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

Highlights

• Tested GMP-compatible activation reagents and media for Treg expansion.

• Tregs that return to resting size (∼8.5 μm) expand more upon restimulation.

• Timing of Treg cryopreservation effects viability, phenotype and function.

• Cryopreserved thymic Tregs could be used as an off-the-shelf cell therapy product.

Abstract

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. Discarded pediatric thymuses are an excellent source of therapeutic Tregs with advantages including cell quantity, homogeneity and stability. 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. 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 with a single stimulation with aAPCs. 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. Restimulation timing was a less critical process parameter than the time between restimulation and cryopreservation. This systematic testing of key variables provides increased certainty regarding methods for in vitro expansion and cryopreservation of Tregs. The ability to cryopreserve expanded Tregs will have broad-ranging applications including enabling centralized manufacturing and long-term storage of cell products.

Introduction

Regulatory T cell (Treg) therapy is a promising approach to prevent or treat graft-versus-host disease following hematopoietic stem cell transplantation, graft rejection following solid organ transplantation, or autoimmune conditions [1]. Several phase 1 clinical trials have been completed [2], [3], [4], [5], [6], [7], [8], [9], [10], [11] and phase 1/2 clinical trials of Treg therapy are underway (reviewed in [12]). 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].

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. 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. Because thymus-derived Tregs are naive and homogenous, they are also resistant to inflammatory cytokine-induced destabilization [14,16], making them a promising autologous cell therapy in children undergoing heart transplantation, or an allogeneic cell therapy in the setting of other diseases [13]. Because ∼1% of children are born with congenital heart defects, many of whom require surgery including removal of the thymus [17], thymuses are routinely discarded. This material could be handled using procedures similar to those used to collect pancreata for islet transplantation and then used as a source of material from which to isolate Tregs.

Multiple protocols have been developed to manufacture Tregs for clinical trials, with variations in almost all key process parameters, including media, activation reagents, interleukin (IL)-2 concentration, restimulation timing and culture duration [2,4,5,[8], [9], [10], [11],[18], [19], [20], [21], [22], [23], [24]]. Recently, a variety of new Good Manufacturing Practice (GMP)–compatible reagents to activate T cells has been released but there are no reported comparisons of the performance of these now commercially available products. The lack of systematic studies to define critical process parameters and optimal reagents leads to uncertainty about the best methods to isolate and expand Tregs for clinical use [13].

Another uncertainty in the Treg manufacturing process is the feasibility of cryopreserving expanded cells. Some studies reported that cryopreservation reduces Treg quality, resulting in decreased FOXP3 expression and impaired suppressive capacity [25,26]. Consequently, to date only a limited number of clinical trials have used cryopreserved Tregs [21,22,27]. Using fresh cells as a clinical treatment creates many logistical hurdles: release testing must be done in a shorter time and there is limited notice of manufacturing failure; there is less infusion time flexibility to accommodate changes in patient health status; and it is difficult to quickly produce cells for rapidly progressing diseases such as acute graft-versus-host disease. Finding ways to cryopreserve clinical-grade Tregs to be used as an "off-the-shelf" product would overcome these hurdles, enable centralized manufacturing and allow a single product to be used to treat multiple patients [28,29]. Indeed, the concept of using “off-the-shelf” allogeneic cells is seen as the future of cell therapy products.

To develop a protocol to manufacture clinical-grade thymus-derived Tregs, we have comprehensively compared multiple process parameters using GMP-compatible reagents. We also report a new protocol to cryopreserve expanded thymus-derived Tregs to enable their use as an “off-the-shelf” cell therapy product.