PROJECT SUMMARY ME/CFS (Myalgic Encephalomyelitis/Chronic Fatigue Syndrome) remains a prevalent, serious and poorly understood disease, impacting millions in the USA alone with problems in concentration, memory and sleep together with musculoskeletal pain. Thus, there is an urgent need to advance our understanding of the basic science of this disease – as an integral step towards an ultimate cure. Despite our lack of understanding of ME/CFS, substantial evidence implicates immune dysregulation either as an underlying cause or major consequence of the disease. Our work has sought to systematically examine gene regulation across the immune system, with the goal of determining which components are dysregulated in ME/CFS. These studies have identified classical monocytes, platelets and certain subsets of T cells as the components of the immune system most severely impacted by ME/CFS. These results establish that immune dysregulation in ME/CFS involves disparate components of the immune system. Project 3 of our ME/CFS Center has three major goals. First, to use multiomic approaches to rigorously determine the gene regulatory changes in monocytes in ME/CFS. To complement these approaches, we will perform monocyte assays to test whether ME/CFS results in alterations in the ability of monocytes to migrate and differentiate into macrophages, a critical function of monocytes. Second, we will examine platelet dysregulation in ME/CFS, as our data identifies platelets as amongst the most dysregulated component of the immune system in ME/CFS, with dysregulation particularly evident in patients undergoing post-exertional malaise. We hypothesize that platelet defects could contribute to circulatory and other symptoms in ME/CFS. In this Aim, we will examine the platelet transcriptome, perform assays to test platelet function, and examine interactions between platelets and other immune cells in ME/CFS. Third, we will systematically identify ME/CFS specific alterations in signaling across the immune system. This goal is important, as identification of molecular changes that impact the immune system broadly are attractive targets for future development of therapies. Achieving these goals has the potential to improve diagnosis, including generating biomarkers, and will identify specific dysregulated pathways in ME/CFS, providing a foundation for future development of therapeutics. For example, our first two goals have the potential to reveal consequential alterations in either (or both) monocytes and platelets in ME/CFS, thereby establishing a rational path towards eventual treatments or a cure. Because the patient and control cohorts examined are common across this Center application, we will generate synergistic data from the constituent projects. In particular, our genomic and functional assays examining monocytes will be paired with profiling of macrophages in muscle (Project I), which are derived from monocytes in circulation. Similarly, Project II will examine alterations to the platelet-derived extracellular vesicle proteome in ME/CFS, complementing our analysis of platelets. Our comprehensive data will be a key resource for all researchers in the field.