Resting CLL T cells have an abnormal mitochondrial redox balance. (A) PBMCs from patients with CLL and HDs were thawed, fixed or not, and stained, after which CD4 T cells were directly analyzed by flow cytometry for expression of total intracellular GLUT-1 storage in fixed CD4 T cells and subsets. Representative histogram of 1 HD (black) and 1 patient with CLL (red), as well as an isotype control (gray), is shown. (B) Analysis of extracellular flux (extracellular acidification rate [ECAR], OCR, and SRC) was performed by fluorescence-activated cell sorting of T cells first (purity >99%) and immediate measurement of the sorted cells for ECAR and OCR; the SRC could be derived from the OCR afterward. (C) Resting CD4 T cells were measured for mitochondrial potential and ROS production. Representative histogram of 1 HD (black), and 1 CLL patient (red) is shown, as well as an isotype control (gray). (D) Mitochondrial mass was analyzed in total resting CD4 T cells and subsets. A representative histogram of 1 HD (black) and 1 patient with CLL (red), as well as an isotype control (gray), is shown. PCG1a (E) and SOD2 (F) were measured in resting total CD4 T cells and subsets. Representative histogram of 1 HD (black) and 1 patient with CLL (red), as well as an isotype control (gray), is shown. (G) Expression of NRF-2 and HO-1 was measured in resting CD4 T cells. Representative histogram of 1 HD (black) and 1 patient with CLL (red), as well as an isotype control (gray), is shown. Each point represents a different HD or CLL patient. Data are presented as mean ± standard error of the mean. *P < .05, **P < .005, ***P < .001, ****P < .0001. MFI, mean fluorescence intensity; ns, not significant; Te (effector), CD27⁻CD45RA⁺; Tm (memory), CD27⁺CD45RA⁻; Tn (naive), CD27⁺CD45RA⁺.