ROS and hypoxia pathways are upregulated in activated CD4 T cells in presence of CLL, whereas OXPHOS and glycolysis are upregulated in absence of CLL. PBMCs from patients with CLL and HDs were thawed, after which CD4 T cells from patients with CLL and HDs were cell sorted or not and subjected to CD3 plus CD28 stimulation for 2 days. GSEA against MSigDB Hallmark gene sets (h.all.v7.4) of 4 patients with CLL resulted in several pathways significantly upregulated in presence (A) and absence (B) of CLL cells. (C) Enrichment plots highlighting hypoxia, ROS, OXPHOS, and glycolysis pathways from analysis depicted in panels A and B. Genes are ranked by differential expression in PBMCs vs isolated cells on the x-axis; red indicates upregulation in PBMCs, and blue indicates upregulation in isolated cells. Curves (green) indicate cumulative enrichment quantified by enrichment score on the y-axis. Tick marks on the x-axis correspond to ranks of genes in the gene set. (D) Heat map showing differentially expressed genes in CLL-derived CD4 T cells stimulated with CD3 plus CD28 antibodies in presence or absence of CLL cells. Genes correspond to hypoxia, ROS, OXPHOS, and glycolysis pathways. Sorted or unsorted CD4 T cells from patients with CLL and HDs were stimulated with CD3 plus CD28 antibodies and analyzed for expression of CD25 and CD71 (E) and GLUT-1 expression and glucose uptake (2-NBDG) (F) using flow cytometry. (D-F) Each point represents a different HD or patient with CLL. Data are presented as mean ± standard error of the mean. ***P < .001, ****P < .0001. FDR, false-discovery rate; MFI, mean fluorescence intensity; ns, not significant.