PROJECT SUMMARY/ABSTRACT Myalgic Encephalomyelitis and Chronic Fatigue Syndrome (ME/CFS) is a debilitating and mysterious chronic illness caused by diverse environmental triggers. Severe disruptions in several immune system components have been described and proposed as drivers of disease pathogenesis and symptoms. However, there is no consensus on the immunological basis for ME/CFS development and sustenance. The two major barriers to progress are the significant patient heterogeneity in symptomatology and disease progression, combined with the lack of quantitative tools to stratify patients and probe the molecular immune underpinnings of disease. The ability to stratify heterogeneous patient groups using reliable, clinically accessible immunological biomarkers would transform efforts to manage ME/CFS clinically and investigate the disease mechanistically. Development of robust multi-parameter biomarker sets would also impact efforts to develop personalized treatment options for ME/CFS patients. The present proposal outlines a multi-disciplinary, systems-biology approach to investigate the immune mechanisms of ME/CFS and to develop ME/CFS-patient specific immune signatures from blood-derived immune cells. The guiding hypothesis is that immune perturbations, particularly to the effector functions of T cell and innate cell (natural killer and myeloid) subsets, contribute to pathogenesis of ME/CFS and that these immune signatures can be used as predictive biomarkers. We will address this hypothesis using a cutting-edge immunogenomics approach based on integrated, high-resolution functional and transcriptomic profiling of immune cell subsets within the blood samples of a large, clinically characterized ME/CFS patient cohort and healthy controls. We will then examine the transcriptional alterations associated with ME/CFS within T and innate cell subsets, with a focus on long non–coding RNAs, owing to their high cell- type-specificity and impact on immune cell development. Our Specific Aims are: 1) To determine the frequencies of immune cell subsets in the blood of a clinically defined ME/CFS patient cohort; 2) To assess functional capacity of memory T cells, innate cells and the differentiation potential of naive T cells during ME/CFS; and 3) To determine the T cell and innate cell subset–specific gene and lncRNA transcripts in ME/CFS patient blood samples. Our goal is to develop a detailed functional and genetic immunological framework that can be used to i) decode the mechanisms of ME/CFS and ii) to develop robust, quantitative immune-biomarker sets for predicting disease susceptibility, stratifying patients and guiding treatment strategies. We have assembled a unique team of experts in human immunology, clinical ME/CFS biology for well-defined patient samples, non-coding RNAs, transcriptomics and bioinformatics, together will contribute to the deep and complimentary expertise necessary to bring about this goal.