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Sample records for improving t-cell assays

  1. A new sensitive and quantitative HTLV-I-mediated cell fusion assay in T cells

    International Nuclear Information System (INIS)

    Pare, Marie-Eve; Gauthier, Sonia; Landry, Sebastien; Sun Jiangfeng; Legault, Eric; Leclerc, Denis; Tanaka, Yuetsu; Marriott, Susan J.; Tremblay, Michel J.; Barbeau, Benoit

    2005-01-01

    Similar to several other viruses, human T cell leukemia virus type I (HTLV-I) induces the formation of multinucleated giant cells (also known as syncytium) when amplified in tissue culture. These syncytia result from the fusion of infected cells with uninfected cells. Due to the intrinsic difficulty of infecting cells with cell-free HTLV-I virions, syncytium formation has become an important tool in the study of HTLV-I infection and transmission. Since most HTLV-I-based cell fusion assays rely on the use of non-T cells, the aim of this study was to optimize a new HTLV-I-induced cell fusion assay in which HTLV-I-infected T cell lines are co-cultured with T cells that have been transfected with an HTLV-I long terminal repeat (LTR) luciferase reporter construct. We demonstrate that co-culture of various HTLV-I-infected T cells with different transfected T cell lines resulted in induction of luciferase activity. Cell-to-cell contact and expression of the viral gp46 envelope protein was crucial for this induction while other cell surface proteins (including HSC70) did not have a significant effect. This quantitative assay was shown to be very sensitive. In this assay, the cell fusion-mediated activation of NF-κB and the HTLV-I LTR occurred through previously described Tax-dependent signaling pathways. This assay also showed that cell fusion could activate Tax-inducible cellular promoters. These results thus demonstrate that this new quantitative HTLV-I-dependent cell fusion assay is versatile, highly sensitive, and can provide an important tool to investigate cellular promoter activation and intrinsic signaling cascades that modulate cellular gene expression

  2. An improved 96-well turbidity assay for T4 lysozyme activity.

    Science.gov (United States)

    Toro, Tasha B; Nguyen, Thao P; Watt, Terry J

    2015-01-01

    T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function. Despite being extensively studied, a reliable, quantitative activity assay for T4L has not been developed. Here, we present an improved T4L turbidity assay as well as an affinity-based T4L expression and purification protocol. This assay is designed for 96-well format and utilizes conditions amenable for both T4L and other lysozymes. This protocol enables easy, efficient, and quantitative characterization of T4L variants and allows comparison between different lysozymes. Our method: •Is applicable for all lysozymes, with enhanced sensitivity for T4 lysozyme compared to other 96-well plate turbidity assays;•Utilizes standardized conditions for comparing T4 lysozyme variants and other lysozymes; and•Incorporates a simplified expression and purification protocol for T4 lysozyme.

  3. Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay

    Science.gov (United States)

    The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...

  4. A robust and scalable TCR-based reporter cell assay to measure HIV-1 Nef-mediated T cell immune evasion.

    Science.gov (United States)

    Anmole, Gursev; Kuang, Xiaomei T; Toyoda, Mako; Martin, Eric; Shahid, Aniqa; Le, Anh Q; Markle, Tristan; Baraki, Bemuluyigza; Jones, R Brad; Ostrowski, Mario A; Ueno, Takamasa; Brumme, Zabrina L; Brockman, Mark A

    2015-11-01

    HIV-1 evades cytotoxic T cell responses through Nef-mediated downregulation of HLA class I molecules from the infected cell surface. Methods to quantify the impact of Nef on T cell recognition typically employ patient-derived T cell clones; however, these assays are limited by the cost and effort required to isolate and maintain primary cell lines. The variable activity of different T cell clones and the limited number of cells generated by re-stimulation can also hinder assay reproducibility and scalability. Here, we describe a heterologous T cell receptor reporter assay and use it to study immune evasion by Nef. Induction of NFAT-driven luciferase following co-culture with peptide-pulsed or virus-infected target cells serves as a rapid, quantitative and antigen-specific measure of T cell recognition of its cognate peptide/HLA complex. We demonstrate that Nef-mediated downregulation of HLA on target cells correlates inversely with T cell receptor-dependent luminescent signal generated by effector cells. This method provides a robust, flexible and scalable platform that is suitable for studies to measure Nef function in the context of different viral peptide/HLA antigens, to assess the function of patient-derived Nef alleles, or to screen small molecule libraries to identify novel Nef inhibitors. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. A novel dendritic cell-based direct ex vivo assay for detection and enumeration of circulating antigen-specific human T cells.

    Science.gov (United States)

    Carrio, Roberto; Zhang, Ge; Drake, Donald R; Schanen, Brian C

    2018-05-07

    Although a variety of assays have been used to examine T cell responses in vitro, standardized ex vivo detection of antigen-specific CD4 + T cells from human circulatory PBMCs remains constrained by low-dimensional characterization outputs and the need for polyclonal, mitogen-induced expansion methods to generate detectable response signals. To overcome these limitations, we developed a novel methodology utilizing antigen-pulsed autologous human dendritic target cells in a rapid and sensitive assay to accurately enumerate antigen-specific CD4 + T cell precursor frequency by multiparametric flow cytometry. With this approach, we demonstrate the ability to reproducibly quantitate poly-functional T cell responses following both primary and recall antigenic stimulation. Furthermore, this approach enables more comprehensive phenotypic profiling of circulating antigen-specific CD4 + T cells, providing valuable insights into the pre-existing polarization of antigen-specific T cells in humans. Combined, this approach permits sensitive and detailed ex vivo detection of antigen-specific CD4 + T cells delivering an important tool for advancing vaccine, immune-oncology and other therapeutic studies.

  6. Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

    Directory of Open Access Journals (Sweden)

    Magdalena Tary-Lehmann

    2012-05-01

    Full Text Available Chronic allograft rejection is in part mediated by host T cells that recognize allogeneic antigens on transplanted tissue. One factor that determines the outcome of a T cell response is clonal size, while another is the effector quality. Studies of alloimmune predictors of transplant graft survival have most commonly focused on only one measure of the alloimmune response. Because differing qualities and frequencies of the allospecific T cell response may provide distinctly different information we analyzed the relationship between frequency of soluble antigen and allo-antigen specific memory IFN-g secreting CD4 and CD8 T cells, their ability to secrete IL-2, and their proliferative capacity, while accounting for cognate and bystander proliferation. The results show proliferative responses primarily reflect on IL-2 production by antigen-specific T cells, and that proliferating cells in such assays entail a considerable fraction of bystander cells. On the other hand, proliferation (and IL-2 production did not reflect on the frequency of IFN-γ producing memory cells, a finding particularly accentuated in the CD8 T cell compartment. These data provide rationale for considering both frequency and effector function of pre-transplant T cell reactivity when analyzing immune predictors of graft rejection.

  7. Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

    Science.gov (United States)

    Anthony, Donald D.; Milkovich, Kimberly A.; Zhang, Wenji; Rodriguez, Benigno; Yonkers, Nicole L.; Tary-Lehmann, Magdalena; Lehmann, Paul V.

    2012-01-01

    Chronic allograft rejection is in part mediated by host T cells that recognize allogeneic antigens on transplanted tissue. One factor that determines the outcome of a T cell response is clonal size, while another is the effector quality. Studies of alloimmune predictors of transplant graft survival have most commonly focused on only one measure of the alloimmune response. Because differing qualities and frequencies of the allospecific T cell response may provide distinctly different information we analyzed the relationship between frequency of soluble antigen and allo-antigen specific memory IFN-γ secreting CD4 and CD8 T cells, their ability to secrete IL-2, and their proliferative capacity, while accounting for cognate and bystander proliferation. The results show proliferative responses primarily reflect on IL-2 production by antigen-specific T cells, and that proliferating cells in such assays entail a considerable fraction of bystander cells. On the other hand, proliferation (and IL-2 production) did not reflect on the frequency of IFN-γ producing memory cells, a finding particularly accentuated in the CD8 T cell compartment. These data provide rationale for considering both frequency and effector function of pre-transplant T cell reactivity when analyzing immune predictors of graft rejection. PMID:24710419

  8. A molecular assay for sensitive detection of pathogen-specific T-cells.

    Directory of Open Access Journals (Sweden)

    Victoria O Kasprowicz

    Full Text Available Here we describe the development and validation of a highly sensitive assay of antigen-specific IFN-γ production using real time quantitative PCR (qPCR for two reporters--monokine-induced by IFN-γ (MIG and the IFN-γ inducible protein-10 (IP10. We developed and validated the assay and applied it to the detection of CMV, HIV and Mycobacterium tuberculosis (MTB specific responses, in a cohort of HIV co-infected patients. We compared the sensitivity of this assay to that of the ex vivo RD1 (ESAT-6 and CFP-10-specific IFN-γ Elispot assay. We observed a clear quantitative correlation between the two assays (P<0.001. Our assay proved to be a sensitive assay for the detection of MTB-specific T cells, could be performed on whole blood samples of fingerprick (50 uL volumes, and was not affected by HIV-mediated immunosuppression. This assay platform is potentially of utility in diagnosis of infection in this and other clinical settings.

  9. Limit-dilution assay and clonal expansion of all T cells capable of proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Chen, W.F.; Wilson, A.; Scollay, R.; Shortman, K. (Walter and Eliza Hall Inst. of Medical Research, Parkville (Australia))

    1982-08-13

    A limit-dilution microculture system is presented in which almost all mature T cells, cultured at a level of about 1 cell/well, grow and expand to clones averaging 60,000 cells over an 8-9 day period. Cloning efficiency is 70-100%, so the set of expanded clones is representative of the starting T-cell population. T cells of all Lyt phenotypes form clones of progeny cells. The system involves culture in flat-bottom microtitre trays, in the presence of concanavalin A as the initiating stimulus, together with appropriately irradiated spleen filler cells and a supplementary source of soluble T cell growth factors. The resultant clones may be screened for cytolytic function, as described in the accompanying paper. The system may be used to assay the level of T cells capable of expansion or precursor function (PTL-p) by using (/sup 3/H)TdR uptake as a readout for the presence or absence of proliferating clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of complicating suppressor or helper effects.

  10. Limit-dilution assay and clonal expansion of all T cells capable of proliferation

    International Nuclear Information System (INIS)

    Chen, W.-F.; Wilson, A.; Scollay, R.; Shortman, K.

    1982-01-01

    A limit-dilution microculture system is presented in which almost all mature T cells, cultured at a level of about 1 cell/well, grow and expand to clones averaging 60,000 cells over an 8-9 day period. Cloning efficiency is 70-100%, so the set of expanded clones is representative of the starting T-cell population. T cells of all Lyt phenotypes form clones of progeny cells. The system involves culture in flat-bottom microtitre trays, in the presence of concanavalin A as the initiating stimulus, together with appropriately irradiated spleen filler cells and a supplementary source of soluble T cell growth factors. The resultant clones may be screened for cytolytic function, as described in the accompanying paper. The system may be used to assay the level of T cells capable of expansion or precursor function (PTL-p) by using [ 3 H]TdR uptake as a readout for the presence or absence of proliferating clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of complicating suppressor or helper effects. (Auth.)

  11. Semi-automated limit-dilution assay and clonal expansion of all T-cell precursors of cytotoxic lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, A.; Chen, W.F.; Scollay, R.; Shortman, K. (Walter and Eliza Hall Inst. of Medical Research, Parkville (Australia))

    1982-08-13

    A limit-dilution microculture system is described, where almost all precursor T cells of the cytotoxic lineage (CTL-p) develop into extended clones of cytotoxic T cells (CTL), which are then detected with a new radio-autographic /sup 111/In-release assay. The principle is to polyclonally activate all T cells with concanavalin A, to expand the resultant clones over an 8-9 day period in cultures saturated with growth factors, then to detect all clones with cytotoxic function by phytohaemagglutinin mediated lysis of P815 tumour cells. The key variables for obtaining high cloning efficiency are the use of flat-bottomed 96-well culture trays, the use of appropriately irradiated spleen filler cells, and the inclusion of a T-cell growth factor supplement. Cultures are set up at input levels of around one T cell per well. Forty percent of T cells then form CTL clones readily detected by the cytotoxic assay. The lytic activity of the average clone is equivalent to 3000 CTL, but clone size appears to be much larger. The precursor cells are predominantly if not entirely from the Lyt 2/sup +/ T-cell subclass and almost all cells of this subclass form cytolytic clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of the CTL-p frequency estimates being distorted by helper or suppressor effects.

  12. Semi-automated limit-dilution assay and clonal expansion of all T-cell precursors of cytotoxic lymphocytes

    International Nuclear Information System (INIS)

    Wilson, A.; Chen, W.-F.; Scollay, R.; Shortman, K.

    1982-01-01

    A limit-dilution microculture system is described, where almost all precursor T cells of the cytotoxic lineage (CTL-p) develop into extended clones of cytotoxic T cells (CTL), which are then detected with a new radio-autographic 111 In-release assay. The principle is to polyclonally activate all T cells with concanavalin A, to expand the resultant clones over an 8-9 day period in cultures saturated with growth factors, then to detect all clones with cytotoxic function by phytohaemagglutinin mediated lysis of P815 tumour cells. The key variables for obtaining high cloning efficiency are the use of flat-bottomed 96-well culture trays, the use of appropriately irradiated spleen filler cells, and the inclusion of a T-cell growth factor supplement. Cultures are set up at input levels of around one T cell per well. Forty percent of T cells then form CTL clones readily detected by the cytotoxic assay. The lytic activity of the average clone is equivalent to 3000 CTL, but clone size appears to be much larger. The precursor cells are predominantly if not entirely from the Lyt 2 + T-cell subclass and almost all cells of this subclass form cytolytic clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of the CTL-p frequency estimates being distorted by helper or suppressor effects. (Auth.)

  13. In vitro immunomodulation of a whole blood IFN-γ release assay enhances T cell responses in subjects with latent tuberculosis infection.

    Directory of Open Access Journals (Sweden)

    Rajiv L Gaur

    Full Text Available Activation of innate immunity via pathogen recognition receptors (PRR modulates adaptive immune responses. PRR ligands are being exploited as vaccine adjuvants and as therapeutics, but their utility in diagnostics has not been explored. Interferon-gamma (IFN-γ release assays (IGRAs are functional T cell assays used to diagnose latent tuberculosis infection (LTBI; however, novel approaches are needed to improve their sensitivity.In vitro immunomodulation of a whole blood IGRA (QuantiFERON®-TB GOLD In-Tube with Toll-like receptor agonists poly(I:C, LPS, and imiquimod was performed on blood from subjects with LTBI and negative controls.In vitro immunomodulation significantly enhanced the response of T cells stimulated with M. tuberculosis antigens from subjects with LTBI but not from uninfected controls. Immunomodulation of IGRA revealed T cell responses in subjects with LTBI whose T cells otherwise do not respond to in vitro stimulation with antigens alone. Similar to their in vivo functions, addition of poly(I:C and LPS to whole blood induced secretion of inflammatory cytokines and IFN-α and enhanced the surface expression of antigen presenting and costimulatory molecules on antigen presenting cells.In vitro immunomodulation of whole blood IGRA may be an effective strategy for enhancing the sensitivity of T cells for diagnosis of LTBI.

  14. An assay to monitor HIV-1 protease activity for the identification of novel inhibitors in T-cells.

    Directory of Open Access Journals (Sweden)

    Brett J Hilton

    Full Text Available The emergence of resistant HIV strains, together with the severe side-effects of existing drugs and lack of development of effective anti-HIV vaccines highlight the need for novel antivirals, as well as innovative methods to facilitate their discovery. Here, we have developed an assay in T-cells to monitor the proteolytic activity of the HIV-1 protease (PR. The assay is based on the inducible expression of HIV-1 PR fused within the Gal4 DNA-binding and transactivation domains. The fusion protein binds to the Gal4 responsive element and activates the downstream reporter, enhanced green fluorescent protein (eGFP gene only in the presence of an effective PR Inhibitor (PI. Thus, in this assay, eGFP acts as a biosensor of PR activity, making it ideal for flow cytometry based screening. Furthermore, the assay was developed using retroviral technology in T-cells, thus providing an ideal environment for the screening of potential novel PIs in a cell-type that represents the natural milieu of HIV infection. Clones with the highest sensitivity, and robust, reliable and reproducible reporter activity, were selected. The assay is easily adaptable to other PR variants, a multiplex platform, as well as to high-throughput plate reader based assays and will greatly facilitate the search for novel peptide and chemical compound based PIs in T-cells.

  15. Large-scale prospective T cell function assays in shipped, unfrozen blood samples

    DEFF Research Database (Denmark)

    Hadley, David; Cheung, Roy K; Becker, Dorothy J

    2014-01-01

    , for measuring core T cell functions. The Trial to Reduce Insulin-dependent diabetes mellitus in the Genetically at Risk (TRIGR) type 1 diabetes prevention trial used consecutive measurements of T cell proliferative responses in prospectively collected fresh heparinized blood samples shipped by courier within...... cell immunocompetence. We have found that the vast majority of the samples were viable up to 3 days from the blood draw, yet meaningful responses were found in a proportion of those with longer travel times. Furthermore, the shipping time of uncooled samples significantly decreased both the viabilities...... North America. In this article, we report on the quality control implications of this simple and pragmatic shipping practice and the interpretation of positive- and negative-control analytes in our assay. We used polyclonal and postvaccination responses in 4,919 samples to analyze the development of T...

  16. Monitoring T-Cell Responses in Translational Studies: Optimization of Dye-Based Proliferation Assay for Evaluation of Antigen-Specific Responses

    Directory of Open Access Journals (Sweden)

    Anja Ten Brinke

    2017-12-01

    Full Text Available Adoptive therapy with regulatory T cells or tolerance-inducing antigen (Ag-presenting cells is innovative and promising therapeutic approach to control undesired and harmful activation of the immune system, as observed in autoimmune diseases, solid organ and bone marrow transplantation. One of the critical issues to elucidate the mechanisms responsible for success or failure of these therapies and define the specificity of the therapy is the evaluation of the Ag-specific T-cell responses. Several efforts have been made to develop suitable and reproducible assays. Here, we focus on dye-based proliferation assays. We highlight with practical examples the fundamental issues to take into consideration for implementation of an effective and sensitive dye-based proliferation assay to monitor Ag-specific responses in patients. The most critical points were used to design a road map to set up and analyze the optimal assay to assess Ag-specific T-cell responses in patients undergoing different treatments. This is the first step to optimize monitoring of tolerance induction, allowing comparison of outcomes of different clinical studies. The road map can also be applied to other therapeutic interventions, not limited to tolerance induction therapies, in which Ag-specific T-cell responses are relevant such as vaccination approaches and cancer immunotherapy.

  17. Comet assay on tetraploid yeast cells

    DEFF Research Database (Denmark)

    Rank, Jette; Syberg, Kristian; Jensen, Klara

    2009-01-01

    Tetraploid yeast cells (Saccharomyces cerevisiae) were used in the comet assay with the intention of developing a new, fast and easy assay for detecting environmental genotoxic agents without using higher organisms. Two DNA-damaging chemicals, H2O2 and acrylamide, together with wastewater from...... three municipal treatment plants were tested for their effect on the yeast-cell DNA. The main problem with using yeast in the comet assay is the necessity to degrade the cell wall. This was achieved by using Zymolase 100 T twice during the procedure, since Zymolase 20 T did not open the cell wall....... Analytical problems that arose due to the small amount of DNA in the yeast nuclei in haploid and diploid cells, which contain 13 Mbp and 26 Mbp DNA per cell, respectively, were solved by using tetraploid yeast cells (52 Mbp) instead. DNA damage was shown after exposure to H2O2 and acrylamide. The lowest dose...

  18. Recovery Profiles of T-Cell Subsets Following Low-Dose Total Body Irradiation and Improvement With Cinnamon

    International Nuclear Information System (INIS)

    Zheng, Xiaodan; Guo, Yuqi; Wang, Lei; Zhang, Honghai; Wang, Shaobo; Wang, Li; An, Lei; Zhou, Xianbin; Li, Xia; Yao, Chengfang

    2015-01-01

    Purpose: Inefficient T-cell reconstitution from x-ray–induced immune damage reduces antitumor response. To understand the profile of T-cell reconstitution after irradiation will overcome the barrier of antitumor immunity. This study aimed to identify the recovery profile of T-cell subsets following x-ray irradiation and to highlight the role of cinnamon on efficient T-cell restoration postexposure in the antitumor response. Methods and Materials: CD3"+, CD8"+, and CD4"+ T cells and Th1, Th2, Th17, and regulatory T (Treg) cells were evaluated at different time points after single low-dose total body irradiation (SLTBI) with or without cinnamon treatments. T-bet, GATA3, RORγt, and Foxp3 signaling specific for Th1, Th2, Th17, and Treg were also analyzed by RT-PCR assay. The effects of cinnamon on efficient T-cell subset reconstitution was confirmed in a lung melanoma model in irradiated mice. Results: Reconstitution of CD4"+ T cells was delayed more than that of CD8"+ T cells in T-cell restoration after SLTBI. The production of IFNγ by Th1 or Tc1 cells was sharply decreased and was accompanied by reduced T-bet mRNA, even when total T-cell numbers had recovered; the frequencies of Th17 and Treg cells and their specific transcription factors (RORγt and Foxp3, respectively) were obviously increased. Irradiation-induced inefficient T-cell reconstitution impaired the antitumor capacities in the lung melanoma model. Pretreatment with cinnamon in irradiated mice accelerated the generation of Th1 and reduced the differentiation of Treg cells by activating T-bet and limiting transcriptions of Foxp3. Improvement resulting from cinnamon pretreatment on the efficient T-cell recovery profile from SLTBI promoted antitumor immunity in the lung melanoma model. Conclusions: T-cell reconstitution from SLTBI was characterized by impaired Th1 and elevated Th17 and Treg cells. Cinnamon effectively improved the imbalance of T-cell subsets by promoting the proliferation of Th1 and

  19. Recovery Profiles of T-Cell Subsets Following Low-Dose Total Body Irradiation and Improvement With Cinnamon

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Xiaodan [Key Laboratory for Tumor Immunology and Traditional Chinese Medicine Immunology, Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan (China); School of Medicine and Life Science, University of Jinan-Shandong Academy of Medical Science, Jinan (China); Guo, Yuqi [Key Laboratory for Tumor Immunology and Traditional Chinese Medicine Immunology, Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan (China); Wang, Lei [Second Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan (China); Zhang, Honghai [Key Laboratory for Tumor Immunology and Traditional Chinese Medicine Immunology, Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan (China); Wang, Shaobo [Shandong University, Jinan (China); Wang, Li [Key Laboratory for Tumor Immunology and Traditional Chinese Medicine Immunology, Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan (China); An, Lei [Key Laboratory for Tumor Immunology and Traditional Chinese Medicine Immunology, Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan (China); School of Medicine and Life Science, University of Jinan-Shandong Academy of Medical Science, Jinan (China); Zhou, Xianbin; Li, Xia [Key Laboratory for Tumor Immunology and Traditional Chinese Medicine Immunology, Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan (China); Yao, Chengfang, E-mail: yaocf9941@163.com [Key Laboratory for Tumor Immunology and Traditional Chinese Medicine Immunology, Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan (China)

    2015-12-01

    Purpose: Inefficient T-cell reconstitution from x-ray–induced immune damage reduces antitumor response. To understand the profile of T-cell reconstitution after irradiation will overcome the barrier of antitumor immunity. This study aimed to identify the recovery profile of T-cell subsets following x-ray irradiation and to highlight the role of cinnamon on efficient T-cell restoration postexposure in the antitumor response. Methods and Materials: CD3{sup +}, CD8{sup +}, and CD4{sup +} T cells and Th1, Th2, Th17, and regulatory T (Treg) cells were evaluated at different time points after single low-dose total body irradiation (SLTBI) with or without cinnamon treatments. T-bet, GATA3, RORγt, and Foxp3 signaling specific for Th1, Th2, Th17, and Treg were also analyzed by RT-PCR assay. The effects of cinnamon on efficient T-cell subset reconstitution was confirmed in a lung melanoma model in irradiated mice. Results: Reconstitution of CD4{sup +} T cells was delayed more than that of CD8{sup +} T cells in T-cell restoration after SLTBI. The production of IFNγ by Th1 or Tc1 cells was sharply decreased and was accompanied by reduced T-bet mRNA, even when total T-cell numbers had recovered; the frequencies of Th17 and Treg cells and their specific transcription factors (RORγt and Foxp3, respectively) were obviously increased. Irradiation-induced inefficient T-cell reconstitution impaired the antitumor capacities in the lung melanoma model. Pretreatment with cinnamon in irradiated mice accelerated the generation of Th1 and reduced the differentiation of Treg cells by activating T-bet and limiting transcriptions of Foxp3. Improvement resulting from cinnamon pretreatment on the efficient T-cell recovery profile from SLTBI promoted antitumor immunity in the lung melanoma model. Conclusions: T-cell reconstitution from SLTBI was characterized by impaired Th1 and elevated Th17 and Treg cells. Cinnamon effectively improved the imbalance of T-cell subsets by promoting the

  20. Large-scale prospective T cell function assays in shipped, unfrozen blood samples: experiences from the multicenter TRIGR trial.

    Science.gov (United States)

    Hadley, David; Cheung, Roy K; Becker, Dorothy J; Girgis, Rose; Palmer, Jerry P; Cuthbertson, David; Krischer, Jeffrey P; Dosch, Hans-Michael

    2014-02-01

    Broad consensus assigns T lymphocytes fundamental roles in inflammatory, infectious, and autoimmune diseases. However, clinical investigations have lacked fully characterized and validated procedures, equivalent to those of widely practiced biochemical tests with established clinical roles, for measuring core T cell functions. The Trial to Reduce Insulin-dependent diabetes mellitus in the Genetically at Risk (TRIGR) type 1 diabetes prevention trial used consecutive measurements of T cell proliferative responses in prospectively collected fresh heparinized blood samples shipped by courier within North America. In this article, we report on the quality control implications of this simple and pragmatic shipping practice and the interpretation of positive- and negative-control analytes in our assay. We used polyclonal and postvaccination responses in 4,919 samples to analyze the development of T cell immunocompetence. We have found that the vast majority of the samples were viable up to 3 days from the blood draw, yet meaningful responses were found in a proportion of those with longer travel times. Furthermore, the shipping time of uncooled samples significantly decreased both the viabilities of the samples and the unstimulated cell counts in the viable samples. Also, subject age was significantly associated with the number of unstimulated cells and T cell proliferation to positive activators. Finally, we observed a pattern of statistically significant increases in T cell responses to tetanus toxin around the timing of infant vaccinations. This assay platform and shipping protocol satisfy the criteria for robust and reproducible long-term measurements of human T cell function, comparable to those of established blood biochemical tests. We present a stable technology for prospective disease-relevant T cell analysis in immunological diseases, vaccination medicine, and measurement of herd immunity.

  1. Immunobiology of T cell responses to Mls-locus-disparate stimulator cells. III. Helper and cytolytic functions of cloned, Mls-reactive T cell lines

    International Nuclear Information System (INIS)

    Katz, M.E.; Tite, J.P.; Janeway, C.A. Jr.

    1986-01-01

    Mls-specific T cell clones derived by limiting dilution were tested for cytotoxic activity in a lectin-dependent 51 Cr-release assay. All the T cell clones tested were cytotoxic in such an assay in apparent contrast to previous reports (1, 2). However, only those target cells sensitive to cytolysis by other L3T4a + cytolytic T cells (3) were killed by Mls-specific T cell clones in short term 51 Cr-release assays, possibly explaining this discrepancy. All the T cell clones tested were L3T4a + ,Lyt-2 - and stimulated B cells from Mls strains of mice to proliferate and secrete immunoglobulin. Furthermore, lysis of innocent bystander targets was observed when the T cells were stimulated with Mls-disparate stimulator cells. These results are consistent with those obtained with L3T4a - T cells specific for protein antigen:self Ia and that express cytotoxic potential (3)

  2. Automation of the ELISpot assay for high-throughput detection of antigen-specific T-cell responses.

    Science.gov (United States)

    Almeida, Coral-Ann M; Roberts, Steven G; Laird, Rebecca; McKinnon, Elizabeth; Ahmed, Imran; Pfafferott, Katja; Turley, Joanne; Keane, Niamh M; Lucas, Andrew; Rushton, Ben; Chopra, Abha; Mallal, Simon; John, Mina

    2009-05-15

    The enzyme linked immunospot (ELISpot) assay is a fundamental tool in cellular immunology, providing both quantitative and qualitative information on cellular cytokine responses to defined antigens. It enables the comprehensive screening of patient derived peripheral blood mononuclear cells to reveal the antigenic restriction of T-cell responses and is an emerging technique in clinical laboratory investigation of certain infectious diseases. As with all cellular-based assays, the final results of the assay are dependent on a number of technical variables that may impact precision if not highly standardised between operators. When studies that are large scale or using multiple antigens are set up manually, these assays may be labour intensive, have many manual handling steps, are subject to data and sample integrity failure and may show large inter-operator variability. Here we describe the successful automated performance of the interferon (IFN)-gamma ELISpot assay from cell counting through to electronic capture of cytokine quantitation and present the results of a comparison between automated and manual performance of the ELISpot assay. The mean number of spot forming units enumerated by both methods for limiting dilutions of CMV, EBV and influenza (CEF)-derived peptides in six healthy individuals were highly correlated (r>0.83, pautomated system compared favourably with the manual ELISpot and further ensured electronic tracking, increased through-put and reduced turnaround time.

  3. An improved haemolytic plaque assay for the detection of cells secreting antibody to bacterial antigens

    DEFF Research Database (Denmark)

    Barington, T; Heilmann, C

    1992-01-01

    Recent advances in the development of conjugate polysaccharide vaccines for human use have stimulated interest in the use of assays detecting antibody-secreting cells (AbSC) with specificity for bacterial antigens. Here we present improved haemolytic plaque-forming cell (PFC) assays detecting Ab......SC with specificity for tetanus and diphtheria toxoid as well as for Haemophilus influenzae type b and pneumococcal capsular polysaccharides. These assays were found to be less time consuming, more economical and yielded 1.9-3.4-fold higher plaque numbers than traditional Jerne-type PFC assays. In the case of anti......-polysaccharide antibodies aggregation of secreted monomeric antibody (IgG) is critical for plaque formation and increases the avidity of binding to target cells....

  4. Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses

    Directory of Open Access Journals (Sweden)

    MacLeod Beth

    2007-09-01

    Full Text Available Abstract Background Standardization of cell-based immunologic monitoring is becoming increasingly important as methods for measuring cellular immunity become more complex. We assessed the ability of two commonly used cell-based assays, tritiated thymidine incorporation (proliferation and IFN-gamma ELISPOT, to predict T cell responses to HER-2/neu, tetanus toxoid (tt, and cytomegalovirus (CMV antigens. These antigens were determined to be low (HER-2/neu, moderate (tt, and robustly (CMV immunogenic proteins. Samples from 27 Stage II, III, and IV HER-2/neu positive breast cancer patients, vaccinated against the HER-2/neu protein and tt, were analyzed by tritiated thymidine incorporation and IFN-gamma ELISPOT for T cell response. Results Linear regression analysis indicates that both stimulation index (SI (p = 0.011 and IFN-gamma secreting precursor frequency (p Conclusion These data underscore the importance of taking into consideration the performance characteristics of assays used to measure T cell immunity. This consideration is particularly necessary when determining which method to utilize for assessing responses to immunotherapeutic manipulations in cancer patients.

  5. Trehalose improves cell proliferation and dehydration tolerance of human HaCaT cells

    Directory of Open Access Journals (Sweden)

    Lee Kyung Eun

    2015-01-01

    Full Text Available Trehalose is a disaccharide molecule that serves as a natural osmotic regulator in halophilic microorganisms and plants but not in mammals. We observed that human HaCaT cells supplied with trehalose improved cell proliferation and extended viability under dehydration. In HaCaT cells, in response to increasing concentrations of exogenous trehalose, the levels of heat shock protein (HSP 70 increased and matrix metalloproteinase (MMP 1 decreased. Proteome analysis of trehalose-treated HaCaT cells revealed remarkable increases in the levels of proteins involved in cell signaling and the cell cycle, including p21 activated kinase I, Sec I family domain protein and elongation factor G. Moreover, the proteins for cell stress resistance, tryptophan hydroxylase, serine/cysteine proteinase inhibitors and vitamin D receptors were also increased. In addition, the proteins responsible for the maintenance of the cytoskeleton and cellular structures including procollagen-lysine dioxygenase, vinculin and ezrin were increased. Proteomic data revealed that trehalose affected HaCaT cells by inducing the proteins involved in cell proliferation. These results suggest that trehalose improves the proliferation and dehydration tolerance of HaCaT cells by inducing proteins involved in cell growth and dehydration protection.

  6. T cell motility as modulator of interactions with dendritic cells

    Directory of Open Access Journals (Sweden)

    Jens Volker Stein

    2015-11-01

    Full Text Available It is well established that the balance of costimulatory and inhibitory signals during interactions with dendritic cells (DCs determines T cell transition from a naïve to an activated or tolerant/anergic status. While many of these molecular interactions are well reproduced in reductionist in vitro assays, the highly dynamic motility of naïve T cells in lymphoid tissue acts as an additional lever to fine-tune their activation threshold. T cell detachment from DCs providing suboptimal stimulation allows them to search for DCs with higher levels of stimulatory signals, while storing a transient memory of short encounters. In turn, adhesion of weakly reactive T cells to DCs presenting pMHC with low affinity is prevented by lipid mediators. Finally, controlled recruitment of CD8+ T cells to cognate DC – CD4+ T cell clusters shapes memory T cell formation and the quality of the immune response. Dynamic physiological lymphocyte motility therefore constitutes a mechanism to mitigate low avidity T cell activation and to improve the search for optimal DCs, while contributing to peripheral tolerance induction in the absence of inflammation.

  7. An improved method for staining cell colonies in clonogenic assays

    OpenAIRE

    Guda, Kishore; Natale, Leanna; Markowitz, Sanford D.

    2007-01-01

    Clonogenic assay is a widely used experimental approach to test for the effects of drugs/genes on the growth and proliferative characteristics of cells in vitro. Accurate quantitation of treatment effects in clonogeneic assays depends on the ability to visualize and count cell colonies precisely. We report a novel method (referred as ETeB) for staining cell colonies grown on plastic and specially coated substrates like collagen. Using colon cancer cell lines grown on plastic and collagen, we ...

  8. Pertussis serology: assessment of IgG anti-PT ELISA for replacement of the CHO cell assay*

    Science.gov (United States)

    DALBY, TINE; SØRENSEN, CHARLOTTE; PETERSEN, JESPER WESTPHAL; KROGFELT, KAREN ANGELIKI

    2010-01-01

    Dalby T, Sørensen C, Petersen JW, Krogfelt KA. Pertussis serology: assessment of IgG anti-PT ELISA for replacement of the CHO cell assay. APMIS 2010; 118: 968–72. Two types of serological assays are commonly used for the assessment of pertussis vaccine-induced antibodies; the Chinese hamster ovary cell (CHO cell) assay and the immunoglobulin G (IgG) anti pertussis toxin (PT) enzyme-linked immunosorbent assay (IgG anti-PT ELISA). Recently, both the techniques have been modified to improve performance with sera with interfering activity (CHO cell assay) or with heat-treated sera (IgG anti-PT ELISA). These two improved techniques were compared by the analysis of 100 individual serum samples from a previous clinical trial and 213 sera from a longitudinal serum collection from 20 Danish adults recently vaccinated with the Danish acellular pertussis vaccine. The comparison showed a significant linear correlation between the results of the two assays with a p-value of ELISA can be used as a replacement for the often troublesome and time-consuming CHO cell assay for the measurement of vaccine-induced human antibodies to PT. PMID:21091778

  9. Analysis of close associations of uropod-associated proteins in human T-cells using the proximity ligation assay

    Directory of Open Access Journals (Sweden)

    Tommy Baumann

    2013-10-01

    Full Text Available We have shown previously that the raft-associated proteins flotillin-1 and -2 are rapidly recruited to the uropods of chemoattractant-stimulated human neutrophils and T-cells and are involved in cell polarization. Other proteins such as the adhesion receptor PSGL-1, the actin-membrane linker proteins ezrin/radixin/moesin (ERM and the signaling enzyme phosphatidylinositol-4-phosphate 5-kinase type Iγ90 (PIPKIγ90 also accumulate in the T-cell uropod. Using the in situ proximity ligation assay (PLA we now have investigated putative close associations of these proteins in human freshly isolated T-cells before and after chemokine addition. The PLA allows in situ subcellular localization of close proximity of endogenous proteins at single-molecule resolution in fixed cells. It allows detection also of weaker and transient complexes that would not be revealed with co-immunoprecipitation approaches. We previously provided evidence for heterodimer formation of tagged flotillin-1 and -2 in T-cells before and after chemokine addition using fluorescence resonance energy transfer (FRET. We now confirm these findings using PLA for the endogenous flotillins in fixed human T-cells. Moreover, in agreement with the literature, our PLA findings confirm a close association of endogenous PSGL-1 and ERM proteins both in resting and chemokine-activated human T-cells. In addition, we provide novel evidence using the PLA for close associations of endogenous activated ERM proteins with PIPKIγ90 and of endogenous flotillins with PSGL-1 in human T-cells, before and after chemokine addition. Our findings suggest that preformed clusters of these proteins coalesce in the uropod upon cell stimulation.

  10. Development of CAR T cells designed to improve antitumor efficacy and safety

    OpenAIRE

    Jaspers, Janneke E.; Brentjens, Renier J.

    2017-01-01

    Chimeric antigen receptor (CAR) T cell therapy has shown promising efficacy against hematologic malignancies. Antitumor activity of CAR T cells, however, needs to be improved to increase therapeutic efficacy in both hematologic and solid cancers. Limitations to overcome are ‘on-target, off-tumor’ toxicity, antigen escape, short CAR T cell persistence, little expansion, trafficking to the tumor and inhibition of T cell activity by an inhibitory tumor microenvironment. Here we will discuss how ...

  11. IL-2 absorption affects IFN-gamma and IL-5, but not IL-4 producing memory T cells in double color cytokine ELISPOT assays.

    Science.gov (United States)

    Quast, Stefan; Zhang, Wenji; Shive, Carey; Kovalovski, Damian; Ott, Patrick A; Herzog, Bernhard A; Boehm, Bernhard O; Tary-Lehmann, Magdalena; Karulin, Alexey Y; Lehmann, Paul V

    2005-09-01

    Cytokine assays are gaining increasing importance for human immune monitoring because they reliably detect antigen-specific T cells in primary PBMC, even at low clonal sizes. Double color ELISPOT assays permit the simultaneous visualization of cells producing two different cytokines. Permitting the simultaneous assessment of type 1 and 2 immunity and due to the limited numbers of PBMC available from human study subjects, double color assays should be particularly attractive for clinical trials. Since the performance of double color assays has not yet been validated, we set out to compare them to single color measurements. Testing the recall antigen-induced cytokine response of PBMC, we found that double color assays regularly provided lower numbers of IFN-gamma and IL-5 spots than single color measurements when IL-2 detection was part of the double color assay. We showed that the inhibitory effect resulted from IL-2 absorption and could be overcome by either antibody free preactivation cultures or by inclusion of anti-CD28 antibody. In contrast, the simultaneous detection of IL-2 did not affect the numbers of IL-4 spots. Therefore, unlike IL-2/IL-4 and IFN-gamma/IL-5 assays, IL-2/IFN-gamma, and IL-2/IL-5 assays require compensation for the IL-2 capture to provide accurate numbers for the frequencies of cytokine producing memory T cells.

  12. Can resting B cells present antigen to T cells

    International Nuclear Information System (INIS)

    Ashwell, J.D.; DeFranco, A.L.; Paul, W.E.; Schwartz, R.H.

    1985-01-01

    Antigen stimulation of T lymphocytes can occur only in the presence of an antigen-presenting cell (APC). An ever-increasing number of cell types have been found to act as APCs; these include macrophages, splenic and lymph node dendritic cells, and Langerhans cells of the skin. Although activated B lymphocytes and B cell lymphomas are known to serve as APCs, it has been generally believed that resting B cells cannot perform this function. However, in recent studies the authors have found that resting B cells can indeed present soluble antigen to T cell clones as well as to antigen-primed T cells. The previous difficulty in demonstrating this activity can be explained by the finding that, in contrast to macrophages and dendritic cells, the antigen-presenting ability of resting B cells is very radiosensitive. Macrophages are usually irradiated with 2000-3300 rads to prevent them from incorporating [ 3 H]thymidine in the T cell proliferation assay. Resting B cells, however, begin to lose presenting function at 1500 rads and have completely lost this activity at 3300 rads. It was also possible to distinguish two distinct T cell clonal phenotypes when resting B cells were used as APCs on the basis of two different assays (T cell proliferation, and B cell proliferation resulting from T cell activation). The majority of T cell clones tested were capable of both proliferating themselves and inducing the proliferation of B cells. Some T cells clones, however, could not proliferate in the presence of antigen and B cell APCs, although they were very good at inducing the proliferation of B cells

  13. Incorporation of a hinge domain improves the expansion of chimeric antigen receptor T cells

    Directory of Open Access Journals (Sweden)

    Le Qin

    2017-03-01

    Full Text Available Abstract Background Multiple iterations of chimeric antigen receptors (CARs have been developed, mainly focusing on intracellular signaling modules. However, the effect of non-signaling extracellular modules on the expansion and therapeutic efficacy of CARs remains largely undefined. Methods We generated two versions of CAR vectors, with or without a hinge domain, targeting CD19, mesothelin, PSCA, MUC1, and HER2, respectively. Then, we systematically compared the effect of the hinge domains on the growth kinetics, cytokine production, and cytotoxicity of CAR T cells in vitro and in vivo. Results During in vitro culture period, the percentages and absolute numbers of T cells expressing the CARs containing a hinge domain continuously increased, mainly through the promotion of CD4+ CAR T cell expansion, regardless of the single-chain variable fragment (scFv. In vitro migration assay showed that the hinges enhanced CAR T cells migratory capacity. The T cells expressing anti-CD19 CARs with or without a hinge had similar antitumor capacities in vivo, whereas the T cells expressing anti-mesothelin CARs containing a hinge domain showed enhanced antitumor activities. Conclusions Hence, our results demonstrate that a hinge contributes to CAR T cell expansion and is capable of increasing the antitumor efficacy of some specific CAR T cells. Our results suggest potential novel strategies in CAR vector design.

  14. Interlaboratory survey for T3 and T4 assays in Italy: results from a two semester period

    International Nuclear Information System (INIS)

    Pilo, A.; Zucchelli, G.C.; Chiesa, M.R.; Piro, M.A.

    1982-01-01

    The usefulness of external quality control schemes (EQCS) is generally acknowledged in clinical chemistry; these schemes allow not only the evaluation of the between-laboratory variability of the assay under study, but also make it possible the improvement of the analytical performances of the participants laboratories. Recently interlaboratory surveys have been extended to radioimmunoassays. Starting from january 1980, a national EQCS hormone assays was organized in Italy; triiodothyronine (T3) and thyroxine (T4) have been the first assays considered due to their large diffusion

  15. ELISPOT Assay for Monitoring Cytotoxic T Lymphocytes (CTL Activity in Cancer Vaccine Clinical Trials

    Directory of Open Access Journals (Sweden)

    Thomas J. Sayers

    2012-05-01

    Full Text Available The profiling and monitoring of immune responses are key elements in the evaluation of the efficacy and development of new biotherapies, and a number of assays have been introduced for analyzing various immune parameters before, during, and after immunotherapy. The choice of immune assays for a given clinical trial depends on the known or suggested immunomodulating mechanisms associated with the tested therapeutic modality. Cell-mediated cytotoxicity represents a key mechanism in the immune response to various pathogens and tumors. Therefore, the selection of monitoring methods for the appropriate assessment of cell-mediated cytotoxicity is thought to be crucial. Assays that can detect both cytotoxic T lymphocytes (CTL frequency and function, such as the IFN-γ enzyme-linked immunospot assay (ELISPOT have gained increasing popularity for monitoring clinical trials and in basic research. Results from various clinical trials, including peptide and whole tumor cell vaccination and cytokine treatment, have shown the suitability of the IFN-γ ELISPOT assay for monitoring T cell responses. However, the Granzyme B ELISPOT assay and Perforin ELISPOT assay may represent a more direct analysis of cell-mediated cytotoxicity as compared to the IFN-γ ELISPOT, since Granzyme B and perforin are the key mediators of target cell death via the granule-mediated pathway. In this review we analyze our own data and the data reported by others with regard to the application of various modifications of ELISPOT assays for monitoring CTL activity in clinical vaccine trials.

  16. The production of lymphokines by primary alloreactive T-cell clones: a co-ordinate analysis of 233 clones in seven lymphokine assays.

    Science.gov (United States)

    Sanderson, C J; Strath, M; Warren, D J; O'Garra, A; Kirkwood, T B

    1985-01-01

    A total of 233 primary alloreactive T-cell clones have been tested for the production of interleukin-2 (IL-2), interleukin-3 (IL-3), immune(gamma) interferon (IFN) and granulocyte-macrophage colony-stimulating factor (CSF-2), B-cell growth factor I and II (BCGFI, BCGFII), and eosinophil differentiation factor (EDF). EDF was assayed by means of the eosinophil differentiation assay (EDA). Two principal correlations were observed: IL-3 was shown to be the major lymphokine detected in the bone marrow proliferation assay (BMPA) used to detect CSF-2, and there was a high correlation between the EDA and BCGFII. Subsequent work has suggested that this latter correlation is because a single factor is responsible for both activities. Apart from these two exceptions, and low level correlations probably due to the fact that different assays detect more than one lymphokine, there was no evidence for co-ordinate expression of lymphokines. There was a large variation in amounts of individual lymphokines produced. More clones produced multiple lymphokines than would be expected from independent control. Taken together, this pattern of regulation is consistent with the hypothesis that antigen stimulation of T cells results in the activation of all the lymphokine genes, but the amount of each produced is determined by secondary controlling mechanisms. PMID:3935571

  17. Improving the efficacy and safety of engineered T cell therapy for cancer.

    Science.gov (United States)

    Shi, Huan; Liu, Lin; Wang, Zhehai

    2013-01-28

    Adoptive T-cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) is a powerful immunotherapeutics approach against metastatic melanoma. The success of TIL therapy has led to novel strategies for redirecting normal T cells to recognize tumor-associated antigens (TAAs) by genetically engineering tumor antigen-specific T cell receptors (TCRs) or chimeric antigen receptor (CAR) genes. In this manner, large numbers of antigen-specific T cells can be rapidly generated compared with the longer term expansion of TILs. Great efforts have been made to improve these approaches. Initial clinical studies have demonstrated that genetically engineered T cells can mediate tumor regression in vivo. In this review, we discuss the development of TCR and CAR gene-engineered T cells and the safety concerns surrounding the use of these T cells in patients. We highlight the importance of judicious selection of TAAs for modified T cell therapy and propose solutions for potential "on-target, off-organ" toxicity. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  18. An improved plating assay for determination of phage titer | Yang ...

    African Journals Online (AJOL)

    In this study, an improved plating assay was developed for detection of the number of recombinant phage Cap-T7 present in a test solution at a certain dilution point by counting the plaque forming units. The data demonstrated that the improved plating assay is fast, useful, and convenient for the determination of the phage ...

  19. Inhibition of clone formation as an assay for T cell-mediated cytotoxicity: short-term kinetics and comparison with 51Cr release

    International Nuclear Information System (INIS)

    Lees, R.K.; MacDonald, H.R.; Sinclair, N.R.; University of Western Ontario London

    1977-01-01

    The short-term kinetics of T cell-mediated cytotoxicity was investigated using a cloning inhibition assay. Murine cytotoxic thymus-derived lymphocytes generated in vitro in mixed leukocyte cultures were incubated for various periods of time at 37degC with allogeneic mastocytoma target cells. The mixtures were then plated in soft agar, and mastocytoma clone formation was assessed after 5-7 days incubation. Using this technique, it was demonstrated that events leading to the loss of cloning ability could be detected after 1-3 min incubation at 37degC, and after 20-30 min, 95% of the clone forming cells had been inactivated. When these results were compared directly with those obtained using the conventional 51 Cr-release assay, it was found that the events leading to loss of cloning ability occurred more rapidly than indicated by the isotope assay. However, a modification of the 51 Cr-release assay involving EDTA addition gave comparable result to the cloning inhibition assay. These results raise the possibility that the events leading to 51 Cr-release of tumor target cells may be related in time to those leading to the loss of cloning ability

  20. Two subpopulations of stem cells for T cell lineage

    International Nuclear Information System (INIS)

    Katsura, Y.; Amagai, T.; Kina, T.; Sado, T.; Nishikawa, S.

    1985-01-01

    An assay system for the stem cell that colonizes the thymus and differentiates into T cells was developed, and by using this assay system the existence of two subpopulations of stem cells for T cell lineage was clarified. Part-body-shielded and 900-R-irradiated C57BL/6 (H-2b, Thy-1.2) recipient mice, which do not require the transfer of pluripotent stem cells for their survival, were transferred with cells from B10 X Thy-1.1 (H-2b, Thy-1.1) donor mice. The reconstitution of the recipient's thymus lymphocytes was accomplished by stem cells in the donor cells and those spared in the shielded portion of the recipient that competitively colonize the thymus. Thus, the stem cell activity of donor cells can be evaluated by determining the proportion of donor-type (Thy-1.1+) cells in the recipient's thymus. Bone marrow cells were the most potent source of stem cells. By contrast, when the stem cell activity was compared between spleen and bone marrow cells of whole-body-irradiated (800 R) C57BL/6 mice reconstituted with B10 X Thy-1.1 bone marrow cells by assaying in part-body-shielded and irradiated C57BL/6 mice, the activity of these two organs showed quite a different time course of development. The results strongly suggest that the stem cells for T cell lineage in the bone marrow comprise at least two subpopulations, spleen-seeking and bone marrow-seeking cells

  1. High-Throughput Flow Cytometric Method for the Simultaneous Measurement of CAR-T Cell Characterization and Cytotoxicity against Solid Tumor Cell Lines.

    Science.gov (United States)

    Martinez, Emily M; Klebanoff, Samuel D; Secrest, Stephanie; Romain, Gabrielle; Haile, Samuel T; Emtage, Peter C R; Gilbert, Amy E

    2018-04-01

    High-throughput flow cytometry is an attractive platform for the analysis of adoptive cellular therapies such as chimeric antigen receptor T cell therapy (CAR-T) because it allows for the concurrent measurement of T cell-dependent cellular cytotoxicity (TDCC) and the functional characterization of engineered T cells with respect to percentage of CAR transduction, T cell phenotype, and measurement of T cell function such as activation in a single assay. The use of adherent tumor cell lines can be challenging in these flow-based assays. Here, we present the development of a high-throughput flow-based assay to measure TDCC for a CAR-T construct co-cultured with multiple adherent tumor cell lines. We describe optimal assay conditions (such as adherent cell dissociation techniques to minimize impact on cell viability) that result in robust cytotoxicity assays. In addition, we report on the concurrent use of T cell transduction and activation antibody panels (CD25) that provide further dissection of engineered T cell function. In conclusion, we present the development of a high-throughput flow cytometry method allowing for in vitro interrogation of solid tumor, targeting CAR-T cell-mediated cytotoxicity, CAR transduction, and engineered T cell characterization in a single assay.

  2. Optimization of interferon gamma ELISPOT assay to detect human cytomegalovirus specific T-cell responses in solid organ transplants.

    Science.gov (United States)

    Abate, Davide; Saldan, Alda; Forner, Gabriella; Tinto, Daniel; Bianchin, Alice; Palù, Giorgio

    2014-02-01

    Assessing the CMV specific CMI in transplant subjects represents a promising strategy to determine the risk of infection on individual basis. In this study 61 adult CMV IgG seropositive solid organ transplant recipients were examined in order to improve the efficacy of CMI detection. For this purpose, pair-wise comparisons were conducted comparing positive control stimuli PWM and PMA/iono and CMV stimuli, pp65 peptide pool and whole CMV particle. Rosette pre-depletion of blood was also investigated for detecting CD4+ or CD8+ T-cell responses using the IFN-g ELISPOT assay. In the time-points 30-180 days after transplantation, PMA/iono produced statistically significant higher responses compared to PWM, probably because PMA/iono activation pathway is independent from the effect of immunosuppressive drugs. The data showed that 11% of transplant patients displayed very low or undetectable responses to pp65 peptide pool antigen while having sustained high responses to whole CMV particle. In addition, in all the subjects analyzed, CMI responses to CMV particle produced a statistically significant higher number of spots compared to pp65 peptide pool antigen. Rosette pre-depletion of whole blood proved to be effective in detecting CD4+ or CD8+ T-cell responses similarly to flow cytometry. Taken together, the following recommendations are suggested to optimize the CMV-ELISPOT for transplantation settings: (1) use PMA/iono as positive control; (2) whole virus particle should be used to avoid peptide-related false negative responses; (3) a rosette pre-depletion step may be useful to detect CD4+ or CD8+ T-cell responses. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society.

    Science.gov (United States)

    Mallone, R; Mannering, S I; Brooks-Worrell, B M; Durinovic-Belló, I; Cilio, C M; Wong, F S; Schloot, N C

    2011-01-01

    Autoimmune T cell responses directed against insulin-producing β cells are central to the pathogenesis of type 1 diabetes (T1D). Detection of such responses is therefore critical to provide novel biomarkers for T1D 'immune staging' and to understand the mechanisms underlying the disease. While different T cell assays are being developed for these purposes, it is important to optimize and standardize methods for processing human blood samples for these assays. To this end, we review data relevant to critical parameters in peripheral blood mononuclear cell (PBMC) isolation, (cryo)preservation, distribution and usage for detecting antigen-specific T cell responses. Based on these data, we propose recommendations on processing blood samples for T cell assays and identify gaps in knowledge that need to be addressed. These recommendations may be relevant not only for the analysis of T cell responses in autoimmune disease, but also in cancer and infectious disease, particularly in the context of clinical trials. © 2010 The Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.

  4. Fine-tuning the CAR spacer improves T-cell potency

    Science.gov (United States)

    Watanabe, Norihiro; Bajgain, Pradip; Sukumaran, Sujita; Ansari, Salma; Heslop, Helen E.; Rooney, Cliona M.; Brenner, Malcolm K.; Leen, Ann M.; Vera, Juan F.

    2016-01-01

    ABSTRACT The adoptive transfer of genetically engineered T cells expressing chimeric antigen receptors (CARs) has emerged as a transformative cancer therapy with curative potential, precipitating a wave of preclinical and clinical studies in academic centers and the private sector. Indeed, significant effort has been devoted to improving clinical benefit by incorporating accessory genes/CAR endodomains designed to enhance cellular migration, promote in vivo expansion/persistence or enhance safety by genetic programming to enable the recognition of a tumor signature. However, our efforts centered on exploring whether CAR T-cell potency could be enhanced by modifying pre-existing CAR components. We now demonstrate how molecular refinements to the CAR spacer can impact multiple biological processes including tonic signaling, cell aging, tumor localization, and antigen recognition, culminating in superior in vivo antitumor activity. PMID:28180032

  5. Human rotavirus specific T cells: quantification by ELISPOT and expression of homing receptors on CD4+ T cells

    International Nuclear Information System (INIS)

    Rojas, Olga Lucia; Gonzalez, Ana Maria; Gonzalez, Rosabel; Perez-Schael, Irene; Greenberg, Harry B.; Franco, Manuel A.; Angel, Juana

    2003-01-01

    Using an intracellular cytokine assay, we recently showed that the frequencies of rotavirus (RV)-specific CD4 + and CD8 + T cells secreting INFγ, circulating in RV infected and healthy adults, are very low compared to the frequencies of circulating cytomegalovirus (CMV) reactive T cells in comparable individuals. In children with acute RV infection, these T cells were barely or not detectable. In the present study, an ELISPOT assay enabled detection of circulating RV-specific INFγ-secreting cells in children with RV diarrhea but not in children with non-RV diarrhea without evidence of a previous RV infection. Using microbead-enriched CD4 + and CD8 + T cell subsets, IFNγ-secreting RV-specific CD8 + but not CD4 + T cells were detected in recently infected children. Using the same approach, both CD4 + and CD8 + RV-specific T cells were detected in healthy adults. Furthermore, stimulation of purified subsets of PBMC that express lymphocyte homing receptors demonstrated that RV-specific INFγ-secreting CD4 + T cells from adult volunteers preferentially express the intestinal homing receptor α4β7, but not the peripheral lymph node homing receptor L-selectin. In contrast, CMV-specific INFγ-secreting CD4 + T cells preferentially express L-selectin but not α4β7. These results suggest that the expression of homing receptors on virus-specific T cells depends on the organ where these cells were originally stimulated and that their capacity to secrete INFγ is independent of the expression of these homing receptors

  6. [Adult T-cell leukemia/lymphoma associated with unusual positivity of anti-ATLA (adult T-cell leukemia-cell-associated antigen) antibodies].

    Science.gov (United States)

    Eto, T; Okamura, H; Okamura, T; Gondo, H; Kudo, J; Shibuya, T; Harada, M; Niho, Y

    1990-03-01

    A 56-year-old female was admitted because of generalized lymphadenopathy. Based upon histological findings of biopsied lymph node, malignant lymphoma, diffuse large cell type was diagnosed. The surface marker analysis showed that malignant cells were positive for CD4 and CD2 but negative for CD8. Although anti-ATLA (adult T-cell leukemia associated antigen) antibody was negative with the use of a gelatin particle agglutination method (P.A.), other methods such as an indirect immunofluorescence assay (I.F.), an enzyme-linked immunosorbent assay (E.I.A.) and a Western blotting assay revealed the positivity for anti-ATLA antibody. Adult T-cell leukemia/lymphoma (ATL/L) was confirmed by the presence of monoclonal integration of HTLV-I proviral DNA in biopsied specimen. This case, showing a pattern of P.A. (-) and I.F. (+), is extremely unusual, because I.F. and P.A. show highly close correlation. Thus, it is important to employ different methods for screening of anti-ATLA antibodies in the diagnosis of ATL/L.

  7. Improve T Cell Therapy in Neuroblastoma

    Science.gov (United States)

    2014-07-01

    relapsed lymphoma following genetic modi - fi cation of tumor-antigen presenting cells and T-lymphocyte transfer. Blood 110:2838–2845 4. Heslop HE et...CD4þCD25þFOXP3þ regulatory T cells of both healthy subjects and type 1 diabetic patients. J Immunol 2006;177:8338–47. 32. HeslopHE, SlobodKS,PuleMA

  8. Enhancement of Human Antigen-Specific Memory T-Cell Responses by Interleukin-7 May Improve Accuracy in Diagnosing Tuberculosis▿ †

    Science.gov (United States)

    Feske, Marsha; Nudelman, Rodolfo J.; Medina, Miguel; Lew, Justin; Singh, Manisha; Couturier, Jacob; Graviss, Edward A.; Lewis, Dorothy E.

    2008-01-01

    Children and immunocompromised adults are at an increased risk of tuberculosis (TB), but diagnosis is more challenging. Recently developed gamma interferon (IFN-γ) release assays provide increased sensitivity and specificity for diagnosis of latent TB, but their use is not FDA approved in immunocompromised or pediatric populations. Both populations have reduced numbers of T cells, which are major producers of IFN-γ. Interleukin 7 (IL-7), a survival cytokine, stabilizes IFN-γ message and increases protein production. IL-7 was added to antigen-stimulated lymphocytes to improve IFN-γ responses as measured by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT) assay. Antigens used were tetanus toxoid (n = 10), p24 (from human immunodeficiency virus [HIV], n = 9), and TB peptides (n = 15). Keyhole limpet hemocyanin was used as a negative control, and phytohemagglutinin was the positive control. IL-7 improved antigen-specific responses to all antigens tested including tetanus toxoid, HIV type 1 p24, and TB peptides (ESAT-6 and CFP-10) with up to a 14-fold increase (mean = 3.8), as measured by ELISA. Increased IFN-γ responses from controls, HIV-positive patients, and TB patients were statistically significant, with P values of <0.05, 0.01, and 0.05, respectively. ELISPOT assay results confirmed ELISA findings (P values of <0.01, 0.02, and 0.03, respectively), with a strong correlation between the two tests (R2 = 0.82 to 0.99). Based on average background levels, IL-7 increased detection of IFN-γ by 39% compared to the level with antigen alone. Increased production of IFN-γ induced by IL-7 improves sensitivity of ELISA and ELISPOT assays for all antigens tested. Further enhancement of IFN-γ-based assays might improve TB diagnosis in those populations at highest risk for TB. PMID:18753334

  9. Limiting dilution analysis of the stem cells for T cell lineage

    International Nuclear Information System (INIS)

    Katsura, Y.; Kina, T.; Amagai, T.; Tsubata, T.; Hirayoshi, K.; Takaoki, Y.; Sado, T.; Nishikawa, S.I.

    1986-01-01

    Stem cell activities of bone marrow, spleen, thymus, and fetal liver cells for T cell lineage were studied comparatively by transferring the cells from these organs through i.v. or intrathymus (i.t.) route into right leg- and tail-shielded (L-T-shielded) and 900 R-irradiated recipient mice, which were able to survive without supplying hemopoietic stem cells. Cells from B10.Thy-1.1 (H-2b, Thy-1.1) mice were serially diluted and were transferred into L-T-shielded and irradiated C57BL/6 (H-2b, Thy-1.2) mice, and 21 days later the thymus cells of recipient mice were assayed for Thy-1.1+ cells by flow cytofluorometry. The percentage of recipient mice possessing donor-type T cells was plotted against the number of cells transferred, and the stem cell activity in each cell source was expressed as the 50% positive value, the number of donor cells required for generating donor-type T cells in the thymuses of 50% of recipient mice. In i.v. transfer experiments, the activity of bone marrow cells was similar to that of fetal liver cells, and about 100 times and nearly 1000 times higher than those of spleen cells and thymus cells, respectively. In i.t. transfer experiments, the number of cells required for generating donor-type T cells was much lower than that in i.v. transfer experiments, although the ratio in 50% positive values between i.v. and i.t. transfers differed among cell sources. In i.t. transfers, the 50% positive value of bone marrow cells was five times, 400 times, and 500 times higher than that of fetal liver cells, spleen cells, and thymus cells, respectively. Our previous finding that stem cells are enriched in the spleens of mice which were whole body-irradiated and marrow-reconstituted 7 days earlier was confirmed also by the present limiting dilution assay carried out in i.v. as well as i.t. transfers

  10. A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: Comparisons to a 4 h 51Cr-release assay

    OpenAIRE

    Kim, GG; Donnenberg, VS; Donnenberg, AD; Gooding, W; Whiteside, TL

    2007-01-01

    Natural killer (NK) cell- or T cell-mediated cytotoxicity traditionally is measured in 4-16h 51Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3−CD16+CD56+). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. ...

  11. An improved method for staining cell colonies in clonogenic assays.

    Science.gov (United States)

    Guda, Kishore; Natale, Leanna; Markowitz, Sanford D

    2007-06-01

    Clonogenic assay is a widely used experimental approach to test for the effects of drugs/genes on the growth and proliferative characteristics of cells in vitro. Accurate quantitation of treatment effects in clonogeneic assays depends on the ability to visualize and count cell colonies precisely. We report a novel method (referred as ETeB) for staining cell colonies grown on plastic and specially coated substrates like collagen. Using colon cancer cell lines grown on plastic and collagen, we compared the colony staining efficiencies of the widely used methylene blue, and Ethidium bromide (ETeB) stains. Results show that the ETeB protocol works well on plastic and is extremely effective for staining colonies on collagen when compared to methylene blue. The key features and advantages of ETeB technique are; (a) reduction in background for colonies grown on collagen and possibly other substrates, (b) the whole procedure takes less than a minute, (c) no post-stain washing step is required which eliminates colony losses for cell lines that are loosely adherent, (d) colony visualization and counting can be done immediately following the staining procedure using a standard UV illuminator and software, and (e) the method works across a wide variety of cell lines. The simplicity and robustness of this procedure should warrant its usage in both small and large-scale clonogenic experiments.

  12. Clonogenic assay: adherent cells.

    Science.gov (United States)

    Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C

    2011-03-13

    The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 1956. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811). Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

  13. Tumor-Infiltrating Merkel Cell Polyomavirus-Specific T Cells Are Diverse and Associated with Improved Patient Survival. | Office of Cancer Genomics

    Science.gov (United States)

    Tumor-infiltrating CD8+ T cells are associated with improved survival of patients with Merkel cell carcinoma (MCC), an aggressive skin cancer causally linked to Merkel cell polyomavirus (MCPyV). However, CD8+ T-cell infiltration is robust in only 4% to 18% of MCC tumors. We characterized the T-cell receptor (TCR) repertoire restricted to one prominent epitope of MCPyV (KLLEIAPNC, "KLL") and assessed whether TCR diversity, tumor infiltration, or T-cell avidity correlated with clinical outcome.

  14. Enteroantigen-presenting B cells efficiently stimulate CD4(+) T cells in vitro

    DEFF Research Database (Denmark)

    Schmidt, Esben Gjerløff Wedebye; Kristensen, Nanna Ny; Claesson, Mogens Helweg

    2011-01-01

    Presentation of enterobacterial antigens by antigen-presenting cells and activation of enteroantigen-specific CD4(+) T cells are considered crucial steps in inflammatory bowel disease (IBD) pathology. The detrimental effects of such CD4(+) T cells have been thoroughly demonstrated in models...... of colitis. Also, we have previously established an in vitro assay where murine enteroantigen-specific colitogenic CD4(+) CD25(-) T cells are activated by splenocytes pulsed with an enterobacterial extract....

  15. An improved microculture-hemolytic spot assay for the study of carrier-specific antibody responses.

    Science.gov (United States)

    Kotkes, P; Weisman, Z; Mozes, E; Bentwich, Z

    1984-11-30

    A microculture system based on limiting dilution and a hemolytic spot assay was adapted for study of the carrier-specific anti-hapten response in vitro. Spleen or lymph node cells from normal mice or mice immunized with NIP-ovalbumin (NIP-OVA) or NIP-human thyroglobulin (NIP-Tg) were cultured for 5 days by the microculture technique. The anti-hapten (anti-NIP) response was measured by assaying the supernatants of the microcultures in a hemolytic spot test with NIP coupled to sheep red blood cells. A micro-ELISA reader was adapted to read the degree of lysis in the spot assay which gives an objective quantitation of the degree of lysis and thus reduces the number of culture replicates. In vivo induced specific helper cells in mice immunized with the carrier protein, human thyroglobulin, as well as carrier-specific T cell factors, gave rise to carrier-specific anti-NIP responses. The microculture system may enhance the expression of T-cell helper function when suppressor cells or their precursors are present in the initial cell preparation.

