Development of GMP-Compatible Protocols for Thymus-Derived Regulatory T Cell Expansion

Abstract

Introduction: Animal and early clinical studies have shown that regulatory T cell (Treg) therapy can prevent graft-versus-host disease (GVHD) following hematopoietic stem cell transplant. Despite these promising results, challenges remain in administering Treg therapy including the difficulty in isolating pure Tregs and expanding them to a therapeutic dose. We have previously found that the thymus, which is routinely removed during pediatric cardiac surgery, is a plentiful source of Tregs. These Tregs can be easily isolated, and can prevent xenoGVHD in mice. However, to use this therapy in humans, standard GMP-compliant methods for isolation and large-scale expansion of thymic Tregs must be developed. Methods: CD4+CD8-CD25+ thymic Tregs were isolated from pediatric thymuses using GMP-compatible magnetic bead-based separation. Tregs were expanded using artificial antigen-presenting cells or GMP-compatible antibody tetramer complexes that stimulate CD3 and CD28, with or without additional activation of CD2. Cells were cultured with rapamycin and IL-2, and were restimulated after 7 days without rapamycin. Results: Thymic Tregs expand in vitro when cultured with tetrameric antibody complexes. About 50-fold expansion was achieved using the anti-CD3/CD28/CD2 antibody tetramers, which represents the expansion that will be necessary to reach a clinically relevant dose. A higher expansion rate was obtained using the anti-CD3/CD28/CD2 antibody tetramer complexes than the anti-CD3/CD28. Cells cultured with these antibody tetramers express high levels of FOXP3, similar levels of Treg-characteristic markers as those expanded with artificial antigen-presenting cells, and do not produce inflammatory cytokines. Conclusion: Thymic Tregs can be isolated and expanded with GMP-compliant methods. Once the protocol is optimized, we can transfer our protocols to a GMP facility for further process development and to generate data required for a clinical trial application. We are the first group to pursue clinical application using allogeneic thymic Tregs, which provide an opportunity for an “off-the-shelf” therapy to treat GVHD. Canadian National Transplant Research Program.