PI3Kδ inhibition enhances mitochondrial activity and memory differentiation of CLL T cells. Analysis of patient samples (A-G). Isolated CLL T cells treated with/without 5 μM idelalisib over a 10-day stimulation period with anti-CD3/anti-CD28 soluble antibodies. (A) Mitochondrial mass (MTG) and membrane potential (MTO) relative to the control (untreated) mean. (B) Extracellular flux analysis (T-cell metabolic profiling test) performed on treated and untreated T cells. Representative OCR kinetic graph is provided. Percentage of SRC, glycolytic capacity, and maximal OCR:ECAR ratio were calculated. When indicated, data were calculated relative to the control mean. (C) CD8+ T-cell subsets assessed according to the surface expression of CD45RA and CD27: naïve, central memory (CM), effector memory (EM), and terminally differentiated effector memory (EMRA). (D) CD8+ Tbetlo-int-hi and Eomeslo-hi populations were identified. (E) The expression levels of Tbet, Eomes, and TOX were quantified relative to the control mean. (F) Frequency of EomesloTCF-1hi and EomeshiTCF-1lo CD8+ T-cell populations. (G) PGC1α levels within the EomesloTCF-1hi and EomeshiTCF-1lo populations. Analysis of murine samples (H-M). Isolated AT Eμ-TCL1 CD8+ T cells were expanded for 6 to 7 days using anti-CD3/anti-CD28 dynabeads with/without 5 μM idelalisib. (H) Representative OCR kinetic graph and tabulated metabolic parameters of expanded CD8+ T cells. (I) Representative histograms and tabulated results of mitochondrial mass (MTG), membrane potential (TMRM), and ROS production (MitoSOX). (J) Representative histogram and tabulated PGC1α expression levels in CD8+ T cells. (K) Quantification of memory CD8+ T-cell phenotypes (naïve, CD44–CD62L+; CM, CD44+CD62L+; EM, CD44+CD62L–; and effector, CD44–CD62L–). (L) Representative histogram and tabulated CD62L expression levels in CD8+ T cells. (M) Quantitative results for the relative expression levels of exhaustion-related markers. Data are cumulative from ≥3 experiments in panels I-M or representative of 3 independent experiments in panel H. Each symbol represents an individual animal in panels I-M. Each data point (idelalisib) was normalized to its individual control (dimethyl sulfoxide [DMSO]) to combine MFI data from multiple independent experiments in panels I-J,L-M. Data are presented as mean ± SEM in panels A-G,I-M; or mean ± SD in panel H. Differences were analyzed using a 2-way ANOVA with Sidak multiple correction test in panels B-F; or a 2-tailed, paired t test in panels A-B,G-M. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. FMO, fluorescence minus one; gMFI, geometric mean fluorescence intensity; ns, non-significant.

PI3Kδ inhibition enhances mitochondrial activity and memory differentiation of CLL T cells. Analysis of patient samples (A-G). Isolated CLL T cells treated with/without 5 μM idelalisib over a 10-day stimulation period with anti-CD3/anti-CD28 soluble antibodies. (A) Mitochondrial mass (MTG) and membrane potential (MTO) relative to the control (untreated) mean. (B) Extracellular flux analysis (T-cell metabolic profiling test) performed on treated and untreated T cells. Representative OCR kinetic graph is provided. Percentage of SRC, glycolytic capacity, and maximal OCR:ECAR ratio were calculated. When indicated, data were calculated relative to the control mean. (C) CD8+ T-cell subsets assessed according to the surface expression of CD45RA and CD27: naïve, central memory (CM), effector memory (EM), and terminally differentiated effector memory (EMRA). (D) CD8+ Tbetlo-int-hi and Eomeslo-hi populations were identified. (E) The expression levels of Tbet, Eomes, and TOX were quantified relative to the control mean. (F) Frequency of EomesloTCF-1hi and EomeshiTCF-1lo CD8+ T-cell populations. (G) PGC1α levels within the EomesloTCF-1hi and EomeshiTCF-1lo populations. Analysis of murine samples (H-M). Isolated AT Eμ-TCL1 CD8+ T cells were expanded for 6 to 7 days using anti-CD3/anti-CD28 dynabeads with/without 5 μM idelalisib. (H) Representative OCR kinetic graph and tabulated metabolic parameters of expanded CD8+ T cells. (I) Representative histograms and tabulated results of mitochondrial mass (MTG), membrane potential (TMRM), and ROS production (MitoSOX). (J) Representative histogram and tabulated PGC1α expression levels in CD8+ T cells. (K) Quantification of memory CD8+ T-cell phenotypes (naïve, CD44–CD62L+; CM, CD44+CD62L+; EM, CD44+CD62L–; and effector, CD44–CD62L–). (L) Representative histogram and tabulated CD62L expression levels in CD8+ T cells. (M) Quantitative results for the relative expression levels of exhaustion-related markers. Data are cumulative from ≥3 experiments in panels I-M or representative of 3 independent experiments in panel H. Each symbol represents an individual animal in panels I-M. Each data point (idelalisib) was normalized to its individual control (dimethyl sulfoxide [DMSO]) to combine MFI data from multiple independent experiments in panels I-J,L-M. Data are presented as mean ± SEM in panels A-G,I-M; or mean ± SD in panel H. Differences were analyzed using a 2-way ANOVA with Sidak multiple correction test in panels B-F; or a 2-tailed, paired t test in panels A-B,G-M. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. FMO, fluorescence minus one; gMFI, geometric mean fluorescence intensity; ns, non-significant.

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