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).