IL-10–producing IgM memory and transitional B cells suppress CD4⁺ T-cell proliferation in a robust and dose-dependent manner. Magnetically selected CD4⁺ T cells were labeled with CFSE (eBioscience) and plated in 96-well flat-bottom tissue culture plates. IgM⁺CD27⁺ memory, CD24^hiCD38^hi transitional, IgM-CD27⁺ switched memory, and IgM⁺CD27^– naïve cells 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) at a concentration of 2 µL/ 8 × 10⁵ cells. 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. Cells were stained with CD4-APC, CD19-PE, and PI (all from BD Biosciences) for 20 minutes and acquired on a BD FACSCalibur instrument. (A) Representative flow cytometry plots of results of CD4⁺ T-cell suppression assay with gating strategy. (B) Proliferation of CD4⁺ T cells cultured alone, with switched-memory B cells, naïve B cells, IgM memory B cells, and transitional B cells at a ratio of 1:1. (C) In vitro suppressive effects of different CD19⁺ B-cell subsets (1:1 ratio) were assayed with anti-CD3/anti-CD28–stimulated CD4⁺ T cells. Bars represent medians and interquartile ranges for the indicated numbers of donors. P < .001 for suppression by both IgM memory and transitional B cells vs positive control by nonparametric ANOVA. (D) Dose-dependent suppression of CD4⁺ T-cell proliferation in the presence of IgM memory (left) and transitional B cells (right) incubated at the indicated B-cell to T-cell ratios. Data are representative of 3 independent experiments with cells from 3 healthy donors. Each data point represents the median and range. (E) Sort-purified IgM memory and transitional B cells and Tregs from healthy individuals were cultured 1:1 with anti-CD3/anti-CD28–stimulated CD4⁺ T cells. Frequencies of IFN-γ⁺ CD4⁺ were assessed relative to the corresponding unstimulated control. (F) Bar graphs show the suppression of TNF-α and IL-2 after coculture with IgM memory, transitional, naïve, and switched memory B cells. Data are representative of 3 independent experiments with cells from 4 healthy donors. Bars represent medians, and whiskers indicate the upper range. *P = .015 vs positive control, **P < .001 vs positive control, for comparisons with both IgM memory and transitional subsets (by nonparametric ANOVA). (G) Frequency of proliferating CD4⁺ T cells cultured with IgM memory, Tregs, or transitional B cells at a ratio of 1:1. (H) Bar graphs show equivalent inhibition of IFN-γ production and proliferation of CD4⁺ T cells by IgM memory, Tregs, and transitional B-cell subsets. Values are medians and upper ranges for 4 healthy donors. *P = .018 for individual comparisons with controls by nonparametric ANOVA; ns, not significant. (I) In vitro suppressive effects of different CD19⁺ B-cell subsets (1:1 ratio) were assayed with anti-CD3/anti-CD28–stimulated CD8⁺ T cells. P < .05 for suppression by both IgM memory and transitional B cells vs positive control by nonparametric ANOVA.