ABSTRACT The immune system is comprised of a complex heterogeneity of cells with dynamic interconnectivity eliciting a diverse array of immunological responses. An individual cell progeny originated from a single clone is programmed to function and behave differently than progenitor or sister cells. Genomics of cells convey the genetic ?potential?, whereas transcriptomics and proteomics describe the phenotypic cellular characteristics. Metabolomics transmits real-time biological activity and energetics, thereby providing a more accurate depiction of the cellular response. Many analytical technologies measure only the average response and fail to quantitate cell autonomy from a highly heterogeneous population of cells. This average measurement undermines the functional individuality of each cell and skews the interpretation of the whole biological system. Our preliminary data have indicated that individual T helper 17 (Th17) cells produce different levels of IL-17, and Th17 cells isolated from non-autoimmune mice preferentially use the ?-oxidation metabolic pathway for energy production. However, very little is known regarding the metabolic pathway that each Th17 cell uses to initiate and sustain the pathogenicity in Sjgren's syndrome (SjS), an autoimmune disease primarily targeting the destruction of lacrimal and salivary glands. As a result, there is pertinent need to examine cellular processes at a single-cell resolution. More importantly, to obtain accurate measurement of functional processes of cellular metabolism, there is an essential need to develop a pragmatic method with high- throughput single-cell metabolomics. To accomplish this goal, we will pursue two major specific aims, Aim 1: Develop a single-cell metabolomics technology that can be applied to profile a single cell's metabolome by integrating single-cell microengraving technique with MALDI-MS, with the hypothesis: Single-cell metabolomics will have sufficient sensitivity to accurately measure metabolome of individual metabolically active cells, Aim 2: Apply single-cell metabolomics technology to compare the metabolome produced by individual Th17 cells in the salivary glands of normal versus SjSs mice, with the hypothesis: Cellular heterogeneity of autoimmune Th17 cells is driven by the changing magnitude of glycolysis versus ?-oxidation of metabolic pathway. Results are expected to generate a single-cell metabolomics tool that could be used to measure the metabolomes of individual cells, and provide a proof-of- concept in deciphering the cellular heterogeneity governed by the magnitude of separate metabolic pathways resulting in diverse autoimmune responses. On a more fundamental level, results should establish the proper direction needed to move this approach forward for elucidating the metabolic pathway of highly autoimmunogenic Th17 cells based on their unique metabolite profile or ?metabolic signature?.