Interrogating in vivo T-cell metabolism in mice using stable isotope labeling metabolomics and rapid cell sorting.

T cells are integral players in the adaptive immune system that readily adapt their metabolism to meet their energetic and biosynthetic needs. A major hurdle to understand physiologic T-cell metabolism has been the differences between in vitro cell culture conditions and the complex in vivo milieu. To address this, we have developed a protocol that merges traditional immunology infection models with whole-body metabolite infusion and mass-spectrometry-based metabolomic profiling to assess T-cell metabolism in vivo. In this protocol, pathogen-infected mice are infused via the tail vein with an isotopically labeled metabolite (2-6 h), followed by rapid magnetic bead isolation to purify T-cell populations (<1 h) and then stable isotope labeling analysis conducted by mass spectrometry (~1-2 d). This procedure enables researchers to evaluate metabolic substrate utilization into central carbon metabolic pathways (i.e., glycolysis and the tricarboxylic acid cycle) by specific T-cell subpopulations in the context of physiological immune responses in vivo.