Figure 7.

Effect of E3/49K on B cell activation and antibody production. (A) Assessment of B cell activation by CD69 expression. Human PBMC (2 × 10⁵/well) were cultured without stimulus, with L23-control or L23-E3/49K cells, with CpG alone, or with CpG plus purified soluble E3/49K (sec49K-His). After 24 h, the cells were stained with the antibody combination CD19/CD69 and analyzed by flow cytometry. A representative experiment showing dot-plots of CD19/CD69 co-expression patterns is depicted. Numbers are percentages of CD69⁺ cells among the CD19⁺ B cell population. The bar graph summarizes percentages of CD19⁺CD69⁺ cells obtained in 13 independent experiments. Statistical significance was calculated by ANOVA. (B) Demonstration of in vitro antibody production. Human PBMC were cultured for 11 days with medium, or stimulated with CpG alone or CpG in the presence of soluble E3/49K. Supernatants (SN) were harvested and titrated on porcine L23-control cells. The presence of antibodies binding to L23 cells was visualized by a second incubation step using FITC-labelled goat anti-human IgM and IgG antibodies and flow cytometry. The numbers in the representative histogram represent mean fluorescence intensity. The bar graph summarizes means obtained in a series of experiments using cells from 5 different blood donors. Statistical significance was calculated by ANOVA.