Fig 1.
Flow cytometry analysis strategy.
Representative dot plots (A) and the corresponding strategy (B) to identify on a gate of circulating Bc (CD19+ CD3-/CD14-) that excludes CD38hi, CD27hi ASC the Bc populations of interest: naïve (CD27- IgD+), IgM mBc (CD27+ IgD+ IgM+) and switched (Sw) mBc (CD27+ IgD- IgM-). Different markers were studied for each Bc subset; shown are representative histograms for CD95. Solid gray histogram represents fluorescence minus one (FMO) and the continuous solid line shows staining with anti-CD95 (C).
Fig 2.
Phenotypic profile of the three Bc subsets.
A summary of the experiments to characterize the phenotype of circulating naïve Bc and IgM and switched (Sw) mBc in PBMC from healthy adult volunteers is presented: CD40 (n = 18), BAFF-R (n = 16), CD21 (n = 6), CD38 (n = 15), CD73 (n = 6), CD95 (n = 7), CD11c (n = 6), CD43 (n = 5), CD122 (n = 6), TLR9 (n = 5), PD–1 (n = 5), and IL-21R (n = 10). Wilcoxon tests were used for evaluating differences among groups and * denotes p<0.05. Individual results are shown (as iMFI) and lines and error bars denote the median and interquartile range, respectively.
Fig 3.
Sorting strategy for purification of the three Bc subsets.
Representative dot plots of a sorting experiment to purify naïve Bc and IgM and switched (Sw) mBc. Dot plots of presort samples are shown in top rows. Dot plots of naïve Bc and IgM and Sw mBc are shown in the second, third, and fourth rows, respectively. Cells were initially gated on CD19+ dump channel- (Aqua, CD3, CD14), first column. After excluding CD38hi, CD27hi ASC (second column), naïve cells were gated as CD27- IgD+ (third column) and the CD27+ subset was sorted as IgM+ or switched (IgM-) mBc (fourth column).
Fig 4.
Proliferation and differentiation to ASC of the three Bc four days after in vitro stimulation.
Representative dot plots of proliferation (first column) and differentiation to ASC (CD38hi CD27hi) (second column) of naïve Bc and IgM and switched mBc four days after stimulation with CpG, a cocktail of cytokines (IL–2, IL–6, and IL–10), and murine fibroblasts (as feeder cells) (A). Summary of experiments for percentages of CFSElow proliferating Bc (B), MFI of CFSElow cells (C), and frequencies of CD38hi CD27hi ASC (D) of each of the Bc studied. Wilcoxon tests were used for evaluating differences among groups and * denotes p<0.05. Individual results are shown and lines and error bars denote the median and interquartile range, respectively.
Fig 5.
Intracellular expression of IgM and IgG of the three Bc subsets four days after in vitro stimulation.
Representative dot plots of an experiment to evaluate intracellular IgM and IgG after stimulation of naïve Bc and IgM and switched mBc (A). Summary of experiments for the percentages of cells expressing IgM (B) and IgG (C) are shown. Wilcoxon tests were used for evaluating differences among groups and * denotes p<0.05. Individual results are shown and lines and error bars denote the median and interquartile range, respectively.