Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics

Abstract

Acute graft-versus-host disease (aGVHD) is associated with high risk of morbidity and mortality and is a common complication after double umbilical cord blood (UCB) transplantation. To reduce these risks, we established a method of CD4(+)CD25(+)FoxP3(+) T regulatory cell (Treg) enrichment from cryopreserved UCB followed by a 18 (+) 1-day expansion culture including anti-CD3/anti-CD28 antibody-coated beads and recombinant human interleukin-2. In a "first-in-human" clinical trial, we evaluated the safety profile of UCB Treg in 23 patients. Patients received a dose of 0.1-30 × 10(5)UCB Treg/kg after double UCB transplantation. The targeted Treg dose was achieved in 74% of cultures, with all products being suppressive in vitro (median 86% suppression at a 1:4 ratio). No infusional toxicities were observed. After infusion, UCB Treg could be detected for 14 days, with the greatest proportion of circulating CD4(+)CD127(-)FoxP3(+) cells observed on day (+)2. Compared with identically treated 108 historical controls without Treg, there was a reduced incidence of grade II-IV aGVHD (43% vs 61%, P = .05) with no deleterious effect on risks of infection, relapse, or early mortality. These results set the stage for a definitive study of UCB Treg to determine its potency in preventing allogeneic aGVHD. This study is registered at http://www.clinicaltrials.gov as NCT00602693.

Figures

Figure 1

Box-plots show the kinetics of CD127-Foxp3+ cells. Proportions and absolute numbers of peripheral blood Treg were determined before and after the infusion of fresh culture-expanded UCB Tregs that were administered 1 day after UCB transplantation (all patients) and again, before and after the infusion of thawed UCB Treg (same donor) 15 days after UCB transplantation (11/17 recipients of CsA/MMF and 6/6 recipients of sirolimus/MMF). In the plot, the cross represents the median value, the box represents the interquartile range, and the vertical line represents the range of the results. Panels A and B show the proportion and absolute number per microliter of peripheral blood CD127-Foxp3+ cells within the CD4+ subset, specifically in recipients of CsA/MMF. Panels C and D show the proportion and absolute number per microliter of peripheral blood CD127-Foxp3+ cells within the CD4+ subset, specifically in recipients of sirolimus/MMF. In a subgroup of 7 patients who had informative HLA discrepancies and received infusions on day +1 and +15 that could be studied by flow cytometry depicting the proportion (E) and absolute numbers per microliter (F) of peripheral blood CD4+CD127−Foxp3+ cells derived from the regulatory T-cell donor unit. (*) In panel B, at the pretransplantation time point the figure excludes one outlier with an absolute Treg count of 152/μL.

Figure 2

Clinical outcomes of patients after nonmyeloablative umbilical cord blood transplantation who received Treg ≥ 30 × 105/kg (dotted line; n = 18) and historical controls (solid line; n = 108). (A) DFS (P = .25), (B) NRM (P = .51), (C) sustained donor engraftment (P = .89), and (D) grade II-IV aGVHD (P = .04).