Massive ex Vivo Expansion of Human Natural Regulatory T Cells (Tregs) with Minimal Loss of in Vivo Functional Activity

A good manufacturing grade–compatible approach generates massive numbers of natural regulatory T cells that retain suppressive function in vivo. Cross-Checking Graft-Versus-Host Disease Fighting in hockey is a long-standing tradition: Stitches and gap-toothed smiles are badges of honor among these aggressive athletes. Yet, a balance must be maintained between the occasional high stick and an all-out melee. Black-and-white striped referees serve to uphold this balance, breaking up fights and preventing the bench-clearing brawl. Regulatory T cells (Tregs) are the referees of the adaptive immune system. They prevent the enforcers, cytotoxic T cells, from an overly exuberant response and, in the case of a bone marrow transplant, from attacking the patient’s own tissues. This process, called graft-versus-host disease (GVHD), is one of the risks of transplantation and differs from organ rejection. However, using Tregs to prevent GVHD has been limited by low Treg numbers and altered function after expansion in vitro. Hippen et al. now report a new way to expand Tregs to numbers much larger than those previously achieved while maintaining their ability to selectively suppress self-attacking cytotoxic T cells in vivo. Umbilical cord blood can be used to expand functional natural Tregs (nTregs); however, the initial number of nTregs in cord blood is limited. Therefore, the authors used peripheral blood as a source of nTregs for expansion. Using good manufacturing practice conditions and artificial antigen-presenting cells designed to stimulate T cell expansion, Hippen et al. expanded nTregs 80-fold after only one stimulation; they then showed that these multiplied cells maintained suppressor function. Stimulation of the nTreg population up to four times expanded the numbers of functional cells ~50 million–fold. When injected into mice at the same time as human T cells, these expanded Tregs significantly reduced mortality resulting from GVHD. Such large numbers of functional nTregs could be used to establish donor banks that would keep human GVHD and autoimmunity in check. Graft-versus-host disease (GVHD) is a frequent and severe complication after hematopoietic cell transplantation. Natural CD4+CD25+ regulatory T cells (nTregs) have proven highly effective in preventing GVHD and autoimmunity in murine models. Yet, clinical application of nTregs has been severely hampered by their low frequency and unfavorable ex vivo expansion properties. Previously, we demonstrated that umbilical cord blood (UCB) nTregs could be purified and expanded in vitro using good manufacturing practice (GMP) reagents; however, the initial number of nTregs in UCB units is limited, and average yield after expansion was only 1 × 109 nTregs. Therefore, we asked whether yield could be increased by using peripheral blood (PB), which contains far larger quantities of nTregs. PB nTregs were purified under GMP conditions and expanded 80-fold to yield 19 × 109 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. nTreg expansion was ~50 million–fold when flow sort–purified nTregs were restimulated four times with aAPCs. Indeed, cryopreserved donor nTregs 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 nTregs could transform the treatment of GVHD and autoimmunity by providing an off-the-shelf, cost-effective, and proven cellular therapy.