Background/Purpose: Anti-dsDNA IgG antibodies are a hallmark of systemic lupus erythematosus (SLE), an autoimmune disease characterized by chronic inflammation and multi-organ damage. Evidence suggests that anti-dsDNA antibodies contribute to a range of end-organ injuries, particularly lupus nephritis. Although the overall expansion of plasma cells is well established in SLE, surprisingly little is known about the phenotype of antigen-specific, dsDNA-reactive B cells and the mechanisms underlying tolerance break towards dsDNA. As insights into these mechanisms are important to understand the emergence of autoantibody responses hallmarking SLE, we aimed to disentangle the cellular phenotype and B cell differentiation pathways involved in the production of anti-dsDNA antibodies.

Methods: PBMCs were isolated from healthy subjects and SLE patients. A novel flow cytometry staining for dsDNA was developed using fluorescent dsDNA tetramers and validated using supernatants of single-cell sorted dsDNA-reactive B cells. Phenotyping of dsDNA-reactive B cells was performed using spectral flow cytometry on PBMCs from healthy subjects (n=10), and anti-dsDNA+ (n=10) and anti-dsDNA- (n=11) SLE patients. Anti-dsDNA positivity in serum and culture supernatants was determined using ELISA.

Results: By index sorting and single cell culture we show that naïve dsDNA-reactive B cells are present in both healthy donors and SLE patients. Spectral flow cytometry revealed that dsDNA-reactive B cells are expanded and activated in anti-dsDNA+ SLE patients only, as compared to negative patients and healthy subjects. The largest expansion was observed in the activated CD21-CXCR5- B cell subsets, pointing to activation through the extrafollicular pathway. While comprising only 2.3% ± 0.2% of the dsDNA-reactive B cells in healthy controls, class-switched CD21-CXCR5- B cells accounted for 21% ± 1.5% of the dsDNA-reactive cells in anti-dsDNA+ SLE patients. These findings indicate a direct relationship between immunological activity of dsDNA-reactive B cells and serological anti-dsDNA positivity, which was further confirmed by the observation that dsDNA-reactive B cells in anti-dsDNA+ versus anti-dsDNA- subjects exhibit increased expression of CD11c, CD95, CD69, Ki67, and BST2, and decreased expression of CXCR5, CD23, and CD72. Interestingly, whereas most CD11c+ cells in the total B cell compartment co-expressed T-bet, CD11c+ dsDNA-reactive B cells largely lacked T-bet expression.

Conclusion: We conclude that dsDNA-reactive B-cells are present within resting naïve B cells in healthy subjects and anti-dsDNA IgG-negative SLE, pointing to defective elimination of dsDNA-reactivity in the human naïve B cell repertoire. In anti-dsDNA+ SLE patients, these cells display markers typifying an extrafollicular pathway maturation and are hallmarked by CD11c expression in the absence of T-bet. These findings identify a novel, unconventional, pathway of CD11c+ B cell differentiation, indicate that human dsDNA-IgG responses in SLE have been licensed to differentiate through an extrafollicular pathway, and offer potential targets for selectively inhibiting these cells in SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/differential-expression-of-activation-markers-on-dsdna-reactive-b-cells-between-healthy-subjects-and-sle-patients-reveals-unconventional-extrafollicular-activation-in-sle/