Challenges in cryopreservation of regulatory T cells (Tregs) for clinical therapeutic applications

Gołąb, Karolina; Leveson-Gower, Dennis B.; Wang, Xiaojun; Grzanka, Jakub; Marek-Trzonkowska, Natalia; Krzystyniak, Adam; Millis, J. Michael; Trzonkowski, Piotr; Witkowski, Piotr

doi:10.1016/j.intimp.2013.02.001

Cited by 46 publications

(40 citation statements)

References 47 publications

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“…Our protocol for expanding baboon Treg, reported herein, not only produces far greater numbers of Treg than those previously reported for this species (approximately 50-fold more), but also compares favorably with other previously reported protocols for expanding human and non-human primate Treg (5, 8, 10–12, 16, 29–31). In contrast to some previous reports, our Treg do not lose their phenotype after cryopreservation, although they do lose suppressive function.…”

Section: Discussionsupporting

confidence: 84%

“…This finding remains surprising given the many ways that cryopreservation can affect Treg. One main factor is the toxic effect of DMSO on Treg and indeed all PBMCs (32), but cryopreservation has also been shown to cause oxidative stress from free radicals, cell membrane and structural damage from ice crystals, osmotic injury, and significant changes in cell metabolism and membrane channels (16). …”

Section: Discussionmentioning

confidence: 99%

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Restimulation After Cryopreservation and Thawing Preserves the Phenotype and Function of Expanded Baboon Regulatory T Cells

Weiner

Duran‐Struuck

Zitsman

et al. 2015

Transplantation Direct

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Background Regulatory T cells (Treg) are being explored for their tolerance-inducing capabilities. Freezing and banking Treg for future use makes this strategy more clinically applicable. We aimed to devise an improved method of expanding and cryopreserving Treg to maximize yield, purity, and function for use in xenotransplantation. Methods Baboon peripheral blood mononuclear cells (PBMC) were isolated from whole blood. CD4+/CD25hi cells were isolated by flow cytometric sorting and expanded for 26 days in culture with IL-2, anti-CD3 antibody, artificial APCs transfected with human CD58, CD32, and CD80, and rapamycin with weekly restimulations. Expanded Treg were frozen for 2 months then thawed and cultured for 48 hours in medium plus 1) no additives, 2) IL-2, 3) anti-CD3 antibody, 4) IL-2 + anti-CD3 antibody, and 5) IL-2 + anti-CD3 antibody + L cells. Phenotype and suppression were assessed after expansion, immediately after thawing, and after culturing. Results We expanded purified baboon Treg more than 10,000-fold. Expanded Treg exhibited excellent suppression in functional assays. Cryopreservation decreased suppressive function without changing phenotype, but increasing amounts of reactivation after thawing produced significantly better viability and suppressive function with a trend towards greater Treg purity. Conclusions We produced numbers of expanded Tregs consistent with clinical use. In contrast to some previous reports, both Treg phenotype and suppressive function were preserved or even enhanced by increasing amounts of restimulation after thawing. Thus, banking of expanded recipient Tregs for in vivo infusion should be possible.

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Section: Discussionsupporting

confidence: 84%

Section: Discussionmentioning

confidence: 99%

Restimulation After Cryopreservation and Thawing Preserves the Phenotype and Function of Expanded Baboon Regulatory T Cells

Weiner

Duran‐Struuck

Zitsman

et al. 2015

Transplantation Direct

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“…Some studies have shown changes in the frequency, phenotype or function on Treg after cryopreservation [19,20]. However, in our hands, Treg retain their suppressive capacity and phenotype after cryopreservation (data not shown).…”

Section: Resultscontrasting

confidence: 57%

Effect of chorioamnionitis on regulatory T cells in moderate/late preterm neonates

et al. 2015

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Regulatory T-cells (Treg) have a protective role for the control of immune activation and tissue damage. The effects of chorioamnionitis (chorio) on Treg in moderate/late preterm newborns are not known. We hypothesized that infants exposed to chorio would have decreased Treg frequency and/or function. We isolated mononuclear cells from adult peripheral blood and cord blood from term and moderate/late preterm infants who were classified for severity of chorio exposure. Mononuclear cells were analyzed by flow cytometry for Treg frequency and phenotype. Treg suppression of activation of conventional T-cells (Tcon) was also quantified. Treg frequencies were similar in all groups of neonates, but lower than that found in adults. Newborn Treg had a naïve phenotype, with decreased levels of CD45RO, HLA-DR, CD39 and TIGIT compared to adult Treg and chorio did not affect the phenotype. Treg from preterm newborns exposed to severe chorio had higher expression of Ki67 compared to the other groups. Treg from preterm newborns were less suppressive than Treg from adults or term, and the level of suppression was reduced with severe chorio. Relative to term, Treg frequency and phenotype were not affected by prematurity and chorio but their functionality was decreased. Lower Treg activity may contribute to inflammation in newborns that is often associated with chorioamnionitis.

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“…There is a wealth of information and large range of protocols available regarding the preservation of cells, including stem cells (the reader is directed to a number of recent reviews: Coopman, 2011;Golab et al, 2013;Hunt, 2011;Robinson et al, 2014;Thirumala et al, 2013). A recent overview of proposals submitted to the FDA for MSCbased products indicated that >80% of 66 submissions considered used cryopreservation to store and transport their product (Mendicino et al, 2014).…”

Section: Can the Cell Therapy Be Preserved And If So How?mentioning

confidence: 99%

From production to patient: challenges and approaches for delivering cell therapies

Coopman

Medcalf

2014

StemBook

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Challenges in cryopreservation of regulatory T cells (Tregs) for clinical therapeutic applications

Cited by 46 publications

References 47 publications

Restimulation After Cryopreservation and Thawing Preserves the Phenotype and Function of Expanded Baboon Regulatory T Cells

Restimulation After Cryopreservation and Thawing Preserves the Phenotype and Function of Expanded Baboon Regulatory T Cells

Effect of chorioamnionitis on regulatory T cells in moderate/late preterm neonates

From production to patient: challenges and approaches for delivering cell therapies

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