PROJECT SUMMARY Systemic lupus erythematosus (SLE) is a multi-organ rheumatologic disease characterized by immune dysregulation and a heterogeneous disease course, with heightened disease severity in pediatric patients. Conventional clinical and laboratory parameters are not sufficiently sensitive or specific for detecting ongoing disease activity or response to specific immunomodulators, underscoring a need to better delineate the underlying immune changes that drive unpredictable flare-ups of disease activity. The lack of knowledge regarding the precise immune cellular and molecular events leading to SLE disease poses a significant hurdle in the effort to develop accurate prognostic disease biomarkers and selective therapeutic agents. Our long- term goal is to identify immune cellular and molecular mediators that could provide targets for therapeutic intervention. The objective of this proposal is to elucidate the mechanisms by which serum-circulating factors including exosomes modulate immune cellular and cytokine derangements in pediatric SLE pathogenesis. The rationale for the proposed research is that understanding these mechanisms could identify novel disease biomarkers that permit accurate prognosis and provide selective therapeutic targets. To achieve this goal, Dr. Hsieh will use a high-dimensional mass cytometry platform, which offers single-cell analysis of over 40 parameters, utilizing rare earth metal isotopes instead of fluorophores as tags bound to antibodies. In a pilot study, Dr. Hsieh established a quantitative and reproducible mass cytometry assay that allows precise monitoring of phenotypic (surface markers) and functional (cytokines/chemokines) immune derangements detectable in peripheral blood of pediatric SLE patients. The data from this study form the basis of Dr. Hsieh's central hypothesis that pediatric SLE patients share a unique multi-parametric monocyte cytokine signature, which drives disease activity and is propagated by serum factors (including exosomes) through activation of the JAK/STAT signaling pathway. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Determine the association of the cytokine signature (and other immune changes) with SLE disease activity, by integrating mass cytometry immune profile data and clinical parameters from a prospective longitudinal pediatric SLE patient cohort; 2) Elucidate immunomodulatory properties of SLE serum-isolated exosomes, by evaluating the ability of diseased serum exosomes to induce the cytokine signature, and identifying exosome proteomic components essential for such activity; and 3) Determine the capacity of ruxolitinib (JAK1/2 inhibitor) to abrogate expression of the cytokine signature, by evaluating ex vivo immunosuppressive effects (including off-target effects) of ruxolitinib on immune derangements observed in pediatric SLE pathogenesis. Dr. Hsieh is an Assistant Professor at the University of Colorado Denver/Ch...