypically and functionally heterogeneous, and further classified into several subsets depending on distinct marker expression and their location. Recent findings indicate the presence of specialized DC subsets that act to expand Natural T-regs or induce Foxp3+ T-regs from Foxp3− CD4+ T cells. For example, two major subsets of DCs in lymphoid organs act differentially in inducing Foxp3+ T-regs from Foxp3− cells or expanding Natural T-regs with model-antigen delivery by anti-DC subset monoclonal antibodies in vivo. Furthermore, DCs expressing CD103 in the intestine induce Foxp3+ T-regs from Foxp3− CD4+ T cells with endogenous TGF-β and retinoic acid. In addition, antigen-presenting DCs have a capacity to generate Foxp3+ T-regs in the oral cavity where many antigens and commensals exist, similar to intestine and skin. In skin and skin-draining lymph nodes, at least six DC subsets have been identified, suggesting a complex DC-T-reg network. Here, we will review the specific activity of DCs in expanding Natural T-regs and inducing Foxp3+ T-regs from Foxp3− precursors, and further discuss the critical function of DCs in maintaining tolerance at various locations including skin and oral cavity. PMID:23801989

  5. Increased sensitivity of antigen-experienced T cells through the enrichment of oligomeric T cell receptor complexes.

    PubMed

    Kumar, Rashmi; Ferez, María; Swamy, Mahima; Arechaga, Ignacio; Rejas, María Teresa; Valpuesta, Jose M; Schamel, Wolfgang W A; Alarcon, Balbino; van Santen, Hisse M

    2011-09-23

    Although memory T cells respond more vigorously to stimulation and they are more sensitive to low doses of antigen than naive T cells, the molecular basis of this increased sensitivity remains unclear. We have previously shown that the T cell receptor (TCR) exists as different-sized oligomers on the surface of resting T cells and that large oligomers are preferentially activated in response to low antigen doses. Through biochemistry and electron microscopy, we now showed that previously stimulated and memory T cells have more and larger TCR oligomers at the cell surface than their naive counterparts. Reconstitution of cells and mice with a point mutant of the CD3ζ subunit, which impairs TCR oligomer formation, demonstrated that the increased size of TCR oligomers was directly responsible for the increased sensitivity of antigen-experienced T cells. Thus, we propose that an "avidity maturation" mechanism underlies T cell antigenic memory.

  6. Inclusion of Strep-Tag II in design of antigen receptors for T cell immunotherapy

    PubMed Central

    Liu, Lingfeng; Sommermeyer, Daniel; Cabanov, Alexandra; Kosasih, Paula; Hill, Tyler; Riddell, Stanley R

    2016-01-01

    The tactical introduction of Strep-tag II into synthetic antigen receptors provides engineered T cells with a marker for identification and rapid purification, and a functional element for selective antibody coated microbead-driven large-scale expansion. Such receptor designs can be applied to chimeric antigen receptors of different ligand specificities and costimulatory domains, and to T cell receptors to facilitate cGMP manufacturing of adoptive T cell therapies to treat cancer and other diseases. PMID:26900664

  7. Murine epidermal antigen-presenting cells in primary and secondary T-cell proliferative responses to a soluble protein antigen in vitro.

    PubMed Central

    Williams, N A; Hill, T J; Hooper, D C

    1990-01-01

    The capacity of epidermal cells (EC) to present antigen to primed and non-immune T cells was investigated using a culture system that supports antigen-specific primary and secondary proliferative responses. Although both naive and bovine serum albumin (BSA)-immune T cells reacted against BSA in the presence of either splenic or epidermal antigen-presenting cells (APC), important differences were noted in the kinetics and the magnitudes of the various responses. Most conspicuous was the relatively poor primary response supported by EC which evidently elicited very few BSA-immune T-helper cells. Despite this, the primed antigen-specific T cells recovered were phenotypically similar to those resulting from the stronger primary responses induced by spleen cells. In contrast to this disparity in the ability to prime, EC and spleen cells stimulated secondary reactions of comparable magnitude. We therefore consider that, in comparison with splenic APC, EC may require some additional stimulus to acquire the capacity to prime. PMID:2148541

  8. Monoclonal antibodies to mouse cell-surface antigens.

    PubMed

    Alaverdi, Noosheen

    2002-05-01

    The cluster of differentiation (CD) nomenclature, originally devised by the International Workshop on Human Leukocyte Differentiation Antigens (HLDA) to classify leukocyte surface antigens, has become more accepted by researchers in nonhuman animal models. The CD homologues from other species are usually identified by multiple studies, including biochemical, functional analysis, cloning, and immunological assays. This unit compiles a complete (as of August, 2001) list of monoclonal antibodies (mAb) to the mouse CD antigens and several non-CD surface antigens that are commonly used as phenotypic and functional markers in the mouse system. There are many reagents recognizing polymorphic epitopes of the mouse MHC and different subunits of TCR; for simplicity, only a few mAbs to the framework determinants of these molecules are listed here.

  9. Spreading the load: antigen transfer between migratory and lymph node-resident dendritic cells promotes T cell priming.

    PubMed

    Mueller, Scott N

    2017-08-28

    Dendritic cells (DC) are specialized in the processing and presentation of antigen for the activation of lymphocytes. Multiple subsets of DCs exist with distinct functions and roles in the initiation of immune responses. DCs found within tissues acquire antigens or become infected by pathogens and migrate to local draining lymph nodes (LN) where they can directly stimulate T cells. These migratory DCs can also transfer antigens to LN-resident DCs and may indirectly enhance T cell priming. In this issue of the European Journal of Immunology, Gurevich et al. [Eur. J. Immunol. 2017. 47: XXXX-XXXX] elegantly demonstrate the influence of the transfer of antigen from migratory DCs to resident DCs on the dynamics of CD8 T cell priming in mice. Using both in vitro imaging to visualise antigen dissemination and intravital 2-photon microscopy to track T cell clustering with migratory and resident DCs, antigen-donor DC were found to efficiently distribute antigen to recipient DC. This process, which involved LFA-1, enhanced the recruitment of CD8(+) T cells into the response and rescued priming when DCs were impaired in presentation capacity. Together, these findings shed light on the dynamics of the transfer of antigens between DCs in vivo for the efficient priming of cytotoxic T cell responses. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  10. Antigen-specific induction of peripheral T cell tolerance in vivo by codelivery of DNA vectors encoding antigen and Fas ligand.

    PubMed

    Georgantas, R W; Leong, K W; August, J T

    2000-04-10

    Fas ligand (FasL, CD95L) induces apoptosis in activated T cells with upregulated Fas (CD95) expression through the process termed activation-induced cell death (AICD). We postulated that coexpression of antigen and FasL within individual antigen-presenting cells would lead to antigen-specific activation of T cells and to their consequent deletion by FasL-mediated AICD. A DNA-gelatin coacervate containing transferrin cell ligand, calcium, and the lysosomatropic agent chloroquine, a formulation previously shown to achieve high-level transfection of immune and muscle cells in vivo, was used to codeliver plasmids encoding FasL and antigen. Mice developed a strong cytolytic T cell response to beta-Gal when injected with DNA encoding beta-galactosidase (LacZ) model antigen, either as naked DNA or DNA nanoparticles, but failed to respond when there was concomitant injection of nanoparticles containing both the LacZ and murine FasL DNA vectors. This loss of T cell response was systemic, specific for beta-Gal, complete when nanoparticles were administered before antigen challenge, and decreased the T cell response from prior immunization with LacZ DNA. In effect, this "tolerization" injection induced antigen-specific peripheral tolerance in study mice, and represents a possible approach to the treatment of autoimmune diseases and transplantation rejection.

  11. Human epidermal Langerhans cells cointernalize by receptor-mediated endocytosis "nonclassical" major histocompatibility complex class I molecules (T6 antigens) and class II molecules (HLA-DR antigens).

    PubMed Central

    Hanau, D; Fabre, M; Schmitt, D A; Garaud, J C; Pauly, G; Tongio, M M; Mayer, S; Cazenave, J P

    1987-01-01

    HLA-DR and T6 surface antigens are expressed only by Langerhans cells and indeterminate cells in normal human epidermis. We have previously demonstrated that T6 antigens are internalized in Langerhans cells and indeterminate cells by receptor-mediated endocytosis. This process is induced by the binding of BL6, a monoclonal antibody directed against T6 antigens. In the present study, using a monoclonal antibody directed against HLA-DR antigens, on human epidermal cells in suspension, we show that the surface HLA-DR antigens are also internalized by receptor-mediated endocytosis in Langerhans and indeterminate cells. Moreover, using immunogold double labeling, we demonstrate that T6 and HLA-DR antigens are internalized through common coated regions of the membrane of Langerhans or indeterminate cells. The receptor-mediated endocytosis that is induced involves coated pits and vesicles, receptosomes, lysosomes, and also, in Langerhans cells, the Birbeck granules. Thus, T6 antigens, which are considered to be "unusual" or "nonclassical" major histocompatibility complex class I molecules, and the major histocompatibility complex class II molecules, HLA-DR, are internalized in Langerhans and indeterminate cells through common receptor-mediated endocytosis organelles. Images PMID:3106979

  12. Red Blood Cell Antigen Genotyping for Sickle Cell Disease, Thalassemia, and Other Transfusion Complications.

    PubMed

    Fasano, Ross M; Chou, Stella T

    2016-10-01

    Since the discovery of the ABO blood group in the early 20th century, more than 300 blood group antigens have been categorized among 35 blood group systems. The molecular basis for most blood group antigens has been determined and demonstrates tremendous genetic diversity, particularly in the ABO and Rh systems. Several blood group genotyping assays have been developed, and 1 platform has been approved by the Food and Drug Administration as a "test of record," such that no phenotype confirmation with antisera is required. DNA-based red blood cell (RBC) phenotyping can overcome certain limitations of hemagglutination assays and is beneficial in many transfusion settings. Genotyping can be used to determine RBC antigen phenotypes in patients recently transfused or with interfering allo- or autoantibodies, to resolve discrepant serologic typing, and/or when typing antisera are not readily available. Molecular RBC antigen typing can facilitate complex antibody evaluations and guide RBC selection for patients with sickle cell disease (SCD), thalassemia, and autoimmune hemolytic anemia. High-resolution RH genotyping can identify variant RHD and RHCE in patients with SCD, which have been associated with alloimmunization. In the future, broader access to cost-efficient, high-resolution RBC genotyping technology for both patient and donor populations may be transformative for the field of transfusion medicine.

  13. Surface Antigen Profiling of Helicobacter pylori-Infected and -Uninfected Gastric Cancer Cells Using Antibody Microarray.

    PubMed

    Sukri, Asif; Hanafiah, Alfizah; Kosai, Nik Ritza; Mohamed Taher, Mustafa; Mohamed Rose, Isa

    2016-10-01

    Comprehensive immunophenotyping cluster of differentiation (CD) antigens in gastric adenocarcinoma, specifically between Helicobacter pylori-infected and -uninfected gastric cancer patients by using DotScan(™) antibody microarray has not been conducted. Current immunophenotyping techniques include flow cytometry and immunohistochemistry are limited to the use of few antibodies for parallel examination. We used DotScan(™) antibody microarray consisting 144 CD antibodies to determine the distribution of CD antigens in gastric adenocarcinoma cells and to elucidate the effect of H. pylori infection toward CD antigen expression in gastric cancer. Mixed leukocytes population derived from gastric adenocarcinoma patients were immunophenotyped using DotScan(™) antibody microarray. AGS cells were infected with H. pylori strains and cells were captured on DotScan(™) slides. Cluster of differentiation antigens involved in perpetuating the tolerance of immune cells to tumor cells was upregulated in gastric adenocarcinoma cells compared to normal cells. CD279 which is essential in T cells apoptosis was found to be upregulated in normal cells. Remarkably, H. pylori-infected gastric cancer patients exhibited upregulated expression of CD27 that important in maintenance of T cells. Infection of cagA+ H. pylori with AGS cells increased CD antigens expression which involved in cancer stem cell while cagA- H. pylori polarized AGS cells to express immune-regulatory CD antigens. Increased CD antigens expression in AGS cells infected with cagA+ H. pylori were also detected in H. pylori-infected gastric cancer patients. This study suggests the tolerance of immune system toward tumor cells in gastric cancer and distinct mechanisms of immune responses exploited by different H. pylori strains. © 2016 John Wiley & Sons Ltd.

  14. Antigen availability determines CD8+ T cell-dendritic cell interaction kinetics and memory fate decisions

    PubMed Central

    Henrickson, Sarah E.; Stutte, Susanne; Quigley, Michael; Alexe, Gabriela; Iannacone, Matteo; Flynn, Michael P.; Omid, Shaida; Jesneck, Jonathan L.; Imam, Sabrina; Mempel, Thorsten R.; Mazo, Irina B.; Haining, William N.; von Andrian, Ulrich H.

