Francisella-primed lymphocytes shift toward glycolysis during control of intramacrophage bacterial growth Lara Mittereder-1, Karen L. Elkins-1 1-Laboratory of Mucosal Pathogens and Cellular Immunology, DBPAP, CBER, FDA Francisella tularensis is a pathogenic intracellular bacterium that causes tularemia via inhalation and has been classified as an agent of bioterrorism. F. tularensis Live Vaccine Strain (LVS) provides partial protection in animals and humans but is not licensed. To study vaccine mechanisms, we have previously established an in vitro co-culture assay that measures control of intramacrophage bacterial replication by immune T cells as a tool to identify potential correlates of protection against Francisella and to further understand protective T cell responses. Previous work suggested that virulent Francisella infection of mouse macrophages inhibits the metabolic shift from oxidative phosphorylation to glycolysis that is required for appropriate activation. To study the role of metabolic shifts in protective T cell responses, we adapted co-cultures to profile metabolic activities of co-cultures containing both LVS-infected macrophages and splenic or peripheral blood lymphocytes from naïve or LVS-primed mice. Further, we studied activities of separated cell populations. Consistent with previous results, co-cultures containing LVS-primed lymphocytes controlled bacterial replication in macrophages and produced high levels of IFN-gamma and nitric oxide. Compared to co-cultures containing naïve lymphocytes, co-cultures with LVS-primed cells gradually shifted metabolically toward glycolysis and exhibited less aerobic respiration. Similar metabolic differences were observed in cell populations separated after co-culture, suggesting that interactions between infected macrophages and LVS-primed lymphocytes induced metabolic shifts in both populations. Further, LVS-primed lymphocytes exhibited a significant loss of respiratory capacity over time in co-culture, indictive of transition to a T effector state. Taken together, we find that LVS-primed lymphocytes override metabolic dysregulation and are better able to achieve effector status to control bacterial infection compared to naïve lymphocytes. These results support future study exploring the use of metabolic intermediates as potential correlates of protection.