IL-10–producing CB-derived CD19⁺ B cells and the naive and transitional B-cell subsets suppress CD4⁺ T-cell proliferation and effector function in a robust and dose-dependent manner. (A) Magnetically selected CD4⁺ T cells were labeled with CFSE (eBioscience) and plated in 96-well flat-bottomed tissue-culture plates. Total CD19⁺ B cells or sort-purified naive and transitional B-cell subsets were added to separate wells at a B-cell to T-cell ratio of 1:1 for 96 hours. T cells were activated with anti-CD3/CD28 Dynabeads (Invitrogen) as per the manufacturer’s instructions. CFSE-stained T cells cultured with no stimulation (negative control) and CFSE-stained T cells cultured with anti-CD3/anti-CD28 beads (positive proliferation control) were included in each experiment. Representative dot plots show the gating strategy for CD4⁺CFSE⁺ T cells. Gates were made on the live cells, lymphocyte population, followed by CD4⁺ T cells and CD4⁺CFSE⁺ T cells. Gating was determined with unstimulated CD4⁺ T cells. (B-C) Proliferation of CD4⁺ T cells cultured alone or with total CD19⁺ B cells, naive B cells, or transitional B cells at a ratio of 1:1. In vitro–suppressive effects of different CD19⁺ B-cell subsets cocultured with anti-CD3/anti-CD28–stimulated CD4⁺ T cells. Bars represent suppressive effects of CB-derived CD19⁺ B-cell subsets or CB Tregs (1:1 ratio) on CD4⁺ T-cell proliferation in vitro (n = 14). Bars represent median values and upper whiskers indicate the range. *P < .05 by nonparametric ANOVA. (D) Dose-dependent suppression of CD4⁺ T-cell proliferation in the presence of total CD19⁺ B cells or naive and transitional subsets cultured at the indicated B-cell to T-cell ratios. Data are plotted as the means and SEs of 4 independent experiments. (E). Bar graphs showing the suppression of IFN-γ, TNF-α, and IL-2 after coculture with CB-derived B-cell subsets and CB-Tregs. Bars indicate median values and upper whiskers represent range from 6 independent experiments. *P < .05 by nonparametric ANOVA. (F) Pretreatment of CB CD19⁺ B cells and B-cell subsets with CpG, CD40, or BCR ligation potentiated the suppressive effect of CB-derived B cells on CD4⁺ T-cell proliferation and effector function, by comparison with their noninduced counterparts (n = 4). Bars indicate median values and ranges (upper whiskers). *P < .05 by paired t test. ns, not significant; SE, standard error.