Key Points• KT64/86 artificial antigen-presenting cells culture stimulation provides marked expansion of Tregs.• In the context of sirolimus, mycophenolate mofetil immunosuppression, adoptive transfer of Tregs resulted in low risk of acute GVHD.We studied the safety and clinical outcomes of patients treated with umbilical cord blood (UCB)-derived regulatory T cells (Tregs) that expanded in cultures stimulated with K562 cells modified to express the high-affinity Fc receptor (CD64) and CD86, the natural ligand of CD28 (KT64/86). Eleven patients were treated with Treg doses from 3-100 3 10 6 Treg/kg.The median proportion of CD41 FoxP3 1 CD127 -in the infused product was 87% (range, 78%-95%), and we observed no dose-limiting infusional adverse events. Clinical outcomes were compared with contemporary controls (n 5 22) who received the same conditioning regimen with sirolimus and mycophenolate mofetil immune suppression. The incidence of grade II-IV acute graft-versus-host disease (GVHD) at 100 days was 9% (95% confidence interval [CI], 0-25) vs 45% (95% CI, 24-67) in controls (P 5 .05). Chronic GVHD at 1 year was zero in Tregs and 14% in controls. Hematopoietic recovery and chimerism, cumulative density of infections, nonrelapse mortality, relapse, and diseasefree survival were similar in the Treg recipients and controls. KT64/86-expanded UCB Tregs were safe and resulted in low risk of acute GVHD.

Graft-versus-host disease (GVHD) is a frequent and severe complication following hematopoietic cell transplantation. Natural CD4+25+ 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 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 re-stimulation increased expansion to ~3,000-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 re-stimulated four times with aAPCs. Indeed, cryopreserved donor nTregs re-stimulated 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.

SUMMARY Clinical application of umbilical cord blood (UCB) as a source of hematopoietic stem cells for transplantation is limited by low CD34+ cell dose, increased risk of graft failure and slow hematopoietic recovery. While the cell dose limitation is partially mitigated by using two UCB units, larger dosed single units would be preferable. We have evaluated the feasibility and safety of StemRegenin-1 (SR-1), an aryl hydrocarbon receptor antagonist that expands CD34+ cells, by placing one of the two units in expansion culture. SR-1 produced a 330-fold increase in CD34+ cells and led to engraftment in 17/17 patients at a median of 15 days for neutrophils and 49 days for platelets, significantly faster than in patients treated with unmanipulated UCB. Taken together, the marked expansion, absence of graft failure and enhanced hematopoietic recovery support testing of SR-1 expansion as a stand-alone graft and suggest it may ameliorate a limitation of UCB transplantation.

Clinical-scale production of NK cells is efficient and can be performed under GMPs. The purified NK cell product results in high NK cell purity with minimal contamination by T cells, monocytes, and B cells, but it requires more time for processing and results in a lower NK cell recovery when compared to NK cell enrichment with CD3 cell depletion alone. Additional laboratory studies and results from clinical trials will identify the best source and type of NK cell product.