Identification of autoreactive B cells with labeled nucleosomes

Abstract

The pathogenesis of autoimmune diseases has not been completely elucidated yet, and only a few specific treatments have been developed so far. In autoimmune diseases mediated by pathogenic autoantibodies, such as systemic lupus erythematosus, the specific detection and analysis of autoreactive B cells is crucial for a better understanding of the physiopathology. Biological characterization of these cells may help to define new therapeutic targets. Very few techniques allowing the precise detection of autoreactive B cells have been described so far. Herein we propose a new flow cytometry technique for specific detection of anti-nucleosome B cells, which secrete autoantibodies in systemic lupus erythematosus, using labeled nucleosomes. We produced different fluorochrome-labeled nucleosomes, characterized them, and finally tested them in flow cytometry. Nucleosomes labeled via the cysteines present in H3 histone specifically bind to autoreactive B cells in the anti-DNA transgenic B6.56R mice model. The present work validates the use of fluorochrome-labeled nucleosomes via cysteines to identify anti-nucleosome B cells and offers new opportunities for the description of autoreactive B cell phenotype.

Phenotyping of autoreactive B cells with labeled nucleosomes in 56R transgenic mice

“…However, only a fraction of IgM a+ B cells have a self-reactive BCR, due to receptor editing of variable regions or pairing of transgenic heavy chain with specific endogenous light chains 19 , 26 . Thus, we decided to further analyse the phenotype of autoreactive B cells using a flow cytometry based-assay using labeled nucleosomes 22 .…”

“…Although these studies provided primordial data regarding tolerance mechanisms, the technology used is not suitable for large scale analysis and does not allow a direct phenotypic approach 20 , 21 . We previously developed a novel flow cytometry-based method to identify self-reactive B cells with fluorescent nucleosomes in B6.56R mice 22 . Nucleosomes constitute the main autoantigen in SLE.…”

“…Therefore, identification of the 56R H chain with only anti-IgM a labeling is not ideal to analyze autoreactive B cells in this model. For these reasons, we used labeled nucleosomes to characterize B cells based on their genuine autoreactivity 22 . In addition, B6.56R mice do not develop illness despite production of anti-dsDNA autoAbs 26 , unless they are crossed with autoimmune prone mice 29 , 30 .…”

Understanding and measuring human B‐cell tolerance and its breakdown in autoimmune disease

“…Theoretically at least, this approach can simultaneously determine the autoreactivity and polyreactivity of large numbers of single cells in multiple B-cell compartments. 73,79 Both rheumatoid factor and anti-cyclic citrullinated peptide (CCP) reactivity of B-cells and PC have been studied by flow cytometry in RA through surface and intracellular staining, respectively. 73,74 In other cases, complete autoantigens frequently expressed as multimers have been used in SLE, rheumatoid arthritis, anti-cardiolipin syndrome, and pemphigus.…”

“…73,74 In other cases, complete autoantigens frequently expressed as multimers have been used in SLE, rheumatoid arthritis, anti-cardiolipin syndrome, and pemphigus. 71,[75][76][77][78][79] In SLE in particular, as further discussed below, flow cytometry has been employed to measure general anti-nuclear reactivity (ANA) 77 ; the expression of the autoreactive 9G4 idiotype 80,81 ; and anti-dsDNA and anti-nucleosome autoreactivity. 73,79 Both rheumatoid factor and anti-cyclic citrullinated peptide (CCP) reactivity of B-cells and PC have been studied by flow cytometry in RA through surface and intracellular staining, respectively.…”