Figure 1.

Proliferation, differentiation, and antibody production after T-cell-dependent in-vitro stimulation and culturing of low numbers of primary human CD19⁺ B cells. (A) Schematic overview of the T-cell-dependent (TD) culture system to induce B-cell differentiation. A total of 25,000, 2,500, or 250 CD19⁺ human B cells (n = 4) were stimulated with a human CD40L-expressing 3T3 feeder layer and recombinant IL-21 (50 ng/ml) enabling conditions I (dark blue), II (cobalt blue), and III (light blue). Cells were analyzed on day 6 and day 9 by flow cytometry to evaluate the (B) number of live CD19⁺ events, (C) amount of proliferation by CTY dilution, and frequency of (D) plasmablast (CD27⁺CD38⁺ B cells) and (E) plasma cell (CD27⁺CD38⁺CD138⁺ B cells). A cutoff of 1,000 events was used to proceed with further analysis. (F) The supernatant was collected on day 6 and day 9 to evaluate IgG, IgA, and IgM production by ELISA (n = 4). Each data point represents the mean of an individual donor with duplicate culture measurements. Mean values are represented by bars and the error bars depict SEM. P-values were calculated using two-way ANOVA with Sidak’s multiple comparison test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.