Effect of Brd4 inhibition on the percentages of naïve, memory, plasmablasts, and plasma cells in healthy human CD19+B cells
To determine whether Brd4 inhibition regulates the differentiation of human B cells, purified CD19+ B cells isolated from healthy donors were activated with combinations of anti-IgM, rCD40L, CpG ODN2006, rIL-4 and rIL-2 in the presence or absence of PFI-1, and then the cells were stained for various indicated surface markers. Flow cytometric analysis was performed to determine the percentages of naïve B cells (CD19+IgD+CD27-), memory B cells (CD19+IgD-CD27+), plasmablasts (CD19+IgD-CD27++) and plasma cells (CD19+CD38++CD138+). As shown in Fig.2A and B, after 5 days in culture, treatment with PFI-1 significantly decreased the percentages of plasmablasts and plasma cells, but did not affect the percentages of naïve and memory B cells. We also observed a similar effect of PFI-1 on B cell differentiation over 7 days in culture (Fig.S2). We also demonstrated that JQ1 treatment reduced the percentages of plasmablasts and plasma cells (data not shown).
To further determine the requirement of Brd4 for terminal B cell differentiation into plasma cells, we used an in vitrocostimulation method known to induce plasmablasts from peripheral B cells (anti-IgM (5 μg/ml), rCD40L (1 μg/ml), CpG ODN2006 (5 μM), rIL-10 (50 ng/ml) and rIL-15 (10 ng/ml)) and examined the effect of PFI-1. We observed that during 5 days in culture, costimulation resulted in an increase in the percentage of CD19+IgD-CD27++and CD19+CD38++CD138+, and this increase could be decreased by treatment with PFI-1 (Fig. 2C and D). Similar results were also obtained over 7 days in culture (Fig. S3). Collectively, our findings suggest that Brd4 inhibition modulates plasmablast-mediated plasma cell differentiation. Furthermore, we evaluated the effect of PFI-1 on the secretion of Ig. Analysis of culture supernatants revealed that costimulation increased the secretion of IgG and IgM; however, this increase was suppressed by treatment with PFI-1 (Fig. 2E).