Ensemble quantitation of absolute metabolite concentrations in T cells reveals conserved features of immunometabolism

Cells regulate metabolite levels to efficiently utilize metabolic networks and avoid toxic buildup. In turn, metabolites dictate metabolic activity by acting as substrates, products, and effectors. Despite their foundational role in cell physiology, kinetics, and thermodynamics, absolute metabolite concentrations are seldom known. Here we develop an ensemble method for absolute metabolite quantitation and quantify 84 metabolites in T cells. Liquid chromatography-mass spectrometry of metabolites co-extracted from T cells and ¹³ C-labeled reference cells reveals absolute concentrations en masse . Across subtypes and individuals, T cell metabolomes resemble one another. T cells possess high adenylate energy charge and favorable redox ratios for energy and biomass production without compromising the forward driving force in glycolysis. Across metabolism, metabolite concentrations exceed their associated Michaelis constants and inhibitor constants two thirds and half of the time, respectively. The conserved features of T cell metabolomes underlie a design principle: metabolite levels prime cells for adaptive immune response.