Direct cell-cell contact contributes to the T-cell–suppressive activity of CB-derived B cells. (A) Representative histograms showing proliferation of CFSE-stained anti-CD3/anti-CD28-stimulated CD4⁺ T cells cultured alone (positive control) or in direct contact with CB-B cells (direct contact) or separated from CB-B cells by Transwell chambers (Transwell). For each of these conditions, CD4⁺ T cells were cultured at a 1:1 ratio with CB-derived total B cells or sort-purified naive or transitional B-cell subsets. Bar graphs illustrate collective data representative from 4 independent experiments. (B) Effect of B:T cell-to-cell contact on CD4⁺ T-cell cytokine production. Anti-CD3/anti-CD28-stimulated CD4⁺ T cells were cultured alone (positive control) or in direct contact with CB-B cells (direct contact) or separated from CB-B cells by Transwell chambers (Transwell). For each of these conditions, CD4⁺ T cells were cultured at a 1:1 ratio with CB-derived total B cells or sort-purified naive or transitional B-cell subsets. Bar graphs illustrate pooled data from 4 independent experiments, comparing the suppressive activity of CB-derived B cells in the presence or absence of direct cell-cell on T-cell cytokine production measured by intracellular cytokine staining and ELISA. (C-D) Effect of B:T-cell-to-cell and IL-10 blocking on CD4⁺ T-cell proliferation (n = 4) (C) and cytokine production (n = 4) (D). Anti-CD3/anti-CD28-stimulated CD4⁺ T cells were cultured alone (positive control) or in direct contact with CB-derived B cells or separated from them by a Transwell chamber in the presence or absence of blocking antibodies against both IL-10 and IL-10R. Bar graphs illustrate collective data from 4 independent experiments. Bars indicate median values and ranges (upper whiskers). *P < .05 by nonparametric ANOVA.