  16. A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.

    Science.gov (United States)

    Kim, G G; Donnenberg, V S; Donnenberg, A D; Gooding, W; Whiteside, T L

    2007-08-31

    Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.

  17. Improve T Cell Therapy in Neuroblastoma

    Science.gov (United States)

    2012-07-01

    Savoldo B, Vigouroux S et al. T lymphocytes redirected against the kappa light chain of human immunoglobulin efficiently kill mature B lymphocyte...Natl Acad Sci U S A. 2004;101(suppl 2):14622–14626. 8. Eghtesad S, Morel PA, Clemens PR. The companions : regulatory T cells and gene therapy...were euthanized and examined for NKT cell localiza- tion to the tumor tissues. Animals treated with anti-CCL2 or anti- CCL20 mAb had lower frequency

  18. Improve T Cell Therapy in Neuroblastoma

    Science.gov (United States)

    2015-09-01

    bioluminescence was then measured overtime. The graph is representative of one of 4 experiments using CMV-CTLs from 4 donors. Panel E. Kaplan-Meier...whole-cell vaccine expressing the iC9 gene and labeled with an enhanced firefly luciferase. Tumor growth was measured by in vivo imaging. Panel E...down regulation in LTE -T cells is not caused by specific culture conditions. T lymphocytes were activated with immobilized OKT3 (1 μg ml) and

  19. High-throughput gene expression profiling of memory differentiation in primary human T cells

    Directory of Open Access Journals (Sweden)

    Russell Kate

    2008-08-01

    Full Text Available Abstract Background The differentiation of naive T and B cells into memory lymphocytes is essential for immunity to pathogens. Therapeutic manipulation of this cellular differentiation program could improve vaccine efficacy and the in vitro expansion of memory cells. However, chemical screens to identify compounds that induce memory differentiation have been limited by 1 the lack of reporter-gene or functional assays that can distinguish naive and memory-phenotype T cells at high throughput and 2 a suitable cell-line representative of naive T cells. Results Here, we describe a method for gene-expression based screening that allows primary naive and memory-phenotype lymphocytes to be discriminated based on complex genes signatures corresponding to these differentiation states. We used ligation-mediated amplification and a fluorescent, bead-based detection system to quantify simultaneously 55 transcripts representing naive and memory-phenotype signatures in purified populations of human T cells. The use of a multi-gene panel allowed better resolution than any constituent single gene. The method was precise, correlated well with Affymetrix microarray data, and could be easily scaled up for high-throughput. Conclusion This method provides a generic solution for high-throughput differentiation screens in primary human T cells where no single-gene or functional assay is available. This screening platform will allow the identification of small molecules, genes or soluble factors that direct memory differentiation in naive human lymphocytes.

  20. A Modified NK Cell Degranulation Assay Applicable for Routine Evaluation of NK Cell Function

    Directory of Open Access Journals (Sweden)

    Snehal Shabrish

    2016-01-01

    Full Text Available Natural killer (NK cells play important role in innate immunity against tumors and viral infections. Studies show that lysosome-associated membrane protein-1 (LAMP-1, CD107a is a marker for degranulation of NK and cytotoxic T cells and its expression is a sensitive marker for the cytotoxic activity determination. The conventional methods of determination of CD107a on NK cells involve use of peripheral blood mononuclear cells (PBMC or pure NK cells and K562 cells as stimulants. Thus, it requires large volume of blood sample which is usually difficult to obtain in pediatric patients and patients with cytopenia and also requires specialized laboratory for maintaining cell line. We have designed a flow cytometric assay to determine CD107a on NK cells using whole blood, eliminating the need for isolation of PBMC or isolate NK cells. This assay uses phorbol-12-myristate-13-acetate (PMA and calcium ionophore (Ca2+-ionophore instead of K562 cells for stimulation and thus does not require specialized cell culture laboratory. CD107a expression on NK cells using modified NK cell degranulation assay compared to the conventional assay was significantly elevated (p<0.0001. It was also validated by testing patients diagnosed with familial hemophagocytic lymphohistiocytosis (FHL with defect in exocytosis. This assay is rapid, cost effective, and reproducible and requires significantly less volume of blood which is important for clinical evaluation of NK cells.

  1. A modified anthrax toxin-based enzyme-linked immunospot assay reveals robust T cell responses in symptomatic and asymptomatic Ebola virus exposed individuals.

    Science.gov (United States)

    Herrera, Bobby Brooke; Hamel, Donald J; Oshun, Philip; Akinsola, Rolake; Akanmu, Alani S; Chang, Charlotte A; Eromon, Philomena; Folarin, Onikepe; Adeyemi, Kayode T; Happi, Christian T; Lu, Yichen; Ogunsola, Folasade; Kanki, Phyllis J

    2018-05-01

    Ebola virus (EBOV) caused more than 11,000 deaths during the 2013-2016 epidemic in West Africa without approved vaccines or immunotherapeutics. Despite its high lethality in some individuals, EBOV infection can produce little to no symptoms in others. A better understanding of the immune responses in individuals who experienced minimally symptomatic and asymptomatic infection could aid the development of more effective vaccines and antivirals against EBOV and related filoviruses. Between August and November 2017, blood samples were collected from 19 study participants in Lagos, Nigeria, including 3 Ebola virus disease (EVD) survivors, 10 individuals with documented close contact with symptomatic EVD patients, and 6 control healthcare workers for a cross-sectional serosurvey and T cell analysis. The Lagos samples, as well as archived serum collected from healthy individuals living in surrounding areas of the 1976 Democratic Republic of Congo (DRC) epidemic, were tested for EBOV IgG using commercial enzyme-linked immunosorbent assays (ELISAs) and Western blots. We detected antibodies in 3 out of 3 Lagos survivors and identified 2 seropositive individuals not known to have ever been infected. Of the DRC samples tested, we detected antibodies in 9 out of 71 (12.7%). To characterize the T cell responses in the Lagos samples, we developed an anthrax toxin-based enzyme-linked immunospot (ELISPOT) assay. The seropositive asymptomatic individuals had T cell responses against EBOV nucleoprotein, matrix protein, and glycoprotein 1 that were stronger in magnitude compared to the survivors. Our data provide further evidence of EBOV exposure in individuals without EVD-like illness and, for the first time, demonstrate that these individuals have T cell responses that are stronger in magnitude compared to severe cases. These findings suggest that T cell immunity may protect against severe EVD, which has important implications for vaccine development.

  2. An improved in vitro micronucleus assay to biological dosimetry

    International Nuclear Information System (INIS)

    Ocampo, Ivette Z.; Okazaki, Kayo; Vieira, Daniel P.

    2013-01-01

    The biological dosimetry is widely used to estimate the absorbed dose in people occupationally or accidentally exposed to the radiation for a better medical treatment, minimizing the harmful effects. Many techniques and methods have been proposed to detect and quantify the radioinduced lesions in genetic material, among them, the micronucleus (MN) assay. In the present study, we proposed an improved in vitro micronucleus technique that is rapid, sensitive and with minor cell manipulations. Assays were carried out with human tumor cells (MCF-7) seeded (3x10 4 cells) in slides placed into Petri dishes. Adherent cells were maintained with RPMI medium, supplemented with fetal calf serum, 1 % antibiotics, cytochalasin B (2 μg/mL), and incubated at 37 deg C in the presence of 5% CO2 for 72h. Cells were pre-treated for 24h with aminoguanidine, a nitric oxide synthase inhibitor. Nitric oxide is an intracellular free-radical, involved in DNA double-strand break repair mechanisms. After incubation, adherent cells on slides were briefly fixed with paraformaldehyde and stained with acridine orange (100 μg/mL) for analysis through fluorescence microscopy. Dye fluorescence permitted accurate discrimination between nuclei and micronuclei (bright green) and cytoplasm (red), and made possible a faster counting of binucleated cells. Aminoguanidine (2 mM) induced significant increase (p< 0.05) in frequencies of binucleated cells with micronuclei and in the number of micronuclei per binucleated cell. Data showed that proposed modifications permit to understand an early aspect of NO inhibition and suggested an improved protocol to MN assays. (author)

  3. IL-7 and CCL19 expression in CAR-T cells improves immune cell infiltration and CAR-T cell survival in the tumor.

    Science.gov (United States)

    Adachi, Keishi; Kano, Yosuke; Nagai, Tomohiko; Okuyama, Namiko; Sakoda, Yukimi; Tamada, Koji

    2018-04-01

    Infiltration, accumulation, and survival of chimeric antigen receptor T (CAR-T) cells in solid tumors is crucial for tumor clearance. We engineered CAR-T cells to express interleukin (IL)-7 and CCL19 (7 × 19 CAR-T cells), as these factors are essential for the maintenance of T-cell zones in lymphoid organs. In mice, 7 × 19 CAR-T cells achieved complete regression of pre-established solid tumors and prolonged mouse survival, with superior anti-tumor activity compared to conventional CAR-T cells. Histopathological analyses showed increased infiltration of dendritic cells (DC) and T cells into tumor tissues following 7 × 19 CAR-T cell therapy. Depletion of recipient T cells before 7 × 19 CAR-T cell administration dampened the therapeutic effects of 7 × 19 CAR-T cell treatment, suggesting that CAR-T cells and recipient immune cells collaborated to exert anti-tumor activity. Following treatment of mice with 7 × 19 CAR-T cells, both recipient conventional T cells and administered CAR-T cells generated memory responses against tumors.

  4. Mammalian cell transformation: Mechanisms of carcinogenesis and assays for carcinogens

    International Nuclear Information System (INIS)

    Barrett, J.C.; Tennant, R.W.

    1985-01-01

    This book contains nine sections, each consisting of several papers. The section titles are: Molecular Changes in Cell Transformation; Differentiation, Growth Control, and Cell Transformation; Mutagenesis and Cell Transformation; Tumor Promotion and Cell Transformation; Mechanisms of Transformation of Human Fibroblasts; Mechanisms of Transformation of Epithelial Cells; Mechanisms of C 3 H 10T12 Cell Transformation; Mechanisms of Radiation-Induced Cell Transformation; and Use of Cell Transformation Assays for Carcinogen Testing

  5. Efficacy of T2 Magnetic Resonance Assay in Monitoring Candidemia after Initiation of Antifungal Therapy: the Serial Therapeutic and Antifungal Monitoring Protocol (STAMP) Trial.

    Science.gov (United States)

    Mylonakis, Eleftherios; Zacharioudakis, Ioannis M; Clancy, Cornelius J; Nguyen, M Hong; Pappas, Peter G

    2018-04-01

    The performance of blood culture for monitoring candidemia clearance is hampered by its low sensitivity, especially during antifungal therapy. The T2 magnetic resonance (T2MR) assay combines magnetic resonance with nanotechnology to identify whole Candida species cells. A multicenter clinical trial studied the performance of T2MR in monitoring candidemia clearance compared to blood culture. Adults with a blood culture positive for yeast were enrolled and had blood cultures and T2MR testing performed on prespecified days. Thirty-one patients completed the trial. Thirteen of the 31 patients (41.9%) had at least one positive surveillance T2MR and/or blood culture result. All positive blood cultures (7/7 [100%]) had an accompanying positive T2MR result with concordance in the identified Candida sp., while only 7/23 (30.4%) T2MR results had an accompanying positive blood culture. There was one case of discordance in species identification between T2MR and the preenrollment blood culture with evidence to support deep-seated infection by the Candida spp. detected by the T2MR assay. Based on the log rank test, there was a statistically significant improvement in posttreatment surveillance using the T2MR assay compared to blood culture ( P = 0.004). Limitations of the study include the small sample size and lack of outcome data. In conclusion, the T2MR assay significantly outperformed blood cultures for monitoring the clearance of candidemia in patients receiving antifungal therapy and may be useful in determining adequate source control, timing for deescalation, and optimal duration of treatment. However, further studies are needed to determine the viability of Candida species cells detected by the T2MR assay and correlate the results with patient outcomes. (This study is registered at ClinicalTrials.gov under registration number NCT02163889.). Copyright © 2018 Mylonakis et al.

  6. Improving CART-Cell Therapy of Solid Tumors with Oncolytic Virus-Driven Production of a Bispecific T-cell Engager.

    Science.gov (United States)

    Wing, Anna; Fajardo, Carlos Alberto; Posey, Avery D; Shaw, Carolyn; Da, Tong; Young, Regina M; Alemany, Ramon; June, Carl H; Guedan, Sonia

    2018-05-01

    T cells expressing chimeric antigen receptors (CART) have shown significant promise in clinical trials to treat hematologic malignancies, but their efficacy in solid tumors has been limited. Oncolytic viruses have the potential to act in synergy with immunotherapies due to their immunogenic oncolytic properties and the opportunity of incorporating therapeutic transgenes in their genomes. Here, we hypothesized that an oncolytic adenovirus armed with an EGFR-targeting, bispecific T-cell engager (OAd-BiTE) would improve the outcome of CART-cell therapy in solid tumors. We report that CART cells targeting the folate receptor alpha (FR-α) successfully infiltrated preestablished xenograft tumors but failed to induce complete responses, presumably due to the presence of antigen-negative cancer cells. We demonstrated that OAd-BiTE-mediated oncolysis significantly improved CART-cell activation and proliferation, while increasing cytokine production and cytotoxicity, and showed an in vitro favorable safety profile compared with EGFR-targeting CARTs. BiTEs secreted from infected cells redirected CART cells toward EGFR in the absence of FR-α, thereby addressing tumor heterogeneity. BiTE secretion also redirected CAR-negative, nonspecific T cells found in CART-cell preparations toward tumor cells. The combinatorial approach improved antitumor efficacy and prolonged survival in mouse models of cancer when compared with the monotherapies, and this was the result of an increased BiTE-mediated T-cell activation in tumors. Overall, these results demonstrated that the combination of a BiTE-expressing oncolytic virus with adoptive CART-cell therapy overcomes key limitations of CART cells and BiTEs as monotherapies in solid tumors and encourage its further evaluation in human trials. Cancer Immunol Res; 6(5); 605-16. ©2018 AACR . ©2018 American Association for Cancer Research.

  7. Epithelial cells as alternative human biomatrices for comet assay.

    Science.gov (United States)

    Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; Valverde, Mahara

    2014-01-01

    The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases.

  8. Low-Dose Growth Hormone for 40 Weeks Induces HIV-1-Specific T-Cell Responses in Patients on Effective Combination Antiretroviral Therapy

    DEFF Research Database (Denmark)

    Herasimtschuk, Anna A; Hansen, Birgitte R; Langkilde, Anne

    2013-01-01

    changes. Subjects with the most robust responses in the ELISpot assays had improved thymic function following rhGH administration, as determined using CD4(+) T-cell receptor rearrangement excision circle (TREC) and thymic density data from the original study. T cells from these robust responders were...

  9. Novel T cells with improved in vivo anti-tumor activity generated by RNA electroporation

    Directory of Open Access Journals (Sweden)

    Xiaojun Liu

    2017-05-01

    Full Text Available ABSTRACT The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell-based adoptive immunotherapy. In this report, we found that OKT3/IL-2-stimulated T cells were phenotypically more heterogeneous, with enhanced anti-tumor activity in vitro and when locally administered in a solid tumor mouse model. To further improve the OKT3/IL-2-based T cell manufacturing procedure, we developed a novel T cell stimulation and expansion method in which peripheral blood mononuclear cells were electroporated with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment against CD3 and the intracellular domains of CD28 and 4-1BB (OKT3-28BB. The expanded T cells were phenotypically and functionally similar to T cells expanded by OKT3/IL-2. Moreover, co-electroporation of CD86 and 4-1BBL could further change the phenotype and enhance the in vivo anti-tumor activity. Although T cells expanded by the co-electroporation of OKT3-28BB with CD86 and 4-1BBL showed an increased central memory phenotype, the T cells still maintained tumor lytic activities as potent as those of OKT3/IL-2 or OKT3-28BB-stimulated T cells. In different tumor mouse models, T cells expanded by OKT3-28BB RNA electroporation showed anti-tumor activities superior to those of OKT3/IL-2 T cells. Hence, T cells with both a less differentiated phenotype and potent tumor killing ability can be generated by RNA electroporation, and this T cell manufacturing procedure can be further optimized by simply co-delivering other splices of RNA, thus providing a simple and cost-effective method for generating high-quality T cells for adoptive immunotherapy.

  10. Specific assay measuring binding of /sup 125/I-Gp 120 from HIV to T4/sup +//CD4/sup +/ cells

    Energy Technology Data Exchange (ETDEWEB)

    Lundin, K.; Nygren, A.; Ramstedt, U.; Gidlund, M.; Wigzell, H.; Arthur, L.O.; Robey, W.G.; Morein, B.

    1987-02-26

    The HIV (HTLV-III) envelope glycoprotein, Gp120, was isolated from virus-infected tissue culture cells using affinity chromatography. A radioimmunoassay was developed to determine the degree of iodinated Gp120 to target CD4/sup +/ (T4/sup +/) cells. /sup 125/I-Gp120 could be shown to selectively bind to CD4/sup +/ cells only. The Gp120 remained bound to these cells after repeated washes. Monoclonal anti-CD4 antibodies block the binding of Gp120 to CD4/sup +/ cells. Monoclonal antibodies to other cell surface components do not interfere with /sup 125/I-Gp120 binding. All IgG antibodies from HIV seropositive donors tested block /sup 125/I-GP120 binding, though with variable titers. The authors believe that this assay provides further proof for the use of CD4 (T4) as a component of the receptor for HIV. It represents a safe, objective and sensitive method for the analysis of Gp120-CD4 interactions, as well as the potential of antibodies to interfere with this binding. (Auth.). 24 refs.; 2 figs.; 8 tabs.

  11. Cell Culture Assay for Human Noroviruses [response

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  12. Whole blood assay to access T cell-immune responses to Mycobacterium tuberculosis antigens in healthy Brazilian individuals

    Directory of Open Access Journals (Sweden)

    Paulo RZ Antas

    2004-02-01

    Full Text Available The production of interferon gamma (IFNgamma guarantees effective T cell-mediated immunity against Mycobacterium tuberculosis infection. In the present study, we simply compare the in vitro immune responses to Mycobacterium antigens in terms of IFNg production in a total of 10 healthy Brazilian volunteers. Whole blood and mononuclear cells were cultivated in parallel with PPD, Ag85B, and M. bovis hsp65, and five-days supernatants were harvested for cytokine detection by ELISA. The inter-assay result was that the overall profile of agreement in response to antigens was highly correlated (r² = 0.9266; p = 0.0102. Potential analysis is in current progress to dictate the usefulness of this method to access the immune responses also in tuberculosis patients and its contacts.

  13. T cell progenitors in the mouse fetal liver

    International Nuclear Information System (INIS)

    Rabinowich, H.; Umiel, T.; Globerson, A.

    1983-01-01

    Fourteen-day mouse fetal liver was found to contain cells capable of giving rise to T as well as B cell functions. The experimental system consisted of congenic C3H/DiSn and (C3H/DiSn X C3H.SW)F1 lethally irradiated (900 R) mice reconstituted with C3H/DiSn fetal liver or bone marrow cells. Assays included thyroid allograft rejection as well as in vitro measurement of reactivity to phytohemagglutinin (PHA) and concanavalin A (Con A) and in a mixed lymphocyte culture (MLC) system in spleen, lymph node, and thymus cells. The fetal liver chimeras were found to become as capable as the bone marrow chimeras in responding in these various assays. The T cell responses lagged behind the responses to the B cell mitogens dextran sulfate (DXS) and lipopolysaccharide (LPS) (30 days after reconstitution, as compared with 14 days for DXS and 21 for LPS). The reacting cells were of the donor genotype, as revealed after treatment with C3H/DiSn (H-2k) anti-C3H.SW (H-2b) congenic sera. T cell responses were not manifest in thymectomized (TX) chimeras. Hence, the liver seems to contain cells capable of developing into T cell lineages in a thymus-dependent process

  14. Human T cell immunosenescence and inflammation in aging.

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    Bektas, Arsun; Schurman, Shepherd H; Sen, Ranjan; Ferrucci, Luigi

    2017-10-01

    The aging process is driven by a finite number of inter-related mechanisms that ultimately lead to the emergence of characteristic phenotypes, including increased susceptibility to multiple chronic diseases, disability, and death. New assays and analytical tools have become available that start to unravel some of these mechanisms. A prevailing view is that aging leads to an imbalance between stressors and stress-buffering mechanisms that causes loss of compensatory reserve and accumulation of unrepaired damage. Central to this paradigm are changes in the immune system and the chronic low-grade proinflammatory state that affect many older individuals, even when they are apparently healthy and free of risk factors. Independent of chronological age, high circulating levels of proinflammatory markers are associated with a high risk of multiple adverse health outcomes in older persons. In this review, we discuss current theories about causes and consequences of the proinflammatory state of aging, with a focus on changes in T cell function. We examine the role of NF-κB activation and its dysregulation and how NF-κB activity differs among subgroups of T cells. We explore emerging hypotheses about immunosenescence and changes in T cell behavior with age, including consideration of the T cell antigen receptor and regulatory T cells (T regs ). We conclude by illustrating how research using advanced technology is uncovering clues at the core of inflammation and aging. Some of the preliminary work in this field is already improving our understanding of the complex mechanisms by which immunosenescence of T cells is intertwined during human aging. © Society for Leukocyte Biology.

  15. Profiling helper T cell subset gene expression in deer mice

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    Hjelle Brian

    2006-08-01

    Full Text Available Abstract Background Deer mice (Peromyscus maniculatus are the most common mammals in North America and are reservoirs for several zoonotic agents, including Sin Nombre virus (SNV, the principal etiologic agent of hantavirus cardiopulmonary syndrome (HCPS in North America. Unlike human HCPS patients, SNV-infected deer mice show no overt pathological symptoms, despite the presence of virus in the lungs. A neutralizing IgG antibody response occurs, but the virus establishes a persistent infection. Limitations of detailed analysis of deer mouse immune responses to SNV are the lack of reagents and methods for evaluating such responses. Results We developed real-time PCR-based detection assays for several immune-related transcription factor and cytokine genes from deer mice that permit the profiling of CD4+ helper T cells, including markers of Th1 cells (T-bet, STAT4, IFNγ, TNF, LT, Th2 cells (GATA-3, STAT6, IL-4, IL-5 and regulatory T cells (Fox-p3, IL-10, TGFβ1. These assays compare the expression of in vitro antigen-stimulated and unstimulated T cells from individual deer mice. Conclusion We developed molecular methods for profiling immune gene expression in deer mice, including a multiplexed real-time PCR assay for assessing expression of several cytokine and transcription factor genes. These assays should be useful for characterizing the immune responses of experimentally- and naturally-infected deer mice.

  16. A Comparison of Real-Time and Endpoint Cell Viability Assays for Improved Synthetic Lethal Drug Validation.

    Science.gov (United States)

    Single, Andrew; Beetham, Henry; Telford, Bryony J; Guilford, Parry; Chen, Augustine

    2015-12-01

    Cell viability assays fulfill a central role in drug discovery studies. It is therefore important to understand the advantages and disadvantages of the wide variety of available assay methodologies. In this study, we compared the performance of three endpoint assays (resazurin reduction, CellTiter-Glo, and nuclei enumeration) and two real-time systems (IncuCyte and xCELLigence). Of the endpoint approaches, both the resazurin reduction and CellTiter-Glo assays showed higher cell viabilities when compared directly to stained nuclei counts. The IncuCyte and xCELLigence real-time systems were comparable, and both were particularly effective at tracking the effects of drug treatment on cell proliferation at sub-confluent growth. However, the real-time systems failed to evaluate contrasting cell densities between drug-treated and control-treated cells at full growth confluency. Here, we showed that using real-time systems in combination with endpoint assays alleviates the disadvantages posed by each approach alone, providing a more effective means to evaluate drug toxicity in monolayer cell cultures. Such approaches were shown to be effective in elucidating the toxicity of synthetic lethal drugs in an isogenic pair of MCF10A breast cell lines. © 2015 Society for Laboratory Automation and Screening.

  17. [Autologous regulatory T cells can suppress the proliferation of lymphoma cell line in vitro].

    Science.gov (United States)

    Ying, Zhi-Tao; Guo, Jun; Ren, Jun; Kong, Yan; Yuan, Zhi-Hong; Liu, Xi-Juan; Zhang, Chen; Zheng, Wen; Song, Yu-Qin; Zhang, Yun-Tao; Zhu, Jun

    2009-06-01

    This study was aimed to investigate the suppressive effect of regulatory T (Treg) cells on the T cell lymphoma EL4 cell line and to explore its mechanism. C57BL/6 Mouse Treg cells were isolated by MACS (magnetic cell sorting). The purity and the expression of Foxp3 were detected by flow cytometry. The suppressive effect of sorted Treg cells on EL4 cells was detected by MTT assay. The secretion of TGF-beta1 and IL-10 was examined by enzyme-linked immunosorbent assay (ELISA). The results showed that CD4(+)CD25(+) T cells could be successfully isolated by MACS with the purity reaching 91.6% and the expression level of Foxp3 was 78.9%. The ratio of viable cells was more than 95%. Regulatory T cells could suppress the proliferation of EL4 cells effectively in the presence of antigen presenting cells (APCs). And the suppressive effect was most significant at 1:1 ratio. In addition, the suppression still existed without APCs. TGF-beta1 and IL-10 could not be detected by ELISA. It is concluded that the Treg cells can suppress T lymphoma cell in vitro. The suppressive effect of Treg cells works in dose-dependent manner, but not in cytokine-dependent manner. The mechanism of this suppression may take effect through cell-cell contact.

  18. Long-term survival of transplanted allogeneic cells engineered to express a T cell chemorepellent.

    Science.gov (United States)

    Papeta, Natalia; Chen, Tao; Vianello, Fabrizio; Gererty, Lyle; Malik, Ashish; Mok, Ying-Ting; Tharp, William G; Bagley, Jessamyn; Zhao, Guiling; Stevceva, Liljana; Yoon, Victor; Sykes, Megan; Sachs, David; Iacomini, John; Poznansky, Mark C

    2007-01-27

    Alloantigen specific T cells have been shown to be required for allograft rejection. The chemokine, stromal cell derived factor-1 (SDF-1) at high concentration, has been shown to act as a T-cell chemorepellent and abrogate T-cell infiltration into a site of antigen challenge in vivo via a mechanism termed fugetaxis or chemorepulsion. We postulated that this mechanism could be exploited therapeutically and that allogeneic cells engineered to express a chemorepellent protein would not be rejected. Allogeneic murine insulinoma beta-TC3 cells and primary islets from BALB/C mice were engineered to constitutively secrete differential levels of SDF-1 and transplanted into allogeneic diabetic C57BL/6 mice. Rejection was defined as the permanent return of hyperglycemia and was correlated with the level of T-cell infiltration. The migratory response of T-cells to SDF-1 was also analyzed by transwell migration assay and time-lapse videomicroscopy. The cytotoxicity of cytotoxic T cell (CTLs) against beta-TC3 cells expressing high levels of SDF-1 was measured in standard and modified chromium-release assays in order to determine the effect of CTL migration on killing efficacy. Control animals rejected allogeneic cells and remained diabetic. In contrast, high level SDF-1 production by transplanted cells resulted in increased survival of the allograft and a significant reduction in blood glucose levels and T-cell infiltration into the transplanted tissue. This is the first demonstration of a novel approach that exploits T-cell chemorepulsion to induce site specific immune isolation and thereby overcomes allograft rejection without the use of systemic immunosuppression.

  19. Automated high-content assay for compounds selectively toxic to Trypanosoma cruzi in a myoblastic cell line.

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    Julio Alonso-Padilla

    2015-01-01

    Full Text Available Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, represents a very important public health problem in Latin America where it is endemic. Although mostly asymptomatic at its initial stage, after the disease becomes chronic, about a third of the infected patients progress to a potentially fatal outcome due to severe damage of heart and gut tissues. There is an urgent need for new drugs against Chagas disease since there are only two drugs available, benznidazole and nifurtimox, and both show toxic side effects and variable efficacy against the chronic stage of the disease.Genetically engineered parasitic strains are used for high throughput screening (HTS of large chemical collections in the search for new anti-parasitic compounds. These assays, although successful, are limited to reporter transgenic parasites and do not cover the wide T. cruzi genetic background. With the aim to contribute to the early drug discovery process against Chagas disease we have developed an automated image-based 384-well plate HTS assay for T. cruzi amastigote replication in a rat myoblast host cell line. An image analysis script was designed to inform on three outputs: total number of host cells, ratio of T. cruzi amastigotes per cell and percentage of infected cells, which respectively provides one host cell toxicity and two T. cruzi toxicity readouts. The assay was statistically robust (Z´ values >0.6 and was validated against a series of known anti-trypanosomatid drugs.We have established a highly reproducible, high content HTS assay for screening of chemical compounds against T. cruzi infection of myoblasts that is amenable for use with any T. cruzi strain capable of in vitro infection. Our visual assay informs on both anti-parasitic and host cell toxicity readouts in a single experiment, allowing the direct identification of compounds selectively targeted to the parasite.

  20. Optimization of methods for the genetic modification of human T cells.

    Science.gov (United States)

    Bilal, Mahmood Y; Vacaflores, Aldo; Houtman, Jon Cd

    2015-11-01

    CD4(+) T cells are not only critical in the fight against parasitic, bacterial and viral infections, but are also involved in many autoimmune and pathological disorders. Studies of protein function in human T cells are confined to techniques such as RNA interference (RNAi) owing to ethical reasons and relative simplicity of these methods. However, introduction of RNAi or genes into primary human T cells is often hampered by toxic effects from transfection or transduction methods that yield cell numbers inadequate for downstream assays. Additionally, the efficiency of recombinant DNA expression is frequently low because of multiple factors including efficacy of the method and strength of the targeting RNAs. Here, we describe detailed protocols that will aid in the study of primary human CD4(+) T cells. First, we describe a method for development of effective microRNA/shRNAs using available online algorithms. Second, we illustrate an optimized protocol for high efficacy retroviral or lentiviral transduction of human T-cell lines. Importantly, we demonstrate that activated primary human CD4(+) T cells can be transduced efficiently with lentiviruses, with a highly activated population of T cells receiving the largest number of copies of integrated DNA. We also illustrate a method for efficient lentiviral transduction of hard-to-transduce un-activated primary human CD4(+) T cells. These protocols will significantly assist in understanding the activation and function of human T cells and will ultimately aid in the development or improvement of current drugs that target human CD4(+) T cells.

  1. [gammadelta T cells stimulated by zoledronate kill osteosarcoma cells].

    Science.gov (United States)

    Jiang, Hui; Xu, Qiang; Yang, Chao; Cao, Zhen-Guo; Li, Zhao-Xu; Ye, Zhao-Ming

    2010-12-01

    To investigate the cytotoxicity of human γδT cells from PBMCs stimulated by zoledronate against osteosarcoma cell line HOS in vitro and in vivo and evaluate the relavent pathways. The peripheral blood mononuclear cells (PBMCs)of healthy donors were stimulated by single dose zoledronate and cultured in the present of IL-2 for two weeks, analysising the percentage of γδT cells on a FACSCalibur cytometer.Study the cytotoxicity of γδT cells against the osteosarcoma line HOS using LDH release assay kit. Pre-treatment of γδT cells with anti-human γδTCR antibody, anti-human NKG2D antibody and concanamycin A to bolck the relavent pathways for evaluating the mechenisms of its cytotoxicity. In vivo, BALB/c mice were inoculated subcutaneously osteosarcoma cell HOS for developing hypodermal tumors. And they were randomized into two groups: unteated group, γδT cell therapy group. Tumor volume and weight of the two groups were compared. After two weeks of culture, γδT cells from zoledronate-stimulated PBMCs could reach (95±3)%. When the E:T as 6:1, 12:1, 25:1, 50:1, the percentage of osteosarcoma cell HOS killed by γδT cells was 26.8%, 31.5%, 37.8%, 40.9%, respectively.When anti-huma γδTCR antibody, anti-human NKG2D antibody and concanamycin A blocked the relavent pathways, the percentage was 32.3%, 4.7%, 16.7% ( E:T as 25:1), respectively. In vivo, the tumor inhibition rate of the group of γδT cell therapy was 42.78%. γδT cells derived from PBMCs stimulated by zoledronate can acquired pure γδT cells. And they show strong cytoxicity against osteosarcoma cell line HOS in vitro and in vivo.

  2. Dual role of miR-21 in CD4+ T-cells: activation-induced miR-21 supports survival of memory T-cells and regulates CCR7 expression in naive T-cells.

    Directory of Open Access Journals (Sweden)

    Katarzyna Smigielska-Czepiel

    Full Text Available Immune cell-type specific miRNA expression patterns have been described but the detailed role of single miRNAs in the function of T-cells remains largely unknown. We investigated the role of miR-21 in the function of primary human CD4+ T-cells. MiR-21 is substantially expressed in T-cells with a memory phenotype, and is robustly upregulated upon αCD3/CD28 activation of both naive and memory T-cells. By inhibiting the endogenous miR-21 function in activated naive and memory T-cells, we showed that miR-21 regulates fundamentally different aspects of T-cell biology, depending on the differentiation status of the T-cell. Stable inhibition of miR-21 function in activated memory T-cells led to growth disadvantage and apoptosis, indicating that the survival of memory T-cells depends on miR-21 function. In contrast, stable inhibition of miR-21 function in activated naive T-cells did not result in growth disadvantage, but led to a significant induction of CCR7 protein expression. Direct interaction between CCR7 and miR-21 was confirmed in a dual luciferase reporter assay. Our data provide evidence for a dual role of miR-21 in CD4+ T cells; Regulation of T-cell survival is confined to activated memory T-cells, while modulation of potential homing properties, through downregulation of CCR7 protein expression, is observed in activated naive T-cells.

  3. Chimeric Antigen Receptor T Cell (Car T Cell Therapy In Hematology

    Directory of Open Access Journals (Sweden)

    Pinar Ataca

    2015-12-01

    Full Text Available It is well demonstrated that 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 (HSCT. Adoptive T cell transfer has been improved to be more specific and potent and cause less off-target toxicities. 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 July 1, 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 beneficiaries 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-clinical studies, effectiveness and drawbacks of this strategy.

  4. Alterations in regulatory T cells induced by specific oligosaccharides improve vaccine responsiveness in mice.

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    Marcel A Schijf

    Full Text Available Prophylactic vaccinations are generally performed to protect naïve individuals with or without suppressed immune responsiveness. In a mouse model for Influenza vaccinations the specific alterations of CD4(+CD25(+Foxp3(+ regulatory T-cells (Tregs in the immune modulation induced by orally supplied oligosaccharides containing scGOS/lcFOS/pAOS was assessed. This dietary intervention increased vaccine specific DTH responses. In addition, a significant increased percentage of T-bet(+ (Th1 activated CD69(+CD4(+ T cells (p<0.001 and reduced percentage of Gata-3(+ (Th2 activated CD69(+CD4(+T cells (p<0.001 was detected in the mesenteric lymph nodes (MLN of mice receiving scGOS/lcFOS/pAOS compared to control mice. Although no difference in the number or percentage of Tregs (CD4(+Foxp3(+ could be determined after scGOS/lcFOS/pAOS intervention, the percentage of CXCR3 (+ /T-bet(+ (Th1-Tregs was significantly reduced (p<0.05 in mice receiving scGOS/lcFOS/pAOS as compared to mice receiving placebo diets. Moreover, although no absolute difference in suppressive capacity could be detected, an alteration in cytokine profile suggests a regulatory T cell shift towards a reducing Th1 suppression profile, supporting an improved vaccination response.These data are indicative for improved vaccine responsiveness due to reduced Th1 suppressive capacity in the Treg population of mice fed the oligosaccharide specific diet, showing compartmentalization within the Treg population. The modulation of Tregs to control immune responses provides an additional arm of intervention using alternative strategies possibly leading to the development of improved vaccines.

  5. miR-664 negatively regulates PLP2 and promotes cell proliferation and invasion in T-cell acute lymphoblastic leukaemia

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Hong; Miao, Mei-hua; Ji, Xue-qiang; Xue, Jun; Shao, Xue-jun, E-mail: xuejunshao@hotmail.com

    2015-04-03

    MicroRNAs (miRNAs) play important roles in the pathogenesis of many types of cancers by negatively regulating gene expression at posttranscriptional level. However, the role of microRNAs in leukaemia, particularly T-cell acute lymphoblastic leukaemia (T-ALL), has remained elusive. Here, we identified miR-664 and its predicted target gene PLP2 were differentially expressed in T-ALL using bioinformatics methods. In T-ALL cell lines, CCK-8 proliferation assay indicated that the cell proliferation was promoted by miR-664, while miR-664 inhibitor could significantly inhibited the proliferation. Moreover, migration and invasion assay showed that overexpression of miR-664 could significantly promoted the migration and invasion of T-ALL cells, whereas miR-664 inhibitor could reduce cell migration and invasion. luciferase assays confirmed that miR-664 directly bound to the 3'untranslated region of PLP2, and western blotting showed that miR-664 suppressed the expression of PLP2 at the protein levels. This study indicated that miR-664 negatively regulates PLP2 and promotes proliferation and invasion of T-ALL cell lines. Thus, miR-664 may represent a potential therapeutic target for T-ALL intervention. - Highlights: • miR-664 mimics promote the proliferation and invasion of T-ALL cells. • miR-664 inhibitors inhibit the proliferation and invasion of T-ALL cells. • miR-664 targets 3′ UTR of PLP2 in T-ALL cells. • miR-664 negatively regulates PLP2 in T-ALL cells.

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

    Science.gov (United States)

    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

    -like receptor (TLR) ligand CpG, induced the most profound antigen-specific T cell response, by both CD4 + and CD8 + T cells, in vivo. Overall, our data reveal the impact of NP composition and surface properties on the type and extension of induced immune responses. Deeper understanding on the NP-immune cell crosstalk can guide the rational development of nano-immunotherapeutic systems with improved and specific therapeutic efficacy and avoiding off-target effects. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Objective scoring of transformed foci in BALB/c 3T3 cell transformation assay by statistical image descriptors.

    Science.gov (United States)

    Urani, C; Corvi, R; Callegaro, G; Stefanini, F M

    2013-09-01

    In vitro cell transformation assays (CTAs) have been shown to model important stages of in vivo carcinogenesis and have the potential to predict carcinogenicity in humans. Advantages of CTAs are their ability of revealing both genotoxic and non-genotoxic carcinogens while reducing both experimental costs and the number of animals used. The endpoint of the CTA is foci formation, and requires classification under light microscopy based on morphology. Thus current limitations for the wide adoption of the assay partially depend on a fair degree of subjectivity in foci scoring. An objective evaluation may be obtained after separating foci from background monolayer in the digital image, and quantifying values of statistical descriptors which are selected to capture eye-scored morphological features. The aim of this study was to develop statistical descriptors to be applied to transformed foci of BALB/c 3T3, which cover foci size, multilayering and invasive cell growth into the background monolayer. Proposed descriptors were applied to a database of 407 foci images to explore the numerical features, and to illustrate open problems and potential solutions. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay

    International Nuclear Information System (INIS)

    Taxvig, Camilla; Olesen, Pelle Thonning; Nellemann, Christine

    2011-01-01

    Although, it is well-established that information on the metabolism of a substance is important in the evaluation of its toxic potential, there is limited experience with incorporating metabolic aspects into in vitro tests for endocrine disrupters. The aim of the current study was a) to study different in vitro systems for biotransformation of ten known endocrine disrupting chemicals (EDs): five azole fungicides, three parabens and 2 phthalates, b) to determine possible changes in the ability of the EDs to bind and activate the thyroid receptor (TR) in the in vitro T-screen assay after biotransformation and c) to investigate the endogenous metabolic capacity of the GH3 cells, the cell line used in the T-screen assay, which is a proliferation assay used for the in vitro detection of agonistic and antagonistic properties of compounds at the level of the TR. The two in vitro metabolizing systems tested the human liver S9 mix and the PCB-induced rat microsomes gave an almost complete metabolic transformation of the tested parabens and phthalates. No marked difference the effects in the T-screen assay was observed between the parent compounds and the effects of the tested metabolic extracts. The GH3 cells themselves significantly metabolized the two tested phthalates dimethyl phthalate (DMP) and diethyl phthalate (DEP). Overall the results and qualitative data from the current study show that an in vitro metabolizing system using liver S9 or microsomes could be a convenient method for the incorporation of metabolic and toxicokinetic aspects into in vitro testing for endocrine disrupting effects.

  9. Discriminating Different Cancer Cells Using a Zebrafish in Vivo Assay

    Directory of Open Access Journals (Sweden)

    Pooja Pardhanani

    2011-10-01

    Full Text Available Despite the expanded understanding of tumor angiogenesis phenomenon and how it impacts cancer treatment outcomes, we have yet to develop a robust assay that can quickly, easily, and quantitatively measure tumor-induced angiogenesis. Since the zebrafish/tumor xenograft represents an emerging tool in this regard, the present study strives to capitalize on the ease, effectiveness, and the adaptability of this model to quantify tumor angiogenesis. In order to test a range of responses, we chose two different tumorigenic cell lines, the human non-small cell lung carcinoma (H1299 and the mouse lung adenocarcinoma (CL13. Non-tumorigenic 3T3-L1 cells served as negative control. The cells were grafted near to the perivitelline space of the zebrafish embryos and the angiogenic response was analyzed using whole-mount alkaline phosphatase (AP vessel staining and fluorescence microscopy. Angiogenic activity was scored based on the length and number of the newly formed ectopic vessels and the percentage of embryos with ectopic vessels. At 2 day-post-implantation, we detected a significant increase in the length and number of ectopic vessels with H1299 cell implantation compared to CL13 cell transplantation, both are higher than 3T3-L1 control. We also observed a significantly higher percentage of embryos with ectopic vessels with H1299 and CL13 transplantation compared to the 3T3-L1 control, but this parameter is not as robust and reliable as measuring the length and number of ectopic vessels. Furthermore, the systemic exposure of zebrafish embryos to an anti-angiogenesis drug (PTK 787, inhibitor of vascular endothelial growth factor receptor tyrosine kinase inhibited tumor-induced angiogenesis, suggesting that the assay can be used to evaluate anti-angiogenic drugs. This study implicates the feasibility of using zebrafish xenotransplantation to perform quantitative measurement of the angiogenic activity of cancer cells which can be further extended to

  10. Discriminating Different Cancer Cells Using a Zebrafish in Vivo Assay

    International Nuclear Information System (INIS)

    Moshal, Karni S.; Ferri-Lagneau, Karine F.; Haider, Jamil; Pardhanani, Pooja; Leung, TinChung

    2011-01-01

    Despite the expanded understanding of tumor angiogenesis phenomenon and how it impacts cancer treatment outcomes, we have yet to develop a robust assay that can quickly, easily, and quantitatively measure tumor-induced angiogenesis. Since the zebrafish/tumor xenograft represents an emerging tool in this regard, the present study strives to capitalize on the ease, effectiveness, and the adaptability of this model to quantify tumor angiogenesis. In order to test a range of responses, we chose two different tumorigenic cell lines, the human non-small cell lung carcinoma (H1299) and the mouse lung adenocarcinoma (CL13). Non-tumorigenic 3T3-L1 cells served as negative control. The cells were grafted near to the perivitelline space of the zebrafish embryos and the angiogenic response was analyzed using whole-mount alkaline phosphatase (AP) vessel staining and fluorescence microscopy. Angiogenic activity was scored based on the length and number of the newly formed ectopic vessels and the percentage of embryos with ectopic vessels. At 2 day-post-implantation, we detected a significant increase in the length and number of ectopic vessels with H1299 cell implantation compared to CL13 cell transplantation, both are higher than 3T3-L1 control. We also observed a significantly higher percentage of embryos with ectopic vessels with H1299 and CL13 transplantation compared to the 3T3-L1 control, but this parameter is not as robust and reliable as measuring the length and number of ectopic vessels. Furthermore, the systemic exposure of zebrafish embryos to an anti-angiogenesis drug (PTK 787, inhibitor of vascular endothelial growth factor receptor tyrosine kinase) inhibited tumor-induced angiogenesis, suggesting that the assay can be used to evaluate anti-angiogenic drugs. This study implicates the feasibility of using zebrafish xenotransplantation to perform quantitative measurement of the angiogenic activity of cancer cells which can be further extended to measure cancer cell

  11. Type 2 innate lymphoid cell suppression by regulatory T cells attenuates airway hyperreactivity and requires inducible T-cell costimulator-inducible T-cell costimulator ligand interaction.

    Science.gov (United States)

    Rigas, Diamanda; Lewis, Gavin; Aron, Jennifer L; Wang, Bowen; Banie, Homayon; Sankaranarayanan, Ishwarya; Galle-Treger, Lauriane; Maazi, Hadi; Lo, Richard; Freeman, Gordon J; Sharpe, Arlene H; Soroosh, Pejman; Akbari, Omid

    2017-05-01

    Atopic diseases, including asthma, exacerbate type 2 immune responses and involve a number of immune cell types, including regulatory T (Treg) cells and the emerging type 2 innate lymphoid cells (ILC2s). Although ILC2s are potent producers of type 2 cytokines, the regulation of ILC2 activation and function is not well understood. In the present study, for the first time, we evaluate how Treg cells interact with pulmonary ILC2s and control their function. ILC2s and Treg cells were evaluated by using in vitro suppression assays, cell-contact assays, and gene expression panels. Also, human ILC2s and Treg cells were adoptively transferred into NOD SCID γC-deficient mice, which were given isotype or anti-inducible T-cell costimulator ligand (ICOSL) antibodies and then challenged with IL-33 and assessed for airway hyperreactivity. We show that induced Treg cells, but not natural Treg cells, effectively suppress the production of the ILC2-driven proinflammatory cytokines IL-5 and IL-13 both in vitro and in vivo. Mechanistically, our data reveal the necessity of inducible T-cell costimulator (ICOS)-ICOS ligand cell contact for Treg cell-mediated ILC2 suppression alongside the suppressive cytokines TGF-β and IL-10. Using a translational approach, we then demonstrate that human induced Treg cells suppress syngeneic human ILC2s through ICOSL to control airway inflammation in a humanized ILC2 mouse model. These findings suggest that peripheral expansion of induced Treg cells can serve as a promising therapeutic target against ILC2-dependent asthma. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  12. A cell-based high-throughput screening assay for radiation susceptibility using automated cell counting

    International Nuclear Information System (INIS)

    Hodzic, Jasmina; Dingjan, Ilse; Maas, Mariëlle JP; Meulen-Muileman, Ida H van der; Menezes, Renee X de; Heukelom, Stan; Verheij, Marcel; Gerritsen, Winald R; Geldof, Albert A; Triest, Baukelien van; Beusechem, Victor W van

    2015-01-01

    Radiotherapy is one of the mainstays in the treatment for cancer, but its success can be limited due to inherent or acquired resistance. Mechanisms underlying radioresistance in various cancers are poorly understood and available radiosensitizers have shown only modest clinical benefit. There is thus a need to identify new targets and drugs for more effective sensitization of cancer cells to irradiation. Compound and RNA interference high-throughput screening technologies allow comprehensive enterprises to identify new agents and targets for radiosensitization. However, the gold standard assay to investigate radiosensitivity of cancer cells in vitro, the colony formation assay (CFA), is unsuitable for high-throughput screening. We developed a new high-throughput screening method for determining radiation susceptibility. Fast and uniform irradiation of batches up to 30 microplates was achieved using a Perspex container and a clinically employed linear accelerator. The readout was done by automated counting of fluorescently stained nuclei using the Acumen eX3 laser scanning cytometer. Assay performance was compared to that of the CFA and the CellTiter-Blue homogeneous uniform-well cell viability assay. The assay was validated in a whole-genome siRNA library screening setting using PC-3 prostate cancer cells. On 4 different cancer cell lines, the automated cell counting assay produced radiation dose response curves that followed a linear-quadratic equation and that exhibited a better correlation to the results of the CFA than did the cell viability assay. Moreover, the cell counting assay could be used to detect radiosensitization by silencing DNA-PKcs or by adding caffeine. In a high-throughput screening setting, using 4 Gy irradiated and control PC-3 cells, the effects of DNA-PKcs siRNA and non-targeting control siRNA could be clearly discriminated. We developed a simple assay for radiation susceptibility that can be used for high-throughput screening. This will aid

  13. Monitoring the initiation and kinetics of human dendritic cell-induced polarization of autologous naive CD4+ T cells.

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    Tammy Oth

    Full Text Available A crucial step in generating de novo immune responses is the polarization of naive cognate CD4+ T cells by pathogen-triggered dendritic cells (DC. In the human setting, standardized DC-dependent systems are lacking to study molecular events during the initiation of a naive CD4+ T cell response. We developed a TCR-restricted assay to compare different pathogen-triggered human DC for their capacities to instruct functional differentiation of autologous, naive CD4+ T cells. We demonstrated that this methodology can be applied to compare differently matured DC in terms of kinetics, direction, and magnitude of the naive CD4+ T cell response. Furthermore, we showed the applicability of this assay to study the T cell polarizing capacity of low-frequency blood-derived DC populations directly isolated ex vivo. This methodology for addressing APC-dependent instruction of naive CD4+ T cells in a human autologous setting will provide researchers with a valuable tool to gain more insight into molecular mechanisms occurring in the early phase of T cell polarization. In addition, it may also allow the study of pharmacological agents on DC-dependent T cell polarization in the human system.

  14. An approach to the unification of suppressor T cell circuits: a simplified assay for the induction of suppression by T cell-derived, antigen-binding molecules (T-ABM).

    Science.gov (United States)

    Chue, B; Ferguson, T A; Beaman, K D; Rosenman, S J; Cone, R E; Flood, P M; Green, D R

    1989-01-01

    A system is presented in which the in vitro response to sheep red blood cells (SRBC) can be regulated using antigenic determinants coupled to SRBC and T cell-derived antigen-binding molecules (T-ABM) directed against the coupled determinants. T suppressor-inducer factors (TsiF's) are composed of two molecules, one of which is a T-ABM and one which bears I-J determinants (I-J+ molecule). Using two purified T-ABM which have not previously been shown to have in vitro activity, we produced antigen-specific TsiF's which were capable of inducing the suppression of the anti-SRBC response. Suppression was found to require both the T-ABM and the I-J+ molecule, SRBC conjugated with the antigen for which the T-ABM was specific, and a population of Ly-2+ T cells in the culture. Two monoclonal TsiF (or TsF1) were demonstrated to induce suppression of the anti-SRBC response in this system, provided the relevant antigen was coupled to the SRBC in culture. The results are discussed in terms of the general functions of T-ABM in the immune system. This model will be useful in direct, experimental comparisons of the function of T-ABM and suppressor T cell factors under study in different systems and laboratories.

  15. [CLINICAL UTILITY OF T-SPOT.TB ASSAY WITH T-Cell Xtend REAGENT FOR ACTIVE TUBERCULOSIS DIAGNOSIS IN THE FIELD TEST AT OUR HOSPITAL].

    Science.gov (United States)

    Nemoto, Kenji; Oh-ishi, Shuji; Taguchi, Masato; Hyodo, Kentaro; Kanazawa, Jun; Miura, Yukiko; Takaku, Takio; Usui, Shingo; Hayashihara, Kenji; Saito, Takefumi

    2016-04-01

    T-SPOT.TB (T-SPOT), an interferon-gamma release assay, has shown promise as a diagnostic tool for active tuberculosis (TB), and its use is expanding. Addition of the T-Cell Xtend (TCX) reagent may allow delayed processing, and this characteristic is important for using this test in the field. However, limited data is available on the usefulness of T-SPOT with TCX as a field test for diagnosing active TB. To investigate the clinical utility of T-SPOT with TCX and the risk factors for a false-negative result in patients with active TB. A total of 57 patients with active TB who underwent the T-SPOT test with TCX prior to treatment were enrolled between May 2013 and May 2015. One patient with an indeterminate result for T-SPOT was excluded; therefore, the data of 56 patients were eventually included in the final analysis. The basic characteristics and clinical findings were compared between the true-positive and false-negative T-SPOT groups. Of the 56 patients, 40 (71.4%), 13 (23.2%), 3 (5.4%) had true-positive, false-negative, and borderline T-SPOT results, respectively. This study did not reveal any significant risk factors for a false-negative T-SPOT result. In this clinical study, the proportion of patients with a false-negative result for T-SPOT with TCX for active TB was higher than that reported previously. Therefore, careful interpretation of a negative result for T-SPOT with TCX is necessary, regardless of the patient's background.

  16. [Regulatory T cells inhibit proliferation of mouse lymphoma cell line EL4 in vitro].

    Science.gov (United States)

    Zhang, Chen; Kong, Yan; Guo, Jun; Ying, Zhi-Tao; Yuan, Zhi-Hong; Zhang, Yun-Tao; Zheng, Wen; Song, Yu-Qin; Li, Ping-Ping; Zhu, Jun

    2010-10-01

    This study was aimed to investigate the effect of regulatory T (Treg) cells on the T cell lymphoma EL4 cells and its mechanism in vitro. C57BL/6 mouse Treg cells were isolated by magnetic cell sorting (MACS). The purity of Treg cells and their expression of Foxp3 were identified by flow cytometry (FCM) and PT-PCR respectively. The suppression of Treg cells on EL4 cells was detected by 3H-TdR method. At the same time, enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of cytokine TGF-β1 and IL-10. The results showed that CD4+CD25+ T cells could be successfully isolated by MACS with the purity reaching 94.52% and the expression of Foxp3 reaching 84.72%. After sorting, the expression of Foxp3 mRNA could be detected by RT-PCR. 3H-TdR assay confirmed that regulatory T cells could suppress the proliferation of EL4 cells with or without antigen presenting cells (APC) or dendritic cells (DC), APC or DC might effectively enhance the suppression. In addition, DC alone also suppressed the proliferation. TGF-β1 and IL-10 could be detected in the supernatant by ELISA. It is concluded that the Treg cells can obviously suppress the proliferation of T cell lymphoma cells in vitro, APC or DC can enhance this suppressive effect, while the DC alone also can suppress the proliferation of EL4 cells, the TGF-β1 and IL-10 cytokine pathway may be one of the mechanisms of suppression.

  17. Pulmonary CCR2+CD4+ T cells are immune regulatory and attenuate lung fibrosis development.

    Science.gov (United States)

    Milger, Katrin; Yu, Yingyan; Brudy, Eva; Irmler, Martin; Skapenko, Alla; Mayinger, Michael; Lehmann, Mareike; Beckers, Johannes; Reichenberger, Frank; Behr, Jürgen; Eickelberg, Oliver; Königshoff, Melanie; Krauss-Etschmann, Susanne

    2017-11-01

    Animal models have suggested that CCR2-dependent signalling contributes to the pathogenesis of pulmonary fibrosis, but global blockade of CCL2 failed to improve the clinical course of patients with lung fibrosis. However, as levels of CCR2 + CD4 + T cells in paediatric lung fibrosis had previously been found to be increased, correlating with clinical symptoms, we hypothesised that distinct CCR2 + cell populations might either increase or decrease disease pathogenesis depending on their subtype. To investigate the role of CCR2 + CD4 + T cells in experimental lung fibrosis and in patients with idiopathic pulmonary fibrosis and other fibrosis. Pulmonary CCR2 + CD4 + T cells were analysed using flow cytometry and mRNA profiling, followed by in silico pathway analysis, in vitro assays and adoptive transfer experiments. Frequencies of CCR2 + CD4 + T cells were increased in experimental fibrosis-specifically the CD62L - CD44 + effector memory T cell phenotype, displaying a distinct chemokine receptor profile. mRNA profiling of isolated CCR2 + CD4 + T cells from fibrotic lungs suggested immune regulatory functions, a finding that was confirmed in vitro using suppressor assays. Importantly, adoptive transfer of CCR2 + CD4 + T cells attenuated fibrosis development. The results were partly corroborated in patients with lung fibrosis, by showing higher percentages of Foxp3 + CD25 + cells within bronchoalveolar lavage fluid CCR2 + CD4 + T cells as compared with CCR2 - CD4 + T cells. Pulmonary CCR2 + CD4 + T cells are immunosuppressive, and could attenuate lung inflammation and fibrosis. Therapeutic strategies completely abrogating CCR2-dependent signalling will therefore also eliminate cell populations with protective roles in fibrotic lung disease. This emphasises the need for a detailed understanding of the functions of immune cell subsets in fibrotic lung disease. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights

  18. Riboflavin Depletion Promotes Tumorigenesis in HEK293T and NIH3T3 Cells by Sustaining Cell Proliferation and Regulating Cell Cycle-Related Gene Transcription.

    Science.gov (United States)

    Long, Lin; He, Jian-Zhong; Chen, Ye; Xu, Xiu-E; Liao, Lian-Di; Xie, Yang-Min; Li, En-Min; Xu, Li-Yan

    2018-05-07

    Riboflavin is an essential component of the human diet and its derivative cofactors play an established role in oxidative metabolism. Riboflavin deficiency has been linked with various human diseases. The objective of this study was to identify whether riboflavin depletion promotes tumorigenesis. HEK293T and NIH3T3 cells were cultured in riboflavin-deficient or riboflavin-sufficient medium and passaged every 48 h. Cells were collected every 5 generations and plate colony formation assays were performed to observe cell proliferation. Subcutaneous tumorigenicity assays in NU/NU mice were used to observe tumorigenicity of riboflavin-depleted HEK293T cells. Mechanistically, gene expression profiling and gene ontology analysis were used to identify abnormally expressed genes induced by riboflavin depletion. Western blot analyses, cell cycle analyses, and chromatin immunoprecipitation were used to validate the expression of cell cycle-related genes. Plate colony formation of NIH3T3 and HEK293T cell lines was enhanced >2-fold when cultured in riboflavin-deficient medium for 10-20 generations. Moreover, we observed enhanced subcutaneous tumorigenicity in NU/NU mice following injection of riboflavin-depleted compared with normal HEK293T cells (55.6% compared with 0.0% tumor formation, respectively). Gene expression profiling and gene ontology analysis revealed that riboflavin depletion induced the expression of cell cycle-related genes. Validation experiments also found that riboflavin depletion decreased p21 and p27 protein levels by ∼20%, and increased cell cycle-related and expression-elevated protein in tumor (CREPT) protein expression >2-fold, resulting in cyclin D1 and CDK4 levels being increased ∼1.5-fold, and cell cycle acceleration. We also observed that riboflavin depletion decreased intracellular riboflavin levels by 20% and upregulated expression of riboflavin transporter genes, particularly SLC52A3, and that the changes in CREPT and SLC52A3 correlated with

  19. Blimp-1 impairs T cell function via upregulation of TIGIT and PD-1 in patients with acute myeloid leukemia.

    Science.gov (United States)

    Zhu, Liuluan; Kong, Yaxian; Zhang, Jianhong; Claxton, David F; Ehmann, W Christopher; Rybka, Witold B; Palmisiano, Neil D; Wang, Ming; Jia, Bei; Bayerl, Michael; Schell, Todd D; Hohl, Raymond J; Zeng, Hui; Zheng, Hong

    2017-06-19

    T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT) and programmed cell death protein 1 (PD-1) are important inhibitory receptors that associate with T cell exhaustion in acute myeloid leukemia (AML). In this study, we aimed to determine the underlying transcriptional mechanisms regulating these inhibitory pathways. Specifically, we investigated the role of transcription factor B lymphocyte-induced maturation protein 1 (Blimp-1) in T cell response and transcriptional regulation of TIGIT and PD-1 in AML. Peripheral blood samples collected from patients with AML were used in this study. Blimp-1 expression was examined by flow cytometry. The correlation of Blimp-1 expression to clinical characteristics of AML patients was analyzed. Phenotypic and functional studies of Blimp-1-expressing T cells were performed using flow cytometry-based assays. Luciferase reporter assays and ChIP assays were applied to assess direct binding and transcription activity of Blimp-1. Using siRNA to silence Blimp-1, we further elucidated the regulatory role of Blimp-1 in the TIGIT and PD-1 expression and T cell immune response. Blimp-1 expression is elevated in T cells from AML patients. Consistent with exhaustion, Blimp-1 + T cells upregulate multiple inhibitory receptors including PD-1 and TIGIT. In addition, they are functionally impaired manifested by low cytokine production and decreased cytotoxicity capacity. Importantly, the functional defect is reversed by inhibition of Blimp-1 via siRNA knockdown. Furthermore, Blimp-1 binds to the promoters of PD-1 and TIGIT and positively regulates their expression. Our study demonstrates an important inhibitory effect of Blimp-1 on T cell response in AML; thus, targeting Blimp-1 and its regulated molecules to improve the immune response may provide effective leukemia therapeutics.

  20. Blimp-1 impairs T cell function via upregulation of TIGIT and PD-1 in patients with acute myeloid leukemia

    Directory of Open Access Journals (Sweden)

    Liuluan Zhu

    2017-06-01

    Full Text Available Abstract Background T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM domain (TIGIT and programmed cell death protein 1 (PD-1 are important inhibitory receptors that associate with T cell exhaustion in acute myeloid leukemia (AML. In this study, we aimed to determine the underlying transcriptional mechanisms regulating these inhibitory pathways. Specifically, we investigated the role of transcription factor B lymphocyte-induced maturation protein 1 (Blimp-1 in T cell response and transcriptional regulation of TIGIT and PD-1 in AML. Methods Peripheral blood samples collected from patients with AML were used in this study. Blimp-1 expression was examined by flow cytometry. The correlation of Blimp-1 expression to clinical characteristics of AML patients was analyzed. Phenotypic and functional studies of Blimp-1-expressing T cells were performed using flow cytometry-based assays. Luciferase reporter assays and ChIP assays were applied to assess direct binding and transcription activity of Blimp-1. Using siRNA to silence Blimp-1, we further elucidated the regulatory role of Blimp-1 in the TIGIT and PD-1 expression and T cell immune response. Results Blimp-1 expression is elevated in T cells from AML patients. Consistent with exhaustion, Blimp-1+ T cells upregulate multiple inhibitory receptors including PD-1 and TIGIT. In addition, they are functionally impaired manifested by low cytokine production and decreased cytotoxicity capacity. Importantly, the functional defect is reversed by inhibition of Blimp-1 via siRNA knockdown. Furthermore, Blimp-1 binds to the promoters of PD-1 and TIGIT and positively regulates their expression. Conclusions Our study demonstrates an important inhibitory effect of Blimp-1 on T cell response in AML; thus, targeting Blimp-1 and its regulated molecules to improve the immune response may provide effective leukemia therapeutics.

  1. Comet assay on mice testicular cells

    Directory of Open Access Journals (Sweden)

    Anoop Kumar Sharma

    2015-05-01

    Full Text Available Heritable mutations may result in a variety of adverse outcomes including genetic disease in the offspring. In recent years the focus on germ cell mutagenicity has increased and the “Globally Harmonized System of Classification and Labelling of Chemicals (GHS” has published classification criteria for germ cell mutagens (Speit et al., 2009. The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cells of mice were investigated. Different classes of chemicals were tested in order to evaluate the sensitivity of the comet assay in testicular cells. The chemicals included environmentally relevant substances such as Bisphenol A, PFOS and Tetrabrombisphenol A. Statistical power calculations will be presented to aid in the design of future Comet assay studies on testicular cells. Power curves were provided with different fold changes in % tail DNA, different number of cells scored and different number of gels (Hansen et al., 2014. An example is shown in Figure 1. A high throughput version of the Comet assay was used. Samples were scored with a fully automatic comet assay scoring system that provided faster scoring of randomly selected cells.

  2. Circumvention of regulatory CD4(+) T cell activity during cross-priming strongly enhances T cell-mediated immunity.

    Science.gov (United States)

    Heit, Antje; Gebhardt, Friedemann; Lahl, Katharina; Neuenhahn, Michael; Schmitz, Frank; Anderl, Florian; Wagner, Hermann; Sparwasser, Tim; Busch, Dirk H; Kastenmüller, Kathrin

    2008-06-01

    Immunization with purified antigens is a safe and practical vaccination strategy but is generally unable to induce sustained CD8(+) T cell-mediated protection against intracellular pathogens. Most efforts to improve the CD8(+) T cell immunogenicity of these vaccines have focused on co-administration of adjuvant to support cross-presentation and dendritic cell maturation. In addition, it has been shown that CD4(+) T cell help during the priming phase contributes to the generation of protective CD8(+) memory T cells. In this report we demonstrate that the depletion of CD4(+) T cells paradoxically enhances long-lasting CD8-mediated protective immunity upon protein vaccination. Functional and genetic in vivo inactivation experiments attribute this enhancement primarily to MHC class II-restricted CD4(+) regulatory T cells (Treg), which appear to physiologically suppress the differentiation process towards long-living effector memory T cells. Since, in functional terms, this suppression by Treg largely exceeds the positive effects of conventional CD4(+) T cell help, even the absence of all CD4(+) T cells or lack of MHC class II-mediated interactions on priming dendritic cells result in enhanced CD8(+) T cell immunogenicity. These findings have important implications for the improvement of vaccines against intracellular pathogens or tumors, especially in patients with highly active Treg.

  3. An In Vitro Potency Assay for Monitoring the Immunomodulatory Potential of Stromal Cell-Derived Extracellular Vesicles

    Directory of Open Access Journals (Sweden)

    Karin Pachler

    2017-07-01

    Full Text Available The regenerative and immunomodulatory activity of mesenchymal stromal cells (MSCs is partially mediated by secreted vesicular factors. Extracellular vesicles (EVs exocytosed by MSCs are gaining increased attention as prospective non-cellular therapeutics for a variety of diseases. However, the lack of suitable in vitro assays to monitor the therapeutic potential of EVs currently restricts their application in clinical studies. We have evaluated a dual in vitro immunomodulation potency assay that reproducibly reports the inhibitory effect of MSCs on induced T-cell proliferation and the alloantigen-driven mixed leukocyte reaction of pooled peripheral blood mononuclear cells in a dose-dependent manner. Phytohemagglutinin-stimulated T-cell proliferation was inhibited by MSC-derived EVs in a dose-dependent manner comparable to MSCs. In contrast, inhibition of alloantigen-driven mixed leukocyte reaction was only observed for MSCs, but not for EVs. Our results support the application of a cell-based in vitro potency assay for reproducibly determining the immunomodulatory potential of EVs. Validation of this assay can help establish reliable release criteria for EVs for future clinical studies.

