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

The phenotypic characterization of self-reactive B cells producing autoantibodies is one of the challenges to get further insight in the physiopathology of autoimmune diseases. We took advantage of our previously developed flow cytometry method, using labeled nucleosomes, prominent autoantigens in systemic lupus erythematosus, to analyze the phenotype of self-reactive B cells in the anti-DNA B6.56R mouse model. We showed that splenic anti-nucleosome B cells express mostly kappa light chains and harbor a marginal zone phenotype. Moreover, these autoreactive B cells fail to acquire a germinal center phenotype and are less abundant in the transitional T3 compartment. In conclusion, the direct detection of autoreactive B cells helped determine their phenotypic characteristics and provided a more direct insight into the B cell tolerance process in B6.56R mice. This method constitutes an interesting new tool to study the mechanisms of B cell tolerance breakdown in B6.56R mice crossed with autoimmune prone models.

Identification of autoreactive B cells with labeled nucleosomes.

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.