    2014-01-01

    Summary T cells are activated by antigen (Ag) bearing dendritic cells (DCs) in lymph nodes in 3 phases. The duration of the initial phase of transient, serial DC-T cell interactions is inversely correlated with Ag dose. The second phase, characterized by stable DC-T cell contacts, is believed to be necessary for full-fledged T cell activation. Here we have shown that this is not the case. CD8+ T cells interacting with DCs presenting low-dose, short-lived Ag did not transition to phase 2, while higher Ag dose yielded phase 2 transition. Both antigenic constellations promoted T cell proliferation and effector differentiation, but yielded different transcriptome signatures at 12h and 24h. T cells that experienced phase 2 developed long-lived memory, whereas conditions without stable contacts yielded immunological amnesia. Thus, T cells make fate decisions within hours after Ag exposure resulting in long-term memory or abortive effector responses, correlating with T cell-DCs interaction kinetics. PMID:24054328

  15. 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.

  16. Selective culling of high avidity antigen-specific CD4+ T cells after virulent Salmonella infection.

    PubMed

    Ertelt, James M; Johanns, Tanner M; Mysz, Margaret A; Nanton, Minelva R; Rowe, Jared H; Aguilera, Marijo N; Way, Sing Sing

    2011-12-01

    Typhoid fever is a persistent infection caused by host-adapted Salmonella strains adept at circumventing immune-mediated host defences. Given the importance of T cells in protection, the culling of activated CD4+ T cells after primary infection has been proposed as a potential immune evasion strategy used by this pathogen. We demonstrate that the purging of activated antigen-specific CD4+ T cells after virulent Salmonella infection requires SPI-2 encoded virulence determinants, and is not restricted only to cells with specificity to Salmonella-expressed antigens, but extends to CD4+ T cells primed to expand by co-infection with recombinant Listeria monocytogenes. Unexpectedly, however, the loss of activated CD4+ T cells during Salmonella infection demonstrated using a monoclonal population of adoptively transferred CD4+ T cells was not reproduced among the endogenous repertoire of antigen-specific CD4+ T cells identified with MHC class II tetramer. Analysis of T-cell receptor variable segment usage revealed the selective loss and reciprocal enrichment of defined CD4+ T-cell subsets after Salmonella co-infection that is associated with the purging of antigen-specific cells with the highest intensity of tetramer staining. Hence, virulent Salmonella triggers the selective culling of high avidity activated CD4+ T-cell subsets, which re-shapes the repertoire of antigen-specific T cells that persist later after infection.

  17. Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

    SciTech Connect

    Kleinhenz, M.E.; Ellner, J.J.

    1985-07-01

    In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the (/sup 3/H)thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced (/sup 3/H)thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% (SD)) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition.

  18. Chimeric antigen receptor T-cell neuropsychiatric toxicity in acute lymphoblastic leukemia.

    PubMed

    Prudent, Vasthie; Breitbart, William S

    2017-01-04

    Chimeric antigen receptor T cells are used in the treatment of B-cell leukemias. Common chimeric antigen receptor T-cell toxicities can range from mild flu-like symptoms, such as fever and myalgia, to a more striking neuropsychiatric toxicity that can present as discrete neurological symptoms and delirium. We report here two cases of chimeric antigen receptor T-cell neuropsychiatric toxicity, one who presented as a mild delirium and aphasia that resolved without intervention, and one who presented with delirium, seizures, and respiratory insufficiency requiring intensive treatment. The current literature on the treatment and proposed mechanisms of this clinically challenging chimeric antigen receptor T-cell complication is also presented.

  19. Skin-Resident Antigen-Presenting Cells: Instruction Manual for Vaccine Development

    PubMed Central

    Fehres, Cynthia M.; Garcia-Vallejo, Juan J.; Unger, Wendy W. J.; van Kooyk, Yvette

    2013-01-01

    The induction of antigen-specific effector T cells is driven by proper antigen presentation and co-stimulation by dendritic cells (DCs). For this reason strategies have been developed to instruct DCs for the induction of CD4+ and CD8+ T cell responses. Since DCs are localized, amongst other locations, in peripheral tissues such as the skin, new vaccines are aiming at targeting antigens to DCs in situ. Optimal skin-DC targeting in combination with adequate adjuvant delivery facilitates DC maturation and migration to draining lymph nodes and enhances antigen cross-presentation and T cell priming. In this review we describe what DC subsets populate the human skin, as well as current vaccination strategies based on targeting strategies and alternative administration for the induction of robust long-lived anti-cancer effector T cells. PMID:23801994

  20. Cancer/testis antigens can be immunological targets in clonogenic CD133+ melanoma cells.

    PubMed

    Gedye, Craig; Quirk, Juliet; Browning, Judy; Svobodová, Suzanne; John, Thomas; Sluka, Pavel; Dunbar, P Rod; Corbeil, Denis; Cebon, Jonathan; Davis, Ian D

    2009-10-01

    "Cancer stem cells" that resist conventional treatments may be a cause of therapeutic failure in melanoma. We report a subpopulation of clonogenic melanoma cells that are characterized by high prominin-1/CD133 expression in melanoma and melanoma cell lines. These cells have enhanced clonogenicity and self-renewal in vitro, and serve as a limited in vitro model for melanoma stem cells. In some cases clonogenic CD133(+) melanoma cells show increased expression of some cancer/testis (CT) antigens. The expression of NY-ESO-1 in an HLA-A2 expressing cell line allowed CD133(+) clonogenic melanoma cells to be targeted for killing in vitro by NY-ESO-1-specific CD8(+) T-lymphocytes. Our in vitro findings raise the hypothesis that if melanoma stem cells express CT antigens in vivo that immune targeting of these antigens may be a viable clinical strategy for the adjuvant treatment of melanoma.

  1. CD8+NKT-like cells regulate the immune response by killing antigen-bearing DCs

    PubMed Central

    Wang, Chao; Liu, Xi; Li, Zhengyuan; Chai, Yijie; Jiang, Yunfeng; Wang, Qian; Ji, Yewei; Zhu, Zhongli; Wan, Ying; Yuan, Zhenglong; Chang, Zhijie; Zhang, Minghui

    2015-01-01

    CD1d-dependent NKT cells have been extensively studied; however, the function of CD8+NKT-like cells, which are CD1d-independent T cells with NK markers, remains unknown. Here, we report that CD1d-independent CD8+NKT-like cells, which express both T cell markers (TCRβ and CD3) and NK cell receptors (NK1.1, CD49b and NKG2D), are activated and significantly expanded in mice immunized with GFP-expressing dendritic cells. Distinct from CD1d-dependent NKT cells, CD8+NKT-like cells possess a diverse repertoire of TCRs and secrete high levels of IFN-gamma but not IL-4. CD8+NKT-like cell development is normal in CD1d−/− mice, which suggests that CD8+NKT-like cells undergo a unique development pathway that differs from iNKT cells. Further functional analyses show that CD8+NKT-like cells suppress T-cell responses through elimination of dendritic cells in an antigen-specific manner. Adoptive transfer of antigen-specific CD8+NKT-like cells into RIP-OVA mice prevented subsequent development of diabetes in the animals induced by activated OT-I CD8 T cells. Our study suggests that CD8+NKT-like cells can function as antigen-specific suppressive cells to regulate the immune response through killing antigen-bearing DCs. Antigen-specific down regulation may provide an active and precise method for constraining an excessive immune response and avoiding bypass suppression of necessary immune responses to other antigens. PMID:26369936

  2. Chitosan Feasibility to Retain Retinal Stem Cell Phenotype and Slow Proliferation for Retinal Transplantation

    PubMed Central

    Srivastava, Girish K.; Rodriguez-Crespo, David; Singh, Amar K.; Casado-Coterillo, Clara; Garcia-Gutierrez, Maria T.; Coronas, Joaquin; Pastor, J. Carlos

    2014-01-01

    Retinal stem cells (RSCs) are promising in cell replacement strategies for retinal diseases. RSCs can migrate, differentiate, and integrate into retina. However, RSCs transplantation needs an adequate support; chitosan membrane (ChM) could be one, which can carry RSCs with high feasibility to support their integration into retina. RSCs were isolated, evaluated for phenotype, and subsequently grown on sterilized ChM and polystyrene surface for 8 hours, 1, 4, and 11 days for analysing cell adhesion, proliferation, viability, and phenotype. Isolated RSCs expressed GFAP, PKC, isolectin, recoverin, RPE65, PAX-6, cytokeratin 8/18, and nestin proteins. They adhered (28 ± 16%, 8 hours) and proliferated (40 ± 20 cells/field, day 1 and 244 ± 100 cells/field, day 4) significantly low (P < 0.05) on ChM. However, they maintained similar viability (>95%) and phenotype (cytokeratin 8/18, PAX6, and nestin proteins expression, day 11) on both surfaces (ChM and polystyrene). RSCs did not express alpha-SMA protein on both surfaces. RSCs express proteins belonging to epithelial, glial, and neural cells, confirming that they need further stimulus to reach a final destination of differentiation that could be provided in in vivo condition. ChM does not alternate RSCs behaviour and therefore can be used as a cell carrier so that slow proliferating RSCs can migrate and integrate into retina. PMID:24719852

  3. ɣδ T cell receptor ligands and modes of antigen recognition

    PubMed Central

    Champagne, Eric

    2011-01-01

    T lymphocytes expressing the γδ-type of T cell receptors for antigens contribute to all aspects of immune responses, including defenses against viruses, bacteria, parasites and tumors, allergy and autoimmunity. Multiple subsets have been individualized in humans as well as in mice and they appear to recognize in a TCR-dependent manner antigens as diverse as small non-peptidic molecules, soluble or membrane-anchored polypeptides and molecules related to MHC antigens on cell surfaces, implying diverse modes of antigen recognition. We review here the γδ TCR ligands which have been identified along the years and their characteristics, with emphasis on a few systems which have been extensively studied such as human γδ T cells responding to phosphoantigens or murine γδ T cells activated by allogeneic MHC antigens. We discuss a speculative model of antigen recognition involving simultaneous TCR recognition of MHC-like and non-MHC ligands which could fit with most available data and shares many similarities with the classical model of MHC-restricted antigen recognition for peptides or lipids by T cells subsets with αβ-type TCRs. PMID:21298486

  4. Identification of Human T Cell Antigens for the Development of Vaccines Against Mycobacterium Tuberculosis

    PubMed Central

    Bertholet, Sylvie; Ireton, Gregory C; Kahn, Maria; Guderian, Jeffrey; Mohamath, Raodoh; Stride, Nicole; Laughlin, Elsa M.; Baldwin, Susan L.; Vedvick, Thomas S.; Coler, Rhea N.; Reed, Steven G.

    2008-01-01

    Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) depends on the identification of antigens that induce appropriate T cell responses. Using bioinformatics, we selected a panel of 94 Mtb genes based on criteria which included growth in macrophages, up- or down-regulation under hypoxic conditions, secretion, membrane association, or because they were members of the PE/PPE or EsX families. Recombinant proteins encoded by these genes were evaluated for IFN-γ recall responses using PBMC from healthy subjects previously exposed to Mtb. From this screen, dominant human T-cell antigens were identified and 49 of these proteins, formulated in CpG, were evaluated as vaccine candidates in a mouse model of tuberculosis (TB). Eighteen of the individual antigens conferred partial protection against challenge with virulent Mtb. A combination of three of these antigens further increased protection against Mtb to levels comparable to those achieved with BCG vaccination. Vaccine candidates that led to reduction in lung bacterial burden following challenge induced pluripotent CD4 and CD8 T cells, including TH1 cell responses characterized by elevated levels of antigen-specific IgG2c, IFN-γ and TNF. Priority vaccine antigens elicited pluripotent CD4 and CD8 T responses in PPD+ donor PBMC. This study identified numerous novel human T cell antigens suitable to be included in subunit vaccines against TB. PMID:19017986

  5. A human T cell clone that mediates the monocyte procoagulant response to specific sensitizing antigen.

    PubMed

    Schwartz, B S; Reitnauer, P J; Hank, J A; Sondel, P M

    1985-09-01

    A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its own, but upon being presented with PPD was able to instruct monocytes to increase their expression of PCA. Antigen presentation could be performed only by autologous monocytes; allogeneic monocytes from donors unrelated to the donor of the reactive clone could not present antigen to cells of the clone in a way that would initiate the procoagulant response. Cells of the reactive clone did not mediate increased monocyte PCA in response to Candida, even though peripheral blood mononuclear cells from the donor demonstrated increased PCA to both Candida and PPD. Thus, the PCA response to specific antigen can be mediated by a single clone of cells that shows specificity in the recognition of both antigen and antigen presenting cell.

