Ex vivo blood B10 and B10pro cells share cell surface markers with memory B cells. (A) Blood B10 cells predominantly exhibit a CD24^hiCD27⁺CD48^hiCD148^hi phenotype. Purified blood B cells were cultured with CpG + PIB for 5 hours before immunofluorescence staining for viability, cell surface molecule expression, and cytoplasmic IL-10. Cell surface CD24, CD27, CD38, CD48, and CD148 expression by IL-10⁺ (thick line) and IL-10⁻ (thin line) CD19⁺ cells was assessed by flow cytometry. (B) Cytoplasmic IL-10 induction does not affect the cell surface phenotype of B cells. CD19⁺ blood B cells were cultured with media on ice (thin line) or with CpG + PIB (thick line) for 5 hours before immunofluorescence staining and flow cytometry analysis as in panel A. (A-B) Shaded histograms represent isotype-matched control mAb staining. Results represent those obtained for 3 persons. (C) Distributions of B10 cells within B-cell subsets defined by CD24, CD27, IgD/CD38, and IgD/CD27 expression. Purified blood B cells were cultured with LPS + PIB for 5 hours before immunofluorescence staining and flow cytometry analysis as in panel A. The horizontal and vertical lines on each contour plot are shown for reference, with the lower left quadrants delineating the IgD⁻CD38⁻ and IgD⁻CD27⁻ subsets determined by control mAb staining. Results represent those obtained for 5 persons. (D) The ex vivo CD24^hiCD27⁺ B-cell subset includes the majority of B10 cells. Purified B cells were cultured with LPS + PIB for 5 hours before immunofluorescence staining for cell surface CD19, CD24, and CD27 expression and cytoplasmic IL-10 expression, with subsequent flow cytometry analysis. (E) B10pro cells derive from the CD24^hiCD27⁺ B-cell subset. Purified blood B cells were sorted into the CD24^hiCD27⁺ and CD24^lowCD27⁻ B-cell subsets, as indicated by the gates shown with purities more than 90% when reanalyzed by flow cytometry. The purified B cells were cultured with CD40L plus either LPS or CpG for 48 hours, with PIB added during the final 5 hours of culture before the relative percentages of IL-10⁺ B cells within the indicated gates was determined. Similar results were obtained in 2 independent experiments. (F) Ex vivo CD24^hiCD27⁺ B cells are the predominant source of secreted IL-10. Purified blood B cells were sorted into the CD24^hiCD27⁺ and CD24^lowCD27⁻ B-cell subsets as in panel E and cultured with the indicated stimuli for 72 hours. IL-10 secreted into the culture supernatant fluid was quantified by ELISA. Bar graphs represent mean IL-10 (± SEM) concentrations from triplicate ELISA determinations. Significant differences between means from CD24^hiCD27⁺ and CD24^lowCD27⁻ B cells are indicated: **P < .01. Differences between means from cells in media or with stimuli are indicated: ##P < .01. (G) B10-cell proliferation in vitro. Blood mononuclear cells were labeled with CFSE and cultured with CD40L and CPG (top panels) or CD40L and LPS (top panels) for 48 to 96 hours, with PIB added for the last 5 hours of culture. Histograms (right) represent CFSE expression by the IL-10⁺ (thick line) or IL-10⁻ (thin line) B-cell subsets. Results are representative of 2 independent experiments.