Extended Data Fig. 5: 6h U-13C-glucose re-exposure following TGR.
From: Metabolic conditioning of CD8+ effector T cells for adoptive cell therapy
WT CD8+ T cells isolated from spleens of C57BL/6 mice were activated with anti-CD3 (5 μg/mL), anti-CD28 (0.5 μg/mL), IL-2 (100 U/mL), expanded for a total of 72 h, and exposed to 10 mM (black) or 1 mM (orange) glucose. Cells were re-exposed to 10 mM U-13C-glucose for 6 h prior to polar metabolite extraction and analysis by LC-MS. 20 h 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, Percent 13C label incorporation into nucleotide monophosphates was analysed. Data are from n = 3 biological replicates. Significance was calculated using 2-tailed Student’s t tests. * p < 0.05. b, Relative 13C label incorporation into serine was analysed. Data are from n = 3 biological replicates. Significance was calculated using 2way ANOVA. Significance is indicated for the 13C portion (open bars). *** p < 0.001. The 12C portion (filled bars) was also significant. ** p < 0.01. c, The GSH/GSSG ratio was calculated from summed isotopologues for each metabolite. Data are from n = 3 biological replicates. Significance was calculated using 2-tailed Student’s t tests, no significant difference was found. d, Relative 13C label incorporation into lactate was analysed. Data are from n = 3 biological replicates. Significance was calculated using 2way ANOVA. Significance is indicated for the 13C portion (open bars), which was not significant. The 12C portion (filled bars) was significant. *** p < 0.001. All error bars show SEM.
