PerC– and Splenic B cells possess different surface markers after activation.

<p>Indicated B cell populations were stained extracellular with mAbs against CD19 and indicated markers after both a 48 h period of stimulation using αCD40+5 hr LPS (+: activated; see materials and methods) and after being isolated freshly (–: non-activated). Shown in A are expression levels of indicated markers on CD19⁺ B cells (representative plots of two identical replicate experiments), in B the average geometric mean fluorescence intensity (GeoMFI) levels from two identical replicate experiments.</p

Activation status of B cells is critical for the effects exerted on T cells.

<p>B- and T cell cultures and subsequent intracellular staining were performed as explained in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088869#pone-0088869-g004" target="_blank">Figure 4</a>. The percentages of T cells producing TNF-α (A) and IFN-γ (B) after co-culture with indicated activated (+) or non-activated (–) B cell subsets is shown. In A and B the mean ± S.D. are depicted of pooled results from two identical replicate experiments (with at least triplicate technical replicates each) that produced similar data. The cytokine levels of indicated B- and T cell cultures (C) were determined from the supernatant using Luminex. Levels shown in C are the mean ± S.D. of pooled results from two identical replicate experiments (with at least triplicate technical replicates each). Statistically significant differences between the various culture conditions were calculated using a one-way ANOVA with Bonferroni post-hoc test. *p<0.05, ** p<0.01, *** p<0.001.</p

Activated PerC B cells suppress TNF-α- an IFN-γ-production by CD4⁺ T cells.

<p>Splenic- and PerC B cells were activated <i>in vitro</i> for 48 h using αCD40+5 hr LPS (see materials and methods), and co-cultured with splenic CD4⁺ T cells that were stimulated polyclonally with anti-CD3 for 72 h. The cells were additionally stimulated with PMA/ionomycin/LPS during the last 5 hours of the co-culture in order to facilitate intracellular TNF-α and IFN-γ staining. Shown are representative plots of TNF–α (A) and IFN-γ (C) production by CD4⁺ T cells, and the percentages of T cells producing TNF-α (B) and IFN-γ (D). E and F additionally show the data for indicated non-activated B cell subsets. Shown in B, D and E are the mean ± S.D. of pooled results from two identical replicate experiments (with at least triplicate technical replicates each) that produced similar data. Statistically significant differences between the various culture conditions (B and D) were calculated using a one-way ANOVA with Bonferroni post-hoc test. ** p<0.01, *** p<0.001.</p

PerC B-1a cells secrete IL-10 and IL-6 similarly to the undifferentiated PerC B cell population.

<p>FACS-purified CD19⁺ CD11b⁺ CD5⁺ PerC B-1a cells were cultured for 48 h in the presence of medium or indicated stimuli. Intracellular IL-10 staining was performed after additional stimulation with PMA/ionomycin/LPS and both representative flow cytometric plots (A) and the percentage of IL-10⁺ B cells (B) are shown, whereas the levels of IL-10 (C) and IL-6 (D) were determined directly from the supernatant (before restimulation took place) using Luminex. Shown in B-D are the mean ± S.D. of pooled results from two identical replicate experiments (with 2–5 technical replicates each) that produced similar data. Statistically significant differences between the various stimuli and the medium control (B-D) of PerC B-1a cells were calculated using a one-way ANOVA with Bonferroni post-hoc test. ** p<0.01, *** p<0.001.</p