Immature monocyte-derived DCs (0.1 × 106 cells per 200 μl per well) with GM-CSF and IL-4 were cultured either alone or with B cells (resting or pre-activated) at the ratio of 1:1 in U-bottom 96-well plates for 48 h. In some experiments, DCs were stimulated with recombinant human TSLP (20 ng per 0.1 × 106 cells, R&D Systems). For transwell experiments, B cells and DCs were kept separated by a 0.4-μm membrane. DCs (0.5 × 106 in 600 μl) were placed in the lower chamber of the transwell plate and pre-activated B cells (0.5 × 106 in 100 μl) in the upper chamber. For the stimulation of DCs using supernatant from activated B cells, immature DCs (0.1 × 106 cells per 250 μl total volume per well) were cultured in GM-CSF and IL-4 alone, or with supernatant from activated B cells (200 μl per well).
In some experiments, resting B cells were directly stimulated in DC–B cell co-culture by using F(ab′)2 of goat anti-human IgM (10 μg ml −1). In these experiments, the role of various surface molecules in contact-dependent induction of maturation of DCs by activated B cells was explored by employing blocking mAbs to anti-CD69 (10 μg ml −1), anti-TACI (10 μg ml −1), anti-CD54 (10 μg ml −1), anti-BAFF-R (10 μg ml −1) and anti-CD11a (10 μg ml −1), or mouse IgG isotype control antibodies (all from R&D systems).
In some experiments, resting B cells were directly stimulated in DC–B cell co-culture by using F(ab′)2 of goat anti-human IgM (10 μg ml −1). In these experiments, the role of various surface molecules in contact-dependent induction of maturation of DCs by activated B cells was explored by employing blocking mAbs to anti-CD69 (10 μg ml −1), anti-TACI (10 μg ml −1), anti-CD54 (10 μg ml −1), anti-BAFF-R (10 μg ml −1) and anti-CD11a (10 μg ml −1), or mouse IgG isotype control antibodies (all from R&D systems).