  4. Simian Immunodeficiency Virus (SIV-Specific Chimeric Antigen Receptor-T Cells Engineered to Target B Cell Follicles and Suppress SIV Replication

    Directory of Open Access Journals (Sweden)

    Kumudhini Preethi Haran

    2018-03-01

    Full Text Available There is a need to develop improved methods to treat and potentially cure HIV infection. During chronic HIV infection, replication is concentrated within T follicular helper cells (Tfh located within B cell follicles, where low levels of virus-specific CTL permit ongoing viral replication. We previously showed that elevated levels of simian immunodeficiency virus (SIV-specific CTL in B cell follicles are linked to both decreased levels of viral replication in follicles and decreased plasma viral loads. These findings provide the rationale to develop a strategy for targeting follicular viral-producing (Tfh cells using antiviral chimeric antigen receptor (CAR T cells co-expressing the follicular homing chemokine receptor CXCR5. We hypothesize that antiviral CAR/CXCR5-expressing T cells, when infused into an SIV-infected animal or an HIV-infected individual, will home to B cell follicles, suppress viral replication, and lead to long-term durable remission of SIV and HIV. To begin to test this hypothesis, we engineered gammaretroviral transduction vectors for co-expression of a bispecific anti-SIV CAR and rhesus macaque CXCR5. Viral suppression by CAR/CXCR5-transduced T cells was measured in vitro, and CXCR5-mediated migration was evaluated using both an in vitro transwell migration assay, as well as a novel ex vivo tissue migration assay. The functionality of the CAR/CXCR5 T cells was demonstrated through their potent suppression of SIVmac239 and SIVE660 replication in in vitro and migration to the ligand CXCL13 in vitro, and concentration in B cell follicles in tissues ex vivo. These novel antiviral immunotherapy products have the potential to provide long-term durable remission (functional cure of HIV and SIV infections.

  5. Different Subsets of T Cells, Memory, Effector Functions, and CAR-T Immunotherapy.

    Science.gov (United States)

    Golubovskaya, Vita; Wu, Lijun

    2016-03-15

    This review is focused on different subsets of T cells: CD4 and CD8, memory and effector functions, and their role in CAR-T therapy--a cellular adoptive immunotherapy with T cells expressing chimeric antigen receptor. The CAR-T cells recognize tumor antigens and induce cytotoxic activities against tumor cells. Recently, differences in T cell functions and the role of memory and effector T cells were shown to be important in CAR-T cell immunotherapy. The CD4⁺ subsets (Th1, Th2, Th9, Th17, Th22, Treg, and Tfh) and CD8⁺ memory and effector subsets differ in extra-cellular (CD25, CD45RO, CD45RA, CCR-7, L-Selectin [CD62L], etc.); intracellular markers (FOXP3); epigenetic and genetic programs; and metabolic pathways (catabolic or anabolic); and these differences can be modulated to improve CAR-T therapy. In addition, CD4⁺ Treg cells suppress the efficacy of CAR-T cell therapy, and different approaches to overcome this suppression are discussed in this review. Thus, next-generation CAR-T immunotherapy can be improved, based on our knowledge of T cell subsets functions, differentiation, proliferation, and signaling pathways to generate more active CAR-T cells against tumors.

  6. Different Subsets of T Cells, Memory, Effector Functions, and CAR-T Immunotherapy

    Directory of Open Access Journals (Sweden)

    Vita Golubovskaya

    2016-03-01

    Full Text Available This review is focused on different subsets of T cells: CD4 and CD8, memory and effector functions, and their role in CAR-T therapy––a cellular adoptive immunotherapy with T cells expressing chimeric antigen receptor. The CAR-T cells recognize tumor antigens and induce cytotoxic activities against tumor cells. Recently, differences in T cell functions and the role of memory and effector T cells were shown to be important in CAR-T cell immunotherapy. The CD4+ subsets (Th1, Th2, Th9, Th17, Th22, Treg, and Tfh and CD8+ memory and effector subsets differ in extra-cellular (CD25, CD45RO, CD45RA, CCR-7, L-Selectin [CD62L], etc.; intracellular markers (FOXP3; epigenetic and genetic programs; and metabolic pathways (catabolic or anabolic; and these differences can be modulated to improve CAR-T therapy. In addition, CD4+ Treg cells suppress the efficacy of CAR-T cell therapy, and different approaches to overcome this suppression are discussed in this review. Thus, next-generation CAR-T immunotherapy can be improved, based on our knowledge of T cell subsets functions, differentiation, proliferation, and signaling pathways to generate more active CAR-T cells against tumors.

  7. Grass-specific CD4+ T-cells exhibit varying degrees of cross-reactivity, implications for allergen-specific immunotherapy

    Science.gov (United States)

    Archila, LD; DeLong, JH; Wambre, E; James, EA; Robinson, DM; Kwok, WW

    2014-01-01

    Background Conceptually, allergic responses may involve cross-reactivity by antibodies or T-cells. While IgE cross-reactivity amongst grass pollen allergens has been observed, cross-reactivity at the allergen-specific T-cell level has been less documented. Identification of the patterns of cross-reactivity may improve our understanding, allowing optimization of better immunotherapy strategies. Objectives We use Phleum pratense as model for the studying of cross-reactivity at the allergen-specific CD4+ T cell level amongst DR04:01 restricted Pooideae grass pollen T-cell epitopes. Methods After In vitro culture of blood mononucleated cells from Grass-pollen allergic subjects with specific Pooideae antigenic epitopes, dual tetramer staining with APC-labeled DR04:01/Phleum pratense tetramers and PE-labeled DR04:01/Pooideae grass homolog tetramers was assessed to identify cross-reactivity amongst allergen-specific DR04:01-restricted T-cells in 6 subjects. Direct ex vivo staining enabled the comparison of frequency and phenotype of different Pooideae grass pollen reactive T-cells. Intracellular cytokine staining (ICS) assays were also used to examine phenotypes of these T-cells. Results T-cells with various degree of cross reactive profiles could be detected. Poa p 1 97-116, Lol p 1 221-240, Lol p 5a 199-218, and Poa p 5a 199-218 were identified as minimally-cross-reactive T-cell epitopes that do not show cross reactivity to Phl p 1 and Phl p 5a epitopes. Ex vivo tetramer staining assays demonstrated T-cells that recognized these minimally-cross reactive T-cell epitopes are present in Grass-pollen allergic subjects. Conclusions Our results suggest that not all Pooideae grass epitopes with sequence homology are cross-reactive. Non-cross reactive T-cells with comparable frequency, phenotype and functionality to Phl p-specific T-cells, suggest that a multiple allergen system should be considered for immunotherapy instead of a mono allergen system. PMID:24708411

  8. Grass-specific CD4(+) T-cells exhibit varying degrees of cross-reactivity, implications for allergen-specific immunotherapy.

    Science.gov (United States)

    Archila, L D; DeLong, J H; Wambre, E; James, E A; Robinson, D M; Kwok, W W

    2014-07-01

    Conceptually, allergic responses may involve cross-reactivity by antibodies or T-cells. While IgE cross-reactivity among grass-pollen allergens has been observed, cross-reactivity at the allergen-specific T-cell level has been less documented. Identification of the patterns of cross-reactivity may improve our understanding, allowing optimization of better immunotherapy strategies. We use Phleum pratense as model for the studying of cross-reactivity at the allergen-specific CD4(+) T cell level among DR04:01 restricted Pooideae grass-pollen T-cell epitopes. After in vitro culture of blood mono-nucleated cells from grass-pollen-allergic subjects with specific Pooideae antigenic epitopes, dual tetramer staining with APC-labelled DR04:01/Phleum pratense tetramers and PE-labelled DR04:01/Pooideae grass homolog tetramers was assessed to identify cross-reactivity among allergen-specific DR04:01-restricted T-cells in six subjects. Direct ex vivo staining enabled the comparison of frequency and phenotype of different Pooideae grass-pollen reactive T-cells. Intracellular cytokine staining (ICS) assays were also used to examine phenotypes of these T-cells. T-cells with various degrees of cross-reactive profiles could be detected. Poa p 1 97-116 , Lol p 1 221-240 , Lol p 5a 199-218 , and Poa p 5a 199-218 were identified as minimally cross-reactive T-cell epitopes that do not show cross-reactivity to Phl p 1 and Phl p 5a epitopes. Ex vivo tetramer staining assays demonstrated T-cells that recognized these minimally cross-reactive T-cell epitopes are present in Grass-pollen-allergic subjects. Our results suggest that not all Pooideae grass epitopes with sequence homology are cross-reactive. Non-cross-reactive T-cells with comparable frequency, phenotype and functionality to Phl p-specific T-cells suggest that a multiple allergen system should be considered for immunotherapy instead of a mono-allergen system. © 2014 John Wiley & Sons Ltd.

  9. Chemokine receptor expression by inflammatory T cells in EAE

    DEFF Research Database (Denmark)

    Mony, Jyothi Thyagabhavan; Khorooshi, Reza; Owens, Trevor

    2014-01-01

    Chemokines direct cellular infiltration to tissues, and their receptors and signaling pathways represent targets for therapy in diseases such as multiple sclerosis (MS). The chemokine CCL20 is expressed in choroid plexus, a site of entry of T cells to the central nervous system (CNS). The CCL20...... receptor CCR6 has been reported to be selectively expressed by CD4(+) T cells that produce the cytokine IL-17 (Th17 cells). Th17 cells and interferon-gamma (IFNγ)-producing Th1 cells are implicated in induction of MS and its animal model experimental autoimmune encephalomyelitis (EAE). We have assessed...... whether CCR6 identifies specific inflammatory T cell subsets in EAE. Our approach was to induce EAE, and then examine chemokine receptor expression by cytokine-producing T cells sorted from CNS at peak disease. About 7% of CNS-infiltrating CD4(+) T cells produced IFNγ in flow cytometric cytokine assays...

  10. Antifibrotic Therapy in Simian Immunodeficiency Virus Infection Preserves CD4+ T-Cell Populations and Improves Immune Reconstitution With Antiretroviral Therapy

    Science.gov (United States)

    Estes, Jacob D.; Reilly, Cavan; Trubey, Charles M.; Fletcher, Courtney V.; Cory, Theodore J.; Piatak, Michael; Russ, Samuel; Anderson, Jodi; Reimann, Thomas G.; Star, Robert; Smith, Anthony; Tracy, Russell P.; Berglund, Anna; Schmidt, Thomas; Coalter, Vicky; Chertova, Elena; Smedley, Jeremy; Haase, Ashley T.; Lifson, Jeffrey D.; Schacker, Timothy W.

    2015-01-01

    Even with prolonged antiretroviral therapy (ART), many human immunodeficiency virus-infected individuals have <500 CD4+ T cells/µL, and CD4+ T cells in lymphoid tissues remain severely depleted, due in part to fibrosis of the paracortical T-cell zone (TZ) that impairs homeostatic mechanisms required for T-cell survival. We therefore used antifibrotic therapy in simian immunodeficiency virus-infected rhesus macaques to determine whether decreased TZ fibrosis would improve reconstitution of peripheral and lymphoid CD4+ T cells. Treatment with the antifibrotic drug pirfenidone preserved TZ architecture and was associated with significantly larger populations of CD4+ T cells in peripheral blood and lymphoid tissues. Combining pirfenidone with an ART regimen was associated with greater preservation of CD4+ T cells than ART alone and was also associated with higher pirfenidone concentrations. These data support a potential role for antifibrotic drug treatment as adjunctive therapy with ART to improve immune reconstitution. PMID:25246534

  11. Peptide pool immunization and CD8+ T cell reactivity

    DEFF Research Database (Denmark)

    Rasmussen, Susanne B; Harndahl, Mikkel N; Buus, Anette Stryhn

    2013-01-01

    Mice were immunized twice with a pool of five peptides selected among twenty 8-9-mer peptides for their ability to form stable complexes at 37°C with recombinant H-2K(b) (half-lives 10-15h). Vaccine-induced immunity of splenic CD8(+) T cells was studied in a 24h IFNγ Elispot assay. Surprisingly...... peptides induced normal peptide immunity i.e. the specific T cell reactivity in the Elispot culture was strictly dependent on exposure to the immunizing peptide ex vivo. However, immunization with two of the peptides, a VSV- and a Mycobacterium-derived peptide, resulted in IFNγ spot formation without...... peptide in the Elispot culture. Immunization with a mixture of the VSV-peptide and a "normal" peptide also resulted in IFNγ spot formation without addition of peptide to the assay culture. Peptide-tetramer staining of CD8(+) T cells from mice immunized with a mixture of VSV-peptide and "normal" peptide...

  12. HIV-specific cytotoxic T lymphocyte precursors exist in a CD28-CD8+ T cell subset and increase with loss of CD4 T cells.

    Science.gov (United States)

    Lewis, D E; Yang, L; Luo, W; Wang, X; Rodgers, J R

    1999-06-18

    To determine whether the CD28-CD8+ T cells that develop during HIV infection contain HIV-specific cytotoxic precursor cells. CD8 subpopulations from six asymptomatic HIV-positive adults, with varying degrees of CD4 T cell loss, were sorted by flow cytometry and HIV-specific precursor cytotoxic T lymphocyte frequencies were measured. Three populations of CD8 T cells were tested: CD28+CD5-- T cells, CD28-CD57+ T cells (thought to be memory cells) and CD28-CD57- T cells (function unknown). Sorted CD8 subsets were stimulated with antigen presenting cells expressing HIV-1 Gag/Pol molecules. Cytotoxic T cell assays on Gag/Pol expressing 51Cr-labeled Epstein-Barr virus transformed autologous B cells lines or control targets were performed after 2 weeks. Specific lysis and precursor frequencies were calculated. Both CD28 positive and CD28-CD57+ populations contained appreciable numbers of precursors (9-1720 per 10(6) CD8+ T cells). However, the CD28-CD57- population had fewer precursors in five out of six people studied. More CD28 positive HIV-specific cytotoxic T lymphocyte precursors were found in patients with CD4:CD8 ratios > 1, whereas more CD28-CD57+ precursors were found in patients whose CD4:CD8 ratios were < 1 (r2, 0.68). Memory HIV-specific precursor cytotoxic T lymphocytes are found in both CD28 positive and CD28-CD8+ cells, however, a CD28-CD57- subpopulation had fewer. Because CD28-CD57+ cells are antigen-driven with limited diversity, the loss of CD28 on CD8 T cells during disease progression may reduce the response to new HIV mutations; this requires further testing.

  13. Antigen specific T-cell responses against tumor antigens are controlled by regulatory T cells in patients with prostate cancer.

    Science.gov (United States)

    Hadaschik, Boris; Su, Yun; Huter, Eva; Ge, Yingzi; Hohenfellner, Markus; Beckhove, Philipp

    2012-04-01

    Immunotherapy is a promising approach in an effort to control castration resistant prostate cancer. We characterized tumor antigen reactive T cells in patients with prostate cancer and analyzed the suppression of antitumor responses by regulatory T cells. Peripheral blood samples were collected from 57 patients with histologically confirmed prostate cancer, 8 patients with benign prostatic hyperplasia and 16 healthy donors. Peripheral blood mononuclear cells were isolated and antigen specific interferon-γ secretion of isolated T cells was analyzed by enzyme-linked immunospot assay. T cells were functionally characterized and T-cell responses before and after regulatory T-cell depletion were compared. As test tumor antigens, a panel of 11 long synthetic peptides derived from a total of 8 tumor antigens was used, including prostate specific antigen and prostatic acid phosphatase. In patients with prostate cancer we noted a 74.5% effector T-cell response rate compared with only 25% in patients with benign prostatic hyperplasia and 31% in healthy donors. In most patients 2 or 3 tumor antigens were recognized. Comparing various disease stages there was a clear increase in the immune response against prostate specific antigens from intermediate to high risk tumors and castration resistant disease. Regulatory T-cell depletion led to a significant boost in effector T-cell responses against prostate specific antigen and prostatic acid phosphatase. Tumor specific effector T cells were detected in most patients with prostate cancer, especially those with castration resistant prostate cancer. Since effector T-cell responses against prostate specific antigens strongly increased after regulatory T-cell depletion, our results indicate that immunotherapy efficacy could be enhanced by decreasing regulatory T cells. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  14. Development of Augmented Leukemia/Lymphoma-Specific T-Cell Immunotherapy for Deployment with Haploidentical, Hematompoietic Progenitor-Cell Transplant

    Science.gov (United States)

    2011-05-01

    cells exhibited redirected specific lysis of a genetically modified EL4 target expressing 92% human CD19 (Figure 6a). T cells not genetically modified...but propagated by crosslinking CD3 using g-irradiated aAPC loaded with OKT3 did not appreciably lyse CD19 EL4 cells (Figure 6b). DISCUSSION We and...by genetically modified T cells. Lysis by chromium release assay of CD19+ EL4 tumor cells compared with CD19 parental EL4 cells by (a) T cells

  15. Universal cytotoxic activity of a HTLV-1 Tax-specific T cell clone from an HLA-A*24:02⁺ patient with adult T-cell leukemia against a variety of HTLV-I-infected T-cells.

    Science.gov (United States)

    Tanaka, Yukie; Yamazaki, Rie; Terasako-Saito, Kiriko; Nakasone, Hideki; Akahoshi, Yu; Nakano, Hirofumi; Ugai, Tomotaka; Wada, Hidenori; Yamasaki, Ryoko; Ishihara, Yuko; Kawamura, Koji; Sakamoto, Kana; Ashizawa, Masahiro; Sato, Miki; Kimura, Shun-ichi; Kikuchi, Misato; Kako, Shinichi; Kanda, Junya; Tanihara, Aki; Nishida, Junji; Kanda, Yoshinobu

    2014-01-01

    Adult T cell leukemia/lymphoma (ATL) is an aggressive mature T cell malignancy that is causally associated with human T cell lymphotropic virus type 1 (HTLV-1) infection. The HTLV-1 regulatory protein Tax aggressively accelerates the proliferation of host cells and is also an important target antigen for CD8(+) cytotoxic T cells (CTLs). We previously reported that several predominant HLA-A*24:02-restricted HTLV-1 Tax301-309-specific CTL clones commonly expressed a particular amino acid sequence motif (P-D-R) in complementarity-determining region 3 of T-cell receptor (TCR)-β chain among unrelated ATL patients who underwent allogeneic stem cell transplantation (allo-HSCT). Furthermore, a PDR-motif(+) CTL clone persistently existed in a long-term survivor as a central CTL clone with strong CTL activities after HSCT. Although a larger analysis of the relationship between PDR-motif(+) CTLs and the clinical course is required, the expression of PDR-motif(+) TCR on CD8(+) T cells may play a critical role in the management of anti-HTLV-1 activities for HLA-A24:02(+) ATL patients. Therefore, in this study, we prepared an HTLV-1 Tax301-309 peptide-specific CTL clone (HT-9) expressing PDR-motif(+) TCR isolated from a long-term survivor after HSCT, and evaluated its CTL activity against a variety of HTLV-1-infected T-cells from HLA-A*24:02(+) ATL patients. Before the assay of CTL function, we confirmed that HT-9 expressed less-differentiated effector-memory phenotypes (CD45RA(-)CCR7(-)CD27(+)CD28(+/-)CD57(+/-)) and T-cell exhaustion marker PD-1(+). In assays of CTL function, HT-9 recognized HTLV-1 Tax in an HLA-restricted fashion and demonstrated strong CTL activities against a variety of HTLV-1-infected T-cells from HLA-A*24:02(+) ATL patients regardless of whether the sources were autologous or allogeneic, but not normal cells. These data indicate that PDR-motif(+) TCR could be an important TCR candidate for TCR-gene immunotherapy for HLA-A24:02(+) ATL patients, provided

  16. Competitive homing assays to study gut-tropic t cell migration.

    Science.gov (United States)

    Villablanca, Eduardo J; Mora, J Rodrigo

    2011-03-01

    In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressions) displayed on endothelial cells (1 2). Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment. Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing

  17. Estimation by limiting dilution analysis of human IL 2-secreting T cells: detection of IL 2 produced by single lymphokine-secreting T cells

    International Nuclear Information System (INIS)

    Vie, H.; Miller, R.A.

    1986-01-01

    We present here a culture method for the estimation, in human blood, of the number of lymphocytes that can respond to mitogen by producing interleukin 2 (IL 2). T cells are cultured at limiting dilutions with PHA or Con A in the presence of Epstein Barr virus-transformed human lymphoblastoid cells (EB-LCL), and supernatants are tested 3 days later for IL 2 content by a cell proliferation assay. The distribution of negative wells follows the expected Poisson single-hit relationship, suggesting that the assay is sensitive to single cells of a single limiting cell type. On average, 16.3% of peripheral blood mononuclear cells can produce IL 2 in such clonal cultures (mean of 12 determinations; SD = 5.6%). Surprisingly, irradiation (up to 2000 rad) of the titrated responder cell population diminishes the estimated frequencies by less than 50%. The ability to detect IL 2 levels in cultures containing only a single, nonproliferating T lymphocyte allows us to estimate the amount of IL 2 generated by an individual effector cell during a 3-day culture interval after mitogen stimulation. The average responding, irradiated T cell generates 0.92 pg of IL 2 (median) within 3 days. The method presented provides a straightforward way to provide independent estimates of responding cell number and of lymphokine production per cell in a variety of clinical situations

  18. Multiple dendritic cell populations activate CD4+ T cells after viral stimulation.

    Directory of Open Access Journals (Sweden)

    Adele M Mount

    2008-02-01

    Full Text Available Dendritic cells (DC are a heterogeneous cell population that bridge the innate and adaptive immune systems. CD8alpha DC play a prominent, and sometimes exclusive, role in driving amplification of CD8(+ T cells during a viral infection. Whether this reliance on a single subset of DC also applies for CD4(+ T cell activation is unknown. We used a direct ex vivo antigen presentation assay to probe the capacity of flow cytometrically purified DC populations to drive amplification of CD4(+ and CD8(+ T cells following infection with influenza virus by different routes. This study examined the contributions of non-CD8alpha DC populations in the amplification of CD8(+ and CD4(+ T cells in cutaneous and systemic influenza viral infections. We confirmed that in vivo, effective immune responses for CD8(+ T cells are dominated by presentation of antigen by CD8alpha DC but can involve non-CD8alpha DC. In contrast, CD4(+ T cell responses relied more heavily on the contributions of dermal DC migrating from peripheral lymphoid tissues following cutaneous infection, and CD4 DC in the spleen after systemic infection. CD4(+ T cell priming by DC subsets that is dependent upon the route of administration raises the possibility that vaccination approaches could be tailored to prime helper T cell immunity.

  19. 7-(O)-Carboxymethyl daidzein conjugated to N-t-Boc-hexylenediamine: a novel compound capable of inducing cell death in epithelial ovarian cancer stem cells.

    Science.gov (United States)

    Green, Jamie M; Alvero, Ayesha B; Kohen, Fortune; Mor, Gil

    2009-09-01

    One of the major difficulties in the treatment of epithelial ovarian cancer (EOC) is the high rate of recurrent disease. This is thought to be due to the survival of a population of chemo-resistant cells within the tumor, the ovarian cancer stem cells (OCSCs), that are able to regenerate the tumor following chemotherapy. Therefore, the identification of a compund that can target the OCSCs is one of the main steps in improving overall survival of ovarian cancer patients. The objective of this study was to determine the effect of N-t-boc-Daidzein, a novel daidzain derivative, on OCSCs. The efficacy of this compound was evaluated in OCSC and mature ovarian cancer cell (mOCC) lines isolated from malignant ovarian cancer asicites. Cells were treated with increasing concentrations of N-t-boc-Daidzein (0.003-10 microM) and cell growth was monitored by "real time in vitro micro-imaging" using the IncuCyte system. Cell viability was measured using the CellTiter 96 Assay. Apoptosis was determined by Caspase-Glo 3/7, 8 and 9 assays. The components of the apoptotic cascade were characterized by western blot analysis. N-t-boc-Daidzein was able to significantly inhibit cell growth and decrease cell viability of OCSC as well as mOCC cells in a dose and time dependent maner. This effect was due to the induction of apoptosis, which is characterized by caspase activation, XIAP and AKT degradation, and mitochondrial depolarization. This study describes a novel compound that can target the OCSCs. These findings may provide vital aide in improving overall survival in patients with EOC.

  20. Measuring T cell-mediated cytotoxicity using fluorogenic caspase substrates.

    Science.gov (United States)

    Chahroudi, A; Silvestri, G; Feinberg, M B

    2003-10-01

    Cytotoxic T lymphocytes (CTLs) play a major role in the immune response against viruses and other intracellular pathogens. In addition, CTLs are implicated in the control of tumor cells in certain settings. Accurate measures of CTL function are of critical importance to study the pathogenesis of infectious diseases and to evaluate the efficacy of new vaccines and immunotherapies. To this end, we have recently developed a flow cytometry-based CTL (FCC) assay that measures the CTL-induced caspase activation within target cells using cell permeable fluorogenic caspase substrates. This novel assay reliably detects, by flow cytometry or fluorescence/confocal microscopy, antigen-specific CTLs in a wide variety of human and murine systems, and is safer and more informative than the standard 51Cr-release assay. In addition, the flow cytometric CTL (FCC) assay provides an alternative method that is often more sensitive and physiologically informative when compared to previously described FCC assays, as it measures a biological indicator of apoptosis within the target cell. The FCC assay may thus represent a useful tool to further understand the molecular and cellular mechanisms that underlie CTL-mediated killing during tumorigenesis or following infection with viruses or other intracellular pathogens.

  1. Immune-mediated bone marrow failure syndromes of progenitor and stem cells: molecular analysis of cytotoxic T cell clones.

    Directory of Open Access Journals (Sweden)

    Ramon Tiu

    2007-03-01

    Full Text Available The unique structure of the T cell receptor (TCR enables molecular identification of individual T cell clones and provides an unique opportunity for the design of molecular diagnostic tests based on the structure of the rearranged TCR chain e.g., using the TCR CDR3 region. Initially, clonal T cell malignancies, including T cell large granular lymphocyte leukemia (T-LGL, mucosis fungoides and peripheral T cell lymphoma were targets for the TCR-based analytic assays such as detection of clonality by T-gamma rearrangement using y-chain-specific PCR or Southern Blotting. Study of these disorders facilitated further analytic concepts and application of rational methods of TCR analysis to investigations of polyclonal T cell-mediated diseases. In hematology, such conditions include graft versus host disease (GvHD and immune-mediated bone marrow failure syndromes. In aplastic anemia (AA, myelodysplastic syndrome (MDS or paroxysmal nocturnal hemoglobinuria (PNH, cytotoxic T cell responses may be directed against certain antigens located on stem or more lineage-restricted progenitor cells in single lineage cytopenias. The nature of the antigenic targets driving polyclonal CTL responses remains unclear. Novel methods of TCR repertoire analysis, include VB flow cytometry, peptide-specific tetramer staining, in vitro stimulation assays and TCR CDR3-specific PCR. Such PCR assay can be either VB family-specific or multiplexed for all VB families. Amplified products can be characterized and quantitated to facilitate detection of the most immunodominant clonotypes. Such clonotypes may serve as markers for the global polyclonal T cell response. Identification of these clonotypes can be performed in blood and tissue biopsy material by various methods. Once immunodominant clonotypes corresponding to pathogenic CTL clones are identified they can serve as surrogate markers for the activity of the pathophysiologic process or even indicate the presence of specific

  2. Rapid assay of intrinsic radiosensitivity based on apoptosis in human CD4 and CD8 T-lymphocytes

    International Nuclear Information System (INIS)

    Ozsahin, Mahmut; Ozsahin, Huelya; Yuquan, Shi; Larsson, Boerje; Wuergler, Friedrich E.; Crompton, Nigel E. A.

    1997-01-01

    Purpose: An assay for radiosensitivity has numerous applications in the clinic. Avoidance of acute responses, prediction of normal tissue toxicity, and individualization of patient radiotherapy are included among these. We have developed a rapid assay (about 24 h) able to predict intrinsic radiosensitivity of CD4 and CD8 T-lymphocytes based on radiation-induced apoptosis. Methods and Materials: Fresh blood samples (1-2 ml in heparinized tubes) were irradiated with 0-, 2-, and 8-Gy X rays at a dose rate of approximately 3 Gy/min. Following irradiation, the cells were collected and prepared for flow-cytometric analysis and cell sorting. In conjunction with the CellQuest software available with the FACSVantage cell sorter (Becton-Dickinson), two T-lymphocyte types were analyzed on the basis of their cell-specific antigens (CD4 and CD8), and DNA was stained with DAPI. Following the separation of these cell types, radiation-induced cell death was assessed. Cytotoxicity was characterized by gradual degradation of internucleosomal DNA which results in a sub-G1 peak on the DNA histogram, and by the associated loss of surface antigens causing an intermediate positive peak in the antibody histogram. Using the assay, we investigated the interdonor variation in a cohort of 45 healthy adult blood donors and 5 children [one had immunodeficiency, centromeric instability, and facial anomalies syndrome (ICF), and one had ataxia telangiectasia (AT)]. Intradonor variation was assessed with 10 different experiments from a single donor. Results: CD4 and CD8 T-lymphocyte radiosensitivities were correlated (r 0.63 and 0.65 for 2 and 8 Gy, respectively) in 45 adult donors. Both for CD4 and CD8 cells, 2 and 8 Gy irradiation responses showed a good correlation (r 0.77 for both). Interdonor variation was significantly higher than intradonor variation (p < 0.0005) for all CD4 and CD8 data. We observed a decrease in the antigen fluorescence of dying cells, a phenomenon referred to as antigen

  3. Transgenic Expression of IL15 Improves Antiglioma Activity of IL13Rα2-CAR T Cells but Results in Antigen Loss Variants.

    Science.gov (United States)

    Krenciute, Giedre; Prinzing, Brooke L; Yi, Zhongzhen; Wu, Meng-Fen; Liu, Hao; Dotti, Gianpietro; Balyasnikova, Irina V; Gottschalk, Stephen

    2017-07-01

    Glioblastoma (GBM) is the most aggressive primary brain tumor in adults and is virtually incurable with conventional therapies. Immunotherapy with T cells expressing GBM-specific chimeric antigen receptors (CAR) is an attractive approach to improve outcomes. Although CAR T cells targeting GBM antigens, such as IL13 receptor subunit α2 (IL13Rα2), HER2, and EGFR variant III (EGFRvIII), have had antitumor activity in preclinical models, early-phase clinical testing has demonstrated limited antiglioma activity. Transgenic expression of IL15 is an appealing strategy to enhance CAR T-cell effector function. We tested this approach in our IL13Rα2-positive glioma model in which limited IL13Rα2-CAR T-cell persistence results in recurrence of antigen-positive gliomas. T cells were genetically modified with retroviral vectors encoding IL13Rα2-CARs or IL15 (IL13Rα2-CAR.IL15 T cells). IL13Rα2-CAR.IL15 T cells recognized glioma cells in an antigen-dependent fashion, had greater proliferative capacity, and produced more cytokines after repeated stimulations in comparison with IL13Rα2-CAR T cells. No autonomous IL13Rα2-CAR.IL15 T-cell proliferation was observed; however, IL15 expression increased IL13Rα2-CAR T-cell viability in the absence of exogenous cytokines or antigen. In vivo , IL13Rα2-CAR.IL15 T cells persisted longer and had greater antiglioma activity than IL13Rα2-CAR T cells, resulting in a survival advantage. Gliomas recurring after 40 days after T-cell injection had downregulated IL13Rα2 expression, indicating that antigen loss variants occur in the setting of improved T-cell persistence. Thus, CAR T cells for GBM should not only be genetically modified to improve their proliferation and persistence, but also to target multiple antigens. Summary: Glioblastoma responds imperfectly to immunotherapy. Transgenic expression of IL15 in T cells expressing CARs improved their proliferative capacity, persistence, and cytokine production. The emergence of antigen

  4. Tissue-specific methylation of human insulin gene and PCR assay for monitoring beta cell death.

    Directory of Open Access Journals (Sweden)

    Mohamed I Husseiny

    Full Text Available The onset of metabolic dysregulation in type 1 diabetes (T1D occurs after autoimmune destruction of the majority of pancreatic insulin-producing beta cells. We previously demonstrated that the DNA encoding the insulin gene is uniquely unmethylated in these cells and then developed a methylation-specific PCR (MSP assay to identify circulating beta cell DNA in streptozotocin-treated mice prior to the rise in blood glucose. The current study extends to autoimmune non-obese diabetic (NOD mice and humans, showing in NOD mice that beta cell death occurs six weeks before the rise in blood sugar and coincides with the onset of islet infiltration by immune cells, demonstrating the utility of MSP for monitoring T1D. We previously reported unique patterns of methylation of the human insulin gene, and now extend this to other human tissues. The methylation patterns of the human insulin promoter, intron 1, exon 2, and intron 2 were determined in several normal human tissues. Similar to our previous report, the human insulin promoter was unmethylated in beta cells, but methylated in all other tissues tested. In contrast, intron 1, exon 2 and intron 2 did not exhibit any tissue-specific DNA methylation pattern. Subsequently, a human MSP assay was developed based on the methylation pattern of the insulin promoter and human islet DNA was successfully detected in circulation of T1D patients after islet transplantation therapy. Signal levels of normal controls and pre-transplant samples were shown to be similar, but increased dramatically after islet transplantation. In plasma the signal declines with time but in whole blood remains elevated for at least two weeks, indicating that association of beta cell DNA with blood cells prolongs the signal. This assay provides an effective method to monitor beta cell destruction in early T1D and in islet transplantation therapy.

  5. Micronucleus Assay in Exfoliated Buccal Epithelial Cells Using Liquid Based Cytology Preparations in Building Construction Workers.

    Science.gov (United States)

    Arul, P; Smitha, Shetty; Masilamani, Suresh; Akshatha, C

    2018-01-01

    Cytogenetic damage in exfoliated buccal epithelial cells due to environmental and occupational exposure is often monitored by micronucleus (MN) assay using liquid based cytology (LBC) preparations. This study was performed to evaluate MN in exfoliated buccal epithelial cells of building construction workers using LBC preparations. LBC preparations of exfoliated buccal epithelial cells from 100 subjects [50 building construction workers (cases) and 50 administrative staffs (controls)] was evaluated by May-Grunwald Giemsa, Hematoxylin and Eosin and Papanicolaou stains. Student's t test was used for statistical analysis and a P value of 5 years) and smokers and non-smokers of cases (P=0.001). However, there were meaningful differences regarding mean frequencies of MN between smokers, non-smokers, those with alcohol consumption or not in cases and controls using various stains (P=0.001). There was an increased risk of cytogenetic damage in building construction workers. However, evaluation of MN of exfoliated buccal epithelial cells in building construction workers serve as a minimally invasive biomarker for cytogenetic damage. LBC preparations can be applied for MN assay as it improves the quality of smears and cell morphology, decreases the confounding factors and reduces false positive results.

  6. Human retinal pigment epithelial cells inhibit proliferation and IL2R expression of activated T cells

    DEFF Research Database (Denmark)

    Kaestel, Charlotte G; Jørgensen, Annette; Nielsen, Mette

    2002-01-01

    -Thymidine incorporation assay, respectively. T cells and RPE cells were cultured directly together or in a transwell system for determination of the effect of cell contact. The importance of cell surface molecules was examined by application of a panel of blocking antibodies (CD2, CD18, CD40, CD40L, CD54, CD58......) in addition to use of TCR negative T cell lines. The expression of IL2R-alpha -beta and -gamma chains of activated T cells was analysed by flow cytometry after incubation of T cells alone or with RPE cells. Human RPE cells were found to inhibit the proliferation of activated T cells by a cell contact......-beta and -gamma chain expression within 24 hr after removal from the coculture. It is concluded that the cultured human adult and foetal RPE cells inhibit the proliferation of activated T cells by a process that does not involve apoptosis. It depends on cell contact but the involved surface molecules were...

  7. TRPV2 activation induces apoptotic cell death in human T24 bladder cancer cells: a potential therapeutic target for bladder cancer.

    Science.gov (United States)

    Yamada, Takahiro; Ueda, Takashi; Shibata, Yasuhiro; Ikegami, Yosuke; Saito, Masaki; Ishida, Yusuke; Ugawa, Shinya; Kohri, Kenjiro; Shimada, Shoichi

    2010-08-01

    To investigate the functional expression of the transient receptor potential vanilloid 2 (TRPV2) channel protein in human urothelial carcinoma (UC) cells and to determine whether calcium influx into UC cells through TRPV2 is involved in apoptotic cell death. The expression of TRPV2 mRNA in bladder cancer cell lines (T24, a poorly differentiated UC cell line and RT4, a well-differentiated UC cell line) was analyzed using reverse transcriptase-polymerase chain reaction. The calcium permeability of TRPV2 channels in T24 cells was investigated using a calcium imaging assay that used cannabidiol (CBD), a relatively selective TRPV2 agonist, and ruthenium red (RuR), a nonselective TRPV channel antagonist. The death of T24 or RT4 cells in the presence of CBD was evaluated using a cellular viability assay. Apoptosis of T24 cells caused by CBD was confirmed using an annexin-V assay and small interfering RNA (siRNA) silencing of TRPV2. TRPV2 mRNA was abundantly expressed in T24 cells. The expression level in UC cells was correlated with high-grade disease. The administration of CBD increased intracellular calcium concentrations in T24 cells. In addition, the viability of T24 cells progressively decreased with increasing concentrations of CBD, whereas RT4 cells were mostly unaffected. Cell death occurred via apoptosis caused by continuous influx of calcium through TRPV2. TRPV2 channels in UC cells are calcium-permeable and the regulation of calcium influx through these channels leads directly to the death of UC cells. TRPV2 channels in UC cells may be a potential new therapeutic target, especially in higher-grade UC cells. Copyright 2010 Elsevier Inc. All rights reserved.

  8. A novel murine T-cell receptor targeting NY-ESO-1.

    Science.gov (United States)

    Rosati, Shannon F; Parkhurst, Maria R; Hong, Young; Zheng, Zhili; Feldman, Steven A; Rao, Mahadev; Abate-Daga, Daniel; Beard, Rachel E; Xu, Hui; Black, Mary A; Robbins, Paul F; Schrump, David A; Rosenberg, Steven A; Morgan, Richard A

    2014-04-01

    Cancer testis antigens, such as NY-ESO-1, are expressed in a variety of prevalent tumors and represent potential targets for T-cell receptor (TCR) gene therapy. DNA encoding a murine anti-NY-ESO-1 TCR gene (mTCR) was isolated from immunized HLA-A*0201 transgenic mice and inserted into a γ-retroviral vector. Two mTCR vectors were produced and used to transduce human PBL. Transduced cells were cocultured with tumor target cell lines and T2 cells pulsed with the NY-ESO-1 peptide, and assayed for cytokine release and cell lysis activity. The most active TCR construct was selected for production of a master cell bank for clinical use. mTCR-transduced PBL maintained TCR expression in short-term and long-term culture, ranging from 50% to 90% efficiency 7-11 days after stimulation and 46%-82% 10-20 days after restimulation. High levels of interferon-γ secretion were observed (1000-12000 pg/mL), in tumor coculture assays and recognition of peptide-pulsed cells was observed at 0.1 ng/mL, suggesting that the new mTCR had high avidity for antigen recognition. mTCR-transduced T cells also specifically lysed human tumor targets. In all assays, the mTCR was equivalent or better than the comparable human TCR. As the functional activity of TCR-transduced cells may be affected by the formation of mixed dimers, mTCRs, which are less likely to form mixed dimers with endogenous hTCRs, may be more effective in vivo. This new mTCR targeted to NY-ESO-1 represents a novel potential therapeutic option for adoptive cell-transfer therapy for a variety of malignancies.

  9. Ibrutinib treatment improves T cell number and function in CLL patients.

    Science.gov (United States)

    Long, Meixiao; Beckwith, Kyle; Do, Priscilla; Mundy, Bethany L; Gordon, Amber; Lehman, Amy M; Maddocks, Kami J; Cheney, Carolyn; Jones, Jeffrey A; Flynn, Joseph M; Andritsos, Leslie A; Awan, Farrukh; Fraietta, Joseph A; June, Carl H; Maus, Marcela V; Woyach, Jennifer A; Caligiuri, Michael A; Johnson, Amy J; Muthusamy, Natarajan; Byrd, John C

    2017-08-01

    Ibrutinib has been shown to have immunomodulatory effects by inhibiting Bruton's tyrosine kinase (BTK) and IL-2-inducible T cell kinase (ITK). The relative importance of inhibiting these 2 kinases has not been examined despite its relevance to immune-based therapies. Peripheral blood mononuclear cells from chronic lymphocytic leukemia (CLL) patients on clinical trials of ibrutinib (BTK/ITK inhibitor; n = 19) or acalabrutinib (selective BTK inhibitor; n = 13) were collected serially. T cell phenotype, immune function, and CLL cell immunosuppressive capacity were evaluated. Ibrutinib markedly increased CD4+ and CD8+ T cell numbers in CLL patients. This effect was more prominent in effector/effector memory subsets and was not observed with acalabrutinib. Ex vivo studies demonstrated that this may be due to diminished activation-induced cell death through ITK inhibition. PD-1 and CTLA-4 expression was significantly markedly reduced in T cells by both agents. While the number of Treg cells remained unchanged, the ratio of these to conventional CD4+ T cells was reduced with ibrutinib, but not acalabrutinib. Both agents reduced expression of the immunosuppressive molecules CD200 and BTLA as well as IL-10 production by CLL cells. Ibrutinib treatment increased the in vivo persistence of activated T cells, decreased the Treg/CD4+ T cell ratio, and diminished the immune-suppressive properties of CLL cells through BTK-dependent and -independent mechanisms. These features provide a strong rationale for combination immunotherapy approaches with ibrutinib in CLL and other cancers. ClinicalTrials.gov NCT01589302 and NCT02029443. Samples described here were collected per OSU-0025. The National Cancer Institute.

  10. The new numerology of immunity mediated by virus-specific CD8(+) T cells.

    Science.gov (United States)

    Doherty, P C

    1998-08-01

    Our understanding of virus-specific CD8(+) T cell responses is currently being revolutionized by peptide-based assay systems that allow flow cytometric analysis of effector and memory cytotoxic T lymphocyte populations. These techniques are, for the first time, putting the analysis of T-cell-mediated immunity on a quantitative basis.

  11. Transformation assay in Bhas 42 cells: a model using initiated cells to study mechanisms of carcinogenesis and predict carcinogenic potential of chemicals.

    Science.gov (United States)

    Sasaki, Kiyoshi; Umeda, Makoto; Sakai, Ayako; Yamazaki, Shojiro; Tanaka, Noriho

    2015-01-01

    Transformation assays using cultured cells have been applied to the study of carcinogenesis. Although various cell systems exist, few cell types such as BALB/c 3T3 subclones and Syrian hamster embryo cells have been used to study chemically induced two-stage carcinogenesis. Bhas 42 cells were established as a clone by the transfection with the v-Ha-ras gene into mouse BALB/c 3T3 A31-1-1 cells and their subsequent selection based on their sensitivity to 12-O-tetradecanoylphorbol-13-acetate. Using Bhas 42 cells, transformed foci were induced by the treatment with nongenotoxic carcinogens, most of which act as tumor promoters. Therefore, Bhas 42 cells were considered to be a model of initiated cells. Subsequently, not only nongenotoxic carcinogens but also genotoxic carcinogens, most of which act as tumor initiators, were found to induce transformed foci by the modification of the protocol. Furthermore, transformation of Bhas 42 cells was induced by the transfection with genes of oncogenic potential. We interpret this high sensitivity of Bhas 42 cells to various types of carcinogenic stimuli to be related to the multistage model of carcinogenesis, as the transfection of v-Ha-ras gene further advances the parental BALB/c 3T3 A31-1-1 cells toward higher transforming potential. Thus, we propose that Bhas 42 cells are a novel and sensitive cell line for the analysis of carcinogenesis and can be used for the detection of not only carcinogenic substances but also gene alterations related to oncogenesis. This review will address characteristics of Bhas 42 cells, the transformation assay protocol, validation studies, and the various chemicals tested in this assay.

  12. Comparison between second and third generation troponin T assay in patients with symptoms suggestive of an acute coronary syndrome but without ST segment elevation.

    Science.gov (United States)

    Jernberg, Tomas; Venge, Per; Lindahl, Bertil

    2003-01-01

    Cardiac troponin T (cTnT) is a useful tool when assessing patients with chest pain and no persistent ST elevation. We compared the 2nd generation with the new, 3rd generation cTnT assay in 750 patients admitted to our coronary care unit because of chest pain. When focusing on patients with cTnT of 0.01-0.20 microg/l, there was a moderate agreement between the methods. According to recent definitions, 35% more patients were classified as having acute myocardial infarction on admission with the 3rd generation assay. The 3rd generation assay also identified a 10% larger low-risk group and was better able to identify patients with signs of very minor myocardial necrosis (cTnT >0.01-0.02 microg/l) and thereby an increased risk of future events. We conclude that the improved precision of the 3rd generation assay does have some clinical implications in terms of improved accuracy of triage and improved prognostic value. Copyright 2003 S. Karger AG, Basel

  13. Simultaneous targeting of prostate stem cell antigen and prostate-specific membrane antigen improves the killing of prostate cancer cells using a novel modular T cell-retargeting system.

    Science.gov (United States)

    Arndt, Claudia; Feldmann, Anja; Koristka, Stefanie; Cartellieri, Marc; Dimmel, Maria; Ehninger, Armin; Ehninger, Gerhard; Bachmann, Michael

    2014-09-01

    Recently, we described a novel modular platform technology in which T cell-recruitment and tumor-targeting domains of conventional bispecific antibodies are split to independent components, a universal effector module (EM) and replaceable monospecific/monovalent target modules (TMs) that form highly efficient T cell-retargeting complexes. Theoretically, our unique strategy should allow us to simultaneously retarget T cells to different tumor antigens by combining the EM with two or more different monovalent/monospecific TMs or even with bivalent/bispecific TMs, thereby overcoming limitations of a monospecific treatment such as the selection of target-negative tumor escape variants. In order to advance our recently introduced prostate stem cell antigen (PSCA)-specific modular system for a dual-targeting of prostate cancer cells, two additional TMs were constructed: a monovalent/monospecific TM directed against the prostate-specific membrane antigen (PSMA) and a bivalent/bispecific TM (bsTM) with specificity for PSMA and PSCA. The functionality of the novel dual-targeting strategies was analyzed by performing T cell activation and chromium release assays. Similar to the PSCA-specific modular system, the novel PSMA-specific modular system mediates an efficient target-dependent and -specific tumor cell lysis at low E:T ratios and picomolar Ab concentrations. Moreover, by combination of the EM with either the bispecific TM directed to PSMA and PSCA or both monospecifc TMs directed to either PSCA or PSMA, dual-specific targeting complexes were formed which allowed us to kill potential escape variants expressing only one or the other target antigen. Overall, the novel modular system represents a promising tool for multiple tumor targeting. © 2014 Wiley Periodicals, Inc.

  14. The influence of the number of cells scored on the sensitivity in the comet assay

    DEFF Research Database (Denmark)

    Sharma, Anoop Kumar; Soussaline, Françoise; Sallette, Jerome

    2012-01-01

    The impact on the sensitivity of the in vitro comet assay by increasing the number of cells scored has only been addressed in a few studies. The present study investigated whether the sensitivity of the assay could be improved by scoring more than 100 cells. Two cell lines and three different che...... of the assay. A two-way ANOVA analysis of variance showed that the contribution from the two variables “the number of cells scored” and “concentration” on the total variation in the coefficients of variance dataset was statistically significant (p...

  15. An improved enzyme-linked immunosorbent assay for whole-cell determination of methanogens in samples from anaerobic reactors

    DEFF Research Database (Denmark)

    Sørensen, A.H.; Ahring, B.K.

    1997-01-01

    An enzyme-linked immunosorbent assay was developed for the detection of whole cells of methanogens in samples from anaerobic continuously stirred tank digesters treating slurries of solid waste. The assay was found to allow for quantitative analysis of the most important groups of methanogens......-quality microtiter plates and the addition of dilute hydrochloric acid to the samples. In an experiment on different digester samples, the test demonstrated a unique pattern of different methanogenic strains present in each sample. The limited preparatory work required for the assay and the simple assay design make...... in samples from anaerobic digesters in a reproducible manner. Polyclonal antisera against eight strains of methanogens were employed in the test, The specificities of the antisera were increased by adsorption with cross-reacting cells. The reproducibility of the assay depended on the use of high...

  16. Characterization of hydroxyurea (HYU) S49 T lymphoma cells

    International Nuclear Information System (INIS)

    Albert, D.A.; Gudas, L.J.

    1986-01-01

    This paper tests the hypotheses that in vivo ribonucleotide reductase activity and consequent deoxyribonucleoside triphosphate production is rate-limited by the availability of M2 activity. The authors selected and characterized cell lines with variable resistance to hydroxyurea and comparing them with wild type S49 T-lymphoma cells. Ribonucleotide reductase assay was measured and in the process C 14-CDP was added in the final volume of assay mixture. It is shown that hydroxourea reversibly binds to the tyrosine radical of the M2 subunit of ribonucleotide reductase that is required for catalytic activity

  17. Assaying Cellular Viability Using the Neutral Red Uptake Assay.

    Science.gov (United States)

    Ates, Gamze; Vanhaecke, Tamara; Rogiers, Vera; Rodrigues, Robim M

    2017-01-01

    The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

  18. CD4+ and CD8+ T cell activation are associated with HIV DNA in resting CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Leslie R Cockerham

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

  19. Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors

    Directory of Open Access Journals (Sweden)

    Daniel C Farley

    Full Text Available It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02. VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN expressed on dendritic cells in vivo. Vector enveloped with E1001 does not transduce T-cell lines used in standard HIV-1-based RCL assays, making current RCL testing formats unsuitable for testing VP02. We therefore developed a novel assay to test for RCL in clinical lots of VP02. This assay, which utilizes a murine leukemia positive control virus and a 293F cell line expressing the E1001 receptor DC-SIGN, meets a series of evaluation criteria defined in collaboration with US regulatory authorities and demonstrates the ability of the assay format to amplify and detect a hypothetical RCL derived from VP02 vector components. This assay was qualified and used to test six independent GMP production lots of VP02, in which no RCL was detected. We propose that the evaluation criteria used to rationally design this novel method should be considered when developing an RCL assay for any lentiviral vector.

  20. Targeting regulatory T cells in cancer.

    LENUS (Irish Health Repository)

    Byrne, William L

    2012-01-31

    Infiltration of tumors by regulatory T cells confers growth and metastatic advantages by inhibiting antitumor immunity and by production of receptor activator of NF-kappaB (RANK) ligand, which may directly stimulate metastatic propagation of RANK-expressing cancer cells. Modulation of regulatory T cells can enhance the efficacy of cancer immunotherapy. Strategies include depletion, interference with function, inhibition of tumoral migration, and exploitation of T-cell plasticity. Problems with these strategies include a lack of specificity, resulting in depletion of antitumor effector T cells or global interruption of regulatory T cells, which may predispose to autoimmune diseases. Emerging technologies, such as RNA interference and tetramer-based targeting, may have the potential to improve selectivity and efficacy.

  1. Measuring Bioenergetics in T Cells Using a Seahorse Extracellular Flux Analyzer.

    Science.gov (United States)

    van der Windt, Gerritje J W; Chang, Chih-Hao; Pearce, Erika L

    2016-04-01

    This unit contains several protocols to determine the energy utilization of T cells in real-time using a Seahorse Extracellular Flux Analyzer (http://www.seahorsebio.com). The advantages to using this machine over traditional metabolic assays include the simultaneous measurement of glycolysis and mitochondrial respiration, in real-time, on relatively small numbers of cells, without any radioactivity. The Basic Protocol describes a standard mitochondrial stress test on the XF(e) 96, which yields information about oxidative phosphorylation and glycolysis, two energy-generating pathways. The alternate protocols provide examples of adaptations to the Basic Protocol, including adjustments for the use of the XF(e) 24. A protocol for real-time bioenergetic responses to T cell activation allows for the analysis of immediate metabolic changes after T cell receptor stimulation. Specific substrate utilization can be determined by the use of differential assay media, or the injection of drugs that specifically affect certain metabolic processes. Accurate cell numbers, purity, and viability are critical to obtain reliable results. Copyright © 2016 John Wiley & Sons, Inc.

  2. Effect of sirolimus on urinary bladder cancer T24 cell line

    Directory of Open Access Journals (Sweden)

    Oliveira Paula A

    2009-01-01

    Full Text Available Abstract Background Sirolimus is recently reported to have antitumour effects on a large variety of cancers. The present study was performed to investigate sirolimus's ability to inhibit growth in T24 bladder cancer cells. Methods T24 bladder cancer cells were treated with various concentrations of sirolimus. MTT assay was used to evaluate the proliferation inhibitory effect on T24 cell line. The viability of T24 cell line was determined by Trypan blue exclusion analysis. Results Sirolimus inhibits the growth of bladder carcinoma cells and decreases their viability. Significant correlations were found between cell proliferation and sirolimus concentration (r = 0.830; p Conclusion Sirolimus has an anti-proliferation effect on the T24 bladder carcinoma cell line. The information from our results is useful for a better understanding sirolimus's anti-proliferative activity in the T24 bladder cancer cell line.

  3. Comparison of Batch Assay and Random Assay Using Automatic Dispenser in Radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Seung Hwan; Jang, Su Jin; Kang, Ji Yeon; Lee, Dong Soo; Chung, June Key; Lee, Myung Chul [Seoul Metropolitan Government Seoul National University Boramae Medical Center, Seoul (Korea, Republic of); Lee, Ho Young; Shin, Sun Young; Min, Gyeong Sun; Lee, Hyun Joo [Seoul National University college of Medicine, Seoul (Korea, Republic of)

    2009-08-15

    Radioimmunoassay (RIA) was usually performed by the batch assay. To improve the efficiency of RIA without increase of the cost and time, random assay could be a choice. We investigated the possibility of the random assay using automatic dispenser by assessing the agreement between batch assay and random assay. The experiments were performed with four items; Triiodothyronine (T3), free thyroxine (fT4), Prostate specific antigen (PSA), Carcinoembryonic antigen (CEA). In each item, the sera of twenty patients, the standard, and the control samples were used. The measurements were done 4 times with 3 hour time intervals by random assay and batch assay. The coefficient of variation (CV) of the standard samples and patients' data in T3, fT4, PSA, and CEA were assessed. ICC (Intraclass correlation coefficient) and coefficient of correlation were measured to assessing the agreement between two methods. The CVs (%) of T3, fT4, PSA, and CEA measured by batch assay were 3.2+-1.7%, 3.9+-2.1%, 7.1+-6.2%, 11.2+-7.2%. The CVs by random assay were 2.1+-1.7%, 4.8+-3.1%, 3.6+-4.8%, and 7.4+-6.2%. The ICC between the batch assay and random assay were 0.9968 (T3), 0.9973 (fT4), 0.9996 (PSA), and 0.9901 (CEA). The coefficient of correlation between the batch assay and random assay were 0.9924(T3), 0.9974 (fT4), 0.9994 (PSA), and 0.9989 (CEA) (p<0.05). The results of random assay showed strong agreement with the batch assay in a day. These results suggest that random assay using automatic dispenser could be used in radioimmunoassay

  4. Comparison of Batch Assay and Random Assay Using Automatic Dispenser in Radioimmunoassay

    International Nuclear Information System (INIS)

    Moon, Seung Hwan; Jang, Su Jin; Kang, Ji Yeon; Lee, Dong Soo; Chung, June Key; Lee, Myung Chul; Lee, Ho Young; Shin, Sun Young; Min, Gyeong Sun; Lee, Hyun Joo

    2009-01-01

    Radioimmunoassay (RIA) was usually performed by the batch assay. To improve the efficiency of RIA without increase of the cost and time, random assay could be a choice. We investigated the possibility of the random assay using automatic dispenser by assessing the agreement between batch assay and random assay. The experiments were performed with four items; Triiodothyronine (T3), free thyroxine (fT4), Prostate specific antigen (PSA), Carcinoembryonic antigen (CEA). In each item, the sera of twenty patients, the standard, and the control samples were used. The measurements were done 4 times with 3 hour time intervals by random assay and batch assay. The coefficient of variation (CV) of the standard samples and patients' data in T3, fT4, PSA, and CEA were assessed. ICC (Intraclass correlation coefficient) and coefficient of correlation were measured to assessing the agreement between two methods. The CVs (%) of T3, fT4, PSA, and CEA measured by batch assay were 3.2±1.7%, 3.9±2.1%, 7.1±6.2%, 11.2±7.2%. The CVs by random assay were 2.1±1.7%, 4.8±3.1%, 3.6±4.8%, and 7.4±6.2%. The ICC between the batch assay and random assay were 0.9968 (T3), 0.9973 (fT4), 0.9996 (PSA), and 0.9901 (CEA). The coefficient of correlation between the batch assay and random assay were 0.9924(T3), 0.9974 (fT4), 0.9994 (PSA), and 0.9989 (CEA) (p<0.05). The results of random assay showed strong agreement with the batch assay in a day. These results suggest that random assay using automatic dispenser could be used in radioimmunoassay

  5. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

    International Nuclear Information System (INIS)

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku; Okada, Naoki

    2016-01-01

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8"+ CAR-T cells had antigen-specific cytotoxic activity. • CD4"+ CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

  6. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

    Energy Technology Data Exchange (ETDEWEB)

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku, E-mail: nakagawa@phs.osaka-u.ac.jp; Okada, Naoki, E-mail: okada@phs.osaka-u.ac.jp

    2016-04-22

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

  7. Redirection to the bone marrow improves T cell persistence and antitumor functions.

    Science.gov (United States)

    Khan, Anjum B; Carpenter, Ben; Santos E Sousa, Pedro; Pospori, Constandina; Khorshed, Reema; Griffin, James; Velica, Pedro; Zech, Mathias; Ghorashian, Sara; Forrest, Calum; Thomas, Sharyn; Gonzalez Anton, Sara; Ahmadi, Maryam; Holler, Angelika; Flutter, Barry; Ramirez-Ortiz, Zaida; Means, Terry K; Bennett, Clare L; Stauss, Hans; Morris, Emma; Lo Celso, Cristina; Chakraverty, Ronjon

    2018-05-01

    A key predictor for the success of gene-modified T cell therapies for cancer is the persistence of transferred cells in the patient. The propensity of less differentiated memory T cells to expand and survive efficiently has therefore made them attractive candidates for clinical application. We hypothesized that redirecting T cells to specialized niches in the BM that support memory differentiation would confer increased therapeutic efficacy. We show that overexpression of chemokine receptor CXCR4 in CD8+ T cells (TCXCR4) enhanced their migration toward vascular-associated CXCL12+ cells in the BM and increased their local engraftment. Increased access of TCXCR4 to the BM microenvironment induced IL-15-dependent homeostatic expansion and promoted the differentiation of memory precursor-like cells with low expression of programmed death-1, resistance to apoptosis, and a heightened capacity to generate polyfunctional cytokine-producing effector cells. Following transfer to lymphoma-bearing mice, TCXCR4 showed a greater capacity for effector expansion and better tumor protection, the latter being independent of changes in trafficking to the tumor bed or local out-competition of regulatory T cells. Thus, redirected homing of T cells to the BM confers increased memory differentiation and antitumor immunity, suggesting an innovative solution to increase the persistence and functions of therapeutic T cells.

  8. The CD8⁺ memory stem T cell (T(SCM)) subset is associated with improved prognosis in chronic HIV-1 infection.

    Science.gov (United States)

    Ribeiro, Susan P; Milush, Jeffrey M; Cunha-Neto, Edecio; Kallas, Esper G; Kalil, Jorge; Somsouk, Ma; Hunt, Peter W; Deeks, Steven G; Nixon, Douglas F; SenGupta, Devi

    2014-12-01

    of the CD8(+) T(SCM) compartment and HIV-1 disease progression in a cohort of chronically infected individuals. Our results suggest that HAART restores a normal frequency of CD8(+) T(SCM) and that the natural preservation of this subset in the setting of untreated HIV-1 infection is associated with improved viral control and immunity. Therefore, the CD8(+) T(SCM) population may represent a correlate of protection in chronic HIV-1 infection that is directly relevant to the design of T cell-based vaccines, adoptive immunotherapy approaches, or the pharmacologic induction of T(SCM). Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  9. Memory T Cell Migration

    OpenAIRE

    Qianqian eZhang; Qianqian eZhang; Fadi G. Lakkis

    2015-01-01

    Immunological memory is a key feature of adaptive immunity. It provides the organism with long-lived and robust protection against infection. In organ transplantation, memory T cells pose a significant threat by causing allograft rejection that is generally resistant to immunosuppressive therapy. Therefore, a more thorough understanding of memory T cell biology is needed to improve the survival of transplanted organs without compromising the host’s ability to fight infections. This review...

  10. EBV induces persistent NF-κB activation and contributes to survival of EBV-positive neoplastic T- or NK-cells.

    Directory of Open Access Journals (Sweden)

    Honami Takada

    Full Text Available Epstein-Barr virus (EBV has been detected in several T- and NK-cell neoplasms such as extranodal NK/T-cell lymphoma nasal type, aggressive NK-cell leukemia, EBV-positive peripheral T-cell lymphoma, systemic EBV-positive T-cell lymphoma of childhood, and chronic active EBV infection (CAEBV. However, how this virus contributes to lymphomagenesis in T or NK cells remains largely unknown. Here, we examined NF-κB activation in EBV-positive T or NK cell lines, SNT8, SNT15, SNT16, SNK6, and primary EBV-positive and clonally proliferating T/NK cells obtained from the peripheral blood of patients with CAEBV. Western blotting, electrophoretic mobility shift assays, and immunofluorescent staining revealed persistent NF-κB activation in EBV-infected cell lines and primary cells from patients. Furthermore, we investigated the role of EBV in infected T cells. We performed an in vitro infection assay using MOLT4 cells infected with EBV. The infection directly induced NF-κB activation, promoted survival, and inhibited etoposide-induced apoptosis in MOLT4 cells. The luciferase assay suggested that LMP1 mediated NF-κB activation in MOLT4 cells. IMD-0354, a specific inhibitor of NF-κB that suppresses NF-κB activation in cell lines, inhibited cell survival and induced apoptosis. These results indicate that EBV induces NF-κB-mediated survival signals in T and NK cells, and therefore, may contribute to the lymphomagenesis of these cells.

  11. T2 Magnetic Resonance Assay-Based Direct Detection of Three Lyme Disease-Related Borrelia Species in Whole-Blood Samples.

    Science.gov (United States)

    Snyder, Jessica L; Giese, Heidi; Bandoski-Gralinski, Cheryl; Townsend, Jessica; Jacobson, Beck E; Shivers, Robert; Schotthoefer, Anna M; Fritsche, Thomas R; Green, Clayton; Callister, Steven M; Branda, John A; Lowery, Thomas J

    2017-08-01

    In early Lyme disease (LD), serologic testing is insensitive and seroreactivity may reflect active or past infection. In this study, we evaluated a novel assay for the direct detection of three species of Borrelia spirochetes in whole blood. The T2 magnetic resonance (T2MR) assay platform was used to amplify Borrelia DNA released from intact spirochetes and to detect amplicon. Analytical sensitivity was determined from blood spiked with known concentrations of spirochetes, and the assay's limit of detection was found to be in the single-cell-per-milliliter range: 5 cells/ml for B. afzelii and 8 cells/ml for Borrelia burgdorferi and Borrelia garinii Clinical samples ( n = 66) from confirmed or suspected early LD patients were also analyzed. B. burgdorferi was detected using T2MR in 2/2 (100%) of blood samples from patients with confirmed early LD, based on the presence of erythema migrans and documentation of seroconversion or a positive real-time blood PCR. T2MR detected B. burgdorferi in blood samples from 17/54 (31%) of patients with probable LD, based on the presence of erythema migrans without documented seroconversion or of documented seroconversion in patients with a compatible clinical syndrome but without erythema migrans. Out of 21 clinical samples tested by real-time PCR, only 1 was positive and 13 were negative with agreement with T2MR. An additional 7 samples that were negative by real-time PCR were positive with T2MR. Therefore, T2MR enables a low limit of detection (LoD) for Borrelia spp. in whole blood samples and is able to detect B. burgdorferi in clinical samples. Copyright © 2017 American Society for Microbiology.

  12. T cell receptor-engineered T cells to treat solid tumors: T cell processing toward optimal T cell fitness

    NARCIS (Netherlands)

    C.H.J. Lamers (Cor); S. van Steenbergen-Langeveld (Sabine); M. van Brakel (Mandy); C.M. Groot-van Ruijven (Corrien); P.M.M.L. van Elzakker (Pascal); B.A. van Krimpen (Brigitte); S. Sleijfer (Stefan); J.E.M.A. Debets (Reno)

    2014-01-01

    textabstractTherapy with autologous T cells that have been gene-engineered to express chimeric antigen receptors (CAR) or T cell receptors (TCR) provides a feasible and broadly applicable treatment for cancer patients. In a clinical study in advanced renal cell carcinoma (RCC) patients with CAR T

  13. Memory T follicular helper CD4 T cells

    Directory of Open Access Journals (Sweden)

    J. Scott eHale

    2015-02-01

    Full Text Available T follicular helper (Tfh cells are the subset of CD4 T helper cells that are required for generation and maintenance of germinal center reactions and the generation of long-lived humoral immunity. This specialized T helper subset provides help to cognate B cells via their expression of CD40 ligand, IL-21, IL-4, and other molecules. Tfh cells are characterized by their expression of the chemokine receptor CXCR5, expression of the transcriptional repressor Bcl6, and their capacity to migrate to the follicle and promote germinal center B cell responses. Until recently, it remained unclear whether Tfh cells differentiated into memory cells and whether they maintain their Tfh commitment at the memory phase. This review will highlight several recent studies that support the idea of Tfh-committed CD4 T cells at the memory stage of the immune response. The implication of these findings is that memory Tfh cells retain their capacity to recall their Tfh-specific effector functions upon reactivation to provide help for B cell responses and play an important role in prime and boost vaccination or during recall responses to infection. The markers that are useful for distinguishing Tfh effector and memory cells, as well as the limitations of using these markers will be discussed. Tfh effector and memory generation, lineage maintenance, and plasticity relative to other T helper lineages (Th1, Th2, Th17, etc will also be discussed. Ongoing discoveries regarding the maintenance and lineage stability versus plasticity of memory Tfh cells will improve strategies that utilize CD4 T cell memory to modulate antibody responses during prime and boost vaccination.

