Extended Data Fig. 6: No effect of PKM2 deletion on fatty acid or glutamine oxidation, but accumulation of glycolytic intermediates and pentose phosphate pathway (PPP) metabolites.
a-d, Activated OT-I+ Thy1.1 + CD8 + T cells electroporated with guides targeting PKM2 (blue) or non-targeting controls (red), were co-cultured with HKP1-ova-GFP, sorted at day 6 post-initial stimulation, and underwent stress tests measuring oxygen consumption (a,c) and extracellular acidification (b,d) for fatty acid oxidation (a,b) or glutamine oxidation (c,d). e–l, Activated OT-I+Thy1.1 + CD8 + T cells were electroporated with guides targeting PKM2 (blue) or non-targeting control red), and either cultured continuously on anti-CD3ε/anti-CD28 coated plates or co-cultured with HKP1-ova-GFP tumor cells. Viable T cells were sorted, and 203 polar metabolites profiled by liquid chromatography/mass spectrometry (LC/MS). Normalized metabolite abundance is presented as fold of control abundance. Metabolites involved in glycolysis (e-h) and the PPP (i–l) are shown for T cells in the listed conditions. m-v, Activated OT-I+Thy1.1 + CD8 + T cells were electroporated with guides targeting PKM2 (blue) or non-targeting control (red), and co-cultured with HKP1-ova-GFP. Viable T cells were sorted on day 4 post-initial stimulation and labeled with 1,2 13C glucose for 2 h, and 204 polar metabolites profiled by LC/MS for abundance and labeling. Normalized metabolite abundance is presented as fold of average control abundance. (m) Metaboanalyst identification of impacted pathways. (n-v) Quantification for all 13C-labeled metabolites in glycolysis (n), the PPP (o), and the tricarboxylic acid (TCA) cycle (p), and different labeled isotopes of metabolites with significantly altered enrichment in PKM2 knockout T cells compared to control: glyceraldehyde-3-phosphate (q), 3-phosphoglyceric acid (r), phosphoenolpyruvic acid (s), ribose-5-phosphate (t), sedoheptulose 7-phosphate (u), and oxoglutaric acid (v). Numbers: Biological replicates: (a-b) n = 5–6 per guide; (c-d) n = 8-9 per guide; (e–l) n = 6 for NTC-2 24 h post, n = 4 for PKM2-8 24 h post, n = 4 for NTC-2 48 h co-culture, n = 4 for PKM2-8 48 h co-culture, n = 4 for NTC-2 96 h plate, n = 4 for PKM2-8 96 h plate, n = 3 for NTC-2 96 h co-culture, and n = 3 for PKM2-8 96 h co-culture, aggregate of two experiments; (m-v) n = 7 per group. Statistics: (e–l,n-v) Multiple unpaired two-tailed t-tests. Data in (a–l,n-v) are presented as mean ± standard deviation.
