Identification of the natural counterpart of CD19^hiFcγIIb^hi B cells in vivo. Splenocytes and lymph node cells enriched with anti-CD19 magnetic beads were double-stained. CD19^hiFcγIIb^hi B cells and CD19^loFcγIIb^lo B cells were sorted by flow cytometry. (A) Numbers in plot regions indicate the percentage of CD19^hiFcγIIb^hi in the gated CD19⁺ B cells. (B) CD19^hiFcγIIb^hi and CD19^loFcγIIb^lo were stimulated with 200 ng/mL LPS, 2 μg/mL CpG ODN, or 2 μg/mL poly I:C for 24 hours; then IL-10 production was measured by ELISA. (C) The phagocytic ability of sorted CD19^hiFcγIIb^hi and CD19^loFcγIIb^lo B cells was assessed by flow cytometry of FITC-OVA-IC phagocytosis. Numbers in the histogram indicate the mean fluorescence intensity of test samples. Ctrl indicates controls (cells incubated with FITC-OVA-IC at 4°C). (D) CD19^hiFcγIIb^hi or CD19^loFcγIIb^lo B cells were cocultured with activated CD4 T cells. In some groups, anti–IL-10 antibody was added into the activated CD4 T/B coculture system. After 5 days, the relative number of viable CD4 T cells in each well was detected by flow cytometry. (E-F) DO11.10 OVA_323-339-specific TCR transgenic × C57BL/6J F1 hybrid mice were killed on days 3, 6, and 9 after immunization with 40 μg OVA plus 10 μg CpG ODN. CD19⁺ cells were sorted from spleen (E) and lymph nodes (F). Left: cell-cycle status of FcγIIb^hi cells analyzed by propidium iodide (numbers indicate percentage of FcγIIb^hi and FcγIIb^lo cells, respectively, in S/M/G₂). Right: percentage of proliferating B cells in FcγIIb^hi or FcγIIb^lo cell population of different phases of immune response by cycle analysis. (B,D) Data are mean ± SD of triplicate cells. *P < .05.