  14. Allogeneic lymphocyte-licensed DCs expand T cells with improved antitumor activity and resistance to oxidative stress and immunosuppressive factors

    Directory of Open Access Journals (Sweden)

    Chuan Jin

    2014-01-01

    Full Text Available Adoptive T-cell therapy of cancer is a treatment strategy where T cells are isolated, activated, in some cases engineered, and expanded ex vivo before being reinfused to the patient. The most commonly used T-cell expansion methods are either anti-CD3/CD28 antibody beads or the “rapid expansion protocol” (REP, which utilizes OKT-3, interleukin (IL-2, and irradiated allogeneic feeder cells. However, REP-expanded or bead-expanded T cells are sensitive to the harsh tumor microenvironment and often short-lived after reinfusion. Here, we demonstrate that when irradiated and preactivated allosensitized allogeneic lymphocytes (ASALs are used as helper cells to license OKT3-armed allogeneic mature dendritic cells (DCs, together they expand target T cells of high quality. The ASAL/DC combination yields an enriched Th1-polarizing cytokine environment (interferon (IFN-γ, IL-12, IL-2 and optimal costimulatory signals for T-cell stimulation. When genetically engineered antitumor T cells were expanded by this coculture system, they showed better survival and cytotoxic efficacy under oxidative stress and immunosuppressive environment, as well as superior proliferative response during tumor cell killing compared to the REP protocol. Our result suggests a robust ex vivo method to expand T cells with improved quality for adoptive cancer immunotherapy.

  15. GLYCAN-DIRECTED CAR-T CELLS.

    Science.gov (United States)

    Steentoft, Catharina; Migliorini, Denis; King, Tiffany R; Mandel, Ulla; June, Carl H; Posey, Avery D

    2018-01-23

    Cancer immunotherapy is rapidly advancing in the treatment of a variety of hematopoietic cancers, including pediatric acute lymphoblastic leukemia and diffuse large B cell lymphoma, with chimeric antigen receptor (CAR)-T cells. CARs are genetically encoded artificial T cell receptors that combine the antigen specificity of an antibody with the machinery of T cell activation. However, implementation of CAR technology in the treatment of solid tumors has been progressing much slower. Solid tumors are characterized by a number of challenges that need to be overcome, including cellular heterogeneity, immunosuppressive tumor microenvironment (TME), and, in particular, few known cancer-specific targets. Post-translational modifications that differentially occur in malignant cells generate valid cell surface, cancer-specific targets for CAR-T cells. We previously demonstrated that CAR-T cells targeting an aberrant O-glycosylation of MUC1, a common cancer marker associated with changes in cell adhesion, tumor growth, and poor prognosis, could control malignant growth in mouse models. Here, we discuss the field of glycan-directed CAR-T cells and review the different classes of antibodies specific for glycan-targeting, including the generation of high affinity O-glycopeptide antibodies. Finally, we discuss historic and recently investigated glycan targets for CAR-T cells and provide our perspective on how targeting the tumor glycoproteome and/or glycome will improve CAR-T immunotherapy. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Curcumin and Viscum album Extract Decrease Proliferation and Cell Viability of Soft-Tissue Sarcoma Cells: An In Vitro Analysis of Eight Cell Lines Using Real-Time Monitoring and Colorimetric Assays.

    Science.gov (United States)

    Harati, K; Behr, B; Daigeler, A; Hirsch, T; Jacobsen, F; Renner, M; Harati, A; Wallner, C; Lehnhardt, M; Becerikli, M

    2017-01-01

    The cytostatic effects of the polyphenol curcumin and Viscum album extract (VAE) were assessed in soft-tissue sarcoma (STS) cells. Eight human STS cell lines were used: fibrosarcoma (HT1080), liposarcoma (SW872, T778, MLS-402), synovial sarcoma (SW982, SYO1, 1273), and malignant fibrous histiocytoma (U2197). Primary human fibroblasts served as control cells. Cell proliferation, viability, and cell index (CI) were analyzed by BrdU assay, MTT assay, and real-time cell analysis (RTCA). As indicated by BrdU and MTT, curcumin significantly decreased the cell proliferation of five cell lines (HT1080, SW872, SYO1, 1273, and U2197) and the viability of two cell lines (SW872 and SW982). VAE led to significant decreases of proliferation in eight cell lines (HT1080, SW872, T778, MLS-402, SW982, SYO1, 1293, and U2197) and reduced viability in seven STS lines (HT1080, SW872, T778, MLS-402, SW982, SYO1, and 1273). As indicated by RTCA for 160 h, curcumin decreased the CI of all synovial sarcoma cell lines as well as T778 and HT1080. VAE diminished the CI in most of the synovial sarcoma (SW982, SYO1) and liposarcoma (SW872, T778) cell lines as well as HT1080. Primary fibroblasts were not affected adversely by the two compounds in RTCA. Curcumin and VAE can inhibit the proliferation and viability of STS cells.

  17. Allograft cytotoxicity co-operation between alloimmune T cells and macrophages

    International Nuclear Information System (INIS)

    Jones, B.; Jones, T.C.

    1978-01-01

    T cells from the spleens of C57BL 10 (H-2sup(b)) mice 7 to 12 days after immunization with P815Y (H-2sup(d)) mastocytoma cells have been shown to co-operate synergistically with an adherent component of non-immune starch induced peritoneal cells in the cytostasis of target cells. Although significant values for synergy could be obtained using the ( 125 I) UdR incorporation assay to measure cytostasis, normal peritoneal cells were incapable of co-operating with T cells in cytolysis as measured by 51 Cr release from pre-labelled target cells. Initially, the synergistic interaction was immunologically specific, but non-specific activity could be induced by challenge with specific antigen. (author)

  18. Reference cells and ploidy in the comet assay

    Directory of Open Access Journals (Sweden)

    Gunnar eBrunborg

    2015-02-01

    Full Text Available In the comet assay, single cells are analyzed with respect to their level of DNA damage. Discrimination of the individual cell or cell type based on DNA content, with concomitant scoring of the DNA damage, is useful since this may allow analysis of mixtures of cells. Different cells can then be characterized based on their ploidy, cell cycle stage, or genome size. We here describe two applications of such a cell type-specific comet assay: (i Testicular cell suspensions, analyzed on the basis of their ploidy during spermatogenesis; and (ii reference cells in the form of fish erythrocytes which can be included as internal standards to correct for inter-assay variations. With standard fluorochromes used in the comet assay, the total staining signal from each cell – whether damaged or undamaged – was found to be associated with the cell’s DNA content. Analysis of the fluorescence intensity of single cells is straightforward since these data are available in scoring systems based on image analysis. The analysis of testicular cell suspensions provides information on cell type specific composition, susceptibility to genotoxicants, and DNA repair. Internal reference cells, either untreated or carrying defined numbers of lesions induced by ionizing radiation, are useful for investigation of experimental factors that can cause variation in comet assay results, and for routine inclusion in experiments to facilitate standardization of methods and comparison of comet assay data obtained in different experiments or in different laboratories. They can also be used - in combination with a reference curve - to quantify the DNA lesions induced by a certain treatment. Fish cells of a range of genome sizes, both greater and smaller than human, are suitable for this purpose and they are inexpensive.

  19. (99) Tc-methylene diphosphonate improves rheumatoid arthritis disease activity by increasing the frequency of peripheral γδ T cells and CD4(+) CD25(+) Foxp3(+) Tregs.

    Science.gov (United States)

    Su, Dinglei; Shen, Minning; Gu, Bingjie; Wang, Xiaoqin; Wang, Dandan; Li, Xia; Sun, Lingyun

    2016-06-01

    γδ T cells exhibit important functions in the pathogenesis of rheumatoid arthritis (RA). In recent years, numerous studies harnessed the γδ T cell-activating capacity of aminobiphosphonates for the treatment of malignant tumors. As (99) Tc-methylene diphosphonate ((99) Tc-MDP) has long been widely used for the treatment of RA in China with good efficacy, we are interested in whether this drug exerts its therapeutic effect on RA by modulating peripheral γδ T cells of RA patients. To investigate the effect of (99) Tc-MDP on the frequency of γδ T cells and CD4(+) CD25(+) Foxp3(+) Tregs in the peripheral blood of patients with active RA. Nineteen patients with active RA were treated with (99) Tc-MDP intravenously at a dose of 20 μg/day consecutively for 10-14 days. Before and after treatment, the main clinical and laboratory parameters for each patient were evaluated. The frequency of CD3(+) γδ(+) T cells and CD4(+) CD25(+) Foxp3(+) Tregs was detected by flow cytometry. Serum levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and transforming growth factor (TGF)-β were measured with enzyme-linked immunosorbent assay. After intravenous (99) Tc-MDP therapy, the frequency of peripheral CD3(+) γδ(+) T cells and CD4(+) CD25(+) Foxp3(+) Tregs were significantly elevated, paralleled with decreased serum levels of TNF-α and IL-6 and increased level of serum TGF-β. The elevation of peripheral CD3(+) γδ(+) T cells was positively correlated with increased serum TGF-β and decreased disease activity. (99) Tc-MDP may improve the activity of RA through upregulating the frequency of peripheral γδ T cells and CD4(+) CD25(+) Foxp3(+) Tregs as well as affecting the serum cytokine environment by increasing TGF-β and decreasing TNF-α and IL-6. © 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd.

  20. Human xenospecific T suppressor cells inhibit T helper cell proliferation to porcine aortic endothelial cells, and NF-kappaB activity in porcine APC.

    Science.gov (United States)

    Ciubotariu, R; Li, J; Colovai, A I; Platt, J L; Cortesini, R; Suciu Foca Cortesini, N

    2001-05-01

    Human T suppressor cells (Ts), capable of preventing autologous T helper cells (Th) from reacting against xenogeneic pig endothelial cells and pig APC can be generated in vitro. Ts derive from a population of CD3(+)CD8(+)CD28(-) T lymphocytes and specifically recognize the MHC class I antigens of the APC used for in vitro immunization. To study the mechanism that underlies suppression, we investigated whether Ts inhibit the expression of costimulatory molecules in xenogeneic professional and semiprofessional APC. We found that Ts down-regulate Th-induced expression of CD86 in pig APC, and that this effect occurs at the level of transcription, as indicated by nuclear run-on and Northern blot assays. EMSA results revealed that inhibition of CD86 expression is mediated by inactivation of transcription factor NF-kappaB. Furthermore, transfection of pig APC with a vector expressing NF-kappaB p65 partially rescued Th-induced expression of the CD86 molecule. These results strongly support the concept that xenospecific Ts inhibit the APC function of xenogeneic cells by preventing activation of NF-kappaB.

  1. Human amniotic epithelial cells inhibit CD4+ T cell activation in acute kidney injury patients by influencing the miR-101-c-Rel-IL-2 pathway.

    Science.gov (United States)

    Liu, Junfeng; Hua, Rong; Gong, Zhangbin; Shang, Bin; Huang, Yongyi; Guo, Lihe; Liu, Te; Xue, Jun

    2017-01-01

    In the pathogenesis of acute kidney injury (AKI), the release of multiple interleukins can lead to increased kidney damage. Human amniotic epithelial cells (HuAECs) can inhibit immune cell activation in vivo and in vitro. We hypothesized that HuAECs could weaken patient-derived peripheral blood CD4+ T-cell activation and decreasing the ability of these cells to express and release IL-2. -Cell proliferation assay revealed that under the same culture conditions, activated AKI patient-derived CD4+ T cells had a significantly reduced proliferation rate when were co-cultured with HuAECs. And the level of IL-2 released was also significantly reduced. Western blot and qRT-PCR assays showed that the expression of c-Rel in the CD4+ T cells was also significantly reduced. However, the expression level of endogenous miR-101 in the CD4+ T cells co-cultured with HuAECs was significantly increased. Luciferase reporter assay results suggested that miR-101 could bind to a specific site in the c-Rel 3' UTR and induce the post-transcriptional silencing of c-Rel. Subsequently, we over-expressed miR-101 in AKI patient-derived CD4+ T cells. The qRT-PCR and western blot assay results revealed that the expression of endogenous c-Rel was significantly reduced, while the ELISA results indicated that the level of IL-2 released was also significantly decreased. Finally, ChIP-PCR assay results showed that the miR-101-overexpressing CD4+ T-cell group and the HuAEC co-culture CD4+ T-cell group exhibited significantly decreased binding capacities between the 'c-Rel-NFκB' complex and the IL-2 gene promoter, and the transcriptional activity of IL-2 was also significantly decreased. Therefore, we confirmed that HuAECs can stimulate miR-101 expression in AKI patient-derived peripheral blood CD4+ T cells, thus inhibiting the expression of the miR-101 target gene c-Rel and leading to a reduction in IL-2 expression and release. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Han, Hui [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Peng, Ji-Run, E-mail: pengjr@medmail.com.cn [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Chen, Peng-Cheng; Gong, Lei [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Qiao, Shi-Shi [Department of Hepatobiliary Surgery, The First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052 (China); Wang, Wen-Zhen; Cui, Zhu-Qingqing; Yu, Xin; Wei, Yu-Hua [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Leng, Xi-Sheng, E-mail: lengxs2003@yahoo.com.cn [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China)

    2011-08-05

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

  4. Cell-patterned glass spray for direct drug assay using mass spectrometry

    International Nuclear Information System (INIS)

    Wu, Jing; Wang, Shiqi; Chen, Qiushui; Jiang, Hao; Liang, Shuping; Lin, Jin-Ming

    2015-01-01

    In this work, the establishment of a glass spray mass spectrometry (GS-MS) platform for direct cell-based drug assay was described. Cell co-culture, drug-induced cell apoptosis, proliferation analysis and intracellular drug absorption measurement were performed simultaneously on this specifically designed platform. Two groups of co-cultured cells (NIH-3T3/HepG2 and HepG2/MCF-7) were cultivated and they showed high viability within 3 days. The biocompatibility of the platform facilitated the subsequent bioassays, in which, cyclophosphamide (CPA) and genistein were used as the model drugs. The distinctions of cell apoptosis and proliferation between the mono-cultured and co-cultured cells were clearly observed and well explained by in situ GS-MS measurements. A satisfactory linearity of the calibration curve between the relative MS intensity and CPA concentrations was obtained using stable isotope labeling method (y = 0.16545 + 0.0985x, R"2 = 0.9937). The variations in the quantity of absorbed drug were detected and the results were consistent with the concentration-dependence of cell apoptosis. All the results demonstrated that direct cell-based drug assay could be performed on the stable isotope labeling assisted GS-MS platform in a facile and quantitative manner. - Highlights: • A versatile glass spray mass spectrometry (GS-MS) platform for direct cell-based drug assay was developed in this paper. • It has characteristics of the atmospheric pressure ionization method. • It is multifunctional for cell co-culture, bioassays, qualitative and quantitative intracellular drug absorption measurement. • GS-MS has the potential to increase the use of mass spectrometry in biological analysis.

  5. Radiosensitivity of CD4 and CD8 positive human T lymphocytes by an in vitro colony formation assay

    International Nuclear Information System (INIS)

    Nakamura, Nori; Kusunoki, Yoichiro; Akiyama, Mitoshi.

    1989-12-01

    The recent development of an in vitro lymphocyte colony assay provides a new opportunity to examine possible variations in human radiosensitivity using peripheral blood lymphocytes (PBL) in place of the hitherto used skin fibroblast assay. Our recent study showed that most of the colonies consisted of lymphocytes bearing CD4 or CD8 antigens. Since the fraction of CD4 + and CD8 + cells in PBL differs among individuals, it was suspected that individual radiosensitivity might be biased by the different subset frequencies if the dose-survival curves of the CD4 + and CD8 + cells differed. In the present study, CD4 + lymphocytes (helper/inducer T cells) and CD8 + lymphocytes (suppressor/cytotoxic T cells) were isolated from PBL and their dose-survival curves were determined. The results showed that the D 10 (the dose required to reduce the surviving fraction to 10 %) was quite similar for these two types of cells (3.13 ± 0.10 Gy [mean ±SD] for CD4 + , 3.34 ± 0.50 Gy for CD8 + and 3.07 ± 0.05 Gy for the unsorted cells), supporting the use of a whole PBL population for screening of individuals with altered radiosensitivity. (author)

  6. Micro-fluidic module for blood cell separation for gene expression radiobiological assays

    International Nuclear Information System (INIS)

    Brengues, Muriel; Gu, Jian; Zenhausern, Frederic

    2015-01-01

    Advances in molecular techniques have improved discovery of biomarkers associated with radiation exposure. Gene expression techniques have been demonstrated as effective tools for biodosimetry, and different assay platforms with different chemistries are now available. One of the main challenges is to integrate the sample preparation processing of these assays into micro-fluidic platforms to be fully automated for point-of-care medical countermeasures in the case of a radiological event. Most of these assays follow the same workflow processing that comprises first the collection of blood samples followed by cellular and molecular sample preparation. The sample preparation is based on the specific reagents of the assay system and depends also on the different subsets of cells population and the type of biomarkers of interest. In this article, the authors present a module for isolation of white blood cells from peripheral blood as a prerequisite for automation of gene expression assays on a micro-fluidic cartridge. For each sample condition, the gene expression platform can be adapted to suit the requirements of the selected assay chemistry (authors)

  7. Use of immunoblotting assay improves the sensitivity of paracoccidioidomycosis diagnosis

    Directory of Open Access Journals (Sweden)

    D. F. Silva

    2008-01-01

    Full Text Available The purpose of this work was to evaluate two serological assays: double immunodiffusion (DI and immunoblotting (IB in immunodiagnosis of paracoccidioidomycosis (PCM. We evaluated by IB assay 23 sera samples from patients with clinical confirmation of PCM, all of them with negative DI results against culture filtrate from Paracoccidioides brasiliensis isolate 113. For IB, as well as for comparative DI assay, we employed soluble components of the cell wall outer surface (SCCWOS from P. brasiliensis isolate 113 cultivated at 36°C in Fava-Neto's agar medium for 5 and 10 days. Among the 20 sera samples analyzed by DI, 13 (65% were negative and 7 (35% were positive against SCCWOS obtained on the 5th and 10th days. By IB assay, 95.4% and 100% of sera reacted against gp43 and gp70 present in SCCWOS from the 5th day and 95.6% recognized these fractions when evaluated against SCCWOS from the 10th day. Our results demonstrated that the use of an immunoenzymatic assay significantly improves the sensitivity of PCM immunodiagnosis and also suggests that at least two serological tests for antibody detection should be adopted in cases of questionable diagnosis.

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

    Science.gov (United States)

    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.

  9. Curcumin improves regulatory T cells in gut-associated lymphoid tissue of colitis mice.

    Science.gov (United States)

    Zhao, Hai-Mei; Xu, Rong; Huang, Xiao-Ying; Cheng, Shao-Min; Huang, Min-Fang; Yue, Hai-Yang; Wang, Xin; Zou, Yong; Lu, Ai-Ping; Liu, Duan-Yong

    2016-06-21

    To explore the probable pathway by which curcumin (Cur) regulates the function of Treg cells by observing the expression of costimulatory molecules of dendritic cells (DCs). Experimental colitis was induced by administering 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)/ethanol solution. Forty male C57BL/6 mice were randomly divided into four groups: normal, TNBS + Cur, TNBS + mesalazine (Mes) and TNBS groups. The mice in the TNBS + Cur and TNBS +Mes groups were treated with Cur and Mes, respectively, while those in the TNBS group were treated with physiological saline for 7 d. After treatment, the curative effect of Cur was evaluated by colonic weight, colonic length, weight index of the colon, and histological observation and score. The levels of CD4(+)CD25+Foxp3(+) T cells (Treg cells) and costimulatory molecules of DCs were measured by flow cytometry. Also, related cytokines were analyzed by enzyme-linked immunosorbent assay. Cur alleviated inflammatory injury of the colonic mucosa, decreased colonic weigh and histological score, and restored colonic length. The number of Treg cells was increased, while the secretion of TNF-α, IL-2, IL-6, IL-12 p40, IL-17 and IL-21 and the expression of costimulatory molecules (CD205, CD54 [ICAM-1], TLR4, CD252[OX40 L], CD256 [RANK] and CD254 [RANK L]) of DCs were notably inhibited in colitis mice treated with Cur. Cur potentially modulates activation of DCs to enhance the suppressive functions of Treg cells and promote the recovery of damaged colonic mucosa in inflammatory bowel disease.

  10. Improving immunological tumor microenvironment using electro-hyperthermia followed by dendritic cell immunotherapy.

    Science.gov (United States)

    Tsang, Yuk-Wah; Huang, Cheng-Chung; Yang, Kai-Lin; Chi, Mau-Shin; Chiang, Hsin-Chien; Wang, Yu-Shan; Andocs, Gabor; Szasz, Andras; Li, Wen-Tyng; Chi, Kwan-Hwa

    2015-10-15

    The treatment of intratumoral dentritic cells (DCs) commonly fails because it cannot evoke immunity in a poor tumor microenvironment (TME). Modulated electro-hyperthermia (mEHT, trade-name: oncothermia) represents a significant technological advancement in the hyperthermia field, allowing the autofocusing of electromagnetic power on a cell membrane to generate massive apoptosis. This approach turns local immunogenic cancer cell death (apoptosis) into a systemic anti-tumor immune response and may be implemented by treatment with intratumoral DCs. The CT26 murine colorectal cancer model was used in this investigation. The inhibition of growth of the tumor and the systemic anti-tumor immune response were measured. The tumor was heated to a core temperature of 42 °C for 30 min. The matured synergetic DCs were intratumorally injected 24 h following mEHT was applied. mEHT induced significant apoptosis and enhanced the release of heat shock protein70 (Hsp70) in CT26 tumors. Treatment with mEHT-DCs significantly inhibited CT26 tumor growth, relative to DCs alone or mEHT alone. The secondary tumor protection effect upon rechallenging was observed in mice that were treated with mEHT-DCs. Immunohistochemical staining of CD45 and F4/80 revealed that mEHT-DC treatment increased the number of leukocytes and macrophages. Most interestingly, mEHT also induced infiltrations of eosinophil, which has recently been reported to be an orchestrator of a specific T cell response. Cytotoxic T cell assay and ELISpot assay revealed a tumor-specific T cell activity. This study demonstrated that mEHT induces tumor cell apoptosis and enhances the release of Hsp70 from heated tumor cells, unlike conventional hyperthermia. mEHT can create a favorable tumor microenvironment for an immunological chain reaction that improves the success rate of intratumoral DC immunotherapy.

  11. Semiallogenic fusions of MSI+ tumor cells and activated B cells induce MSI-specific T cell responses

    International Nuclear Information System (INIS)

    Garbe, Yvette; Klier, Ulrike; Linnebacher, Michael

    2011-01-01

    Various strategies have been developed to transfer tumor-specific antigens into antigen presenting cells in order to induce cytotoxic T cell responses against tumor cells. One approach uses cellular vaccines based on fusions of autologous antigen presenting cells and allogeneic tumor cells. The fusion cells combine antigenicity of the tumor cell with optimal immunostimulatory capacity of the antigen presenting cells. Microsatellite instability caused by mutational inactivation of DNA mismatch repair genes results in translational frameshifts when affecting coding regions. It has been shown by us and others that these mutant proteins lead to the presentation of immunogenic frameshift peptides that are - in principle - recognized by a multiplicity of effector T cells. We chose microsatellite instability-induced frameshift antigens as ideal to test for induction of tumor specific T cell responses by semiallogenic fusions of microsatellite instable carcinoma cells with CD40-activated B cells. Two fusion clones of HCT116 with activated B cells were selected for stimulation of T cells autologous to the B cell fusion partner. Outgrowing T cells were phenotyped and tested in functional assays. The fusion clones expressed frameshift antigens as well as high amounts of MHC and costimulatory molecules. Autologous T cells stimulated with these fusions were predominantly CD4 + , activated, and reacted specifically against the fusion clones and also against the tumor cell fusion partner. Interestingly, a response toward 6 frameshift-derived peptides (of 14 tested) could be observed. Cellular fusions of MSI + carcinoma cells and activated B cells combine the antigen-presenting capacity of the B cell with the antigenic repertoire of the carcinoma cell. They present frameshift-derived peptides and can induce specific and fully functional T cells recognizing not only fusion cells but also the carcinoma cells. These hybrid cells may have great potential for cellular immunotherapy and

  12. Clinical assessment of anti-viral CD8+ T cell immune monitoring using QuantiFERON-CMV® assay to identify high risk allogeneic hematopoietic stem cell transplant patients with CMV infection complications.

    Directory of Open Access Journals (Sweden)

    Siok-Keen Tey

    Full Text Available The reconstitution of anti-viral cellular immunity following hematopoietic stem cell transplantation (HSCT is crucial in preventing cytomegalovirus (CMV-associated complications. Thus immunological monitoring has emerged as an important tool to better target pre-emptive anti-viral therapies. However, traditional laboratory-based assays are too cumbersome and complicated to implement in a clinical setting. Here we conducted a prospective study of a new whole blood assay (referred to as QuantiFERON-CMV® to determine the clinical utility of measuring CMV-specific CD8+ T-cell responses as a prognostic tool. Forty-one evaluable allogeneic HSCT recipients underwent weekly immunological monitoring from day 21 post-transplant and of these 21 (51.2% showed CMV reactivation and 29 (70.7% developed acute graft-versus-host disease (GvHD. Patients with acute GvHD (grade ≥ 2 within 6 weeks of transplant showed delayed reconstitution of CMV-specific T-cell immunity (p = 0.013 and a higher risk of CMV viremia (p = 0.026. The median time to stable CMV-specific immune reconstitution was 59 days and the incidence of CMV reactivation was lower in patients who developed this than those who did not (27% versus 65%; p = 0.031. Furthermore, a failure to reconstitute CMV-specific immunity soon after the onset of CMV viraemia was associated with higher peak viral loads (5685 copies/ml versus 875 copies/ml; p = 0.002. Hence, QuantiFERON-CMV® testing in the week following CMV viremia can be useful in identifying HSCT recipients at risk of complicated reactivation.

  13. Relative Contributions of B Cells and Dendritic Cells from Lupus-Prone Mice to CD4+ T Cell Polarization.

    Science.gov (United States)

    Choi, Seung-Chul; Xu, Zhiwei; Li, Wei; Yang, Hong; Roopenian, Derry C; Morse, Herbert C; Morel, Laurence

    2018-05-01

    Mouse models of lupus have shown that multiple immune cell types contribute to autoimmune disease. This study sought to investigate the involvement of B cells and dendritic cells in supporting the expansion of inflammatory and regulatory CD4 + T cells that are critical for lupus pathogenesis. We used lupus-prone B6.NZM2410.Sle1.Sle2.Sle3 (TC) and congenic C57BL/6J (B6) control mice to investigate how the genetic predisposition of these two cell types controls the activity of normal B6 T cells. Using an allogeneic in vitro assay, we showed that TC B1-a and conventional B cells expanded Th17 cells significantly more than their B6 counterparts. This expansion was dependent on CD86 and IL-6 expression and mapped to the Sle1 lupus-susceptibility locus. In vivo, TC B cells promoted greater differentiation of CD4 + T cells into Th1 and follicular helper T cells than did B6 B cells, but they limited the expansion of Foxp3 regulatory CD4 + T cells to a greater extent than did B6 B cells. Finally, when normal B6 CD4 + T cells were introduced into Rag1 -/- mice, TC myeloid/stromal cells caused their heightened activation, decreased Foxp3 regulatory CD4 + T cell differentiation, and increased renal infiltration of Th1 and Th17 cells in comparison with B6 myeloid/stromal cells. The results show that B cells from lupus mice amplify inflammatory CD4 + T cells in a nonredundant manner with myeloid/stromal cells. Copyright © 2018 by The American Association of Immunologists, Inc.

  14. Cytotoxicity of arctigenin and matairesinol against the T-cell lymphoma cell line CCRF-CEM.

    Science.gov (United States)

    Su, Shan; Cheng, Xinlai; Wink, Michael

    2015-09-01

    Arctigenin and matairesinol possess a diversity of bioactivities. Here we investigated the cytotoxicity of arctigenin and matairesinol against a T-cell lymphoma cell line CCRF-CEM and the underlying mechanisms that have not been explored before. The cytotoxic activity was investigated using MTT assay. The cell cycle arrest and reactive oxygen species (ROS) accumulation were determined by flow cytometric analysis. The apoptosis induction was assessed using Annexin V/Propidium Iodide assay. The gene quantification analysis was measured through real-time polymerase chain reaction. Arctigenin and matairesinol exhibited significant antiproliferative activity against CCRF-CEM cells after 72 h treatment with IC50 values of 1.21 ± 0.15 μm and 4.27 ± 0.41 μm, respectively. In addition, both lignans arrest CCRF-CEM cells in the S phase. Furthermore, they could induce apoptosis in CCRF-CEM cells in a concentration- and time-dependent manner. Interestingly, the lignans differentially regulated the expression of several key genes involved in apoptosis pathways, including Bax, Bad and caspase-9. Moreover, both lignans could increase ROS levels in CCRF-CEM cells. Our study provides an insight into the potential of arctigenin and matairesinol as good candidates for the development of novel agents against T-cell lymphoma. © 2015 Royal Pharmaceutical Society.

  15. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Yan; Hirane, Miku; Araki, Mutsumi [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

    2014-04-04

    Highlights: • LPA{sub 5} inhibits the cell growth and motile activities of 3T3 cells. • LPA{sub 5} suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA{sub 5} on the cell motile activities inhibited by LPA{sub 1} in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA{sub 5} in 3T3 cells. • LPA signaling via LPA{sub 5} acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA{sub 1}–LPA{sub 6}) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA{sub 1} inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA{sub 5} in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA{sub 1} and LPA{sub 5} on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA{sub 5} may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA{sub 1}.

  16. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    International Nuclear Information System (INIS)

    Dong, Yan; Hirane, Miku; Araki, Mutsumi; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2014-01-01

    Highlights: • LPA 5 inhibits the cell growth and motile activities of 3T3 cells. • LPA 5 suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA 5 on the cell motile activities inhibited by LPA 1 in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA 5 in 3T3 cells. • LPA signaling via LPA 5 acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA 1 –LPA 6 ) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA 1 inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA 5 in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA 1 and LPA 5 on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA 5 may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA 1

  17. Induced pluripotent stem cells-derived myeloid-derived suppressor cells regulate the CD8+ T cell response

    Directory of Open Access Journals (Sweden)

    Daniel Joyce

    2018-05-01

    Full Text Available Myeloid-derived suppressor cells (MDSCs are markedly increased in cancer patients and tumor-bearing mice and promote tumor growth and survival by inhibiting host innate and adaptive immunity. In this study, we generated and characterized MDSCs from murine-induced pluripotent stem cells (iPSCs. The iPSCs were co-cultured with OP9 cells, stimulated with GM-CSF, and became morphologically heterologous under co-culturing with hepatic stellate cells. Allogeneic and OVA-specific antigen stimulation demonstrated that iPS-MDSCs have a T-cell regulatory function. Furthermore, a popliteal lymph node assay and autoimmune hepatitis model showed that iPS-MDSCs also regulate immune responsiveness in vivo and have a therapeutic effect against hepatitis. Taken together, our results demonstrated a method of generating functional MDSCs from iPSCs and highlighted the potential of iPS-MDSCs as a key cell therapy resource for transplantation and autoimmune diseases. Keywords: Myeloid-derived suppressor cells, Induced pluripotent stem cells, T cell response

  18. Harnessing the Power of T Cells: The Promising Hope for a Universal Influenza Vaccine

    Directory of Open Access Journals (Sweden)

    E. Bridie Clemens

    2018-03-01

    Full Text Available Next-generation vaccines that utilize T cells could potentially overcome the limitations of current influenza vaccines that rely on antibodies to provide narrow subtype-specific protection and are prone to antigenic mismatch with circulating strains. Evidence from animal models shows that T cells can provide heterosubtypic protection and are crucial for immune control of influenza virus infections. This has provided hope for the design of a universal vaccine able to prime against diverse influenza virus strains and subtypes. However, multiple hurdles exist for the realisation of a universal T cell vaccine. Overall primary concerns are: extrapolating human clinical studies, seeding durable effective T cell resident memory (Trm, population human leucocyte antigen (HLA coverage, and the potential for T cell-mediated immune escape. Further comprehensive human clinical data is needed during natural infection to validate the protective role T cells play during infection in the absence of antibodies. Furthermore, fundamental questions still exist regarding the site, longevity and duration, quantity, and phenotype of T cells needed for optimal protection. Standardised experimental methods, and eventually simplified commercial assays, to assess peripheral influenza-specific T cell responses are needed for larger-scale clinical studies of T cells as a correlate of protection against influenza infection. The design and implementation of a T cell-inducing vaccine will require a consensus on the level of protection acceptable in the community, which may not provide sterilizing immunity but could protect the individual from severe disease, reduce the length of infection, and potentially reduce transmission in the community. Therefore, increasing the standard of care potentially offered by T cell vaccines should be considered in the context of pandemic preparedness and zoonotic infections, and in combination with improved antibody vaccine targeting methods

  19. Improved laboratory assays of Pu and U for SRP purification and finishing processes

    International Nuclear Information System (INIS)

    Holland, M.K.; Dorsett, R.S.

    1986-01-01

    Significant improvements have been made in routine assay techniques for uranium and plutonium as part of an effort to improve accountability at the Savannah River Plant (SRP). Emphasis was placed on input/output accountability points and key physical inventory tanks associated with purification and finishing processes. Improvements were made in existing assay methods; new methods were implemented; and the application of these methods was greatly expanded. Prior to assays, samples were validated via density measurements. Digital density meters precise to four, five, and six decimal places were used to meet specific requirements. Improved plutonium assay techniques are now in routine use: controlled-potential coulometry, ion-exchange coulometry, and Pu(III) diode-array spectrophotometry. A new state-of-the-art coulometer was fabricated and used to ensure maximum accuracy in verifying standards and in measuring plutonium in product streams. The diode-array spectrophotometer for Pu(III) measurements was modified with fiber optics to facilitate remote measurements; rapid, precise measurements made the technique ideally suited for high-throughput assays. For uranium assays, the isotope-dilution mass spectrometric (IDMS) method was converted to a gravimetric basis. The IDMS method and the existing Davies-Gray titration (gravimetric basis) have met accountability requirements for uranium. More recently, a Pu(VI) diode-array spectrophotometric method was used on a test basis to measure plutonium in shielded-cell input accountability samples. In addition, tests to measure uranium via diode-array spectrophotometry were initiated. This rapid, precise method will replace IDMS for certain key sample points

  20. Human leucocyte antigen class I-redirected anti-tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells.

    Science.gov (United States)

    Tan, M P; Dolton, G M; Gerry, A B; Brewer, J E; Bennett, A D; Pumphrey, N J; Jakobsen, B K; Sewell, A K

    2017-01-01

    CD4 + T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour-specific CD4 + T cells occur in low frequency, express relatively low-affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4 + T cells with tumour-specific HLA class I-restricted TCRs prior to adoptive transfer. The lack of help from the co-receptor CD8 glycoprotein in CD4 + cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4 + and CD8 + T cells expressing wild-type and a range of affinity-enhanced TCRs specific for the HLA A*0201-restricted NY-ESO-1- and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4 + T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4 + T cells than CD8 + T cells. These results indicate that the CD4 + T cell component of current adoptive therapies using TCRs optimized for CD8 + T cells is below par and that there is room for substantial improvement. © 2016 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.

  1. c-MPL provides tumor-targeted T-cell receptor-transgenic T cells with costimulation and cytokine signals.

    Science.gov (United States)

    Nishimura, Christopher D; Brenner, Daniel A; Mukherjee, Malini; Hirsch, Rachel A; Ott, Leah; Wu, Meng-Fen; Liu, Hao; Dakhova, Olga; Orange, Jordan S; Brenner, Malcolm K; Lin, Charles Y; Arber, Caroline

    2017-12-21

    Adoptively transferred T-cell receptor (TCR)-engineered T cells depend on host-derived costimulation and cytokine signals for their full and sustained activation. However, in patients with cancer, both signals are frequently impaired. Hence, we developed a novel strategy that combines both essential signals in 1 transgene by expressing the nonlymphoid hematopoietic growth factor receptor c-MPL (myeloproliferative leukemia), the receptor for thrombopoietin (TPO), in T cells. c-MPL signaling activates pathways shared with conventional costimulatory and cytokine receptor signaling. Thus, we hypothesized that host-derived TPO, present in the tumor microenvironment, or pharmacological c-MPL agonists approved by the US Food and Drug Administration could deliver both signals to c-MPL-engineered TCR-transgenic T cells. We found that c-MPL + polyclonal T cells expand and proliferate in response to TPO, and persist longer after adoptive transfer in immunodeficient human TPO-transgenic mice. In TCR-transgenic T cells, c-MPL activation enhances antitumor function, T-cell expansion, and cytokine production and preserves a central memory phenotype. c-MPL signaling also enables sequential tumor cell killing, enhances the formation of effective immune synapses, and improves antileukemic activity in vivo in a leukemia xenograft model. We identify the type 1 interferon pathway as a molecular mechanism by which c-MPL mediates immune stimulation in T cells. In conclusion, we present a novel immunotherapeutic strategy using c-MPL-enhanced transgenic T cells responding to either endogenously produced TPO (a microenvironment factor in hematologic malignancies) or c-MPL-targeted pharmacological agents. © 2017 by The American Society of Hematology.

  2. Immortalization of human neural stem cells with the c-myc mutant T58A.

    Directory of Open Access Journals (Sweden)

    Lidia De Filippis

    turn, carries a single and well characterized mutation. From a different perspective, these data report on a safe approach to increase human neural stem cells propagation in culture, without altering their basic properties. These T-IhNSC line provides a versatile model for the elucidation of the mechanisms involved in human neural stem cells expansion and for development of high throughput assays for both basic and translational research on human neural cell development. The improved proclivity of T-IhNSC to generate human oligodendrocytes propose T-IhNSC as a feasible candidate for the design of experimental and, perhaps, therapeutic approaches in demyelinating diseases.

  3. Erythrocytes and cell line-based assays to evaluate the cytoprotective activity of antioxidant components obtained from natural sources.

    Science.gov (United States)

    Botta, Albert; Martínez, Verónica; Mitjans, Montserrat; Balboa, Elena; Conde, Enma; Vinardell, M Pilar

    2014-02-01

    Oxidative stress can damage cellular components including DNA, proteins or lipids, and may cause several skin diseases. To protect from this damage and addressing consumer's appeal to natural products, antioxidants obtained from algal and vegetal extracts are being proposed as antioxidants to be incorporated into formulations. Thus, the development of reliable, quick and economic in vitro methods to study the cytoactivity of these products is a meaningful requirement. A combination of erythrocyte and cell line-based assays was performed on two extracts from Sargassum muticum, one from Ulva lactuca, and one from Castanea sativa. Antioxidant properties were assessed in erythrocytes by the TBARS and AAPH assays, and cytotoxicity and antioxidant cytoprotection were assessed in HaCaT and 3T3 cells by the MTT assay. The extracts showed no antioxidant activity on the TBARS assay, whereas their antioxidant capacity in the AAPH assay was demonstrated. On the cytotoxicity assays, extracts showed low toxicity, with IC50 values higher than 200μg/mL. C. sativa extract showed the most favourable antioxidant properties on the antioxidant cytoprotection assays; while S. muticum and U. lactuca extracts showed a slight antioxidant activity. This battery of methods was useful to characterise the biological antioxidant properties of these natural extracts. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Clinical-Grade-Expanded Regulatory T Cells Prevent Graft-versus-Host Disease While Allowing a Powerful T Cell-Dependent Graft-versus-Leukemia Effect in Murine Models.

    Science.gov (United States)

    Del Papa, Beatrice; Ruggeri, Loredana; Urbani, Elena; Baldoni, Stefano; Cecchini, Debora; Zei, Tiziana; Iacucci Ostini, Roberta; Crescenzi, Barbara; Carotti, Alessandra; Pierini, Antonio; Sportoletti, Paolo; Di Bartolomeo, Paolo; Falzetti, Franca; Mecucci, Cristina; Velardi, Andrea; Martelli, Massimo F; Di Ianni, Mauro

    2017-11-01

    We developed a good manufacturing practices-compatible expansion protocol to improve number and purity of regulatory T cells (Tregs) available for clinical trials. Six clinical-grade separation procedures were performed, followed by expansion with high-dose interleukin (IL)-2, anti-CD3/anti-CD28 TCR stimulation, and rapamycin for 19 days achieving a median of 8.5-fold (range, 6.25 to 13.7) expansion. FOXP3 expression was stably maintained over the culture period, while the percentage of CD127 was significantly reduced. The in vitro suppression assay showed a strong Mixed Lymphocytes Reaction inhibition. In vitro amplification did not induce any karyotypic modification. To evaluate the graft-versus-host disease (GVHD)/graft-versus-leukemia (GVL) bifunctional axis, expanded Tregs and conventional T cells (Tcons) were tested in NOD/SCID/IL2Rgnull mice injected with primary acute myeloid leukemia (AML) cells, AML cell line, acute lymphoid leukemia Philadelphia cell line, or Burkitt-like lymphoma cell line. All mice that received leukemia cells together with expanded Tregs and Tcons were rescued from leukemia and survived without GVHD, showing that Treg expansion procedure did not compromise GVHD control and the strong Tcon-mediated GVL activity. This report might represent the basis for treating high-risk leukemia and/or relapsed/refractory leukemia patients with high-dose Treg/Tcons. Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  5. Improved Insulin Sensitivity despite Increased Visceral Adiposity in Mice Deficient for the Immune Cell Transcription Factor T-bet

    Science.gov (United States)

    Stolarczyk, Emilie; Vong, Chi Teng; Perucha, Esperanza; Jackson, Ian; Cawthorne, Michael A.; Wargent, Edward T.; Powell, Nick; Canavan, James B.; Lord, Graham M.; Howard, Jane K.

    2013-01-01

    Summary Low-grade inflammation in fat is associated with insulin resistance, although the mechanisms are unclear. We report that mice deficient in the immune cell transcription factor T-bet have lower energy expenditure and increased visceral fat compared with wild-type mice, yet paradoxically are more insulin sensitive. This striking phenotype, present in young T-bet−/− mice, persisted with high-fat diet and increasing host age and was associated with altered immune cell numbers and cytokine secretion specifically in visceral adipose tissue. However, the favorable metabolic phenotype observed in T-bet-deficient hosts was lost in T-bet−/− mice also lacking adaptive immunity (T-bet−/−xRag2−/−), demonstrating that T-bet expression in the adaptive rather than the innate immune system impacts host glucose homeostasis. Indeed, adoptive transfer of T-bet-deficient, but not wild-type, CD4+ T cells to Rag2−/− mice improved insulin sensitivity. Our results reveal a role for T-bet in metabolic physiology and obesity-associated insulin resistance. PMID:23562076

  6. Mucorales-Specific T Cells in Patients with Hematologic Malignancies.

    Science.gov (United States)

    Potenza, Leonardo; Vallerini, Daniela; Barozzi, Patrizia; Riva, Giovanni; Gilioli, Andrea; Forghieri, Fabio; Candoni, Anna; Cesaro, Simone; Quadrelli, Chiara; Maertens, Johan; Rossi, Giulio; Morselli, Monica; Codeluppi, Mauro; Mussini, Cristina; Colaci, Elisabetta; Messerotti, Andrea; Paolini, Ambra; Maccaferri, Monica; Fantuzzi, Valeria; Del Giovane, Cinzia; Stefani, Alessandro; Morandi, Uliano; Maffei, Rossana; Marasca, Roberto; Narni, Franco; Fanin, Renato; Comoli, Patrizia; Romani, Luigina; Beauvais, Anne; Viale, Pier Luigi; Latgè, Jean Paul; Lewis, Russell E; Luppi, Mario

    2016-01-01

    Invasive mucormycosis (IM) is an emerging life-threatening fungal infection. It is difficult to obtain a definite diagnosis and to initiate timely intervention. Mucorales-specific T cells occur during the course of IM and are involved in the clearance of the infection. We have evaluated the feasibility of detecting Mucorales-specific T cells in hematological patients at risk for IM, and have correlated the detection of such cells with the clinical conditions of the patients. By using an enzyme linked immunospot assay, the presence of Mucorales-specific T cells in peripheral blood (PB) samples has been investigated at three time points during high-dose chemotherapy for hematologic malignancies. Mucorales-specific T cells producing interferon-γ, interleukin-10 and interleukin-4 were analysed in order to detect a correlation between the immune response and the clinical picture. Twenty-one (10.3%) of 204 patients, accounting for 32 (5.3%) of 598 PB samples, tested positive for Mucorales-specific T cells. Two groups could be identified. Group 1, including 15 patients without signs or symptoms of invasive fungal diseases (IFD), showed a predominance of Mucorales-specific T cells producing interferon-gamma. Group 2 included 6 patients with a clinical picture consistent with invasive fungal disease (IFD): 2 cases of proven IM and 4 cases of possible IFD. The proven patients had significantly higher number of Mucorales-specific T cells producing interleukin-10 and interleukin-4 and higher rates of positive samples by using derived diagnostic cut-offs when compared with the 15 patients without IFD. Mucorales-specific T cells can be detected and monitored in patients with hematologic malignancies at risk for IM. Mucorales-specific T cells polarized to the production of T helper type 2 cytokines are associated with proven IM and may be evaluated as a surrogate diagnostic marker for IM.

  7. Mucorales-Specific T Cells in Patients with Hematologic Malignancies.

    Directory of Open Access Journals (Sweden)

    Leonardo Potenza

    Full Text Available Invasive mucormycosis (IM is an emerging life-threatening fungal infection. It is difficult to obtain a definite diagnosis and to initiate timely intervention. Mucorales-specific T cells occur during the course of IM and are involved in the clearance of the infection. We have evaluated the feasibility of detecting Mucorales-specific T cells in hematological patients at risk for IM, and have correlated the detection of such cells with the clinical conditions of the patients.By using an enzyme linked immunospot assay, the presence of Mucorales-specific T cells in peripheral blood (PB samples has been investigated at three time points during high-dose chemotherapy for hematologic malignancies. Mucorales-specific T cells producing interferon-γ, interleukin-10 and interleukin-4 were analysed in order to detect a correlation between the immune response and the clinical picture. Twenty-one (10.3% of 204 patients, accounting for 32 (5.3% of 598 PB samples, tested positive for Mucorales-specific T cells. Two groups could be identified. Group 1, including 15 patients without signs or symptoms of invasive fungal diseases (IFD, showed a predominance of Mucorales-specific T cells producing interferon-gamma. Group 2 included 6 patients with a clinical picture consistent with invasive fungal disease (IFD: 2 cases of proven IM and 4 cases of possible IFD. The proven patients had significantly higher number of Mucorales-specific T cells producing interleukin-10 and interleukin-4 and higher rates of positive samples by using derived diagnostic cut-offs when compared with the 15 patients without IFD.Mucorales-specific T cells can be detected and monitored in patients with hematologic malignancies at risk for IM. Mucorales-specific T cells polarized to the production of T helper type 2 cytokines are associated with proven IM and may be evaluated as a surrogate diagnostic marker for IM.

  8. Susceptibility of Hep3B cells in different phases of cell cycle to tBid.

    Science.gov (United States)

    Ma, Shi-Hong; Chen, George G; Ye, Caiguo; Leung, Billy C S; Ho, Rocky L K; Lai, Paul B S

    2011-01-01

    tBid is a pro-apoptotic molecule. Apoptosis inducers usually act in a cell cycle-specific fashion. The aim of this study was to elucidate whether effect of tBid on hepatocellular carcinoma (HCC) Hep3B cells was cell cycle phase specific. We synchronized Hep3B cells at G0/G1, S or G2/M phases by chemicals or flow sorting and tested the susceptibility of the cells to recombinant tBid. Cell viability was measured by MTT assay and apoptosis by TUNEL. The results revealed that tBid primarily targeted the cells at G0/G1 phase of cell cycle, and it also increased the cells at the G2/M phase. 5-Fluorouracil (5-FU), on the other hand, arrested Hep3B cells at the G0/G1 phase, but significantly reduced cells at G2/M phase. The levels of cell cycle-related proteins and caspases were altered in line with the change in the cell cycle. The combination of tBid with 5-FU caused more cells to be apoptotic than either agent alone. Therefore, the complementary effect of tBid and 5-FU on different phases of the cell cycle may explain their synergistric effect on Hep3B cells. The elucidation of the phase-specific effect of tBid points to a possible therapeutic option that combines different phase specific agents to overcome resistance of HCC. Copyright © 2010 Elsevier B.V. All rights reserved.

  9. Identification of genuine primary pulmonary NK cell lymphoma via clinicopathologic observation and clonality assay.

    Science.gov (United States)

    Gong, Li; Wei, Long-Xiao; Huang, Gao-Sheng; Zhang, Wen-Dong; Wang, Lu; Zhu, Shao-Jun; Han, Xiu-Juan; Yao, Li; Lan, Miao; Li, Yan-Hong; Zhang, Wei

    2013-08-19

    Extranodal natural killer (NK)/T-cell lymphoma, nasal type, is an uncommon lymphoma associated with the Epstein-Barr virus (EBV). It most commonly involves the nasal cavity and upper respiratory tract. Primary pulmonary NK/T cell lymphoma is extremely rare. If a patient with a NK or T-cell tumor has an unusual reaction to treatment or an unusual prognosis, it is wise to differentiate NK from T-cell tumors. The clinicopathologic characteristics, immunophenotype, EBV in situ hybridization, and T cell receptor (TCR) gene rearrangement of primary pulmonary NK cell lymphoma from a 73-year-old Chinese woman were investigated and the clonal status was determined using female X-chromosomal inactivation mosaicism and polymorphisms at the phosphoglycerate kinase (PGK) gene. The lesion showed the typical histopathologic characteristics and immunohistochemical features of NK/T cell lymphoma. However, the sample was negative for TCR gene rearrangement. A clonality assay demonstrated that the lesion was monoclonal. It is concluded that this is the first recorded case of genuine primary pulmonary NK cell lymphoma. The purpose of the present work is to recommend that pathologists carefully investigate the whole lesion to reduce the likelihood that primary pulmonary NK cell lymphoma will be misdiagnosed as an infectious lesion. In addition, TCR gene rearrangement and clonal analysis, which is based on female X-chromosomal inactivation mosaicism and polymorphisms at PGK and androgen receptor (AR) loci, were found to play important roles in differentiating NK cell lymphoma from T cell lymphoma. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5205300349457729.

  10. In Vitro Priming of Naı̈ve T-cells with p-Phenylenediamine and Bandrowski's Base.

    Science.gov (United States)

    Gibson, Andrew; Kim, Seung-Hyun; Faulkner, Lee; Evely, Jane; Pirmohamed, Munir; Park, Kevin B; Naisbitt, Dean J

    2015-10-19

    p-Phenylenediamine (PPD) is a component of hair dye formulations that is associated with T-cell mediated allergic contact dermatitis. Antigen-specific T-cells from allergic contact dermatitis patients are activated with either PPD or the oxidation product, Bandrowski's base. In nonallergic individuals, T-cells that are activated by Bandrowski's base, but not by PPD, are readily detectable. The aim of the current study was to use an in vitro T-cell priming assay to assess the activation of memory and naı̈ve T-cells from healthy donors with PPD and Bandrowski's base, and to compare these responses to those observed from allergic patients. Both PPD and Bandrowski's base-responsive clones were generated from allergic patients. The majority of Bandrowski's base-responsive clones were CD4+ and displayed a lack of PPD reactivity. In contrast, CD4+ and CD8+ clones displaying PPD reactivity were detected. Approximately 25% of these displayed low levels of reactivity to Bandrowski's base. Clones from the allergic patients secreted a range of cytokines including IFN-γ, Il-13, and Il-22. In healthy donors, Bandrowski's base-specific T-cell proliferative responses and cytokine secretion were detected with both naı̈ve and memory T-cells. T-cell clones generated from the Bandrowski's base-responsive cultures responded to Bandrowski's base but not PPD. PPD-specific naı̈ve and memory T-cell responses were not detected from healthy donors. These data show that Bandrowski's base stimulates pre-existing memory T-cells isolated from healthy donors and primes naı̈ve T-cells when the chemical is bound to autologous dendritic cells. Priming naı̈ve T-cells against PPD failed, suggesting an important individual susceptibility factor is missing from the in vitro T-cell priming assay.

  11. Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition

    Science.gov (United States)

    Cherkassky, Leonid; Morello, Aurore; Villena-Vargas, Jonathan; Feng, Yang; Dimitrov, Dimiter S.; Jones, David R.; Sadelain, Michel; Adusumilli, Prasad S.

    2016-01-01

    Following immune attack, solid tumors upregulate coinhibitory ligands that bind to inhibitory receptors on T cells. This adaptive resistance compromises the efficacy of chimeric antigen receptor (CAR) T cell therapies, which redirect T cells to solid tumors. Here, we investigated whether programmed death-1–mediated (PD-1–mediated) T cell exhaustion affects mesothelin-targeted CAR T cells and explored cell-intrinsic strategies to overcome inhibition of CAR T cells. Using an orthotopic mouse model of pleural mesothelioma, we determined that relatively high doses of both CD28- and 4-1BB–based second-generation CAR T cells achieved tumor eradication. CAR-mediated CD28 and 4-1BB costimulation resulted in similar levels of T cell persistence in animals treated with low T cell doses; however, PD-1 upregulation within the tumor microenvironment inhibited T cell function. At lower doses, 4-1BB CAR T cells retained their cytotoxic and cytokine secretion functions longer than CD28 CAR T cells. The prolonged function of 4-1BB CAR T cells correlated with improved survival. PD-1/PD-1 ligand [PD-L1] pathway interference, through PD-1 antibody checkpoint blockade, cell-intrinsic PD-1 shRNA blockade, or a PD-1 dominant negative receptor, restored the effector function of CD28 CAR T cells. These findings provide mechanistic insights into human CAR T cell exhaustion in solid tumors and suggest that PD-1/PD-L1 blockade may be an effective strategy for improving the potency of CAR T cell therapies. PMID:27454297

  12. Circulating CXCR5+CD4+ T cells participate in the IgE accumulation in allergic asthma.

    Science.gov (United States)

    Gong, Fang; Zhu, Hua-Yan; Zhu, Jie; Dong, Qiao-Jing; Huang, Xuan; Jiang, Dong-Jin

    2018-05-01

    The pathogenesis of allergic asthma is primarily characterized by abnormality in immunoglobin(Ig)E pathway, suggesting a possible role for follicular helper T cells (Tfh) in the genesis of excessive IgE accumulation. The blood chemokine (C-X-C motif) receptor 5 (CXCR)5 + CD4 + T cells, known as "circulating" Tfh, share common functional characteristics with Tfh cells from germinal centers. The aim of this study was to determine the phenotypes and functions of circulating CXCR5 + CD4 + T cells in allergic asthmatics. Here we found the frequency of the circulating CXCR5 + CD4 + T cells was raised in allergic asthma compared with healthy control (HC). Phenotypic assays showed that activated circulating CXCR5 + CD4 + T cells display the key features of Tfh cells, including invariably coexpressed programmed cell death (PD)-1 and inducible costimulator (ICOS). The frequency of interleukin IL-4 + -, IL-21 + -producing CXCR5 + CD4 + T cells was increased in allergic asthma patients compared with HC. Furthermore, sorted circulating CXCR5 + CD4 + T cells from allergic asthma patients boosted IgE production in coculture assay which could be inhibited by IL-4 or IL-21 blockage. Interestingly, IL-4 + -, IL-21 + -CXCR5 + CD4 + T cells positively correlated with total IgE in the blood. Our data indicated that circulating CXCR5 + CD4 + T cells may have a significant role in facilitating IgE production in allergic asthma patients. Copyright © 2018 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  13. [Evaluation of T-SPOT.TB assay in the diagnosis of pulmonary tuberculosis within different age groups].

    Science.gov (United States)

    Pan, Liping; Jia, Hongyan; Liu, Fei; Gao, Mengqiu; Sun, Huishan; Du, Boping; Sun, Qi; Xing, Aiying; Wei, Rongrong; Zhang, Zongde

    2015-12-01

    To evaluate the value of T-SPOT.TB assay in the diagnosis of pulmonary tuberculosis within different age groups. We analyzed 1 518 suspected pulmonary tuberculosis (PTB) patients who were admitted to the Beijing Chest Hospital from November 2012 to February 2014 and had valid T-SPOT.TB tests before anti-tuberculosis therapy. The 599 microbiologically and/or histopathologically-confirmed PTB patients (16-89 years old, 388 males and 211 females) and 235 non-TB patients (14-85 years old, 144 males and 91 females) were enrolled for the analysis of diagnostic performance of T-SPOT.TB, while patients with uncertain diagnosis or diagnosis based on clinical impression (n=684) were excluded from the analysis. The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio of the T-SPOT.TB were analyzed according to the final diagnosis. Furthermore, the diagnostic performance of T-SPOT.TB assay in the younger patients (14-59 years old) and elderly patients (60-89 years old) were also analyzed respectively. Categorical variables were compared by Pearson's Chi-square test, while continuous variables were compared by the Mann-Whitney U-test. The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio of the T-SPOT.TB in diagnosis of PTB were 90.1% (540/599), 65.5% (154/235), 86.9% (540/621), 72.3% (154/213), 2.61, and 0.15, respectively. The sensitivity and specificity of T-SPOT.TB assay were 92.6% (375/405) and 75.6% (99/131), respectively in the younger patients, and 85.0% (165/194), 52.9% (55/104) respectively in the elderly patients. The sensitivity and specificity of T-SPOT.TB assay in the younger patients were significantly higher than those in the elderly patients (Pspot forming cells in the younger PTB patients were significantly higher than in the elderly PTB patients [300 (126, 666)/10(6) PBMCs vs. 258 (79, 621

  14. Comparison of cell-based assays for the identification and evaluation of competitive CXCR4 inhibitors.

    Directory of Open Access Journals (Sweden)

    Anneleen Van Hout

    Full Text Available The chemokine receptor CXCR4 is activated by its unique chemokine ligand CXCL12 and regulates many physiological and developmental processes such as hematopoietic cell trafficking. CXCR4 is also one of the main co-receptors for human immunodeficiency virus (HIV entry. Dysfunction of the CXCL12/CXCR4 axis contributes to several human pathologies, including cancer and inflammatory diseases. Consequently, inhibition of CXCR4 activation is recognized as an attractive target for therapeutic intervention. In this regard, numerous agents modifying CXCR4 activity have been evaluated in in vitro experimental studies and pre-clinical models. Here, we evaluated a CXCL12 competition binding assay for its potential as a valuable initial screen for functional and competitive CXCR4 inhibitors. In total, 11 structurally diverse compounds were included in a side-by-side comparison of in vitro CXCR4 cell-based assays, such as CXCL12 competition binding, CXCL12-induced calcium signaling, CXCR4 internalization, CXCL12-guided cell migration and CXCR4-specific HIV-1 replication experiments. Our data indicated that agents that inhibit CXCL12 binding, i.e. the anti-CXCR4 peptide analogs T22, T140 and TC14012 and the small molecule antagonists AMD3100, AMD3465, AMD11070 and IT1t showed inhibitory activity with consistent relative potencies in all further applied CXCR4-related assays. Accordingly, agents exerting no or very weak receptor binding (i.e., CTCE-9908, WZ811, Me6TREN and gambogic acid showed no or very poor anti-CXCR4 inhibitory activity. Thus, CXCL12 competition binding studies were proven to be highly valuable as an initial screening assay and indicative for the pharmacological and functional profile of competitive CXCR4 antagonists, which will help the design of new potent CXCR4 inhibitors.

  15. Blocking of PDL-1 interaction enhances primary and secondary CD8 T cell response to herpes simplex virus-1 infection.

    Directory of Open Access Journals (Sweden)

    Rudragouda Channappanavar

    Full Text Available The blocking of programmed death ligand-1 (PDL-1 has been shown to enhance virus-specific CD8 T cell function during chronic viral infections. Though, how PDL-1 blocking at the time of priming affects the quality of CD8 T cell response to acute infections is not well understood and remains controversial. This report demonstrates that the magnitude of the primary and secondary CD8 T cell responses to herpes simplex virus-1 (HSV-1 infection is subject to control by PDL-1. Our results showed that after footpad HSV-1 infection, PD-1 expression increases on immunodominant SSIEFARL peptide specific CD8 T cells. Additionally, post-infection, the level of PDL-1 expression also increases on CD11c+ dendritic cells. Intraperitoneal administration of anti-PDL-1 monoclonal antibody given one day prior to and three days after cutaneous HSV-1 infection, resulted in a marked increase in effector and memory CD8 T cell response to SSIEFARL peptide. This was shown by measuring the quantity and quality of SSIEFARL-specific CD8 T cells by making use of ex-vivo assays that determine antigen specific CD8 T cell function, such as intracellular cytokine assay, degranulation assay to measure cytotoxicity and viral clearance. Our results are discussed in terms of the beneficial effects of blocking PDL-1 interactions, while giving prophylactic vaccines, to generate a more effective CD8 T cell response to viral infection.

  16. Autoreactive T Cells in Human Smokers Is Predictive of Clinical Outcome

    Directory of Open Access Journals (Sweden)

    Chuang eXu

    2012-08-01

    Full Text Available Cross-sectional studies have suggested a role for activation of adaptive immunity in smokers with emphysema, but the clinical application of these findings has not been explored. Here we examined the utility of detecting autoreactive T cells as a screening tool for emphysema in an at-risk population of smokers. We followed 156 former and current (ever-smokers for two years to assess whether peripheral blood CD4 T cell cytokine responses to lung elastin fragments (EFs could discriminate between those with and without emphysema, and to evaluate the relevance of autoreactive T cells to predict changes during follow-up in lung physiological parameters. Volunteers underwent baseline complete phenotypic assessment with pulmonary function tests, quantitative chest CT, yearly six minutes walk distance (6MWD testing, and annual measurement of CD4 T cell cytokine responses to EFs. The areas under the receiver operating characteristic curve to predict emphysema for interferon gamma (IFN-γ, and interleukin 6 (IL-6 responses to EFs were 0.81 (95% CI of 0.74 to 0.88 and 0.79 (95% CI of 0.72 to 0.86 respectively. We developed a dual cytokine enzyme-linked immunocell spot assay, the γ-6 Spot, using CD4 T cell IFN-γ and IL-6 responses and found that it discriminated emphysema with 90% sensitivity. After adjusting for potential confounders, the presence of autoreactive T cells was predictive of a decrease in 6MWD over two years (decline in 6MWD, -19 meters (m per fold change in IFN-γ; P=0.026, and -26 m per fold change in IL-6; P=0.003. These findings collectively suggest that the EF specific autoreactive CD4 T cell assay, γ-6 Spot, could provide a non-invasive diagnostic tool with potential application to large-scale screening to discriminate emphysema in ever-smokers, and predict early relevant physiological outcomes in those at risk.

  17. tRNA modification profiles of the fast-proliferating cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Chao; Niu, Leilei; Song, Wei [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Department of Obstetrics and Gynecology, Peking University Third Hospital, Peking University, Beijing 100191 (China); Xiong, Xin; Zhang, Xianhua [Departmentof Pharmacy, Peking University Third Hospital, Peking University, Beijing 100191 (China); Zhang, Zhenxi; Yang, Yi; Yi, Fan [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Department of Obstetrics and Gynecology, Peking University Third Hospital, Peking University, Beijing 100191 (China); Zhan, Jun; Zhang, Hongquan [Department of Anatomy, Histology and Embryology, Laboratory of Molecular Cell Biology and Tumor Biology, Peking University, Beijing 100191 (China); Yang, Zhenjun; Zhang, Li-He [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Department of Obstetrics and Gynecology, Peking University Third Hospital, Peking University, Beijing 100191 (China); Zhai, Suodi [Departmentof Pharmacy, Peking University Third Hospital, Peking University, Beijing 100191 (China); Li, Hua, E-mail: huali88@sina.com [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Department of Obstetrics and Gynecology, Peking University Third Hospital, Peking University, Beijing 100191 (China); Ye, Min, E-mail: yemin@bjmu.edu.cn [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Department of Obstetrics and Gynecology, Peking University Third Hospital, Peking University, Beijing 100191 (China); Du, Quan, E-mail: quan.du@pku.edu.cn [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Department of Obstetrics and Gynecology, Peking University Third Hospital, Peking University, Beijing 100191 (China)

    2016-08-05

    Despite the recent progress in RNA modification study, a comprehensive modification profile is still lacking for mammalian cells. Using a quantitative HPLC/MS/MS assay, we present here a study where RNA modifications are examined in term of the major RNA species. With paired slow- and fast-proliferating cell lines, distinct RNA modification profiles are first revealed for diverse RNA species. Compared to mRNAs, increased ribose and nucleobase modifications are shown for the highly-structured tRNAs and rRNAs, lending support to their contribution to the formation of high-order structures. This study also reveals a dynamic tRNA modification profile in the fast-proliferating cells. In addition to cultured cells, this unique tRNA profile has been further confirmed with endometrial cancers and their adjacent normal tissues. Taken together, the results indicate that tRNA is a actively regulated RNA species in the fast-proliferating cancer cells, and suggest that they may play a more active role in biological process than expected. -- Highlights: •RNA modifications were first examined in term of the major RNA species. •A dynamic tRNA modifications was characterized for the fast-proliferating cells. •The unique tRNA profile was confirmed with endometrial cancers and their adjacent normal tissues. •tRNA was predicted as an actively regulated RNA species in the fast-proliferating cancer cells.

  18. tRNA modification profiles of the fast-proliferating cancer cells

    International Nuclear Information System (INIS)

    Dong, Chao; Niu, Leilei; Song, Wei; Xiong, Xin; Zhang, Xianhua; Zhang, Zhenxi; Yang, Yi; Yi, Fan; Zhan, Jun; Zhang, Hongquan; Yang, Zhenjun; Zhang, Li-He; Zhai, Suodi; Li, Hua; Ye, Min; Du, Quan

    2016-01-01

    Despite the recent progress in RNA modification study, a comprehensive modification profile is still lacking for mammalian cells. Using a quantitative HPLC/MS/MS assay, we present here a study where RNA modifications are examined in term of the major RNA species. With paired slow- and fast-proliferating cell lines, distinct RNA modification profiles are first revealed for diverse RNA species. Compared to mRNAs, increased ribose and nucleobase modifications are shown for the highly-structured tRNAs and rRNAs, lending support to their contribution to the formation of high-order structures. This study also reveals a dynamic tRNA modification profile in the fast-proliferating cells. In addition to cultured cells, this unique tRNA profile has been further confirmed with endometrial cancers and their adjacent normal tissues. Taken together, the results indicate that tRNA is a actively regulated RNA species in the fast-proliferating cancer cells, and suggest that they may play a more active role in biological process than expected. -- Highlights: •RNA modifications were first examined in term of the major RNA species. •A dynamic tRNA modifications was characterized for the fast-proliferating cells. •The unique tRNA profile was confirmed with endometrial cancers and their adjacent normal tissues. •tRNA was predicted as an actively regulated RNA species in the fast-proliferating cancer cells.

