Background/Purpose:  B cell hyperactivation and autoantibody (auto-Ab) production are hallmarks of SLE. Auto-Abs, reactive to RNA-containing antigens are found in a fraction of SLE patients, particularly those with severe disease activity. Toll-like receptor 7 (TLR7), an intracellular TLR that recognizes ssRNA, has been highly implicated in the generation of pathogenic anti-RNA autoantibodies in SLE, however the cellular origin of anti-RNA Ab–secreting cells is not well defined. Human transitional (TR) B cells have been previously shown to be enriched in polyreactive specificities and increase of TR B cells have been described in SLE patients. This study was undertaken to determine a possible link between the transitional B cell expansion and increase in TLR7 expression in SLE.

Methods: Human peripheral blood mononuclear cells (PBMCs) were collected from both SLE patients and healthy donors (HDs) and analyzed by multicolor flow cytometry. Frequencies of TR B cells within the CD19⁺ cell population were determined based on the relative expression of IgD, CD10, CD24, CD27, and CD38 surface markers. Expression levels of TLR7 and TLR9 were measured by real-time PCR. Additionally, CD19⁺ CD38⁺⁺CD24⁺⁺ TR B cells from human cord blood were isolated by cell sorting. These cells were stimulated with IFNα, followed by stimulation with anti-IgM and/or TLR7 agonist R848. Changes in mRNA levels of TLR7 were analyzed 3-6 hours post-stimulation. Plasma cell differentiation was assessed by flow cytometry after 5 days of in vitro cell culture; antibody production was analyzed by ELISA, and the expression of cytokines was measured by bead-based immunoassay.

Results:  Analysis of the B cell compartment revealed a significant increase in the frequencies of circulating CD19⁺CD27^–IgD⁺CD10⁺CD38⁺⁺CD24⁺⁺ TR B cells in SLE patients, compared to HDs. We found a significant positive correlation between the levels of TLR7, but not TLR9 expression, and the frequencies of circulating TR B cells. SLE patients who carry a risk G allele (TLR7 polymorphism rs3853839, associated with increased TLR7 expression) show a trend toward increased TR cell frequencies compared to non-risk allele carriers. SLE patients with high frequencies of TR B cells were also more likely to been found positive for pathogenic Sm/RNP auto-Abs in the clinic. IFNα treatment induced a nearly 10-fold increase in the expression of TLR7 and IRF7 in TR B cells isolated from umbilical cord blood. IFNα-primed TR B cells become highly responsive to in vitro stimulation with R848 and differentiated into CD27⁺⁺CD38⁺⁺CD24^–plasmablast in the absence of any other stimuli. Stimulation of TR B cells with R848 promoted IgM production, whereas stimulation with anti-IgM plus R848 induced IFN-γ, IL-6 and IL-10 cytokines.

Conclusion: Our findings show a direct correlation between TLR7 expression levels and the expansion of TR B cells in SLE patients. Upon IFNα exposure, human TR B cells become hyper-responsive to stimulation through TLR7. Our study suggest that human TR B cells might be an important source of auto-Abs and provide a new link between innate IFN and TLR7 signaling and B cell activation in SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/expansion-of-transitional-b-cells-in-sle-patients-correlates-with-increased-toll-like-receptor-7-expression/