Project Summary/Abstract T cells become activated when they encounter an antigen presenting cell and receive three signals: TCR signaling via peptide-MHC, co-stimulatory signaling and cytokines. Activation drives a host of metabolically demanding processes such as proliferation, differentiation, migration, and effector functions. Thus, T cells switch from oxidative phosphorylation (OXPHOS), when naïve, to aerobic glycolysis upon activation to generate enough ATP to accommodate these processes. We recently showed that T cells engage aerobic glycolysis within minutes of TCR signaling, independent of co-stimulatory signaling. We found that this mechanism was mediated by pyruvate dehydrogenase kinase 1 (PDHK1), a mitochondrial enzyme that associated with LCK and migrated to the T cell synapse upon activation. These data suggested that aerobic glycolysis could be spatially regulated at the T cell synapse during activation. Using pH sensitive fluorescent systems, we were able to generate data visualizing aerobic glycolysis restricted to the T cell synapse. These data suggest that the mitochondrial positioning in T cells could regulate the initiation of aerobic glycolysis at the immunological synapse. Further, our lab and others have shown that glycolytic enzymes, GAPDH and LDH, are mRNA binding proteins and repress their cytokine translation in naïve T cells, and activation of glycolysis via TCR stimulus promotes the dissociation of glycolytic enzymes from cytokines mRNA. Therefore, we hypothesize that mitochondrial migration to the IS, regulated by mitochondrial LCK, enables localized aerobic glycolysis and subsequent synapse restricted cytokine translation for directed effector functions. To address this hypothesis we will, (1) Identify the role of mitochondrial LCK in driving mitochondrial migration to the T cell synapse and the initiation of aerobic glycolis. Using pH sensitive fluorescence systems, and mitochondrial and LCK reporters, we will visualize the dynamics of aerobic glycolysis, mitochondria and LCK at the T cell synapse when TCR is stimulated by an APC. Moreover, we will (2) Investigate the role of site restricted aerobic glycolysis in promoting localized cytokine translation at the T cell synapse. Using modulators of glycolytic metabolism, we will determine whether aerobic glycolysis could enhance the production of effector cytokines in activated T cells, and whether aerobic glycolysis predicts sites where cytokines are being translated. By better understanding the early signals that promote metabolic reprogramming and drive effector functions we can develop therapeutic targets that will allow us to metabolically modulate T cell activity.