(595d) Ensemble Absolute Metabolite Quantitation in T Cells Reveals Conserved Features of Immunometabolism

Metabolism plays a key role in immune cell proliferation, activation, and persistence. Thus, quantitative understanding of immunometabolism underlies improving immune functions and developing successful immunotherapies. However, comprehensive quantitation of T-cell metabolic state remains elusive. Here we developed a simple and accessible technique for comprehensive absolute metabolite quantitation in T cells. Absolute, not relative, metabolite concentrations govern the kinetics and the thermodynamics of enzyme-catalyzed reactions, but absolute metabolite quantification has been inaccessible due to the need for laborious analytical procedures involving various internal standards. As a result, absolute metabolite quantification has only been done in select model systems E. coli, yeast, and mammalian epithelial cells. To overcome this shortcoming, we innovated an isotope-ratio-based approach that leverages the knowledge of absolute concentrations in the three model systems, ¹³C labeling, and liquid chromatography-mass spectrometry (LC-MS) to quantify T-cell metabolome en masse. Our approach involves simultaneous extraction of unlabeled T cells and ¹³C-labeled reference cells to distinguish metabolite origins and to quantify their ratios, which we used to infer the absolute metabolite concentrations in T cells. We quantified absolute metabolite concentrations in both Jurkat T cells and human primary T cells. For the latter, we isolated CD4+ and CD8+ T cells from peripheral blood mononuclear cells (PBMCs) and quantified their metabolism within a few hours of blood collection. Across these three T cell types (Jurkat, CD4+, and CD8+), we obtained ¹²C-to-¹³C enrichment ratios for individual metabolites using LC-MS and quantified absolute metabolite concentrations of ~80 metabolites. When compared across donors, most primary T cell metabolites, including central carbon metabolites and nucleotides, lay along the line of unity, indicating that this comprehensive dataset is suitable for a wide range of fundamental and applied research into immunometabolism and immunotherapy. Within the same donor, CD4+ and CD8+ T cell metabolite concentrations had near perfect correlation (r>0.9), which eliminates the need for CD4+ and CD8+ T cell isolation in CAR-T cell therapies. On the other hand, despite Jurkat cells being CD4+ T-lymphocyte-derived, their metabolome compared to that of primary CD4+ T cells displayed only a modest correlation (r=0.6). Absolute metabolite concentrations facilitate the integration of metabolomics with proteomics and fluxomics using kinetic and thermodynamic laws. Using absolute concentrations, we obtained overall Gibbs free energy changes across glycolysis in both CD4+ (ΔG = –48 kJ/mol) and CD8+ (ΔG = –46 kJ/mol) T cells. Comparison of metabolite concentrations to the Km and Ki constants of cognate enzymes revealed high active site occupancy but low inhibitor binding site occupancy in all three T-cell types. Collectively, our kinetic and thermodynamic analyses manifested efficient enzyme utilization with sufficient forward driving force. Thus, our comprehensive absolute metabolite quantitation offers fundamental systems-level insights into immunometabolism and a solid foundation for quantitative modeling of immunometabolism.