Time-course analysis of proliferation, activation, and upregulation of checkpoint inhibitors of T cells in parallel to RNA sequencing reveals their delayed responses associated with perturbed metabolism. (A) Representative graphs showing the percentage of CD4⁺ or CD8⁺ T cells with upregulated CD25 surface expression after stimulation with CD3/CD28 beads and percentage of proliferation by measuring the dilution of VPD450 in CD4⁺ and CD8⁺ T cells from patients and 3 HCs at basal state (unstimulated), and on days 1, 2, 3, 4, 7, and 8 (and day 10 for PD1 and GITR) after stimulation. Marker up/downregulation was measured via flow cytometry at basal state (before stimulation), and on days 1, 2, 3, 4, 7, and 8 (and day 10 for PD1 and GITR) after stimulation. (B) Summary graphs showing the percentage of CD8⁺ T cells that have upregulated PD1, LAG3, AITR/GITR surface expression after stimulation with CD3/CD28 beads measured via flow cytometry at basal state (unstimulated), and on days 1, 2, 3, 4, 7, and 8 (and day 10 for PD1 and GITR) after stimulation. (C) PBMCs from patients and 4 HCs were stimulated with CD3/CD28 beads, and on the third day after stimulation CD8⁺ T cells were sorted via flow cytometry. Bulk RNA sequencing was performed on sorted CD8⁺ T cells. Plots show the gene set enrichment analysis of 50 hallmark functions based on calculated NES. Pathways are stratified for the top 10 most significantly upregulated pathways upon stimulation comparing patients and the HCs. (D) Plots showing the calculation of ranking of NES within gene set enrichment analysis from hallmark databases representing the stimulation-induced changes in expression of CD8⁺ T cells in patients and HCs with regard to OXPHOS (right) and glycolysis gene sets (left). (E) Heatmap showing the expression of genes involved in glucose metabolism extracted from bulk RNA-sequencing analysis. Significance for gene expressions is indicated in panel E.