PGA-1 induced hypoxic signature in T cells is due to pseudohypoxia and correlates with HIF-1α stabilization and expression of its target genes. (A) PBMCs were thawed and cultured with and without PGA-1 cells and stimulated with anti-CD3/28. 48 hours after stimulation, CD4⁺ T cells were sorted and lysed for RNA sequencing and purified using magnetic beads for intracellular metabolite analysis. Heat map showing the expression of HIF-1, HIF-2, and common targets. (B) HIF-1α assessment by flow cytometry 24 hours after stimulation, when the expression of CD25 in activated T cells is comparable between stimulation in the presence or absence of cell lines. (C) Gene expression of HIF1A, VEGFA, PDK1, and LDHA was evaluated by qPCR at 24 hours to confirm RNA sequencing data. In parallel, PBMCs were stimulated with anti-CD3/28 in hypoxia (HYP, PO₂ = 1%), as a control. (D) Fraction of pS6⁺ T cells was evaluated by flow cytometry. (E) Intracellular α-KG / SA (arbitrary units, assuming equal ionization efficiency of α-KG and SA, according to similarity in their structure) in CD4⁺ T cells stimulated in the presence or absence of PGA-1. (F) Intracellular NAD⁺ / NADH ratio represented in arbitrary units. (G) Regulation of HIF-1α stability by oxygen and pseudohypoxia. Data are presented as mean ± SEM. ∗∗∗P < .001; ∗P < .05; ns (1-way ANOVA followed by Tukey multiple comparison test). α-KG / SA, α-ketoglutarate–to–succinate ratio; ns, not significant; PHD, prolyl hydroxylase. Scheme was generated with BioRender.com.