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Objective: Antinuclear antibodies (ANAs) are diagnostic in several autoimmune disorders, yet the failure to achieve B cell tolerance in these diseases is still poorly understood. Although secreted ANAs detected by an indirect immunofluorescence assay are the gold standard for autoreactivity, there has been no convenient assay with which to measure the frequency of circulating B cells that recognize nuclear antigens (ANA+ B cells) in patients. The aim of this study was to generate an assay to easily identify these B cells and to examine its utility in a study of autoreactive B cells in systemic lupus erythematosus (SLE)., Methods: We developed and validated a novel flow cytometry-based assay that identifies ANA+ B cells using biotinylated nuclear extracts, and utilized it to examine B cell tolerance checkpoints in peripheral blood mononuclear cells obtained from SLE patients and healthy controls., Results: We observed progressive selection against ANA+ B cells as they matured from transitional to naive to CD27+IgD- and CD27+IgD+ memory cells in both healthy subjects and SLE patients; however, ANA+ naive B cells in SLE patients were not anergized to the same extent as in healthy individuals. We also showed that anergy induction is restored in SLE patients treated with belimumab, an inhibitor of BAFF., Conclusion: This assay will enable studies of large populations to identify potential genetic or environmental factors affecting B cell tolerance checkpoints in healthy subjects and patients with autoimmune disease and permit monitoring of the B cell response to therapeutic interventions., (© 2016, American College of Rheumatology.)

A strong gender bias is seen in many autoimmune diseases including systemic lupus erythematosus (SLE). To investigate the basis for the female preponderance in SLE, we have been studying BALB/c mice in which B cells express the R4A heavy chain of an anti-DNA antibody in association with an endogenous light chain repertoire (R4Atg mice). In unmanipulated mice, approximately 5% of B cells express the R4A transgene. R4Atg mice do not spontaneously develop elevated serum titers of anti-DNA antibodies. Administration of either estradiol (E2) or prolactin (Pr) results in escape from tolerance of autoreactive B cells, expressed as an increase in transgene-expressing B cells and elevated serum titers of anti-DNA antibodies. We previously demonstrated that autoreactive B cells maturing in an estrogenic milieu develop as marginal zone (MZ) B cells; when these same B cells mature in the presence of increased prolactin, they develop as follicular (Fo) B cells. To determine the long term consequence of this differential maturation of DNA-reactive B cells, we treated R4Atg BALB/c mice with E2 or Pr for 6 weeks until serum titers of anti-DNA antibody were high, at which time hormonal exposure was discontinued. In E2-treated mice, the anti-DNA titers remained high even 3 months after discontinuation of hormone exposure. Nascent B cells underwent normal tolerance induction, but existing autoreactive MZ B cells persisted and continued to secrete autoantibody. In contrast, Pr caused only a short-term increase in anti-DNA antibody titers. By 3 months after cessation of hormone treatment, serum anti-DNA antibody titers and B cell subsets were indistinguishable from those in placebo (P) treated mice. These findings suggest that autoantibody responses are sustained for variable lengths of time depending on the B cell subset producing the autoantibodies. This observation may be relevant to understanding the heterogeneous presentation of patients with SLE and to the design of therapies targeting specific B-cell populations in autoimmune disease., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

It is clear that estrogen can accelerate and exacerbate disease in some lupus-prone mouse strains. It also appears that estrogen can contribute to disease onset or flare in a subset of patients with lupus. We have previously shown estrogen alters B-cell development to decrease lymphopoiesis and increase the frequency of marginal zone B cells. Furthermore, estrogen diminishes B-cell receptor signaling and allows for the increased survival of high-affinity DNA-reactive B cells. Here, we analyze the contribution of estrogen receptor α or β engagement to the altered B-cell maturation and selection mediated by increased exposure to estrogen. We demonstrate that engagement of either estrogen receptor α or β can alter B-cell maturation, but only engagement of estrogen receptor α is a trigger for autoimmunity. Thus, maturation and selection are regulated differentially by estrogen. These observations have therapeutic implications.

The prevalence of systemic lupus erythematosus (SLE) is far higher in females than in males and numerous investigations to understand this gender bias have been performed, which propose as casual actors genetic predispositions and sex hormones effects. We will describe in this review how the sex hormones estrogen and prolactin influence B cell maturation and selection, permitting B cells to mature to immunocompetence in a mouse model of lupus. Finally, we will discuss the relevance and implications of these results for human disease.

The prevalence of systemic lupus erythematosus (SLE) is far higher in females than in males and numerous investigations to understand this gender bias have been conducted. While it is plausible that some sex-linked genes may contribute to the genetic predisposition for the disease, other likely culprits are the sex hormones estrogen and prolactin. In this chapter we review studies that have addressed the influence of sex hormones in SLE activity and discuss the recent data established in a BALB/c mouse transgenic for the heavy chain of an anti-DNA antibody. These mice are prone to develop lupus following exposure to exogenous sex hormones. We describe how estrogen and prolactin influence B cell maturation and selection, permitting B cells to mature to immunocompetence. Finally, we discuss the relevance and implications of these data for human disease.