Figure 7.

IFNα priming of transitional B cells promotes TLR7 expression, cell differentiation and cytokine production in response to TLR7-ligand stimulation. Cord-blood (CB) transitional (TR) B cells were treated with IFNα and analyzed for the expression of TLR7 and responsiveness to R848 stimulation in vitro. (A) Representative flow plots of B cell subsets in cord blood; shown is the gating of TR and follicular mature (FM) B cells based on CD24 and CD38 expression (B) TR or FM B cells were sort-purified and left untreated or stimulated with 1000 μ/ml IFNα for 6 h. The expression of TLR7, IRF7, and UNC93B in each subset was quantified by RT-PCR; data are presented as fold-change of mRNA levels relative to unstimulated cell (summary of three independent experiments). (C) Purified CB TR B cells were loaded with CFSE and left untreated or primed with IFNα and cultured for 3 days in RPMI medium. Representative histograms show the percentage of proliferating (CFSE^lo) cells (data is representative of two independent experiments). (D-G) Purified CB TR B cells were primed with IFNα and stimulated with R848 (50 ng/ml) or a combination of R848 plus F(ab′)2 anti-human IgM (anti-IgM) (10 μg/ml) and cultured for 5 days. (D) Representative flow plots showing the percentages of TR B and plasma/plasmablast (PL/PLB) under different stimulatory conditions. (E) Summary data showing the percentage of CD24⁻CD38⁺⁺ cells of live cells in cultures. (F) IgM titers in cell culture supernatants measured by ELISA. (G) Concentrations of IL-6 and IL-10 in cell culture supernatants measured by LEGENDPlex bead-based assay and presented as IL-6 to IL-10 ratios. Summary data of three independent experiments. No-priming and IFNα-priming conditions were compared using a 2-way repeated measure ANOVA.