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  1. Article ; Online: An optimized retroviral toolbox for overexpression and genetic perturbation of primary lymphocytes

    Lieve E. H. van der Donk / Jet van der Spek / Tom van Duivenvoorde / Marieke S. ten Brink / Teunis B. H. Geijtenbeek / Coenraad P. Kuijl / Jeroen W. J. van Heijst / Louis S. Ates

    Biology Open, Vol 11, Iss

    2022  Volume 2

    Abstract: Genetic manipulation of primary lymphocytes is crucial for both clinical purposes and fundamental research. Despite their broad use, we encountered a paucity of data on systematic comparison and optimization of retroviral vectors, the workhorses of ... ...

    Abstract Genetic manipulation of primary lymphocytes is crucial for both clinical purposes and fundamental research. Despite their broad use, we encountered a paucity of data on systematic comparison and optimization of retroviral vectors, the workhorses of genetic modification of primary lymphocytes. Here, we report the construction and validation of a versatile range of retroviral expression vectors. These vectors can be used for the knockdown or overexpression of genes of interest in primary human and murine lymphocytes, in combination with a wide choice of selection and reporter strategies. By streamlining the vector backbone and insert design, these publicly available vectors allow easy interchangeability of the independent building blocks, such as different promoters, fluorescent proteins, surface markers and antibiotic resistance cassettes. We validated these vectors and tested the optimal promoters for in vitro and in vivo overexpression and knockdown of the murine T cell antigen receptor. By publicly sharing these vectors and the data on their optimization, we aim to facilitate genetic modification of primary lymphocytes for researchers entering this field.
    Keywords retrovirus ; t cell ; lymphocytes ; overexpression ; Science ; Q ; Biology (General) ; QH301-705.5
    Subject code 006
    Language English
    Publishing date 2022-02-01T00:00:00Z
    Publisher The Company of Biologists
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: RD5-mediated lack of PE_PGRS and PPE-MPTR export in BCG vaccine strains results in strong reduction of antigenic repertoire but little impact on protection.

    Louis S Ates / Fadel Sayes / Wafa Frigui / Roy Ummels / Merel P M Damen / Daria Bottai / Marcel A Behr / Jeroen W J van Heijst / Wilbert Bitter / Laleh Majlessi / Roland Brosch

    PLoS Pathogens, Vol 14, Iss 6, p e

    2018  Volume 1007139

    Abstract: Tuberculosis is the deadliest infectious disease worldwide. Although the BCG vaccine is widely used, it does not efficiently protect against pulmonary tuberculosis and an improved tuberculosis vaccine is therefore urgently needed. Mycobacterium ... ...

    Abstract Tuberculosis is the deadliest infectious disease worldwide. Although the BCG vaccine is widely used, it does not efficiently protect against pulmonary tuberculosis and an improved tuberculosis vaccine is therefore urgently needed. Mycobacterium tuberculosis uses different ESX/Type VII secretion (T7S) systems to transport proteins important for virulence and host immune responses. We recently reported that secretion of T7S substrates belonging to the mycobacteria-specific Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins of the PGRS (polymorphic GC-rich sequences) and MPTR (major polymorphic tandem repeat) subfamilies required both a functional ESX-5 system and a functional PPE38/71 protein for secretion. Inactivation of ppe38/71 and the resulting loss of PE_PGRS/PPE-MPTR secretion were linked to increased virulence of M. tuberculosis strains. Here, we show that a predicted total of 89 PE_PGRS/PPE-MPTR surface proteins are not exported by certain animal-adapted strains of the M. tuberculosis complex including M. bovis. This Δppe38/71-associated secretion defect therefore also occurs in the M. bovis-derived tuberculosis vaccine BCG and could be partially restored by introduction of the M. tuberculosis ppe38-locus. Epitope mapping of the PPE-MPTR protein PPE10, further allowed us to monitor T-cell responses in splenocytes from BCG/M. tuberculosis immunized mice, confirming the dependence of PPE10-specific immune-induction on ESX-5/PPE38-mediated secretion. Restoration of PE_PGRS/PPE-MPTR secretion in recombinant BCG neither altered global antigenic presentation or activation of innate immune cells, nor protective efficacy in two different mouse vaccination-infection models. This unexpected finding stimulates a reassessment of the immunomodulatory properties of PE_PGRS/PPE-MPTR proteins, some of which are contained in vaccine formulations currently in clinical evaluation.
    Keywords Immunologic diseases. Allergy ; RC581-607 ; Biology (General) ; QH301-705.5
    Subject code 570
    Language English
    Publishing date 2018-06-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: A gamma interferon independent mechanism of CD4 T cell mediated control of M. tuberculosis infection in vivo.

