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  • A strategy to purify, expand and gene-mark regulatory T cells (tregs) for use in the treatment of chronic Graft-Versus-Host Disease (GVHD)

    Author(s)
    Au, R
    Ping, Z
    Hutchins, C
    Mudie, K
    Western, R
    Kennedy, G
    Tey, S
    Hill, G
    Griffith University Author(s)
    Tey, Siok
    Year published
    2019
    Metadata
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    Abstract
    Background & Aim Chronic Graft-Versus-Host Disease (GVHD) is a major complication of bone marrow transplantation. Regulatory T cells (Tregs) have been shown to ameliorate GVHD in pre-clinical studies and early phase clinical trials. However, Treg adoptive transfer is currently limited by cell dose and purity; and strategies to combine this with in vivo expansion are being actively pursued. In this project, we optimized a clinically applicable method to purify Tregs by Fluorescent-Activated Cell Sorting (FACS). We also developed a method to gene-mark Tregs to enable clinical fate-tracking. Methods, Results & Conclusion We ...
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    Background & Aim Chronic Graft-Versus-Host Disease (GVHD) is a major complication of bone marrow transplantation. Regulatory T cells (Tregs) have been shown to ameliorate GVHD in pre-clinical studies and early phase clinical trials. However, Treg adoptive transfer is currently limited by cell dose and purity; and strategies to combine this with in vivo expansion are being actively pursued. In this project, we optimized a clinically applicable method to purify Tregs by Fluorescent-Activated Cell Sorting (FACS). We also developed a method to gene-mark Tregs to enable clinical fate-tracking. Methods, Results & Conclusion We could enrich Tregs to high purity: from 6.6% (±1.4%) in buffy coat samples to 54.1% (±20.9%) after CD25-immunomagnetic selection on a CliniMACS Plus device (Miltenyi Biotec), and to 95.3% (± 1.8%) purity after multiparametric FACS-sorting for CD4+CD25+CD127low Tregs (MacsQuant Tyto, Miltenyi Biotec). The purified Tregs could be expanded with CD3/CD28 activation in medium supplemented with interleukin-2 and rapamycin. We showed that Tregs could be successfully gene-marked with a clinically approved retroviral vector, SFG.iCasp9.2A.ΔCD19. The expanded gene-marked Tregs maintained a high level of FOXP3 expression (92% ± 0.7%) and FOXP3 promoter demethylation, which is a hallmark of bona fide Tregs. Importantly, gene-marking did not alter FOXP3 expression or Treg suppressor function. We will now scale up this process using leukapheresis samples from healthy volunteers in preparation for a phase I clinical study in patients with chronic GVHD using Treg transfer in combination with low-dose interleukin-2 for in vivo expansion. Gene-marking will enable analysis of the contribution of adoptive transfer (gene-marked) versus in vivoexpansion (non-gene marked) Tregs, thus informing rational Treg-directed combination therapy.
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    Conference Title
    CYTOTHERAPY
    Volume
    21
    Issue
    5
    DOI
    https://doi.org/10.1016/j.jcyt.2019.04.026
    Subject
    Clinical sciences
    Science & Technology
    Life Sciences & Biomedicine
    Cell & Tissue Engineering
    Biotechnology & Applied Microbiology
    Cell Biology
    Publication URI
    http://hdl.handle.net/10072/392792
    Collection
    • Conference outputs

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