Date: Saturday, May 2, 2015
Session Time: 5:30pm-7:30pm
Presentation Time: 5:30pm-7:30pm
Location: Exhibit Hall E
Feasibility of clinical application of regulatory T cells (Tregs) in clinical transplantation would be improved if Tregs or pre-enriched cells for Treg isolation could be cryopreserved until the right time for infusion. In this study we tested two strategies: 1) cryopreservation of CD4+ cells for Treg isolation/expansion and 2) cryopreservation of already expanded Tregs followed by another expansion.
Human CD4+ cells were isolated from leukapheresis products, frozen and kept in liquid nitrogen. After 1 year, cells were thawed and cultured overnight. Cell viability/apoptosis and expression of CD25, CD127, FoxP3 markers were checked. Next, Tregs were sorted and expanded for 13 days. Suppression of proliferation assay was performed at the end of expansion.
Another portion of fresh CD4+ cells was used directly to sort and expand Tregs. Cells were cryopreserved after expansion and stored for 1 year. Then, they were processed in the same way as CD4+ cells for thawing and expanded again.
Results. The average cell recovery was 76% and 32% just after thawing, but decreased to 38% and 8% after overnight culture for CD4+ and Treg cells, respectively. That might be related to 25% of CD4+ cells and 55% of Tregs indicated apoptosis/necrosis. The mean % of CD25hiCD127- cells in fresh CD4+ cells was 7.13% and decreased to 5.86% after thawing. Also, mean % of FoxP3+ cells decreased from 7.1% to 6%. After o/n culture, these proportions increased to 6.82% and 9.47%, respectively. After Treg thawing % of CD25hiCD127- cells decreased from 97.4% to 67.2%. No change was observed in % of FoxP3+ (>75%). However, after o/n culture % of CD25hiCD127- and FoxP3+ cells decreased to 42.9% and 46.4%, respectively. Tregs obtained after sorting from thawed CD4 cell expanded well, but failed to present in vitro suppressive abilities in contrast to thawed Tregs after their expansion.
Conclusion. Tregs obtained from cryopreserved CD4+ cells didn't show suppressive properties, which might be related to instability of key Treg markers upon cryopreservation and subsequent inability to sort pure cells. In contrast, cryopreserved Tregs after expansion demonstrated suppressive abilities. These results indicate that expanded Tregs can be cryopreserved and used again as therapy product upon stimulation and expansion to overcome cell loss and instability of their markers.
To cite this abstract in AMA style:Gołąb K, Savari O, Ramachandran S, Tekin Z, Tibudan M, Millis J, Trzonkowski P, Witkowski P. Feasibility of Regulatory T Cell Cryopreservation and Banking for Clinical Therapeutic Applications [abstract]. Am J Transplant. 2015; 15 (suppl 3). https://atcmeetingabstracts.com/abstract/feasibility-of-regulatory-t-cell-cryopreservation-and-banking-for-clinical-therapeutic-applications/. Accessed April 20, 2021.
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