Project Summary/Abstract A considerable body of evidence implicates type I interferons (IFNs) in systemic lupus erythematosus (SLE); however, the “interferon signature” does not address the source and the key pathogenic type I IFNs in SLE, making it challenging to design effective precision medicine for SLE. We propose the novel hypothesis that it is the endogenous production of IFNβ by B cells that plays a defining role in shaping the autoantibody response and disease activity in a substantial subset of patients with SLE. We have found, in humans and mice, that the transitional (Tr) stage of B-cell development is characterized by heterogeneity in IFNβ expression at the single- cell level. Our preliminary data further suggest that: (i) Tr B cells with high endogenous production of IFNβ undergo strong IFNβ autocrine signaling and changes in the transcriptome signature in a manner that supports RNA/RNP-associated TLR7 responses. This imprinted signature promotes subsequent survival and development of RNP-reactive B cells including their differentiation into long-lived memory B cells; and (ii) Anti- dsDNA autoAb producing cells are derived from Tr cells that do not express high levels of endogenous IFNβ expression; however, high levels of endogenous IFNβ in memory B cells play a critical role in the differentiation of short-lived anti-dsDNA memory B cells. The model is supported by compelling preliminary data including analyses of PBMCs from SLE patients that indicated significantly higher IFNβ expression in B cells from SLE patients with renal involvement and significantly higher expression of intracellular IFNβ in B cells from female African-American (AA) SLE patients as compared with non-AA patients and healthy controls. In Aim 1, we will establish if lupus flare is associated with increases in IFNβhi memory B cells and if this phenotype is more common in AA SLE patients. High-throughput single-cell transcriptome analysis coupled with state-of-the-art computational analysis will be used to identify transcriptional signatures in DNA- and RNA-reactive B cells to refine this marker; to establish if, as predicted, there is commonality between the RNP-reactive Tr and memory B cell transcriptome; and to identify the association of enhanced IFNβ signaling with stage-specific dysregulation of downstream pathways. Functional and target-specific analysis of sorted human PBMCs will then be used to determine if endogenous B-cell IFNβ and its downstream pathways are essential for RNA- vs DNA-ligand mediated Tr and memory B-cell activation and survival. In Aim 2, we will use the same approaches in the BXD2 lupus model and BCR transgenic mice to determine if endogenous IFNβ is essential for, and differentially regulates, anti-RNP and anti-DNA B cell development in vivo. This hypothetical model does not negate the contributions of other cells and factors to the pathogenesis of SLE but reframes the current paradigm. The data generated will provide novel insights ...