Figure 4.

Ectopic expression of BCL-6 represses memory B cell differentiation in vitro. Plasma and memory B cell cultures were infected with lentivirus encoding GFP alone (Empty Vector, Control) or a fusion protein of GFP and BCL-6 (BCL-6) on day 2 and harvested for analyses on day 5. (A) Cells were first gated for GFP expression and analyzed with antibodies to CD20 and CD38. Percentages represent proportion of cell types (memory B cells and plasma cells) in each gated population. Data are representative of three experiments. (B) Graphical representation of the fraction of GFP⁺CD20⁺CD38⁻ memory B cells in infected IL-4 B cell cultures 3 and 7 d after infection (5 and 9 total days in culture). Data shown are the mean of four experiments for day 5 and 3 experiments for day 9. Error bars correspond to the standard deviation. (C) BCL-6–infected memory B cell cultures are similarly proliferative compared with memory B cells infected with the control virus. The fold induction in proliferation was calculated by dividing the number of GFP⁺ cells 7 d after infection by the number 3 d after infection from in vitro memory B cells infected with the control or BCL-6 virus. Shown are two independent experiments. (D) Memory B cell cultures with forced expression of BCL-6 and control cells have similar amounts of cell death. Infected memory B cell cultures were harvested on day 5, 6 (for Expt #2), 7, and 9. Cells were analyzed for apoptosis with annexin V and 7-AAD staining of GFP⁺ cells. Graphical representation of annexinV⁺7AAD⁻GFP⁺ cells 3, 4, 5, and 7 d after infection. Shown are two independent experiments.