Adaeze Precious Ekwe , Raymond Au , Ping Zhang , Benjamin A. McEnroe , Mei Ling Tan , Alda Saldan , Andrea S. Henden , Cheryl J. Hutchins , Ashleigh Henderson , Kari Mudie , Keri Kerr , Madonna Fuery , Glen A. Kennedy , Geoffrey R. Hill , Siok-Keen Tey
{"title":"Clinical grade multiparametric cell sorting and gene-marking of regulatory T cells","authors":"Adaeze Precious Ekwe , Raymond Au , Ping Zhang , Benjamin A. McEnroe , Mei Ling Tan , Alda Saldan , Andrea S. Henden , Cheryl J. Hutchins , Ashleigh Henderson , Kari Mudie , Keri Kerr , Madonna Fuery , Glen A. Kennedy , Geoffrey R. Hill , Siok-Keen Tey","doi":"10.1016/j.jcyt.2024.02.023","DOIUrl":null,"url":null,"abstract":"<div><h3>Background aims</h3><p>Regulatory T cells (Tregs) are the main mediators of peripheral tolerance. Treg-directed therapy has shown promising results in preclinical studies of diverse immunopathologies. At present, the clinical applicability of adoptive Treg transfer is limited by difficulties in generating Tregs at sufficient cell dose and purity.</p></div><div><h3>Methods</h3><p>We developed a Good Manufacturing Practice (GMP) compliant method based on closed-system multiparametric Fluorescence-Activated Cell Sorting (FACS) to purify Tregs, which are then expanded <em>in vitro</em> and gene-marked with a clinical grade retroviral vector to enable <em>in vivo</em> fate tracking. Following small-scale optimization, we conducted four clinical-scale processing runs.</p></div><div><h3>Results</h3><p>We showed that Tregs could be enriched to 87– 92% purity following FACS-sorting, and expanded and transduced to yield clinically relevant cell dose of 136–732×10<sup>6</sup> gene-marked cells, sufficient for a cell dose of at least 2 × 10<sup>6</sup> cells/kg. The expanded Tregs were highly demethylated in the <em>FOXP3</em> Treg-specific demethylated region (TSDR), consistent with bona fide natural Tregs. They were suppressive <em>in vitro</em>, but a small percentage could secrete proinflammatory cytokines, including interferon-γ and interleukin-17A.</p></div><div><h3>Conclusions</h3><p>This study demonstrated the feasibility of isolating, expanding and gene-marking Tregs in clinical scale, thus paving the way for future phase I trials that will advance knowledge about the <em>in vivo</em> fate of transferred Tregs and its relationship with concomitant Treg-directed pharmacotherapy and clinical response.</p></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":null,"pages":null},"PeriodicalIF":3.7000,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cytotherapy","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1465324924000665","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Background aims
Regulatory T cells (Tregs) are the main mediators of peripheral tolerance. Treg-directed therapy has shown promising results in preclinical studies of diverse immunopathologies. At present, the clinical applicability of adoptive Treg transfer is limited by difficulties in generating Tregs at sufficient cell dose and purity.
Methods
We developed a Good Manufacturing Practice (GMP) compliant method based on closed-system multiparametric Fluorescence-Activated Cell Sorting (FACS) to purify Tregs, which are then expanded in vitro and gene-marked with a clinical grade retroviral vector to enable in vivo fate tracking. Following small-scale optimization, we conducted four clinical-scale processing runs.
Results
We showed that Tregs could be enriched to 87– 92% purity following FACS-sorting, and expanded and transduced to yield clinically relevant cell dose of 136–732×106 gene-marked cells, sufficient for a cell dose of at least 2 × 106 cells/kg. The expanded Tregs were highly demethylated in the FOXP3 Treg-specific demethylated region (TSDR), consistent with bona fide natural Tregs. They were suppressive in vitro, but a small percentage could secrete proinflammatory cytokines, including interferon-γ and interleukin-17A.
Conclusions
This study demonstrated the feasibility of isolating, expanding and gene-marking Tregs in clinical scale, thus paving the way for future phase I trials that will advance knowledge about the in vivo fate of transferred Tregs and its relationship with concomitant Treg-directed pharmacotherapy and clinical response.
期刊介绍:
The journal brings readers the latest developments in the fast moving field of cellular therapy in man. This includes cell therapy for cancer, immune disorders, inherited diseases, tissue repair and regenerative medicine. The journal covers the science, translational development and treatment with variety of cell types including hematopoietic stem cells, immune cells (dendritic cells, NK, cells, T cells, antigen presenting cells) mesenchymal stromal cells, adipose cells, nerve, muscle, vascular and endothelial cells, and induced pluripotential stem cells. We also welcome manuscripts on subcellular derivatives such as exosomes. A specific focus is on translational research that brings cell therapy to the clinic. Cytotherapy publishes original papers, reviews, position papers editorials, commentaries and letters to the editor. We welcome "Protocols in Cytotherapy" bringing standard operating procedure for production specific cell types for clinical use within the reach of the readership.