CD80 and CD86 costimulatory signaling contributes to the suppressive effect of IgM memory and transitional B cells. (A) Purified CD4⁺ T cells cultured with sorted CD19⁺ B-cell subsets from healthy volunteers and stimulated with anti-CD3/anti-CD28 beads in the presence or absence of CD80- and CD86-blocking antibodies. Stimulated CD4⁺ T cells labeled with CFSE and cultured alone served as a positive control. (B) Purified CD4⁺ T cells cultured with sorted CD19⁺ B-cell subsets from healthy volunteers and stimulated with anti-CD3/anti-CD28 beads in the presence of CD80- and CD86-blocking antibodies and in the presence or absence of IL-10 blocking. Stimulated CD4⁺ T cells labeled with CFSE and cultured alone served as a positive control. (C-D) A combination of cell-cell contact blocking (with either a transwell block or CD80/CD86 antibodies) and antibodies to IL-10/IL-10R fully reverses the suppressive capacity of IgM memory and transitional B-cell subsets. In (A-D), bars represent the medians, and whiskers indicate the upper ranges of values from 3 independent experiments with 5 samples per group. *P < .05 by nonparametric ANOVA; ns, not significant.