CD138^highMHCII⁺ cells preferentially express CXCR3, produce IgG autoantibodies and encompass some long-lived cells.

<p>A) CD138-enriched spleen cells of diseased NZB/W mice were analyzed for CXCR3 expression by flow cytometry. Cells were co-stained with anti-B220, anti-CD138, anti-MHCII and anti-CXCR3 Ab. Among CD138^high cells, two subsets can be defined: one which does not express CXCR3 (e) and one which does (f) (see FACS histogram; grey area corresponds to the staining obtained with an isotype control Ab). All CXCR3⁺ cells belong to the MHCII⁺ subset of CD138^high cells (dot-plot, right). FACS data are derived from one representative NZB/W mouse out of eleven tested 32–36 week-old mice. B) Spleen-derived plasma cells (CD138^high, c+d, upper dot-plot) from 30 week-old control BALB/c mice were analyzed for MHCII and CXCR3 expression (lower dot-plot). FACS data are derived from one representative BALB/c mouse out of 3. C) The four spleen cell subsets (a to d, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058140#pone-0058140-g001" target="_blank">Figure 1</a>) were sorted by flow cytometry from the spleen of diseased NZB/W mice. Total RNA was isolated from each subset and the expression levels of <i>cxcr3</i> (black bars) and <i>cxcr4</i> (white bars) transcripts were evaluated by quantitative real-time PCR. Results are expressed as the fold induction of gene transcription as compared to the “a” subset (B220⁺ cells). D) The profile of Ig isotypes produced by CXCR3<b>⁻</b> and CXCR3⁺ CD138^high cells, respectively, was assessed by specific IgM and IgG intracellular stainings. Surface stainings were performed with anti-CD138 and anti-CXCR3 Ab. Mean percentages of IgM- and IgG-producing cells among CD138^highCXCR3<b>⁻</b> and CD138^highCXCR3⁺ cell subsets are shown. Results are derived from eight individual NZB/W mice. E) CXCR3<b>⁻</b> (e) and CXCR3⁺ (f) cell subsets were sorted by flow cytometry from the spleen of diseased NZB/W mice and they were tested for spontaneous secretion of anti-histone IgG by ELISpot. Results are expressed as the number of ASC per 1×10⁶ cells (mean +/− SEM). Each dot corresponds to a different number of plated cells. Results are derived from three representative diseased NZB/W mice. F) Identification of short-lived and long-lived plasma cells within the CXCR3<b>⁻</b> (e) and CXCR3⁺ (f) subsets. Upon intra-peritoneal injection and two weeks feeding with BrdU, 36–38 week-old NZB/W mice were killed and spleen cell subsets were analyzed for BrdU incorporation by flow cytometry. FACS histograms are representative of one NZB/W mouse out of five. Percentages of long-lived (BrdU<b>⁻</b>) cells are expressed as the mean +/− SD (n = 5). *statistically significant differences (<i>p</i><0.05, Mann-Whitney U test).