Program Director/Principal Investigator (Last, First, Middle): PD: Pascual, V. / PI: Project 1 Pascual, V. Project Summary Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by widespread inflammation and development of autoantibodies against nuclear antigens. SLE is clinically heterogeneous and molecularly diverse. This heterogeneity might contribute to the high occurrence of clinical trial failures, underscoring the need for biomarkers to stratify patients according to individual pathogenic drivers of disease. In an attempt to understand the complexity of SLE, we established a pediatric cohort and have followed it for the past decade using validated clinical disease activity (DA) measures as well as blood gene expression profiles during flares and remissions. Our studies confirm the prevalence of IFN, neutrophil/myeloid and plasmablast gene signatures and their correlation with DA at the cohort level. Personalized immunomonitoring revealed, however, significant heterogeneity in how these major signatures correlate with DA at the individual patient level. We hypothesize that decoding the cellular and/or molecular components of these signatures in well- defined groups of patients will enable development of biomarkers and computational tools for stratification, which will enable rational clinical trial design. Towards this goal, we are proposing two aims: 1) to establish the origin and composition of three major SLE blood signatures at the single cell level. We will examine the cells that give rise to these signatures using high definition immunophenotyping and transcriptional profiling at the population and single cell levels; 2) to determine if molecular DA markers correlate with altered cytosolic and/or endosomal nucleic acid (NA) sensing pathways in ex vivo patient blood cells and in vitro assays. Here, we first propose to apply a sensitive and robust assay to quantify the endogenous activity of cGAS, a universal cytosolic DNA sensor, in PBMCs from patients during flares and remissions. Second, we will test the response of patient cells to relevant endosomal and cytosolic nucleic acid ligands in vitro using multi-dimensional readouts. Through the implementation of our aims, we will i) reveal the source of SLE molecular signatures; ii) understand the extent of heterogeneity of blood SLE myeloid cells and plasma cells; iii) determine which cell subsets/molecular pathways and/or NA sensors contribute to immune activation leading to SLE flares. Understanding SLE heterogeneity and developing tools to assess it in the clinical setting will ultimately open new paths towards personalized therapeutic approaches.