HD preculture of PBMCs affects TCR signaling status and metabolism of CD8 T cells. (A) Phosphorylation of proximal TCR signaling components were compared between isolated CD8 T cells from fresh and precultured PBMCs by western blotting. Membranes were probed with antibodies against CD3ζ p-Y142, CD3ζ, Src p-Y394, and Lck. ERK1/2 served as a loading control. (B) Differentially expressed probe sets assigned to the metabolic pathways, glycolysis, fructolysis, and pentose phosphate pathway from a comparison of CD8⁺CD45R0⁺ T cells from fresh and HD-precultured PBMCs are shown in a heatmap of scaled expression values. (C) Differentially expressed glycolysis pathway genes of the same experiment are shown in a KEGG pathway map with color-coded log-fold changes. (D) High concordance of changes in glycolysis-related gene expression changes between CD8⁺CD45R0⁺ T cells from fresh PBMCs (presented in B-C) and pan T cells from LD precultured PBMCs as shown by Venn diagram display. F, fresh; HD, high density; LD, low density; P, precultured.