Figure 2

Functional consequences of SpA binding to B cells. (A) B cells from two donors were cultured for 3 d in medium with or without graded doses of SAC (left panel), Mod-SpA or goat anti-human IgM Ab (right panel), with or without IL-2. Proliferative response was measured by incorporation of ³H-thymidine during the last 16 h of the culture. Triplicates of one donor are showed and expressed as mean cpm ± SEM. Data have been analyzed with the paired t test (*p < 0.05; ***p < 0.001). (B) Calcium mobilization assay was performed on BJAB cells (2 × 10⁶/mL) loaded with Indo/AM and stimulated at 37°C with ionomycin (discontinued line), anti-IgM Ab (gray line) or biotinylated Mod-SpA plus streptavidin (black line). Indo-1 fluorescence was monitored during 350 s and the ratio (emission at 405/20 nm/emission at 500/11) was depicted. One representative experiment of two is shown. (C, D) B cells were cultured for 2 d in medium, IL-4 and/or CD40L in the presence of medium alone (open bars), 10 µg/mL goat anti-human IgM Ab (hatched bars) or 10 µg/mL Mod-SpA (black bars). Production of IL-6 (C, n = 3) and MIP1 β (D, n = 2) was assessed by ELISA in cell-free supernatants. Data are expressed as mean ± SEM pg/mL. One-tailed Mann-Whitney test was used to determine statistical significance and p values are indicated as *p < 0.05.