Background/Purpose:

B cell homeostasis is perturbed in SLE patients; in particular many patients with active disease have a large expansion of IgD- CD27- B cells (DN). The DN population is heterogeneous for CXCR5 expression, and CXCR5- DN are a the majority population in SLE patients with expanded DN but not in HCD. To further understand how these expanded cells differ from other B cells subsets and how they may be dysregulated in SLE, we analyzed gene transcription through RNA sequencing of sorted purified naïve, switched memory (SWM), CXCR5+ DN and CXCR5- DN (DNN) B cells from HCD and SLE patients with expanded DNN.

Methods:

RNA from sorted B cells subsets was isolated, amplified and sequenced. The resulting reads were aligned to the human genome and transcripts were assembled and quantified using edgeR. Transcripts are expressed as reads per kilobase per million (RPKM), genes that showed at least a two fold difference and a false discover rate < 0.05 were considered differentially expressed. TLR7 signalling after stimulation with R848 was measured by staining with anti- phospho-tyrosine specific anti-ERK. Changes in gene expression were evaluated by flow cytometry of purified B cells after overnight R848 stimulation. 

Results:

Genes differentially regulated between SLE and HCD B cells were primarily interferon regulated genes and were up-regulated in all B cell subsets, including several positive regulators of viral pathogen sensing and TLR signaling. DNN showed several commonalities in gene expression with naïve B cells that differentiated them from memory B cells and CXCR5+ DN. These included higher expression of BCL6, IL21 receptor, and CD72 and decreased expression of the high affinity IL2 receptor, CD25, and genes associated with active cell division. Significantly, a negative regulator of TLR signaling, TRAF5, was uniquely down regulated in DNN in both HCD and SLE patients. Consistent with decreased expression of negative regulators of TLR signaling, DNN had enhanced in vitro sensitivity to TLR7 antagonist R848 as measured by ERK phosphorylation. Furthermore, R848 increased expressions of genes necessary for antigen presentation including HLA-DR and CD86 but decreased expression of the inhibitory receptors CD32b and CD72 in DN B cells but not naïve B cells. 

Conclusion:

DNN represent a separate B cell lineage with a distinct origin and function as they differed from other B cells subsets both in uniquely expressing several genes and and a lack expression of other genes. One of the genes that is down regulated in DNN relative to all other B cell subsets is TRAF5, which in mice has been shown to negatively regulate TLR responses in B cells. Supporting the hypothesis that this gene may have a similar function in human B cells, both TLR7 signaling and responsiveness was enhanced in DNN. RNA from apoptotic cells can be internalized as immune complexes and through B cell receptors specific for auto-antigens. These antigens are powerful stimulants of TLR7 and DNN are uniquely sensitive to this signal. Furthermore, increased expression of HLA-DR and CD86 in DNN after TLR will enhance antigen presentation to CD4 T cells. DNN likely play an important role in SLE pathogenesis by linking innate TLR signaling with B cell mediated adaptive immunity.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/decreased-expression-of-negative-regulators-of-toll-like-receptor-signaling-and-increased-tlr7-responsiveness-in-expanded-igd-cd27-b-cells-from-systemic-lupus-erythematosus-patients/