In the first generation of studies (referred to as CB 1.0), investigators at the University of Minnesota where nearly 2000 CBTs have been performed, identified the CD34 cell dose threshold [3], pioneered the double CBT platform [456], and optimized conditioning regimens specifically for CBT, i.e. total body irradiation (TBI) 1320 cGy, cyclophosphamide (CY) 120 mg/kg and fludarabine (FLU) 75 mg/m² (TCF) [7]. In 2012, we initiated a first in human trial sponsored by Novartis and Magenta Therapeutics to test the safety and efficacy of ex vivo expansion culture to overcome the obstacle of low cell dose using an aryl hydrocarbon antagonist (AHRa) in the presence of stimulatory cytokines. In the initial studies, using the double CB platform with one unit expanded and the other unit unmodified as an additional safety measure, we showed that 18 of 18 patients engrafted at a median of 14.5 days (range, 7–23 days) after myeloablative conditioning (MAC) with TCF, substantially better than identically-treated historical controls (P<0.01) [8]. Based on these results, 18 additional patients received an expanded CB product alone, 9 with the same MAC and another 9 treated with non-myelablative conditioning (NMAC) consisting of CY 50 mg/kg, FLU 200 mg/m² and TBI 200 cGy (Wagner et al., ASH abstract 2017).

Similar to prior reports, expansion culture yielded a median of 1.2×10⁹ CD34+ cells, markedly greater than the input number of 4.2×10⁶-a 324-fold (range, 42–1,643 fold) expansion of CD34+ cells. As transplant results vary by intensity of the conditioning regimen, patient outcomes were compared to similarly treated historical cohorts (N=151 MAC; 132 NMAC). For both groups, demographics were similar. For recipients of MAC, CB after expansion culture engrafted in 100% at a median of 14 days (range, 7–32 days), better than that in the historical cohort transplanted with one or two CB units without expansion where engraftment occurred in 89% at a median of 23 days (range, 19–31 days) (P<0.01). Complete chimerism was rapid for both T and myeloid cells with no late graft failures. For recipients of NMAC, the expanded CB also engrafted in 100% but at a median of 7 days (range, 6–14 days) as compared to 95% at a median of 15 days (range, 7–22 days) after unmodified double CBT. In conclusion, the use of an AHRa-based HSC expansion culture accelerates hematopoietic recovery and reduces the risk of graft failure after CBT. In addition, such robust expansion permits us to reconsider the cell dose threshold when selecting the CB graft. Rather than a lower limit of 3.0×10⁷ nucleated cell/kg, future studies will allow a lower limit of 1.0×10⁷ nucleated cells/kg, widening the pool of potential donor units for consideration and therefore, increasing the chance of a better HLA match.

While engraftment has been the principal challenge for CBT, GVHD has been a long standing obstacle for all HSC sources. T cells mediate GVHD which leads to direct tissue damage and the need for prolonged courses of pharmacological immune suppression. Ex vivo T cell depletion is an effective strategy for reducing the risk of GVHD but it is associated with increased risks of graft failure, opportunistic infection and relapse [9], as conventional CD4+ and CD8+ T cells facilitate engraftment, accelerate immune reconstitution, and play a crucial role in the graft-versus-malignancy (GVM) effect of allogeneic HSC. Therefore, we and others have investigated alternative approaches. One strategy has been to isolate a rare population of thymic-derived CD4+CD25+FoxP3+ T cells [referred to as regulatory T cells (Treg)] found to be critical in maintaining self-tolerance and immune homeostasis [1011121314]. In several murine models of allogeneic HSCT across MHC class I and II barriers, lethal GVHD was suppressed when freshly isolated or ex vivo-expanded polyclonal FoxP3+ Treg were co-infused with T cells. On the basis of these compelling data, we developed a method for isolating and expanding the CD4+CD25+FoxP3+ Treg population in CB that could be used with any HSC source [15]. Crucial for maintaining maternal-fetal tolerance, their high frequency in CB make it an ideal source for adoptive immunotherapy. Following a 18±1-day expansion culture incorporating anti-CD3/anti-CD28 antibody-coated beads and IL-2, we initiated a “first-in-human” dose escalation of CB Treg from 0.01 to 3×10⁶ Treg/kg in double CBT patients [16]. There were no infusional toxicities and GVHD was significantly reduced (43% vs. 61%; P=0.05) with no obvious adverse effect on overall risks of infection and relapse with >2 years of follow-up. Based on distinguishing HLA markers that permitted detection of the culture-expanded Treg, Treg were detectable in the PB for up to 14 days (peak on day +2). However, greater numbers of Treg were needed to prevent GVHD. We subsequently developed a more robust expansion strategy, using the K562 cell-line that stably expressed CD64 FcR to allow loading with anti-CD3 for T cell signaling and CD86 for T cell co-stimulation. In a second dose escalation trial, we infused 3 to 100×10⁶ Treg/kg (ie, ratio up to 1:10). Here, we significantly reduced the risk of GVHD from 48% in Siro/MMF treated recipients to 12% (P<0.01) [17]. In conclusion, this is the first clear demonstration that ex vivo expanded Treg are potent suppressors of acute GVHD in humans and sets the stage for future clinical trials using CB Treg for prevention of GVHD after PBSCT. While an important HSC source for transplantation, CB may also be an important source of T cell populations for adoptive immunotherapy.

Today, at the 30th anniversary of CBT, CB holds substantial promise both as a source of HSC and lymphocyte subsets for adoptive cellular therapies. While costs of cell acquisition and longer initial hospitalizations have long been barriers to its wider use, CB is being re-evaluated as more reports demonstrate high survival rates in patients with hematologic malignancy [7], potent GVM in patients with minimal residual disease [18], and rapid engraftment, fewer hospital days and lesser healthcare resource utilization after ex vivo expansion culture by various methods. In the next generation of studies (CB 2.0) we will focus on the broader use of CB as a source of adoptive cellular therapy to further improve outcomes after HSCT regardless of HSC source and in the treatment of new indications.