Decreased glycolytic compensation in human T cells lacking AMPK. A total of 6 × 106 day 10 human T cells were transplanted with 1 × 106 autologous non–T cells into irradiated NSG recipients. Human CD3+ T cells were recovered from spleens of NSG recipients on day 25 after transplant and purified via magnetic selection. (A) Genomic DNA was purified from day 25 T cells followed by PCR amplification, Sanger sequencing, and decomposition analysis. (B-E) T cells recovered on day 25 were placed into the Seahorse metabolic analyzer, where the OCR (B) was measured simultaneously with ECAR (C). ECAR response to oligomycin (D) and maximal ECAR values (E) were calculated as in Figure 1. (F-H) Similar to panels A-E, 6 × 106 day-10 human T cells were transplanted with 1 × 106 autologous non–T cells into irradiated NSG recipients, recovered on day 28, and CD4 vs CD8 percentages quantitated by flow cytometry (F). These values were then used to calculate the posttransplant CD4/CD8 ratio (G). Pretransplant (post-CRISPR) CD4 and CD8 percentages are shown for comparison (H). Plots in panels B-E represent data from 2 pooled samples per group (3-4 mice in each pool; 6-8 mice total). Eleven mice per group in panels F-G. Each experiment was performed at least twice. Data in panel G represent the mean ± standard error of the mean. ∗P < .05; ∗∗∗P < .001.