  19. Chronic Inflammation and  T Cells

    Directory of Open Access Journals (Sweden)

    Nathan S Fay

    2016-05-01

    Full Text Available The epithelial tissues of the skin, lungs, reproductive tract, and intestines are the largest physical barriers the body has to protect against infection. Epithelial tissues are woven with a matrix of immune cells programmed to mobilize the host innate and adaptive immune responses. Included among these immune cells are  T cells that are unique in their TCR usage, location, and functions in the body. Stress reception by  T cells as a result of traumatic epithelial injury, malignancy, and/or infection induces  T cell activation. Once activated,  T cells function to repair tissue, induce inflammation, recruit leukocytes, and lyse cells. Many of these functions are mediated via the production of cytokines and growth factors upon  T cell activation. Pathogenesis of many chronic inflammatory diseases involve  T cells; some of which are exacerbated by their presence, while others are improved.  T cells require a delicate balance between their need for acute inflammatory mediators to function normally and the detrimental impact imparted by chronic inflammation. This review will focus on the recent progress made in understanding how epithelial  T cells influence the pathogenesis of chronic inflammatory diseases and how a balance between acute and chronic inflammation impacts  T cell function. Future studies will be important to understand how this balance is achieved.

  20. Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    International Nuclear Information System (INIS)

    Diaz, Jason; Wang, Xin; Tsang, Sabrina H.; Jiao, Jing; You, Jianxin

    2014-01-01

    Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells

  1. Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    Energy Technology Data Exchange (ETDEWEB)

    Diaz, Jason; Wang, Xin; Tsang, Sabrina H. [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States); Jiao, Jing [Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104 (United States); You, Jianxin, E-mail: jianyou@mail.med.upenn.edu [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States)

    2014-07-08

    Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells.

  2. Cell-free assay measuring repair DNA synthesis in human fibroblasts

    International Nuclear Information System (INIS)

    Ciarrocchi, G.; Linn, S.

    1978-01-01

    Osmotic disruption of confluent cultured human fibroblasts that have been irradiated or exposed to chemical carcinogens allows the specific measurement of repair DNA synthesis using dTTP as a precursor. Fibroblasts similarly prepared from various xeroderma pigmentosum cell lines show the deficiencies of uv-induced DNA synthesis predicted from in vivo studies, while giving normal responses to methylmethanesulfonate. A pyrimidine-dimer-specific enzyme, T4 endonuclease V, stimulated the rate of uv-induced repair synthesis with normal and xeroderma pigmentosum cell lines. This system should prove useful for identifying agents that induce DNA repair, and cells that respond abnormally to such induction. It should also be applicable to an in vitro complementation assay with repair-defective cells and proteins obtained from repair-proficient cells. Finally, by using actively growing fibroblasts and thymidine in the system, DNA replication can be measured and studied in vitro

  3. Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays

    International Nuclear Information System (INIS)

    Walter, M.N.M.; Wright, K.T.; Fuller, H.R.; MacNeil, S.; Johnson, W.E.B.

    2010-01-01

    We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-β1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.

  4. 224-T Transuranic Waste Storage and Assay Facility dangerous waste permit application

    International Nuclear Information System (INIS)

    1992-01-01

    Westinghouse Hanford Company is a major contractor to the US Department of Energy Richland Field Office and serves as cooperator of the 224-T Transuranic Waste Storage and Assay Facility, the storage unit addressed in this permit application. At the time of submission of this portion of the Hanford Facility. Dangerous Waste Permit Application covering the 224-T Transuranic Waste Storage and Assay Facility, many issues identified in comments to the draft Hanford Facility Dangerous Waste Permit remain unresolved. This permit application reflects the positions taken by the US Department of Energy, Company on the draft Hanford Facility Dangerous Waste Permit and may not be read to conflict with those comments. The 224-T Transuranic Waste Storage and Assay Facility Dangerous Waste Permit Application (Revision 0) consists of both a Part A and Part B permit application. An explanation of the Part A revisions associated with this unit, including the Part A revision currently in effect, is provided at the beginning of the Part A section. The Part B consists of 15 chapters addressing the organization and content of the Part B Checklist prepared by the Washington State Department of Ecology (Ecology 1987). The 224-T Transuranic Waste Storage and Assay Facility Dangerous Waste Permit Application contains information current as of March 1, 1992

  5. Regulation of the O-glycan-type Sialyl-Lewis X (sLex) Bio-synthesis Pathway during Cell Transformation Programs: Epithelial-Mesenchymal Transition (EMT) and Molecular Subtypes in Breast Carcinoma and Human T Cell Activation

    KAUST Repository

    AbuElela, Ayman

    2017-12-01

    the promoters of the GTs genes. Moreover, they showed differential binding of pioneer and nonpioneer transcription factors (TFs). We proposed a model for the regulation of FUT7 during T cell activation that relies on the interplay between chromatin-remodeling and cell-fate-specifying TFs. Furthermore, we developed a fluorescent multiplex cell rolling (FMCR) assay to study the cell adhesion properties under physiological conditions. Compared to the conventional parallel plate flow chamber (PPFC) assay, the novel technique posses a high-throughput capacity which helps eliminate the inter-experimental variation problem by running multiple samples simultaneously and under competitive settings. We also developed a real-time analysis pipeline that enhanced the statistical power of the assay. Overall these modifications to the traditional parallel plate assay improves the reliability and results along with saving time and effort.

  6. Validation and comparison of two NGS assays for the detection of EGFR T790M resistance mutation in liquid biopsies of NSCLC patients.

    Science.gov (United States)

    Vollbrecht, Claudia; Lehmann, Annika; Lenze, Dido; Hummel, Michael

    2018-04-06

    Analysis of circulating cell-free DNA (cfDNA) derived from peripheral blood ("liquid biopsy") is an attractive alternative to identify non-small cell lung cancer (NSCLC) patients with the EGFR T790M mutation eligible for 3rd generation tyrosine kinase inhibitor therapy. We evaluated two PCR-based next generation sequencing (NGS) approaches, one including unique molecular identifiers (UMI), with focus on highly sensitive EGFR T790M mutation detection. Therefore, we extracted and sequenced cfDNA from synthetic plasma samples spiked with mutated DNA at decreasing allele frequencies and from 21 diagnostic NSCLC patients. Data evaluation was performed to determine the limit of detection (LoD), accuracy, specificity and sensitivity of both assays. Considering all tested reference dilutions and mutations the UMI assay performed best in terms of LoD (1% vs. 5%), sensitivity (95.8% vs. 81.3%), specificity (100% vs. 93.8%) and accuracy (96.9% vs. 84.4%). Comparing mutation status of diagnostic samples with both assays showed 81.3% concordance with primary mutation verifiable in 52% of cases. EGFR T790M was detected concordantly in 6/7 patients with allele frequencies from 0.1% to 27%. In one patient, the T790M mutation was exclusively detectable with the UMI assay. Our data demonstrate that both assays are applicable as multi-biomarker NGS tools enabling the simultaneous detection of primary EGFR driver and resistance mutations. However, for mutations with low allelic frequencies the use of NGS panels with UMI facilitates a more sensitive and reliable detection.

  7. Activated CD4+ T cells enhance radiation effect through the cooperation of interferon-γ and TNF-α

    International Nuclear Information System (INIS)

    Wang, Yixiang; Radfar, Soroosh; Khong, Hung T

    2010-01-01

    Approaches that enhance radiation effect may lead to improved clinical outcome and decrease toxicity. Here we investigated whether activated CD4+ T cells (aCD4) can serve as an effective radiosensitizer. CD4+ T cells were activated with anti-CD3 and anti-CD28 mAbs. Hela cells were presensitized with aCD4 or conditioned supernatant (aCD4S) or recombinant cytokines for 2 days, followed γ-irradiation. The treated cells were cultured for an additional 2 to 5 days for cell proliferation, cell cycle, and western blot assays. For confirmation, other cancer cell lines were also used. Presensitization of tumor cells with aCD4 greatly increased tumor cell growth inhibition. Soluble factors secreted from activated CD4 + T cells were primarily responsible for the observed effect. IFN-γ seemed to play a major role. TNF-α, though inactive by itself, significantly augmented the radiosensitizing activity of IFN-γ. aCD4S, but not IFN-γ or IFN-γ/TNF-α combination, was found to enhance the γ-irradiation-induced G2/M phase arrest. Bax expression was highly upregulated in Hela cells presensitized with aCD4S followed by γ-irradiation. The radio-sensitizing activity of aCD4 is not uniquely observed with Hela cell line, but also seen with other cancer cell lines of various histology. Our findings suggest possible molecular and cellular mechanisms that may help explain the radio-sensitization effect of activated lymphocytes, and may provide an improved strategy in the treatment of cancer with radiotherapy

  8. Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia.

    Science.gov (United States)

    Fraietta, Joseph A; Beckwith, Kyle A; Patel, Prachi R; Ruella, Marco; Zheng, Zhaohui; Barrett, David M; Lacey, Simon F; Melenhorst, Jan Joseph; McGettigan, Shannon E; Cook, Danielle R; Zhang, Changfeng; Xu, Jun; Do, Priscilla; Hulitt, Jessica; Kudchodkar, Sagar B; Cogdill, Alexandria P; Gill, Saar; Porter, David L; Woyach, Jennifer A; Long, Meixiao; Johnson, Amy J; Maddocks, Kami; Muthusamy, Natarajan; Levine, Bruce L; June, Carl H; Byrd, John C; Maus, Marcela V

    2016-03-03

    Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is highly promising but requires robust T-cell expansion and engraftment. A T-cell defect in chronic lymphocytic leukemia (CLL) due to disease and/or therapy impairs ex vivo expansion and response to CAR T cells. To evaluate the effect of ibrutinib treatment on the T-cell compartment in CLL as it relates to CAR T-cell generation, we examined the phenotype and function of T cells in a cohort of CLL patients during their course of treatment with ibrutinib. We found that ≥5 cycles of ibrutinib therapy improved the expansion of CD19-directed CAR T cells (CTL019), in association with decreased expression of the immunosuppressive molecule programmed cell death 1 on T cells and of CD200 on B-CLL cells. In support of these findings, we observed that 3 CLL patients who had been treated with ibrutinib for ≥1 year at the time of T-cell collection had improved ex vivo and in vivo CTL019 expansion, which correlated positively together and with clinical response. Lastly, we show that ibrutinib exposure does not impair CAR T-cell function in vitro but does improve CAR T-cell engraftment, tumor clearance, and survival in human xenograft models of resistant acute lymphocytic leukemia and CLL when administered concurrently. Our collective findings indicate that ibrutinib enhances CAR T-cell function and suggest that clinical trials with combination therapy are warranted. Our studies demonstrate that improved T-cell function may also contribute to the efficacy of ibrutinib in CLL. These trials were registered at www.clinicaltrials.gov as #NCT01747486, #NCT01105247, and #NCT01217749. © 2016 by The American Society of Hematology.

  9. Epigenetic control of CD8+ T cell differentiation.

    Science.gov (United States)

    Henning, Amanda N; Roychoudhuri, Rahul; Restifo, Nicholas P

    2018-05-01

    Upon stimulation, small numbers of naive CD8 + T cells proliferate and differentiate into a variety of memory and effector cell types. CD8 + T cells can persist for years and kill tumour cells and virally infected cells. The functional and phenotypic changes that occur during CD8 + T cell differentiation are well characterized, but the epigenetic states that underlie these changes are incompletely understood. Here, we review the epigenetic processes that direct CD8 + T cell differentiation and function. We focus on epigenetic modification of DNA and associated histones at genes and their regulatory elements. We also describe structural changes in chromatin organization that affect gene expression. Finally, we examine the translational potential of epigenetic interventions to improve CD8 + T cell function in individuals with chronic infections and cancer.

  10. Palindromic nucleotide analysis in human T cell receptor rearrangements.

    Directory of Open Access Journals (Sweden)

    Santosh K Srivastava

    Full Text Available Diversity of T cell receptor (TCR genes is primarily generated by nucleotide insertions upon rearrangement from their germ line-encoded V, D and J segments. Nucleotide insertions at V-D and D-J junctions are random, but some small subsets of these insertions are exceptional, in that one to three base pairs inversely repeat the sequence of the germline DNA. These short complementary palindromic sequences are called P nucleotides. We apply the ImmunoSeq deep-sequencing assay to the third complementarity determining region (CDR3 of the β chain of T cell receptors, and use the resulting data to study P nucleotides in the repertoire of naïve and memory CD8(+ and CD4(+ T cells. We estimate P nucleotide distributions in a cross section of healthy adults and different T cell subtypes. We show that P nucleotide frequency in all T cell subtypes ranges from 1% to 2%, and that the distribution is highly biased with respect to the coding end of the gene segment. Classification of observed palindromic sequences into P nucleotides using a maximum conditional probability model shows that single base P nucleotides are very rare in VDJ recombination; P nucleotides are primarily two bases long. To explore the role of P nucleotides in thymic selection, we compare P nucleotides in productive and non-productive sequences of CD8(+ naïve T cells. The naïve CD8(+ T cell clones with P nucleotides are more highly expanded.

  11. Ulinastatin Reduces T Cell Apoptosis in Rats with Severe Acute ...

    African Journals Online (AJOL)

    T cell apoptosis was determined by Annexin-V/PI double-staining. Oxidative stress was evaluated by examining changes in the levels of reactive oxygen species (ROS). Total superoxide dismutase (SOD) in serum was tested by hydroxylamine colorimetric assay, and malondialdehyde levels were examined by thiobarbituric ...

  12. Discovery of Highly Potent Tyrosinase Inhibitor, T1, with Significant Anti-Melanogenesis Ability by zebrafish in vivo Assay and Computational Molecular Modeling

    Science.gov (United States)

    Chen, Wang-Chuan; Tseng, Tien-Sheng; Hsiao, Nai-Wan; Lin, Yun-Lian; Wen, Zhi-Hong; Tsai, Chin-Chuan; Lee, Yu-Ching; Lin, Hui-Hsiung; Tsai, Keng-Chang

    2015-01-01

    Tyrosinase is involved in melanin biosynthesis and the abnormal accumulation of melanin pigments leading to hyperpigmentation disorders that can be treated with depigmenting agents. A natural product T1, bis(4-hydroxybenzyl)sulfide, isolated from the Chinese herbal plant, Gastrodia elata, is a strong competitive inhibitor against mushroom tyrosinase (IC50 = 0.53 μM, Ki = 58 +/- 6 nM), outperforms than kojic acid. The cell viability and melanin quantification assay demonstrate that 50 μM of T1 apparently attenuates 20% melanin content of human normal melanocytes without significant cell toxicity. Moreover, the zebrafish in vivo assay reveals that T1 effectively reduces melanogenesis with no adverse side effects. The acute oral toxicity study evidently confirms that T1 molecule is free of discernable cytotoxicity in mice. Furthermore, the molecular modeling demonstrates that the sulfur atom of T1 coordinating with the copper ions in the active site of tyrosinase is essential for mushroom tyrosinase inhibition and the ability of diminishing the human melanin synthesis. These results evident that T1 isolated from Gastrodia elata is a promising candidate in developing pharmacological and cosmetic agents of great potency in skin-whitening.

  13. Meningeal mast cell-T cell crosstalk regulates T cell encephalitogenicity.

    Science.gov (United States)

    Russi, Abigail E; Walker-Caulfield, Margaret E; Guo, Yong; Lucchinetti, Claudia F; Brown, Melissa A

    2016-09-01

    GM-CSF is a cytokine produced by T helper (Th) cells that plays an essential role in orchestrating neuroinflammation in experimental autoimmune encephalomyelitis, a rodent model of multiple sclerosis. Yet where and how Th cells acquire GM-CSF expression is unknown. In this study we identify mast cells in the meninges, tripartite tissues surrounding the brain and spinal cord, as important contributors to antigen-specific Th cell accumulation and GM-CSF expression. In the absence of mast cells, Th cells do not accumulate in the meninges nor produce GM-CSF. Mast cell-T cell co-culture experiments and selective mast cell reconstitution of the meninges of mast cell-deficient mice reveal that resident meningeal mast cells are an early source of caspase-1-dependent IL-1β that licenses Th cells to produce GM-CSF and become encephalitogenic. We also provide evidence of mast cell-T cell co-localization in the meninges and CNS of recently diagnosed acute MS patients indicating similar interactions may occur in human demyelinating disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. A novel microculture kinetic assay (MiCK assay) for malignant cell growth and chemosensitivity.

    Science.gov (United States)

    Kravtsov, V D

    1994-01-01

    The THERMOmax microplate reader was adapted for monitoring the growth kinetics of human leukaemic OCI/AML-2 and mouse tumour J-774.1 cell lines in continuous culture. Fluid evaporation from wells, CO2 escape and contamination were prevented by hermetic sealing of the microcultures in wells of a 96-well microplate, thus enabling the cells to grow exponentially for 72 h under the conditions of the incubated microplate reader. For both OCI/AML-2 cells, which grow in suspension, and adherent J-774.1 cells, a linear correlation was demonstrated between the number of unstained cells seeded in a given microplate well and the optical density (OD) of that well. Therefore, the OD/time curve of the culture could be deemed to be its growth curve. By the use of the linear fit equation, the actual number of the cells in the wells was computable at any time point of the assay. In the chemosensitivity test, an inhibitory effect of ARA-C on the growth of the cells could be estimated by viewing of the growth curves plotted on the screen. The maximum kinetic rates (Vmax) of the curves in the control and the ARA-C-treated wells were compared, yielding a growth inhibition index (GII). Comparison of results of the kinetic chemosensitivity assay with those of a [3H]thymidine incorporation assay revealed that the novel assay is suitable for precise quantitation of the cell chemosensitivity, is more informative and has the added technical advantage of performance without recourse to radioactive or chemically hazardous substances.

  15. Human cord blood suppressor T lymphocytes. II. Characterization of inducer of suppressor cells

    International Nuclear Information System (INIS)

    Cheng, H.; Delespesse, G.

    1986-01-01

    Previously, we reported an antigen nonspecific inducer of T suppressor cell factor (TisF) produced by cord blood mononuclear cells (MNC) in 48-hr, two-way mixed lymphocyte cultures (MLC). The target of this factor was a radiosensitive, T4+ (T8-) adult suppressor T cell subset. The cellular origin of this TisF was examined in the present study. IgG production by pokeweed mitogen (PWM)-stimulated adult MNC was used as an assay for TisF activity. It was found that TisF-producing cells formed rosettes with sheep erythrocytes (E+) and were independent of adherent cells (AC) in the production of TisF. They were resistant to irradiation (2500 rads) and phenotypic characterization with T cell reactive monoclonal antibodies indicated that they resided in the T8- (T4+) population. Furthermore, both TQ1- and TQ1+ cells were required for the production of TisF activity and such activity could not be reconstituted by supernatants from TQ1- MLC and TQ1+ MLC. These results indicate that the production of TisF is dependent upon interactions between radioresistant E+, T8-, TQ1- and radioresistant E+, T8-, TQ1+ cells

  16. Routine detection of Epstein-Barr virus specific T-cells in the peripheral blood by flow cytometry

    Science.gov (United States)

    Crucian, B. E.; Stowe, R. P.; Pierson, D. L.; Sams, C. F.

    2001-01-01

    The ability to detect cytomegalovirus-specific T-cells (CD4(+)) in the peripheral blood by flow cytometry has been recently described by Picker et al. In this method, cells are incubated with viral antigen and responding (cytokine producing) T-cells are then identified by flow cytometry. To date, this technique has not been reliably used to detect Epstein-Barr virus (EBV)-specific T-cells primarily due to the superantigen/mitogenic properties of the virus which non-specifically activate T-cells. By modifying culture conditions under which the antigens are presented, we have overcome this limitation and developed an assay to detect and quantitate EBV-specific T-cells. The detection of cytokine producing T-cells by flow cytometry requires an extremely strong signal (such as culture in the presence of PMA and ionomycin). Our data indicate that in modified culture conditions (early removal of viral antigen) the non-specific activation of T-cells by EBV is reduced, but antigen presentation will continue uninhibited. Using this method, EBV-specific T-cells may be legitimately detected using flow cytometry. No reduction in the numbers of antigen-specific T-cells was observed by the early removal of target antigen when verified using cytomegalovirus antigen (a virus with no non-specific T-cell activation properties). In EBV-seropositive individuals, the phenotype of the EBV-specific cytokine producing T-cells was evaluated using four-color flow cytometry and found to be CD45(+), CD3(+), CD4(+), CD45RA(-), CD69(+), CD25(-). This phenotype indicates the stimulation of circulating previously unactivated memory T-cells. No cytokine production was observed in CD4(+) T-cells from EBV-seronegative individuals, confirming the specificity of this assay. In addition, the use of four color cytometry (CD45, CD3, CD69, IFNgamma/IL-2) allows the total quantitative assessment of EBV-specific T-cells while monitoring the interference of EBV non-specific mitogenic activity. This method may

  17. Transporters for Antiretroviral Drugs in Colorectal CD4+ T Cells and Circulating α4β7 Integrin CD4+ T Cells: Implications for HIV Microbicides.

    Science.gov (United States)

    Mukhopadhya, Indrani; Murray, Graeme I; Duncan, Linda; Yuecel, Raif; Shattock, Robin; Kelly, Charles; Iannelli, Francesco; Pozzi, Gianni; El-Omar, Emad M; Hold, Georgina L; Hijazi, Karolin

    2016-09-06

    CD4+ T lymphocytes in the colorectal mucosa are key in HIV-1 transmission and dissemination. As such they are also the primary target for antiretroviral (ARV)-based rectal microbicides for pre-exposure prophylaxis. Drug transporters expressed in mucosal CD4+ T cells determine ARV distribution across the cell membrane and, most likely, efficacy of microbicides. We describe transporters for antiretroviral drugs in colorectal mucosal CD4+ T lymphocytes and compare gene expression with circulating α4β7+CD4+ T cells, which traffic to the intestine and have been shown to be preferentially infected by HIV-1. Purified total CD4+ T cells were obtained from colorectal tissue and blood samples by magnetic separation. CD4+ T cells expressing α4β7 integrin were isolated by fluorescence-activated cell sorting from peripheral blood mononuclear cells of healthy volunteers. Expressions of 15 efflux and uptake drug transporter genes were quantified using Taqman qPCR assays. Expression of efflux transporters MRP3, MRP5, and BCRP and uptake transporter CNT2 were significantly higher in colorectal CD4+ T cells compared to circulating CD4+ T cells (p = 0.01-0.03). Conversely, circulating α4β7+CD4+ T cells demonstrated significantly higher expression of OATPD compared to colorectal CD4+ T cells (p = 0.001). To the best of our knowledge this is the first report of drug transporter gene expression in colorectal CD4+ and peripheral α4β7+CD4+ T cells. The qualitative and quantitative differences in drug transporter gene expression profiles between α4β7+CD4+ T cells and total mucosal CD4+ T cells may have significant implications for the efficacy of rectally delivered ARV-microbicides. Most notably, we have identified efflux drug transporters that could be targeted by selective inhibitors or beneficial drug-drug interactions to enhance intracellular accumulation of antiretroviral drugs.

  18. Pha-induced T-cell cytotoxity. Mechanism and application in haemodialysis and renal transplant patients.

    NARCIS (Netherlands)

    Huges-Wirawan, Gladys Ratna Widhi Indrati

    1978-01-01

    This thesis describes a method to measure PHA-incluced cytotoxicity of human lymphocytes (nonspecific T-cell cytotoxicity), using 3H-thymidine prelabelled target cells (HeLa cells). The method has some advantages over the widely used 51Cr-release assay. Its application in two clinical conditions is

  19. Task-shifting of CD4 T cell count monitoring by the touchscreen-based Muse™ Auto CD4/CD4% single-platform system for CD4 T cell numeration: Implication for decentralization in resource-constrained settings.

    Science.gov (United States)

    Kouabosso, André; Mossoro-Kpinde, Christian Diamant; Bouassa, Ralph-Sydney Mboumba; Longo, Jean De Dieu; Mbeko Simaleko, Marcel; Grésenguet, Gérard; Bélec, Laurent

    2018-04-01

    The accuracy of CD4 T cell monitoring by the recently developed flow cytometry-based CD4 T cell counting Muse™ Auto CD4/CD4% Assay analyzer (EMD Millipore Corporation, Merck Life Sciences, KGaA, Darmstadt, Germany) was evaluated in trained lay providers against laboratory technicians. After 2 days of training on the Muse™ Auto CD4/CD4% analyzer, EDTA-blood samples from 6 HIV-positive and 4 HIV-negative individuals were used for CD4 T cell counting in triplicate in parallel by 12 trained lay providers as compared to 10 lab technicians. Mean number of CD4 T cells in absolute number was 829 ± 380 cells/μl by lay providers and 794 ± 409 cells/μl by technicians (P > 0.05); and in percentage 36.2 ± 14.8%CD4 by lay providers and 36.1 ± 15.0%CD4 by laboratory technician (P > 0.05). The unweighted linear regression and Passing-Bablok regression analyses on CD4 T cell results expressed in absolute count revealed moderate correlation between CD4 T cell counts obtained by lay providers and lab technicians. The mean absolute bias measured by Bland-Altman analysis between CD4 T cell/μl obtained by lay providers and lab technicians was -3.41 cells/μl. Intra-assay coefficient of variance (CV) of Muse™ Auto CD4/CD4% in absolute number was 10.1% by lay providers and 8.5% by lab technicians (P > 0.05), and in percentage 5.5% by lay providers and 4.4% by lab technicians (P > 0.05). The inter-assay CV of Muse™ Auto CD4/CD4% in absolute number was 13.4% by lay providers and 10.3% by lab technicians (P > 0.05), and in percentage 7.8% by lay providers and 6.9% by lab technicians (P > 0.05). The study demonstrates the feasibility of CD4 T cell counting using the alternative flow cytometer Muse™ Auto CD4/CD4% analyzer by trained lay providers and therefore the practical possibility of decentralization CD4 T cell counting to health community centers. Copyright © 2018. Published by Elsevier B.V.

  20. Low interleukin-2 concentration favors generation of early memory T cells over effector phenotypes during chimeric antigen receptor T-cell expansion.

    Science.gov (United States)

    Kaartinen, Tanja; Luostarinen, Annu; Maliniemi, Pilvi; Keto, Joni; Arvas, Mikko; Belt, Heini; Koponen, Jonna; Loskog, Angelica; Mustjoki, Satu; Porkka, Kimmo; Ylä-Herttuala, Seppo; Korhonen, Matti

    2017-06-01

    Adoptive T-cell therapy offers new options for cancer treatment. Clinical results suggest that T-cell persistence, depending on T-cell memory, improves efficacy. The use of interleukin (IL)-2 for in vitro T-cell expansion is not straightforward because it drives effector T-cell differentiation but does not promote the formation of T-cell memory. We have developed a cost-effective expansion protocol for chimeric antigen receptor (CAR) T cells with an early memory phenotype. Lymphocytes were transduced with third-generation lentiviral vectors and expanded using CD3/CD28 microbeads. The effects of altering the IL-2 supplementation (0-300 IU/mL) and length of expansion (10-20 days) on the phenotype of the T-cell products were analyzed. High IL-2 levels led to a decrease in overall generation of early memory T cells by both decreasing central memory T cells and augmenting effectors. T memory stem cells (T SCM , CD95 + CD45RO - CD45RA + CD27 + ) were present variably during T-cell expansion. However, their presence was not IL-2 dependent but was linked to expansion kinetics. CD19-CAR T cells generated in these conditions displayed in vitro antileukemic activity. In summary, production of CAR T cells without any cytokine supplementation yielded the highest proportion of early memory T cells, provided a 10-fold cell expansion and the cells were functionally potent. The number of early memory T cells in a T-cell preparation can be increased by simply reducing the amount of IL-2 and limiting the length of T-cell expansion, providing cells with potentially higher in vivo performance. These findings are significant for robust and cost-effective T-cell manufacturing. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  1. Human adipose tissue-derived mesenchymal stem cells inhibit T-cell lymphoma growth in vitro and in vivo.

    Science.gov (United States)

    Ahn, Jin-Ok; Chae, Ji-Sang; Coh, Ye-Rin; Jung, Woo-Sung; Lee, Hee-Woo; Shin, Il-Seob; Kang, Sung-Keun; Youn, Hwa-Young

    2014-09-01

    Human mesenchymal stem cells (hMSCs) are thought to be one of the most reliable stem cell sources for a variety of cell therapies. This study investigated the anti-tumor effect of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) on EL4 murine T-cell lymphoma in vitro and in vivo. The growth-inhibitory effect of hAT-MSCs on EL4 tumor cells was evaluated using a WST-1 cell proliferation assay. Cell-cycle arrest and apoptosis were investigated by flow cytometry and western blot. To evaluate an anti-tumor effect of hAT-MSCs on T-cell lymphoma in vivo, CM-DiI-labeled hAT-MSCs were circumtumorally injected in tumor-bearing nude mice, and tumor size was measured. hAT-MSCs inhibited T-cell lymphoma growth by altering cell-cycle progression and inducing apoptosis in vitro. hAT-MSCs inhibited tumor growth in tumor-bearing nude mice and prolonged survival time. Immunofluorescence analysis showed that hAT-MSCs migrated to tumor sites. hAT-MSCs suppress the growth of T-cell lymphoma, suggesting a therapeutic option for T-cell lymphoma. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  2. Cellular prion protein and γ-synuclein overexpression in LS 174T colorectal cancer cell drives endothelial proliferation-to-differentiation switch

    Directory of Open Access Journals (Sweden)

    Sing-Hui Ong

    2018-03-01

    Full Text Available Background Tumor-induced angiogenesis is an imperative event in pledging new vasculature for tumor metastasis. Since overexpression of neuronal proteins gamma-synuclein (γ-Syn and cellular prion protein (PrPC is always detected in advanced stages of cancer diseases which involve metastasis, this study aimed to investigate whether γ-Syn or PrPC overexpression in colorectal adenocarcinoma, LS 174T cells affects angiogenesis of endothelial cells, EA.hy 926 (EA. Methods EA cells were treated with conditioned media (CM of LS 174T-γ-Syn or LS 174T-PrP, and their proliferation, invasion, migration, adhesion and ability to form angiogenic tubes were assessed using a range of biological assays. To investigate plausible background mechanisms in conferring the properties of EA cells above, nitrite oxide (NO levels were measured and the expression of angiogenesis-related factors was assessed using a human angiogenesis antibody array. Results EA proliferation was significantly inhibited by LS 174T-PrP CM whereas its telomerase activity was reduced by CM of LS 174T-γ-Syn or LS 174T-PrP, as compared to EA incubated with LS 174T CM. Besides, LS 174T-γ-Syn CM or LS 174T-PrP CM inhibited EA invasion and migration in Boyden chamber assay. Furthermore, LS 174T-γ-Syn CM significantly inhibited EA migration in scratch wound assay. Gelatin zymography revealed reduced secretion of MMP-2 and MMP-9 by EA treated with LS 174T-γ-Syn CM or LS 174T-PrP CM. In addition, cell adhesion assay showed lesser LS 174T-γ-Syn or LS 174T-PrP cells adhered onto EA, as compared to LS 174T. In tube formation assay, LS 174T-γ-Syn CM or LS 174T-PrP CM induced EA tube formation. Increased NO secretion by EA treated with LS 174T-γ-Syn CM or LS 174T-PrP CM was also detected. Lastly, decreased expression of pro-angiogenic factors like CXCL16, IGFBP-2 and amphiregulin in LS 174T-γ-Syn CM or LS 174T-PrP CM was detected using the angiogenesis antibody array. Discussion These results

  3. Inhibition of tumor growth in syngenetic chimeric mice mediated by a depletion of suppressor T cells

    International Nuclear Information System (INIS)

    Rotter, V.; Trainin, N.

    1975-01-01

    Syngeneic chimeric (lethally irradiated and reconstituted with syngeneic bone marrow cells) mice manifested an increased resistance to the development of Lewis lung carcinoma. In addition, these mice had a higher response to polyvinylpyrrolidone and a reduced reactivity to T mitogens. The present findings suggest that syngeneic chimeric mice lack suppressor T cells shown to regulate the development of Lewis lung tumor and the response to polyvinylpyrrolidone. Other components of the T cell population, such as helper cells responding to sheep red blood cells or cells involved in allograft rejection, assayed in these syngeneic chimeras were found unaffected. The fact that chimeric mice are deficient in a certain suppressor T cell population whereas other T activities are normal suggests the existence of different cell lines within the T cell population. (U.S.)

  4. Human immunodeficiency virus long terminal repeat responds to T-cell activation signals

    International Nuclear Information System (INIS)

    Tong-Starksen, S.E.; Luciw, P.A.; Peterlin, B.M.

    1987-01-01

    Human immunodeficiency virus (HIV), the causative agent of AIDS, infects and kills lymphoid cells bearing the CD4 antigen. In an infected cell, a number of cellular as well as HIV-encoded gene products determine the levels of viral gene expression and HIV replication. Efficient HIV replication occurs in activated T cells. Utilizing transient expression assays, the authors show that gene expression directed by the HIV long terminal repeat (LTR) increases in response to T-cell activation signals. The effects of T-cell activation and of the HIV-encoded trans-activator (TAT) are multiplicative. Analysis of mutations and deletions in the HIV LTR reveals that the region responding to T-cell activation signals is located at positions -105 to -80. These sequences are composed of two direct repeats, which are homologous to the core transcriptional enhancer elements in the simian virus 40 genome. The studies reveal that these elements function as the HIV enhancer. By acting directly on the HIV LTR, T-cell activation may play an important role in HIV gene expression and in the activation of latent HIV

  5. Attachment and spreadout study of 3T3 cells onto PP track etched films

    International Nuclear Information System (INIS)

    Smolko, Eduardo; Mazzei, Ruben; Tadey, Daniel; Lombardo, Daniel

    2001-01-01

    Polymer surface modifications are obtained by the application of radiation treatments and other physico-chemical methods: fission fragment (ff) irradiation and etching. The biocompatibility of the surface is then observed by cell seeding and cell adhesion experiments. Approaches to improvement of the cell adhesion are obtained by different methods: for example, in PS, cell adhesion is improved after ion implantation; in PMMA, after bombarding the polymer, the surface is reconditioned with surfactants and proteins and in PVDF, cell adhesion is assayed on nuclear tracks membranes. In this work, we obtained important cell adhesion improvements in PP films by irradiation with swift heavy ions and subsequent etching of the nuclear tracks. We use BOPP (isotactic -25 μm thickness). Irrradiations were performed with a Cf-252 californium ff source. The source has a heavy ff and a light one, with 160-200 MeV energy divided among them corresponding to ff energies between 1 and 2 MeV/amu. A chemical etching procedure consisting of a solution of sulphuric acid and chromium three oxide at 85 deg. C was used. The 3T3 NIH fibroblast cell line was used for the cell adhesion experiment. Here we report for the first time, the results of a series of experiments by varying the ff fluence and the etching time showing that attachment and spreadout of cells are very much improved in this cell line according to the number of pores and the pore size

  6. Changing T cell specificity by retroviral T cell receptor display

    NARCIS (Netherlands)

    Kessels, H. W.; van den Boom, M. D.; Spits, H.; Hooijberg, E.; Schumacher, T. N.

    2000-01-01

    The diversity of the T cell receptor (TCR) repertoire is limited, because of the processes of positive and negative T cell selection. To obtain T cells with specificities beyond the immune system's capacity, we have developed a strategy for retroviral TCR display. In this approach, a library of T

  7. Imbalance between IL-17A-Producing Cells and Regulatory T Cells during Ischemic Stroke

    Directory of Open Access Journals (Sweden)

    Yuehua Hu

    2014-01-01

    Full Text Available Immune responses and inflammation are key elements in the pathogenesis of ischemic stroke (IS. Although the involvement of IL-17A in IS has been demonstrated using animal models, the involvement of IL-17A and IL-17-secreting T cell subsets in IS patients has not been verified, and whether the balance of Treg/IL-17-secreting T cells is altered in IS patients remains unknown. In the present study, we demonstrated that the proportion of peripheral Tregs and the levels of IL-10 and TGF-β were reduced in patients with IS compared with controls using flow cytometry (FCM, real-time PCR, and ELISA assays. However, the proportions of Th17 and γδ T cells, the primary IL-17A-secreting cells, increased dramatically, and these effects were accompanied by increases in the levels of IL-17A, IL-23, IL-6, and IL-1β in IS patients. These studies suggest that the increase in IL-17A-producing cells and decrease in Treg cells might contribute to the pathogenesis of IS. Manipulating the balance between Tregs and IL-17A-producing cells might be helpful for the treatment of IS.

  8. Improving immunological tumor microenvironment using electro-hyperthermia followed by dendritic cell immunotherapy

    International Nuclear Information System (INIS)

    Tsang, Yuk-Wah; Huang, Cheng-Chung; Yang, Kai-Lin; Chi, Mau-Shin; Chiang, Hsin-Chien; Wang, Yu-Shan; Andocs, Gabor; Szasz, Andras; Li, Wen-Tyng; Chi, Kwan-Hwa

    2015-01-01

    The treatment of intratumoral dentritic cells (DCs) commonly fails because it cannot evoke immunity in a poor tumor microenvironment (TME). Modulated electro-hyperthermia (mEHT, trade-name: oncothermia) represents a significant technological advancement in the hyperthermia field, allowing the autofocusing of electromagnetic power on a cell membrane to generate massive apoptosis. This approach turns local immunogenic cancer cell death (apoptosis) into a systemic anti-tumor immune response and may be implemented by treatment with intratumoral DCs. The CT26 murine colorectal cancer model was used in this investigation. The inhibition of growth of the tumor and the systemic anti-tumor immune response were measured. The tumor was heated to a core temperature of 42 °C for 30 min. The matured synergetic DCs were intratumorally injected 24 h following mEHT was applied. mEHT induced significant apoptosis and enhanced the release of heat shock protein70 (Hsp70) in CT26 tumors. Treatment with mEHT-DCs significantly inhibited CT26 tumor growth, relative to DCs alone or mEHT alone. The secondary tumor protection effect upon rechallenging was observed in mice that were treated with mEHT-DCs. Immunohistochemical staining of CD45 and F4/80 revealed that mEHT-DC treatment increased the number of leukocytes and macrophages. Most interestingly, mEHT also induced infiltrations of eosinophil, which has recently been reported to be an orchestrator of a specific T cell response. Cytotoxic T cell assay and ELISpot assay revealed a tumor-specific T cell activity. This study demonstrated that mEHT induces tumor cell apoptosis and enhances the release of Hsp70 from heated tumor cells, unlike conventional hyperthermia. mEHT can create a favorable tumor microenvironment for an immunological chain reaction that improves the success rate of intratumoral DC immunotherapy. The online version of this article (doi:10.1186/s12885-015-1690-2) contains supplementary material, which is available to

  9. A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts

    Directory of Open Access Journals (Sweden)

    Bendinelli Mauro

    2009-03-01

    Full Text Available Abstract Background Cytotoxic T lymphocytes play a crucial role in the immunological control of microbial infections and in the design of vaccines and immunotherapies. Measurement of cytotoxic T lymphocyte activity requires that the test antigen is presented by target cells having the same or compatible class I major hystocompatibility complex antigens as the effector cells. Conventional assays use target cells labeled with 51chromium and infer cytotoxic T lymphocyte activity by measuring the isotope released by the target cells lysed following incubation with antigen-specific cytotoxic T lymphocytes. This assay is sensitive but needs manipulation and disposal of hazardous radioactive reagents and provides a bulk estimate of the reporter released, which may be influenced by spontaneous release of the label and other poorly controllable variables. Here we describe a novel method for producing target in outbred hosts and assessing cytotoxic T lymphocyte activity by flow cytometry. Results The method consists of culturing skin fibroblasts, immortalizing them with a replication defective clone of simian virus 40, and finally transducing them with a bicistronic vector encoding the target antigen and the reporter green fluorescent protein. When used in a flow cytometry-based assay, the target cells obtained with this method proved valuable for assessing the viral envelope protein specific cytotoxic T lymphocyte activity in domestic cats acutely or chronically infected with feline immunodeficiency virus, a lentivirus similar to human immunodeficiency virus and used as animal model for AIDS studies. Conclusion Given the versatility of the bicistronic vector used, its ability to deliver multiple and large transgenes in target cells, and its extremely wide cell specificity when pseudotyped with the vesicular stomatitis virus envelope protein, the method is potentially exploitable in many animal species.

  10. Measuring Bioenergetics in T Cells Using a Seahorse Extracellular Flux Analyzer

    NARCIS (Netherlands)

    van der Windt, Gerritje J. W.; Chang, Chih-Hao; Pearce, Erika L.

    2016-01-01

    This unit contains several protocols to determine the energy utilization of T cells in real-time using a Seahorse Extracellular Flux Analyzer (http://www.seahorsebio.com). The advantages to using this machine over traditional metabolic assays include the simultaneous measurement of glycolysis and

  11. Virally inactivated human platelet concentrate lysate induces regulatory T cells and immunosuppressive effect in a murine asthma model.

    Science.gov (United States)

    Lee, Yueh-Lun; Lee, Lin-Wen; Su, Chen-Yao; Hsiao, George; Yang, Yi-Yuan; Leu, Sy-Jye; Shieh, Ying-Hua; Burnouf, Thierry

    2013-09-01

    Platelet concentrate lysates (PCLs) are increasingly used in regenerative medicine. We have developed a solvent/detergent (S/D)-treated PCL. The functional properties of this preparation should be unveiled. We hypothesized that, due to transforming growth factor-β1 (TGF-β1) content, PCLs may exert immunosuppressive and anti-inflammatory functions. PCL was prepared by S/D treatment, oil extraction, and hydrophobic interaction chromatography. The content of TGF-β in PCL was determined by enzyme-linked immunosorbent assay. Cultured CD4+ T cells were used to investigate the effects of PCL on expression of transcription factor forkhead box P3 (Foxp3), the inhibition of T-cell proliferation, and cytokine production. The regulatory function of PCL-converted CD4+ T cells was analyzed by suppressive assay. The BALB/c mice were given PCL-converted CD4+ T cells before ovalbumin (OVA) sensitization and challenge using an asthma model. Inflammatory parameters, such as the level of immunoglobulin E (IgE), airway hyperresponsiveness (AHR), bronchial lavage fluid eosinophils, and cytokines were assayed. Recombinant human (rHu) TGF-β1 was used as control. PCL significantly enhanced the development of CD4+Foxp3+-induced regulatory T cells (iTregs). Converted iTregs produced neither Th1 nor Th2 cytokines and inhibited normal T-cell proliferation. PCL- and rHuTGF-β-converted CD4+ T cells prevented OVA-induced asthma. PCL- and rHuTGF-β-modified T cells both significantly reduced expression levels of OVA-specific IgE and significantly inhibited the development of AHR, airway eosinophilia, and Th2 responses in mice. S/D-treated PCL promotes Foxp3+ iTregs and exerts immunosuppressive and anti-inflammatory properties. This finding may help to understand the clinical properties of platelet lysates. © 2013 American Association of Blood Banks.

  12. CYTOKINESIS-BLOCK MICRONUCLEUS ASSAY IN HUMAN GLIOMA CELLS EXPOSED TO RADIATION

    Directory of Open Access Journals (Sweden)

    Jerzy Slowinski

    2011-05-01

    Full Text Available Biological tests are efficient in reflecting the biological influences of several types of generally harmful exposures. The micronucleus assay is widely used in genotoxicity studies or studies on genomic damage in general. We present methodological aspects of cytokinesis-block micronucleus assay performed in human gliomas irradiated in vitro. Eight human glioblastoma cell lines obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany were gamma-irradiated (60Co over a dose range of 0-10 Gy. Cytokinesis-block micronucleus assay was performed to quantitate cytogenetic damage. The cells were fixed directly on dishes, stained with fluorochrome DAPI and evaluated under fluorescent and phase contrast microscope. The micronucleus frequency was expressed as a micronuclei (MN per binucleated cell (BNC ratio, calculated after scoring at least 100 BNC per dish. The frequency of spontaneous MN ranged from 0.17 to 0.613 (mean: 0.29 ± 0.14. After irradiation increase of MN frequency in the range of 0.312 - 2.241 (mean: 0.98 ± 0.68 was found at 10 Gy. Gliomas are extremely heterogenous in regard to cytogenetic effects of irradiation, as shown in this study by cytokinesis-block micronucleus assay. This test is easily performed on irradiated glioma cell lines and can assist in determining their radiosensitivity. However, in order to obtain reliable and reproducible results, precise criteria for MN scoring must be strictly followed. Simultaneous use of fluorescent and phase contrast equipment improves imaging of morphological details and can further optimize MN scoring.

  13. Single-cell nanotoxicity assays of superparamagnetic iron oxide nanoparticles.

    Science.gov (United States)

    Eustaquio, Trisha; Leary, James F

    2012-01-01

    Properly evaluating the nanotoxicity of nanoparticles involves much more than bulk-cell assays of cell death by necrosis. Cells exposed to nanoparticles may undergo repairable oxidative stress and DNA damage or be induced into apoptosis. Exposure to nanoparticles may cause the cells to alter their proliferation or differentiation or their cell-cell signaling with neighboring cells in a tissue. Nanoparticles are usually more toxic to some cell subpopulations than others, and toxicity often varies with cell cycle. All of these facts dictate that any nanotoxicity assay must be at the single-cell level and must try whenever feasible and reasonable to include many of these other factors. Focusing on one type of quantitative measure of nanotoxicity, we describe flow and scanning image cytometry approaches to measuring nanotoxicity at the single-cell level by using a commonly used assay for distinguishing between necrotic and apoptotic causes of cell death by one type of nanoparticle. Flow cytometry is fast and quantitative, provided that the cells can be prepared into a single-cell suspension for analysis. But when cells cannot be put into suspension without altering nanotoxicity results, or if morphology, attachment, and stain location are important, a scanning image cytometry approach must be used. Both methods are described with application to a particular type of nanoparticle, a superparamagnetic iron oxide nanoparticle (SPION), as an example of how these assays may be applied to the more general problem of determining the effects of nanomaterial exposure to living cells.

  14. Functionality of Dengue Virus Specific Memory T Cell Responses in Individuals Who Were Hospitalized or Who Had Mild or Subclinical Dengue Infection

    Science.gov (United States)

    Jeewandara, Chandima; Adikari, Thiruni N.; Gomes, Laksiri; Fernando, Samitha; Fernando, R. H.; Perera, M. K. T.; Ariyaratne, Dinuka; Kamaladasa, Achala; Salimi, Maryam; Prathapan, Shamini

    2015-01-01

    Background Although antibody responses to dengue virus (DENV) in naturally infected individuals have been extensively studied, the functionality of DENV specific memory T cell responses in relation to clinical disease severity is incompletely understood. Methodology/Principal findings Using ex vivo IFNγ ELISpot assays, and by determining cytokines produced in ELISpot supernatants, we investigated the functionality of DENV-specific memory T cell responses in a large cohort of individuals from Sri Lanka (n=338), who were naturally infected and were either hospitalized due to dengue or had mild or sub clinical dengue infection. We found that T cells of individuals with both past mild or sub clinical dengue infection and who were hospitalized produced multiple cytokines when stimulated with DENV-NS3 peptides. However, while DENV-NS3 specific T cells of those with mild/sub clinical dengue infection were more likely to produce only granzyme B (p=0.02), those who were hospitalized were more likely to produce both TNFα and IFNγ (p=0.03) or TNFα alone. We have also investigated the usefulness of a novel T cell based assay, which can be used to determine the past infecting DENV serotype. 92.4% of DENV seropositive individuals responded to at least one DENV serotype of this assay and none of the seronegatives responded. Individuals who were seronegative, but had received the Japanese encephalitis vaccine too made no responses, suggesting that the peptides used in this assay did not cross react with the Japanese encephalitis virus. Conclusions/significance The types of cytokines produced by DENV-specific memory T cells appear to influence the outcome of clinical disease severity. The novel T cell based assay, is likely to be useful in determining the past infecting DENV serotype in immune-epidemiological studies and also in dengue vaccine trials. PMID:25875020

  15. Pre-transplant donor-specific T-cell alloreactivity is strongly associated with early acute cellular rejection in kidney transplant recipients not receiving T-cell depleting induction therapy.

    Directory of Open Access Journals (Sweden)

    Elena Crespo

    Full Text Available Preformed T-cell immune-sensitization should most likely impact allograft outcome during the initial period after kidney transplantation, since donor-specific memory T-cells may rapidly recognize alloantigens and activate the effector immune response, which leads to allograft rejection. However, the precise time-frame in which acute rejection is fundamentally triggered by preformed donor-specific memory T cells rather than by de novo activated naïve T cells is still to be established. Here, preformed donor-specific alloreactive T-cell responses were evaluated using the IFN-γ ELISPOT assay in a large consecutive cohort of kidney transplant patients (n = 90, to assess the main clinical variables associated with cellular sensitization and its predominant time-frame impact on allograft outcome, and was further validated in an independent new set of kidney transplant recipients (n = 67. We found that most highly T-cell sensitized patients were elderly patients with particularly poor HLA class-I matching, without any clinically recognizable sensitizing events. While one-year incidence of all types of biopsy-proven acute rejection did not differ between T-cell alloreactive and non-alloreactive patients, Receiver Operating Characteristic curve analysis indicated the first two months after transplantation as the highest risk time period for acute cellular rejection associated with baseline T-cell sensitization. This effect was particularly evident in young and highly alloreactive individuals that did not receive T-cell depletion immunosuppression. Multivariate analysis confirmed preformed T-cell sensitization as an independent predictor of early acute cellular rejection. In summary, monitoring anti-donor T-cell sensitization before transplantation may help to identify patients at increased risk of acute cellular rejection, particularly in the early phases after kidney transplantation, and thus guide decision-making regarding the use of induction

  16. An Enzyme-Linked Immunosorbent Spot Assay Measuring Borrelia burgdorferi B31-Specific Interferon Gamma-Secreting T Cells Cannot Discriminate Active Lyme Neuroborreliosis from Past Lyme Borreliosis: a Prospective Study in the Netherlands.

    Science.gov (United States)

    van Gorkom, T; Sankatsing, S U C; Voet, W; Ismail, D M; Muilwijk, R H; Salomons, M; Vlaminckx, B J M; Bossink, A W J; Notermans, D W; Bouwman, J J M; Kremer, K; Thijsen, S F T

    2018-04-01

    Two-tier serology testing is most frequently used for the diagnosis of Lyme borreliosis (LB); however, a positive result is no proof of active disease. To establish a diagnosis of active LB, better diagnostics are needed. Tests investigating the cellular immune system are available, but studies evaluating the utility of these tests on well-defined patient populations are lacking. Therefore, we investigated the utility of an enzyme-linked immunosorbent spot (ELISpot) assay to diagnose active Lyme neuroborreliosis. Peripheral blood mononuclear cells (PBMCs) of various study groups were stimulated by using Borrelia burgdorferi strain B31 and various recombinant antigens, and subsequently, the number of Borrelia -specific interferon gamma (IFN-γ)-secreting T cells was measured. We included 33 active and 37 treated Lyme neuroborreliosis patients, 28 healthy individuals treated for an early manifestation of LB in the past, and 145 untreated healthy individuals. The median numbers of B. burgdorferi B31-specific IFN-γ-secreting T cells/2.5 × 10 5 PBMCs did not differ between active Lyme neuroborreliosis patients (6.0; interquartile range [IQR], 0.5 to 14.0), treated Lyme neuroborreliosis patients (4.5; IQR, 2.0 to 18.6), and treated healthy individuals (7.4; IQR, 2.3 to 14.9) ( P = 1.000); however, the median number of B. burgdorferi B31-specific IFN-γ-secreting T cells/2.5 × 10 5 PBMCs among untreated healthy individuals was lower (2.0; IQR, 0.5 to 3.9) ( P ≤ 0.016). We conclude that the Borrelia ELISpot assay, measuring the number of B. burgdorferi B31-specific IFN-γ-secreting T cells/2.5 × 10 5 PBMCs, correlates with exposure to the Borrelia bacterium but cannot be used for the diagnosis of active Lyme neuroborreliosis. Copyright © 2018 van Gorkom et al.

  17. T-cell help permits memory CD8(+) T-cell inflation during cytomegalovirus latency.

    Science.gov (United States)

    Walton, Senta M; Torti, Nicole; Mandaric, Sanja; Oxenius, Annette

    2011-08-01

    CD4(+) T cells are implied to sustain CD8(+) T-cell responses during persistent infections. As CD4(+) T cells are often themselves antiviral effectors, they might shape CD8(+) T-cell responses via help or via controlling antigen load. We used persistent murine CMV (MCMV) infection to dissect the impact of CD4(+) T cells on virus-specific CD8(+) T cells, distinguishing between increased viral load in the absence of CD4(+) T cells and CD4(+) T-cell-mediated helper mechanisms. Absence of T-helper cells was associated with sustained lytic MCMV replication and led to a slow and gradual reduction of the size and function of the MCMV-specific CD8(+) T-cell pool. However, when virus replication was controlled in the absence of CD4(+) T cells, CD8(+) T-cell function was comparably impaired, but in addition CD8(+) T-cell inflation, a hallmark of CMV infection, was completely abolished. Thus, CD8(+) T-cell inflation during latent CMV infection is strongly dependent on CD4(+) T-cell helper functions, which can partially be compensated by ongoing lytic viral replication in the absence of CD4(+) T cells. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. SU-E-T-427: Cell Surviving Fractions Derived From Tumor-Volume Variation During Radiotherapy for Non-Small Cell Lung Cancer: Comparison with Predictive Assays

    Energy Technology Data Exchange (ETDEWEB)

    Chvetsov, A; Schwartz, J; Mayr, N [University of Washington, Seattle, WA (United States); Yartsev, S [London Health Sciences Centre, London, Ontario (Canada)

    2014-06-01

    Purpose: To show that a distribution of cell surviving fractions S{sub 2} in a heterogeneous group of patients can be derived from tumor-volume variation curves during radiotherapy for non-small cell lung cancer. Methods: Our analysis was based on two data sets of tumor-volume variation curves for heterogeneous groups of 17 patients treated for nonsmall cell lung cancer with conventional dose fractionation. The data sets were obtained previously at two independent institutions by using megavoltage (MV) computed tomography (CT). Statistical distributions of cell surviving fractions S{sup 2} and cell clearance half-lives of lethally damaged cells T1/2 have been reconstructed in each patient group by using a version of the two-level cell population tumor response model and a simulated annealing algorithm. The reconstructed statistical distributions of the cell surviving fractions have been compared to the distributions measured using predictive assays in vitro. Results: Non-small cell lung cancer presents certain difficulties for modeling surviving fractions using tumor-volume variation curves because of relatively large fractional hypoxic volume, low gradient of tumor-volume response, and possible uncertainties due to breathing motion. Despite these difficulties, cell surviving fractions S{sub 2} for non-small cell lung cancer derived from tumor-volume variation measured at different institutions have similar probability density functions (PDFs) with mean values of 0.30 and 0.43 and standard deviations of 0.13 and 0.18, respectively. The PDFs for cell surviving fractions S{sup 2} reconstructed from tumor volume variation agree with the PDF measured in vitro. Comparison of the reconstructed cell surviving fractions with patient survival data shows that the patient survival time decreases as the cell surviving fraction increases. Conclusion: The data obtained in this work suggests that the cell surviving fractions S{sub 2} can be reconstructed from the tumor volume

  19. SU-E-T-427: Cell Surviving Fractions Derived From Tumor-Volume Variation During Radiotherapy for Non-Small Cell Lung Cancer: Comparison with Predictive Assays

    International Nuclear Information System (INIS)

    Chvetsov, A; Schwartz, J; Mayr, N; Yartsev, S

    2014-01-01

    Purpose: To show that a distribution of cell surviving fractions S 2 in a heterogeneous group of patients can be derived from tumor-volume variation curves during radiotherapy for non-small cell lung cancer. Methods: Our analysis was based on two data sets of tumor-volume variation curves for heterogeneous groups of 17 patients treated for nonsmall cell lung cancer with conventional dose fractionation. The data sets were obtained previously at two independent institutions by using megavoltage (MV) computed tomography (CT). Statistical distributions of cell surviving fractions S 2 and cell clearance half-lives of lethally damaged cells T1/2 have been reconstructed in each patient group by using a version of the two-level cell population tumor response model and a simulated annealing algorithm. The reconstructed statistical distributions of the cell surviving fractions have been compared to the distributions measured using predictive assays in vitro. Results: Non-small cell lung cancer presents certain difficulties for modeling surviving fractions using tumor-volume variation curves because of relatively large fractional hypoxic volume, low gradient of tumor-volume response, and possible uncertainties due to breathing motion. Despite these difficulties, cell surviving fractions S 2 for non-small cell lung cancer derived from tumor-volume variation measured at different institutions have similar probability density functions (PDFs) with mean values of 0.30 and 0.43 and standard deviations of 0.13 and 0.18, respectively. The PDFs for cell surviving fractions S 2 reconstructed from tumor volume variation agree with the PDF measured in vitro. Comparison of the reconstructed cell surviving fractions with patient survival data shows that the patient survival time decreases as the cell surviving fraction increases. Conclusion: The data obtained in this work suggests that the cell surviving fractions S 2 can be reconstructed from the tumor volume variation curves measured

  20. Narcolepsy Type 1 Is Associated with a Systemic Increase and Activation of Regulatory T Cells and with a Systemic Activation of Global T Cells.

    Science.gov (United States)

    Lecendreux, Michel; Churlaud, Guillaume; Pitoiset, Fabien; Regnault, Armelle; Tran, Tu Anh; Liblau, Roland; Klatzmann, David; Rosenzwajg, Michelle

    2017-01-01

    Narcolepsy is a rare neurologic disorder characterized by excessive daytime sleepiness, cataplexy and disturbed nocturnal sleep patterns. Narcolepsy type 1 (NT1) has been shown to result from a selective loss of hypothalamic hypocretin-secreting neurons with patients typically showing low CSF-hypocretin levels (NT1 could be an immune-mediated pathology. Moreover, susceptibility to NT1 has recently been associated with several pathogens, particularly with influenza A H1N1 virus either through infection or vaccination. The goal of this study was to compare peripheral blood immune cell populations in recent onset pediatric NT1 subjects (post or non-post 2009-influenza A H1N1 vaccination) to healthy donors. We demonstrated an increased number of central memory CD4+ T cells (CD62L+ CD45RA-) associated to an activated phenotype (increase in CD69 and CD25 expression) in NT1 patients. Percentage and absolute count of regulatory T cells (Tregs) in NT1 patients were increased associated with an activated phenotype (increase in GITR and LAP expression), and of activated memory phenotype. Cytokine production by CD4+ and CD8+ T cells after activation was not modified in NT1 patients. In H1N1 vaccinated NT1 patients, absolute counts of CD3+, CD8+ T cells, and B cells were increased compared to non-vaccinated NT1 patients. These results support a global T cell activation in NT1 patients and thus support a T cell-mediated autoimmune origin of NT1, but do not demonstrate the pathological role of H1N1 prophylactic vaccination. They should prompt further studies of T cells, particularly of Tregs (such as suppression and proliferation antigen specific assays, and also T-cell receptor sequencing), in NT1.

  1. Citrus aurantium L. dry extracts promote C/ebpβ expression and improve adipocyte differentiation in 3T3-L1 cells.

    Science.gov (United States)

    Raciti, Gregory Alexander; Fiory, Francesca; Campitelli, Michele; Desiderio, Antonella; Spinelli, Rosa; Longo, Michele; Nigro, Cecilia; Pepe, Giacomo; Sommella, Eduardo; Campiglia, Pietro; Formisano, Pietro; Beguinot, Francesco; Miele, Claudia

    2018-01-01

    Metabolic and/or endocrine dysfunction of the white adipose tissue (WAT) contribute to the development of metabolic disorders, such as Type 2 Diabetes (T2D). Therefore, the identification of products able to improve adipose tissue function represents a valuable strategy for the prevention and/or treatment of T2D. In the current study, we investigated the potential effects of dry extracts obtained from Citrus aurantium L. fruit juice (CAde) on the regulation of 3T3-L1 cells adipocyte differentiation and function in vitro. We found that CAde enhances terminal adipocyte differentiation of 3T3-L1 cells raising the expression of CCAAT/enhancer binding protein beta (C/Ebpβ), peroxisome proliferator activated receptor gamma (Pparγ), glucose transporter type 4 (Glut4) and fatty acid binding protein 4 (Fabp4). CAde improves insulin-induced glucose uptake of 3T3-L1 adipocytes, as well. A focused analysis of the phases occurring in the pre-adipocytes differentiation to mature adipocytes furthermore revealed that CAde promotes the early differentiation stage by up-regulating C/ebpβ expression at 2, 4 and 8 h post the adipogenic induction and anticipating the 3T3-L1 cell cycle entry and progression during mitotic clonal expansion (MCE). These findings provide evidence that the exposure to CAde enhances in vitro fat cell differentiation of pre-adipocytes and functional capacity of mature adipocytes, and pave the way to the development of products derived from Citrus aurantium L. fruit juice, which may improve WAT functional capacity and may be effective for the prevention and/or treatment of T2D.

  2. Improved radioiodination of biomolecules using exhaustive Chloramine-T oxidation

    International Nuclear Information System (INIS)

    Robles, Ana M.; Balter, Henia S.; Oliver, Patricia; Welling, Mick M.; Pauwels, Ernest K.J.

    2001-01-01

    To improve standardization in analytical reagents we investigated Chloramine-T radioiodination ( 125 I) of several biomolecules based on the use of a single amount of the oxidizing agent Chloramine-T as the limiting reagent being exhausted during the course of the reaction. Whenever the labeling yield resulted in less than one atom 125 I/molecule, a second amount of the oxidizing agent was added. Thereafter, the integrity of the various biomolecules was assessed using radioimmunoassays, radioreceptor binding assays, or radioimmunometric assays. Purification yields were done by gel permeation (56%±19%, n=230) or by precipitation with trichloroacetic acid (59%±19%, n=230). Specific activity (117±61 MBq/nmol) and the degree of iodine incorporation (1.4±0.8 atoms of 125 I/molecule) were achieved after 300 sec of incubation. A second addition of Chloramine-T resulted in an increased labeling yield of all biomolecules tested by a mean factor of 1.8±0.9. After the second addition of Chloramine-T, we observed for some biomolecules a significant (p<0.001) decreased effect in biological performance. In conclusion, the use of Chloramine-T as a limiting reagent resulted in molecules with appropriate immunological and biological performance. In general, tracers were minimally damaged and assessment of the shelf life as well as storing conditions showed the usefulness of the standardization of biomolecule labeling

  3. MicroRNA-101 regulates T-cell acute lymphoblastic leukemia progression and chemotherapeutic sensitivity by targeting Notch1.

    Science.gov (United States)

    Qian, Lu; Zhang, Wanggang; Lei, Bo; He, Aili; Ye, Lianhong; Li, Xingzhou; Dong, Xin

    2016-11-01

    The present study aimed to investigate the role of microRNA (miR)-101 in acute lymphoblastic leukemia progression and chemoresistance. Furthermore, a novel target gene of miR-101 was identified. Here, we confirmed that miR-101 was significantly downregulated in the blood samples of patients with T-cell acute lymphoblastic leukemia (T-ALL) compared with the healthy controls, as determined by reverse transcription quantitative polymerase chain reaction (RTqPCR) analysis. The in vitro experiments demonstrated that miR-101 significantly repressed the proliferation and invasion, and induced potent apoptosis in Jurkat cells, as determined by CCK-8, flow cytometer and cell invasion assays. Luciferase assay confirmed that Notch1 was a target gene of miR-101, and western blotting showed that miR-101 suppressed the expression of Notch1 at the protein level. Moreover, functional restoration assays revealed that Notch1 mediates the effects of miR-101 on Jurkat cell proliferation, apoptosis and invasion. miR-101 enhanced the sensitivity of Jurkat cells to the chemotherapeutic agent adriamycin. Taken together, our results show for the first time that miR-101 acts as a tumor suppressor in T-cell acute lymphoblastic leukaemia and it could enhance chemotherapeutic sensitivity. Furthermore, Notch1 was identified to be a novel target of miR-101. This study indicates that miR-101 may represent a potential therapeutic target for T-cell acute lymphoblastic leukemia intervention.

  4. In silico and cell-based analyses reveal strong divergence between prediction and observation of T-cell-recognized tumor antigen T-cell epitopes.

    Science.gov (United States)

    Schmidt, Julien; Guillaume, Philippe; Dojcinovic, Danijel; Karbach, Julia; Coukos, George; Luescher, Immanuel

    2017-07-14

    Tumor exomes provide comprehensive information on mutated, overexpressed genes and aberrant splicing, which can be exploited for personalized cancer immunotherapy. Of particular interest are mutated tumor antigen T-cell epitopes, because neoepitope-specific T cells often are tumoricidal. However, identifying tumor-specific T-cell epitopes is a major challenge. A widely used strategy relies on initial prediction of human leukocyte antigen-binding peptides by in silico algorithms, but the predictive power of this approach is unclear. Here, we used the human tumor antigen NY-ESO-1 (ESO) and the human leukocyte antigen variant HLA-A*0201 (A2) as a model and predicted in silico the 41 highest-affinity, A2-binding 8-11-mer peptides and assessed their binding, kinetic complex stability, and immunogenicity in A2-transgenic mice and on peripheral blood mononuclear cells from ESO-vaccinated melanoma patients. We found that 19 of the peptides strongly bound to A2, 10 of which formed stable A2-peptide complexes and induced CD8 + T cells in A2-transgenic mice. However, only 5 of the peptides induced cognate T cells in humans; these peptides exhibited strong binding and complex stability and contained multiple large hydrophobic and aromatic amino acids. These results were not predicted by in silico algorithms and provide new clues to improving T-cell epitope identification. In conclusion, our findings indicate that only a small fraction of in silico -predicted A2-binding ESO peptides are immunogenic in humans, namely those that have high peptide-binding strength and complex stability. This observation highlights the need for improving in silico predictions of peptide immunogenicity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Evaluation of a new T2 Magnetic Resonance assay for rapid detection of emergent fungal pathogen Candida auris on clinical skin swab samples.

    Science.gov (United States)

    Sexton, D Joseph; Bentz, Meghan L; Welsh, Rory M; Litvintseva, Anastasia P

    2018-06-25

    Candida auris is a multidrug-resistant pathogenic yeast whose recent emergence is of increasing public-health concern. C. auris can colonize multiple body sites, including patients' skin, and survive for weeks in the healthcare environment, facilitating patient-to-patient transmission and fueling healthcare-associated outbreaks. Rapid and accurate detection of C. auris colonization is essential for timely implementation of infection control measures and prevent transmission. Currently, axilla/groin composite swabs, used to assess colonization status, are processed using a culture-based method that is sensitive and specific but requires 14 days. This delay limits the opportunity to respond and highlights the need for a faster alternative. The culture-independent T2 Magnetic Resonance (T2MR) system is a rapid diagnostic platform shown to detect target pathogens of interest from unprocessed blood samples in T2 assay was evaluated for screening of the skin surveillance samples. Inclusivity and limit of detection of the T2 C. auris assay were assessed with spiked samples in a representative skin flora background. The T2 C. auris assay recognized isolates from each of the 4 known clades of C. auris and consistently detected cells at 5 CFU/mL. Finally, 89 clinical axilla/groin swab samples were processed with the T2 C. auris assay. The culture-based diagnostic assay was used as a gold standard to determine performance statistics including sensitivity (0.89) and specificity (0.98). Overall, the T2 C. auris assay performed well as a rapid diagnostic and could help expedite the detection of C. auris in patient skin swabs. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  6. Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

    Science.gov (United States)

    Alvarez-Vallina, L; Yañez, R; Blanco, B; Gil, M; Russell, S J

    2000-04-01

    Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.

