Fig. 3. PBC micelle–Ag complexes enhance B cell proliferation and antibody production.
(A) Schematic of experiment showing stimulation of murine spleen cells following RBC lysis with the following treatment groups: anti-IgM F(ab′)2 (10 μg/ml) as positive control, Ag only [Ag: hen egg white lysozyme (HEL) or ovalbumin (OVA)], PBC micelles only (10 μg/ml), or PBC micelle–Ag (10 μg/ml each). Anti-CD40 (5 μg/ml) was added to all the treatment groups except unstimulated controls. Corresponding representative histograms for the stimulation groups with CFSE expression (blue lines) for viable B cells (Zombie−CD3−CD19+B220+) generated 4 days after proliferation. The number of daughter populations is indicated in a row on top of each histogram (i.e., zero = nonproliferating parent peak). Peaks for CFSE-unstained cells (light gray, filled) are also shown. (B) Percent proliferated viable B cells from spleens as determined by CFSElo gating for young WT mice. (C) Anti-HEL IgM titers and (D) anti-OVA IgM titers in the supernatants of cells stimulated with different treatment groups. For cell culture, splenic cells (0.5 × 106 cells per well) were stimulated in 96 well U-bottom plates. Experiments were performed with cells from both C57BL/6 and BALB/c mice. Data are represented as mean ± SEM. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test; n = 4. In addition to the significance denoted in the image with the respective P values, mean values for anti-F(ab′)2 and PBC micelle–Ag groups in (B) are also statistically significant from control with P < 0.0001. PBC micelle, HEL, and OVA are not significant from the medium-only control in (B) to (D).