Figure 5. Effect of TCDD on CD40L-induced protein expression of plasmacytic differentiation regulators in mouse B cells.

Freshly isolated mouse B cells (1 × 10⁶/ml) were treated with TCDD at 1(not shown), 3, 10 (not shown), or 30 nM, and/or vehicle (VH). In addition, B cells not treated with VH (NA) served as a control for VH treatment. The B cells were then co-cultured with irradiated CD40L-L cells (5 × 10⁴ cell/well) in the presence of recombinant mouse IL-2 (10 U/ml), IL-6 (100 U/ml), and IL-10 (20 ng/ml) for 3 days. Cells were transferred to new plates in the absence of CD40L-L cells and cultured for three additional days. Cells were harvested right after isolation (Day 0, naive), and on Day 2 (not shown) and Day 6 (CD40L + VH, CD40L + TCDD 3 nM, and CD40L + TCDD 30 nM), and then assessed by multiparametric flow cytometry for surface expression of CD138 and intracellular expression of Blimp-1 and Pax5. Dead cells were identified by staining with Live/Dead near-infrared Staining Kit, and are not included in these data graphs (no significant differences in cell viability were observed between different treatment groups). Results depicted are fluorescent signals for Blimp-1, Pax5, and CD138. Percentages of cells within each gate, mean ± SE of three replicates, are shown in the center. *, p < 0.05, **, p < 0.01, compared to the VH-treated group. Data are representative of two separate experiments with three replicates per group and are concatenated (combined) from three experimental replicates.