Accumulation of defective depolarized mitochondria in T cells from patients with CLL and Eμ-TCL1 model. Analysis of patient samples (A-G). (A) Mitochondrial mass (MTG) and mitochondrial membrane potential (MTO) in CD8+ T cells from HDs and patients with CLL. MTO:MTG (potential-to-mass) ratio normalized to HD mean values was determined. (B) Fold increase in MTG and MTO levels 48 hours after stimulation using anti-CD3/anti-CD28 soluble antibodies, relative to unstimulated (unstim) cells. (C-F) Extracellular flux analysis (T-cell metabolic profiling test) on T cells isolated after 48 hours with/without anti-CD3/anti-CD28 stimulation. (C) Representative OCR kinetic graph, alongside basal OCR and SRC from independent experiments. (D) Representative ECAR kinetic graph, alongside basal ECAR and glycolytic capacity from independent experiments. (E) Basal OCR and ECAR energy map, showing metabolic stages from independent experiments. (F) ATP production rates from glycolysis (glycoATP) and oxidative phosphorylation (mitoATP). (G) T-cell activation levels measured by surface levels of interleukin-2 receptor (CD25) after 48-hour anti-CD3/anti-CD28 stimulation. Analysis of murine samples (H-L). (H) Quantification of MTG, TMRM (MTO alternative), and TMRM:MTG (potential-to-mass) ratio in WT or AT Eμ-TCL1 splenic CD8+ T cells (data normalized to WT mean values). (I) Flow cytometric quantification of mitochondrial ROS (MitoSOX) and cellular glucose uptake (2-NBDG) in CD8+ T cells. (J) Representative transmission electron microscope images of CD8+ T cells from WT or AT Eμ-TCL1 spleens (red arrows indicate mitochondria), alongside measurements of mitochondrial parameters (each symbol represents a separate replicate image). (K-L) Extracellular flux analysis (mitochondrial stress test) on isolated splenic CD8+ T cells after 5 days stimulation using anti-CD3/anti-CD28 dynabeads. (K) Representative OCR kinetic graph and tabulated metabolic parameters. (L) Basal OCR and ECAR energy map. Data are cumulative from ≥3 experiments in panels H-I or representative of 3 independent experiments in panels K-L. Each symbol represents an individual animal in panels H-I. Isolated CD8+ T cells were pooled from 3 different mice spleens in panels J-L. Data are presented as mean ± standard error of the mean (SEM) in panels A-I; or mean ± standard deviation (SD) in panels J-L. Differences were analyzed using a 2-way analysis of variance (ANOVA) with Sidak multiple correction test in panels C-D,F or a 2-tailed unpaired t test in panels A-B,G-K. ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001. 2-NBDG, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose; ATP, adenosine triphosphate; gMFI, geometric mean fluorescence intensity; ns, non-significant.

Accumulation of defective depolarized mitochondria in T cells from patients with CLL and Eμ-TCL1 model. Analysis of patient samples (A-G). (A) Mitochondrial mass (MTG) and mitochondrial membrane potential (MTO) in CD8+ T cells from HDs and patients with CLL. MTO:MTG (potential-to-mass) ratio normalized to HD mean values was determined. (B) Fold increase in MTG and MTO levels 48 hours after stimulation using anti-CD3/anti-CD28 soluble antibodies, relative to unstimulated (unstim) cells. (C-F) Extracellular flux analysis (T-cell metabolic profiling test) on T cells isolated after 48 hours with/without anti-CD3/anti-CD28 stimulation. (C) Representative OCR kinetic graph, alongside basal OCR and SRC from independent experiments. (D) Representative ECAR kinetic graph, alongside basal ECAR and glycolytic capacity from independent experiments. (E) Basal OCR and ECAR energy map, showing metabolic stages from independent experiments. (F) ATP production rates from glycolysis (glycoATP) and oxidative phosphorylation (mitoATP). (G) T-cell activation levels measured by surface levels of interleukin-2 receptor (CD25) after 48-hour anti-CD3/anti-CD28 stimulation. Analysis of murine samples (H-L). (H) Quantification of MTG, TMRM (MTO alternative), and TMRM:MTG (potential-to-mass) ratio in WT or AT Eμ-TCL1 splenic CD8+ T cells (data normalized to WT mean values). (I) Flow cytometric quantification of mitochondrial ROS (MitoSOX) and cellular glucose uptake (2-NBDG) in CD8+ T cells. (J) Representative transmission electron microscope images of CD8+ T cells from WT or AT Eμ-TCL1 spleens (red arrows indicate mitochondria), alongside measurements of mitochondrial parameters (each symbol represents a separate replicate image). (K-L) Extracellular flux analysis (mitochondrial stress test) on isolated splenic CD8+ T cells after 5 days stimulation using anti-CD3/anti-CD28 dynabeads. (K) Representative OCR kinetic graph and tabulated metabolic parameters. (L) Basal OCR and ECAR energy map. Data are cumulative from ≥3 experiments in panels H-I or representative of 3 independent experiments in panels K-L. Each symbol represents an individual animal in panels H-I. Isolated CD8+ T cells were pooled from 3 different mice spleens in panels J-L. Data are presented as mean ± standard error of the mean (SEM) in panels A-I; or mean ± standard deviation (SD) in panels J-L. Differences were analyzed using a 2-way analysis of variance (ANOVA) with Sidak multiple correction test in panels C-D,F or a 2-tailed unpaired t test in panels A-B,G-K. ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001. 2-NBDG, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose; ATP, adenosine triphosphate; gMFI, geometric mean fluorescence intensity; ns, non-significant.

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