Project Summary Generating antibodies, refining their qualities, and creating durable humoral memory are crucial parts of adaptive immunity. The capacity of vaccines to protect against microbes draws on each facet of these processes, but even for some approved and useful vaccines the efficacy is needs to be better. Accordingly, it is vital to decipher key cellular and molecular processes that affect antibody (Ab) qualities. Major efforts are directed toward the identification of ways in which intracellular sensors, mediators of intermediary metabolism, and metabolites proper alter immune cell differentiation or function. It has long been known that malnutrition undermines immune defenses against infection, and a body of work has suggested that protein deficiency may decrease effective Ab responses. Nutrient supply is intrinsically linked to intracellular sensors such as mTORC1 and programming of cellular metabolism in immune cells. For instance, experimental models of isolated protein deficiency have documented decreases in venous concentrations of amino acids (a.a.) and lower mTORC1 activity in freshly isolated organs from the malnourished rodents. Our work on mTORC1 in B cells found antibody responses to be altered by B cell-restricted haplo-insufficiency of Raptor, with partially reduced activity similar in magnitude to that reported in the setting of protein deprivation. Moreover, we – and others in parallel – uncovered evidence of variegated hypoxia in the white pulp and lymphoid follicles after immunization and formation of germinal centers. Preliminary in vitro and in vivo experiments provide evidence that (a) glutamine, at the physiological concentration of non-inflamed interstitia, is limiting for fully efficient switching to IgG1 and for plasma cell differentiation, and (b) glutaminolysis (the conversion of glutamine to glutamate, and then α-ketoglutarate) can be limiting for these processes. These findings are the premise for the overarching model of this application: that nutrients may be present in follicles at concentrations where either increases or further decreases alter the nature of the antibody response as it draws on lymphocyte proliferation and function. Accordingly, in Aim 1 we will test the impact on Ab responses of reducing a.a. supply to or utilization by mature B cells. Aim 2 will identify consequences for metabolic and epigenetic programming of the B cells in which glutamine supply or glutaminolysis are restricted, alone or with reduced glucose oxidation capacity. An implication of the model is that increased circulating a.a. – or even just glutamine – could enhance outcomes of immunization. In Aim 3, we will use a newly identified endocrine approach to test if hyperaminoacidemia increases interstitial glutamine and yields greater Ab responses or humoral memory. As a novel facet of the experiments, we will leverage a state-of the-art development in imaging mass spectrometry (IMS) to assess glutamine and selected metabo...