Extended Data Fig. 4. TGR T_E have altered glucose reallocation upon re-exposure to drive anabolic metabolism.

WT CD8⁺ T cells isolated from spleens of C7Bl/6 mice were activated with anti-CD3 (5 mg/mL), anti-CD28 (0.5 mg/mL), IL-2 (100 U/mL), expanded for a total of 72 hours, and exposed to 10mM (control) or 1mM (TGR) glucose during the final 5 minutes cells were re-exposed to normal (U-¹²C) glucose or heavy labeled (U-¹³C) glucose. 20h cultures were started at 1×10⁶ (control) and 1.5×10⁶ (TGR) cells per/ml to generate similar end concentrations for glucose pulse experiments. (a) Table showing the results of a Kegg-pathway analysis of the significantly increased and decreased metabolite pools in TGR T_E after normal (U-¹²C) glucose re-feeding when compared to re-fed control T_E. (b) Table shows the results of a Kegg pathway analysis of the metabolites that exhibited increased glucose-derived U-¹³C assimilation (as determined by X13CMS software) after re-feeding TGR T_E with heavy labeled (U-¹³C) glucose compared to re-fed control T_E. Data are from 3 biological replicates, representative of 3 independent experiments. Kegg Pathways were ranked according to the number of metabolite hits in that pathway (from highest to lowest). Metabolites in the highest ranked pathways were further validated by targeted analysis. (c) Polar metabolites were extracted and untargeted metabolomic analysis was performed using XCMS on cells re-exposed to normal (U-¹²C) glucose. Volcano plot depicts relative metabolite pools compared between control and TGR cells. Orange circle represents a pentose phosphate pathway metabolite Pentose-phosphate, significantly increased in glucose re-fed TGR T_E. Statistical significance was calculated using a Welch’s t-test, comparing relative intensities of each isotopologue in labeled samples of control T_E versus those of TGR T_E.