Fig. 3

Suppressive function of UCB-Tregs expanded 18 days and 42 days. (A) Representative histograms of cell proliferation assay. (B) CFSE-labeled PBMCs were cocultured with UCB-Tregs by adding anti-CD3/28 beads at the indicated PBMC:Treg ratios of 1:1, 1:2, 1:4, and 1:8. CFSE alone: CFSE-labeled PBMCs without anti-CD3/28 beads. Positive control was CFSE-labeled PBMCs with anti-CD3/28 beads but without Tregs. Proliferation was determined via CFSE dilution on day 3; measurements were performed in duplicates. The proliferation of PBMCs, CD4+ T cells, and CD8+ T cells was directly measured via flow cytometry or after labeling with fluorescently conjugated anti-CD4 and anti-CD8 antibodies to distinguish the populations. The suppressive capacity was significantly different among the different proportions of expanded Tregs. The figures on the left show the coculture results of UCB-Tregs expanded for 18 days and the figures on the right depict the co-culture results of UCB-Tregs expanded for 42 days. Data are presented as the mean ± SEM. One-way ANOVA, followed by Tukey’s post hoc test was used; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 8–15. (C) No significant differences were observed in the suppressive function of d18 and d42 UCB-Tregs against PBMCs, CD4+ T cells, and CD8+ T cells. On the left is the coculture results of PBMCs with UCB-Tregs, in the middle is the coculture results of CD4+ T cells with UCB-Tregs, and on the right is the coculture results of CD8+ T cells with UCB-Tregs. Two independent sample t-test or Mann-Whitney test was used; n = 8–15