  6. Focused specificity of intestinal TH17 cells towards commensal bacterial antigens.

    PubMed

    Yang, Yi; Torchinsky, Miriam B; Gobert, Michael; Xiong, Huizhong; Xu, Mo; Linehan, Jonathan L; Alonzo, Francis; Ng, Charles; Chen, Alessandra; Lin, Xiyao; Sczesnak, Andrew; Liao, Jia-Jun; Torres, Victor J; Jenkins, Marc K; Lafaille, Juan J; Littman, Dan R

    2014-06-05

    T-helper-17 (TH17) cells have critical roles in mucosal defence and in autoimmune disease pathogenesis. They are most abundant in the small intestine lamina propria, where their presence requires colonization of mice with microbiota. Segmented filamentous bacteria (SFB) are sufficient to induce TH17 cells and to promote TH17-dependent autoimmune disease in animal models. However, the specificity of TH17 cells, the mechanism of their induction by distinct bacteria, and the means by which they foster tissue-specific inflammation remain unknown. Here we show that the T-cell antigen receptor (TCR) repertoire of intestinal TH17 cells in SFB-colonized mice has minimal overlap with that of other intestinal CD4(+) T cells and that most TH17 cells, but not other T cells, recognize antigens encoded by SFB. T cells with antigen receptors specific for SFB-encoded peptides differentiated into RORγt-expressing TH17 cells, even if SFB-colonized mice also harboured a strong TH1 cell inducer, Listeria monocytogenes, in their intestine. The match of T-cell effector function with antigen specificity is thus determined by the type of bacteria that produce the antigen. These findings have significant implications for understanding how commensal microbiota contribute to organ-specific autoimmunity and for developing novel mucosal vaccines.

  7. Identification of a cell-surface antigen selectively expressed on the natural killer cell

    PubMed Central

    1977-01-01

    We have studied the cell-surface phenotype of natural killer (NK) cells of NZB and B6 mice which react to an MuLV+ lymphoid tumor. (a) NK cells do not express Thy1, Ly2, or Ig surface markers. (b) NK cells express an antigen recognized by C3H anti-CE antiserum ('anti-Ly1.2 antiserum'). Inasmuch as NK activity of spleen cells from B6 and B6/Ly1.1 congenic strains were both equally sensitive to C3H anti-CE antiserum, the NK antigen is distinct from Ly1.2. This point was confirmed by the observation that alphaNK activity was removed by absorption of C3H anti-CE antiserum with spleen cells from either B6 or B6/Ly1.1 congenic strains. Absorption of C3H alphaCE serum with BALB/c thymocytes and spleen cells (which are Ly1.2+NK-) removed anti-Ly1.2 activity and left anti-NK activity intact. This absorption step could be circumvented by inserting the BALB/c genotype into the recipient immunized to CE cells (i.e., (C3H X BALB/c)F1 alphaCE spleen cells). This antiserum, provisionally termed 'anti-NK', defines a new subclass of lymphocytes which may play a central role in the immunosurveillance against tumors. PMID:187714

  8. Development of an enzyme-linked immunosorbent assay for studying Vibrio cholerae cell surface antigens.

    PubMed Central

    Cryz, S J; Fürer, E; Germanier, R

    1982-01-01

    An enzyme-linked immunosorbent assay for the measurement of antibodies directed against cell surface antigens of Vibrio cholerae (CSA ELISA) was developed. NaN3-killed whole cells of V. cholerae, adsorbed to polystyrene tubes, were used as immobilized antigens. The assay was capable of detecting antibodies directed against lipopolysaccharide and non-lipopolysaccharide surface antigens. In addition, the CSA ELISA was capable of detecting non-vibriocidal antibody. An antiserum raised in rabbits by immunization with live V. cholerae 1418 (Ogawa, El Tor) was capable of reacting with various heterologous strains of V. cholerae used as immobilized antigens. Therefore, common antigens shared by V. cholerae strains could be detected by using the CSA ELISA. PMID:7107860

  9. Low molecular weight antigen arrays delete high affinity memory B cells without affecting specific T-cell help.

    PubMed

    Reim, J W; Symer, D E; Watson, D C; Dintzis, R Z; Dintzis, H M

    1996-12-01

    An ongoing, T-cell dependent, secondary antibody response to an epitope can be suppressed in vivo by low molecular weight, soluble polymers, bearing multiple copies of the same epitope. This study illustrates that such suppressive T-cell independent antigen arrays target the epitope-specific, high affinity, memory B cells for long-term functional elimination. Splenocytes from hyperimmune unsuppressed donors, when adoptively transferred into irradiated recipients will readily reconstitute a secondary anti-hapten response after antigenic challenge. No such response was observed with splenocytes transferred from hyperimmune donors suppressed with antigen arrays. The extent of suppression depended on antigen array dose and duration of exposure in the donor animals. The suppressive antigen array carryover from the donors into the recipients was negligible and insufficient to account for the observed suppression. B cells from hyperimmune mice producing high affinity anti-fluorescein antibodies, generated by multiple fluoresceinated ovalbumin (FL-OVA) injections, were helped efficiently by T cells from hyperimmune donors, which were either unsuppressed or suppressed with antigen arrays. Accordingly, help from T cells, specific for the carrier protein remains intact after such suppression. Neither lipopolysaccharide (LPS), nor additional transferred carrier-primed T cells could reverse the unresponsiveness of adoptively transferred splenocytes from suppressed animals. Flow cytometry showed that the number of hapten-specific B cells was markedly reduced after suppression. Collectively, these data show that the long term elimination of an ongoing T-cell dependent antibody response by suppressive exogenous antigen arrays is due to the functional deletion of high affinity, antigen-specific B cells, even in the presence of adequate T-cell help. The long-term nature of such functional deletion strongly suggests physical deletion of the antigen-specific B cell population.

  10. Stage-specific embryonic antigen-4 identifies human dental pulp stem cells.

    PubMed

    Kawanabe, Noriaki; Murata, Satoko; Fukushima, Hiroaki; Ishihara, Yoshihito; Yanagita, Takeshi; Yanagita, Emmy; Ono, Mitsuaki; Kurosaka, Hiroshi; Kamioka, Hiroshi; Itoh, Tomoo; Kuboki, Takuo; Yamashiro, Takashi

    2012-03-10

    Embryonic stem cell-associated antigens are expressed in a variety of adult stem cells as well as embryonic stem cells. In the present study, we investigated whether stage-specific embryonic antigen (SSEA)-4 can be used to isolate dental pulp (DP) stem cells. DP cells showed plastic adherence, specific surface antigen expression, and multipotent differentiation potential, similar to mesenchymal stem cells (MSC). SSEA-4+ cells were found in cultured DP cells in vitro as well as in DP tissue in vivo. Flow cytometric analysis demonstrated that 45.5% of the DP cells were SSEA-4+. When the DP cells were cultured in the presence of all-trans-retinoic acid, marked downregulation of SSEA-3 and SSEA-4 and the upregulation of SSEA-1 were observed. SSEA-4+ DP cells showed a greater telomere length and a higher growth rate compared to ungated and SSEA-4- cells. A clonal assay demonstrated that 65.5% of the SSEA-4+ DP cells had osteogenic potential, and the SSEA-4+ clonal DP cells showed multilineage differentiation potential toward osteoblasts, chondrocytes, and neurons in vitro. In addition, the SSEA-4+ DP cells had the capacity to form ectopic bone in vivo. Thus, our results suggest that SSEA-4 is a specific cell surface antigen that can be used to identify DP stem cells.

  11. JC virus T-antigen regulates glucose metabolic pathways in brain tumor cells.

    PubMed

    Noch, Evan; Sariyer, Ilker Kudret; Gordon, Jennifer; Khalili, Kamel

    2012-01-01

    Recent studies have reported the detection of the human neurotropic virus, JCV, in a significant population of brain tumors, including medulloblastomas. Accordingly, expression of the JCV early protein, T-antigen, which has transforming activity in cell culture and in transgenic mice, results in the development of a broad range of tumors of neural crest and glial origin. Evidently, the association of T-antigen with a range of tumor-suppressor proteins, including p53 and pRb, and signaling molecules, such as β-catenin and IRS-1, plays a role in the oncogenic function of JCV T-antigen. We demonstrate that T-antigen expression is suppressed by glucose deprivation in medulloblastoma cells and in glioblastoma xenografts that both endogenously express T-antigen. Mechanistic studies indicate that glucose deprivation-mediated suppression of T-antigen is partly influenced by 5'-activated AMP kinase (AMPK), an important sensor of the AMP/ATP ratio in cells. In addition, glucose deprivation-induced cell cycle arrest in the G1 phase is blocked with AMPK inhibition, which also prevents T-antigen downregulation. Furthermore, T-antigen prevents G1 arrest and sustains cells in the G2 phase during glucose deprivation. On a functional level, T-antigen downregulation is partially dependent on reactive oxygen species (ROS) production during glucose deprivation, and T-antigen prevents ROS induction, loss of ATP production, and cytotoxicity induced by glucose deprivation. Additionally, we have found that T-antigen is downregulated by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and the pentose phosphate inhibitors, 6-aminonicotinamide and oxythiamine, and that T-antigen modulates expression of the glycolytic enzyme, hexokinase 2 (HK2), and the pentose phosphate enzyme, transaldolase-1 (TALDO1), indicating a potential link between T-antigen and metabolic regulation. These studies point to the possible involvement of JCV T-antigen in medulloblastoma proliferation and the metabolic

  12. Human T helper cells specific for antigens of typhus group rickettsiae enhance natural killer cell activity in vitro.

    PubMed Central

    Carl, M; Martin, E E; Dasch, G A

    1986-01-01

    The peripheral blood mononuclear cells (PBMC) from 5 individuals immune to typhus group rickettsiae and from 13 nonimmune individuals were stimulated in vitro for 7 days with typhus group rickettsial antigen (TGRA). At the end of day 7, lysis of the natural killer (NK)-susceptible target K562 by these PBMC was determined. As controls, PBMC from both groups of donors were cultured in vitro for 7 days without antigen or were freshly isolated, and lysis of the K562 target was determined. There was no significant difference between the level of NK activity in freshly isolated PBMC from immune and nonimmune donors. PBMC from immune donors which were stimulated with antigen for 7 days exhibited significantly greater NK activity than did the control population, which was cultured for 7 days without antigen. PBMC from immune donors which were stimulated with TGRA demonstrated significantly higher NK activity than the same PBMC stimulated with antigen derived from an antigenically unrelated rickettsia, Coxiella burnetii. There was no significant difference, however, in the level of NK activity of nonimmune antigen-stimulated PBMC compared with that of the same PBMC population cultured without antigen. Most of the antigen-stimulated NK activity was mediated by Leu-11-positive cells as determined by electronic cell sorting. The ability of TGRA to sustain the NK activity of PBMC from immune donors was abolished when the T4/Leu-3-positive population of lymphocytes was eliminated by positive or negative selection prior to antigen stimulation. The ability of TGRA to sustain the NK activity of PBMC from immune donors was also significantly decreased in the presence of antibodies against human interleukin-2. The results suggest that the activity of human NK cells can be sustained in vitro by antigen-specific T helper cells and that the effect of the T helper cell is mediated, at least in part, by interleukin-2. PMID:2945787

  13. The Lewis-Y carbohydrate antigen is expressed by many human tumors and can serve as a target for genetically redirected T cells despite the presence of soluble antigen in serum.

    PubMed

    Westwood, Jennifer A; Murray, William K; Trivett, Melanie; Haynes, Nicole M; Solomon, Benjamin; Mileshkin, Linda; Ball, David; Michael, Michael; Burman, Angela; Mayura-Guru, Preethi; Trapani, Joseph A; Peinert, Stefan; Hönemann, Dirk; Miles Prince, H; Scott, Andrew M; Smyth, Mark J; Darcy, Phillip K; Kershaw, Michael H

    2009-04-01

    In this study we aimed to determine the suitability of the Lewis-Y carbohydrate antigen as a target for immunotherapy using genetically redirected T cells. Using the 3S193 monoclonal antibody and immunohistochemistry, Lewis-Y was found to be expressed on a range of tumors including 42% squamous cell lung carcinoma, 80% lung adenocarcinoma, 25% ovarian carcinoma, and 25% colorectal adenocarcinoma. Expression levels varied from low to intense on between 1% and 90% of tumor cells. Lewis- was also found in soluble form in sera from both normal donors and cancer patients using a newly developed enzyme-linked immunosorbent assay. Serum levels in patients was often less than 1 ng/mL, similar to normal donors, but approximately 30% of patients had soluble Lewis-Y levels exceeding 1 ng/mL and up to 9 ng/mL. Lewis-Y-specific human T cells were generated by genetic modification with a chimeric receptor encoding a single-chain humanized antibody linked to the T-cell signaling molecules, T-cell receptor-zeta, and CD28. T cells responded against the Lewis-Y antigen by cytokine secretion and cytolysis in response to tumor cells. Importantly, the T-cell response was not inhibited by patient serum containing soluble Lewis-Y. This study demonstrates that Lewis-Y is expressed on a large number of tumors and Lewis-Y-specific T cells can retain antitumor function in the presence of patient serum, indicating that this antigen is a suitable target for this form of therapy.

