Molecular Characterization of Circulating Plasma Cells in Patients with Active Systemic Lupus Erythematosus

<div><p>Systemic lupus erythematosus (SLE) is a generalized autoimmune disease characterized by abnormal B cell activation and the occurrence of increased frequencies of circulating plasma cells (PC). The molecular characteristics and nature of circulating PC and B cells in SLE have not been completely characterized. Microarray analysis of gene expression was used to characterize circulating PC in subjects with active SLE. Flow cytometry was used to sort PC and comparator B cell populations from active SLE blood, normal blood and normal tonsil. The gene expression profiles of the sorted B cell populations were then compared.</p> <p>SLE PC exhibited a similar gene expression signature as tonsil PC. The differences in gene expression between SLE PC and normal tonsil PC and tonsil plasmablasts (PB) suggest a mature Ig secreting cell phenotype in the former population. Despite this, SLE PC differed in expression of about half the genes from previously published gene expression profiles of normal bone marrow PC, indicating that these cells had not achieved a fully mature status. Abnormal expression of several genes, including <em>CXCR4</em> and <em>S1P₁</em>, suggests a mechanism for the persistence of SLE PC in the circulation. All SLE B cell populations revealed an interferon (IFN) gene signature previously only reported in unseparated SLE peripheral blood mononuclear cells. These data indicate that SLE PC are a unique population of Ig secreting cells with a gene expression profile indicative of a mature, but not fully differentiated phenotype.</p> </div

Proposed plasma cell trafficking in SLE based on gene expression profiling.

<p>Normal B cell traffic into germinal center reactions appears unchanged in SLE from normal. Plasma cells in SLE differ from tonsil plasma cells in expression of cell surface markers that may affect plasma cell homing to bone marrow. Plasma cells continue to mature in the blood and have a gene program upregulated similar to bone marrow plasma cells with long-lived potential. Downregulated gene program include continued and more pronounced decrease in expression of CXCR4, MHC class II and cell surface markers. Endogenous factors such as IFN<i>α</i> in SLE may contribute to these changes.</p

Gene expression profiling of circulating SLE B cells populations demonstrating the differential gene expression in SLE PC compared with SLE naïve and memory B cells.

<p><b>A</b>. 780 genes are differentially expressed in SLE PC compared to SLE naive and memory B cells identified using SAM. 571 genes shown in the heatmap are upregulated and 209 genes are downregulated in SLE PC compared to SLE naïve and memory B cells. <b>B</b>. The genes differentially expressed in SLE PC were then compared gene by gene for corresponding expression in sorted tonsillar B cell populations.</p

Gene expression profiling of sorted tonsil B cell populations.

<p>The heatmap shown represents the differential gene expression identified using- SAM in tonsil PC (TPC), tonsil naïve, pre-GC/germinal center founder (GCF), dark zone/germinal center (GC) and memory B cells. A total of 1858 genes are shown. 1690 are upregulated and 168 are downregulated. The majority of the gene signature is shared with tonsil PB (TPB).</p

Relative expression of genes involved in lymphocyte trafficking.

<p>S1P₁, CD69 and CXCR4 were examined for gene expression in IgSC populations (tonsil PC and SLE PC). A significant difference (p<0.06) in gene expression among the populations is denoted in the figure and was determined by a student's t-test with a correction for multiple comparisons using Bonferroni correction.</p

TPC differentially expressed genes and comparative expression in TPB.

<p>Selected list of differentially expressed genes (using SAM) in tonsil plasma cells (CD19⁺CD38⁺⁺⁺IgD⁻) compared to tonsil non-Ig secreting cells (naïve cells CD19⁺CD38⁺IgD⁺, memory cells CD19⁺CD38^+/−IgD⁻, dark zone cells CD19⁺CD38⁺⁺IgD⁻ and pre-GC/germinal center founder cells CD19⁺CD38⁺⁺IgD⁺) and the relative expression in tonsil plasmablast (CD19⁺CD38⁺⁺⁺IgD⁺). This list depicts the upregulated or downregulated genes in tonsil PC compared to tonsil PB. Bold typeface denotes a differentially upregulated gene in TPC compared with non IgSC from tonsil. Italics typeface denotes a differentially downregulated gene in TPC compared with non IgSC from tonsil. Gene function adapted from publicly available databases NCBI Gene and HPRD (Human Protein Reference Database).</p

Differentially expressed genes in SLE PC compared to tonsil Ig secreting cells.

<p>77 differentially expressed genes in SLE PC compared to tonsil PC and 72 differentially expressed genes in SLE PC compared to tonsil PB, both identified by SAM. The bolded genes represent genes upregulated in SLE PC compared to tonsil Ig secreting cells. The genes in italics represent downregulated genes compared with tonsil Ig secreting cells. The asterisk denotes genes common to both comparisons. Gene function adapted from NCBI Gene and HPRD (Human Protein Reference Database).</p

Comparative expression of bone marrow PC discriminating genes in SLE PC and tonsil PC.

<p>51 genes previously reported to discriminate bone marrow PC from tonsil PC (<b>A</b>) were compared to gene expression profiles of SLE PC and tonsil PC from the current data set (<b>B</b>). The blue line in the figure divides the 51 genes into those that are similarly (top) and dissimilarly (bottom) expressed by SLE PC and bone marrow PC. Twenty-seven genes demonstrate a similar pattern of expression in SLE PC and bone marrow PC and 24 genes exhibit a dissimilar pattern of expression in bone marrow and SLE PC.</p

Unique gene expression pattern of HLA class II genes in SLE PC in comparison to tonsil PC and PB.

<p>Expression of HLA Class II genes in SLE PC, tonsil PC and tonsil PB. Differences in gene expression are shown as mean expression value log2 transformed data. Significance determined by student's t-test is denoted by asterisk(s) in the figure. Correction for multiple comparisons performed using Bonferroni correction.</p