Multiple unit pooled umbilical cord blood is a viable source of therapeutic regulatory T cells

Abstract

Background: Regulatory T cells (Treg) are potentially a useful therapeutic option for the treatment of immunopathological conditions including graft-versus-host disease. Umbilical cord blood (UCB) offers certain advantages over adult peripheral blood (APB) as a source of Treg for cellular therapy but yields far fewer Treg per unit. Pooling of Treg from multiple donors may overcome this challenge.

Methods: In this study, we assessed the in vitro and in vivo efficacy of multiple donor pooled UCB or APB-derived Treg.

Results: In vitro, pooled freshly isolated UCB-derived Treg were as suppressive as APB-derived Treg. However, in a mouse model of human skin allodestruction, pooled UCB-derived Treg were more potent at suppressing alloresponses and prolonging skin survival compared with pooled APB-derived Treg. Improved survival of UCB Treg in an in vivo cell survival assay and their lower expression of human leukocyte antigen-ABC suggested that lower immunogenicity may account for their superior efficacy in vivo.

Conclusion: Multiple-unit UCB is therefore a viable source of human Treg for cellular therapy, and pooling of Treg from multiple donors offers a useful strategy for achieving required therapeutic doses.

Figures

FIGURE 1

Phenotype of UCB-derived and APB-derived CD25+ Treg. Treg freshly isolated from UCB or APB were immunostained for CD4, CD25, CD127, FOXP3, and CD45RA (A–C). The percentage of cells or mean fluorescence intensity (MFI) values are plotted for each donor, with means indicated by horizontal lines. Percentage of CD4+ cells within each isolate expressing FOXP3+CD127lo plotted for 10 UCB donors and nine adult donors (A). MFI associated with FOXP3 expression among freshly isolated CD4+ T cells plotted for 10 UCB donors and nine adult donors (B). Percentage of freshly isolated Treg expressing CD45RA plotted for seven UCB donors and eight adult donors, gated on CD25+CD127lo cells (C). Expression of CD39 (D) and perforin (E) by αCD3 and αCD28 bead stimulated or unstimulated UCB-derived or APB-derived Treg cultured for 48 hr in the presence of IL-2. IL-10 levels in supernatants from 48-hr cultures of αCD3 and αCD28 bead stimulated or unstimulated UCB-derived or APB-derived Treg (F). Four different donors with up to four repeats were tested in each group (D–F). *P<0.05.

FIGURE 2

Suppressive capacity of UCB-derived versus APB-derived Treg. Treg freshly isolated from UCB were cultured with 5×104 irradiated allogeneic adult PBMCs (P) at various Treg-to-responder ratios, in the presence of allogeneic stimulators (A). Responder proliferation was measured by a 3H-thymidine incorporation assay. Percentage proliferation is taken as the number of responders undergoing proliferation in the presence of Treg as a percentage of the number of stimulated responders proliferating in the absence of Treg. Mean values±standard deviation (SD) from six replicates at each Treg-to-responder ratio are plotted for three UCB donors. Treg freshly isolated from APB were cultured in conditions identical to those used for UCB-derived Treg (B). Percentage proliferation of responders, relative to stimulated responders without Treg, is plotted as the mean of six replicates at each Treg-to-responder ratio, from one representative experiment including three adult donors. Data are represented as mean values±SD. Treg from the same three UCB or APB samples as those depicted in (A) and (B), respectively, were pooled and subjected to the in vitro suppression assay under identical conditions as Treg of single-donor origin (C). Data are represented as mean±SD for six replicates at each Treg-to-responder ratio.

FIGURE 3

Prolongation of human skin allograft survival by adoptively transferred UCB-derived and APB-derived Treg in a humanized mouse model. BALB/c Rag2−/−cγ−/− mice were transplanted with human skin and 35 days later received 5×106 PBMCs alone or together with 1×106 freshly isolated UCB-derived or APB-derived Treg, pooled from multiple donors. Percentage of grafts surviving (i.e., exhibiting no macroscopic indicators of rejection) is plotted over time. Treatment with UCB-derived Treg extended human skin allograft survival time significantly in comparison to PBMC alone (P=0.0323), achieving a MST of 73 days (two independent in vivo assays, Treg pooled from four to six donors).

FIGURE 4

Survival of APB-derived and UCB-derived Treg in vivo after adoptive transfer. 5×106 PBMCs were injected intraperitoneally into BALB/c Rag2−/−cγ−/− mice alone (n=5 mice) or with 1×106 freshly isolated, CFSE-stained Treg pooled from either five adult donors (n=3 mice) or five UCB donors (n=5 mice) (A). Cells were extracted by peritoneal lavage on day 7 after adoptive transfer and Treg (identified as CFSE+ cells) were enumerated. Mean cell numbers are plotted±SD. Freshly isolated Treg from UCB and APB were immunostained for HLA-DR (B) and HLA-ABC (C), and the MFI among CD4+ cells from each source was determined. Data are represented as mean values from three UCB and three APB Treg donors±SD.