  7. TNP-specific Lyt-2+ cytolytic T cell clones preferentially respond to TNP-conjugated epidermal cells

    International Nuclear Information System (INIS)

    Shimada, S.; Katz, S.I.

    1985-01-01

    A most effective method for the induction of hapten-specific allergic contact sensitivity (CS) is via epicutaneous application of the hapten. Another effective method is by the administration of haptenated epidermal cells (EC) subcutaneously. The latter method induces more intense and longer lasting CS than does the subcutaneous administration of haptenated spleen cells (SC). Thus, there may be something unique about EC which, when haptenated, allows them to generate effector cells more effectively than do SC. The authors therefore, attempted to generate T cell clones that were both hapten- and epidermal-specific. Four days after painting mice with 7% trinitrochlorobenzene, draining lymph node cells were obtained and T cells were purified. These cells were co-cultured with trinitrophenylated (TNP) Langerhans cell-enriched EC. After 4 days, cells were harvested and rested on non-TNP-conjugated EC. The cells were restimulated and rested three times, and were then cloned by limiting dilution with added interleukin 2, which was then continually added. Proliferation of T cells was assessed by [ 3 H]-thymidine incorporation. Cytotoxicity assays utilized TNP-conjugated concanavalin A SC blasts or EC as targets. Clones A-2 and E-4 are Thy-1+, Lyt-2+, and L3T4-, and TNP-specific. In contrast to noncloned TNP-specific T cells, the clones proliferate preferentially in response to TNP-EC rather than TNP-SC. Also in contrast to noncloned T cells, the clones were preferentially cytotoxic for TNP-EC; compared to TNP-SC, there was an eight- to 32-fold increase in killing when TNP-EC were used as targets. Clones A-2 and E-4 therefore exhibit hapten and epidermal specificity

  8. T cell receptor sequencing of early-stage breast cancer tumors identifies altered clonal structure of the T cell repertoire.

    Science.gov (United States)

    Beausang, John F; Wheeler, Amanda J; Chan, Natalie H; Hanft, Violet R; Dirbas, Frederick M; Jeffrey, Stefanie S; Quake, Stephen R

    2017-11-28

    Tumor-infiltrating T cells play an important role in many cancers, and can improve prognosis and yield therapeutic targets. We characterized T cells infiltrating both breast cancer tumors and the surrounding normal breast tissue to identify T cells specific to each, as well as their abundance in peripheral blood. Using immune profiling of the T cell beta-chain repertoire in 16 patients with early-stage breast cancer, we show that the clonal structure of the tumor is significantly different from adjacent breast tissue, with the tumor containing ∼2.5-fold greater density of T cells and higher clonality compared with normal breast. The clonal structure of T cells in blood and normal breast is more similar than between blood and tumor, and could be used to distinguish tumor from normal breast tissue in 14 of 16 patients. Many T cell sequences overlap between tissue and blood from the same patient, including ∼50% of T cells between tumor and normal breast. Both tumor and normal breast contain high-abundance "enriched" sequences that are absent or of low abundance in the other tissue. Many of these T cells are either not detected or detected with very low frequency in the blood, suggesting the existence of separate compartments of T cells in both tumor and normal breast. Enriched T cell sequences are typically unique to each patient, but a subset is shared between many different patients. We show that many of these are commonly generated sequences, and thus unlikely to play an important role in the tumor microenvironment. Copyright © 2017 the Author(s). Published by PNAS.

  9. A new DPYD genotyping assay for improving the safety of 5-fluorouracil therapy.

    Science.gov (United States)

    Sistonen, Johanna; Smith, Chingying; Fu, Yung-Kang; Largiadèr, Carlo R

    2012-12-24

    Chemotherapeutic use of 5-fluorouracil (5FU) is compromised by 10-20% of patients developing severe toxicity. Recently described genetic variation in dihydropyrimidine dehydrogenase (DPYD) has been shown to be a major predictor of 5FU toxicity. Here, we describe a new genotyping assay for routine clinical use that covers all the major DPYD risk variants. Genomic regions targeting DPYD risk variants (c.1129-5923C>G, c.1679T>G/A, c.1905+1G>A, c.2846A>T) and additional markers (c.234-123G>C, c.496A>G, c.775A>G) were amplified in a multiplex PCR reaction. The subsequent steps including allele-specific primer extension, hybridization of the primers to a microarray, scanning of the array, and data analysis were automated within the INFINITI® Analyzer (AutoGenomics). The assay was validated by analyzing 107 blood samples obtained from patients previously re-sequenced for the DPYD. The genotypes obtained with the developed assay were 100% concordant with the re-sequencing. The procedure is suitable for routine clinical use since the results are obtained within one day. For heterozygous risk variant carriers (~7% of Europeans), the treatment can be adjusted by 5FU dose reduction, whereas carriers of two risk alleles should be treated with an alternative therapy. The developed assay provides a novel tool to improve the safety of commonly used 5FU-based chemotherapies. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Impaired T-lymphocyte colony formation by cord blood mononuclear cells

    International Nuclear Information System (INIS)

    Herrod, H.G.; Valenski, W.R.

    1982-01-01

    When compared to adult mononuclear cells, cord blood mononuclear cells demonstrated significantly decreased T-lymphocyte colony formation (1351 +/- 643 vs 592 +/- 862, P less than 0.01). This diminished colony-forming activity did not appear to be associated with impaired responsiveness to the stimulant phytohemagglutinin or with excessive suppressor-cell activity. Irradiation reduced the colony-forming capacity of cord blood mononuclear cells more than it did that of adult mononuclear cells. Depletion of adherent cells reduced cord blood mononuclear-cell colony-forming capacity by 40%, while similar treatment reduced adult colony formation by 10%. Lymphocyte proliferation in liquid culture of cord and adult cells was minimally affected by these procedures. The colony-forming capacity of cord blood could be enhanced by the addition of irradiated adult cells (284 +/- 72 vs 752 +/- 78, P less than 0.01). This enhancement was demonstrated to be due to a soluble factor produced by a population of irradiated adult cells depleted of the OKT8+ subpopulation of lymphocytes. These results indicate that the progenitor cells of T-lymphocyte colonies in cord blood have distinct biologic characteristics when compared to colony progenitors present in adult blood. This assay may prove to be useful in our efforts to understand the differentiation of T-cell function in man

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

    International Nuclear Information System (INIS)

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

    1991-01-01

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

  12. CD4+ T-cell lines used to evaluate a Mycobacterium avium subsp. paratuberculosis (MAP) peptide vaccine

    DEFF Research Database (Denmark)

    Lybeck, Kari; Sjurseth, Siri K.; Al-Touama, Zainab

    The aim of the study was to establish a protocol for generation of MAP-specific T-cell lines and to use these lines for evaluation of a peptide vaccine. A protocol for culturing T-cell lines from peripheral blood of goats naturally infected with MAP was established. CD4+ T cells were positively...... selected using an anti CD4 mAb and Dynabeads. Sorted CD4+ cells were cultivated with purified protein derivative from MAP (PPDj) or E. coli sonicate, IL-2, and IL-15. After two cultivation cycles, T cells were tested for recall responses in a proliferative T-cell assay. T-cell line responses were...... in average 92 % for PPDj, and -3 % for E. coli sonicate. CD4+ T-cell lines stimulated with PPDj showed a 6 fold increase in IFN- γ production compared to controls. These results indicated that the T-cell lines were MAP-specific. The protocol was subsequently used to evaluate MAP-specific peptides as vaccine...

  13. Validation of an improved enzyme-linked immunosorbent assay for the diagnosis of trypanosomal antibodies in Ghanaian cattle

    International Nuclear Information System (INIS)

    Doku, C.K.; Seidu, I.B.M.

    2000-01-01

    The validation of an enzyme-linked immunosorbent assay (Ab-ELISA) for the detection of antibodies to pathogenic trypanosomes in cattle is described. Two hundred known negative sera obtained from the tsetse-free zone of Dori (Burkina Faso) were analyzed using microtitre plates pre-coated with crude antigen lysates of Trypanosoma congolense and T. vivax. A pre-test optimization was carried out and a percent positivity (PP) of 50% was chosen (specificity: >82%) for assaying field sera. A total of 440 serum samples collected from cattle in areas of known and unknown disease prevalence were assayed. For all animals the packed red cell volume (PCV) was determined and the buffy coat technique (BCT) and blood smears were examined to detect trypanosomes at the species level. A comparison of the BCT and Ab-ELISA results showed there was a much higher prevalence of antibodies to both species than the parasite prevalence as shown by the BCT (10 fold). The rate of agreement between BCT-positive and Ab-ELISA-positive samples for both species was low (<10%). No conclusion could be drawn from this finding because of the low number of known BCT positive cases that were identified. There was a better, albeit highly variable, agreement between BCT-negative and Ab-ELISA-negative samples (30-70%). Proposals for further improvement of the Ab-ELISA and prospects for the use of the assay in the monitoring of trypanosomosis control in Ghana are discussed. (author)

  14. Blimp-1–mediated CD4 T cell exhaustion causes CD8 T cell dysfunction during chronic toxoplasmosis

    Science.gov (United States)

    Cobb, Dustin A.; Bhadra, Rajarshi

    2016-01-01

    CD8, but not CD4, T cells are considered critical for control of chronic toxoplasmosis. Although CD8 exhaustion has been previously reported in Toxoplasma encephalitis (TE)–susceptible model, our current work demonstrates that CD4 not only become exhausted during chronic toxoplasmosis but this dysfunction is more pronounced than CD8 T cells. Exhausted CD4 population expressed elevated levels of multiple inhibitory receptors concomitant with the reduced functionality and up-regulation of Blimp-1, a transcription factor. Our data demonstrates for the first time that Blimp-1 is a critical regulator for CD4 T cell exhaustion especially in the CD4 central memory cell subset. Using a tamoxifen-dependent conditional Blimp-1 knockout mixed bone marrow chimera as well as an adoptive transfer approach, we show that CD4 T cell–intrinsic deletion of Blimp-1 reversed CD8 T cell dysfunction and resulted in improved pathogen control. To the best of our knowledge, this is a novel finding, which demonstrates the role of Blimp-1 as a critical regulator of CD4 dysfunction and links it to the CD8 T cell dysfunctionality observed in infected mice. The critical role of CD4-intrinsic Blimp-1 expression in mediating CD4 and CD8 T cell exhaustion may provide a rational basis for designing novel therapeutic approaches. PMID:27481131

  15. T cell activation inhibitors reduce CD8+ T cell and pro-inflammatory macrophage accumulation in adipose tissue of obese mice.

    Directory of Open Access Journals (Sweden)

    Vince N Montes

    Full Text Available Adipose tissue inflammation and specifically, pro-inflammatory macrophages are believed to contribute to insulin resistance (IR in obesity in humans and animal models. Recent studies have invoked T cells in the recruitment of pro-inflammatory macrophages and the development of IR. To test the role of the T cell response in adipose tissue of mice fed an obesogenic diet, we used two agents (CTLA-4 Ig and anti-CD40L antibody that block co-stimulation, which is essential for full T cell activation. C57BL/6 mice were fed an obesogenic diet for 16 weeks, and concomitantly either treated with CTLA-4 Ig, anti-CD40L antibody or an IgG control (300 µg/week. The treatments altered the immune cell composition of adipose tissue in obese mice. Treated mice demonstrated a marked reduction in pro-inflammatory adipose tissue macrophages and activated CD8+ T cells. Mice treated with anti-CD40L exhibited reduced weight gain, which was accompanied by a trend toward improved IR. CTLA-4 Ig treatment, however, was not associated with improved IR. These data suggest that the presence of pro-inflammatory T cells and macrophages can be altered with co-stimulatory inhibitors, but may not be a significant contributor to the whole body IR phenotype.

  16. T-cell receptor transfer into human T cells with ecotropic retroviral vectors.

    Science.gov (United States)

    Koste, L; Beissert, T; Hoff, H; Pretsch, L; Türeci, Ö; Sahin, U

    2014-05-01

    Adoptive T-cell transfer for cancer immunotherapy requires genetic modification of T cells with recombinant T-cell receptors (TCRs). Amphotropic retroviral vectors (RVs) used for TCR transduction for this purpose are considered safe in principle. Despite this, TCR-coding and packaging vectors could theoretically recombine to produce replication competent vectors (RCVs), and transduced T-cell preparations must be proven free of RCV. To eliminate the need for RCV testing, we transduced human T cells with ecotropic RVs so potential RCV would be non-infectious for human cells. We show that transfection of synthetic messenger RNA encoding murine cationic amino-acid transporter 1 (mCAT-1), the receptor for murine retroviruses, enables efficient transient ecotropic transduction of human T cells. mCAT-1-dependent transduction was more efficient than amphotropic transduction performed in parallel, and preferentially targeted naive T cells. Moreover, we demonstrate that ecotropic TCR transduction results in antigen-specific restimulation of primary human T cells. Thus, ecotropic RVs represent a versatile, safe and potent tool to prepare T cells for the adoptive transfer.

  17. Frequency of mutant T lymphocytes defective in the expression of the T-cell antigen receptor gene among radiation-exposed people

    International Nuclear Information System (INIS)

    Kyoizumi, Seishi; Umeki, Shigeko; Akiyama, Mitoshi

    1991-06-01

    The frequency of mutant T lymphocytes defective in T-cell receptor gene (α or β) expression was measured using the two-color flow cytometric technique. Results for a total of 203 atomic bomb survivors, 78 of whom were proximally exposed (DS86 doses of ≥ 1.5 Gy) and 125 of whom were distally exposed (DS86 doses of 228 Th formerly used for radiodiagnosis. In addition, thyroid disease patients treated with 131 I showed a dose-related increase of mutant frequency. It was suggested that the present T-cell receptor mutation assay has a unique characteristic as a biological dosimeter for the measurement of recent exposures to genotoxic agents. (author)

  18. Selection and characterization of T-cell variants lacking molecules involved in T-cell activation (T3 T-cell receptor, T44, and T11): analysis of the functional relationship among different pathways of activation

    International Nuclear Information System (INIS)

    Moretta, A.; Poggi, A.; Olive, D.; Bottino, C.; Fortis, C.; Pantaleo, G.; Moretta, L.

    1987-01-01

    A clone of the interleukin 2-producing Jurkat leukemia cell line termed JA3 (surface phenotype, T3 + , Ti + , T44 + , T11 + , T40 + ) has been used to induce and select cell variants lacking surface molecules involved in T-cell activation. Following 200 rad of γ-radiation (1 rad = 0.01 Gy), cells were treated with monoclonal antibodies (mAbs) directed to T3, Ti, T44, or T11 antigen and complement. After growth of the residual cells in culture, negative cells were cloned under limiting conditions. Depending on the specificity of the mAb used for the immunoselection, three groups of variants were obtained. (i) The use of mAbs directed to T3 or Ti resulted in cell variants that expressed the T3 - Ti - T44 + Leu1 + T11 + T40 + 4F2 + HLA class I + surface phenotype. (ii) Immunoselection with anti-T44 mAb resulted in 2 variants that shared the T3 - Ti - T44 - Leu1 - T11 - T40 - 4F2 - HLA class I + phenotype. (iii) Cell treatment with anti-T11 mAb resulted in 15 variants characterized by the lack of T11 antigen expression and of all the other T-cell-specific surface antigens. Therefore, it appears that the different sets of JA3 cell variants, like T cells at discrete stages of intrathymic differentiation, may follow a coordinated expression of surface differentiation antigens. Analysis of the functional responsiveness of the three distinct groups of JA3 cell variants to different stimuli showed that all produced interleukin 2 in response to A23187 calcium ionophore plus phorbol 12-myristate 13-acetate

  19. FOXO3 regulates CD8 T cell memory by T cell-intrinsic mechanisms.

    Directory of Open Access Journals (Sweden)

    Jeremy A Sullivan

    2012-02-01

    Full Text Available CD8 T cell responses have three phases: expansion, contraction, and memory. Dynamic alterations in proliferation and apoptotic rates control CD8 T cell numbers at each phase, which in turn dictate the magnitude of CD8 T cell memory. Identification of signaling pathways that control CD8 T cell memory is incomplete. The PI3K/Akt signaling pathway controls cell growth in many cell types by modulating the activity of FOXO transcription factors. But the role of FOXOs in regulating CD8 T cell memory remains unknown. We show that phosphorylation of Akt, FOXO and mTOR in CD8 T cells occurs in a dynamic fashion in vivo during an acute viral infection. To elucidate the potentially dynamic role for FOXO3 in regulating homeostasis of activated CD8 T cells in lymphoid and non-lymphoid organs, we infected global and T cell-specific FOXO3-deficient mice with Lymphocytic Choriomeningitis Virus (LCMV. We found that FOXO3 deficiency induced a marked increase in the expansion of effector CD8 T cells, preferentially in the spleen, by T cell-intrinsic mechanisms. Mechanistically, the enhanced accumulation of proliferating CD8 T cells in FOXO3-deficient mice was not attributed to an augmented rate of cell division, but instead was linked to a reduction in cellular apoptosis. These data suggested that FOXO3 might inhibit accumulation of growth factor-deprived proliferating CD8 T cells by reducing their viability. By virtue of greater accumulation of memory precursor effector cells during expansion, the numbers of memory CD8 T cells were strikingly increased in the spleens of both global and T cell-specific FOXO3-deficient mice. The augmented CD8 T cell memory was durable, and FOXO3 deficiency did not perturb any of the qualitative attributes of memory T cells. In summary, we have identified FOXO3 as a critical regulator of CD8 T cell memory, and therapeutic modulation of FOXO3 might enhance vaccine-induced protective immunity against intracellular pathogens.

  20. Drak2 Does Not Regulate TGF-β Signaling in T Cells.

    Directory of Open Access Journals (Sweden)

    Tarsha L Harris

    Full Text Available Drak2 is a serine/threonine kinase expressed highest in T cells and B cells. Drak2-/- mice are resistant to autoimmunity in mouse models of type 1 diabetes and multiple sclerosis. Resistance to these diseases occurs, in part, because Drak2 is required for the survival of autoreactive T cells that induce disease. However, the molecular mechanisms by which Drak2 affects T cell survival and autoimmunity are not known. A recent report demonstrated that Drak2 negatively regulated transforming growth factor-β (TGF-β signaling in tumor cell lines. Thus, increased TGF-β signaling in the absence of Drak2 may contribute to the resistance to autoimmunity in Drak2-/- mice. Therefore, we examined if Drak2 functioned as a negative regulator of TGF-β signaling in T cells, and whether the enhanced susceptibility to death of Drak2-/- T cells was due to augmented TGF-β signaling. Using several in vitro assays to test TGF-β signaling and T cell function, we found that activation of Smad2 and Smad3, which are downstream of the TGF-β receptor, was similar between wildtype and Drak2-/- T cells. Furthermore, TGF-β-mediated effects on naïve T cell proliferation, activated CD8+ T cell survival, and regulatory T cell induction was similar between wildtype and Drak2-/- T cells. Finally, the increased susceptibility to death in the absence of Drak2 was not due to enhanced TGF-β signaling. Together, these data suggest that Drak2 does not function as a negative regulator of TGF-β signaling in primary T cells stimulated in vitro. It is important to investigate and discern potential molecular mechanisms by which Drak2 functions in order to better understand the etiology of autoimmune diseases, as well as to validate the use of Drak2 as a target for therapeutic treatment of these diseases.

  1. Norovirus-specific memory T cell responses in adult human donors

    Directory of Open Access Journals (Sweden)

    Maria Malm

    2016-10-01

    Full Text Available Norovirus (NoV is a leading cause of acute gastroenteritis in people of all ages worldwide. NoV specific serum antibodies which block the binding of NoV virus-like particles (VLPs to the cell receptors have been thoroughly investigated. In contrast, only a few publications are available on the NoV capsid VP1 protein-specific T cell responses in humans naturally infected with the virus. Freshly isolated peripheral blood mononuclear cells of eight healthy adult human donors previously exposed to NoV were stimulated with purified VLPs derived from NoV GII.4-1999, GII.4-2012 (Sydney, and GI.3, and IFN-g production was measured by an ELISPOT assay. In addition, 76 overlapping synthetic peptides spanning the entire 539 amino acid sequence of GII.4 VP1 were pooled into two-dimensional matrices and used to identify putative T cell epitopes. Seven of the eight subjects produced IFN-g in response to the peptides and five subjects produced IFN-g in response to the VLPs of the same origin. In general, stronger T cell responses were induced with the peptides in each donor compared to the VLPs. A CD8+ T cell epitope in the shell domain of the VP1 (134SPSQVTMFPHIIVDVRQL151 was identified in two subjects, both having human leukocyte antigen (HLA-A*02:01 allele. To our knowledge, this is the first report using synthetic peptides to study NoV-specific T cell responses in human subjects and identify T cell epitopes.

  2. Implementation and Use of State-of-the-Art, Cell-Based In Vitro Assays.

    Science.gov (United States)

    Langer, Gernot

    2016-01-01

    The impressive advances in the generation and interpretation of functional omics data have greatly contributed to a better understanding of the (patho-)physiology of many biological systems and led to a massive increase in the number of specific targets and phenotypes to investigate in both basic and applied research. The obvious complexity revealed by these studies represents a major challenge to the research community and asks for improved target characterisation strategies with the help of reliable, high-quality assays. Thus, the use of living cells has become an integral part of many research activities because the cellular context more closely represents target-specific interrelations and activity patterns. Although still predominant, the use of traditional two-dimensional (2D) monolayer cell culture models has been gradually complemented by studies based on three-dimensional (3D) spheroid (Sutherland 1988) and other 3D tissue culture systems (Santos et al. 2012; Matsusaki et al. 2014) in an attempt to employ model systems more closely representing the microenvironment of cells in the body. Hence, quite a variety of state-of-the-art cell culture models are available for the generation of novel chemical probes or the identification of starting points for drug development in translational research and pharma drug discovery. In order to cope with these information-rich formats and their increasing technical complexity, cell-based assay development has become a scientific research topic in its own right and is used to ensure the provision of significant, reliable and high-quality data outlasting any discussions related to the current "irreproducibility epidemic" (Dolgin 2014; Prinz et al. 2011; Schatz 2014). At the same time the use of cells in microplate assay formats has become state of the art and greatly facilitates rigorous cell-based assay development by providing the researcher with the opportunity to address the multitude of factors affecting the actual

  3. The single-cell gel electrophoresis assay to determine apoptosis ...

    African Journals Online (AJOL)

    When the frequency of appearance of apoptotic cells following was observed over a period of time, there was a significant increase in appearance of apoptosis when using single cell gel electrophoresis assay. The present report demonstrates that the characteristic pattern of apoptotic comets detected by the comet assay ...

  4. Toward precision manufacturing of immunogene T-cell therapies.

    Science.gov (United States)

    Xu, Jun; Melenhorst, J Joseph; Fraietta, Joseph A

    2018-05-01

    Cancer can be effectively targeted using a patient's own T cells equipped with synthetic receptors, including chimeric antigen receptors (CARs) that redirect and reprogram these lymphocytes to mediate tumor rejection. Over the past two decades, several strategies to manufacture genetically engineered T cells have been proposed, with the goal of generating optimally functional cellular products for adoptive transfer. Based on this work, protocols for manufacturing clinical-grade CAR T cells have been established, but these complex methods have been used to treat only a few hundred individuals. As CAR T-cell therapy progresses into later-phase clinical trials and becomes an option for more patients, a major consideration for academic institutions and industry is developing robust manufacturing processes that will permit scaling-out production of immunogene T-cell therapies in a reproducible and efficient manner. In this review, we will discuss the steps involved in cell processing, the major obstacles surrounding T-cell manufacturing platforms and the approaches for improving cellular product potency. Finally, we will address the challenges of expanding CAR T-cell therapy to a global patient population. Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  5. Donor Vδ1+ γδ T cells expand after allogeneic hematopoietic stem cell transplantation and show reactivity against CMV-infected cells but not against progressing B-CLL.

    Science.gov (United States)

    Prinz, Immo; Thamm, Kristina; Port, Matthias; Weissinger, Eva M; Stadler, Michael; Gabaev, Ildar; Jacobs, Roland; Ganser, Arnold; Koenecke, Christian

    2013-05-11

    γδ T lymphocytes play an important role in immune reactions towards infections and malignancies. In particular, Vγ9-Vδ1+ T lymphocytes are thought to play protective antiviral roles in human CMV infection. Recently, Vδ1+ T lymphocytes were proposed to also have anti- B-CLL reactivity. Here we report a case of 48-year-old man who received allogeneic stem cell transplantation for progressive B-CLL. Within one year after transplantation, lymphoma relapsed despite a dramatic increase of Vδ1+ T cells in the patient's blood. In vitro killing assays revealed activity of patient's γδ cells against CMV target cells, but not against the relapsing lymphoma-cells. This argues for a contribution of Vδ1+ cells in the immune reaction against CMV reactivation, but does not support a strong correlation of expanded Vδ1+ T cells and favorable disease outcome in B-CLL patients.

  6. Humoral and cellular CMV responses in healthy donors; identification of a frequent population of CMV-specific, CD4+ T cells in seronegative donors

    DEFF Research Database (Denmark)

    Loeth, Nina; Assing, Kristian; Madsen, Hans O

    2012-01-01

    .e., T lymphocyte) assays. Here, we have analyzed the CMV status of 100 healthy blood bank donors using both serology and cellular assays. About half (56%) were found to be CMV seropositive, and they all mounted strong CD8+ and/or moderate CD4+ T cell responses ex vivo against the immunodominant CMV...... protein, pp65. Of the 44 seronegative donors, only five (11%) mounted ex vivo T cell responses; surprisingly, 33 (75%) mounted strong CD4+ T cell responses after a brief in vitro peptide stimulation culture. This may have significant implications for the analysis and selection of HCT donors.......CMV status is an important risk factor in immune compromised patients. In hematopoeitic cell transplantations (HCT), both donor and recipient are tested routinely for CMV status by serological assays; however, one might argue that it might also be of relevance to examine CMV status by cellular (i...

  7. An indicator cell assay for blood-based diagnostics.

    Directory of Open Access Journals (Sweden)

    Samuel A Danziger

    Full Text Available We have established proof of principle for the Indicator Cell Assay Platform™ (iCAP™, a broadly applicable tool for blood-based diagnostics that uses specifically-selected, standardized cells as biosensors, relying on their innate ability to integrate and respond to diverse signals present in patients' blood. To develop an assay, indicator cells are exposed in vitro to serum from case or control subjects and their global differential response patterns are used to train reliable, disease classifiers based on a small number of features. In a feasibility study, the iCAP detected pre-symptomatic disease in a murine model of amyotrophic lateral sclerosis (ALS with 94% accuracy (p-Value = 3.81E-6 and correctly identified samples from a murine Huntington's disease model as non-carriers of ALS. Beyond the mouse model, in a preliminary human disease study, the iCAP detected early stage Alzheimer's disease with 72% cross-validated accuracy (p-Value = 3.10E-3. For both assays, iCAP features were enriched for disease-related genes, supporting the assay's relevance for disease research.

  8. Repression of COUP-TFI Improves Bone Marrow-Derived Mesenchymal Stem Cell Differentiation into Insulin-Producing Cells

    Directory of Open Access Journals (Sweden)

    Tao Zhang

    2017-09-01

    Full Text Available Identifying molecular mechanisms that regulate insulin expression in bone marrow-derived mesenchymal stem cells (bmMSCs can provide clues on how to stimulate the differentiation of bmMSCs into insulin-producing cells (IPCs, which can be used as a therapeutic approach against type 1 diabetes (T1D. As repression factors may inhibit differentiation, the efficiency of this process is insufficient for cell transplantation. In this study, we used the mouse insulin 2 (Ins2 promoter sequence and performed a DNA affinity precipitation assay combined with liquid chromatography-mass spectrometry to identify the transcription factor, chicken ovalbumin upstream promoter transcriptional factor I (COUP-TFI. Functionally, bmMSCs were reprogrammed into IPCs via COUP-TFI suppression and MafA overexpression. The differentiated cells expressed higher levels of genes specific for islet endocrine cells, and they released C-peptide and insulin in response to glucose stimulation. Transplantation of IPCs into streptozotocin-induced diabetic mice caused a reduction in hyperglycemia. Mechanistically, COUP-TFI bound to the DR1 (direct repeats with 1 spacer element in the Ins2 promoter, thereby negatively regulating promoter activity. Taken together, the data provide a novel mechanism by which COUP-TFI acts as a negative regulator in the Ins2 promoter. The differentiation of bmMSCs into IPCs could be improved by knockdown of COUP-TFI, which may provide a novel stem cell-based therapy for T1D. Keywords: siRNAs, differentiation, stem cell transplantation, diabetes, mesenchymal stem cells

  9. Single Cell Assay for Analyzing Single Cell Exosome and Endocrine Secretion and Cancer Markers

    Science.gov (United States)

    Chiu, Yu-Jui

    To understand the inhomogeneity of cells in biological systems, there is a growing demand for the capability to characterize the properties of individual single cells. Since single cell studies require continuous monitoring of the cell behaviors instead of a snapshot test at a single time point, an effective single-cell assay that can support time lapsed studies in a high throughput manner is desired. Most currently available single-cell technologies cannot provide proper environments to sustain cell growth and cannot provide, for appropriate cell types, proliferation of single cells and convenient, non-invasive tests of single cell behaviors from molecular markers. In this dissertation, I present a highly versatile single-cell assay that can accommodate different cellular types, enable easy and efficient single cell loading and culturing, and be suitable for the study of effects of in-vitro environmental factors in combination with drug screening. The salient features of the assay are the non-invasive collection and surveying of single cell secretions at different time points and massively parallel translocation of single cells by user defined criteria, producing very high compatibility to the downstream process such as single cell qPCR and sequencing. Above all, the acquired information is quantitative -- for example, one of the studies is measured by the number of exosomes each single cell secretes for a given time period. Therefore, our single-cell assay provides a convenient, low-cost, and enabling tool for quantitative, time lapsed studies of single cell properties.

  10. Human T cell colony formation in microculture: analysis of growth requirements and functional activities.

    Science.gov (United States)

    Gelfand, E W; Lee, J W; Dosch, H M; Price, G B

    1981-03-01

    A microculture method in methylcellulose has been developed for the study of human T cell colony formation. The technique is simple, reliable, does not require preincubation with lectin and requires small numbers of cells. Colony formation was dependent on the presence of phytohemagglutin-conditioned medium, a T colony precursor cell (TCPC), and a "helper" or accessory T cell. Plating efficiency was increased 10-fold in the presence of irradiated feeder cells. Progenitors of the T colony cells were identified in peripheral blood, tonsil, and spleen but not in thymus or thoracic duct. They were isolated in the E-rosetting, theophylline-resistant, Fc-IgG-negative cell populations. In peripheral blood the frequency of TCPC and accessory cells, the T colony forming unit, was estimated to be 8 X 10(-3). Colony cells proliferated in response to lectins and allogeneic cells. Forty to 80% of the cells were Ia-positive and stimulated both autologous and allogeneic mixed lymphocyte responses. They were incapable of mediating antibody-dependent cytotoxicity. In contrast, they were effective in assays of spontaneous cytotoxicity but only against certain target cells. This method for the analysis of T colony formation should prove valuable in the functional analysis of T cell subsets in immunodeficiency states or the transplant recipient.

  11. Identifying activated T cells in reconstituted RAG deficient mice using retrovirally transduced Pax5 deficient pro-B cells.

    Directory of Open Access Journals (Sweden)

    Nadesan Gajendran

    Full Text Available Various methods have been used to identify activated T cells such as binding of MHC tetramers and expression of cell surface markers in addition to cytokine-based assays. In contrast to these published methods, we here describe a strategy to identify T cells that respond to any antigen and track the fate of these activated T cells. We constructed a retroviral double-reporter construct with enhanced green fluorescence protein (EGFP and a far-red fluorescent protein from Heteractis crispa (HcRed. LTR-driven EGFP expression was used to enrich and identify transduced cells, while HcRed expression is driven by the CD40Ligand (CD40L promoter, which is inducible and enables the identification and cell fate tracing of T cells that have responded to infection/inflammation. Pax5 deficient pro-B cells that can give rise to different hematopoietic cells like T cells, were retrovirally transduced with this double-reporter cassette and were used to reconstitute the T cell pool in RAG1 deficient mice that lack T and B cells. By using flow cytometry and histology, we identified activated T cells that had developed from Pax5 deficient pro-B cells and responded to infection with the bacterial pathogen Listeria monocytogenes. Microscopic examination of organ sections allowed visual identification of HcRed-expressing cells. To further characterize the immune response to a given stimuli, this strategy can be easily adapted to identify other cells of the hematopoietic system that respond to infection/inflammation. This can be achieved by using an inducible reporter, choosing the appropriate promoter, and reconstituting mice lacking cells of interest by injecting gene-modified Pax5 deficient pro-B cells.

  12. Engineering CAR-T cells.

    Science.gov (United States)

    Zhang, Cheng; Liu, Jun; Zhong, Jiang F; Zhang, Xi

    2017-01-01

    Chimeric antigen receptor redirected T cells (CAR-T cells) have achieved inspiring outcomes in patients with B cell malignancies, and are now being investigated in other hematologic malignancies and solid tumors. CAR-T cells are generated by the T cells from patients' or donors' blood. After the T cells are expanded and genetically modified, they are reinfused into the patients. However, many challenges still need to be resolved in order for this technology to gain widespread adoption. In this review, we first discuss the structure and evolution of chimeric antigen receptors. We then report on the tools used for production of CAR-T cells. Finally, we address the challenges posed by CAR-T cells.

  13. Critical biological parameters modulate affinity as a determinant of function in T-cell receptor gene-modified T-cells.

    Science.gov (United States)

    Spear, Timothy T; Wang, Yuan; Foley, Kendra C; Murray, David C; Scurti, Gina M; Simms, Patricia E; Garrett-Mayer, Elizabeth; Hellman, Lance M; Baker, Brian M; Nishimura, Michael I

    2017-11-01

    T-cell receptor (TCR)-pMHC affinity has been generally accepted to be the most important factor dictating antigen recognition in gene-modified T-cells. As such, there is great interest in optimizing TCR-based immunotherapies by enhancing TCR affinity to augment the therapeutic benefit of TCR gene-modified T-cells in cancer patients. However, recent clinical trials using affinity-enhanced TCRs in adoptive cell transfer (ACT) have observed unintended and serious adverse events, including death, attributed to unpredicted off-tumor or off-target cross-reactivity. It is critical to re-evaluate the importance of other biophysical, structural, or cellular factors that drive the reactivity of TCR gene-modified T-cells. Using a model for altered antigen recognition, we determined how TCR-pMHC affinity influenced the reactivity of hepatitis C virus (HCV) TCR gene-modified T-cells against a panel of naturally occurring HCV peptides and HCV-expressing tumor targets. The impact of other factors, such as TCR-pMHC stabilization and signaling contributions by the CD8 co-receptor, as well as antigen and TCR density were also evaluated. We found that changes in TCR-pMHC affinity did not always predict or dictate IFNγ release or degranulation by TCR gene-modified T-cells, suggesting that less emphasis might need to be placed on TCR-pMHC affinity as a means of predicting or augmenting the therapeutic potential of TCR gene-modified T-cells used in ACT. A more complete understanding of antigen recognition by gene-modified T-cells and a more rational approach to improve the design and implementation of novel TCR-based immunotherapies is necessary to enhance efficacy and maximize safety in patients.

  14. First experiences with the AMERLEX-MAB FREE T4 assay

    International Nuclear Information System (INIS)

    Nijhof, W.A.; Penders, T.J.

    1989-01-01

    The new Amerlex-MAB FT 4 is a quick direct free T 4 assay with good reproducability. The correlation between the Amerlex-MAB FT 4 and the free T 4 of Byk is good. In the non-thyreoidal illness patient group no deviation for the values were found. Amerlex-MAB FT 4 is cheaper, because no total T4 has to be measured. More research has to be done for special patient sera. Disturbing influences as free fatty acids, heparin and auto-antibodies have to be checked. (R.B.). 3 refs.; 3 figs.; 4 tabs

  15. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    International Nuclear Information System (INIS)

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A.

    2014-01-01

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication

  16. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Gagnon, David [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Gjoerup, Ole [Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA 02111 (United States); Archambault, Jacques [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Bullock, Peter A., E-mail: Peter.Bullock@tufts.edu [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States)

    2014-11-15

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication.

  17. Use of high throughput qPCR screening to rapidly clone low frequency tumour specific T-cells from peripheral blood for adoptive immunotherapy

    Directory of Open Access Journals (Sweden)

    Serrano Oscar K

    2008-10-01

    Full Text Available Abstract Background The adoptive transfer of autologous tumor reactive lymphocytes can mediate significant tumor regression in some patients with refractory metastatic cancer. However, a significant obstacle for this promising therapy has been the availability of highly efficient methods to rapidly isolate and expand a variety of potentially rare tumor reactive lymphocytes from the natural repertoire of cancer patients. Methods We developed a novel in vitro T cell cloning methodology using high throughput quantitative RT-PCR (qPCR assay as a rapid functional screen to detect and facilitate the limiting dilution cloning of a variety of low frequency T cells from bulk PBMC. In preclinical studies, this strategy was applied to the isolation and expansion of gp100 specific CD8+ T cell clones from the peripheral blood of melanoma patients. Results In optimization studies, the qPCR assay could detect the reactivity of 1 antigen specific T cell in 100,000 background cells. When applied to short term sensitized PBMC microcultures, this assay could detect T cell reactivity against a variety of known melanoma tumor epitopes. This screening was combined with early limiting dilution cloning to rapidly isolate gp100154–162 reactive CD8+ T cell clones. These clones were highly avid against peptide pulsed targets and melanoma tumor lines. They had an effector memory phenotype and showed significant proliferative capacity to reach cell numbers appropriate for adoptive transfer trials (~1010 cells. Conclusion This report describes a novel high efficiency strategy to clone tumor reactive T cells from peripheral blood for use in adoptive immunotherapy.

  18. MMSET deregulation affects cell cycle progression and adhesion regulons in t(4;14) myeloma plasma cells

    Science.gov (United States)

    Brito, Jose L.R.; Walker, Brian; Jenner, Matthew; Dickens, Nicholas J.; Brown, Nicola J.M.; Ross, Fiona M.; Avramidou, Athanasia; Irving, Julie A.E.; Gonzalez, David; Davies, Faith E.; Morgan, Gareth J.

    2009-01-01

    Background The recurrent immunoglobulin translocation, t(4;14)(p16;q32) occurs in 15% of multiple myeloma patients and is associated with poor prognosis, through an unknown mechanism. The t(4;14) up-regulates fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET) genes. The involvement of MMSET in the pathogenesis of t(4;14) multiple myeloma and the mechanism or genes deregulated by MMSET upregulation are still unclear. Design and Methods The expression of MMSET was analyzed using a novel antibody. The involvement of MMSET in t(4;14) myelomagenesis was assessed by small interfering RNA mediated knockdown combined with several biological assays. In addition, the differential gene expression of MMSET-induced knockdown was analyzed with expression microarrays. MMSET gene targets in primary patient material was analyzed by expression microarrays. Results We found that MMSET isoforms are expressed in multiple myeloma cell lines, being exclusively up-regulated in t(4;14)-positive cells. Suppression of MMSET expression affected cell proliferation by both decreasing cell viability and cell cycle progression of cells with the t(4;14) translocation. These findings were associated with reduced expression of genes involved in the regulation of cell cycle progression (e.g. CCND2, CCNG1, BRCA1, AURKA and CHEK1), apoptosis (CASP1, CASP4 and FOXO3A) and cell adhesion (ADAM9 and DSG2). Furthermore, we identified genes involved in the latter processes that were differentially expressed in t(4;14) multiple myeloma patient samples. Conclusions In conclusion, dysregulation of MMSET affects the expression of several genes involved in the regulation of cell cycle progression, cell adhesion and survival. PMID:19059936

  19. A Cancer Cell-Activatable Aptamer-Reporter System for One-Step Assay of Circulating Tumor Cells

    Directory of Open Access Journals (Sweden)

    Zihua Zeng

    2014-01-01

    Full Text Available The current antibody-mediated numeration assays of circulating tumor cells (CTCs require multiple steps and are time-consuming. To overcome these technical limitations, a cancer cell-activatable aptamer-reporter was formulated by conjugating a biomarker-specific aptamer sequence with paired fluorochrome-quencher molecules. In contrast to the antibody probes, the intact aptamer-reporter was optically silent in the absence of cells of interest. However, when used in an assay, the aptamer selectively targeted cancer cells through interaction with a specific surface biomarker, which triggered internalization of the aptamer-reporter and, subsequently, into cell lysosomes. Rapid lysosomal degradation of the aptamer-reporter resulted in separation of the paired fluorochrome-quencher molecules. The released fluorochrome emitted bright fluorescent signals exclusively within the targeted cancer cells, with no background noise in the assay. Thus, the assays could be completed in a single step within minutes. By using this one-step assay, CTCs in whole blood and marrow aspirate samples of patients with lymphoma tumors were selectively highlighted and rapidly detected with no off-target signals from background blood cells. The development of the cancer cell-activatable aptamer-reporter system allows for the possibility of a simple and robust point-of-care test for CTC detection, which is currently unavailable.

  20. Narcolepsy Type 1 Is Associated with a Systemic Increase and Activation of Regulatory T Cells and with a Systemic Activation of Global T Cells.

    Directory of Open Access Journals (Sweden)

    Michel Lecendreux

    Full Text Available Narcolepsy is a rare neurologic disorder characterized by excessive daytime sleepiness, cataplexy and disturbed nocturnal sleep patterns. Narcolepsy type 1 (NT1 has been shown to result from a selective loss of hypothalamic hypocretin-secreting neurons with patients typically showing low CSF-hypocretin levels (<110 pg/ml. This specific loss of hypocretin and the strong association with the HLA-DQB1*06:02 allele led to the hypothesis that NT1 could be an immune-mediated pathology. Moreover, susceptibility to NT1 has recently been associated with several pathogens, particularly with influenza A H1N1 virus either through infection or vaccination. The goal of this study was to compare peripheral blood immune cell populations in recent onset pediatric NT1 subjects (post or non-post 2009-influenza A H1N1 vaccination to healthy donors. We demonstrated an increased number of central memory CD4+ T cells (CD62L+ CD45RA- associated to an activated phenotype (increase in CD69 and CD25 expression in NT1 patients. Percentage and absolute count of regulatory T cells (Tregs in NT1 patients were increased associated with an activated phenotype (increase in GITR and LAP expression, and of activated memory phenotype. Cytokine production by CD4+ and CD8+ T cells after activation was not modified in NT1 patients. In H1N1 vaccinated NT1 patients, absolute counts of CD3+, CD8+ T cells, and B cells were increased compared to non-vaccinated NT1 patients. These results support a global T cell activation in NT1 patients and thus support a T cell-mediated autoimmune origin of NT1, but do not demonstrate the pathological role of H1N1 prophylactic vaccination. They should prompt further studies of T cells, particularly of Tregs (such as suppression and proliferation antigen specific assays, and also T-cell receptor sequencing, in NT1.

  1. Antibodies to the human T-cell lymphoma/leukemia virus type I in Dutch haemophiliacs

    NARCIS (Netherlands)

    Goudsmit, J.; Miedema, F.; Breederveld, C.; Terpstra, F.; Roos, M.; Schellekens, P.; Melief, C.

    1986-01-01

    95 Dutch haemophiliacs were tested for antibodies to membrane antigens on cells infected with human T-cell leukemia virus type I (HTLV-I-MA) by indirect immunofluorescence and to purified HTLV-I by enzyme-linked immunosorbent assay. Antibodies to HTLV-I-MA were present in 8 of 95 (8%) haemophiliacs,

  2. Multifunctional chitosan/polyvinyl pyrrolidone/45S5 Bioglass® scaffolds for MC3T3-E1 cell stimulation and drug release

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Qingqing [Institute of Advanced Materials for Nano-Bio Applications, School of Ophthalmology & Optometry, Wenzhou Medical University, 270 Xueyuan Xi Road, Wenzhou, Zhejiang 325027 (China); Li, Wei [Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Cauerstrasse 6, Erlangen 91058 (Germany); Yu, Shanshan; Ma, Liwei [Institute of Advanced Materials for Nano-Bio Applications, School of Ophthalmology & Optometry, Wenzhou Medical University, 270 Xueyuan Xi Road, Wenzhou, Zhejiang 325027 (China); Jin, Dayong [Institute for Biomedical Materials and Devices, Faculty of Science, University of Technology Sydney, NSW 2007 (Australia); Advanced Cytometry Labs, ARC Center of Excellence for Nanoscale BioPhotonics, Macquarie University, Sydney, NSW 2109 (Australia); Boccaccini, Aldo R., E-mail: Aldo.Boccaccini@ww.uni-erlangen.de [Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Cauerstrasse 6, Erlangen 91058 (Germany); Liu, Yong, E-mail: yongliu1980@hotmail.com [Institute of Advanced Materials for Nano-Bio Applications, School of Ophthalmology & Optometry, Wenzhou Medical University, 270 Xueyuan Xi Road, Wenzhou, Zhejiang 325027 (China); Advanced Cytometry Labs, ARC Center of Excellence for Nanoscale BioPhotonics, Macquarie University, Sydney, NSW 2109 (Australia)

    2015-11-01

    Novel chitosan–polyvinyl pyrrolidone/45S5 Bioglass® (CS-PVP/BG) scaffolds were prepared via foam replication and chemical cross-linking techniques. The pristine BG, CS-PVP coated BG and genipin cross-linked CS-PVP/BG (G-CS-PVP/BG) scaffolds were synthesized and characterized in terms of chemical composition, physical structure and morphology respectively. Resistance to enzymatic degradation of the scaffold is improved significantly with the use of genipin cross-linked CS-PVP. The bio-effects of scaffolds on MC3T3-E1 osteoblast-like cells were evaluated by studying cell viability, adhesion and proliferation. The CCK-8 assay shows that cell viability on the resulting G-CS-PVP/BG scaffold is improved obviously after cross-linking of genipin. Cell skeleton images exhibit that well-stretched F-actin bundles are obtained on the G-CS-PVP/BG scaffold. SEM results present significant improvement on the cell adhesion and proliferation for cells cultured on the G-CS-PVP/BG scaffold. The drug release performance on the as-synthesized scaffold was studied in a phosphate buffered saline (PBS) solution. Vancomycin is found to be released in burst fashion within 24 h from the pristine BG scaffold, however, the release period from the G-CS-PVP/BG scaffold is enhanced to 7 days, indicating improved drug release properties of the G-CS-PVP/BG scaffold. Our results suggest that the G-CS-PVP/BG scaffolds possess promising physicochemical properties, sustained drug release capability and good biocompatibility for MC3T3-E1 cells' proliferation and adhesion, suggesting their potential applications in areas such as MC3T3-E1 cell stimulation and bone tissue engineering. - Highlights: • Novel genipi–chitosan–polyvinyl pyrrolidone/45S5 Bioglass® scaffolds are prepared. • Resistance to enzymatic degradation of the scaffold is improved significantly. • The resulting scaffold shows enhanced MC3T3-E1 cell adhesion and proliferation. • Release of antibiotic vancomycin from the

  3. Multifunctional chitosan/polyvinyl pyrrolidone/45S5 Bioglass® scaffolds for MC3T3-E1 cell stimulation and drug release

    International Nuclear Information System (INIS)

    Yao, Qingqing; Li, Wei; Yu, Shanshan; Ma, Liwei; Jin, Dayong; Boccaccini, Aldo R.; Liu, Yong

    2015-01-01

    Novel chitosan–polyvinyl pyrrolidone/45S5 Bioglass® (CS-PVP/BG) scaffolds were prepared via foam replication and chemical cross-linking techniques. The pristine BG, CS-PVP coated BG and genipin cross-linked CS-PVP/BG (G-CS-PVP/BG) scaffolds were synthesized and characterized in terms of chemical composition, physical structure and morphology respectively. Resistance to enzymatic degradation of the scaffold is improved significantly with the use of genipin cross-linked CS-PVP. The bio-effects of scaffolds on MC3T3-E1 osteoblast-like cells were evaluated by studying cell viability, adhesion and proliferation. The CCK-8 assay shows that cell viability on the resulting G-CS-PVP/BG scaffold is improved obviously after cross-linking of genipin. Cell skeleton images exhibit that well-stretched F-actin bundles are obtained on the G-CS-PVP/BG scaffold. SEM results present significant improvement on the cell adhesion and proliferation for cells cultured on the G-CS-PVP/BG scaffold. The drug release performance on the as-synthesized scaffold was studied in a phosphate buffered saline (PBS) solution. Vancomycin is found to be released in burst fashion within 24 h from the pristine BG scaffold, however, the release period from the G-CS-PVP/BG scaffold is enhanced to 7 days, indicating improved drug release properties of the G-CS-PVP/BG scaffold. Our results suggest that the G-CS-PVP/BG scaffolds possess promising physicochemical properties, sustained drug release capability and good biocompatibility for MC3T3-E1 cells' proliferation and adhesion, suggesting their potential applications in areas such as MC3T3-E1 cell stimulation and bone tissue engineering. - Highlights: • Novel genipi–chitosan–polyvinyl pyrrolidone/45S5 Bioglass® scaffolds are prepared. • Resistance to enzymatic degradation of the scaffold is improved significantly. • The resulting scaffold shows enhanced MC3T3-E1 cell adhesion and proliferation. • Release of antibiotic vancomycin from the

  4. Nonmalignant T cells stimulate growth of T-cell lymphoma cells in the presence of bacterial toxins

    DEFF Research Database (Denmark)

    Woetmann, Anders; Lovato, Paola; Eriksen, Karsten W

    2007-01-01

    Bacterial toxins including staphylococcal enterotoxins (SEs) have been implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCLs). Here, we investigate SE-mediated interactions between nonmalignant T cells and malignant T-cell lines established from skin and blood of CTCL patients....... The malignant CTCL cells express MHC class II molecules that are high-affinity receptors for SE. Although treatment with SE has no direct effect on the growth of the malignant CTCL cells, the SE-treated CTCL cells induce vigorous proliferation of the SE-responsive nonmalignant T cells. In turn, the nonmalignant...... T cells enhance proliferation of the malignant cells in an SE- and MHC class II-dependent manner. Furthermore, SE and, in addition, alloantigen presentation by malignant CTCL cells to irradiated nonmalignant CD4(+) T-cell lines also enhance proliferation of the malignant cells. The growth...

  5. Regulatory activity of azabisphosphonate-capped dendrimers on human CD4+ T cell proliferation enhances ex-vivo expansion of NK cells from PBMCs for immunotherapy

    Directory of Open Access Journals (Sweden)

    Caminade Anne-Marie

    2009-09-01

    Full Text Available Abstract Background Adoptive cell therapy with allogenic NK cells constitutes a promising approach for the treatment of certain malignancies. Such strategies are currently limited by the requirement of an efficient protocol for NK cell expansion. We have developed a method using synthetic nanosized phosphonate-capped dendrimers allowing such expansion. We are showing here that this is due to a specific inhibitory activity towards CD4+ T cell which could lead to further medical applications of this dendrimer. Methods Mononuclear cells from human peripheral blood were used to investigate the immunomodulatory effects of nanosized phosphonate-capped dendrimers on interleukin-2 driven CD4+T cell expansion. Proliferation status was investigated using flow cytometry analysis of CFSE dilution and PI incorporation experiments. Magnetic bead cell sorting was used to address activity towards individual or mixed cell sub-populations. We performed equilibrium binding assay to assess the interaction of fluorescent dendrimers with pure CD4+ T cells. Results Phosphonate-capped dendrimers are inhibiting the activation, and therefore the proliferation; of CD4+ T cells in IL-2 stimulated PBMCs, without affecting their viability. This allows a rapid enrichment of NK cells and further expansion. We found that dendrimer acts directly on T cells, as their regulatory property is maintained when stimulating purified CD4+ T cells with anti-CD3/CD28 microbeads. Performing equilibrium binding assays using a fluorescent analogue, we show that the phosphonate capped-dendrimers are specifically interacting with purified CD4+ T cells. Ultimately, we found that our protocol prevents the IL-2 related expansion of regulatory T cells that would be deleterious for the activity of infused NK cells. Conclusion High yield expansion of NK cells from human PBMCs by phosphonate-capped dendrimers and IL-2 occurs through the specific inhibition of the CD4+ lymphocyte compartment. Given the

  6. Developmental heterogeneity in DNA packaging patterns influences T-cell activation and transmigration.

    Directory of Open Access Journals (Sweden)

    Soumya Gupta

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

  7. Newborn Screening for Severe Combined Immunodeficiency-A History of the TREC Assay

    Directory of Open Access Journals (Sweden)

    Mary T. Bausch-Jurken

    2017-06-01

    Full Text Available Infants born with T cell lymphopenias, especially severe combined immunodeficiency (SCID are at risk for serious, often fatal infections without intervention within the first year or two of life. The majority of these disorders can be detected through the use of the T cell recombination excision circle assay (TREC assay. The TREC assay detects the presence of non-replicating, episomal DNA that is formed during T cell development. This assay initially developed to measure thymic output during aging and HIV infection, has undergone modifications for the purpose of newborn screening (NBS for SCID. To meet the requirements for inclusion on NBS panels, the assay needed to utilize blood from dried blood spots on NBS cards, and be both sensitive and specific, avoiding the costs of false positives. Currently, the assay relies upon real time, quantitative PCR (RT-qPCR to detect TRECs in punches taken from dried blood spots. This review seeks to highlight some of the early work leading up to the initial implementation of the TREC assay for SCID detection, and the subsequent revisions made to optimize the assay.

  8. Induction of Mitochondria Mediated Apoptosis in Human Breast Cancer Cells (T-47D) by Annona reticulata L. Leaves Methanolic Extracts.

    Science.gov (United States)

    Roham, Pratiksha H; Kharat, Kiran R; Mungde, Priyanka; Jadhav, Mahadev A; Makhija, Surinder J

    2016-01-01

    Annona reticulata Linn. (Common name: Bullock's-heart) (Annonaceae family) is a semi-evergreen and small deciduous tree. The extracts of various parts of Annona reticulata L. have been reported as cytotoxic to many cancer cells. Annona reticulata L. leaves' methanolic extract (ARME) was prepared and used against the breast cancer cells. The breast cancer cells (T-47D) viability and IC50 were evaluated by Vybrant® MTT Cell Proliferation Assay Kit. Detection of phosphatidylserine on membranes of apoptotic cells was done by Attune flow cytometer. RNA transcripts were quantified in ARME treated and untreated cells. Finally, the Vybrant® FAM Poly Caspases assay kit was used for analysis of polycaspases activity in T-47D cells. The IC50 (5 ± 0.5 µg/mL) of the ARME was found against breast cancer cells (T-47D). The Paclitaxel was used as a control standard drug for the study. The downregulation of Bcl-2 and upregulation of Bax and Bak, and caspases activation suggested induction of apoptosis in T-47D cells by ARME through mitochondrial pathway. The cell cycle halted at G2/M phase in the ARME treated cells. The ARME was found to be effective against Breast cancer cells (T-47D).

  9. Application of liquid-based cytology preparation in micronucleus assay of exfoliated buccal epithelial cells in road construction workers.

    Science.gov (United States)

    Arul, P

    2017-01-01

    Asphalts are bitumens that consist of complex of hydrocarbon mixtures and it is used mainly in road construction and maintenance. This study was undertaken to evaluate the micronucleus (MN) assay of exfoliated buccal epithelial cells in road construction workers using liquid-based cytology (LBC) preparation. Three different stains (May-Grunwald Giemsa, hematoxylin and eosin, and Papanicolaou) were used to evaluate the frequency of MN in exfoliated buccal epithelial of 100 participants (fifty road construction workers and fifty administrative staff) using LBC preparation. Statistical analysis was performed with Student's t-test, and Proad construction exhibit a higher frequency of MN in exfoliated buccal epithelial cells and they are under the significant risk of cytogenetic damage. LBC preparation has potential application for the evaluation of frequency of MN. This technique may be advocated in those who are occupationally exposed to potentially carcinogenic agents in view of improvement in the smear quality and visualization of cell morphology.

  10. CD52 expression on CD4+ T cells in HIV-positive individuals on cART

    DEFF Research Database (Denmark)

    Vojdeman, Fie Juhl; Gaardbo, Julie Christine; Hartling, Hans Jakob

    2018-01-01

    BACKGROUND: Human immune defect virus (HIV) persists in a latent state in quiescent CD4+ T cells preventing eradication of HIV. CD52 is a surface molecule modulated by HIV. We aimed at examining factors related to CD52 expression on CD4+ T cells in HIV-positive individuals and the impact...... of initiation of combination antiretroviral therapy (cART). METHODS: Peripheral blood mononuclear cells (PBMC) from 18 HIV-positive individuals and 10 uninfected age and gender matched controls were examined by flow cytometry for CD38 and CD52 expression on CD4+ T cells. Stimulation assays were performed on 8...... healthy blood donors to determine a cut-off for CD52 expression. RESULTS: All examined CD4+ T cells expressed CD52. However, both CD4+ T cells with higher (CD52++) and with lower CD52 expression (CD52dim) were found in HIV-positive individuals compared to uninfected controls. Two % CD52dim cells defined...

  11. Standardization of the CFU-GM assay: Advantages of plating a fixed number of CD34+ cells in collagen gels.

    Science.gov (United States)

    Dobo, Irène; Pineau, Danielle; Robillard, Nelly; Geneviève, Frank; Piard, Nicole; Zandecki, Marc; Hermouet, Sylvie

    2003-10-01

    We investigated whether plating a stable amount of CD34(+) cells improves the CFU-GM assay. Data of CFU-GM assays performed with leukaphereses products in two transplant centers using a commercial collagen-based medium and unified CFU-GM scoring criteria were pooled and analyzed according to the numbers of CD34(+) cells plated. A first series of 113 CFU-GM assays was performed with a fixed number of mononuclear cells (i.e., a variable number of CD34(+) cells). In these cultures the CFU-GM/CD34 ratio varied according to the number of CD34(+) cells plated: median CFUGM/CD34 ratios were 1/6.2 to 1/6.6 for grafts containing or =2% CD34(+) cells. The median CFU-GM/CD34 ratio also varied depending on pathology: 1/9.3 for multiple myeloma (MM), 1/6.8 for Hodgkin's disease (HD), 1/6.5 for non-Hodgkin lymphoma (NHL), and 1/4.5 for solid tumors (ST). A second series of 95 CFU-GM assays was performed with a fixed number of CD34(+) cells (220/ml). The range of median CFU-GM/CD34 ratios was narrowed to 1/7.0 to 1/5.2, and coefficients of variation for CFU-GM counts decreased by half to 38.1% (NHL), 36.1% (MM), 49.9% (HD), and 22.4% (ST). In addition, CFU-GM scoring was facilitated as the percentages of cultures with >50 CFU/GM/ml decreased from 6.7% to 43.8% when a variable number of CD34(+) cells was plated, to 4.5% to 16.7% when 220 CD34(+) cells/ml were plated. Hence, plating a fixed number of CD34(+) cells in collagen gels improves the CFU-GM assay by eliminating cell number-related variability and reducing pathology-related variability in colony growth.

  12. Cell adhesion-mediated radioresistance (CAM-RR). Extracellular matrix-dependent improvement of cell survival in human tumor and normal cells in vitro

    International Nuclear Information System (INIS)

    Cordes, N.; Meineke, V.

    2003-01-01

    Background: Cell-extracellular matrix (ECM) contact is thought to have great impact on cellular mechanisms resulting in increased cell survival upon exposure to ionizing radiation. Several human tumor cell lines and normal human fibroblastic cell strains of different origin, all of them expressing the wide-spread and important integrin subunit β1, were irradiated, and clonogenic cell survival, β1-integrin cell surface expression, and adhesive functionality were investigated. Material and Methods: Human tumor cell lines A172 (glioblastoma), PATU8902 (pancreas carcinoma), SKMES1 (lung carcinoma), A549 (lung carcinoma), and IPC298 (melanoma) as well as normal human skin (HSF1) and lung fibroblasts (CCD32) and human keratinocytes (HaCaT) were irradiated with 0-8 Gy. Besides colony formation assays, β1-integrin cell surface expression by flow cytometry and adhesive functionality by adhesion assays were analyzed. Results: All cell lines showed improved clonogenic survival after irradiation in the presence of fibronectin as compared to plastic. Irradiated cells exhibited a significant, dose-dependent increase in β1-integrin cell surface expression following irradiation. As a parameter of the adhesive functionality of the β1-integrin, a radiation-dependent elevation of cell adhesion to fibronectin in comparison with adhesion to plastic was demonstrated. Conclusion: The in vitro cellular radiosensitivity is highly influenced by fibronectin according to the phenomenon of cell adhesion-mediated radioresistance. Additionally, our emerging data question the results of former and current in vitro cytotoxicity studies performed in the absence of an ECM. These findings might also be important for the understanding of malignant transformation, anchorage-independent cell growth, optimization of radiotherapeutic regimes and the prevention of normal tissue side effects on the basis of experimental radiobiological data. (orig.)

  13. The production of KIR-Fc fusion proteins and their use in a multiplex HLA class I binding assay.

    Science.gov (United States)

    Hilton, Hugo G; Moesta, Achim K; Guethlein, Lisbeth A; Blokhuis, Jeroen; Parham, Peter; Norman, Paul J

    2015-10-01

    Soluble recombinant proteins that comprise the extracellular part of a surface expressed receptor attached to the Fc region of an IgG antibody have facilitated the determination of ligand specificity for an array of immune system receptors. Among such receptors is the family of killer cell immunoglobulin-like receptors (KIR) that recognize HLA class I ligands. These receptors, expressed on natural killer (NK) cells and T cells, play important roles in both immune defense and placental development in early pregnancy. Here we describe a method for the production of two domain KIR-Fc fusion proteins using baculovirus infected insect cells. This method is more scalable than traditional mammalian cell expression systems and produces efficiently folded proteins that carry posttranslational modifications found in native KIR. We also describe a multiplex binding assay using the Luminex platform that determines the avidity and specificity of two domain KIR-Fc for a panel of microbeads, each coated with one of 97 HLA class I allotypes. This assay is simple to perform, and represents a major improvement over the assays used previously, which were limited in the number of KIR and HLA class I combinations that could be assayed at any one time. The results obtained from this assay can be used to predict the response of NK cell and T cells when their KIR recognize HLA class I. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Role of very late antigen-1 in T-cell-mediated immunity to systemic viral infection

    DEFF Research Database (Denmark)

    Ørding Kauffmann, Susanne; Thomsen, Allan Randrup; Christensen, Jan Pravsgaard

    2006-01-01

    or their distribution between lymphoid and nonlymphoid organs. Regarding a functional role of VLA-1, we found that intracerebral infection of both VLA-1(-/-) and wild-type (wt) mice resulted in lethal T-cell-mediated meningitis, and quantitative and qualitative analyses of the cellular exudate did not reveal any...... significant differences between the two strains. Expression of VLA-1 was also found to be redundant regarding the ability of effector T cells to eliminate virus from internal organs of i.v. infected mice. Using delayed-type hypersensitivity (DTH) assays to evaluate subdermal CD8(+) T......, the current findings indicate that the expression of VLA-1 is not pivotal for T-cell-mediated antiviral immunity to a systemic infection....

  15. Cell based assays for anti-Plasmodium activity evaluation.

    Science.gov (United States)

    Mokgethi-Morule, Thabang; N'Da, David D

    2016-03-10

    Malaria remains one of the most common and deadly infectious diseases worldwide. The severity of this global public health challenge is reflected by the approximately 198 million people, who were reportedly infected in 2013 and by the more than 584,000 related deaths in that same year. The rising emergence of drug resistance towards the once effective artemisinin combination therapies (ACTs) has become a serious concern and warrants more robust drug development strategies, with the objective of eradicating malaria infections. The intricate biology and life cycle of Plasmodium parasites complicate the understanding of the disease in such a way that would enhance the development of more effective chemotherapies that would achieve radical clinical cure and that would prevent disease relapse. Phenotypic cell based assays have for long been a valuable approach and involve the screening and analysis of diverse compounds with regards to their activities towards whole Plasmodium parasites in vitro. To achieve the Millennium Development Goal (MDG) of malaria eradication by 2020, new generation drugs that are active against all parasite stages (erythrocytic (blood), exo-erythrocytic (liver stages and gametocytes)) are needed. Significant advances are being made in assay development to overcome some of the practical challenges of assessing drug efficacy, particularly in the liver and transmission stage Plasmodium models. This review discusses primary screening models and the fundamental progress being made in whole cell based efficacy screens of anti-malarial activity. Ongoing challenges and some opportunities for improvements in assay development that would assist in the discovery of effective, safe and affordable drugs for malaria treatments are also discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Linearization of the Bradford Protein Assay

    OpenAIRE

    Ernst, Orna; Zor, Tsaffrir

    2010-01-01

    Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, t...

  17. Neuron-mediated generation of regulatory T cells from encephalitogenic T cells suppresses EAE

    DEFF Research Database (Denmark)

    Liu, Yawei; Teige, Ingrid; Birnir, Bryndis

    2006-01-01

    Neurons have been neglected as cells with a major immune-regulatory function because they do not express major histocompatibility complex class II. Our data show that neurons are highly immune regulatory, having a crucial role in governing T-cell response and central nervous system (CNS) inflamma......Neurons have been neglected as cells with a major immune-regulatory function because they do not express major histocompatibility complex class II. Our data show that neurons are highly immune regulatory, having a crucial role in governing T-cell response and central nervous system (CNS......) inflammation. Neurons induce the proliferation of activated CD4+ T cells through B7-CD28 and transforming growth factor (TGF)-beta1-TGF-beta receptor signaling pathways, resulting in amplification of T-cell receptor signaling through phosphorylated ZAP-70, interleukin (IL)-2 and IL-9. The interaction between...... neurons and T cells results in the conversion of encephalitogenic T cells to CD25+ TGF-beta1+ CTLA-4+ FoxP3+ T regulatory (Treg) cells that suppress encephalitogenic T cells and inhibit experimental autoimmune encephalomyelitis. Suppression is dependent on cytotoxic T lymphocyte antigen (CTLA)-4...

