CD80/CD86 and CTLA-4 coreceptor signaling is a prerequisite for the suppressive effect of CB-derived Bregs. (A) CD80/86 blockade significantly inhibits the ability of CB B-cell subsets to suppress the effector function and proliferation of peripheral CD4⁺ T cells. Cumulative data show the effect of CD80 and CD86 coreceptor blockade in cultures of purified CFSE-stained proliferating CD4⁺ T cells and sorted CB-derived CD19⁺ B-cell subsets at a 1:1 ratio. Bar graphs illustrate collective data from 4 independent experiments. (B) CTLA-4 blockade significantly inhibits the ability of CB B-cell subsets to suppress the effector function and proliferation of peripheral CD4⁺ T cells. The effects of CTLA-4 blockade were assessed in cultures of purified CFSE-stained proliferating CD4⁺ T cells and sorted CB-derived CD19⁺ B-cell subsets as compared with the corresponding positive control. Bar charts compare the effect of CTLA-4 blocking on CD4⁺ T-cell proliferation and IFN-γ, TNF-α, and IL-2 production at a 1:1 B-cell to T-cell ratio (n = 4). (C) CTLA-4 blockade has no significant impact on the ability of PB-derived Breg subsets to suppress the proliferation of peripheral CD4⁺ T cells. The effects of CTLA-4 blockade were assessed in cultures of purified CFSE-stained proliferating CD4⁺ T cells and sort-purified PB-derived CD19⁺ B-cell subsets as compared with the corresponding positive control (n = 4). (D) A combination of blocking antibodies to IL-10, CTLA-4, CD80, and CD86 is sufficient to fully reverse the ability of CB-Breg subsets to suppress CD4⁺ T-cell proliferation in vitro (n = 4). Bars indicate median values and ranges (upper whiskers). *P < .05 by nonparametric ANOVA.