Impaired glycolytic switch in CLL-derived CD4 T cells. PBMCs from patients with CLL and HDs were thawed, and T cells were stimulated for 2 or 5 days using either CD3, or CD3 plus CD28 antibodies. Subsequently, CD4 T cells were analyzed by flow cytometry for CD25, CD71, and 4-1BB expression after 2 days (A). Proliferation (B) and PD-1 expression (C) of CD4 T cells were analyzed after 5 days of CD3 plus CD28 stimulation using CellTrace Violet. (D) Glucose transporter GLUT-1 was measured on resting and activated T cells derived from patients with CLL and HDs after 2 days of CD4 T-cell activation. GLUT-1 expression was measured on activated (CD25⁺) T cells (right). (E) Glucose uptake (2-NBDG) was measured in activated CD4 T cells after stimulation using CD3 or CD3 plus CD28 antibodies for 2 days. Mitochondrial membrane potential (MitoTracker Orange) (F) and mitochondrial reactive oxygen species (ROS; MitoSOX) (G) was measured in T cells activated for 2 days with CD3 plus CD28 antibodies. (H) Mitochondrial biogenesis (calculated as ratio of MitoTracker Green mean fluorescence intensity (MFI) in T cells 2 days after activation over unstimulated T cells). Each dot represents a different HD or patient with CLL. Data are presented as mean ± standard error of the mean. *P < .05, **P < .005, ***P < .001, ****P < .0001. ns, not significant.