  18. Decline of influenza-specific CD8+ T cell repertoire in healthy geriatric donors

    Directory of Open Access Journals (Sweden)

    Ramachandra Lakshmi

    2011-08-01

    Full Text Available Abstract Background While influenza vaccination results in protective antibodies against primary infections, clearance of infection is primarily mediated through CD8+ T cells. Studying the CD8+ T cell response to influenza epitopes is crucial in understanding the disease associated morbidity and mortality especially in at risk populations such as the elderly. We compared the CD8+ T cell response to immunodominant and subdominant influenza epitopes in HLA-A2+ control, adult donors, aged 21-42, and in geriatric donors, aged 65 and older. Results We used a novel artificial Antigen Presenting Cell (aAPC based stimulation assay to reveal responses that could not be detected by enzyme-linked immunosorbent spot (ELISpot. 14 younger control donors and 12 geriatric donors were enrolled in this study. The mean number of influenza-specific subdominant epitopes per control donor detected by ELISpot was only 1.4 while the mean detected by aAPC assay was 3.3 (p = 0.0096. Using the aAPC assay, 92% of the control donors responded to at least one subdominant epitopes, while 71% of control donors responded to more than one subdominant influenza-specific response. 66% of geriatric donors lacked a subdominant influenza-specific response and 33% of geriatric donors responded to only 1 subdominant epitope. The difference in subdominant response between age groups is statistically significant (p = 0.0003. Conclusion Geriatric donors lacked the broad, multi-specific response to subdominant epitopes seen in the control donors. Thus, we conclude that aging leads to a decrease in the subdominant influenza-specific CTL responses which may contribute to the increased morbidity and mortality in older individuals.

  19. Cytotoxic Effect of the Genus Sinularia Extracts on Human SCC25 and HaCaT Cells

    International Nuclear Information System (INIS)

    Wang, G.H.; Chou, T.H.; Liang, C.H.; Lin, R.J.; Sheu, J.H.; Wang, S.H.

    2009-01-01

    Soft corals of the genus Sinularia are being increasingly adopted to treat a wide variety of disease processes. However, the mechanism underlying its activity against human oral cancer cells is poorly understood. This study evaluates the cyototoxicity effects of the genus Sinularia extracts (S. grandilobata, S. parva, S. triangula, S. scabra, S. nanolobata and S. gibberosa) by SCC25 and HaCaT cells. The cell adhesion assay indicates that extracts reduce the cell attachment. Extracts exhibit a dose-dependent cytotoxic effect using MTS assay.Treatment of extracts to observe the morphological alterations in cells, membrane blebbing, nuclear condensation, and apoptotic bodies is demonstrated. Flow cytometry shows that extracts sensitized the cells in the G0/G1 and G2/M phases with a concomitant significantly increased sub-G1 fraction, suggesting cell death by apoptosis. Extracts of the genus Sinularia thus apparently cause apoptosis of SCC25 and HaCaT cells, and warrant further research investigating the possible antioral cancer compounds in these soft corals.

  20. Genome-Based In Silico Identification of New Mycobacterium tuberculosis Antigens Activating Polyfunctional CD8+ T Cells in Human Tuberculosis

    DEFF Research Database (Denmark)

    Tang, Sheila Tuyet; van Meijgaarden, Krista E.; Caccamo, Nadia

    2011-01-01

    8(+) T cell proliferation assays (CFSE dilution) in 41 M. tuberculosis-responsive donors identified 70 new M. tuberculosis epitopes. Using HLA/peptide tetramers for the 18 most prominently recognized HLA-A*0201-binding M. tuberculosis peptides, recognition by cured TB patients' CD8(+) T cells......-epitope/Ag repertoire for human CD8(+) T cells is much broader than hitherto suspected, and the newly identified M. tuberculosis Ags are recognized by (poly) functional CD8(+) T cells during control of infection. These results impact on TB-vaccine design and biomarker identification. The Journal of Immunology, 2011...

  1. The immune checkpoint regulator PD-L1 is a specific target for naturally occurring CD4(+) T cells

    DEFF Research Database (Denmark)

    Munir, Shamaila; Andersen, Gitte Holmen; Svane, Inge Marie

    2013-01-01

    Programmed cell death 1 ligand 1 (PD-L1) is an important regulator of T-cell responses and may consequently limit anticancer immunity. We have recently identified PD-L1-specific, cytotoxic CD8(+) T cells. In the present study, we develop these findings and report that CD4(+) helper T cells...... spontaneously recognize PD-L1. We examined the locality of a previously identified HLA-A*0201-restricted PD-L1-epitope for the presence of possible CD4(+) T-cell epitopes. Thus, we identified naturally occurring PD-L1-specific CD4(+) T cells among the peripheral blood lymphocytes of cancer patients...... and - to lesser extents - healthy donors, by means of ELISPOT assays. PD-L1-specific CD4(+) T cells appeared to be TH17 cells exhibiting an effector T-cell cytokine profile. Hence, PD-L1-specific CD4(+) T cells released interferon γ (IFNγ), tumor necrosis factor α (TNFα) and interleukin-17 (IL-17) in response...

  2. CD4+ T cell effects on CD8+ T cell location defined using bioluminescence.

    Directory of Open Access Journals (Sweden)

    Mitra Azadniv

    2011-01-01

    Full Text Available T lymphocytes of the CD8+ class are critical in delivering cytotoxic function and in controlling viral and intracellular infections. These cells are "helped" by T lymphocytes of the CD4+ class, which facilitate their activation, clonal expansion, full differentiation and the persistence of memory. In this study we investigated the impact of CD4+ T cells on the location of CD8+ T cells, using antibody-mediated CD4+ T cell depletion and imaging the antigen-driven redistribution of bioluminescent CD8+ T cells in living mice. We documented that CD4+ T cells influence the biodistribution of CD8+ T cells, favoring their localization to abdominal lymph nodes. Flow cytometric analysis revealed that this was associated with an increase in the expression of specific integrins. The presence of CD4+ T cells at the time of initial CD8+ T cell activation also influences their biodistribution in the memory phase. Based on these results, we propose the model that one of the functions of CD4+ T cell "help" is to program the homing potential of CD8+ T cells.

  3. Phenotypic and functional profiling of CD4 T cell compartment in distinct populations of healthy adults with different antigenic exposure.

    Directory of Open Access Journals (Sweden)

    Sophie Roetynck

    Full Text Available Multiparameter flow cytometry has revealed extensive phenotypic and functional heterogeneity of CD4 T cell responses in mice and humans, emphasizing the importance of assessing multiple aspects of the immune response in correlation with infection or vaccination outcome. The aim of this study was to establish and validate reliable and feasible flow cytometry assays, which will allow us to characterize CD4 T cell population in humans in field studies more fully.We developed polychromatic flow cytometry antibody panels for immunophenotyping the major CD4 T cell subsets as well as broadly characterizing the functional profiles of the CD4 T cells in peripheral blood. We then validated these assays by conducting a pilot study comparing CD4 T cell responses in distinct populations of healthy adults living in either rural or urban Kenya. This study revealed that the expression profile of CD4 T cell activation and memory markers differed significantly between African and European donors but was similar amongst African individuals from either rural or urban areas. Adults from rural Kenya had, however, higher frequencies and greater polyfunctionality among cytokine producing CD4 T cells compared to both urban populations, particularly for "Th1" type of response. Finally, endemic exposure to malaria in rural Kenya may have influenced the expansion of few discrete CD4 T cell populations with specific functional signatures.These findings suggest that environmentally driven T cell activation does not drive the dysfunction of CD4 T cells but is rather associated with greater magnitude and quality of CD4 T cell response, indicating that the level or type of microbial exposure and antigenic experience may influence and shape the functionality of CD4 T cell compartment. Our data confirm that it is possible and mandatory to assess multiple functional attributes of CD4 T cell response in the context of infection.

  4. Prostate Cancer Cells Express More Androgen Receptor (AR) Following Androgen Deprivation, Improving Recognition by AR-Specific T Cells.

    Science.gov (United States)

    Olson, Brian M; Gamat, Melissa; Seliski, Joseph; Sawicki, Thomas; Jeffery, Justin; Ellis, Leigh; Drake, Charles G; Weichert, Jamey; McNeel, Douglas G

    2017-12-01

    Androgen deprivation is the primary therapy for recurrent prostate cancer, and agents targeting the androgen receptor (AR) pathway continue to be developed. Because androgen-deprivation therapy (ADT) has immmunostimulatory effects as well as direct antitumor effects, AR-targeted therapies have been combined with other anticancer therapies, including immunotherapies. Here, we sought to study whether an antigen-specific mechanism of resistance to ADT (overexpression of the AR) may result in enhanced AR-specific T-cell immune recognition, and whether this might be strategically combined with an antitumor vaccine targeting the AR. Androgen deprivation increased AR expression in human and murine prostate tumor cells in vitro and in vivo The increased expression persisted over time. Increased AR expression was associated with recognition and cytolytic activity by AR-specific T cells. Furthermore, ADT combined with vaccination, specifically a DNA vaccine encoding the ligand-binding domain of the AR, led to improved antitumor responses as measured by tumor volumes and delays in the emergence of castrate-resistant prostate tumors in two murine prostate cancer models (Myc-CaP and prostate-specific PTEN-deficient mice). Together, these data suggest that ADT combined with AR-directed immunotherapy targets a major mechanism of resistance, overexpression of the AR. This combination may be more effective than ADT combined with other immunotherapeutic approaches. Cancer Immunol Res; 5(12); 1074-85. ©2017 AACR . ©2017 American Association for Cancer Research.

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

    Science.gov (United States)

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

    2016-09-08

    Inflammation is paramount in pancreatic oncogenesis. We identified a uniquely activated γδT cell population, which constituted ∼40% of tumor-infiltrating T cells in human pancreatic ductal adenocarcinoma (PDA). Recruitment and activation of γδT cells was contingent on diverse chemokine signals. Deletion, depletion, or blockade of γδT cell recruitment was protective against PDA and resulted in increased infiltration, activation, and Th1 polarization of αβT cells. Although αβT cells were dispensable to outcome in PDA, they became indispensable mediators of tumor protection upon γδT cell ablation. PDA-infiltrating γδT cells expressed high levels of exhaustion ligands and thereby negated adaptive anti-tumor immunity. Blockade of PD-L1 in γδT cells enhanced CD4(+) and CD8(+) T cell infiltration and immunogenicity and induced tumor protection suggesting that γδT cells are critical sources of immune-suppressive checkpoint ligands in PDA. We describe γδT cells as central regulators of effector T cell activation in cancer via novel cross-talk. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Development of an androgen reporter gene assay (AR-LUX) utilizing a human cell line with an endogenously regulated androgen receptor

    NARCIS (Netherlands)

    Blankvoort, B.M.G.; Groene, E.M. de; Meeteren-Kreikamp, A.P. van; Witkamp, R.F.; Rodenburg, R.J.T.; Aarts, J.M.M.J.G.

    2001-01-01

    The aim of the work described in this report is to develop and characterize a cell-based androgen reporter assay. For this purpose, the androgen receptor (AR) expressing human breast cancer cell line T47D was stably transfected with a luciferase gene under transcriptional control of the PB-ARE-2

  7. Rapamycin sensitizes T-ALL cells to dexamethasone-induced apoptosis

    Directory of Open Access Journals (Sweden)

    Mu Dezhi

    2010-11-01

    Full Text Available Abstract Background Glucocorticoid (GC resistance is frequently seen in acute lymphoblastic leukemia of T-cell lineage (T-ALL. In this study we investigate the potential and mechanism of using rapamycin to restore the sensitivity of GC-resistant T-ALL cells to dexamethasone (Dex treatment. Methods Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl- 2,5-diphenyltetrazolium bromide (MTT assay. Fluorescence-activated cell sorting (FACS analysis was used to analyze apoptosis and cell cycles. Western blot analysis was performed to test the expression of the downstream effector proteins of mammalian target of rapamycin (mTOR, the cell cycle regulatory proteins, and apoptosis associated proteins. Results 10 nM rapamycin markedly increased GC sensitivity in GC-resistant T-ALL cells and this effect was mediated, at least in part, by inhibition of mTOR signaling pathway. Cell cycle arrest was associated with modulation of G1-S phase regulators. Both rapamycin and Dex can induce up-regulation of cyclin-dependent kinase (CDK inhibitors of p21 and p27 and co-treatment of rapamycin with Dex resulted in a synergistic induction of their expressions. Rapamycin did not obviously affect the expression of cyclin A, whereas Dex induced cyclin A expression. Rapamycin prevented Dex-induced expression of cyclin A. Rapamycin had a stronger inhibition of cyclin D1 expression than Dex. Rapamycin enhanced GC-induced apoptosis and this was not achieved by modulation of glucocorticoid receptor (GR expression, but synergistically up-regulation of pro-apoptotic proteins like caspase-3, Bax, and Bim, and down-regulation of anti-apoptotic protein of Mcl-1. Conclusion Our data suggests that rapamycin can effectively reverse GC resistance in T-ALL and this effect is achieved by inducing cell cycles arrested at G0/G1 phase and activating the intrinsic apoptotic program. Therefore, combination of mTOR inhibitor rapamycin with GC containing protocol might be an attracting

  8. A glimpse into the interactions of cells in a microenvironment: the modulation of T cells by mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Choi J

    2014-05-01

    Full Text Available Jonghoon Choi,1,2 Mintai P Hwang,3 Jong-Wook Lee,3 Kwan Hyi Lee3,41Institute of Research Strategy and Development (IRSD, Seoul, Republic of Korea; 2Department of Bionano Engineering, Hanyang University, Ansan, Republic of Korea; 3Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Republic of Korea; 4Department of Biomedical Engineering, University of Science and Technology, Seoul, Republic of KoreaAbstract: Mesenchymal stem cells (MSCs have been thought to hold potential as a mode of therapy for immuno-related pathologies, particularly for autoimmune diseases. Despite their potential, the interaction between MSCs and T cells, key players in the pathophysiology of autoimmune diseases, is not yet well understood, thereby preventing further clinical progress. A major obstacle is the highly heterogeneous nature of MSCs in vitro. Unfortunately, bulk assays do not provide information with regard to cell–cell contributions that may play a critical role in the overall cellular response. To address these issues, we investigated the interaction between smaller subsets of MSCs and CD4 T cells in a microwell array. We demonstrate that MSCs appear capable of modulating the T cell proliferation rate in response to persistent cell–cell interactions, and we anticipate the use of our microwell array in the classification of subpopulations within MSCs, ultimately leading to specific therapeutic interventions.Keywords: mesenchymal stem cell, T cell, microwell array

  9. Single-cell analysis reveals a link between CD3- and CD59-mediated signaling pathways in Jurkat T cells

    International Nuclear Information System (INIS)

    Lipp, A. M.

    2012-01-01

    Elevation of intracellular free calcium concentration ([Ca2+]i) is a key signal during T cell activation and is commonly used as a read-out parameter for stimulation of T cell signaling. Upon T cell stimulation a variety of calcium signals is produced by individual cells of the T cell population and the type of calcium signal strongly influences cell fate decisions. The heterogeneous nature of T cells is masked in ensemble measurements, which highlights the need for single-cell measurements. In this study we used single-cell calcium measurements in Jurkat cells to investigate signaling pathways, which are triggered by different proteins, namely CD3 and CD59. By application of an automated cluster algorithm the presented assay provides unbiased analysis of a large data set of individual calcium time traces generated by the whole cell population. By using this method we could demonstrate that the Jurkat population generates heterogeneous calcium signals in a stimulus-dependent manner. Furthermore, our data revealed the existence of a link between CD3- and CD59-mediated signaling pathways. Single-cell calcium measurements in Jurkat cells expressing different levels of the T cell receptor (TCR) complex indicated that CD59-mediated calcium signaling is critically dependent on TCR surface expression levels. In addition, triggering CD59-mediated calcium signaling resulted in down-regulation of TCR surface expression levels, which is known to happen upon direct TCR triggering too. Moreover, by using siRNA-mediated protein knock-downs and protein knock-out Jurkat mutants we could show that CD3- and CD59-mediated calcium signaling require identical key proteins. We therefore explored by which mechanism CD59-mediated signaling couples into TCR-mediated signaling. Fluorescence recovery after photobleaching (FRAP) experiments and live-cell protein-protein interaction assays provided no evidence of a direct physical interaction between CD3- and CD59-mediated signaling pathways

  10. Unraveling Natural Killer T-Cells Development

    Directory of Open Access Journals (Sweden)

    Sabrina Bianca Bennstein

    2018-01-01

    Full Text Available Natural killer T-cells are a subset of innate-like T-cells with the ability to bridge innate and adaptive immunity. There is great interest in harnessing these cells to improve tumor therapy; however, greater understanding of invariant NKT (iNKT cell biology is needed. The first step is to learn more about NKT development within the thymus. Recent studies suggest lineage separation of murine iNKT cells into iNKT1, iNKT2, and iNKT17 cells instead of shared developmental stages. This review will focus on these new studies and will discuss the evidence for lineage separation in contrast to shared developmental stages. The author will also highlight the classifications of murine iNKT cells according to identified transcription factors and cytokine production, and will discuss transcriptional and posttranscriptional regulations, and the role of mammalian target of rapamycin. Finally, the importance of these findings for human cancer therapy will be briefly discussed.

  11. Intra-laboratory validation of a human cell based in vitro angiogenesis assay for testing angiogenesis modulators

    Directory of Open Access Journals (Sweden)

    Jertta-Riina Sarkanen

    2011-01-01

    Full Text Available The developed standardized human cell based in vitro angiogenesis assay was intra-laboratory validated to verify that the method is reliable and relevant for routine testing of modulators of angiogenesis e.g. pharmaceuticals and industrial chemicals. This assay is based on the earlier published method but it was improved and shown to be more sensitive and rapid than the previous assay. The performance of the assay was assessed by using 6 reference chemicals, which are widely used pharmaceuticals that inhibit angiogenesis: acetyl salicylic acid, erlotinib, 2-methoxyestradiol, levamisole, thalidomide, and anti-vascular endothelial growth factor. In the intra-laboratory validation, the sensitivity of the assay (upper and lower limits of detection and linearity of response in tubule formation, batch to batch variation in tubule formation between different Master cell bank batches, and precision as well as the reliability of the assay (reproducibility and repeatability were tested. The pre-set acceptance criteria for the intra-laboratory validation study were met. The relevance of the assay in man was investigated by comparing the effects of reference chemicals and their concentrations to the published human data. The comparison showed a good concordance, which indicates that this human cell based angiogenesis model predicts well the effects in man and has the potential to be used to supplement and/or replace of animal tests.

  12. Human dendritic cells sequentially matured with CD4(+) T cells as a secondary signal favor CTL and long-term T memory cell responses.

    Science.gov (United States)

    Simon, Thomas; Tanguy-Royer, Séverine; Royer, Pierre-Joseph; Boisgerault, Nicolas; Frikeche, Jihane; Fonteneau, Jean-François; Grégoire, Marc

    2012-01-01

    Dendritic cells (DCs) are professional antigen-presenting cells involved in the control and initiation of immune responses. In vivo, DCs exposed at the periphery to maturation stimuli migrate to lymph nodes, where they receive secondary signals from CD4+ T helper cells. These DCs become able to initiate CD8+ cytotoxic T lymphocyte (CTL) responses. However, in vitro investigations concerning human monocyte-derived DCs have never focused on their functional properties after such sequential maturation. Here, we studied human DC phenotypes and functions according to this sequential exposure to maturation stimuli. As first signals, we used TNF-α/polyI:C mimicking inflammatory and pathogen stimuli and, as second signals, we compared activated CD4+ T helper cells to a combination of CD40-L/ IFN-γ. Our results show that a sequential activation with activated CD4+ T cells dramatically increased the maturation of DCs in terms of their phenotype and cytokine secretion compared to DCs activated with maturation stimuli delivered simultaneously. Furthermore, this sequential maturation led to the induction of CTL with a long-term effector and central memory phenotypes. Thus, sequential delivery of maturation stimuli, which includes CD4+ T cells, should be considered in the future to improve the induction of long-term CTL memory in DC-based immunotherapy.

  13. Characterization of CD4 T Cell Epitopes of Infliximab and Rituximab Identified from Healthy Donors

    Directory of Open Access Journals (Sweden)

    Moustafa Hamze

    2017-05-01

    Full Text Available The chimeric antibodies anti-CD20 rituximab (Rtx and anti-TNFα infliximab (Ifx induce antidrug antibodies (ADAs in many patients with inflammatory diseases. Because of the key role of CD4 T lymphocytes in the initiation of antibody responses, we localized the CD4 T cell epitopes of Rtx and Ifx. With the perspective to anticipate immunogenicity of therapeutic antibodies, identification of the CD4 T cell epitopes was performed using cells collected in healthy donors. Nine T cell epitopes were identified in the variable chains of both antibodies by deriving CD4 T cell lines raised against either Rtx or Ifx. The T cell epitopes often exhibited a good affinity for human leukocyte antigen (HLA-DR molecules and were part of the peptides identified by MHC-associated peptide proteomics assay from HLA-DR molecules of dendritic cells (DCs loaded with the antibodies. Two-third of the T cell epitopes identified from the healthy donors stimulated peripheral blood mononuclear cells from patients having developed ADAs against Rtx or Ifx and promoted the secretion of a diversity of cytokines. These data emphasize the predictive value of evaluating the T cell repertoire of healthy donors and the composition of peptides bound to HLA-DR of DCs to anticipate and prevent immunogenicity of therapeutic antibodies.

  14. Automated image-based assay for evaluation of HIV neutralization and cell-to-cell fusion inhibition.

    Science.gov (United States)

    Sheik-Khalil, Enas; Bray, Mark-Anthony; Özkaya Şahin, Gülsen; Scarlatti, Gabriella; Jansson, Marianne; Carpenter, Anne E; Fenyö, Eva Maria

    2014-08-30

    Standardized techniques to detect HIV-neutralizing antibody responses are of great importance in the search for an HIV vaccine. Here, we present a high-throughput, high-content automated plaque reduction (APR) assay based on automated microscopy and image analysis that allows evaluation of neutralization and inhibition of cell-cell fusion within the same assay. Neutralization of virus particles is measured as a reduction in the number of fluorescent plaques, and inhibition of cell-cell fusion as a reduction in plaque area. We found neutralization strength to be a significant factor in the ability of virus to form syncytia. Further, we introduce the inhibitory concentration of plaque area reduction (ICpar) as an additional measure of antiviral activity, i.e. fusion inhibition. We present an automated image based high-throughput, high-content HIV plaque reduction assay. This allows, for the first time, simultaneous evaluation of neutralization and inhibition of cell-cell fusion within the same assay, by quantifying the reduction in number of plaques and mean plaque area, respectively. Inhibition of cell-to-cell fusion requires higher quantities of inhibitory reagent than inhibition of virus neutralization.

  15. Improving shuffler assay accuracy

    International Nuclear Information System (INIS)

    Rinard, P.M.

    1995-01-01

    Drums of uranium waste should be disposed of in an economical and environmentally sound manner. The most accurate possible assays of the uranium masses in the drums are required for proper disposal. The accuracies of assays from a shuffler are affected by the type of matrix material in the drums. Non-hydrogenous matrices have little effect on neutron transport and accuracies are very good. If self-shielding is known to be a minor problem, good accuracies are also obtained with hydrogenous matrices when a polyethylene sleeve is placed around the drums. But for those cases where self-shielding may be a problem, matrices are hydrogenous, and uranium distributions are non-uniform throughout the drums, the accuracies are degraded. They can be greatly improved by determining the distributions of the uranium and then applying correction factors based on the distributions. This paper describes a technique for determining uranium distributions by using the neutron count rates in detector banks around the waste drum and solving a set of overdetermined linear equations. Other approaches were studied to determine the distributions and are described briefly. Implementation of this correction is anticipated on an existing shuffler next year

  16. Supernatural T cells: genetic modification of T cells for cancer therapy.

    Science.gov (United States)

    Kershaw, Michael H; Teng, Michele W L; Smyth, Mark J; Darcy, Phillip K

    2005-12-01

    Immunotherapy is receiving much attention as a means of treating cancer, but complete, durable responses remain rare for most malignancies. The natural immune system seems to have limitations and deficiencies that might affect its ability to control malignant disease. An alternative to relying on endogenous components in the immune repertoire is to generate lymphocytes with abilities that are greater than those of natural T cells, through genetic modification to produce 'supernatural' T cells. This Review describes how such T cells can circumvent many of the barriers that are inherent in the tumour microenvironment while optimizing T-cell specificity, activation, homing and antitumour function.

  17. [Effects of sintered bone modified with surface mineralization/P24 peptide composite biomaterial on the adhesion, proliferation and osteodifferentiation of MC3T3-E1 cells].

    Science.gov (United States)

    Li, Jingfeng; Zheng, Qixin; Guo, Xiaodong; Chen, Liaobin

    2014-10-01

    In the present research, the effects of sintered bone modified with surface mineralization/P24 peptide composite biomaterials on the adhesion, proliferation and osteodifferentiation of MC3T3-E1 cells were investigated. The experiments were divided into three groups due to biomaterials used: Group A (composite materials of sintered bone modified with surface mineralization and P24, a peptide of bone morphogenetic protein-2); Group B (sintered bone modified with surface mineralization) and Group C (sintered bone only). The three groups were observed by scanning electron microscopy (SEM) before the experiments, respectively. Then MC3T3-E1 cells were cultured on the surfaces of the three kinds of material, respectively. The cell adhesion rate was assessed by precipitation method. The proliferative ability of MC3T3-E1 cells were measured with MTT assay. And the ALP staining and measurement of alkaline phosphatase (ALP) activity were performed to assess the differentiation of cells into osteoblasts. The SEM results showed that the materials in the three groups retained the natural pore structure and the pore sizes were in the range between 200-850 μm. The adhesive ratio measurements and MTT assay suggested that adhesion and proliferation of MC3T3-E1 cells in Group A were much higher than those in Group B and Group C (P bone modified with surface mineralization/P24 composite material was confirmed to improve the adhesion rate and proliferation and osteodifferentiation of MC3T3-E1 cells, and maintained their morphology.

  18. Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high-throughput screening of Wnt inhibitors.

    Science.gov (United States)

    Yamaguchi, Kiyoshi; Zhu, Chi; Ohsugi, Tomoyuki; Yamaguchi, Yuko; Ikenoue, Tsuneo; Furukawa, Yoichi

    2017-12-01

    Constitutive activation of Wnt signaling plays an important role in colorectal and liver tumorigenesis. Cell-based assays using synthetic TCF/LEF (T-cell factor/lymphoid enhancer factor) reporters, as readouts of β-catenin/TCF-dependent transcriptional activity, have contributed greatly to the discovery of small molecules that modulate Wnt signaling. In the present study, we report a novel screening method, called a bidirectional dual reporter assay. Integrated transcriptome analysis identified a histidine ammonia-lyase gene (HAL) that was negatively regulated by β-catenin/TCF-dependent transcriptional activity. We leveraged a promoter region of the HAL gene as another transcriptional readout of Wnt signaling. Cells stably expressing both an optimized HAL reporter and the TCF/LEF reporter enabled bidirectional reporter activities in response to Wnt signaling. Increased HAL reporter activity and decreased TCF/LEF reporter activity were observed simultaneously in the cells when β-catenin/TCF7L2 was inhibited. Notably, this method could decrease the number of false positives observed when screening an inhibitor library compared with the conventional TCF/LEF assay. We found that Brefeldin A, a disruptor of the Golgi apparatus, inhibited the Wnt/β-catenin signaling pathway. The utility of our system could be expanded to examine other disease-associated pathways beyond the Wnt/β-catenin signaling pathway. © 2017 Wiley Periodicals, Inc.

  19. STANDARDIZATION OF A FLUORESCENT-BASED QUANTITATIVE ADHESION ASSAY TO STUDY ATTACHMENT OF Taenia solium ONCOSPHERE TO EPITHELIAL CELLS In Vitro

    Science.gov (United States)

    Chile, Nancy; Evangelista, Julio; Gilman, Robert H.; Arana, Yanina; Palma, Sandra; Sterling, Charles R; Garcia, Hector H.; Gonzalez, Armando; Verastegui, Manuela

    2012-01-01

    To fully understand the preliminary stages of Taenia solium oncosphere attachment in the gut, adequate tools and assays are necessary to observe and quantify this event that leads to infection. A fluorescent-based quantitative adhesion assay, using biotinylated activated-oncospheres and monolayers of Chinese hamster ovary cells (CHO-K1) or human intestinal monolayer cells (INT-407, HCT-8 or HT-29), was developed to study initial events during the infection of target cells and to rapidly quantify the in vitro adhesion of T. solium oncospheres. Fluorescein streptavidin was used to identify biotinylated activated-oncospheres adhered to cells. This adherence was quantified using an automated fluorescence plate reader, and the results were expressed as fluorescence intensity values. A series of three assays were performed. The first was to identify the optimum number of biotinylated activated-oncospheres to be used in the adhesion assay. The goal of the second assay was to validate this novel method with the established oncosphere-binding system using the immunofluorescent-antibody assay (IFA) method to quantify oncosphere adhesion. A total of 10,000 biotinylated activated-oncospheres were utilized to assess the role of sera and laminin (LM) in oncosphere adherence to a CHO-K1 cell monolayer. The findings that sera and LM increase the adhesion of oncospheres to monolayer cells were similar to results that were previously obtained using the IFA method. The third assay compared the adherence of biotinylated activated-oncospheres to different types of human intestinal monolayer cells. In this case, the fluorescence intensity was greatest when using the INT-407 cell monolayer. We believe this new method of quantification offers the potential for rapid, large-scale screening to study and elucidate specific molecules and mechanisms involved in oncosphere-host cell attachment. PMID:22178422

  20. SU-E-T-253: Open-Source Automatic Software for Quantifying Biological Assays of Radiation Effects

    International Nuclear Information System (INIS)

    Detappe, A; Korideck, H; Makrigiorgos, G; Berbeco, R

    2014-01-01

    Purpose: Clonogenic cell survival is a common assay for quantifying the effect of drugs and radiation. Manual counting of surviving colonies can take 30–90seconds per plate, a major limitation for large studies. Currently available automatic counting tools are not easily modified for radiation oncology research. Our goal is to provide an open-source toolkit for precise, accurate and fast analysis of biological assays in radiation oncology. Methods: As an example analysis, we used HeLa cells incubated with gadolinium nanoparticles prior to irradiation. After treatment, the cells are grown for 14days to allow for colony formation. To analyze the colony growth, we capture images of each dish for archiving and automatic computer-based analysis. A FujifilmX20 camera is placed at the top of a box setup, 20cm above the sample, which is backlit by a LED lamp placed at the bottom of the box. We use a Gaussian filter (width=1.3mm) and color threshold (19–255). The minimum size for a colony to be counted is 1mm. For this example, 20 dishes with a large range of colonies were analyzed. Each dish was counted 3 times manually by 3 different users and then compared to our counter. Results: Automatic counting of cell colonies takes an average of 7seconds, enabling the analysis process to be accelerated 4–12 times. The average precision of the automatic counter was 1.7%. The Student t-test demonstrated the non-significant differences between the two counting methods (p=0.64). The ICC demonstrated the reliability of each method with ICC>0.999 (automatic) and ICC=0.95 (manual). Conclusion: We developed an open-source automatic toolkit for the analysis of biological assays in radiation oncology and demonstrated the accuracy, precision and effort savings for clonogenic cell survival quantification. This toolkit is currently being used in two laboratories for routine experimental analysis and will be made freely available on our departmental website

  1. A PDMS Device Coupled with Culture Dish for In Vitro Cell Migration Assay.

    Science.gov (United States)

    Lv, Xiaoqing; Geng, Zhaoxin; Fan, Zhiyuan; Wang, Shicai; Pei, WeiHua; Chen, Hongda

    2018-04-30

    Cell migration and invasion are important factors during tumor progression and metastasis. Wound-healing assay and the Boyden chamber assay are efficient tools to investigate tumor development because both of them could be applied to measure cell migration rate. Therefore, a simple and integrated polydimethylsiloxane (PDMS) device was developed for cell migration assay, which could perform quantitative evaluation of cell migration behaviors, especially for the wound-healing assay. The integrated device was composed of three units, which included cell culture dish, PDMS chamber, and wound generation mold. The PDMS chamber was integrated with cell culture chamber and could perform six experiments under different conditions of stimuli simultaneously. To verify the function of this device, it was utilized to explore the tumor cell migration behaviors under different concentrations of fetal bovine serum (FBS) and transforming growth factor (TGF-β) at different time points. This device has the unique capability to create the "wound" area in parallel during cell migration assay and provides a simple and efficient platform for investigating cell migration assay in biomedical application.

  2. CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients.

    Science.gov (United States)

    Turtle, Cameron J; Hanafi, Laïla-Aïcha; Berger, Carolina; Gooley, Theodore A; Cherian, Sindhu; Hudecek, Michael; Sommermeyer, Daniel; Melville, Katherine; Pender, Barbara; Budiarto, Tanya M; Robinson, Emily; Steevens, Natalia N; Chaney, Colette; Soma, Lorinda; Chen, Xueyan; Yeung, Cecilia; Wood, Brent; Li, Daniel; Cao, Jianhong; Heimfeld, Shelly; Jensen, Michael C; Riddell, Stanley R; Maloney, David G

    2016-06-01

    T cells that have been modified to express a CD19-specific chimeric antigen receptor (CAR) have antitumor activity in B cell malignancies; however, identification of the factors that determine toxicity and efficacy of these T cells has been challenging in prior studies in which phenotypically heterogeneous CAR-T cell products were prepared from unselected T cells. We conducted a clinical trial to evaluate CD19 CAR-T cells that were manufactured from defined CD4+ and CD8+ T cell subsets and administered in a defined CD4+:CD8+ composition to adults with B cell acute lymphoblastic leukemia after lymphodepletion chemotherapy. The defined composition product was remarkably potent, as 27 of 29 patients (93%) achieved BM remission, as determined by flow cytometry. We established that high CAR-T cell doses and tumor burden increase the risks of severe cytokine release syndrome and neurotoxicity. Moreover, we identified serum biomarkers that allow testing of early intervention strategies in patients at the highest risk of toxicity. Risk-stratified CAR-T cell dosing based on BM disease burden decreased toxicity. CD8+ T cell-mediated anti-CAR transgene product immune responses developed after CAR-T cell infusion in some patients, limited CAR-T cell persistence, and increased relapse risk. Addition of fludarabine to the lymphodepletion regimen improved CAR-T cell persistence and disease-free survival. Immunotherapy with a CAR-T cell product of defined composition enabled identification of factors that correlated with CAR-T cell expansion, persistence, and toxicity and facilitated design of lymphodepletion and CAR-T cell dosing strategies that mitigated toxicity and improved disease-free survival. ClinicalTrials.gov NCT01865617. R01-CA136551; Life Science Development Fund; Juno Therapeutics; Bezos Family Foundation.

  3. Bifenthrin activates homotypic aggregation in human T-cell lines.

    Science.gov (United States)

    Hoffman, Nataly; Tran, Van; Daniyan, Anthony; Ojugbele, Olutosin; Pryor, Stephen C; Bonventre, Josephine A; Flynn, Katherine; Weeks, Benjamin S

    2006-03-01

    Here, we addressed the concern that, despite the lack of overt toxicity, exposure to low levels of the common household pyrethroid pesticide, bifenthrin, could cause harm to the immune system. To do this, we measure the effect of bifenthrin on phytohemagglutinin (PHA) activation of homotypic aggregation in human T-cell lines. The human CD4+ H9, and Jurkat cell lines and the human promonocyte U937 cell line, were exposed to varying concentrations of bifenthrin. Cell viability was determined using the AlmarBlue Toxicity Assay. Concentrations of bifenthrin which did not reduce cell viability were determined and these concentrations were tested for the effect of bifenthrin on PHA-mediated homotypic aggregation. Blocking antibodies to ICAM and LFA-1 were used to disrupt aggregation and a nonspecific IgG was used as a control. Bifenthrin was found to be nontoxic at concentrations ranging from 10(-4) to 10(-13) M. Bifenthrin did not inhibit PHA induced cell aggregation in all cell lines tested. However, at 10(-4) M, bifenthrin to form aggregates stimulated homotypic aggregation in the H9 and Jurkat T-cell lines. The bifenthrin-induced aggregate formation, like that seen with PHA, was blocked by treating the cells with antibodies to either LFA-1 or ICAM. The results here show that bifenthrin activates T-cell function by stimulating ICAM/LFA-1 mediated homotypic aggregation. This data suggests that exposure to bifenthrin, even at "acceptable" limits, can increase the risk for and frequency of inflammatory responses and diseases such as asthma.

  4. WRN-targeted therapy using inhibitors NSC 19630 and NSC 617145 induce apoptosis in HTLV-1-transformed adult T-cell leukemia cells

    Directory of Open Access Journals (Sweden)

    R. Moles

    2016-11-01

    Full Text Available Abstract Background Human T-cell leukemia virus type 1 (HTLV-1 infection is associated with adult T-cell leukemia/lymphoma (ATLL, a lymphoproliferative malignancy with a dismal prognosis and limited therapeutic options. Recent evidence shows that HTLV-1-transformed cells present defects in both DNA replication and DNA repair, suggesting that these cells might be particularly sensitive to treatment with a small helicase inhibitor. Because the “Werner syndrome ATP-dependent helicase” encoded by the WRN gene plays important roles in both cellular proliferation and DNA repair, we hypothesized that inhibition of WRN activity could be used as a new strategy to target ATLL cells. Methods Our analysis demonstrates an apoptotic effect induced by the WRN helicase inhibitor in HTLV-1-transformed cells in vitro and ATL-derived cell lines. Inhibition of cellular proliferation and induction of apoptosis were demonstrated with cell cycle analysis, XTT proliferation assay, clonogenic assay, annexin V staining, and measurement of mitochondrial transmembrane potential. Results Targeted inhibition of the WRN helicase induced cell cycle arrest and apoptosis in HTLV-1-transformed leukemia cells. Treatment with NSC 19630 (WRN inhibitor induces S-phase cell cycle arrest, disruption of the mitochondrial membrane potential, and decreased expression of anti-apoptotic factor Bcl-2. These events were associated with activation of caspase-3-dependent apoptosis in ATL cells. We identified some ATL cells, ATL-55T and LMY1, less sensitive to NSC 19630 but sensitive to another WRN inhibitor, NSC 617145. Conclusions WRN is essential for survival of ATL cells. Our studies suggest that targeting the WRN helicase with small inhibitors is a novel promising strategy to target HTLV-1-transformed ATL cells.

  5. Mesenchymal stem cells increase T-regulatory cells and improve healing following trauma and hemorrhagic shock.

    Science.gov (United States)

    Gore, Amy V; Bible, Letitia E; Song, Kimberly; Livingston, David H; Mohr, Alicia M; Sifri, Ziad C

    2015-07-01

    Rodent lungs undergo full histologic recovery within 1 week following unilateral lung contusion (LC). However, when LC is followed by hemorrhagic shock (HS), healing is impaired. We hypothesize that the intravenous administration of mesenchymal stem cells (MSCs) in animals undergoing combined LC followed by HS (LCHS) will improve wound healing. Male Sprague-Dawley rats (n = 5-6 per group) were subjected to LCHS with or without the injection of a single intravenous dose of 5 × 10 MSCs following return of shed blood after HS. Rats were sacrificed 7 days following injury. Flow cytometry was used to determine the T-regulatory cell (Treg) population in peripheral blood. Lung histology was graded using a well-established lung injury score (LIS). Components of the LIS include average inflammatory cells per high-power field over 30 fields, interstitial edema, pulmonary edema, and alveolar integrity, with total scores ranging from 0 to 11. Data were analyzed by analysis of variance followed by Tukey's multiple comparison test, expressed as mean (SD). p healing with an LIS unchanged from naive. The addition of HS resulted in a persistently elevated LIS score, whereas the addition of MSCs to LCHS decreased the LIS score back to naive levels. The change in LIS was driven by a significant decrease in edema scores. In rats undergoing LC alone, 10.5% (3.3%) of CD4 cells were Tregs. The addition of HS caused no significant change in Treg population (9.3% [0.7%]), whereas LCHS + MSC significantly increased the population to 18.2% (6.8%) in peripheral blood (p healing following trauma and HS is improved by a single dose of MSCs given immediately after injury. This enhanced healing is associated with an increase in the Treg population and a significant decrease in lung edema score as compared with animals undergoing LCHS. Further study into the role of Tregs in MSC-mediated wound healing is warranted.

  6. [Amplification of γδ T cells in PBMCs of healthy donors and osteosarcoma patients stimulated by zoledronate].

    Science.gov (United States)

    Li, Zhao-xu; Sun, Ling-ling; Cheng, Rui-lin; Sun, Zheng-wang; Ye, Zhao-ming

    2012-08-01

    To investigate the amplification and cytotoxicity of γδ T cells in peripheral blood mononuclear cells (PBMCs) of healthy donors and osteosarcoma patients stimulated by zoledronate (Zol) and IL-2. PBMCs from healthy donors and osteosarcoma patients were stimulated with IL-2 and Zol+IL-2, respectively. After 14-day culture, the purity of γδ T cells was assessed by flow cytometry. The cytotoxicity of γδ T cells against target cells was analyzed using a standard lactate dehydrogenase release assay with γδ T lymphocyte-sensitive Daudi cells, γδ T lymphocyte-resistant Raji cells and human osteoblast cell line, hFOB, as the target cells. After 2-week culture ex vivo of PBMCs from healthy donors and osteosarcoma patients, compared with stimulation of IL-2, Zol+IL-2 significantly promoted the amplification of γδ T cells. In addition, γδ T cells showed the higher cytotoxicity against Daudi cells, but no cytotoxic effect on normal cells like hFOB. γδ T cells of high purity and high cytotoxicity can be obtained by the stimulation of Zol combined with IL-2 on PBMCs from healthy donors and osteosarcoma patients.

  7. Low standby leakage 12T SRAM cell characterisation

    Science.gov (United States)

    Yadav, Arjun; Nakhate, Sangeeta

    2016-09-01

    In this work, a low power and variability-aware static random access memory (SRAM) architecture based on a twelve-transistor (12T) cell is proposed. This cell obtains low static power dissipation due to a parallel global latch (G-latch) and storage latch (S-latch), along with a global wordline (GWL), which offer a high cell ratio and pull-up ratio for reliable read and write operations and a low cell ratio and pull-up ratio during idle mode to reduce the standby power dissipation. In the idle state, only the S-latch stores bits, while the G-latch is isolated from the S-latch and the GWL is deactivated. The leakage power consumption of the proposed SRAM cell is thereby reduced by 38.7% compared to that of the conventional six-transistor (6T) SRAM cell. This paper evaluates the impact of the chip supply voltage and surrounding temperature variations on the standby leakage power and observes considerable improvement in the power dissipation. The read/write access delay, read static noise margin (SNM) and write SNM were evaluated, and the results were compared with those of the standard 6T SRAM cell. The proposed cell, when compared with the existing cell using the Monte Carlo method, shows an appreciable improvement in the standby power dissipation and layout area.

  8. CD19 CAR–T cells of defined CD4+:CD8+ composition in adult B cell ALL patients

    Science.gov (United States)

    Turtle, Cameron J.; Hanafi, Laïla-Aïcha; Berger, Carolina; Gooley, Theodore A.; Cherian, Sindhu; Hudecek, Michael; Sommermeyer, Daniel; Melville, Katherine; Pender, Barbara; Budiarto, Tanya M.; Robinson, Emily; Steevens, Natalia N.; Chaney, Colette; Soma, Lorinda; Chen, Xueyan; Li, Daniel; Cao, Jianhong; Heimfeld, Shelly; Jensen, Michael C.; Riddell, Stanley R.; Maloney, David G.

    2016-01-01

    BACKGROUND. T cells that have been modified to express a CD19-specific chimeric antigen receptor (CAR) have antitumor activity in B cell malignancies; however, identification of the factors that determine toxicity and efficacy of these T cells has been challenging in prior studies in which phenotypically heterogeneous CAR–T cell products were prepared from unselected T cells. METHODS. We conducted a clinical trial to evaluate CD19 CAR–T cells that were manufactured from defined CD4+ and CD8+ T cell subsets and administered in a defined CD4+:CD8+ composition to adults with B cell acute lymphoblastic leukemia after lymphodepletion chemotherapy. RESULTS. The defined composition product was remarkably potent, as 27 of 29 patients (93%) achieved BM remission, as determined by flow cytometry. We established that high CAR–T cell doses and tumor burden increase the risks of severe cytokine release syndrome and neurotoxicity. Moreover, we identified serum biomarkers that allow testing of early intervention strategies in patients at the highest risk of toxicity. Risk-stratified CAR–T cell dosing based on BM disease burden decreased toxicity. CD8+ T cell–mediated anti-CAR transgene product immune responses developed after CAR–T cell infusion in some patients, limited CAR–T cell persistence, and increased relapse risk. Addition of fludarabine to the lymphodepletion regimen improved CAR–T cell persistence and disease-free survival. CONCLUSION. Immunotherapy with a CAR–T cell product of defined composition enabled identification of factors that correlated with CAR–T cell expansion, persistence, and toxicity and facilitated design of lymphodepletion and CAR–T cell dosing strategies that mitigated toxicity and improved disease-free survival. TRIAL REGISTRATION. ClinicalTrials.gov NCT01865617. FUNDING. R01-CA136551; Life Science Development Fund; Juno Therapeutics; Bezos Family Foundation. PMID:27111235

  9. Accurate Region-of-Interest Recovery Improves the Measurement of the Cell Migration Rate in the In Vitro Wound Healing Assay.

    Science.gov (United States)

    Bedoya, Cesar; Cardona, Andrés; Galeano, July; Cortés-Mancera, Fabián; Sandoz, Patrick; Zarzycki, Artur

    2017-12-01

    The wound healing assay is widely used for the quantitative analysis of highly regulated cellular events. In this essay, a wound is voluntarily produced on a confluent cell monolayer, and then the rate of wound reduction (WR) is characterized by processing images of the same regions of interest (ROIs) recorded at different time intervals. In this method, sharp-image ROI recovery is indispensable to compensate for displacements of the cell cultures due either to the exploration of multiple sites of the same culture or to transfers from the microscope stage to a cell incubator. ROI recovery is usually done manually and, despite a low-magnification microscope objective is generally used (10x), repositioning imperfections constitute a major source of errors detrimental to the WR measurement accuracy. We address this ROI recovery issue by using pseudoperiodic patterns fixed onto the cell culture dishes, allowing the easy localization of ROIs and the accurate quantification of positioning errors. The method is applied to a tumor-derived cell line, and the WR rates are measured by means of two different image processing software. Sharp ROI recovery based on the proposed method is found to improve significantly the accuracy of the WR measurement and the positioning under the microscope.

  10. Adoptive immunotherapy using PRAME-specific T cells in medulloblastoma.

    Science.gov (United States)

    Orlando, Domenico; Miele, Evelina; De Angelis, Biagio; Guercio, Marika; Boffa, Iolanda; Sinibaldi, Matilde; Po, Agnese; Caruana, Ignazio; Abballe, Luana; Carai, Andrea; Caruso, Simona; Camera, Antonio; Moseley, Annemarie; Hagedoorn, Renate S; Heemskerk, Mirjam H M; Giangaspero, Felice; Mastronuzzi, Angela; Ferretti, Elisabetta; Locatelli, Franco; Quintarelli, Concetta

    2018-04-03

    Medulloblastoma is the most frequent malignant childhood brain tumor with a high morbidity. Identification of new therapeutic targets would be instrumental in improving patient outcomes. We evaluated the expression of the tumor-associated antigen PRAME in biopsies from 60 medulloblastoma patients. PRAME expression was detectable in 82% of tissues independent of molecular and histopathologic subgroups. High PRAME expression also correlated with worse overall survival. We next investigated the relevance of PRAME as a target for immunotherapy. Medulloblastoma cells were targeted using genetically modified T cells with a PRAME-specific TCR (SLL TCR T cells). SLL TCR T cells efficiently killed medulloblastoma HLA-A*02+ DAOY cells as well as primary HLA-A*02+ medulloblastoma cells. Moreover, SLL TCR T cells controlled tumor growth in an orthotopic mouse model of medulloblastoma. To prevent unexpected T cell-related toxicity,an inducible caspase 9 (iC9) gene was introduced in frame with the SLL TCR; this safety switch triggered prompt elimination of genetically-modified T cells. Altogether, these data indicate that T cells genetically modified with a high-affinity, PRAME-specific TCR and iC9 may represent a promising innovative approach for treating HLA-A*02+ medulloblastoma patients. Copyright ©2018, American Association for Cancer Research.

  11. A novel method to generate T-cell receptor-deficient chimeric antigen receptor T cells.

    Science.gov (United States)

    Kamiya, Takahiro; Wong, Desmond; Png, Yi Tian; Campana, Dario

    2018-03-13

    Practical methods are needed to increase the applicability and efficacy of chimeric antigen receptor (CAR) T-cell therapies. Using donor-derived CAR-T cells is attractive, but expression of endogenous T-cell receptors (TCRs) carries the risk for graft-versus-host-disease (GVHD). To remove surface TCRαβ, we combined an antibody-derived single-chain variable fragment specific for CD3ε with 21 different amino acid sequences predicted to retain it intracellularly. After transduction in T cells, several of these protein expression blockers (PEBLs) colocalized intracellularly with CD3ε, blocking surface CD3 and TCRαβ expression. In 25 experiments, median TCRαβ expression in T lymphocytes was reduced from 95.7% to 25.0%; CD3/TCRαβ cell depletion yielded virtually pure TCRαβ-negative T cells. Anti-CD3ε PEBLs abrogated TCRαβ-mediated signaling, without affecting immunophenotype or proliferation. In anti-CD3ε PEBL-T cells, expression of an anti-CD19-41BB-CD3ζ CAR induced cytokine secretion, long-term proliferation, and CD19 + leukemia cell killing, at rates meeting or exceeding those of CAR-T cells with normal CD3/TCRαβ expression. In immunodeficient mice, anti-CD3ε PEBL-T cells had markedly reduced GVHD potential; when transduced with anti-CD19 CAR, these T cells killed engrafted leukemic cells. PEBL blockade of surface CD3/TCRαβ expression is an effective tool to prepare allogeneic CAR-T cells. Combined PEBL and CAR expression can be achieved in a single-step procedure, is easily adaptable to current cell manufacturing protocols, and can be used to target other T-cell molecules to further enhance CAR-T-cell therapies. © 2018 by The American Society of Hematology.

  12. Chimeric Antigen Receptor-Engineered T Cells for Immunotherapy of Cancer

    Directory of Open Access Journals (Sweden)

    Marc Cartellieri

    2010-01-01

    Full Text Available CD4+ and CD8+ T lymphocytes are powerful components of adaptive immunity, which essentially contribute to the elimination of tumors. Due to their cytotoxic capacity, T cells emerged as attractive candidates for specific immunotherapy of cancer. A promising approach is the genetic modification of T cells with chimeric antigen receptors (CARs. First generation CARs consist of a binding moiety specifically recognizing a tumor cell surface antigen and a lymphocyte activating signaling chain. The CAR-mediated recognition induces cytokine production and tumor-directed cytotoxicity of T cells. Second and third generation CARs include signal sequences from various costimulatory molecules resulting in enhanced T-cell persistence and sustained antitumor reaction. Clinical trials revealed that the adoptive transfer of T cells engineered with first generation CARs represents a feasible concept for the induction of clinical responses in some tumor patients. However, further improvement is required, which may be achieved by second or third generation CAR-engrafted T cells.

  13. Effect of low-dose irradiation upon T cell subsets involved in the response of primed A/J mice to SaI cells

    International Nuclear Information System (INIS)

    Anderson, R.E.; Williams, W.L.; Tokuda, Sei

    1988-01-01

    A/Jax (A/J) mice primed to Sarcoma I (SaI) exhibit an augmented response in association with low-dose (0.15 Gy) irradiation. This phenomenon is best demonstrated in tumour neutralization (Winn assay) or cell transfer experiments utilizing mice depleted of thymus-derived (T) cells. It is particularly dependent upon the duration of priming and the growth characteristics of the tumour in the primary host. The importance of these two variables appears to relate to their influence upon the cell types responsible for the host response, and includes both an effector and a suppressor component. Radiation-induced inhibition of the suppressor component appears responsible for low-dose augmentation and results in injury to a T cell of the Lyt-1 - 2 + phenotype. In Winn assays employing equal numbers of immune spleen cells and SaI cells, the smallest tumours are associated with Lyt-1-positive (Lyt-1 + 2 - and Lyt-1 + 2 + ) cells and exposure to 0.15 Gy markedly inhibits their anti-SaI activity. Thus, even though the effect is in the opposite direction, both the effector and suppressor components of the anti-SaI response in A/J mice are exceedingly radiosensitive. (author)

  14. CD4+ T Cells Mediate Aspergillosis Vaccine Protection.

    Science.gov (United States)

    Diaz-Arevalo, Diana; Kalkum, Markus

    2017-01-01

    Adaptive effector CD4 + T cells play essential roles in the defense against fungal infections, especially against invasive aspergillosis (IA). Such protective CD4 + T cells can be generated through immunization with specialized antifungal vaccines, as has been demonstrated for pulmonary Aspergillus fumigatus infections in mouse experiments. Adaptive transfer of fungal antigen-specific CD4 + T cells conferred protection onto non-immunized naive mice, an experimental approach that could potentially become a future treatment option for immunosuppressed IA patients, focusing on the ultimate goal to improve their otherwise dim chances for survival. Here, we describe the different techniques to analyze CD4 + T cell immune responses after immunization with a recombinant fungal protein. We present three major methods that are used to analyze the role of CD4 + T cells in protection against A. fumigatus challenge. They include (1) transplantation of CD4 + T cells from vaccinated mice into immunosuppressed naive mice, observing increasing protection of the cell recipients, (2) depletion of CD4 + T cells from vaccinated mice, which abolishes vaccine protection, and (3) T cell proliferation studies following stimulation with overlapping synthetic peptides or an intact protein vaccine. The latter can be used to validate immunization status and to identify protective T cell epitopes in vaccine antigens. In the methods detailed here, we used versions of the well-studied Asp f3 protein expressed in a bacterial host, either as the intact full length protein or its N-terminally truncated version, comprised of residues 15-168. However, these methods are generally applicable and can well be adapted to study other protein-based subunit vaccines.

  15. Discriminating modes of toxic action in mice using toxicity in BALB/c mouse fibroblast (3T3) cells.

    Science.gov (United States)

    Huang, Tao; Yan, Lichen; Zheng, Shanshan; Wang, Yue; Wang, Xiaohong; Fan, Lingyun; Li, Chao; Zhao, Yuanhui; Martyniuk, Christopher J

    2017-12-01

    The objective of this study was to determine whether toxicity in mouse fibroblast cells (3T3 cells) could predict toxicity in mice. Synthesized data on toxicity was subjected to regression analysis and it was observed that relationship of toxicities between mice and 3T3 cells was not strong (R 2  = 0.41). Inclusion of molecular descriptors (e.g. ionization, pKa) improved the regression to R 2  = 0.56, indicating that this relationship is influenced by kinetic processes of chemicals or specific toxic mechanisms associated to the compounds. However, to determine if we were able to discriminate modes of action (MOAs) in mice using the toxicities generated from 3T3 cells, compounds were first classified into "baseline" and "reactive" guided by the toxic ratio (TR) for each compound in mice. Sequence, binomial and recursive partitioning analyses provided strong predictions of MOAs in mice based upon toxicities in 3T3 cells. The correct classification of MOAs based on these methods was 86%. Nearly all the baseline compounds predicted from toxicities in 3T3 cells were identified as baseline compounds from the TR in mice. The incorrect assignment of MOAs for some compounds is hypothesized to be due to experimental uncertainty that exists in toxicity assays for both mice and 3T3 cells. Conversely, lack of assignment can also arise because some reactive compounds have MOAs that are different in mice compared to 3T3 cells. The methods developed here are novel and contribute to efforts to reduce animal numbers in toxicity tests that are used to evaluate risks associated with organic pollutants in the environment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. T-cells fighting B-cell lymphoproliferative malignancies: the emerging field of CD19 CAR T-cell therapy

    NARCIS (Netherlands)

    Heijink, D. M.; Kater, A. P.; Hazenberg, M. D.; Hagenbeek, A.; Kersten, M. J.

    2016-01-01

    CAR T-cells are autologous T-cells transduced with a chimeric antigen receptor (CAR). The CAR contains an antigen recognition part (originating from an antibody), a T-cell receptor transmembrane and cytoplasmic signalling part, and one or more co-stimulatory domains. While CAR T-cells can be

  17. Prognostic implications of high-sensitivity cardiac troponin T assay in a real-world population with non-ST-elevation acute coronary syndrome.

    Science.gov (United States)

    Magnoni, Marco; Gallone, Guglielmo; Ceriotti, Ferruccio; Vergani, Vittoria; Giorgio, Daniela; Angeloni, Giulia; Maseri, Attilio; Cianflone, Domenico

    2018-09-01

    High-sensitivity cardiac troponin T (hsTnT) was recently approved for clinical use by the Food and Drug Administration. The transition from contemporary to hsTnT assays requires a thorough understanding of the clinical differences between these assays. HsTnT may provide a more accurate prognostic stratification than contemporary cardiac troponin I (cTnI) in patients with non-ST-segment elevation acute coronary syndromes (NSTE-ACS). HsTnT and cTnI were measured in 644 patients with CK-MB negative NSTE-ACS who were enrolled in the prospective multicenter SPAI (Stratificazione Prognostica dell'Angina Instabile) study. Patients were stratified at the 99th percentile reference limit for each assay. The primary endpoint was cardiovascular death (CVD) or non-fatal myocardial infarction (MI); the secondary endpoint was the occurrence of unstable angina (UA). Follow-up lasted 180 days. Patients with hsTnT ≥99th percentile were at higher risk of CVD/MI (30-day: 5.9% vs 0.8%, p  = 0.001; 180-day: 11.1% vs 4.7%, p  = 0.004), also after adjusting for TIMI Risk Score. No significant difference in CVD/MI at 180-day was found between hsTnT-positive/cTnI-negative and hsTnT-negative/cTnI-negative patients (adjHR 1.61, 95% CI 0.74-3.49, p  = 0.232). Occurrence of UA was not differently distributed between hsTnT groups dichotomized at the 99th percentile (12.4% vs 12.5% p  = 0.54). Our investigation on a real-world NSTE-ACS population showed good prognostic performance of hsTnT in the risk stratification of the hard endpoint, but did not demonstrate the improved prognostic ability of hsTnT over contemporary cTn. Neither troponin assay predicted the recurrence of UA, suggesting the acute rise of cardiac troponin as a marker of severity, but not the occurrence of future coronary instability.

  18. CAR T Cells Targeting Podoplanin Reduce Orthotopic Glioblastomas in Mouse Brains.

    Science.gov (United States)

    Shiina, Satoshi; Ohno, Masasuke; Ohka, Fumiharu; Kuramitsu, Shunichiro; Yamamichi, Akane; Kato, Akira; Motomura, Kazuya; Tanahashi, Kuniaki; Yamamoto, Takashi; Watanabe, Reiko; Ito, Ichiro; Senga, Takeshi; Hamaguchi, Michinari; Wakabayashi, Toshihiko; Kaneko, Mika K; Kato, Yukinari; Chandramohan, Vidyalakshmi; Bigner, Darell D; Natsume, Atsushi

    2016-03-01

    Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor in adults with a 5-year overall survival rate of less than 10%. Podoplanin (PDPN) is a type I transmembrane mucin-like glycoprotein, expressed in the lymphatic endothelium. Several solid tumors overexpress PDPN, including the mesenchymal type of GBM, which has been reported to present the worst prognosis among GBM subtypes. Chimeric antigen receptor (CAR)-transduced T cells can recognize predefined tumor surface antigens independent of MHC restriction, which is often downregulated in gliomas. We constructed a lentiviral vector expressing a third-generation CAR comprising a PDPN-specific antibody (NZ-1-based single-chain variable fragment) with CD28, 4-1BB, and CD3ζ intracellular domains. CAR-transduced peripheral blood monocytes were immunologically evaluated by calcein-mediated cytotoxic assay, ELISA, tumor size, and overall survival. The generated CAR T cells were specific and effective against PDPN-positive GBM cells in vitro. Systemic injection of the CAR T cells into an immunodeficient mouse model inhibited the growth of intracranial glioma xenografts in vivo. CAR T-cell therapy that targets PDPN would be a promising adoptive immunotherapy to treat mesenchymal GBM. ©2016 American Association for Cancer Research.

  19. Dysfunction of irradiated thymus for the development of helper T cells

    International Nuclear Information System (INIS)

    Amagai, T.; Kina, T.; Hirokawa, K.; Nishikawa, S.; Imanishi, J.; Katsura, Y.

    1987-01-01

    The development of cytotoxic T cells and helper T cells in an intact or irradiated thymus was investigated. C57BL/6 (H-2b, Thy-1.2) mice were whole body-irradiated, or were irradiated with shielding over either the thymus or right leg and tail, and were transferred with 1.5 X 10(7) bone marrow cells from B10.Thy-1.1 mice (H-2b, Thy-1.1). At various days after reconstitution, thymus cells from the recipient mice were harvested and a peanut agglutinin low-binding population was isolated. This population was further treated with anti-Thy-1.2 plus complement to remove host-derived cells and was assayed for the frequency of cytotoxic T cell precursors (CTLp) and for the activity of helper T cells (Th). In the thymus of thymus-shielded and irradiated mice, Th activity reached normal control level by day 25, whereas CTLp frequency remained at a very low level during these days. In the thymus of whole body-irradiated mice, generation of CTLp was highly accelerated while that of Th was retarded, the period required for reconstitution being 25 days and more than 42 days for CTLp and Th, respectively. Preferential development of CTLp was also seen in right leg- and tail-shielded (L-T-shielded) and irradiated recipients. Histological observation indicated that Ia+ nonlymphoid cells were well preserved in the thymus of thymus-shielded and irradiated recipients, whereas in L-T-shielded and irradiated recipients, such cells in the medulla were markedly reduced in number. These results suggest strongly that the generation of Th but not CTLp is dependent on radiosensitive thymic component(s), and that such components may represent Ia+ cells themselves in the medulla or some microenvironment related to Ia+ cells

  20. T cell immunity

    OpenAIRE

    Emel Bülbül Başkan

    2013-01-01

    Since birth, our immune system is constantly bombarded with self-antigens and foreign pathogens. To stay healthy, complex immune strategies have evolved in our immune system to maintain self-tolerance and to defend against foreign pathogens. Effector T cells are the key players in steering the immune responses to execute immune functions. While effector T cells were initially identified to be immune promoting, recent studies unraveled negative regulatory functions of effector T cells...

  1. Subcutaneous panniculitis-like T-cell lymphoma

    Directory of Open Access Journals (Sweden)

    Francis Abel

    2010-01-01

    Full Text Available This case report describes a 38 year-old lady with the clinical, histopathological, and immunohistochemical (IHC changes of subcutaneous panniculitis-like T-cell lymphoma (SPTCL. The IHC findings revealed CD8 + and CD56 - cells, which are indicative of tumors which have an indolent course. Our patient is being managed with tapering doses of corticosteroids for the last nine months with good improvement.

  2. Anti-regulatory T cells

    DEFF Research Database (Denmark)

    Andersen, Mads Hald

    2017-01-01

    responses to tumours or inhibiting autoimmunity development. However, recent studies report the discovery of self-reactive pro-inflammatory T cells—termed anti-regulatory T cells (anti-Tregs)—that target immune-suppressive cells. Thus, regulatory cells can now be defined as both cells that suppress immune...... reactions as well as effector cells that counteract the effects of suppressor cells and support immune reactions. Self-reactive anti-Tregs have been described that specifically recognize human leukocyte antigen-restricted epitopes derived from proteins that are normally expressed by regulatory immune cells......Our initial understanding of immune-regulatory cells was based on the discovery of suppressor cells that assure peripheral T-cell tolerance and promote immune homeostasis. Research has particularly focused on the importance of regulatory T cells (Tregs) for immune modulation, e.g. directing host...

  3. Self-reactive T cells

    DEFF Research Database (Denmark)

    Becker, Jürgen C; thor Straten, Per; Andersen, Mads Hald

    2014-01-01

    -proteins expressed in regulatory immune cells have been reported, especially in patients with cancer. The seemingly lack of tolerance toward such proteins is interesting, as it suggests a regulatory function of self-reactive T (srT) cells, which may be important for the fine tuning of the immune system......The immune system is a tightly regulated and complex system. An important part of this immune regulation is the assurance of tolerance toward self-antigens to maintain immune homeostasis. However, in recent years, antigen-specific cellular immune responses toward several normal self....... In particular, surprising has been the description of cytotoxic srT cells that are able to eliminate normal regulatory immune cells. Such srT cells may be important as effector cells that suppress regulatory suppressor cells. The current knowledge of the nature and function of srT cells is still limited. Still...

  4. Enhanced gastric cancer growth potential of mesenchymal stem cells derived from gastric cancer tissues educated by CD4+ T cells.

    Science.gov (United States)

    Xu, Rongman; Zhao, Xiangdong; Zhao, Yuanyuan; Chen, Bin; Sun, Li; Xu, Changgen; Shen, Bo; Wang, Mei; Xu, Wenrong; Zhu, Wei

    2018-04-01

    Gastric cancer mesenchymal stem cells (GC-MSCs) can promote the development of tumour growth. The tumour-promoting role of tumour-associated MSCs and T cells has been demonstrated. T cells as the major immune cells may influence and induce a pro-tumour phenotype in MSCs. This study focused on whether CD4 + T cells can affect GC-MSCs to promote gastric cancer growth. CD4 + T cells upregulation of programmed death ligand 1 (PD-L1) expression in GC-MSCs through the phosphorylated signal transducer and activator of transcription (p-STAT3) signalling pathway was confirmed by immunofluorescence, western blotting and RT-PCR. Migration of GC cells was detected by Transwell migration assay, and apoptosis of GC cells was measured by flow cytometry using annexin V/propidium iodide double staining. CD4 + T cell-primed GC-MSCs promoted GC growth in a subcutaneously transplanted tumour model in BALB/c nu/nu mice. Gastric cancer mesenchymal stem cells stimulated by activated CD4 + T cells promoted migration of GC cells and enhanced GC growth potential in BALB/c nu/nu xenografts. PD-L1 upregulation of GC-MSCs stimulated by CD4 + T cells was mediated through the p-STAT3 signalling pathway. CD4 + T cells-primed GC-MSCs have greater GC volume and growth rate-promoting role than GC-MSCs, with cancer cell-intrinsic PD-1/mammalian target of rapamycin (mTOR) signalling activation. This study showed that GC-MSCs are plastic. The immunophenotype of GC-MSCs stimulated by CD4 + T cells has major changes that may influence tumour cell growth. This research was based on the interaction between tumour cells, MSCs and immune cells, providing a new understanding of the development and immunotherapy of GC. © 2017 John Wiley & Sons Ltd.

