PGA-1 cells induce a hypoxic gene signature and profound metabolic alterations in T cells, skewing the fate of pyruvate into aerobic glycolysis. PBMCs were cultured with and without PGA-1 cells and stimulated with αCD3/CD28. After stimulation, CD4⁺ T cells were sorted and lysed for RNA sequencing (48 hours after stimulation), metabolomics (48 hours after stimulation), and extracellular flux analysis (24 hours after stimulation). (A-B) Enrichment plot showing hypoxic gene signature and downregulation of OXPHOS in CD4⁺ T cells exposed to PGA-1. On the x-axis, red indicates the presence of PGA-1 cells, whereas blue represents PBMC stimulated alone. Curves (green) indicate cumulative enrichment quantified by enrichment score on the y-axis. Tick marks on the x-axis correspond to the rank of genes in the gene set. (C) Heat map representing the abundance of metabolites in CD4⁺ T cells unstimulated and stimulated in presence or absence of PGA-1. Metabolomics data were analyzed with Tercen software and scaled and hierarchical clustered by rows and columns, and significantly different metabolites in T cells stimulated in the presence of PGA-1 compared with the stimulated control (paired t test) were plotted. Metabolites abundance in unstimulated cells were plotted as a comparison. Two main clusters were identified: metabolites increasing after T-cell stimulation (gray box) and metabolites increasing when T cells are stimulated in the presence of PGA-1 cells (purple box). (D) Extracellular flux analysis showing extracellular acidification rate and oxygen consumption rate of sorted unstimulated CD4⁺ T cells, stimulated for 24 hours in the presence or absence of PGA-1 and stimulated in hypoxia (HYP; 1% O₂) as a positive control. FDR, false discovery rate; NES, normalized enrichment score; UNSTIM, unstimulated.