FIGURE 1. STAT3 activation is required for the centrocyte (CC) to pre-plasmablast (Pre-PB) transition of cultured GC B cells.

Purified CD20^hiCD38^hi centroblasts (CB) were differentiated in vitro in the presence of IL-2, IL-4, and CD40L-CD8 fusion on HK cells for 12 days. In parallel cultures, AG490 (5 uM) or IL-21 (50 ng/ml) was included throughout the assay to either inhibit or further activate Jak/STAT signaling, respectively. A. On indicated days, aliquots of cells from the control (ctrl) and AG490 cultures were stained and B cell subsets identified based on surface CD20 and CD38 expression. B. Changes in the expression of indicated transcription factors were analyzed by Western Blot. n.s., a non-specific band recognized by the polyclonal Blimp-1 Ab. The Mum1 Ab recognizes a conformational epitope on the IRF4 protein enriched in the nucleus (30). GAPDH is used as a loading control. C. Culture supernatants were harvested on the days indicated from control, AG490, and IL-21 cultures, and analyzed by ELISA for total IgG and IgM Ab production. Plotted in the graph are mean ± s.d. of two duplicate cultures in the same experiment. Note the different y-axis scale for IgM and IgG. D. Expression of Jak3 and IRF4 mRNA was measured by qRT-PCR on RNA samples prepared from the control and AG490 cultures on the days indicated. Data are representative of 3 or more independent experiments.