CD86 expression by B cells cultured with plasmacytoid dendritic cells and RNA-containing immune complexes.

<p>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.</p

Frequency of switched memory and naïve B cells in presence of plasmacytoid dendritic cells and RNA-containing immune complexes.

<p>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.</p

Twenty one genes differentially expressed by double negative CD27^-IgD^- B cells and switched memory CD27⁺IgD^- B cells.

<p>Heat map of gene expression levels by double negative CD27^-IgD^- B cells (CD27^negIgD^neg) and switched memory B cells (CD27^posIgD^neg) (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 CD27^negIgD^neg and CD27^posIgD^neg 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.</p

Plasmacytoid dendritic cells and RNA-containing immune complexes increase the frequency of CD27^-IgD^- B cells.

<p>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), CD27^hiIgD^- 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.</p

Analysis of <i>IRF5</i> transcripts containing the Ex6 in/del.

<p>Using estimated transcript read counts obtained from MMSeq, expression of transcripts containing vs. those missing the Ex6c deletion was compared in healthy donors (n = 4) and SLE patients carrying the H2 risk haplotype (n = 4). Shown are the total number of reads aligned per group of patients. ^aχ² = 1377726 with <i>p</i><2.2×10⁻¹⁶; <i>p</i> values were obtained by the Pearson’s χ²-test with Yates continuity correction. Totaled reads mapping to the Ex6C deletion were similarly compared across grouped healthy vs. SLE (risk) patients; ^bχ² = 16.0379 with <i>p</i><6.209×10⁻¹⁵. Σ = sum.</p

Pile-up view of reads from deep sequencing of PCR-amplified <i>IRF5</i> fragments.

<p><i>A,</i> Pile-up view of <i>IRF5</i> sequencing reads mapped to the <i>IRF5</i> genomic sequence was generated with IGV. Peak heights represent the relative frequency of mappings to the indicated regions - hills reflect a high mapping frequency of reads to a particular region and valleys indicate low mapping frequency or deletions (example shown with star); arrows point to areas of reads aligning to previously unannotated exons. Results shown are from 4 independent healthy donors and 5 independent SLE patients. <i>B,</i> Reads from healthy donors and SLE patients that mapped to exon 7, shown by the star in <i>A</i> above, and reflect true internal deletions of exon 7 are shown. Junction reads are aligned by position and labeled as to whether it was detected in a healthy or SLE sample; the exact number of reads per sample that contained the junction is shown in parentheses.</p

Correlation between Gibbs expression estimates and clone count.

<p>Gibbs expression estimates and Monte Carlo standard errors were generated by MMSeq. Bar graphs show Gibbs expression measures from next-generation sequencing versus clone counts from traditional cloning and sequencing. Data from n = 2 healthy donors and n = 5 SLE patients is shown; at least 20 <i>IRF5</i>-positive clones from each donor were used for this analysis.</p

Workflow for enrichment and analysis of single gene profiling by RNA-Seq.

<p>Following PCR amplification of RNA of a single gene, cDNA is sequenced by next-generation sequencing and reads are mapped to sequences of alternatively spliced transcripts to obtain expression estimates and to compare transcript expression profiles across samples.</p

Expression estimates of <i>IRF5</i> alternatively spliced transcripts in healthy donors and SLE patients.

<p><i>A,</i> Gibbs expression estimates were obtained with MMSeq and converted to raw read counts, then normalized to total read counts. Normalized read counts for each transcript in 5 SLE patients and a representative healthy donor (#1) are plotted; read counts were compared between the two sets using a student's <i>t</i>-test. *<i>p</i><0.05; **<i>p</i><0.01. <i>B,</i> Scatter plot showing the correlation between MMSeq-derived expression estimates and true (assigned) expression levels of the 18 transcript variants. R² = 0.6532 at alpha = 0.05 and <i>p</i><0.0001 indicating a highly significant correlation.</p

Top four most abundant transcripts are shared by SLE patients with the H2 full risk haplotype.

<p><i>A,</i> Relative abundance ranking of transcripts from 4 healthy donors, 1 SLE patient with the H3 full protective haplotype, and 4 SLE patients with the H2 full risk haplotype is shown. Transcripts were ranked from most to least abundant using MMSeq-generated Gibbs expression estimates; individual transcripts are represented by distinct colors. <i>B,</i> Hierarchial clustering was performed on the nine samples using expression profiles of the 18 <i>IRF5</i> transcript variants. Height represents the difference between samples. Red values are AU <i>p</i>-values and green values are BP values. Clusters with AU larger than 95% are statistically significant with a <i>p</i><0.05.</p