    Alena M Gallegos / Jeroen W J van Heijst / Miriam Samstein / Xiaodi Su / Eric G Pamer / Michael S Glickman

    PLoS Pathogens, Vol 7, Iss 5, p e

    2011  Volume 1002052

    Abstract: CD4 T cell deficiency or defective IFNγ signaling render humans and mice highly susceptible to Mycobacterium tuberculosis (Mtb) infection. The prevailing model is that Th1 CD4 T cells produce IFNγ to activate bactericidal effector mechanisms of infected ... ...

    Abstract CD4 T cell deficiency or defective IFNγ signaling render humans and mice highly susceptible to Mycobacterium tuberculosis (Mtb) infection. The prevailing model is that Th1 CD4 T cells produce IFNγ to activate bactericidal effector mechanisms of infected macrophages. Here we test this model by directly interrogating the effector functions of Th1 CD4 T cells required to control Mtb in vivo. While Th1 CD4 T cells specific for the Mtb antigen ESAT-6 restrict in vivo Mtb growth, this inhibition is independent of IFNγ or TNF and does not require the perforin or FAS effector pathways. Adoptive transfer of Th17 CD4 T cells specific for ESAT-6 partially inhibited Mtb growth while Th2 CD4 T cells were largely ineffective. These results imply a previously unrecognized IFNγ/TNF independent pathway that efficiently controls Mtb and suggest that optimization of this alternative effector function may provide new therapeutic avenues to combat Mtb through vaccination.
    Keywords Immunologic diseases. Allergy ; RC581-607 ; Biology (General) ; QH301-705.5
    Subject code 570
    Language English
    Publishing date 2011-05-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article: Heterogeneous Differentiation Patterns of Individual CD8⁺ T Cells

    Gerlach, Carmen / Arno Velds / Heinz Jacobs / Jan C. Rohr / Jeroen W. J. van Heijst / Joost B. Beltman / Jos Urbanus / Leïla Perié / Nienke van Rooij / Rob J. de Boer / Shalin H. Naik / Ton N. M. Schumacher

    Science. 2013 May 3, v. 340, no. 6132

    2013  

    Abstract: Dynamic Protection During an immune response, CD8 ⁺ T cells are recruited to provide protection. Most cells differentiate into short-lived effectors that help to clear the pathogen, whereas others form long-lived memory cells to protect against ... ...

    Abstract Dynamic Protection During an immune response, CD8 ⁺ T cells are recruited to provide protection. Most cells differentiate into short-lived effectors that help to clear the pathogen, whereas others form long-lived memory cells to protect against reinfection. Gerlach et al. (p. 635, published online 14 March) and Buchholz et al. (p. 630, published online 14 March) used in vivo fate mapping of mouse T cells with a defined specificity during a bacterial infection to show that the dynamics of the single-cell response are not uniform. The response of a particular T cell population is the average of a small number of clones that expand greatly (“large clones”) and many clones that only proliferate at low amounts (“small clones”). The memory pool arises largely from small clones whereas effectors are primarily made up of large clones.
    Keywords bacterial infections ; CD8-positive T-lymphocytes ; clones ; immune response ; memory ; mice ; pathogens
    Language English
    Dates of publication 2013-0503
    Size p. 635-639.
    Publishing place American Association for the Advancement of Science
    Document type Article
    ZDB-ID 128410-1
    ISSN 1095-9203 ; 0036-8075
    ISSN (online) 1095-9203
    ISSN 0036-8075
    DOI 10.1126/science.1235487
    Database NAL-Catalogue (AGRICOLA)

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