Mild hypothermia provides Treg stability

Marek-Trzonkowska, Natalia; Piekarska, Karolina; Filipowicz, Natalia; Piotrowski, Arkadiusz; Gucwa, Magdalena; Vogt, Katrin; Sawitzki, Birgit; Siebert, Janusz; Trzonkowski, Piotr

doi:10.1038/s41598-017-10151-1

Cited by 22 publications

(18 citation statements)

References 64 publications

(89 reference statements)

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“…Our standard large scale clinical-grade manual Treg expansion using G-Rex100 vessels resulted in cellular products with a Treg mean purity of 94.7% (range 89.5–98.1, n = 11) after 12–14 days of expansion (Figure 2C, upper panel). As published by us and other investigators, Treg expansion capacity is largely variable between donors (6, 13, 14, 17, 18, 25, 26). For direct comparison, isolated Treg enriched populations were split 1:10 after QC sampling to allow for a manual medium scale expansion culture using GRex10 devices (10 cm 2 ) in parallel to each CliniMACS Prodigy® culture (100 cm 2 ).…”

Section: Resultsmentioning

confidence: 60%

Automated Clinical Grade Expansion of Regulatory T Cells in a Fully Closed System

et al. 2019

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Adoptive transfer of T regulatory cells (Treg) has been successfully exploited in the context of graft-versus-host disease, transplantation, and autoimmune disease. For the majority of applications, clinical administration of Treg requires laborious ex vivo expansion and typically involves open handling for culture feeds and repetitive sampling. Here we show results from our approach to translate manual Treg manufacturing to the fully closed automated CliniMACS Prodigy® system reducing contamination risk, hands-on time, and quality variation from human intervention. Polyclonal Treg were isolated from total nucleated cells obtained through leukapheresis of healthy donors by CD8+ cell depletion and subsequent CD25high enrichment. Treg were expanded with the CliniMACS Prodigy® device using clinical-grade cell culture medium, rapamycin, IL-2, and αCD3/αCD28 beads for 13–14 days. We successfully integrated expansion bead removal and final formulation into the automated procedure, finalizing the process with a ready to use product for bedside transfusion. Automated Treg expansion was conducted in parallel to an established manual manufacturing process using G-Rex cell culture flasks. We could prove similar expansion kinetics leading to a cell yield of up to 2.12 × 109 cells with the CliniMACS Prodigy® and comparable product phenotype of >90% CD4+CD25highCD127lowFOXP3+ cells that had similar in vitro immunosuppressive function. Efficiency of expansion bead depletion was comparable to the CliniMACS® Plus system and the final ready-to-infuse product had phenotype stability and high vitality after overnight storage. We anticipate this newly developed closed system expansion approach to be a starting point for the development of enhanced throughput clinical scale Treg manufacture, and for safe automated generation of antigen-specific Treg grafted with a chimeric antigen receptor (CAR Treg).

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Section: Resultsmentioning

confidence: 60%

Automated Clinical Grade Expansion of Regulatory T Cells in a Fully Closed System

et al. 2019

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“…Given the low frequency of Tregs in the periphery, most clinical applications require an in vitro cell expansion culturing step classifying them as advanced therapy medicinal products. A growing number of culturing methods are being developed and published aiming at Treg induction, enhanced ex vivo expansion, alloreactivity and more recently, the implementation of specific T cell receptors or chimeric antigen receptors ( 17 , 18 , 25 , 31 – 39 ). We are thus at a point where protocol diversity is growing exponentially, emphasizing the necessity to harmonize reporting regimens as a prerequisite of reproducibility and quality assurance.…”

Section: Discussionmentioning

confidence: 99%

Minimum Information about T Regulatory Cells: A Step toward Reproducibility and Standardization

et al. 2018

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Cellular therapies with CD4+ T regulatory cells (Tregs) hold promise of efficacious treatment for the variety of autoimmune and allergic diseases as well as posttransplant complications. Nevertheless, current manufacturing of Tregs as a cellular medicinal product varies between different laboratories, which in turn hampers precise comparisons of the results between the studies performed. While the number of clinical trials testing Tregs is already substantial, it seems to be crucial to provide some standardized characteristics of Treg products in order to minimize the problem. We have previously developed reporting guidelines called minimum information about tolerogenic antigen-presenting cells, which allows the comparison between different preparations of tolerance-inducing antigen-presenting cells. Having this experience, here we describe another minimum information about Tregs (MITREG). It is important to note that MITREG does not dictate how investigators should generate or characterize Tregs, but it does require investigators to report their Treg data in a consistent and transparent manner. We hope this will, therefore, be a useful tool facilitating standardized reporting on the manufacturing of Tregs, either for research purposes or for clinical application. This way MITREG might also be an important step toward more standardized and reproducible testing of the Tregs preparations in clinical applications.

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“…found that culturing T regs at 33 ° C, compared to 37 ° C, yields a product with higher expression of CD25 and FOXP3, higher frequency of demethylated T reg ‐specific demethylated region (TSDR) and higher suppressive capacity . This group has since switched to using 33 ° C for manufacturing clinical grade cells . Mixed results have been reported for the effect of oxygen on T reg stability and function in culture.…”

Section: Modulating Treg Function or Stability With Culture Conditionsmentioning

confidence: 99%

Methods to manufacture regulatory T cells for cell therapy

MacDonald

Piret

Levings

2019

Clinical and Experimental Immunology

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Regulatory T cell (T reg ) therapy has shown promise in early clinical trials for treating graft-versus-host disease, transplant rejection and autoimmune disorders. A challenge has been to isolate sufficiently pure T regs and expand them to a clinical dose. However, there has been considerable progress in the development and optimization of these methods, resulting in a variety of manufacturing protocols being tested in clinical trials. In this review, we summarize methods that have been used to manufacture T regs for clinical trials, including the choice of cell source and protocols for cell isolation and expansion. We also discuss alternative culture or genome editing methods for modulating T reg specificity, function or stability that could be applied to future clinical manufacturing protocols to increase the efficacy of T reg therapy.

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Mild hypothermia provides Treg stability

Cited by 22 publications

References 64 publications

Automated Clinical Grade Expansion of Regulatory T Cells in a Fully Closed System

Automated Clinical Grade Expansion of Regulatory T Cells in a Fully Closed System

Minimum Information about T Regulatory Cells: A Step toward Reproducibility and Standardization

Methods to manufacture regulatory T cells for cell therapy

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