Fig 1.
Plasmacytoid dendritic cells and RNA-containing immune complexes increase the frequency of CD27-IgD- B cells.
Flow cytometric analysis of peripheral blood B cells isolated from SLE patients (SLE) or from healthy individuals (HC). The CD19+ B cells were stained without culturing (fresh) or either cultured alone or together with plasmacytoid dendritic cells (pDC) for 6 days, in absence or presence of RNA-containing immune complexes (RNA-IC). All cultures were supplemented with IL-3 and GM-CSF (Cyt). (A) Representative plots and gating strategy of B cells stained for the cell surface expression of CD27 and IgD, and categorized as: CD27+IgD- switched memory cells (SM), CD27hiIgD- plasmablasts (PC), CD27+IgD+ non-switched memory cells (NSM), CD27-IgD+ naive cells (N) and CD27-IgD- double negative B cells (DN). The plots show B cells fom healthy individuals stained without culturing (left plot), cultured in presence of IL-3/GM-CSF (middle plot), or stimulated with RNA-IC+ IL-3/GM-CSF (right plot). (B) The frequency of CD27-IgD- B cells in the total CD19+ B cell population. (C) The frequency of CD27-IgD-CD95+ B cells in the total CD19+ B cell population. (B, C) Individual values (dots) and the mean (horizontal bars) are shown. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001. Statistical analyses were performed by Mann Whitney test or Wilcoxon signed rank test.
Fig 2.
Frequency of switched memory and naïve B cells in presence of plasmacytoid dendritic cells and RNA-containing immune complexes.
Flow cytometric analysis of B cells, without culturing (fresh) or stimulated for 6 days with IL-3/GM-CSF (Cyt) in absence or presence of RNA-containing immune complexes (RNA-IC). The CD19+ B cells were either stimulated alone or in co-cultures with plasmacytoid dendritic cells (pDC). (A) The percentage of CD27+IgD- isotype switched memory (SM) B cells. (B) The percentage of CD27-IgD+ naïve B cells. Individual values (dots) and mean values (horizontal bars) are shown * = p<0.05, ** = p<0.01. Statistical analyses were performed by Friedman test.
Fig 3.
CD86 expression by B cells cultured with plasmacytoid dendritic cells and RNA-containing immune complexes.
Flow cytometric analysis of CD86 expression by (A) CD19+ B cells and (B) subsets of B cells. A) The B cells were analyzed without culturing (fresh) or after culturing for 6 days with IL-3/GM-CSF (Cyt), in absence or presence of RNA-containing immune complexes (RNA-IC) and plasmacytoid dendritic cells (pDCs). (B) The CD86 expression was further analyzed on B cell subsets characterized as switched memory cells (CD27+IgD-), non-switched memory (CD27+IgD+), double negative (CD27-IgD-) or naïve (CD27-IgD+) B cells. The median fluorescence intensity (MFI) of CD86 expression is based on (A) 9 and (B) 4–6 individual donors, respectively. * = p<0.05, ** = p<0.01. Statistical analyses were performed by Wilcoxon signed rank test.
Fig 4.
Increased IL-6 and IL-10 production by co-cultivated B cells and plasmacytoid dendritic cells.
B cells and pDCs were isolated from healthy donor PBMCs. (A) The B cells were cultivated alone or (A, B) in co-cultures with pDCs for 6 days with IL-3/GM-CSF (Cyt), and in absence or presence of RNA-containing immune complexes (RNA-IC). Production of the cytokines (A) IL-6 and (B) IL-10 in the cell cultures were determined by immunoassays. (C, D) The frequency of (C) IL-6 or (D) IL-10 producing pDCs and B cells in the co-cultures was determines by flow cytometry. Mean values ± SEM based on 7–11 individual donors are shown in (A) and (B). The frequencies of IL-6 and IL-10 positive cells are based on (C) 3–6 and (D) 7–9 individual donors * = p<0.05, ** = p<0.01, *** = p<0.001. Statistical analyses were performed by Wilcoxon signed rank test.
Fig 5.
Twenty one genes differentially expressed by double negative CD27-IgD- B cells and switched memory CD27+IgD- B cells.
Heat map of gene expression levels by double negative CD27-IgD- B cells (CD27negIgDneg) and switched memory B cells (CD27posIgDneg) (genes with >2-fold difference, p≤0.001). Plasmacytoid dendritic cells and CD19+ B cells were isolated from peripheral blood of healthy individuals (n = 10) and co-cultivated in the presence of RNA-containing immune complexes (RNA-IC) for four days. The CD27negIgDneg and CD27posIgDneg B cell subsets were isolated by flow cytometric cell sorting after staining with antibodies to CD123, CD19, CD27 and IgD. The mRNA expression of totally 614 genes were analyzed by nCounter gene expression platform and nSolverAnalysis Software 3.0. Each column represents a sample and rows represent differentially expressed genes. Median differences between the groups were analyzed by using Wilcoxon signed rank test.