  14. Design, engineering, and production of human recombinant t cell receptor ligands derived from human leukocyte antigen DR2.

    PubMed

    Chang, J W; Mechling, D E; Bächinger, H P; Burrows, G G

    2001-06-29

    Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers on the surface of antigen-presenting cells that bind the T cell receptor, initiating a cascade of interactions that results in antigen-specific activation of clonal populations of T cells. Susceptibility to multiple sclerosis is associated with certain MHC class II haplotypes, including human leukocyte antigen (HLA) DR2. Two DRB chains, DRB5*0101 and DRB1*1501, are co-expressed in the HLA-DR2 haplotype, resulting in the formation of two functional cell surface heterodimers, HLA-DR2a (DRA*0101, DRB5*0101) and HLA-DR2b (DRA*0101, DRB1*1501). Both isotypes can present an immunodominant peptide of myelin basic protein (MBP-(84-102)) to MBP-specific T cells from multiple sclerosis patients. We have previously demonstrated that the peptide binding/T cell recognition domains of rat MHC class II (alpha1 and beta1 domains) could be expressed as a single exon for structural and functional characterization; Burrows, G. G., Chang, J. W., Bächinger, H.-P., Bourdette, D. N., Wegmann, K. W., Offner, H., and Vandenbark A. A. (1999) Protein Eng. 12, 771-778; Burrows, G. G., Adlard, K. L., Bebo, B. F., Jr., Chang, J. W., Tenditnyy, K., Vandenbark, A. A., and Offner, H. (2000) J. Immunol. 164, 6366-6371). Single-chain human recombinant T cell receptor ligands (RTLs) of approximately 200 amino acid residues derived from HLA-DR2b were designed using the same principles and have been produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides. Structural characterization using circular dichroism predicted that these molecules retained the antiparallel beta-sheet platform and antiparallel alpha-helices observed in the native HLA-DR2 heterodimer. The proteins exhibited a cooperative two-state thermal unfolding transition, and DR2-derived RTLs with a covalently linked MBP peptide (MBP-(85-99)) showed increased stability to thermal unfolding relative to the

  15. Sequential induction of MHC antigens on autochthonous cells of ileum affected by Crohn's disease.

    PubMed Central

    Koretz, K.; Momburg, F.; Otto, H. F.; Möller, P.

    1987-01-01

    Changes were examined in the expression of Class I and II major histocompatibility complex (MHC) antigens by autochthonous cells of the terminal ileum affected by Crohn's disease. The study was based on the analysis of transmural specimens from terminal ileum segments obtained in the course of ileocolectomy for colon cancer and Crohn's disease. Serial sections were immunostained using monoclonal antibodies directed against monomorphic determinants of HLA-A,B,C, DR, DP, DQ, and the invariant chain (Ii) associated with Class II molecules. Compared with the normal state, the only change in Class I antigen expression occurring in Crohn's disease was the induction of HLA-A,B,C antigens in lymphatic endothelium. Changes in Class II antigen expression were more substantial. Enhancement of HLA-DR expression was found in enterocytes; DR induction was observed in glial cells of the visceral nervous plexus and in venular and venous endothelium. HLA-DP and DQ antigens were induced in enterocytes, glial cells, and capillary and venular endothelium, although this induction was restricted to areas of moderate or high inflammatory activity. The tissue distribution of Ii closely resembled that of HLA-DR, although this association was not strict: on the one hand, arterial endothelium contained low amounts of Ii in the absence of DR antigens; on the other hand, glial cells expressed Class II molecules in the absence of Ii. The extent of local enhancement/induction of MHC antigens was positively correlated with the local density of the cellular infiltrate. These data suggest that altered MHC antigen expression by autochthonous structures might be mediated by factors released from the lymphohistiocytic infiltrate, which is itself attracted by an unknown signal. In conjunction with an unknown antigen, the enhanced expression of Class II antigens might trigger an autoaggressive immune response. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:3425689

  16. Chicken histone genes retain nuclear matrix association throughout the cell cycle.

    PubMed Central

    Dalton, S; Younghusband, H B; Wells, J R

    1986-01-01

    The association between histone genes and the nuclear matrix (NM) during periods of high (S-phase) and low (non-S-phase) transcriptional activity has been investigated with synchronized cells from a chicken erythroid cell line (abbreviated ts34). By DNase I and restriction enzyme analysis, these studies reveal that both core and linker histone genes (represented by H2A and H1 genes respectively) are attached to the NM independent of their transcriptional activity during the cell-cycle. The tissue-specific histone gene H5, expressed constitutively, is nuclear matrix (NM)-associated in ts34 cells but is found in the supernatant (S/N) fractions of a non-erythroid T-cell line. Furthermore, we show that DNA sequences necessary for NM-attachment of the H5 gene lie within a 780 base pair region spanning part of the coding and 5' non-translated region. Of the three non-histone genes investigated, beta-actin sequences are expressed and are NM-attached, feather keratin genes are not expressed and predominate in the S/N, and beta-globin genes although not expressed in the ts34 cell line used were found in the NM fraction. In this case the association may be fortuitous or may reflect an early event prior to transcription of globin genes in differentiating erythroid cells. These results generally support the notion that actively transcribed genes are NM-attached, but that attachment per se is not synonymous with transcription. Images PMID:2428014

  17. Some aspects of oncogenic virus-host cell and virus-tumor cell antigenic relationships.

    PubMed

    Nastac, E

    1982-01-01

    Some viewpoints are presented as regards the virus-host cell relationship within the framework of carcinogenesis. Data are reviewed which point out the possibility of the transfer of cellular antigenic fractions from the tumor cell to the virus that grows in it, as well as of a hybridization between the virus genome and the genome of the tumoral host cell. Such a hybridization may have multiple consequences, among which the appearance of new oncogenic variants of viruses so far known to be nononcogenic ones.

  18. Investigation of red blood cell antigens with highly fluorescent and stable semiconductor quantum dots.

    PubMed

    de Farias, Patrícia Maria Albuquerque; Santos, Beate Saegesser; de Menezes, Frederico Duarte; de Carvalho Ferreira, Ricardo; Barjas-Castro, Maria Lourdes; Castro, Vagner; Lima, Paulo Roberto Moura; Fontes, Adriana; Cesar, Carlos Lenz

    2005-01-01

    We report a new methodology for red blood cell antigen expression determination by a simple labeling procedure employing luminescent semiconductor quantum dots. Highly luminescent and stable core shell cadmium sulfide/cadmium hydroxide colloidal particles are obtained, with a predominant size of 9 nm. The core-shell quantum dots are functionalized with glutaraldehyde and conjugated to a monoclonal anti-A antibody to target antigen-A in red blood cell membranes. Erythrocyte samples of blood groups A+, A2+, and O+ are used for this purpose. Confocal microscopy images show that after 30 min of conjugation time, type A+ and A2+ erythrocytes present bright emission, whereas the O+ group cells show no emission. Fluorescence intensity maps show different antigen expressions for the distinct erythrocyte types. The results obtained strongly suggest that this simple labeling procedure may be employed as an efficient tool to investigate quantitatively the distribution and expression of antigens in red blood cell membranes.

  19. B lymphocytes as direct antigen-presenting cells for anti-tumor DNA vaccines

    PubMed Central

    Colluru, Viswa Teja; McNeel, Douglas G.

    2016-01-01

    In spite of remarkable preclinical efficacy, DNA vaccination has demonstrated low immunogenicity in humans. While efforts have focused on increasing cross-presentation of DNA-encoded antigens, efforts to increase DNA vaccine immunogenicity by targeting direct presentation have remained mostly unexplored. In these studies, we compared the ability of different APCs to present antigen to T cells after simple co-culture with plasmid DNA. We found that human primary peripheral B lymphocytes, and not monocytes or in vitro derived dendritic cells (DCs), were able to efficiently encode antigen mRNA and expand cognate tumor antigen-specific CD8 T cells ex vivo. Similarly, murine B lymphocytes co-cultured with plasmid DNA, and not DCs, were able to prime antigen-specific T cells in vivo. Moreover, B lymphocyte-mediated presentation of plasmid antigen led to greater Th1-biased immunity and was sufficient to elicit an anti-tumor effect in vivo. Surprisingly, increasing plasmid presentation by B cells, and not cross presentation of peptides by DCs, further augmented traditional plasmid vaccination. Together, these data suggest that targeting plasmid DNA to B lymphocytes, for example through transfer of ex vivo plasmidloaded B cells, may be novel means to achieve greater T cell immunity from DNA vaccines. PMID:27661128

  20. Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape.

    PubMed

    Hegde, Meenakshi; Mukherjee, Malini; Grada, Zakaria; Pignata, Antonella; Landi, Daniel; Navai, Shoba A; Wakefield, Amanda; Fousek, Kristen; Bielamowicz, Kevin; Chow, Kevin K H; Brawley, Vita S; Byrd, Tiara T; Krebs, Simone; Gottschalk, Stephen; Wels, Winfried S; Baker, Matthew L; Dotti, Gianpietro; Mamonkin, Maksim; Brenner, Malcolm K; Orange, Jordan S; Ahmed, Nabil

    2016-08-01

    In preclinical models of glioblastoma, antigen escape variants can lead to tumor recurrence after treatment with CAR T cells that are redirected to single tumor antigens. Given the heterogeneous expression of antigens on glioblastomas, we hypothesized that a bispecific CAR molecule would mitigate antigen escape and improve the antitumor activity of T cells. Here, we created a CAR that joins a HER2-binding scFv and an IL13Rα2-binding IL-13 mutein to make a tandem CAR exodomain (TanCAR) and a CD28.ζ endodomain. We determined that patient TanCAR T cells showed distinct binding to HER2 or IL13Rα2 and had the capability to lyse autologous glioblastoma. TanCAR T cells exhibited activation dynamics that were comparable to those of single CAR T cells upon encounter of HER2 or IL13Rα2. We observed that TanCARs engaged HER2 and IL13Rα2 simultaneously by inducing HER2-IL13Rα2 heterodimers, which promoted superadditive T cell activation when both antigens were encountered concurrently. TanCAR T cell activity was more sustained but not more exhaustible than that of T cells that coexpressed a HER2 CAR and an IL13Rα2 CAR, T cells with a unispecific CAR, or a pooled product. In a murine glioblastoma model, TanCAR T cells mitigated antigen escape, displayed enhanced antitumor efficacy, and improved animal survival. Thus, TanCAR T cells show therapeutic potential to improve glioblastoma control by coengaging HER2 and IL13Rα2 in an augmented, bivalent immune synapse that enhances T cell functionality and reduces antigen escape.

  1. Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape

    PubMed Central

    Mukherjee, Malini; Grada, Zakaria; Pignata, Antonella; Landi, Daniel; Navai, Shoba A.; Wakefield, Amanda; Bielamowicz, Kevin; Chow, Kevin K.H.; Brawley, Vita S.; Byrd, Tiara T.; Krebs, Simone; Gottschalk, Stephen; Wels, Winfried S.; Baker, Matthew L.; Dotti, Gianpietro; Mamonkin, Maksim; Brenner, Malcolm K.

    2016-01-01

    In preclinical models of glioblastoma, antigen escape variants can lead to tumor recurrence after treatment with CAR T cells that are redirected to single tumor antigens. Given the heterogeneous expression of antigens on glioblastomas, we hypothesized that a bispecific CAR molecule would mitigate antigen escape and improve the antitumor activity of T cells. Here, we created a CAR that joins a HER2-binding scFv and an IL13Rα2-binding IL-13 mutein to make a tandem CAR exodomain (TanCAR) and a CD28.ζ endodomain. We determined that patient TanCAR T cells showed distinct binding to HER2 or IL13Rα2 and had the capability to lyse autologous glioblastoma. TanCAR T cells exhibited activation dynamics that were comparable to those of single CAR T cells upon encounter of HER2 or IL13Rα2. We observed that TanCARs engaged HER2 and IL13Rα2 simultaneously by inducing HER2-IL13Rα2 heterodimers, which promoted superadditive T cell activation when both antigens were encountered concurrently. TanCAR T cell activity was more sustained but not more exhaustible than that of T cells that coexpressed a HER2 CAR and an IL13Rα2 CAR, T cells with a unispecific CAR, or a pooled product. In a murine glioblastoma model, TanCAR T cells mitigated antigen escape, displayed enhanced antitumor efficacy, and improved animal survival. Thus, TanCAR T cells show therapeutic potential to improve glioblastoma control by coengaging HER2 and IL13Rα2 in an augmented, bivalent immune synapse that enhances T cell functionality and reduces antigen escape. PMID:27427982

  2. Hodgkin and sternberg-reed cell antigen(s) detected by an antiserum to a cell line (L428) derived from Hodgkin's disease.

