Figure 4. Limited access to Glc and O₂ forces CD8⁺ TILs to enhance FA catabolism.

(A–G) Vaccine model. (A) Glc concentrations in plasma, early and late stage B16Braf_V600E tumors (n=3). (B) Relative transcript levels: Upper: CD8⁺ T cells in Gal- or Glc+2-DG- vs. Glc-medium under hypoxia. Middle: specific CD8⁺ TILs from day 30 vs. 14 tumors. Lower: specific CD8⁺ T harvested from day 90 vs. 14 Spl (n=3). Color code below 3rd and 4th row: comparisons between in vitro and in vivo samples. (C) Experimental set-up: ¹³C₆-Glc in vivo tracing. (D) Intensity of glycolysis metabolites in CD44⁺CD8⁺ TILs. (E) Experimental set-up: ¹³C₁₆-palmitate in vivo tracing. (F) Intensity of FA metabolites in CD44⁺CD8⁺ TILs. (G) Relative contribution of ¹³C₆-Glc- and ¹³C₁₆- palmitate-derived carbons to citrate and malate, calculated by dividing labeling carbon numbers from ¹³C₁₆-palmitate by the numbers from ¹³C₆-Glc. Data of CD44⁺CD8⁺ T cells from day 30 tumors are normalized to those from day 14 tumors (left) or day 30 Spl (middle); Spl data: day 30 normalized to 14 (right). (D, F, G) n=2–3, pooled from ~30 mice/sample. (H–I) OT-1 transfer model. (H) Normalized contribution of ¹³C₁₆-palmitate to TCA cycle metabolites and L-palmitoylcarnitine in CD44⁺CD8⁺ TILs from day 30 vs. 14 tumors or day 30 Spl. (I) Intensities of FA metabolites. (H–I) n=2, pooled from ~20 mice/sample. (D, F, G–I) Data show as mean values. (J–L) Bar graphs: Relative intensity of free FA species in tumor interstitial fluid, dashed lines show ratio of 1. Pie charts: Abundance of different FA species. Total numbers: combined FA intensity. (J–K) day 30 over 14 B16Braf_V600E tumors (J, n=3) or PDX melanomas (K, n = 5). (L) Human melanoma metastases vs. human sera (n = 4). (D, F, G–L) Representative of two assays. (A, J–L) mean - SEM. See also Figures S4, S5.