Massive ex vivo expansion of human natural regulatory T cells (T _regs) with minimal loss of in vivo functional activity

Graft-versus-host disease (GVHD) is a frequent and severe complication after hematopoietic cell transplantation. Natural CD4⁺CD25 ⁺ regulatory T cells (nT_regs) have proven highly effective in preventing GVHD and autoimmunity in murine models. Yet, clinical application of nT_regs has been severely hampered by their low frequency and unfavorable ex vivo expansion properties. Previously, we demonstrated that umbilical cord blood (UCB) nT_regs could be purified and expanded in vitro using good manufacturing practice (GMP) reagents; however, the initial number of nT_regs in UCB units is limited, and average yield after expansion was only 1 × 10⁹ nT_regs. Therefore, we asked whether yield could be increased by using peripheral blood (PB), which contains far larger quantities of nT_regs. PB nT_regs were purified under GMP conditions and expanded 80-fold to yield 19 × 10 ⁹ cells using anti-CD3 antibody-loaded, cell-based artificial antigen-presenting cells (aAPCs) that expressed the high-affinity Fc receptor and CD86. A single restimulation increased expansion to ∼3000-fold and yield to >600 × 10⁹ cells while maintaining Foxp3 expression and suppressor function. nT_reg expansion was ∼50 million-fold when flow sort-purified nT_regs were restimulated four times with aAPCs. Indeed, cryopreserved donor nT_regs restimulated four times significantly reduced GVHD lethality induced by the infusion of human T cells into immune-deficient mice. The capability to efficiently produce donor cell banks of functional nT_regs could transform the treatment of GVHD and autoimmunity by providing an off-the-shelf, cost-effective, and proven cellular therapy.