Specifically, thymus-derived Tregs are abundant ( 14), and because in the thymus CD25 expression on CD4 +CD8 − T cells exclusively marks Tregs ( 15), a homogeneous population of Tregs can be recovered by magnetic bead-based separation of CD25 + and CD8 − cells. We have recently shown that discarded pediatric thymuses are a feasible source of clinically-applicable Tregs, with numerous advantages over blood- or cord blood-derived cells. However, significant obstacles remain to the broad use of Treg-based therapies, including developing and optimizing manufacturing protocols to generate large numbers of cells with the desired phenotype and function, while limiting manufacturing complexity and cost ( 13). Several Phase I clinical trials have been completed ( 2– 11) and Phase I/II clinical trials of Treg therapy are underway (reviewed in ( 12)). Regulatory T cell (Treg) therapy is a promising approach to prevent or treat graft-versus-host disease (GVHD) following hematopoietic stem cell transplantation, graft rejection following solid organ transplantation or autoimmune conditions ( 1). The ability to cryopreserve expanded Tregs will have broad-ranging applications including enabling centralized manufacturing and long-term storage of cell products. This systematic testing of key variables provides increased certainty regarding methods for in vitro expansion and cryopreservation of Tregs. Restimulation timing was a less critical process parameter than the time between restimulation and cryopreservation. Cryopreservation tests revealed a critical timing effect: only cells cryopreserved 1–3 days, but not >3 days, after restimulation maintained high viability and FOXP3 expression upon thawing. By comparing activation reagents, including soluble antibody tetramers, antibody-conjugated beads and artificial antigen-presenting cells (aAPCs), and different media, we found that the combination of Dynabeads Treg Xpander and ImmunoCult-XF medium preserved FOXP3 expression and suppressive function, and resulted in expansion that was comparable to a single stimulation with aAPCs. Here we report systematic testing of activation reagents, cell culture media, restimulation timing, and cryopreservation to develop a good-manufacturing-practice (GMP)-compatible method to expand and cryopreserve Tregs. Discarded pediatric thymuses are an excellent source of therapeutic Tregs with advantages including cell quantity, homogeneity and stability. Regulatory T cells (Tregs) are a promising therapy for several immune-mediated conditions but manufacturing a homogeneous and consistent product, especially one that includes cryopreservation, has been challenging. MKL: secured funding, conceived and designed experiments, provided overall direction and interpretation, and wrote the manuscript. BRB: secured funding, conceived and designed experiments and critically reviewed the manuscript. JMP: secured funding, provided critical feedback throughout the project, and critically reviewed the manuscript. LJW: secured funding, provided critical feedback throughout the project and critically reviewed the manuscript. IED: provided critical feedback throughout the project and critically reviewed the manuscript. MGH and GZ conducted experiments and analyzed data. REH: provided critical feedback throughout the project and critically reviewed the manuscript. KLH: conceived and designed experiments, analyzed data and critically reviewed the manuscript. SI: conceived, designed and conducted experiments, analyzed data and critically reviewed the manuscript. Author contributionsKNM: conceived, designed and conducted experiments, analyzed data, and wrote the manuscript.
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