Figure 6
Figure 6. Regulatory DCs induce CD19hiFcγIIbhi regulatory B-cell generation from T1, T2, MZ, and B1 B cells. (A) Splenic B cells cocultured with regulatory DCs were stained for CD19, FcγRIIb, CD5, and CD1d. Cells within the CD19hiFcγRIIbhi B-cell gate were assessed for CD1d and CD5 expression. Cells within the CD5+CD1dhi B-cell gate were assessed for CD19 and FcγRIIb expression. (B) Splenic B cells cocultured with regulatory DCs were stained for CD19, FcγRIIb, and TIM-1. Cells within the CD19hiFcγRIIbhi B-cell gate were assessed for TIM-1 expression. (C) Splenic T1 (CD93+IgMhiCD23lo), T2 (CD93+IgMhiCD23hi), T3 (CD93+IgMloCD23hi), MZ (CD93−CD21hiCD23lo), FO (CD93−CD21loCD23hi), and B1 (B220+CD5+) B cells were sorting-purified, respectively, and cultured with or without regulatory DCs for 48 hours. Culture supernatants were collected for IL-10 assay. (D) Splenic T1, T2, MZ, or B1 B cells cultured with regulatory DCs were stained for CD19 and CD16/CD32 and analyzed by flow cytometry. (E) Splenic T1, T2, MZ, or B1 B cells were cultured with regulatory DCs. CD19+IL-10+ B cells were gated and assessed for FcγIIb expression. Gray lines represent the expression of FcγIIb on T1, T2, MZ, and B1 B cells before coculture. (F) Splenic T1, T2, MZ, or B1 B cells were cultured with regulatory DCs for 48 hours. CD19hiFcγRIIbhi B cells derived from T1, T2, MZ, and B1 B cells were sorted by flow cytometry, respectively. The phagocytic ability of freshly isolated T1, T2, MZ, and B1 B cells and sorted CD19hiFcγIIbhi populations derived from T1, T2, MZ, and B1 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). (G) Freshly isolated T1, T2, MZ, and B1 B cells or sorted CD19hiFcγIIbhi populations derived from T1, T2, MZ, and B1 B cells were cocultured with activated CD4 T cells (naive peptide-specific CD4 T cells were stimulated by maDCs for 24 hours). In some groups, neutralizing 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. Results are representative of 3 independent experiments. (C,G) Data are mean ± SD of triplicate cells. *P < .05. **P < .01. NS indicates not significant.

Regulatory DCs induce CD19hiFcγIIbhi regulatory B-cell generation from T1, T2, MZ, and B1 B cells. (A) Splenic B cells cocultured with regulatory DCs were stained for CD19, FcγRIIb, CD5, and CD1d. Cells within the CD19hiFcγRIIbhi B-cell gate were assessed for CD1d and CD5 expression. Cells within the CD5+CD1dhi B-cell gate were assessed for CD19 and FcγRIIb expression. (B) Splenic B cells cocultured with regulatory DCs were stained for CD19, FcγRIIb, and TIM-1. Cells within the CD19hiFcγRIIbhi B-cell gate were assessed for TIM-1 expression. (C) Splenic T1 (CD93+IgMhiCD23lo), T2 (CD93+IgMhiCD23hi), T3 (CD93+IgMloCD23hi), MZ (CD93−CD21hiCD23lo), FO (CD93−CD21loCD23hi), and B1 (B220+CD5+) B cells were sorting-purified, respectively, and cultured with or without regulatory DCs for 48 hours. Culture supernatants were collected for IL-10 assay. (D) Splenic T1, T2, MZ, or B1 B cells cultured with regulatory DCs were stained for CD19 and CD16/CD32 and analyzed by flow cytometry. (E) Splenic T1, T2, MZ, or B1 B cells were cultured with regulatory DCs. CD19+IL-10+ B cells were gated and assessed for FcγIIb expression. Gray lines represent the expression of FcγIIb on T1, T2, MZ, and B1 B cells before coculture. (F) Splenic T1, T2, MZ, or B1 B cells were cultured with regulatory DCs for 48 hours. CD19hiFcγRIIbhi B cells derived from T1, T2, MZ, and B1 B cells were sorted by flow cytometry, respectively. The phagocytic ability of freshly isolated T1, T2, MZ, and B1 B cells and sorted CD19hiFcγIIbhi populations derived from T1, T2, MZ, and B1 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). (G) Freshly isolated T1, T2, MZ, and B1 B cells or sorted CD19hiFcγIIbhi populations derived from T1, T2, MZ, and B1 B cells were cocultured with activated CD4 T cells (naive peptide-specific CD4 T cells were stimulated by maDCs for 24 hours). In some groups, neutralizing 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. Results are representative of 3 independent experiments. (C,G) Data are mean ± SD of triplicate cells. *P < .05. **P < .01. NS indicates not significant.

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