Altered mitochondrial homeostasis in CLL-derived CD8⁺ T cells. (A) KEGG pathway analysis showing top 10 of most significantly different pathways in CD8⁺ T cells from HDs vs CLL, and heatmap of differentially expressed genes in OXPHOS pathway (Gene Expression Omnibus [GEO] accession number GSE8835; P = 3.4 × 10⁻¹⁵ for OXPHOS pathway). PBMCs from CLL patients and age-matched HDs were analyzed by flow cytometry (C,E-H) or Seahorse EFA (B,D) either directly after thawing (B-D,H), 2 days of culturing (E-F), or 3 days of culturing (G). (B) Cells were analyzed for OCR (indicating OXPHOS; CLL, n = 7; HD, n = 6). (C) Mitochondrial mass was determined by flow cytometry (Mitotracker Green; CLL, n = 29; HD, n = 29) and by qPCR by calculating the mitochondrial DNA (mtDNA)-to-nuclear DNA (nDNA) ratio (CLL, n = 8; HD, n = 5). (D) SRC was calculated as the ratio of maximum OCR over basal OCR (CLL, n = 6; HD, n = 6). (E) ΔΨm was determined by flow cytometry (Mitotracker Orange; CLL, n = 11; HD, n = 12; data are the same as unstimulated control in Figure 5D). (F) Mitochondrial ROS (mitoSOX; CLL, n = 11; HD, n = 12; data are the same as unstimulated control in Figure 5E). (G) Mitochondrial ROS as well as mitochondrial potential were analyzed in HD CD8 T cells cocultured with HD-derived B cells or CLL (HD, n = 8). (H) PGC-1α, SOD1, and SOD2 (CLL, n = 8; HD, n = 8), HO-1, NRF-2, and ERRα (CLL, n = 8; HD, n = 4). Data were normalized to HD to compile multiple independent experiments (B,D). Normality was determined by a D’Agostino and Pearson normality test. The P value was calculated by a Welch test (E-F), Mann-Whitney test (B-D,G-H), or an unpaired Student t test (H). Data are presented as mean plus or minus SEM. *P < .05; **P < .005; ****P < .0001.