    PubMed

    Stein, H; Gerdes, J; Kirchner, H; Schaadt, M; Diehl, V

    1981-10-15

    Antisera to the cell line L428, derived from Hodgkin's disease, were raised in rabbits by injecting L428 cells intravenously and subcutaneously. The anti-L428 cell serum that did not react with HLA-DR was absorbed with tonsil cell plus acute myeloid leukemia cells or tonsil cells plus neutrophils, monocytes, and blood lymphocytes. Then it was tested for its ability to discriminate between L428 cells, Hodgkin and Sternberg-Reed cells, and various other cells. It was found that the anti L428 cell serum absorbed with tonsil cells plus acute myeloid leukemia cells stained only L428 cells, Hodgkin and Sternberg-Reed cells, and neutrophils. The anti L428 cell serum absorbed with tonsil cell plus neutrophils, monocytes, and blood lymphocytes reacted with L428 cells and Hodgkin and sternberg-Reed cells from 13 cases of Hodgkin's disease. It did not react with any other cell type present in the blood or in lymphoid tissue or with cells from five cases of non-Hodgkin's lymphoma. The absorbed anti-L428 cell serum also failed to stain Daudi and HRIK cell line cells. We conclude that the anti-L428 cell serum defines an antigen that is apparently restricted in expression to L428 cells and Hodgkin and Sternberg-Reed cells. This is a strong indication that the L428 cell line cells are derived from Hodgkin and Sternberg-Reed cells.

  3. The B-cell tumor–associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor

    PubMed Central

    Schmitt, Thomas M.; Baskar, Sivasubramanian; Lupo-Stanghellini, Maria Teresa; Nishida, Tetsuya; Yamamoto, Tori N.; Bleakley, Marie; Turtle, Cameron J.; Chang, Wen-Chung; Greisman, Harvey A.; Wood, Brent; Maloney, David G.; Jensen, Michael C.; Rader, Christoph; Riddell, Stanley R.

    2010-01-01

    Monoclonal antibodies and T cells modified to express chimeric antigen receptors specific for B-cell lineage surface molecules such as CD20 exert antitumor activity in B-cell malignancies, but deplete normal B cells. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) was identified as a highly expressed gene in B-cell chronic lymphocytic leukemia (B-CLL), but not normal B cells, suggesting it may serve as a tumor-specific target for therapy. We analyzed ROR1-expression in normal nonhematopoietic and hematopoietic cells including B-cell precursors, and in hematopoietic malignancies. ROR1 has characteristics of an oncofetal gene and is expressed in undifferentiated embryonic stem cells, B-CLL and mantle cell lymphoma, but not in major adult tissues apart from low levels in adipose tissue and at an early stage of B-cell development. We constructed a ROR1-specific chimeric antigen receptor that when expressed in T cells from healthy donors or CLL patients conferred specific recognition of primary B-CLL and mantle cell lymphoma, including rare drug effluxing chemotherapy resistant tumor cells that have been implicated in maintaining the malignancy, but not mature normal B cells. T-cell therapies targeting ROR1 may be effective in B-CLL and other ROR1-positive tumors. However, the expression of ROR1 on some normal tissues suggests the potential for toxi-city to subsets of normal cells. PMID:20702778

  4. The B-cell tumor-associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor.

    PubMed

    Hudecek, Michael; Schmitt, Thomas M; Baskar, Sivasubramanian; Lupo-Stanghellini, Maria Teresa; Nishida, Tetsuya; Yamamoto, Tori N; Bleakley, Marie; Turtle, Cameron J; Chang, Wen-Chung; Greisman, Harvey A; Wood, Brent; Maloney, David G; Jensen, Michael C; Rader, Christoph; Riddell, Stanley R

    2010-11-25

    Monoclonal antibodies and T cells modified to express chimeric antigen receptors specific for B-cell lineage surface molecules such as CD20 exert antitumor activity in B-cell malignancies, but deplete normal B cells. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) was identified as a highly expressed gene in B-cell chronic lymphocytic leukemia (B-CLL), but not normal B cells, suggesting it may serve as a tumor-specific target for therapy. We analyzed ROR1-expression in normal nonhematopoietic and hematopoietic cells including B-cell precursors, and in hematopoietic malignancies. ROR1 has characteristics of an oncofetal gene and is expressed in undifferentiated embryonic stem cells, B-CLL and mantle cell lymphoma, but not in major adult tissues apart from low levels in adipose tissue and at an early stage of B-cell development. We constructed a ROR1-specific chimeric antigen receptor that when expressed in T cells from healthy donors or CLL patients conferred specific recognition of primary B-CLL and mantle cell lymphoma, including rare drug effluxing chemotherapy resistant tumor cells that have been implicated in maintaining the malignancy, but not mature normal B cells. T-cell therapies targeting ROR1 may be effective in B-CLL and other ROR1-positive tumors. However, the expression of ROR1 on some normal tissues suggests the potential for toxi-city to subsets of normal cells.

  5. Granuloma cells in chronic inflammation express CD205 (DEC205) antigen and harbor proliferating T lymphocytes: similarity to antigen-presenting cells.

    PubMed

    Ohtani, Haruo

    2013-02-01

    Granulomas are classified as immune or foreign body granulomas. Of these, the immune granulomas, a hallmark of granulomatous inflammation, are closely related to cell-mediated immune responses. The aim of the present study is to characterize immune granuloma cells in 33 patients with granulomatous inflammation focusing on the expression of CD205 (DEC205), a cell surface marker of antigen presenting cells, and their spatial relationship to T cells. CD205 was frequently expressed by immune granuloma cells, in contrast to foreign body granuloma cells that lacked CD205 expression. T cells were not only distributed in a lymphocyte collar around the granuloma, but also present among the granuloma cells (termed 'intra-granuloma T cells'). Intra-granuloma T cells stained positive for Ki-67 (median positivity = 9.4%) by double immunostaining for CD3 and Ki-67. This indicated the presence of proliferative stimuli within the granuloma that could activate the intra-granuloma T cells. The labeling index of Ki-67 in intra-granuloma T cells was significantly higher than that of T cells in the lymphocyte collar (P < 0.0001) or T cells in the T cell zone (paracortex) of chronic tonsillitis or reactive lymphadenitis (P = 0.002). These data indicate a close similarity between immune granulomas and antigen presenting cells.

  6. Antigen-specific CD4{sup +} effector T cells: Analysis of factors regulating clonal expansion and cytokine production

    SciTech Connect

    Ohnuki, Kazunobu; Watanabe, Yuri; Takahashi, Yusuke; Kobayashi, Sakiko; Watanabe, Shiho; Ogawa, Shuhei; Kotani, Motoko; Kozono, Haruo; Tanabe, Kazunari; Abe, Ryo

    2009-03-20

    In order to fully understand T cell-mediated immunity, the mechanisms that regulate clonal expansion and cytokine production by CD4{sup +} antigen-specific effector T cells in response to a wide range of antigenic stimulation needs clarification. For this purpose, panels of antigen-specific CD4{sup +} T cell clones with different thresholds for antigen-induced proliferation were generated by repeated stimulation with high- or low-dose antigen. Differences in antigen sensitivities did not correlate with expression of TCR, CD4, adhesion or costimulatory molecules. There was no significant difference in antigen-dependent cytokine production by TG40 cells transfected with TCR obtained from either high- or low-dose-responding T cell clones, suggesting that the affinity of TCRs for their ligands is not primary determinant of T cell antigen reactivity. The proliferative responses of all T cell clones to both peptide stimulation and to TCR{beta} crosslinking revealed parallel dose-response curves. These results suggest that the TCR signal strength of effector T cells and threshold of antigen reactivity is determined by an intrinsic property, such as the TCR signalosome and/or intracellular signaling machinery. Finally, the antigen responses of high- and low-peptide-responding T cell clones reveal that clonal expansion and cytokine production of effector T cells occur independently of antigen concentration. Based on these results, the mechanisms underlying selection of high 'avidity' effector and memory T cells in response to pathogen are discussed.

  7. Adipose-derived stem cells retain their regenerative potential after methotrexate treatment

    SciTech Connect

    Beane, Olivia S.; Fonseca, Vera C.; Darling, Eric M.

    2014-10-01

    In musculoskeletal tissues like bone, chemotherapy can impair progenitor cell differentiation and proliferation, resulting in decreased bone growth and mineralization throughout a patient's lifetime. In the current study, we investigated the effects of chemotherapeutics on adipose-derived stem cell (ASC) function to determine whether this cell source could be a candidate for repairing, or even preventing, chemotherapy-induced tissue damage. Dose-dependent proliferation rates of ASCs and normal human fibroblasts (NHFs) were quantified after treatment with cytarabine (CY), etoposide (ETO), methotrexate (MTX), and vincristine (VIN) using a fluorescence-based assay. The influence of MTX on the multipotency of ASCs and freshly isolated stromal vascular fraction (SVF) cells was also evaluated using lineage-specific stains and spectrophotometry. ASC and NHF proliferation were equally inhibited by exposure to CY and ETO; however, when treated with MTX and VIN, ASCs exhibited greater resistance. This was especially apparent for MTX-treated samples, with ASC proliferation showing no inhibition for clinically relevant MTX doses ranging from 0.1 to 50 μM. Additional experiments revealed that the differentiation potential of ASCs was not affected by MTX treatment and that upregulation of dihydrofolate reductase possibly contributed to this response. Moreover, SVF cells, which include ASCs, exhibited similar resistance to MTX impairment, with respect to cellular proliferation, clonogenicity, and differentiation capability. Therefore, we have shown that the regenerative properties of ASCs resist the cytotoxicity of MTX, identifying these cells as a potential key for repairing musculoskeletal damage in patients undergoing chemotherapy. - Highlights: • Long-term effects of chemotherapeutics can include musculoskeletal dysfunction. • A screen of common drugs showed disparate effects on ASCs and fibroblasts. • One drug, methotrexate, did not impair ASC growth characteristics

  8. Antigenic differences between AKR lymphoma and thymus cells leading to detection of a tumor antigen associated with immunological enhancement.

    PubMed

    Laguens, R P; Colmerauer, M E; Segal, A; Pasqualini, C D

    1978-06-15

    In an experimental model conditioning for enhancement, an AKR lymphoma was made to grow in BALB/c mice, permitting the simultaneous comparison of tumor-bearing (progressor) and tumor-rejecting (regressor) animals. By immunofluorescence using as target AKR lymphoma and normal thymus cells, both acetone-fixed and unfixed, it was observed that the allogeneic progressor serum contained three antibodies, two of which could be asborbed by thymocytes while the other combined selectively with the acetone-fixed lymphoma target. This tumor-specific antibody could not be detected in regressor serum which, on the other hand, could be completely absorbed by thymocytes. The identification of this acetone-resistant tumor antigen led to the preparation of aceton-treated acellular lymphoma extracts: a precipitate was obtained which upon inoculation in BALB/c mice produced an antiserum that combined selectively with lymphoma targets. In vivo experiments showed that pretreatment with this antigen led to a significant increase in allogeneic tumor incidence, 76% as compared to 37% in the controls. It is concluded that in this allogeneic model, an acetone-resistant tumor-specific antigen and the corresponding antibody are involved in tumor enhancement.

  9. Antigens protected functional red blood cells by the membrane grafting of compact hyperbranched polyglycerols.

    PubMed

    Chapanian, Rafi; Constantinescu, Iren; Brooks, Donald E; Scott, Mark D; Kizhakkedathu, Jayachandran

    2013-01-02

    Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen, glucose, and ions. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane and mask RBC surface antigens.

  10. A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface.

    PubMed

    de Jong, Rob N; Beurskens, Frank J; Verploegen, Sandra; Strumane, Kristin; van Kampen, Muriel D; Voorhorst, Marleen; Horstman, Wendy; Engelberts, Patrick J; Oostindie, Simone C; Wang, Guanbo; Heck, Albert J R; Schuurman, Janine; Parren, Paul W H I

    2016-01-01

    IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. The identified IgG1 Fc backbones provide a novel platform for the generation of therapeutics with enhanced effector functions that only become activated upon binding to target cell-expressed antigen.

  11. Chimeric Antigen Receptor–Modified T Cells for Acute Lymphoid Leukemia

    PubMed Central

    Barrett, David; Aplenc, Richard; Porter, David L.; Rheingold, Susan R.; Teachey, David T.; Chew, Anne; Hauck, Bernd; Wright, J. Fraser; Milone, Michael C.; Levine, Bruce L.; June, Carl H.

