Duvelisib added during CART cell culture increased mitochondrial fusion with increased mitochondrial fusion proteins but did not change the OCRs. (A) T cells cultured with or without PI3K inhibitors were stained with NAO and assessed by flow cytometry, with representative NAO fluorescence shown on the CD4⁺ and CD8⁺ T-cell subsets. (B) Transmission electron microscopic images of nontransduced and CAR transduced T cells at day 14 of culture, with cell cross-sectional areas and mitochondrial cross-sectional area as a percentage of total cell area shown below each image. Images with cell size and mitochondrial area representative of the mean for each group are shown. FIJI software measured T-cell sizes across >10 replicate images for each patient and condition. Representative images from >420 acquired images are shown. Red arrows, mitochondria. (C) T-cell size, shown as cross-sectional area, over time in culture without or with duvelisib. (D) Differences in cross-sectional area of cytoplasm and nucleus at day 14 of culture in control and duvelisib T cell cultures. (E) Violin plots showing mitochondrial area as a percentage of T cell cross-sectional area for nontransduced and CART transduced cells. (F) Representative western blot analysis of mitochondrial fusion proteins MFN1 and MFN2. (G) Evaluation of oxygen consumption rates using a Seahorse bioenergetics bioanalyzer of OCR and extracellular acidification rate (ECAR) for CLL T-cell cultures. Cells were plated at 4 × 10⁵ per well for both control CART and Duv-CART. Results from 4 representative CLL samples from 12 replicates are shown. (H) Quantified western blots (MFN1, MFN2, and p.DRP1) from 3 to 5 CLL donors for select proteins. *P < .05; **P ≤ .01; ****P ≤ .0001; ns, not significant.