(A) Schematic of the experimental model used to study the effect of TB-PE on CD8⁺ T cell functionality. (B) Heatmap showing hierarchical clustering of the top 100 variable genes between TB-PE–treated and control CD8⁺ T cells stimulated with anti-CD3/CD28 antibodies. (C) Volcano plot of differentially expressed genes (DEGs). (D) Gene set enrichment analysis (GSEA) of DEGs (P < 0.05 and absolute log fold change > 1) comparing TB-PE–treated and control activated CD8⁺ T cells. Dot plots illustrate the top 10 upregulated and downmodulated enriched Gene Ontology biological process terms. (E) Heatmap of normalized expression of selected genes involved in T cell activation. (F) Gene expression levels of GZMB, IFNG, TNF, IL21, and CD69 across conditions. (G) CD8⁺ T cells were stimulated with anti-CD3/CD28 antibodies in the presence of TB-PE; cells activated in the absence of PE served as controls. Expression of the activation markers CD69, CD25, and HLA-DR was measured by flow cytometry after 24 hours (CD69, CD25) or 48 hours (HLA-DR). Representative flow plots (left) and quantification of activated cells (right) are shown. (H) Proportions of CD69⁺ (left) or CD25⁺ (right) cells stimulated in the presence of an additional TB-PE pool, 3 independent TB-PE samples (red), or HF-PE (gray). Values are relative to the non-PE control condition (blue). Data are shown as mean ± SD. Each dot represents an independent donor. Statistical significance was determined by paired 2-tailed t test or 1-way ANOVA followed by Tukey’s honestly significant difference (HSD) post-test. *P ≤ 0.05, **P ≤ 0.01. (I) HIF-1α expression in activated CD8⁺ T cells determined by flow cytometry. gMFI, geometric mean fluorescence intensity. Each dot represents an independent donor. Statistical significance was determined by paired 2-tailed t test or 1-way ANOVA followed by Tukey’s HSD post-test. *P ≤ 0.05, **P ≤ 0.01.