    2014-01-01

    Summary Chimeric antigen receptor–modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre–B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×106 to 1.2×107 CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce anti-leukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor–modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL. PMID:23527958

  12. Major histocompatibility complex class II antigen expression in B and T cell non-Hodgkin's lymphoma.

    PubMed Central

    Smith, M E; Holgate, C S; Williamson, J M; Grigor, I; Quirke, P; Bird, C C

    1987-01-01

    An immunohistochemical study of 46 B and T cell non-Hodgkin's lymphomas, using monoclonal antibodies to the products of the major histocompatibility complex (MHC) class II antigen subregions, DP, DQ, and DR, showed that most B and T cell lymphomas express these antigens. Both coordinate and non-coordinate expression of MHC class II antigens was observed, but this did not correlate with immunological phenotype, morphological grade, or proliferation index as determined by flow cytometry. Images Fig 1 Fig 2 Fig 3 PMID:3546388

  13. Dendritic Cell Targeting of Bacillus anthracis Protective Antigen Expressed by Lactobacillus acidophilus Protects Mice from Lethal Challenge

    DTIC Science & Technology

    2008-10-28

    Dendritic cell targeting of Bacillus anthracis protective antigen expressed by Lactobacillus acidophilus protects mice from lethal challenge M...lethal chal- lenge. A vaccine strategy was established by using Lactobacillus acidophilus to deliver Bacillus anthracis protective antigen (PA) via...4. TITLE AND SUBTITLE Dendritic cell targeting of Bacillus anthracis protective antigen expressed by Lactobacillus acidophilus protects mice

  14. Effect of antigen/antibody ratio on macrophage uptake, processing, and presentation to T cells of antigen complexed with polyclonal antibodies

    PubMed Central

    1991-01-01

    Activation of a galactosidase-specific murine T hybridoma clone and of a human tetanus toxoid-specific T clone by antigen-presenting cells (APC) was used to evaluate the regulatory function of antibodies complexed with the relevant antigen. Complexed antigen, in fact, is taken up with high efficiency thanks to Fc receptors borne by APC. Antibody/antigen ratio in the complexes proved to be a critical parameter in enhancing antigen presentation. Complexes in moderate antibody excess provided optimal T cell activation independently of the physical state of the complexes (precipitated by a second antibody or solubilized by complement). Complexes in extreme antibody excess, on the contrary, did not yield T cell activation although taken up by APC efficiently. The effect of antibodies at extreme excess was observed with substimulatory dose of antigen (loss of potentiation) and with optimal dose of antigen (loss of stimulation). An excess of specific polyclonal antibodies hampers proteolytic degradation of antigen in vitro, supporting the view that a similar mechanism may operate within the APC that have internalized immune complexes in extreme antibody excess. The possibility that immune complex forming in extreme antibody excess may turn off the T cell response is proposed as a regulatory mechanism. PMID:1985125

  15. How Antigen Quantity and Quality Determine T-Cell Decisions in Lymphoid Tissue▿ †

    PubMed Central

    Zheng, Huan; Jin, Bo; Henrickson, Sarah E.; Perelson, Alan S.; von Andrian, Ulrich H.; Chakraborty, Arup K.

    2008-01-01

    T lymphocytes (T cells) express T-cell receptor (TCR) molecules on their surface that can recognize peptides (p) derived from antigenic proteins bound to products of the major histocompatibility complex (MHC) genes. The pMHC molecules are expressed on the surface of antigen-presenting cells, such as dendritic cells (DCs). T cells first encounter antigen on DCs in lymph nodes (LN). Intravital microscopy experiments show that upon entering the LN containing antigen, CD8+ T cells first move rapidly. After a few hours, they stop and make extended contacts with DCs. The factors that determine when and how this transition occurs are not well understood. We report results from computer simulations that suggest that the duration of phase one is related to the low probability of productive interactions between T cells and DCs. This is demonstrated by our finding that the antigen dose and type determine when such a transition occurs. These results are in agreement with experimental observations. TCR-pMHC binding characteristics and the antigen dose determine the time required for a productive T-cell-DC encounter (resulting in sustained contact). We find that the ratio of this time scale and the half-life of the pMHC complex itself provide a consolidated measure of antigen quantity and type. Results obtained upon varying different measures of antigen quantity and type fall on one curve when graphed against this ratio of time scales. Thus, we provide a mechanism for how the effects of varying one set of parameters are influenced by other prevailing conditions. This understanding should help guide future experimentation. PMID:18426917

  16. Porcine Adipose-Derived Mesenchymal Stem Cells Retain Their Stem Cell Characteristics and Cell Activities While Enhancing the Expression of Liver-Specific Genes after Acute Liver Failure.

    PubMed

    Hu, Chenxia; Zhou, Ning; Li, Jianzhou; Shi, Ding; Cao, Hongcui; Li, Jun; Li, Lanjuan

    2016-01-05

    Acute liver failure (ALF) is a kind of complicated syndrome. Furthermore, adipose-derived mesenchymal stem cells (ADMSCs) can serve as a useful cell resource for autotransplantation due to their abundance and micro-invasive accessability. However, it is unknown how ALF will influence the characteristics of ADMSCs and whether ADMSCs from patients suffering from end-stage liver diseases are potential candidates for autotransplantation. This study was designed to compare various properties of ALF-derived ADMSCs with normal ADMSCs in pig models, with regard to their cellular morphology, cell proliferative ability, cell apoptosis, expression of surface antigens, mitochondrial and lysosomal activities, multilineage potency, and expression of liver-specific genes. Our results showed that ALF does not influence the stem cell characteristics and cell activities of ADMSCs. Intriguingly, the expression levels of several liver-specific genes in ALF-derived ADMSCs are higher than in normal ADMSCs. In conclusion, our findings indicate that the stem cell characteristics and cell activities of ADMSCs were not altered by ALF and these cells can serve as a new source for regenerative medicine.

  17. Equine-Induced Pluripotent Stem Cells Retain Lineage Commitment Toward Myogenic and Chondrogenic Fates

    PubMed Central

    Quattrocelli, Mattia; Giacomazzi, Giorgia; Broeckx, Sarah Y.; Ceelen, Liesbeth; Bolca, Selin; Spaas, Jan H.; Sampaolesi, Maurilio

    2016-01-01

    Summary Induced pluripotent stem cells (iPSCs) hold great potential not only for human but also for veterinary purposes. The equine industry must often deal with health issues concerning muscle and cartilage, where comprehensive regenerative strategies are still missing. In this regard, a still open question is whether equine iPSCs differentiate toward muscle and cartilage, and whether donor cell type influences their differentiation potential. We addressed these questions through an isogenic system of equine iPSCs obtained from myogenic mesoangioblasts (MAB-iPSCs) and chondrogenic mesenchymal stem cells (MSC-iPSCs). Despite similar levels of pluripotency characteristics, the myogenic differentiation appeared enhanced in MAB-iPSCs. Conversely, the chondrogenic differentiation was augmented in MSC-iPSCs through both teratoma and in vitro differentiation assays. Thus, our data suggest that equine iPSCs can differentiate toward the myogenic and chondrogenic lineages, and can present a skewed differentiation potential in favor of the source cell lineage. PMID:26771353

  18. Asymmetric cell division of T cells upon antigen presentation uses multiple conserved mechanisms.

    PubMed

    Oliaro, Jane; Van Ham, Vanessa; Sacirbegovic, Faruk; Pasam, Anupama; Bomzon, Ze'ev; Pham, Kim; Ludford-Menting, Mandy J; Waterhouse, Nigel J; Bots, Michael; Hawkins, Edwin D; Watt, Sally V; Cluse, Leonie A; Clarke, Chris J P; Izon, David J; Chang, John T; Thompson, Natalie; Gu, Min; Johnstone, Ricky W; Smyth, Mark J; Humbert, Patrick O; Reiner, Steven L; Russell, Sarah M

    2010-07-01

    Asymmetric cell division is a potential means by which cell fate choices during an immune response are orchestrated. Defining the molecular mechanisms that underlie asymmetric division of T cells is paramount for determining the role of this process in the generation of effector and memory T cell subsets. In other cell types, asymmetric cell division is regulated by conserved polarity protein complexes that control the localization of cell fate determinants and spindle orientation during division. We have developed a tractable, in vitro model of naive CD8(+) T cells undergoing initial division while attached to dendritic cells during Ag presentation to investigate whether similar mechanisms might regulate asymmetric division of T cells. Using this system, we show that direct interactions with APCs provide the cue for polarization of T cells. Interestingly, the immunological synapse disseminates before division even though the T cells retain contact with the APC. The cue from the APC is translated into polarization of cell fate determinants via the polarity network of the Par3 and Scribble complexes, and orientation of the mitotic spindle during division is orchestrated by the partner of inscuteable/G protein complex. These findings suggest that T cells have selectively adapted a number of evolutionarily conserved mechanisms to generate diversity through asymmetric cell division.

  19. B Cells use Conserved Polarity Cues to Regulate Their Antigen Processing and Presentation Functions

    PubMed Central

    Yuseff, Maria-Isabel; Lennon-Duménil, Ana Maria

    2015-01-01

    The ability of B cells to produce high-affinity antibodies and to establish immunological memory in response to a wide range of pathogenic antigens is an essential part of the adaptive immune response. The initial step that triggers a humoral immune response involves the acquisition of antigens by B cells via their surface immunoglobulin, the B cell receptor (BCR). BCR-engaged antigens are transported into specialized lysosomal compartments where proteolysis and production of MHC class II-peptide complexes occur, a process referred to as antigen processing. Expression of MHC class II complexes at the B cell surface allows them to interact with T cells and to receive their help to become fully activated. In this review, we describe how B cells rely on conserved cell polarity mechanisms to coordinate local proteolytic secretion and mechanical forces at the B cell synapse enabling them to efficiently acquire and present extracellular antigens. We foresee that the mechanisms that dictate B cell activation can be used to tune B cell responses in the context of autoimmune diseases and cancer. PMID:26074919

  20. Identification and visualization of multidimensional antigen-specific T-cell populations in polychromatic cytometry data.

    PubMed

    Lin, Lin; Frelinger, Jacob; Jiang, Wenxin; Finak, Greg; Seshadri, Chetan; Bart, Pierre-Alexandre; Pantaleo, Giuseppe; McElrath, Julie; DeRosa, Steve; Gottardo, Raphael

    2015-07-01

    An important aspect of immune monitoring for vaccine development, clinical trials, and research is the detection, measurement, and comparison of antigen-specific T-cells from subject samples under different conditions. Antigen-specific T-cells compose a very small fraction of total T-cells. Developments in cytometry technology over the past five years have enabled the measurement of single-cells in a multivariate and high-throughput manner. This growth in both dimensionality and quantity of data continues to pose a challenge for effective identification and visualization of rare cell subsets, such as antigen-specific T-cells. Dimension reduction and feature extraction play pivotal role in both identifying and visualizing cell populations of interest in large, multi-dimensional cytometry datasets. However, the automated identification and visualization of rare, high-dimensional cell subsets remains challenging. Here we demonstrate how a systematic and integrated approach combining targeted feature extraction with dimension reduction can be used to identify and visualize biological differences in rare, antigen-specific cell populations. By using OpenCyto to perform semi-automated gating and features extraction of flow cytometry data, followed by dimensionality reduction with t-SNE we are able to identify polyfunctional subpopulations of antigen-specific T-cells and visualize treatment-specific differences between them.

  1. Signal transduction-associated and cell activation-linked antigens expressed in human mast cells.

    PubMed

    Valent, Peter; Ghannadan, Minoo; Hauswirth, Alexander W; Schernthaner, Gerit-Holger; Sperr, Wolfgang R; Arock, Michel

    2002-05-01

    Mast cells (MCs) are multifunctional hematopoietic effector cells that produce and release an array of biologically active mediator substances. Growth and functions of MCs are regulated by cytokines, other extracellular factors, surface and cytoplasmic receptors, oncogene products, and a complex network of signal transduction cascades. Key regulators of differentiation of MCs appear to be stem cell factor (SCF) and its tyrosine kinase receptor KIT (c-kit proto-oncogene product=CD117), downstream-acting elements, and the mi transcription factor (MITF). Signaling through KIT is negatively regulated by the signal regulatory protein (SIRP)-alpha (CD172a)-SHP-1-pathway that is disrupted in neoplastic MCs in MC proliferative disorders. Both KIT and FcepsilonRI are involved in MC activation and mediator release. Activation of MCs through FcepsilonRI is associated with increased expression of activation-linked membrane antigens as well as with signaling events involving Lyn and Syk kinases, the phosphatidylinositol-3-kinase-pathway, Ras pathway, and the phospholipase C-protein kinase C pathway. A similar network of signaling is found in SCF-activated MCs. The current article gives an overview on signal transduction-associated and activation-linked antigens expressed in human MCs. Wherever possible the functional implication of signaling pathways and antigen expression are discussed.