  5. Simultaneous assessment of cytotoxic T lymphocyte responses against multiple viral infections by combined usage of optimal epitope matrices, anti- CD3 mAb T-cell expansion and "RecycleSpot"

    Directory of Open Access Journals (Sweden)

    Wong Johnson T

    2005-05-01

    Full Text Available Abstract The assessment of cellular anti-viral immunity is often hampered by the limited availability of adequate samples, especially when attempting simultaneous, high-resolution determination of T cell responses against multiple viral infections. Thus, the development of assay systems, which optimize cell usage, while still allowing for the detailed determination of breadth and magnitude of virus-specific cytotoxic T lymphocyte (CTL responses, is urgently needed. This study provides an up-to-date listing of currently known, well-defined viral CTL epitopes for HIV, EBV, CMV, HCV and HBV and describes an approach that overcomes some of the above limitations through the use of peptide matrices of optimally defined viral CTL epitopes in combination with anti-CD3 in vitro T cell expansion and re-use of cells from negative ELISpot wells. The data show that, when compared to direct ex vivo cell preparations, antigen-unspecific in vitro T cell expansion maintains the breadth of detectable T cell responses and demonstrates that harvesting cells from negative ELISpot wells for re-use in subsequent ELISpot assays (RecycleSpot, further maximized the use of available cells. Furthermore when combining T cell expansion and RecycleSpot with the use of rationally designed peptide matrices, antiviral immunity against more than 400 different CTL epitopes from five different viruses can be reproducibly assessed from samples of less than 10 milliliters of blood without compromising information on the breadth and magnitude of these responses. Together, these data support an approach that facilitates the assessment of cellular immunity against multiple viral co-infections in settings where sample availability is severely limited.

  6. Gap junctions at the dendritic cell-T cell interface are key elements for antigen-dependent T cell activation.

    Science.gov (United States)

    Elgueta, Raul; Tobar, Jaime A; Shoji, Kenji F; De Calisto, Jaime; Kalergis, Alexis M; Bono, Maria R; Rosemblatt, Mario; Sáez, Juan C

    2009-07-01

    The acquired immune response begins with Ag presentation by dendritic cells (DCs) to naive T cells in a heterocellular cell-cell contact-dependent process. Although both DCs and T cells are known to express connexin43, a gap junction protein subunit, the role of connexin43 on the initiation of T cell responses remains to be elucidated. In the present work, we report the formation of gap junctions between DCs and T cells and their role on T cell activation during Ag presentation by DCs. In cocultures of DCs and T cells, Lucifer yellow microinjected into DCs is transferred to adjacent transgenic CD4(+) T cells, only if the specific antigenic peptide was present at least during the first 24 h of cocultures. This dye transfer was sensitive to gap junction blockers, such as oleamide, and small peptides containing the extracellular loop sequences of conexin. Furthermore, in this system, gap junction blockers drastically reduced T cell activation as reflected by lower proliferation, CD69 expression, and IL-2 secretion. This lower T cell activation produced by gap junction blockers was not due to a lower expression of CD80, CD86, CD40, and MHC-II on DCs. Furthermore, gap junction blocker did not affect polyclonal activation of T cell induced with anti-CD3 plus anti-CD28 Abs in the absence of DCs. These results strongly suggest that functional gap junctions assemble at the interface between DCs and T cells during Ag presentation and that they play an essential role in T cell activation.

  7. Cytotoxic effects of the synthetic oestrogens and androgens on Balb/c 3T3 and HepG2 cells

    Directory of Open Access Journals (Sweden)

    Minta Maria

    2014-12-01

    Full Text Available The aim of the study was to test and compare the cytotoxic potential of two synthetic oestrogens: diethylstilboestrol (DES and ethinyloestradiol (EE2 and two androgens: testosterone propionate (TP and trenbolone (TREN on two cell lines. The fibroblast cell line Balb/c 3T3 and the hepatoma cell line HepG2 were selected. To get more insight into the mode of toxic action, four methods were used, which evaluated different biochemical endpoints: mitochondrial activity (3-(4,5-dimethylthiazol-2-yl- 2,5-diphenyltetrazolium bromide reduction assay, lysosomal activity (neutral red uptake assay, total protein content, and lactate dehydrogenase release. Cytotoxicity was assessed after 24, 48, and 72 h exposure to eight concentrations ranging from 0.78 to 100 μg/mL. Concentration- and time- dependent effects were observed. Depending on the line and assay used, half maximal effective concentration after 72 h (EC50-72h values ranged as follows: DES 1-13.7 μg/mL (Balb/c 3T3 and 3.7-5.2 μg/mL (HepG2; EE2 2.1-14.3 μg/mL (Balb/c 3T3 and 1.8-7.8 μg/mL (HepG2; TP-14.9-17.5 μg/mL (Balb/c 3T3, and 63.9- 100 μg/mL (HepG2; and TREN 11.3-31.4 μg/mL (Balb/c 3T3 and 12.5-59.4 μg/mL (HepG2. The results revealed that oestrogens were more toxic than androgens and the most affected endpoint was mitochondrial activity. In contrast to oestrogens, for which EC50-72h values were similar in both lines and by all assays used, Balb/c 3T3 cells were more sensitive than HepG2 cells to TP.

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

    Science.gov (United States)

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

    2016-03-31

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

  9. Genetic modification of T cells improves the effectiveness of adoptive tumor immunotherapy.

    Science.gov (United States)

    Jakóbisiak, Marek; Gołab, Jakub

    2010-10-01

    Appropriate combinations of immunotherapy and gene therapy promise to be more effective in the treatment of cancer patients than either of these therapeutic approaches alone. One such treatment is based on the application of patients' cytotoxic T cells, which can be activated, expanded, and genetically engineered to recognize particular tumor-associated antigens (TAAs). Because T cells recognizing TAAs might become unresponsive in the process of tumor development as a result of tumor evasion strategies, immunogenic viral antigens or alloantigens could be used for the expansion of cytotoxic T cells and then redirected through genetic engineering. This therapeutic approach has already demonstrated promising results in melanoma patients and could be used in the treatment of many other tumors. The graft-versus-leukemia, or more generally graft-versus-tumor, reaction based on the application of a donor lymphocyte infusion can also be ameliorated through the incorporation of suicide genes into donor lymphocytes. Such lymphocytes could be safely and more extensively used in tumor patients because they could be eliminated should a severe graft-versus-host reaction develop.

  10. Analysis of Chemopredictive Assay for Targeting Cancer Stem Cells in Glioblastoma Patients

    Directory of Open Access Journals (Sweden)

    Candace M. Howard

    2017-04-01

    Full Text Available Introduction: The prognosis of glioblastoma (GBM treated with standard-of-care maximal surgical resection and concurrent adjuvant temozolomide (TMZ/radiotherapy remains very poor (less than 15 months. GBMs have been found to contain a small population of cancer stem cells (CSCs that contribute to tumor propagation, maintenance, and treatment resistance. The highly invasive nature of high-grade gliomas and their inherent resistance to therapy lead to very high rates of recurrence. For these reasons, not all patients with similar diagnoses respond to the same chemotherapy, schedule, or dose. Administration of ineffective anticancer therapy is not only costly but more importantly burdens the patient with unnecessary toxicity and selects for the development of resistant cancer cell clones. We have developed a drug response assay (ChemoID that identifies the most effective chemotherapy against CSCs and bulk of tumor cells from of a panel of potential treatments, offering great promise for individualized cancer management. Providing the treating physician with drug response information on a panel of approved drugs will aid in personalized therapy selections of the most effective chemotherapy for individual patients, thereby improving outcomes. A prospective study was conducted evaluating the use of the ChemoID drug response assay in GBM patients treated with standard of care. Methods: Forty-one GBM patients (mean age 54 years, 59% male, all eligible for a surgical biopsy, were enrolled in an Institutional Review Board–approved protocol, and fresh tissue samples were collected for drug sensitivity testing. Patients were all treated with standard-of-care TMZ plus radiation with or without maximal surgery, depending on the status of the disease. Patients were prospectively monitored for tumor response, time to recurrence, progression-free survival (PFS, and overall survival (OS. Odds ratio (OR associations of 12-month recurrence, PFS, and OS outcomes

  11. Human dendritic cells sequentially matured with CD4+ T cells as a secondary signal favor CTL and long-term T memory cell responses

    Directory of Open Access Journals (Sweden)

    Thomas Simon

    2012-01-01

    Full Text Available Dendritic cells (DCs are professional antigen-presenting cells involved in the control and initiation of immune responses. In vivo, DCs exposed at the periphery to maturation stimuli migrate to lymph nodes, where they receive secondary signals from CD4+ T helper cells. These DCs become able to initiate CD8+ cytotoxic T lymphocyte (CTL responses. However, in vitro investigations concerning human monocyte-derived DCs have never focused on their functional properties after such sequential maturation. Here, we studied human DC phenotypes and functions according to this sequential exposure to maturation stimuli. As first signals, we used TNF-α/polyI:C mimicking inflammatory and pathogen stimuli and, as second signals, we compared activated CD4+ T helper cells to a combination of CD40-L/ IFN-γ. Our results show that a sequential activation with activated CD4+ T cells dramatically increased the maturation of DCs in terms of their phenotype and cytokine secretion compared to DCs activated with maturation stimuli delivered simultaneously. Furthermore, this sequential maturation led to the induction of CTL with a long-term effector and central memory phenotypes. Thus, sequential delivery of maturation stimuli, which includes CD4+ T cells, should be considered in the future to improve the induction of long-term CTL memory in DC-based immunotherapy.

  12. T cell-mediated hepatitis in mice infected with lymphocytic choriomeningitis virus. Liver cell destruction by H-2 class I-restricted virus-specific cytotoxic T cells as a physiological correlate of the 51Cr-release assay

    International Nuclear Information System (INIS)

    Zinkernagel, R.M.; Haenseler, E.; Leist, T.; Cerny, A.; Hengartner, H.; Althage, A.

    1986-01-01

    A model for immunologically T cell-mediated hepatitis was established in mice infected with lymphocytic choriomeningitis virus (LCMV). The severity of hepatitis was monitored histologically and by determination of changes in serum levels of the enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH), and alkaline phosphatase (AP). Kinetics of histological disease manifestations, increases of liver enzyme levels in the serum, and cytotoxic T cell activities in livers and spleens all correlated and were dependent upon several parameters: LCMV-isolate; LCMV-WE caused extensive hepatitis, LCMV-Armstrong virtually none. Virus dose. Route of infection; i.v. or i.p. infection caused hepatitis, whereas infection into the footpad did not. The general genetic background of the murine host; of the strains tested, Swiss mice and A-strain mice were more susceptible than C57BL or CBA mice; BALB/c and DBA/2 mice were least susceptible. The degree of immunocompetence of the murine host; T cell deficient nu/nu mice never developed hepatitis, whereas nu/+ or +/+ mice always did. B cell-depleted anti-IgM-treated mice developed immune-mediated hepatitis comparably or even more extensively than control mice. Local cytotoxic T cell activity; mononuclear cells isolated from livers during the period of overt hepatitis were two to five times more active than equal numbers of spleen cells. Adoptive transfer of nylon wool-nonadherent anti-Thy-1.2 and anti-Lyt-2 plus C-sensitive, anti-L3T4 plus C-resistant lymphocytes into irradiated mice preinfected with LCMV-WE caused a rapid time- and dose-dependent linear increase of serum enzyme levels. This increase was caused by adoptive transfer of lymphocytes if immune cell donors and recipient mice shared class I, but not when they shared class II histocompatibility antigens

  13. Biomonitoring of genotoxic risk in radar facility workers: comparison of the comet assay with micronucleus assay and chromatid breakage assay

    International Nuclear Information System (INIS)

    Garaj-Vrhovac, V.; Kopjar, N.

    2003-01-01

    Genotoxic risks of occupational exposure in a radar facility were evaluated by using alkaline comet assay, micronucleus assay and chromatid breakage assay on peripheral blood leukocytes in exposed subjects and corresponding controls. Results show that occupational exposure to microwave radiation correlates with an increase of genome damage in somatic cells. The levels of DNA damage in exposed subjects determined by using alkaline comet assay were increased compared to control and showed interindividual variations. Incidence of micronuclei was also significantly increased compared to baseline control values. After short exposure of cultured lymphocytes to bleomycin, cells of occupationally exposed subjects responded with high numbers of chromatid breaks. Although the level of chromosome damage generated by bleomycin varied greatly between individuals, in exposed subjects a significantly elevated number of chromatid breaks was observed. Our results support data reported in literature indicating that microwave radiation represents a potential DNA-damaging hazard. Alkaline comet assay is confirmed as a sensitive and highly reproducible technique for detection of primary DNA damage inflicted in somatic cells. Micronucleus assay was confirmed as reliable bio-markers of effect and chromatid breakage assay as sensitive bio-marker of individual cancer susceptibility. The results obtained also confirm the necessity to improve measures and to perform accurate health surveillance of individuals occupationally exposed to microwave radiation

  14. [Improvement of local lymph node assay for cosmetics safety evaluation].

    Science.gov (United States)

    Liu, Zhen; Liu, Junping; Wang, Fei; Xu, Guifeng; Hou, Juan; Wan, Xuying; Zhang, Tianbao

    2009-09-01

    To improve the local lymph node assay (LLNA) as an alternative method to detect chemicals for both sensitization and irritation. The following chemicals: one negative control: 4-Aminobenzoic Acid, three sensitizers: 2,4-dinitrochlorobenzene (DNCB), Hexyl cinnamic aldehyde (HCA), 2-Aminophenol (2-APC) and two irritations: potassium hydroxide (KOH), sodium lauryl sulphate (SLS) were selected. According to the normal LLNA, groups of female Balb/c mice were treated with test solutions. The thickness of each ear was measured and each auricle was weighed. On the sixth day, the bilateral draining auricular lymph nodes were excised and weighed. The single cell suspensions were prepared, the lymphocyte were counted and the proliferations of lymph cells were detected by cell counting kit-8 (CCK-8). Significant increase in ear thickness and weight were found in groups of KOH, SLS and DNCB (above 0.5%) (P LLNA using auricle thickness and weighing as observed markers for irritation, and using lymph nodes weighing and proliferation of lymphocyte as observed markers for sensitization, could evaluate both sensitization and irritation at the same time.

  15. Challenges and prospects of chimeric antigen receptor T cell therapy in solid tumors.

    Science.gov (United States)

    Jindal, Vishal; Arora, Ena; Gupta, Sorab

    2018-05-05

    Chimeric antigen receptor (CAR) T cell therapy is a novel and innovative immunotherapy. CAR-T cells are genetically engineered T cells, carrying MHC independent specific antigen receptor and co-stimulatory molecule which can activate an immune response to a cancer specific antigen. This therapy showed great results in hematological malignancies but were unable to prove their worth in solid tumors. Likely reasons for their failure are lack of antigens, poor trafficking, and hostile tumor microenvironment. Excessive amount of research is going on to improve the efficacy of CAR T cell therapy in solid tumors. In this article, we will discuss the challenges faced in improving the outcome of CAR T cell therapy in solid tumors and various strategies adopted to curb them.

  16. Bone marrow function. I. Peripheral T cells are responsible for the increased auto-antiidiotype response of older mice

    International Nuclear Information System (INIS)

    Kim, Y.T.; Goidl, E.A.; Samarut, C.; Weksler, M.E.; Thorbecke, G.J.; Siskind, G.W.

    1985-01-01

    After immunization with trinitrophenyl (TNP)-Ficoll, mice produced both anti-TNP antibodies and auto-anti-idiotype (auto-anti-Id) antibodies specific for the anti-TNP antibody. Older animals produced more auto-anti-Id than did young animals. When mice were exposed to a normally lethal dose of irradiation while their bone marrow (BM) was partially shielded, they survived and slowly (6 wk) regained immune function, as indicated by the number of nucleated cells in their spleen and the in vitro primary plaque-forming cell (PFC) response of their spleen cells to TNP-treated aminoethylated polyacrylamide beads. Recovery is presumably the result of repopulation of the peripheral lymphoid system by cells originating in the BM. By enzyme-linked immunosorbent assay (ELISA), and by hapten-augmentable PFC assay, the authors show that, after recovery from irradiation with their BM shielded, old animals produce low auto-anti-Id responses, like those of young animals. The transfer of splenic T cells into mice irradiated with their BM shielded provided evidence that the magnitude of the auto-anti-Id response is controlled by the peripheral T cells. Thus, mice that received splenic T cells from aged donors produced high levels of auto-anti-Id while those that received splenic T cells from young donors produce low levels of auto-anti-Id

  17. IL-15 improves the cytotoxicity of cytokine-induced killer cells against leukemia cells by upregulating CD3+CD56+ cells and downregulating regulatory T cells as well as IL-35.

    Science.gov (United States)

    Tao, Qianshan; Chen, Tianping; Tao, Lili; Wang, Huiping; Pan, Ying; Xiong, Shudao; Zhai, Zhimin

    2013-01-01

    Cytokine-induced killer (CIK) cells are usually generated from peripheral blood mononuclear cells with the stimulation of IL-2 in vitro. Unlike the conventional IL-2-stimulated CIK cells (IL-2-CIK cells), we investigated the characteristics and potential mechanism of IL-15-stimulated CIK cells (IL-15-CIK cells) in this study. Compared with IL-2-CIK cells, the percentage of CD3CD56 cells was significantly increased in IL-15-CIK cells, but the expression of regulatory T (Treg) cells and IL-35 was significantly decreased in IL-15-CIK cells. Meanwhile, the in vitro cytotoxicity against human myeloid leukemia cells K562 of IL-15-CIK cells was significantly augmented compared with IL-2-CIK cells. These data suggest that IL-15 may improve the cytotoxicity of CIK cells against leukemia cells by upregulating CD3CD56 cells and downregulating Treg cells and IL-35.

  18. Cell-mediated cytotoxicity for melanome tumor cells: detection by a (3H)proline release assay

    International Nuclear Information System (INIS)

    Saal, J.G.; Rieber, E.P.; Riethmueller, G.

    1976-01-01

    An in vitro lymphocyte-mediated cytotoxicity assay using [ 3 H]proline-labelled target cells is described. The assay, modified from an original procedure of Bean et al., assesses the release of [ 3 H]proline by filtering the total culture fluid containing both trypsinised tumor cells and effector cells. Filtration is performed with a semiautomatic harvesting device using low suction pressure and large-diameter glass filters. Pretreatment of filters with whole serum diminishes adsorption of cell-free radioactive material considerably and thus increases the sensitivity of the assay. Nearly 100% of the radioactivity could be recovered with this harvesting device. The technique allowed the detection of cytolytic activities of lymphocytes after 6 h of incubation. Lymphocytes from patients with primary malignant melanoma showed a significantly higher cytolytic reactivity (p > 0.001) than normal donors' lymphocytes against three different melanoma cell lines. In a series of parallel experiments on 36 patients and 18 normal donors, this modification of the [ 3 ]proline test was compared with three different assays: the conventional microcytotoxicity test of Takasugi and Klein, the original [ 3 H]proline microcytotoxicity test of Bean et al., and the viability count of tumor cells. (Auth.)

  19. Towards immunotherapy with redirected T cells in a large animal model: Ex vivo activation, expansion, and genetic modification of canine T cells

    Science.gov (United States)

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

    2014-01-01

    Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) has shown promising anti-tumor activity in early phase clinical studies, especially for hematological malignancies. However, most preclinical models do not reliably mimic human disease. We reasoned that developing an adoptive T-cell therapy approach for spontaneous osteosarcoma (OS) occurring in dogs would more closely reproduce the condition in human cancer. To generate CAR-expressing canine T cells we developed expansion and transduction protocols that allow for the generation of sufficient numbers of CAR-expressing canine T cells for future clinical studies in dogs within 2 weeks of ex vivo culture. To evaluate the functionality of CAR-expressing canine T cells we targeted HER2-positive OS. We demonstrate that canine OS is positive for HER2, and that canine T cells expressing a HER2-specific CAR with human-derived transmembrane and CD28.ζ signaling domains recognize and kill HER2-positive canine OS cell lines in an antigen-dependent manner. To reduce the potential immunogenicity of the CAR we evaluated a CAR with canine-derived transmembrane and signaling domains, and found no functional difference between human and canine CARs. Hence, we have successfully developed a strategy to generate CAR-expressing canine T cells for future preclinical studies in dogs. Testing T-cell therapies in an immunocompetent, outbred animal model may improve our ability to predict their safety and efficacy prior to conducting studies in humans. PMID:25198528

  20. Cell transformation assays for prediction of carcinogenic potential: state of the science and future research needs

    Science.gov (United States)

    Creton, Stuart; Aardema, Marilyn J.; Carmichael, Paul L.; Harvey, James S.; Martin, Francis L.; Newbold, Robert F.; O’Donovan, Michael R.; Pant, Kamala; Poth, Albrecht; Sakai, Ayako; Sasaki, Kiyoshi; Scott, Andrew D.; Schechtman, Leonard M.; Shen, Rhine R.; Tanaka, Noriho; Yasaei, Hemad

    2012-01-01

    Cell transformation assays (CTAs) have long been proposed as in vitro methods for the identification of potential chemical carcinogens. Despite showing good correlation with rodent bioassay data, concerns over the subjective nature of using morphological criteria for identifying transformed cells and a lack of understanding of the mechanistic basis of the assays has limited their acceptance for regulatory purposes. However, recent drivers to find alternative carcinogenicity assessment methodologies, such as the Seventh Amendment to the EU Cosmetics Directive, have fuelled renewed interest in CTAs. Research is currently ongoing to improve the objectivity of the assays, reveal the underlying molecular changes leading to transformation and explore the use of novel cell types. The UK NC3Rs held an international workshop in November 2010 to review the current state of the art in this field and provide directions for future research. This paper outlines the key points highlighted at this meeting. PMID:21852270

  1. γ/δ T cell subsets in human aging using the classical α/β T cell model.

    Science.gov (United States)

    Vasudev, Anusha; Ying, Crystal Tan Tze; Ayyadhury, Shamini; Puan, Kia Joo; Andiappan, Anand Kumar; Nyunt, Ma Shwe Zin; Shadan, Nurhidaya Binte; Mustafa, Seri; Low, Ivy; Rotzschke, Olaf; Fulop, Tamas; Ng, Tze Pin; Larbi, Anis

    2014-10-01

    Aging is associated with an increased susceptibility to infections and diseases. It has also been associated with reduced functionality and altered distribution of immune cells, especially T cells. Whereas classical α/β T cells, especially CD8(+) T cells, were shown to be highly susceptible to aging, the effects of viral persistent stimulations on the fate of γ/δ T cells are much less documented. Healthy, elderly individuals of Chinese ethnical background were recruited under the aegis of SLAS-II. In this observational study, γ/δ T cell populations were characterized by flow cytometry and compared with the α/β CD4(+) and CD8(+) T cells in elderly and young controls. In our study, we identified a reduced frequency of γ/δ T cells but not α/β T cells with aging. The classical markers of α/β T cell aging, including CD28, CD27, and CD57, did not prove significant for γ/δ T cells. The extreme range of expression of these markers in γ/δ T cells was responsible for the lack of relationship between γ/δ T cell subsets, CD4/CD8 ratio, and anti-CMV titers that was significant for α/β T cells and, especially, CD8(+) T cells. Although markers of aging for γ/δ T cells are not clearly identified, our data collectively suggest that the presence of CD27 γ/δ T cells is associated with markers of α/β T cell aging. © 2014 Society for Leukocyte Biology.

  2. Eosinophils from Murine Lamina Propria Induce Differentiation of Naïve T Cells into Regulatory T Cells via TGF-β1 and Retinoic Acid.

    Directory of Open Access Journals (Sweden)

    Hong-Hu Chen

    Full Text Available Treg cells play a crucial role in immune tolerance, but mechanisms that induce Treg cells are poorly understood. We here have described eosinophils in lamina propria (LP that displayed high aldehyde dehydrogenase (ALDH activity, a rate-limiting step during all-trans retinoic acid (ATRA synthesis, and expressed TGF-β1 mRNA and high levels of ATRA. Co-incubation assay confirmed that LP eosinophils induced the differentiation of naïve T cells into Treg cells. Differentiation promoted by LP eosinophils were inhibited by blocked either TGF-β1 or ATRA. Peripheral blood (PB eosinophils did not produce ATRA and could not induce Treg differentiation. These data identifies LP eosinophils as effective inducers of Treg cell differentiation through a mechanism dependent on TGF-β1 and ATRA.

  3. ArtinM Mediates Murine T Cell Activation and Induces Cell Death in Jurkat Human Leukemic T Cells

    Science.gov (United States)

    Oliveira-Brito, Patrícia Kellen Martins; Gonçalves, Thiago Eleutério; Vendruscolo, Patrícia Edivânia; Roque-Barreira, Maria Cristina

    2017-01-01

    The recognition of cell surface glycans by lectins may be critical for the innate and adaptive immune responses. ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus, activates antigen-presenting cells by recognizing TLR2 N-glycans and induces Th1 immunity. We recently demonstrated that ArtinM stimulated CD4+ T cells to produce proinflammatory cytokines. Here, we further studied the effects of ArtinM on adaptive immune cells. We showed that ArtinM activates murine CD4+ and CD8+ T cells, augmenting their positivity for CD25, CD69, and CD95 and showed higher interleukin (IL)-2 and interferon (IFN)-γ production. The CD4+ T cells exhibited increased T-bet expression in response to ArtinM, and IL-2 production by CD4+ and CD8+ T cells depended on the recognition of CD3εγ-chain glycans by ArtinM. The ArtinM effect on aberrantly-glycosylated neoplastic lymphocytes was studied in Jurkat T cells, in which ArtinM induced IL-2, IFN-γ, and IL-1β production, but decreased cell viability and growth. A higher frequency of AnnexinV- and propidium iodide-stained cells demonstrated the induction of Jurkat T cells apoptosis by ArtinM, and this apoptotic response was reduced by caspases and protein tyrosine kinase inhibitors. The ArtinM effects on murine T cells corroborated with the immunomodulatory property of lectin, whereas the promotion of Jurkat T cells apoptosis may reflect a potential applicability of ArtinM in novel strategies for treating lymphocytic leukemia. PMID:28665310

  4. Mixture models for single-cell assays with applications to vaccine studies

    OpenAIRE

    Finak, Greg; McDavid, Andrew; Chattopadhyay, Pratip; Dominguez, Maria; De Rosa, Steve; Roederer, Mario; Gottardo, Raphael

    2013-01-01

    Blood and tissue are composed of many functionally distinct cell subsets. In immunological studies, these can be measured accurately only using single-cell assays. The characterization of these small cell subsets is crucial to decipher system-level biological changes. For this reason, an increasing number of studies rely on assays that provide single-cell measurements of multiple genes and proteins from bulk cell samples. A common problem in the analysis of such data is to identify biomarkers...

  5. Rapid quantification of the latent reservoir for HIV-1 using a viral outgrowth assay.

    Directory of Open Access Journals (Sweden)

    Gregory M Laird

    Full Text Available HIV-1 persists in infected individuals in a stable pool of resting CD4(+ T cells as a latent but replication-competent provirus. This latent reservoir is the major barrier to the eradication of HIV-1. Clinical trials are currently underway investigating the effects of latency-disrupting compounds on the persistence of the latent reservoir in infected individuals. To accurately assess the effects of such compounds, accurate assays to measure the frequency of latently infected cells are essential. The development of a simpler assay for the latent reservoir has been identified as a major AIDS research priority. We report here the development and validation of a rapid viral outgrowth assay that quantifies the frequency of cells that can release replication-competent virus following cellular activation. This new assay utilizes bead and column-based purification of resting CD4(+ T cells from the peripheral blood of HIV-1 infected patients rather than cell sorting to obtain comparable resting CD4(+ T cell purity. This new assay also utilizes the MOLT-4/CCR5 cell line for viral expansion, producing statistically comparable measurements of the frequency of latent HIV-1 infection. Finally, this new assay employs a novel quantitative RT-PCR specific for polyadenylated HIV-1 RNA for virus detection, which we demonstrate is a more sensitive and cost-effective method to detect HIV-1 replication than expensive commercial ELISA detection methods. The reductions in both labor and cost make this assay suitable for quantifying the frequency of latently infected cells in clinical trials of HIV-1 eradication strategies.

  6. Cytotoxicity of Titanate-Calcium Complexes to MC3T3 Osteoblast-Like Cells

    Science.gov (United States)

    Drury, Jeanie L.; Moussi, Joelle; Taylor-Pashow, Kathryn M. L.

    2016-01-01

    Monosodium titanates (MST) are a relatively novel form of particulate titanium dioxide that have been proposed for biological use as metal sorbents or delivery agents, most recently calcium (II). In these roles, the toxicity of the titanate or its metal complex is crucial to its biological utility. The aim of this study was to determine the cytotoxicity of MST and MST-calcium complexes with MC3T3 osteoblast-like cells; MST-Ca(II) complexes could be useful to promote bone formation in various hard tissue applications. MC3T3 cells were exposed to native MST or MST-Ca(II) complexes for 24–72 h. A CellTiter-Blue® assay was employed to assess the metabolic activity of the cells. The results showed that MST and MST-Ca(II) suppressed MC3T3 metabolic activity significantly in a dose-, time-, and cell-density-dependent fashion. MST-Ca(II) suppressed MC3T3 metabolism in a statistically identical manner as native MST at all concentrations. We concluded that MST and MST-Ca(II) are significantly cytotoxic to MC3T3 cells through a mechanism yet unknown; this is a potential problem to the biological utility of these complexes. PMID:28044136

  7. Cytotoxicity of Titanate-Calcium Complexes to MC3T3 Osteoblast-Like Cells

    Directory of Open Access Journals (Sweden)

    Yen-Wei Chen

    2016-01-01

    Full Text Available Monosodium titanates (MST are a relatively novel form of particulate titanium dioxide that have been proposed for biological use as metal sorbents or delivery agents, most recently calcium (II. In these roles, the toxicity of the titanate or its metal complex is crucial to its biological utility. The aim of this study was to determine the cytotoxicity of MST and MST-calcium complexes with MC3T3 osteoblast-like cells; MST-Ca(II complexes could be useful to promote bone formation in various hard tissue applications. MC3T3 cells were exposed to native MST or MST-Ca(II complexes for 24–72 h. A CellTiter-Blue® assay was employed to assess the metabolic activity of the cells. The results showed that MST and MST-Ca(II suppressed MC3T3 metabolic activity significantly in a dose-, time-, and cell-density-dependent fashion. MST-Ca(II suppressed MC3T3 metabolism in a statistically identical manner as native MST at all concentrations. We concluded that MST and MST-Ca(II are significantly cytotoxic to MC3T3 cells through a mechanism yet unknown; this is a potential problem to the biological utility of these complexes.

  8. Chimeric Antigen Receptor (CAR) T cells: Lessons Learned from Targeting of CD19 in B cell malignancies

    Science.gov (United States)

    Hay, Kevin A; Turtle, Cameron J

    2017-01-01

    Adoptive immunotherapy with chimeric antigen receptor-modified T (CAR-T) cells is a rapidly growing therapeutic approach to treating patients with refractory cancer, with over 100 clinical trials in various malignancies in progress. The enthusiasm for CAR-T cells has been driven by the clinical success of CD19-targeted CAR-T therapy in B-cell acute lymphoblastic leukemia, and the promising data in B-cell non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. Despite the success of targeting CD19 with CAR-T cells in early clinical studies, many challenges remain to improve outcomes, reduce toxicity, and determine the appropriate settings for CAR-T cell immunotherapy. Reviewing the lessons learned thus far in CD19 CAR-T cell trials and how some of these challenges may be overcome will help guide the development of CAR-T cell therapy for malignancies of B-cell origin, as well as for other hematopoietic and non-hematopoietic cancers. PMID:28110394

  9. Indoleamine 2,3-dioxygenase-expressing leukemic dendritic cells impair a leukemia-specific immune response by inducing potent T regulatory cells.

    Science.gov (United States)

    Curti, Antonio; Trabanelli, Sara; Onofri, Chiara; Aluigi, Michela; Salvestrini, Valentina; Ocadlikova, Darina; Evangelisti, Cecilia; Rutella, Sergio; De Cristofaro, Raimondo; Ottaviani, Emanuela; Baccarani, Michele; Lemoli, Roberto M

    2010-12-01

    The immunoregulatory enzyme indoleamine 2,3-dioxygenase, which catalyzes the conversion of tryptophan into kynurenine, is expressed in a significant subset of patients with acute myeloid leukemia, resulting in the inhibition of T-cell proliferation and the induction of regulatory T cells. Acute myeloid leukemia cells can be differentiated into dendritic cells, which have increased immunogenicity and have been proposed as vaccines against leukemia. Leukemic dendritic cells were generated from acute myeloid leukemia cells and used as stimulators in functional assays, including the induction of regulatory T cells. Indoleamine 2,3-dioxygenase expression in leukemic dendritic cells was evaluated at molecular, protein and enzymatic levels. We demonstrate that, after differentiation into dendritic cells, both indoleamine 2,3-dioxygenase-negative and indoleamine 2,3-dioxygenase-positive acute myeloid leukemia samples show induction and up-regulation of indoleamine 2,3-dioxygenase gene and protein, respectively. Indoleamine 2,3-dioxygenase-positive acute myeloid leukemia dendritic cells catabolize tryptophan into kynurenine metabolite and inhibit T-cell proliferation through an indoleamine 2,3-dioxygenase-dependent mechanism. Moreover, indoleamine 2,3-dioxygenase-positive leukemic dendritic cells increase the number of allogeneic and autologous CD4(+)CD25(+) Foxp3(+) T cells and this effect is completely abrogated by the indoleamine 2,3-dioxygenase-inhibitor, 1-methyl tryptophan. Purified CD4(+)CD25(+) T cells obtained from co-culture with indoleamine 2,3-dioxygenase-positive leukemic dendritic cells act as regulatory T cells as they inhibit naive T-cell proliferation and impair the complete maturation of normal dendritic cells. Importantly, leukemic dendritic cell-induced regulatory T cells are capable of in vitro suppression of a leukemia-specific T cell-mediated immune response, directed against the leukemia-associated antigen, Wilms' tumor protein. These data identify

  10. The cell biology of T-dependent B cell activation

    DEFF Research Database (Denmark)

    Owens, T; Zeine, R

    1989-01-01

    The requirement that CD4+ helper T cells recognize antigen in association with class II Major Histocompatibility Complex (MHC) encoded molecules constrains T cells to activation through intercellular interaction. The cell biology of the interactions between CD4+ T cells and antigen-presenting cells...... includes multipoint intermolecular interactions that probably involve aggregation of both polymorphic and monomorphic T cell surface molecules. Such aggregations have been shown in vitro to markedly enhance and, in some cases, induce T cell activation. The production of T-derived lymphokines that have been...... implicated in B cell activation is dependent on the T cell receptor for antigen and its associated CD3 signalling complex. T-dependent help for B cell activation is therefore similarly MHC-restricted and involves T-B intercellular interaction. Recent reports that describe antigen-independent B cell...

  11. Novel T lymphocyte proliferation assessment using whole mouse cryo-imaging

    Science.gov (United States)

    Wuttisarnwattana, Patiwet; Raza, Syed A.; Eid, Saada; Cooke, Kenneth R.; Wilson, David L.

    2014-03-01

    New imaging technologies enable one to assess T-cell proliferation, an important feature of the immunological response. However, none of the traditional imaging modalities allow one to examine quantiatively T-cell function with microscopic resolution and single cell sensitivity over an entire mouse. To address this need, we established T-cells proliferation assays using 3D microscopic cryo-imaging. Assays include: (1) biodistribution of T-cells, (2) secondary lymphoid organ (SLO) volume measurement, (3) carboxyfluorescein succinimidyl ester (CFSE) dilution per cell as cells divide. To demonstrate the application, a graft-versus-host-disease (GVHD) model was used. 3D visualization show that T-cells specifically homed to the SLOs (spleen and lymph nodes) as well as GVHD target organs (such as GI-tract, liver, skin and thymus).The spleen was chosen as representative of the SLOs. For spleen size analysis, volumes of red and white pulp were measured. Spleen volumes of the allogeneic mice (with GVHD) were significantly larger than those of the syngeneic mice (without GVHD) at 72 to 120 hours post-transplant. For CFSE dilution approach, we employed color-coded volume rendering and probability density function (PDF) of single cell intensity to assess T-cell proliferation in the spleen. As compared to syngeneic T-cells, the allogeneic T-cells quickly aggregated in the spleen as indicated by increasing of CFSE signal over the first 48 hours. Then they rapidly proliferated as evidenced by reduced CFSE intensity (at 48-96 hours). Results suggest that assays can be used to study GVHD treatments using T-cell proliferation and biodistibution as assays. In summary, this is the first time that we are able to track and visualize T-cells in whole mouse with single cell sensitivity. We believe that our technique can be an alternative choice to traditional in vitro immunological proliferation assays by providing assessment of proliferation in an in vivo model.

  12. CD4+CD62L+ Central Memory T Cells Can Be Converted to Foxp3+ T Cells

    Science.gov (United States)

    Zhang, Xiaolong; Chang Li, Xian; Xiao, Xiang; Sun, Rui; Tian, Zhigang; Wei, Haiming

    2013-01-01

    The peripheral Foxp3+ Treg pool consists of naturally arising Treg (nTreg) and adaptive Treg cells (iTreg). It is well known that naive CD4+ T cells can be readily converted to Foxp3+ iTreg in vitro, and memory CD4+ T cells are resistant to conversion. In this study, we investigated the induction of Foxp3+ T cells from various CD4+ T-cell subsets in human peripheral blood. Though naive CD4+ T cells were readily converted to Foxp3+ T cells with TGF-β and IL-2 treatment in vitro, such Foxp3+ T cells did not express the memory marker CD45RO as do Foxp3+ T cells induced in the peripheral blood of Hepatitis B Virus (HBV) patients. Interestingly, a subset of human memory CD4+ T cells, defined as CD62L+ central memory T cells, could be induced by TGF-β to differentiate into Foxp3+ T cells. It is well known that Foxp3+ T cells derived from human CD4+CD25- T cells in vitro are lack suppressive functions. Our data about the suppressive functions of CD4+CD62L+ central memory T cell-derived Foxp3+ T cells support this conception, and an epigenetic analysis of these cells showed a similar methylation pattern in the FOXP3 Treg-specific demethylated region as the naive CD4+ T cell-derived Foxp3+ T cells. But further research showed that mouse CD4+ central memory T cells also could be induced to differentiate into Foxp3+ T cells, such Foxp3+ T cells could suppress the proliferation of effector T cells. Thus, our study identified CD4+CD62L+ central memory T cells as a novel potential source of iTreg. PMID:24155942

  13. Alkaline Comet Assay for Assessing DNA Damage in Individual Cells.

    Science.gov (United States)

    Pu, Xinzhu; Wang, Zemin; Klaunig, James E

    2015-08-06

    Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies. Copyright © 2015 John Wiley & Sons, Inc.

  14. CAR T-cell therapy for glioblastoma: ready for the next round of clinical testing?

    Science.gov (United States)

    Prinzing, Brooke L; Gottschalk, Stephen M; Krenciute, Giedre

    2018-05-01

    The outcome for patients with glioblastoma (GBM) remains poor, and there is an urgent need to develop novel therapeutic approaches. T cells genetically modified with chimeric antigen receptors (CARs) hold the promise to improve outcomes since they recognize and kill cells through different mechanisms than conventional therapeutics. Areas covered: This article reviews CAR design, tumor associated antigens expressed by GBMs that can be targeted with CAR T cells, preclinical and clinical studies conducted with CAR T cells, and genetic approaches to enhance their effector function. Expert commentary: While preclinical studies have highlighted the potent anti-GBM activity of CAR T cells, the initial foray of CAR T-cell therapies into the clinic resulted only in limited benefits for GBM patients. Additional genetic modification of CAR T cells has resulted in a significant increase in their anti-GBM activity in preclinical models. We are optimistic that clinical testing of these enhanced CAR T cells will be safe and result in improved anti-glioma activity in GBM patients.

  15. CCL22-specific T Cells

    DEFF Research Database (Denmark)

    Martinenaite, Evelina; Munir Ahmad, Shamaila; Hansen, Morten

    2016-01-01

    Tumor cells and tumor-infiltrating macrophages produce the chemokine CCL22, which attracts regulatory T cells (Tregs) into the tumor microenvironment, decreasing anticancer immunity. Here, we investigated the possibility of targeting CCL22-expressing cells by activating specific T cells. We...... analyzed the CCL22 protein signal sequence, identifying a human leukocyte antigen A2- (HLA-A2-) restricted peptide epitope, which we then used to stimulate peripheral blood mononuclear cells (PMBCs) to expand populations of CCL22-specific T cells in vitro. T cells recognizing an epitope derived from...... the signal-peptide of CCL22 will recognize CCL22-expressing cells even though CCL22 is secreted out of the cell. CCL22-specific T cells recognized and killed CCL22-expressing cancer cells. Furthermore, CCL22-specific T cells lysed acute monocytic leukemia cells in a CCL22 expression-dependent manner. Using...

  16. An automated cell-counting algorithm for fluorescently-stained cells in migration assays

    Directory of Open Access Journals (Sweden)

    Novielli Nicole M

    2011-10-01

    Full Text Available Abstract A cell-counting algorithm, developed in Matlab®, was created to efficiently count migrated fluorescently-stained cells on membranes from migration assays. At each concentration of cells used (10,000, and 100,000 cells, images were acquired at 2.5 ×, 5 ×, and 10 × objective magnifications. Automated cell counts strongly correlated to manual counts (r2 = 0.99, P

  17. Chimeric Antigen Receptor (CAR) T Cells: Lessons Learned from Targeting of CD19 in B-Cell Malignancies.

    Science.gov (United States)

    Hay, Kevin A; Turtle, Cameron J

    2017-03-01

    Adoptive immunotherapy with chimeric antigen receptor-modified (CAR)-T cells is a rapidly growing therapeutic approach to treating patients with refractory cancer, with over 100 clinical trials in various malignancies in progress. The enthusiasm for CAR-T cells has been driven by the clinical success of CD19-targeted CAR-T cell therapy in B-cell acute lymphoblastic leukemia, and the promising data in B-cell non-Hodgkin's lymphoma and chronic lymphocytic leukemia. Despite the success of targeting CD19 with CAR-T cells in early clinical studies, many challenges remain to improve outcomes, reduce toxicity, and determine the appropriate settings for CAR-T cell immunotherapy. Reviewing the lessons learned thus far in CD19 CAR-T cell trials and how some of these challenges may be overcome will help guide the development of CAR-T cell therapy for malignancies of B-cell origin, as well as for other hematopoietic and non-hematopoietic cancers.

  18. Modulation of Trypanosoma cruzi-specific T-cell responses after chemotherapy for chronic Chagas disease

    Directory of Open Access Journals (Sweden)

    María Cecilia Albareda

    2015-05-01

    Full Text Available The aim of this review is to describe the contributions of the knowledge of T-cell responses to the understanding of the physiopathology and the responsiveness to etiological treatment during the chronic phase of Chagas disease. T-helper (Th1 and interleukin (IL-10 Trypanosoma cruzi-specific T-cells have been linked to the asymptomatic phase or to severe clinical forms of the disease, respectively or vice versa, depending on the T. cruzi antigen source, the patient’s location and the performed immunological assays. Parasite-specific T-cell responses are modulated after benznidazole (BZ treatment in chronically T. cruzi-infected subjects in association with a significant decrease in T. cruzi-specific antibodies. Accumulating evidence has indicated that treatment efficacy during experimental infection with T. cruzi results from the combined action of BZ and the activation of appropriate immune responses in the host. However, strong support of this interaction in T. cruzi-infected humans remains lacking. Overall, the quality of T-cell responses might be a key factor in not only disease evolution, but also chemotherapy responsiveness. Immunological parameters are potential indicators of treatment response regardless of achievement of cure. Providing tools to monitor and provide early predictions of treatment success will allow the development of new therapeutic options.

  19. Modulation of Autoimmune T-Cell Memory by Stem Cell Educator Therapy: Phase 1/2 Clinical Trial.

    Science.gov (United States)

    Delgado, Elias; Perez-Basterrechea, Marcos; Suarez-Alvarez, Beatriz; Zhou, Huimin; Revuelta, Eva Martinez; Garcia-Gala, Jose Maria; Perez, Silvia; Alvarez-Viejo, Maria; Menendez, Edelmiro; Lopez-Larrea, Carlos; Tang, Ruifeng; Zhu, Zhenlong; Hu, Wei; Moss, Thomas; Guindi, Edward; Otero, Jesus; Zhao, Yong

    2015-12-01

    Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that causes a deficit of pancreatic islet β cells. The complexities of overcoming autoimmunity in T1D have contributed to the challenges the research community faces when devising successful treatments with conventional immune therapies. Overcoming autoimmune T cell memory represents one of the key hurdles. In this open-label, phase 1/phase 2 study, Caucasian T1D patients (N = 15) received two treatments with the Stem Cell Educator (SCE) therapy, an approach that uses human multipotent cord blood-derived multipotent stem cells (CB-SCs). SCE therapy involves a closed-loop system that briefly treats the patient's lymphocytes with CB-SCs in vitro and returns the "educated" lymphocytes (but not the CB-SCs) into the patient's blood circulation. This study is registered with ClinicalTrials.gov, NCT01350219. Clinical data demonstrated that SCE therapy was well tolerated in all subjects. The percentage of naïve CD4(+) T cells was significantly increased at 26 weeks and maintained through the final follow-up at 56 weeks. The percentage of CD4(+) central memory T cells (TCM) was markedly and constantly increased at 18 weeks. Both CD4(+) effector memory T cells (TEM) and CD8(+) TEM cells were considerably decreased at 18 weeks and 26 weeks respectively. Additional clinical data demonstrated the modulation of C-C chemokine receptor 7 (CCR7) expressions on naïve T, TCM, and TEM cells. Following two treatments with SCE therapy, islet β-cell function was improved and maintained in individuals with residual β-cell function, but not in those without residual β-cell function. Current clinical data demonstrated the safety and efficacy of SCE therapy in immune modulation. SCE therapy provides lasting reversal of autoimmune memory that could improve islet β-cell function in Caucasian subjects. Obra Social "La Caixa", Instituto de Salud Carlos III, Red de Investigación Renal, European Union FEDER Funds, Principado de

  20. Quantifying rates of cell migration and cell proliferation in co-culture barrier assays reveals how skin and melanoma cells interact during melanoma spreading and invasion.

    Science.gov (United States)

    Haridas, Parvathi; Penington, Catherine J; McGovern, Jacqui A; McElwain, D L Sean; Simpson, Matthew J

    2017-06-21

    Malignant spreading involves the migration of cancer cells amongst other native cell types. For example, in vivo melanoma invasion involves individual melanoma cells migrating through native skin, which is composed of several distinct subpopulations of cells. Here, we aim to quantify how interactions between melanoma and fibroblast cells affect the collective spreading of a heterogeneous population of these cells in vitro. We perform a suite of circular barrier assays that includes: (i) monoculture assays with fibroblast cells; (ii) monoculture assays with SK-MEL-28 melanoma cells; and (iii) a series of co-culture assays initiated with three different ratios of SK-MEL-28 melanoma cells and fibroblast cells. Using immunostaining, detailed cell density histograms are constructed to illustrate how the two subpopulations of cells are spatially arranged within the spreading heterogeneous population. Calibrating the solution of a continuum partial differential equation to the experimental results from the monoculture assays allows us to estimate the cell diffusivity and the cell proliferation rate for the melanoma and the fibroblast cells, separately. Using the parameter estimates from the monoculture assays, we then make a prediction of the spatial spreading in the co-culture assays. Results show that the parameter estimates obtained from the monoculture assays lead to a reasonably accurate prediction of the spatial arrangement of the two subpopulations in the co-culture assays. Overall, the spatial pattern of spreading of the melanoma cells and the fibroblast cells is very similar in monoculture and co-culture conditions. Therefore, we find no clear evidence of any interactions other than cell-to-cell contact and crowding effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Drug-permeability and transporter assays in Caco-2 and MDCK cell lines.

    Science.gov (United States)

    Volpe, Donna A

    2011-12-01

    The human colon adenocarcinoma Caco-2 and Madin-Darby canine kidney epithelial cell lines provide in vitro tools to assess a drug's permeability and transporter interactions during discovery and development. The cells, when cultured on semiporous filters, form confluent monolayers that model the intestinal epithelial barrier for permeability, transporter and drug-interaction assays. The applications of these assays in pharmaceutical research include qualitative prediction and ranking of absorption, determining mechanism(s) of permeability, formulation effects on drug permeability, and the potential for transporter-mediated drug-drug interactions. This review focuses on recent examples of Caco-2 and Madin-Darby canine kidney cells assays for drug permeability including transfected and knock-down cells, miniaturization and automation, and assay combinations to better understand and predict intestinal drug absorption.

  2. Accessory signals in T-T cell interactions between antigen- and alloantigen-specific, human memory T cells generated in vitro

    DEFF Research Database (Denmark)

    Odum, N; Ryder, L P; Georgsen, J

    1990-01-01

    The potential of activated HLA class II-positive T cells as antigen-/alloantigen-presenting cells remains controversial. In our model system we use in vitro-primed, HLA class II-specific T cells of the memory T-cell phenotype, CD4+, CD29+ (4B4+), and CD45RO+ (UCHL-1). We have previously shown......), or a calcium ionophore (A23187) enabled Ta to elicit alloantigen-specific memory T-cell responses and to present purified protein derivative (PPD) to PPD-specific T-cell lines. The addition of irradiated, Epstein-Barr virus-transformed B-cell lines (EBV-LCL) (but not their supernatants) had a similar but less...

  3. Non-Catalytic Functions of Pyk2 and Fyn Regulate Late Stage Adhesion in Human T Cells

    Science.gov (United States)

    Houtman, Jon C. D.

    2012-01-01

    T cell activation drives the protective immune response against pathogens, but is also critical for the development of pathological diseases in humans. Cytoskeletal changes are required for downstream functions in T cells, including proliferation, cytokine production, migration, spreading, and adhesion. Therefore, investigating the molecular mechanism of cytoskeletal changes is crucial for understanding the induction of T cell-driven immune responses and for developing therapies to treat immune disorders related to aberrant T cell activation. In this study, we used a plate-bound adhesion assay that incorporated near-infrared imaging technology to address how TCR signaling drives human T cell adhesion. Interestingly, we observed that T cells have weak adhesion early after TCR activation and that binding to the plate was significantly enhanced 30–60 minutes after receptor activation. This late stage of adhesion was mediated by actin polymerization but was surprisingly not dependent upon Src family kinase activity. By contrast, the non-catalytic functions of the kinases Fyn and Pyk2 were required for late stage human T cell adhesion. These data reveal a novel TCR-induced signaling pathway that controls cellular adhesion independent of the canonical TCR signaling cascade driven by tyrosine kinase activity. PMID:23300847

  4. Chimeric Antigen Receptor-Modified T Cells Redirected to EphA2 for the Immunotherapy of Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Ning Li

    2018-02-01

    Full Text Available Erythropoietin-producing hepatocellular carcinoma A2 (EphA2 is overexpressed in more than 90% of non-small cell lung cancer (NSCLC but not significantly in normal lung tissue. It is therefore an important tumor antigen target for chimeric antigen receptors (CAR-T-based therapy in NSCLC. Here, we developed a specific CAR targeted to EphA2, and the anti-tumor effects of this CAR were investigated. A second generation CAR with co-stimulatory receptor 4-1BB targeted to EphA2 was developed. The functionality of EphA2-specific T cells in vitro was tested with flow cytometry and real-time cell electronic sensing system assays. The effect in vivo was evaluated in xenograft SCID Beige mouse model of EphA2 positive NSCLC. These EphA2-specifc T cells can cause tumor cell lysis by producing the cytokines IFN-γ when cocultured with EphA2-positive targets, and the cytotoxicity effects was specific in vitro. In vivo, the tumor signals of mice treated with EphA2-specifc T cells presented the tendency of decrease, and was much lower than the mice treated with non-transduced T cells. The anti-tumor effects of this CAR-T technology in vivo and vitro had been confirmed. Thus, EphA2-specific T-cell immunotherapy may be a promising approach for the treatment of EphA2-positive NSCLC.

  5. Lactobacillus rhamnosus CRL1505 nasal administration improves recovery of T-cell mediated immunity against pneumococcal infection in malnourished mice.

    Science.gov (United States)

    Barbieri, N; Herrera, M; Salva, S; Villena, J; Alvarez, S

    2017-05-30

    Immunobiotic lactic acid bacteria have become an interesting alternative for the prevention of respiratory infections. Previously, we demonstrated that the nasal administration of Lactobacillus rhamnosus CRL1505, during repletion of malnourished mice, resulted in diminished susceptibility to the challenge with the respiratory pathogen Streptococcus pneumoniae. Considering the known alterations induced by malnutrition on T lymphocytes and the importance of this cell population on the protection against respiratory pathogens, we aimed to study the effect of L. rhamnosus CRL1505 nasal administration on the recovery of T cell-mediated defences against pneumococcal infection in malnourished mice under nutritional recovery. Malnourished mice received a balanced conventional diet (BCD) for seven days or BCD for seven days with nasal L. rhamnosus CRL1505 supplementation during last two days of the treatment. After the treatments mice were infected with S. pneumoniae. Flow cytometry studies were carried out in bone marrow, thymus, spleen and lung to study T cells, and Th 1 /Th 2 cytokine profiles were determined in broncho-alveolar lavages and serum. The administration of CRL1505 strain to malnourished mice under recovery reduced quantitative and qualitative alterations of CD4 + T cells in the bone marrow, thymus, spleen and lung induced by malnutrition. In addition, CRL1505 treatment augmented Th 2 -cytokines (interleukin 10 and 4) in respiratory and systemic compartments after pneumococcal infection. These results show that modulation of CD4 + T lymphocytes induced by L. rhamnosus CRL1505 has an important role in the beneficial effect induced by this strain on the recovery of malnourished mice. These data also indicate that nasally administered L. rhamnosus CRL1505 may represent a non-invasive alternative to modulate and improve the T cell-mediated immunity against respiratory pathogens in immunocompromised malnourished hosts.

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

    Science.gov (United States)

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

    2005-05-01

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

  7. CD4+ T-Cell Reactivity to Orexin/Hypocretin in Patients With Narcolepsy Type 1.

    Science.gov (United States)

    Ramberger, Melanie; Högl, Birgit; Stefani, Ambra; Mitterling, Thomas; Reindl, Markus; Lutterotti, Andreas

    2017-03-01

    Narcolepsy type 1 is accompanied by a selective loss of orexin/hypocretin (hcrt) neurons in the lateral hypothalamus caused by yet unknown mechanisms. Epidemiologic and genetic associations strongly suggest an immune-mediated pathogenesis of the disease. We compared specific T-cell reactivity to orexin/hcrt peptides in peripheral blood mononuclear cells of narcolepsy type 1 patients to healthy controls by a carboxyfluorescein succinimidyl ester proliferation assay. Orexin/hcrt-specific T-cell reactivity was also determined by cytokine (interferon gamma and granulocyte-macrophage colony-stimulating factor) analysis. Individuals were considered as responders if the cell division index of CD3+CD4+ T cells and both stimulation indices of cytokine secretion exceeded the cutoff 3. Additionally, T-cell reactivity to orexin/hcrt had to be confirmed by showing reactivity to single peptides present in different peptide pools. Using these criteria, 3/15 patients (20%) and 0/13 controls (0%) showed orexin/hcrt-specific CD4+ T-cell proliferation (p = .2262). The heterogeneous reactivity pattern did not allow the identification of a preferential target epitope. A significant role of orexin/hcrt-specific T cells in narcolepsy type 1 patients could not be confirmed in this study. Further studies are needed to assess the exact role of CD4+ T cells and possible target antigens in narcolepsy type 1 patients. © Sleep Research Society 2016. Published by Oxford University Press [on behalf of the Sleep Research Society].

  8. HBsAg-redirected T cells exhibit antiviral activity in HBV-infected human liver chimeric mice.

    Science.gov (United States)

    Kruse, Robert L; Shum, Thomas; Tashiro, Haruko; Barzi, Mercedes; Yi, Zhongzhen; Whitten-Bauer, Christina; Legras, Xavier; Bissig-Choisat, Beatrice; Garaigorta, Urtzi; Gottschalk, Stephen; Bissig, Karl-Dimiter

    2018-04-06

    Chronic hepatitis B virus (HBV) infection remains incurable. Although HBsAg-specific chimeric antigen receptor (HBsAg-CAR) T cells have been generated, they have not been tested in animal models with authentic HBV infection. We generated a novel CAR targeting HBsAg and evaluated its ability to recognize HBV+ cell lines and HBsAg particles in vitro. In vivo, we tested whether human HBsAg-CAR T cells would have efficacy against HBV-infected hepatocytes in human liver chimeric mice. HBsAg-CAR T cells recognized HBV-positive cell lines and HBsAg particles in vitro as judged by cytokine production. However, HBsAg-CAR T cells did not kill HBV-positive cell lines in cytotoxicity assays. Adoptive transfer of HBsAg-CAR T cells into HBV-infected humanized mice resulted in accumulation within the liver and a significant decrease in plasma HBsAg and HBV-DNA levels compared with control mice. Notably, the fraction of HBV core-positive hepatocytes among total human hepatocytes was greatly reduced after HBsAg-CAR T cell treatment, pointing to noncytopathic viral clearance. In agreement, changes in surrogate human plasma albumin levels were not significantly different between treatment and control groups. HBsAg-CAR T cells have anti-HBV activity in an authentic preclinical HBV infection model. Our results warrant further preclinical exploration of HBsAg-CAR T cells as immunotherapy for HBV. Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  9. T-cell receptor and K-deleting recombination excision circles in newborn screening of T- and B-cell defects: review of the literature and future challenges

    Directory of Open Access Journals (Sweden)

    Marco Chiarini

    2013-05-01

    Full Text Available Since its introduction as a public health programme in the United States in the early 1960s, newborn blood screening (NBS has evolved from the detection of phenylalanine levels on filter paper to the application of DNA-based technologies to identify T-cell lymphopenia in infants with severe combined immunodeficiency. This latter use of NBS has required the development of an assay for T-cell lymphopenia based on the quantification of T-cell receptor excision circles (TRECs that could be performed on dried blood spots routinely collected from newborn infants. The TREC-based NBS was developed six years ago, and there have already been 7 successful pilot studies since then. Similarly, efforts are now being made to establish a screen for B-cell defects, in particular agammaglobulinaemia, taking advantage of the introduction of the method for the quantification of K-deleting recombination excision circles (KRECs. A further achievement of NBS could be the simultaneous recognition of T- and B-cell defects using the combined quantification of TRECs and KRECs from Guthrie card blood spots. This approach may help the early identification of infants with T- and B-cell deficiencies so that they can then be referred to specialised paediatric centres, where a precise diagnosis of severe combined immunodeficiency and agammaglobulinaemia can be performed, and where then they can immediately receive specific therapy. Simultaneous TREC and KREC quantification should also allow classification of patients into subgroups and help identify children with less serious primary immunodeficiencies. This would help avoid the opportunistic infections and frequent hospitalisations that result from a late or lack of diagnosis.

  10. Methods to Improve Adoptive T-Cell Therapy for Melanoma

    DEFF Research Database (Denmark)

    Donia, Marco; Hansen, Morten; Sendrup, Sarah L

    2013-01-01

    desirable. In this study, we demonstrated that a high in vitro tumor reactivity of infusion products was associated with clinical responses upon adoptive transfer. In addition, we systematically characterized the responses of a series of TIL products to relevant autologous short term-cultured melanoma cell...... lines from 12 patients. We provide evidence that antitumor reactivity of both CD8(+) and CD4(+) T cells could be enhanced in most TIL products by autologous melanoma sensitization by pretreatment with low-dose IFN-γ. IFN-γ selectively enhanced responses to tumor-associated antigens other than melanoma...... differentiation antigens. In addition, IFN-γ treatment was invariably associated with restored/increased cancer immunogenicity as demonstrated by upregulation of major histocompatibility complex molecules. These findings suggest a potential synergism between IFN-γ and ACT, and have important implications...

  11. Characterisation of the Immunomodulatory Effects of Meningococcal Opa Proteins on Human Peripheral Blood Mononuclear Cells and CD4+ T Cells.

    Directory of Open Access Journals (Sweden)

    Claire Jones

    Full Text Available Opa proteins are major surface-expressed proteins located in the Neisseria meningitidis outer membrane, and are potential meningococcal vaccine candidates. Although Opa proteins elicit high levels of bactericidal antibodies following immunisation in mice, progress towards human clinical trials has been delayed due to previous findings that Opa inhibits T cell proliferation in some in vitro assays. However, results from previous studies are conflicting, with different Opa preparations and culture conditions being used. We investigated the effects of various Opa+ and Opa- antigens from N. meningitidis strain H44/76 in a range of in vitro conditions using peripheral blood mononuclear cells (PBMCs and purified CD4+ T cells, measuring T cell proliferation by CFSE dilution using flow cytometry. Wild type recombinant and liposomal Opa proteins inhibited CD4+ T cell proliferation after stimulation with IL-2, anti-CD3 and anti-CD28, and these effects were reduced by mutation of the CEACAM1-binding region of Opa. These effects were not observed in culture with ex vivo PBMCs. Opa+ and Opa- OMVs did not consistently exert a stimulatory or inhibitory effect across different culture conditions. These data do not support a hypothesis that Opa proteins would be inhibitory to T cells if given as a vaccine component, and T cell immune responses to OMV vaccines are unlikely to be significantly affected by the presence of Opa proteins.

  12. Specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells in vivo

    International Nuclear Information System (INIS)

    Tanaka, K.; Hayakawa, H.; Sekiguchi, M.; Okada, Y.

    1977-01-01

    The specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells was examined using an in vivo assay system with hemagglutinating virus of Japan (Sendai virus) inactivated by uv light. A clear dose response was observed between the level of uv-induced unscheduled DNA synthesis of xeroderma pigmentosum cells and the amount of T4 endonuclease V activity added. The T4 enzyme was unstable in human cells, and its half-life was 3 hr. Fractions derived from an extract of Escherichia coli infected with T4v 1 , a mutant defective in the endonuclease V gene, showed no ability to restore the uv-induced unscheduled DNA synthesis of xeroderma pigmentosum cells. However, fractions derived from an extract of T4D-infected E. coli with endonuclease V activity were effective. The T4 enzyme was effective in xeroderma pigmentosum cells on DNA damaged by uv light but not in cells damaged by 4-nitroquinoline 1-oxide. The results of these experiments show that the T4 enzyme has a specific action on human cell DNA in vivo. Treatment with the T4 enzyme increased the survival of group A xeroderma pigmentosum cells after uv irradiation

  13. CXCR5-Dependent Entry of CD8 T Cells into Rhesus Macaque B-Cell Follicles Achieved through T-Cell Engineering.

    Science.gov (United States)

    Ayala, Victor I; Deleage, Claire; Trivett, Matthew T; Jain, Sumiti; Coren, Lori V; Breed, Matthew W; Kramer, Joshua A; Thomas, James A; Estes, Jacob D; Lifson, Jeffrey D; Ott, David E

    2017-06-01

    Follicular helper CD4 T cells, T FH , residing in B-cell follicles within secondary lymphoid tissues, are readily infected by AIDS viruses and are a major source of persistent virus despite relative control of viral replication. This persistence is due at least in part to a relative exclusion of effective antiviral CD8 T cells from B-cell follicles. To determine whether CD8 T cells could be engineered to enter B-cell follicles, we genetically modified unselected CD8 T cells to express CXC chemokine receptor 5 (CXCR5), the chemokine receptor implicated in cellular entry into B-cell follicles. Engineered CD8 T cells expressing human CXCR5 (CD8 hCXCR5 ) exhibited ligand-specific signaling and chemotaxis in vitro Six infected rhesus macaques were infused with differentially fluorescent dye-labeled autologous CD8 hCXCR5 and untransduced CD8 T cells and necropsied 48 h later. Flow cytometry of both spleen and lymph node samples revealed higher frequencies of CD8 hCXCR5 than untransduced cells, consistent with preferential trafficking to B-cell follicle-containing tissues. Confocal fluorescence microscopy of thin-sectioned lymphoid tissues demonstrated strong preferential localization of CD8 hCXCR5 T cells within B-cell follicles with only rare cells in extrafollicular locations. CD8 hCXCR5 T cells were present throughout the follicles with some observed near infected T FH In contrast, untransduced CD8 T cells were found in the extrafollicular T-cell zone. Our ability to direct localization of unselected CD8 T cells into B-cell follicles using CXCR5 expression provides a strategy to place highly effective virus-specific CD8 T cells into these AIDS virus sanctuaries and potentially suppress residual viral replication. IMPORTANCE AIDS virus persistence in individuals under effective drug therapy or those who spontaneously control viremia remains an obstacle to definitive treatment. Infected follicular helper CD4 T cells, T FH , present inside B-cell follicles represent a

  14. The regulation of CD5 expression in murine T cells

    Directory of Open Access Journals (Sweden)

    Herzenberg Leonard A

    2001-05-01

    Full Text Available Abstract Background CD5 is a pan-T cell surface marker that is also present on a subset of B cells, B-1a cells.Functional and developmental subsets of T cells express characteristic CD5 levels that vary over roughly a 30-fold range. Previous investigators have cloned a 1.7 Kb fragment containing the CD5 promoter and showed that it can confer similar lymphocyte-specific expression pattern as observed for endogenous CD5 expression. Results We further characterize the CD5 promoter and identify minimal and regulatory regions on the CD5 promoter. Using a luciferase reporter system, we show that a 43 bp region on the CD5 promoter regulates CD5 expression in resting mouse thymoma EL4 T cells and that an Ets binding site within the 43 bp region mediates the CD5 expression. In addition, we show that Ets-1, a member of the Ets family of transcription factors, recognizes the Ets binding site in the electrophoretic mobility shift assay (EMSA. This Ets binding site is directly responsible for the increase in reporter activity when co-transfected with increasing amounts of Ets-1 expression plasmid. We also identify two additional evolutionarily-conserved regions in the CD5 promoter (CD5X and CD5Y and demonstrate the respective roles of the each region in the regulation of CD5 transcription. Conclusion Our studies define a minimal and regulatory promoter for CD5 and show that the CD5 expression level in T cells is at least partially dependent on the level of Ets-1 protein. Based on the findings in this report, we propose a model of CD5 transcriptional regulation in T cells.