Figure 5.

Increased frequencies of TNFα-producing CD19⁺CD24^hiCD27⁺ B cells in patients with Crohn’s disease (CD). (A–D) CD19⁺CD24^hiCD27⁺ B cells from healthy controls (HCs) and CD patients were sorted and stimulated with 100 nM CpG oligonucleotides for 48 h; then, FACS was used to measure TNFα and interleukin-10 (IL-10) expression in the cells from (A) HCs and (B) CD patients, N = 10. (C) The percentages of CD19⁺TNFα⁺ B cells from HCs and CD patients are shown. (D) The ratios of Fr.I (IL-10⁺TNFα⁺ B cells) to Fr.II (IL-10⁺TNFα⁻ B cells) from HCs and CD patients are shown. (E) Sorted CD19⁺CD24^hiCD27⁺ B cells from HCs (N = 3) were stimulated with 100 nM CpG oligonucleotides for 48 h; then, the IL-10⁺TNFα⁻ B cells and IL-10⁺TNFα⁺ B cells were sorted, and the expression of IL-10 and TNFα was analyzed by Q-PCR. (F) Sorted IL-10⁺TNFα⁻ B cells and IL-10⁺TNFα⁺ B cells were labeled with CFSE and cultured with 100 nM CpG oligonucleotides for 72 h. The CFSE dilution was measured by FACS, and the percentages of CFSE-labeled proliferating B cells are shown (right panel). (G–I) FACS was used to sort IL-10⁺TNFα^-, IL-10⁺TNFα⁺ IL-10^-TNFα⁺ and IL-10^-TNFα⁻ B cell subsets, which were then cultured with CD4⁺CD25⁻ T cells isolated from HCs and stimulated with 1 µg/mL anti-CD3 for 72 h. (G) Foxp3 expression and (H) IFNγ expression levels were measured by flow cytometry. In addition, CD14⁺ monocytes isolated from HCs were cultured for 24 h and then stimulated with 100 µg/mL LPS before the cytoplasmic TNFα expression was assessed. The data are shown as the mean ± SEM (N = 3). *P < 0.05, **P < 0.01.