  2. Aberrant Cosmc genes result in Tn antigen expression in human colorectal carcinoma cell line HT-29

    PubMed Central

    Yu, Xiaofeng; Du, Zhenzhen; Sun, Xuhong; Shi, Chuanqin; Zhang, Huaixiang; Hu, Tao

    2015-01-01

    The Tn antigen, which arises from mutation in the Cosmc gene is one of the most common tumor associated carbohydrate antigens. Cosmc resides in X24 encoded by a single gene and functions as a specific molecular chaperone for T-synthase. While the Tn antigen cannot be detected in normal cells, Cosmc mutations inactivate T-synthase and consequently result in Tn antigen expression within certain cancers. In addition to this Cosmc mutation-induced expression, the Tn antigen is also expressed in such cell lines as Jurkat T, LSC and LS174T. Whether the Cosmc mutation is present in the colon cancer cell line HT-29 is still unclear. Here, we isolate HT-29-Tn+ cells from HT-29 cells derived from a female colon cancer patient. These HT-29-Tn+ cells show a loss of the Cosmc gene coding sequence (CDS) leading to an absence of T-synthase activity and Tn antigen expression. Additionally, almost no methylation of Cosmc CpG islands was detected in HT-29-Tn+ as well as in HT-29-Tn- and Tn- tumor cells from male patients. In contrast, the methylation frequency of CpG island of Cosmc in normal female cells was ~50%. Only one active allele of Cosmc existed in HT-29-Tn+ and HT-29-Tn- cells as based upon detection of SNP sites. These results indicate that Tn antigens expression and T-synthase inactivity in HT-29-Tn+ cells can be related to the absence of CDS in Cosmc active alleles, while an inactive allele deletion of Cosmc in HT-29 cells has no influence on Cosmc function. PMID:26045765

  3. Targeted delivery of antigen to hamster nasal lymphoid tissue with M-cell-directed lectins.

    PubMed Central

    Giannasca, P J; Boden, J A; Monath, T P

    1997-01-01

    The nasal cavity of a rodent is lined by an epithelium organized into distinct regional domains responsible for specific physiological functions. Aggregates of nasal lymphoid tissue (NALT) located at the base of the nasal cavity are believed to be sites of induction of mucosal immune responses to airborne antigens. The epithelium overlying NALT contains M cells which are specialized for the transcytosis of immunogens, as demonstrated in other mucosal tissues. We hypothesized that NALT M cells are characterized by distinct glycoconjugate receptors which influence antigen uptake and immune responses to transcytosed antigens. To identify glycoconjugates that may distinguish NALT M cells from other cells of the respiratory epithelium (RE), we performed lectin histochemistry on sections of the hamster nasal cavity with a panel of lectins. Many classes of glycoconjugates were found on epithelial cells in this region. While most lectins bound to sites on both the RE and M cells, probes capable of recognizing alpha-linked galactose were found to label the follicle-associated epithelium (FAE) almost exclusively. By morphological criteria, the FAE contains >90% M cells. To determine if apical glycoconjugates on M cells were accessible from the nasal cavity, an M-cell-selective lectin and a control lectin in parallel were administered intranasally to hamsters. The M-cell-selective lectin was found to specifically target the FAE, while the control lectin did not. Lectin bound to M cells in vivo was efficiently endocytosed, consistent with the role of M cells in antigen transport. Intranasal immunization with lectin-test antigen conjugates without adjuvant stimulated induction of specific serum immunoglobulin G, whereas antigen alone or admixed with lectin did not. The selective recognition of NALT M cells by a lectin in vivo provides a model for microbial adhesin-host cell receptor interactions on M cells and the targeted delivery of immunogens to NALT following intranasal

  4. Inhibiting DNA methylation activates cancer testis antigens and expression of the antigen processing and presentation machinery in colon and ovarian cancer cells.

    PubMed

    Siebenkäs, Cornelia; Chiappinelli, Katherine B; Guzzetta, Angela A; Sharma, Anup; Jeschke, Jana; Vatapalli, Rajita; Baylin, Stephen B; Ahuja, Nita

    2017-01-01

    Innovative therapies for solid tumors are urgently needed. Recently, therapies that harness the host immune system to fight cancer cells have successfully treated a subset of patients with solid tumors. These responses have been strong and durable but observed in subsets of patients. Work from our group and others has shown that epigenetic therapy, specifically inhibiting the silencing DNA methylation mark, activates immune signaling in tumor cells and can sensitize to immune therapy in murine models. Here we show that colon and ovarian cancer cell lines exhibit lower expression of transcripts involved in antigen processing and presentation to immune cells compared to normal tissues. In addition, treatment with clinically relevant low doses of DNMT inhibitors (that remove DNA methylation) increases expression of both antigen processing and presentation and Cancer Testis Antigens in these cell lines. We confirm that treatment with DNMT inhibitors upregulates expression of the antigen processing and presentation molecules B2M, CALR, CD58, PSMB8, PSMB9 at the RNA and protein level in a wider range of colon and ovarian cancer cell lines and treatment time points than had been described previously. In addition, we show that DNMTi treatment upregulates many Cancer Testis Antigens common to both colon and ovarian cancer. This increase of both antigens and antigen presentation by epigenetic therapy may be one mechanism to sensitize patients to immune therapies.

  5. Do ABO Blood Group Antigens Hamper the Therapeutic Efficacy of Mesenchymal Stromal Cells?

    PubMed Central

    Moll, Guido; Hult, Annika; von Bahr, Lena; Alm, Jessica J.; Heldring, Nina; Hamad, Osama A.; Stenbeck-Funke, Lillemor; Larsson, Stella; Teramura, Yuji; Roelofs, Helene; Nilsson, Bo; Fibbe, Willem E.; Olsson, Martin L.; Le Blanc, Katarina

    2014-01-01

    Investigation into predictors for treatment outcome is essential to improve the clinical efficacy of therapeutic multipotent mesenchymal stromal cells (MSCs). We therefore studied the possible harmful impact of immunogenic ABO blood groups antigens – genetically governed antigenic determinants – at all given steps of MSC-therapy, from cell isolation and preparation for clinical use, to final recipient outcome. We found that clinical MSCs do not inherently express or upregulate ABO blood group antigens after inflammatory challenge or in vitro differentiation. Although antigen adsorption from standard culture supplements was minimal, MSCs adsorbed small quantities of ABO antigen from fresh human AB plasma (ABP), dependent on antigen concentration and adsorption time. Compared to cells washed in non-immunogenic human serum albumin (HSA), MSCs washed with ABP elicited stronger blood responses after exposure to blood from healthy O donors in vitro, containing high titers of ABO antibodies. Clinical evaluation of hematopoietic stem cell transplant (HSCT) recipients found only very low titers of anti-A/B agglutination in these strongly immunocompromised patients at the time of MSC treatment. Patient analysis revealed a trend for lower clinical response in blood group O recipients treated with ABP-exposed MSC products, but not with HSA-exposed products. We conclude, that clinical grade MSCs are ABO-neutral, but the ABP used for washing and infusion of MSCs can contaminate the cells with immunogenic ABO substance and should therefore be substituted by non-immunogenic HSA, particularly when cells are given to immunocompentent individuals. PMID:24454787

  6. Constraints in antigen presentation severely restrict T cell recognition of the allogeneic fetus

    PubMed Central

    Erlebacher, Adrian; Vencato, Daniela; Price, Kelly A.; Zhang, Dorothy; Glimcher, Laurie H.

    2007-01-01

    How the fetus escapes rejection by the maternal immune system remains one of the major unsolved questions in transplantation immunology. Using a system to visualize both CD4+ and CD8+ T cell responses during pregnancy in mice, we find that maternal T cells become aware of the fetal allograft exclusively through “indirect” antigen presentation, meaning that T cell engagement requires the uptake and processing of fetal/placental antigen by maternal APCs. This reliance on a relatively minor allorecognition pathway removes a major threat to fetal survival, since it avoids engaging the large number of T cells that typically drive acute transplant rejection through their ability to directly interact with foreign MHC molecules. Furthermore, CD8+ T cells that indirectly recognize fetal/placental antigen undergo clonal deletion without priming for cytotoxic effector function and cannot induce antigen-specific fetal demise even when artificially activated. Antigen presentation commenced only at mid-gestation, in association with the endovascular invasion of placental trophoblasts and the hematogenous release of placental debris. Our results suggest that limited pathways of antigen presentation, in conjunction with tandem mechanisms of immune evasion, contribute to the unique immunological status of the fetus. The pronounced degree of T cell ignorance of the fetus also has implications for the pathophysiology of immune-mediated early pregnancy loss. PMID:17446933

  7. Atypical natural killer T-cell receptor recognition of CD1d–lipid antigens

    PubMed Central

    Le Nours, Jérôme; Praveena, T.; Pellicci, Daniel G.; Gherardin, Nicholas A.; Ross, Fiona J.; Lim, Ricky T.; Besra, Gurdyal S.; Keshipeddy, Santosh; Richardson, Stewart K.; Howell, Amy R.; Gras, Stephanie; Godfrey, Dale I.; Rossjohn, Jamie; Uldrich, Adam P.

    2016-01-01

    Crucial to Natural Killer T (NKT) cell function is the interaction between their T-cell receptor (TCR) and CD1d-antigen complex. However, the diversity of the NKT cell repertoire and the ensuing interactions with CD1d-antigen remain unclear. We describe an atypical population of CD1d–α-galactosylceramide (α-GalCer)-reactive human NKT cells that differ markedly from the prototypical TRAV10-TRAJ18-TRBV25-1+ type I NKT cell repertoire. These cells express a range of TCR α- and β-chains that show differential recognition of glycolipid antigens. Two atypical NKT TCRs (TRAV21-TRAJ8-TRBV7–8 and TRAV12-3-TRAJ27-TRBV6-5) bind orthogonally over the A′-pocket of CD1d, adopting distinct docking modes that contrast with the docking mode of all type I NKT TCR-CD1d-antigen complexes. Moreover, the interactions with α-GalCer differ between the type I and these atypical NKT TCRs. Accordingly, diverse NKT TCR repertoire usage manifests in varied docking strategies and specificities towards CD1d–α-GalCer and related antigens, thus providing far greater scope for diverse glycolipid antigen recognition. PMID:26875526

  8. Regulation of Sialyl Lewis Antigen Expression in Colon Cancer Cells by Sialidase NEU4*

    PubMed Central

    Shiozaki, Kazuhiro; Yamaguchi, Kazunori; Takahashi, Kohta; Moriya, Setsuko; Miyagi, Taeko

    2011-01-01

    Sialyl Lewis antigens, sialyl Lewis a and sialyl Lewis x, are utilized as tumor markers, and their increase in cancer is associated with tumor progression by enhancement of cancer cell adhesion to endothelial E-selectin. However, regulation mechanisms are not fully understood. We previously demonstrated that NEU4 is the only sialidase efficiently acting on mucins and it is down-regulated in colon cancer. To elucidate the significance of NEU4 down-regulation, we investigated sialyl Lewis antigens as endogenous substrates for the sialidase. NEU4 was found to hydrolyze the antigens in vitro and decrease cell surface levels much more effectively than other sialidases. Western blot, thin layer chromatography, and metabolic inhibition studies of desialylation products revealed NEU4 to preferentially catalyze sialyl Lewis antigens expressed on O-glycans. Cell adhesion to and motility and growth on E-selectin were significantly reduced by NEU4. E-selectin stimulation of colon cancer cells enhanced cell motility through activation of the p38/Hsp27/actin reorganization pathway, whereas NEU4 attenuated the signaling. On immunocytochemical analysis, some NEU4 molecules were localized at cell surfaces. Under hypoxia conditions whereby the antigens were increased concomitantly with several sialyl- and fucosyltransferases, NEU4 expression was markedly decreased. These results suggest that NEU4 plays an important role in control of sialyl Lewis antigen expression and its impairment in colon cancer. PMID:21521691

  9. Common Ewing sarcoma-associated antigens fail to induce natural T cell responses in both patients and healthy individuals.

