Type I Interferons Promote Development of Flares in Systemic Lupus Erythematosus by Enhancing B-Cell Activation and Differentiation of Age-Associated B Cells

Objectives Systemic lupus erythematosus (SLE) is characterized by unpredictable flares interspersed with periods of disease quiescence. Elevated interferon (IFN) levels increase the likelihood of flares, but the precise immunologic mechanisms by which this occurs are unclear. In mice, IFN exposure expands age-associated B cells (ABCs), a population enriched for autoreactive and ANA-secreting cells. In this study, we examined the role of IFN in the activation and differentiation of B-cell subsets in flaring and quiescent SLE patients. Methods A CyTOF panel was developed to quantify IFN-induced proteins (IIPs) across peripheral blood immune populations. A composite IIP score (mean expression of 6 IIPs) was generated for each cell population as a surrogate for IFN exposure. 15 healthy controls (HCs), 26 quiescent (clinical SLEDAI > 0 for 1 year with no increase in immunosuppressive treatment, ≤ 10 mg prednisone) and 42 recently flaring (<1 month, change in clinical SLEDAI-2K ≥ 1 requiring escalation of therapy) were analyzed. To assess the direct effects of IFN, naïve B cells from HCs were isolated and stimulated with IFNα, IFNβ, or IFNγ. Cells were cultured under conditions that either promoted or inhibited ABC differentiation, including IL-21, anti-CD40, Fab2, CpG, or IL-4. Results CyTOF identified 7 B cell subsets, all of which exhibited higher IIP levels and greater activation in flaring vs quiescent patients (Figure 1A). ABCs were more abundant in flaring patients, and their frequency correlated with the global IIP signature (Figure 1B,C). Expression of activation markers (CD86, TLR7, TLR9, HLA-DR, Ki67) was strongly associated with IIP score, but not disease status, indicating that IFN, rather than flare alone, drives B cell activation (Figure 1D). Importantly, the association between activation and IFN exposure was evident even within individual patients, where the top 10% of IFN-experienced B cells had significantly higher activation than the bottom 10%. In vitro, IFNα and IFNβ directly induced expression of activation markers within 18-24 hours. In isolated naïve B cells, all 3 IFNs increased ABC differentiation, even without canonical ABC-inducing signals (Figure 1E). Notably, IFN overcame IL-4-mediated suppression of ABC differentiation, in part by reducing IL-4Rα and inducing TLR7 expression. In SLE patients treated with the IFN-blocking therapy Anifrolumab, ABC frequency and IIP signatures decreased (Figure 1F). Figure 1. A) IIP score for HCs, quiescent, and flaring SLE patients in B cell subsets. Higher IIP scores were found in flaring versus quiescent, and SLE patients versus HCs. (Mann Whitney U test with BH correction for multiple tests) B) Frequency of B cell subsets displayed as the proportion of CD19+ B cells. Flaring patients had more ABCs than quiescent patients and HCs. (Mann Whitney U test with BH correction for multiple tests) C) Correlation between cellular abundance and IFN signature. The proportion of ABCs were correlated with IIP score as well as IFN-stimulated genes (ISGs). IIP score also correlated with the proportion of PBs. (Spearman correlation) D) Correlation of IIP score and markers of activation. The expression of activation markers correlated with IIP score for SLE patients. (Spearman correlation) E) ABC differentiation from purified naïve B cells. 5 days of incubation with IFNα, IFNβ, or IFNγ caused significantly more differentiation of ABCs, irrespective of the incubation conditions (+/− IL-21, IL-4, anti-CD40, CpG). Results are displayed as the fold change from the respective non-IFN conditions. (Student’s T test with Holm correction for multiple comparisons post Shapiro-Wilk test to assess for normality) F) The ABC profile of patients treated with Anifrolumab versus standard-of-care. Both the IIP score and proportion of ABCs were significantly reduced in Anifrolumab-treated patients. (Mann Whitney U test) In Anifrolumab-treated patients, the frequency of ABCs showed a trend to correlation with the IIP score. (Spearman correlation) Conclusion IFN exposure drives human B-cell activation and promotes differentiation of ABCs. Our results identify a mechanistic link between IFN signaling and pathogenic B-cell development, and support IFN blockade as a strategy to reduce pathogenic ABCs and prevent SLE flares.