CD19^hiFcγRIIb^hi B cells inhibit CD4 T-cell proliferation both in vitro and in vivo. (A) The effect of CD19^hiFcγIIb^hi B cells on in vitro maDC-primed proliferation of activated CD4 T cells was assayed. CD4 T cells were activated by maDCs for 24 hours in the presence of OVA_323-339 peptide, and then CD19^hiFcγIIb^hi B cells were added. Five days later, the cells were collected and double-stained with anti-CD4-allophycocyanin and 7-AAD and counted by flow cytometry. (B-C) The effect of CD19^hiFcγIIb^hi B cells on in vivo maDC-initiated proliferation of CD4 T cells was assayed. CD4 T cells from OT-2 × CD45.1 F1 hybrid mice were labeled with 5μM CFSE and then transferred intravenously into C57BL/6J mice on day −1. On day 0, antigen-loaded maDCs were transferred subcutaneously into the left footpad. On day 1, CD19^hiFcγIIb^hi B cells or CD19^loFcγIIb^lo B cells were also transferred into the left footpads. On day 5, mononuclear suspensions from the left popliteal lymph nodes were double-stained with anti–CD4-peridinin chlorophyll protein-Cy5.5 and anti–CD45.1-allophycocyanin for flow cytometry analysis. Representative dot plots showing the percentage of peptide-specific CD4 T cells (CD45.1⁺) among total CD4 T cells (B, left), and the percentage of peptide-specific CD4 T cells among total CD4 T cells was calculated (n = 3; B, right). Representative histograms showing CFSE expression by the CD4⁺CD45.1⁺ T cells (C, left), and the proliferation of CD4⁺CD45.1⁺ T cells was analyzed (n = 3; C, right). (D) As described in panel A, neutralizing anti–IL-10 antibody was added into the culture system. Data are mean ± SD. *P < .05. **P < .01.