    PubMed

    Altvater, Bianca; Kailayangiri, Sareetha; Theimann, Nadine; Ahlmann, Martina; Farwick, Nicole; Chen, Christiane; Pscherer, Sibylle; Neumann, Ilka; Mrachatz, Gabriele; Hansmeier, Anna; Hardes, Jendrik; Gosheger, Georg; Juergens, Heribert; Rossig, Claudia

    2014-10-01

    Disseminated or relapsed Ewing sarcoma (EwS) has remained fatal in the majority of patients. A promising approach to preventing relapse after conventional therapy is to establish tumor antigen-specific immune control. Efficient and specific T cell memory against the tumor depends on the expansion of rare T cells with native specificity against target antigens overexpressed by the tumor. Candidate antigens in EwS include six-transmembrane epithelial antigen of the prostate-1 (STEAP1), and the human cancer/testis antigens X-antigen family member 1 (XAGE1) and preferentially expressed antigen in melanoma (PRAME). Here, we screened normal donors and EwS patients for the presence of circulating T cells reactive with overlapping peptide libraries of these antigens by IFN-γ Elispot analysis. The majority of 22 healthy donors lacked detectable memory T cell responses against STEAP1, XAGE1 and PRAME. Moreover, ex vivo detection of T cells specific for these antigens in both blood and bone marrow were limited to a minority of EwS patients and required nonspecific T cell prestimulation. Cytotoxic T cells specific for the tumor-associated antigens were efficiently and reliably generated by in vitro priming using professional antigen-presenting cells and optimized cytokine stimulation; however, these T cells failed to interact with native antigen processed by target cells and with EwS cells expressing the antigen. We conclude that EwS-associated antigens fail to induce efficient T cell receptor (TCR)-mediated antitumor immune responses even under optimized conditions. Strategies based on TCR engineering could provide a more effective means to manipulating T cell immunity toward targeted elimination of tumor cells.

  10. Extraocular muscle satellite cells are high performance myo-engines retaining efficient regenerative capacity in dystrophin deficiency.

    PubMed

    Stuelsatz, Pascal; Shearer, Andrew; Li, Yunfei; Muir, Lindsey A; Ieronimakis, Nicholas; Shen, Qingwu W; Kirillova, Irina; Yablonka-Reuveni, Zipora

    2015-01-01

    Extraocular muscles (EOMs) are highly specialized skeletal muscles that originate from the head mesoderm and control eye movements. EOMs are uniquely spared in Duchenne muscular dystrophy and animal models of dystrophin deficiency. Specific traits of myogenic progenitors may be determinants of this preferential sparing, but very little is known about the myogenic cells in this muscle group. While satellite cells (SCs) have long been recognized as the main source of myogenic cells in adult muscle, most of the knowledge about these cells comes from the prototypic limb muscles. In this study, we show that EOMs, regardless of their distinctive Pax3-negative lineage origin, harbor SCs that share a common signature (Pax7(+), Ki67(-), Nestin-GFP(+), Myf5(nLacZ+), MyoD-positive lineage origin) with their limb and diaphragm somite-derived counterparts, but are remarkably endowed with a high proliferative potential as revealed in cell culture assays. Specifically, we demonstrate that in adult as well as in aging mice, EOM SCs possess a superior expansion capacity, contributing significantly more proliferating, differentiating and renewal progeny than their limb and diaphragm counterparts. These robust growth and renewal properties are maintained by EOM SCs isolated from dystrophin-null (mdx) mice, while SCs from muscles affected by dystrophin deficiency (i.e., limb and diaphragm) expand poorly in vitro. EOM SCs also retain higher performance in cell transplantation assays in which donor cells were engrafted into host mdx limb muscle. Collectively, our study provides a comprehensive picture of EOM myogenic progenitors, showing that while these cells share common hallmarks with the prototypic SCs in somite-derived muscles, they distinctively feature robust growth and renewal capacities that warrant the title of high performance myo-engines and promote consideration of their properties for developing new approaches in cell-based therapy to combat skeletal muscle wasting.

  11. Mapping of BrdU label-retaining dental pulp cells in growing teeth and their regenerative capacity after injuries.

    PubMed

    Ishikawa, Yuko; Ida-Yonemochi, Hiroko; Suzuki, Hironobu; Nakakura-Ohshima, Kuniko; Jung, Han-Sung; Honda, Masaki J; Ishii, Yumiko; Watanabe, Nobukazu; Ohshima, Hayato

    2010-09-01

    Recent studies have demonstrated that human dental pulp contains adult stem cells. A pulse of the thymidine analog BrdU given to young animals at the optimal time could clarify where slow-cycling long-term label-retaining cells (LRCs), putative adult stem cells, reside in the pulp tissue. This study focuses on the mapping of LRCs in growing teeth and their regenerative capacity after tooth injuries. Two to seven peritoneal injections of BrdU into pregnant Wistar rats revealed slow-cycling long-term dense LRCs in the mature tissues of born animals. Numerous dense LRCs were postnatally decreased in number and reached a plateau at 4 weeks after birth when they mainly resided in the center of the dental pulp, associating with blood vessels. Mature dental pulp cells were stained with Hoechst 33342 and sorted into (<0.76%) side population cells using FACS, which included dense LRCs. Some dense LRCs co-expressed mesenchymal stem cell markers such as STRO-1 or CD146. Tooth injuries caused degeneration of the odontoblast layer, and newly differentiated odontoblast-like cells contained LRCs. Thus, dense LRCs in mature pulp tissues were supposed to be dental pulp stem cells possessing regenerative capacity for forming newly differentiated odontoblast-like cells. The present study proposes the new hypothesis that both granular and dense LRCs are equipped in the dental pulp and that the dense LRCs with proliferative capacity play crucial roles in the pulpal healing process following exogenous stimuli in cooperation with the granular LRCs.

  12. Msx1-modulated muscle satellite cells retain a primitive state and exhibit an enhanced capacity for osteogenic differentiation.

    PubMed

    Ding, Ke; Liu, Wen-Ying; Zeng, Qiang; Hou, Fang; Xu, Jian-Zhong; Yang, Zhong

    2017-03-01

    Multipotent muscle satellite cells (MuSCs) have been identified as potential seed cells for bone tissue engineering. However, MuSCs exhibit a rapid loss of stemness after in vitro culturing, thereby compromising their therapeutic efficiency. Muscle segment homeobox gene 1 (msx1) has been found to induce the dedifferentiation of committed progenitor cells, as well as terminally differentiated myotubes. In this study, a Tet-off retroviral gene delivery system was used to modulate msx1 expression. After ten passages, MuSCs that did not express msx-1 (e.g., the non-msx1 group) were compared with MuSCs with induced msx-1 expression (e.g., the msx1 group). The latter group exhibited a more juvenile morphology, it contained a significantly lower percentage of senescent cells characterized by positive β-galactosidase staining, and it exhibited increased proliferation and a higher proliferation index. Immunocytochemical stainings further detected a more primitive gene expression profile for the msx1 group, while osteogenic differentiation assays and ectopic bone formation assays demonstrated an improved capacity for the msx1 group to undergo osteogenic differentiation. These results suggest that transient expression of msx1 in MuSCs can retain a primitive state, thereby enhancing their capacity for osteogenic differentiation and restoring the potential for MuSCs to serve as seed cells for bone tissue engineering.

  13. Cell-mediated immunity to hepatitis B virus antigens in mice: correlation of in vivo and in vitro assays.

    PubMed Central

    De Moerloose, P A; Frazer, I H; Sewell, W A; Collins, E J; Mackay, I R

    1986-01-01

    Cell mediated immunity (CMI) to hepatitis B viral antigens was studied in BALB/mice after immunization with purified hepatitis B surface antigen (HBsAg), or core antigen (HBcAg), with adjuvants. The two in vitro assays for cell-mediated immunity (CMI), utilizing lymph node cells, were release of interferon after exposure to antigen, and blast transformation of lymphocytes, and the in vivo assay was ear swelling at 24 h after local injection of antigen. Immunization with HBsAg or HBcAg with adjuvants induced antigen-specific cutaneous reactivity; if no adjuvants were given, immunization with HBcAg, but not HBsAg, induced cutaneous reactivity. CMI could be adoptively transferred by lymph node cells, but for only a limited period after immunization with HbsAg or HBcAg. The ability of lymph node cells from mice immunized with HBV antigens to transfer adoptively CMI correlated well with their production of interferon after challenge with antigen in vitro, but less well with blastogenesis after challenge with antigen in vitro, or with cutaneous reactivity to antigen in the donor mouse. Reliable antigen-specific lymphokine release assays, rather than blast transformation of lymphocytes or cutaneous reactivity after antigen challenge, are required to assess CMI to HBV antigens in the mouse and, by inference, in man. Images Fig. 1 PMID:3091300

  14. Dissecting the Tumor Myeloid Compartment Reveals Rare Activating Antigen Presenting Cells, Critical for T cell Immunity

    PubMed Central

    Broz, Miranda; Binnewies, Mikhail; Boldajipour, Bijan; Nelson, Amanda; Pollock, Joshua; Erle, David; Barczak, Andrea; Rosenblum, Michael; Daud, Adil; Barber, Diane; Amigorena, Sebastian; van’t Veer, Laura J.; Sperling, Anne; Wolf, Denise; Krummel, Matthew F.

    2014-01-01

    SUMMARY It is well understood that antigen-presenting cells (APC) within tumors typically do not maintain cytotoxic T cell (CTL) function, despite engaging them. Across multiple mouse tumor models and human tumor biopsies, we have delineated the intratumoral dendritic-cell (DC) populations as distinct from macrophage populations. Within these, CD103+ DCs are extremely sparse and yet remarkably capable CTL stimulators. These are uniquely dependent upon IRF8, Zbtb46 and Batf3 transcription factors and generated by GM-CSF and Flt3L cytokines. Regressing tumors have higher proportions of these cells, T-cell dependent immune clearance relies upon them, and abundance of their transcripts in human tumors correlates with clinical outcome. This cell type presents opportunities for prognostic and therapeutic approaches across multiple cancer types. PMID:25446897

  15. Antigen-specific regulation of IgE antibodies by non-antigen-specific γδ T cells1

    PubMed Central

    Huang, Yafei; Aydintug, M. Kemal; Loomis, Joshua; MacLeod, Megan K.; McKee, Amy S.; Kirchenbaum, Greg; Jakubzick, Claudia V.; Kedl, Ross M.; Sun, Deming; Jacobelli, Jordan; O'Brien, Rebecca L.; Born, Willi K.

    2013-01-01

    We re-examined the observation that γδ T cells, when transferred from mice tolerized to an inhaled conventional antigen (Ag), suppress the allergic IgE response to this Ag specifically. Using ovalbumin and hen egg lysozyme in crisscross fashion, we confirmed the Ag-specific IgE regulatory effect of the γδ T cells. Although only Vγ4+ γδ T cells are regulators, the Ag specificity does not stem from specificity of their γδ TCRs. Instead, the Vγ4+ γδ T cells failed to respond to either Ag, but rapidly acquired Ag-specific regulatory function in vivo following i.v. injection of non-T cells derived from the spleen of Ag-tolerized mice. This correlated with their in vivo Ag acquisition from i.v. injected Ag-loaded splenic non-T cells, and in vivo transfer of membrane label provided evidence for direct contact between the injected splenic non-T cells and the Vγ4+ γδ T cells. Together, our data suggest that Ag itself, when acquired by γδ T cells, directs the specificity of their IgE suppression. PMID:23275606

  16. NY-ESO-1 antigen-reactive T cell receptors exhibit diverse therapeutic capability

    PubMed Central

    Sommermeyer, Daniel; Conrad, Heinke; Krönig, Holger; Gelfort, Haike; Bernhard, Helga; Uckert, Wolfgang

    2013-01-01

    The cancer-testis antigen NY-ESO-1 has been used as a target for different immunotherapies like vaccinations and adoptive transfer of antigen-specific cytotoxic T cells, as it is expressed in various tumor types and has limited expression in normal cells. The in vitro generation of T cells with defined antigen specificity by T cell receptor (TCR) gene transfer is an established method to create cells for immunotherapy. However, an extensive characterization of TCR which are candidates for treatment of patients is crucial for successful therapies. The TCR has to be efficiently expressed, their affinity to the desired antigen should be high enough to recognize low amounts of endogenously processed peptides on tumor cells, and the TCR should not be cross-reactive to other antigens. We characterized three NY-ESO-1 antigen-reactive cytotoxic T lymphocyte clones which were generated by different approaches of T cell priming (autologous, allogeneic), and transferred their TCR into donor T cells for more extensive evaluations. Although one TCR most efficiently bound MHC-multimers loaded with NY-ESO-1 peptide, T cells expressing this transgenic TCR were not able to recognize endogenously processed antigen. A second TCR recognized HLA-A2 independent of the bound peptide beside its much stronger recognition of NY-ESO-1 bound to HLA-A2. A third TCR displayed an intermediate but peptide-specific performance in all functional assays and, therefore, is the most promising candidate TCR for further clinical development. Our data indicate that multiple parameters of TCR gene-modified T cells have to be evaluated to identify an optimal TCR candidate for adoptive therapy. PMID:22907642

  17. 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.

  18. Discovery of chemotherapy-associated ovarian cancer antigens by interrogating memory T cells.

    PubMed

    Paroli, M