Cryopreservation timing is a critical process parameter in a thymic regulatory T-cell therapy manufacturing protocol

MacDonald, Katherine N.; Ivison, Sabine; Hippen, Keli L.; Hoeppli, R.; Hall, Maurice C.; Zheng, Grace; Dijke, I. Esmé; Aklabi, Mohammed Al; Freed, Darren H.; Rebeyka, Ivan M.; Gandhi, Sanjiv K.; West, Lori J.; Piret, James M.; Blazar, Bruce R.; Levings, Megan K.

doi:10.1016/j.jcyt.2019.10.011

Cited by 19 publications

(23 citation statements)

References 50 publications

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“…Repigmentation can occur in patients treated with JAK inhibitors to suppress Teff activity, though supportive treatment by UV light may be required (65). For intermittent treatment of vitiligo, it will be beneficial to store autologous GD3 CAR Tregs for later use (66).…”

Section: Discussionmentioning

confidence: 99%

Antigen Specificity Enhances Disease Control by Tregs in Vitiligo

et al. 2020

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Vitiligo is an autoimmune skin disease characterized by melanocyte destruction. Regulatory T cells (Tregs) are greatly reduced in vitiligo skin, and replenishing peripheral skin Tregs can provide protection against depigmentation. Ganglioside D3 (GD3) is overexpressed by perilesional epidermal cells, including melanocytes, which prompted us to generate GD3-reactive chimeric antigen receptor (CAR) Tregs to treat vitiligo. Mice received either untransduced Tregs or GD3-specific Tregs to test the hypothesis that antigen specificity contributes to reduced autoimmune reactivity in vitro and in vivo. CAR Tregs displayed increased IL-10 secretion in response to antigen, provided superior control of cytotoxicity towards melanocytes, and supported a significant delay in depigmentation compared to untransduced Tregs and vehicle control recipients in a TCR transgenic mouse model of spontaneous vitiligo. The latter findings were associated with a greater abundance of Tregs and melanocytes in treated mice versus both control groups. Our data support the concept that antigen-specific Tregs can be prepared, used, and stored for long-term control of progressive depigmentation.

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Section: Discussionmentioning

confidence: 99%

Antigen Specificity Enhances Disease Control by Tregs in Vitiligo

et al. 2020

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“…Others have shown that thawing of cryopreserved expanded Tregs followed by restimulation can overcome the detrimental effects of cryopreservation on Treg number and phenotype [ 215 ]. In other studies, higher Treg viability and Foxp3 expression were obtained when cells were cryopreserved 1–3 days, but not >3 days after last restimulation [ 216 ]. The ability to cryopreserve and thaw expanded Tregs will have broad-ranging implications when designing clinical trials.…”

Section: Translational Challengesmentioning

confidence: 99%

Regulatory T Cell Therapy of Graft-versus-Host Disease: Advances and Challenges

Hefazi

Bolivar-Wagers

Blazar

2021

IJMS

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Graft-versus-host disease (GVHD) is the leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Immunomodulation using regulatory T cells (Tregs) offers an exciting option to prevent and/or treat GVHD as these cells naturally function to maintain immune homeostasis, can induce tolerance following HSCT, and have a tissue reparative function. Studies to date have established a clinical safety profile for polyclonal Tregs. Functional enhancement through genetic engineering offers the possibility of improved potency, specificity, and persistence. In this review, we provide the most up to date preclinical and clinical data on Treg cell therapy with a particular focus on GVHD. We discuss the different Treg subtypes and highlight the pharmacological and genetic approaches under investigation to enhance the application of Tregs in allo-HSCT. Lastly, we discuss the remaining challenges for optimal clinical translation and provide insights as to future directions of the field.

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“…Moreover, naive and memory Tregs (nTregs and mTregs, defined by the presence or absence of CD45RA, respectively) have distinct characteristics (Miyara et al, 2009) and expansion potential (Hoffmann et al, 2006(Hoffmann et al, , 2009Marek et al, 2011). We thus investigated the ability of CD3/CD28/CD2 tetramers to expand nTregs vs. mTregs in comparison to an artificial antigenpresenting cell (aAPC)-based method we previously found to be optimal for nTreg expansion (Dijke et al, 2016;MacDonald et al, 2019). aAPC-based pre-activation resulted in a substantial and consistent increase in nTreg and mTreg yield at day 5 ( Figure 1C).…”

Section: Optimization Of Crispr and Hdr Delivery To Human Tregsmentioning

confidence: 99%

Optimized CRISPR-mediated gene knock-in reveals FOXP3-independent control of human Treg identity

Lam

Lin

Gillies

et al. 2021

Preprint

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SummaryTreg cell therapy is a promising curative approach for a variety of immune-mediated conditions. CRISPR-based genome editing allows precise insertion of transgenes through homology-directed repair, but use in human Tregs has been limited. We report an optimized protocol for CRISPR-mediated gene knock-in in human Tregs with high-yield expansion. To establish a benchmark of human Treg dysfunction, we targeted the master transcription factor FOXP3 in naive and memory Tregs. Although FOXP3-knockout Tregs upregulated cytokine expression, effects on suppressive capacity manifested slowly and primarily in memory Tregs. Moreover, FOXP3-knockout Tregs retained their characteristic phenotype and had few changes in their DNA methylation landscape, with FOXP3 maintaining methylation at regions enriched for AP-1 binding sites. Thus, while FOXP3 is important for human Treg development, it has a limited role in maintaining mature Treg identity. Optimized gene knock-in with human Tregs will enable mechanistic studies and the development of tailored, next-generation Treg cell therapies.

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Cryopreservation timing is a critical process parameter in a thymic regulatory T-cell therapy manufacturing protocol

Cited by 19 publications

References 50 publications

Antigen Specificity Enhances Disease Control by Tregs in Vitiligo

Antigen Specificity Enhances Disease Control by Tregs in Vitiligo

Regulatory T Cell Therapy of Graft-versus-Host Disease: Advances and Challenges

Optimized CRISPR-mediated gene knock-in reveals FOXP3-independent control of human Treg identity

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