Culture with PI3Kis increased the frequencies of CD8⁺ naive and memory T cells and decreased the frequencies of senescent CD8⁺ T cells from untreated patients with CLL. Mononuclear cells collected from CLL donors were analyzed by flow cytometry. Cryopreserved cells were thawed, and T cells were isolated and expanded for 9 days with duvelisib or idelalisib added to the culture medium every 3 days (q3d). (A) T cells from healthy donors (n = 5) and patients with CLL (n = 18) assessed by flow cytometry for expression of costimulatory molecules (CD27 and CD28) on the CD8⁺ and CD4⁺ subsets. (B) The frequencies of CD8⁺CD27⁺CD45RO⁻ T cells in patients with CLL compared with healthy controls. The dashed line indicates the threshold frequency of CD8+ naive T-cells previously associated with complete remission in patients with CLL treated with CD19-targeted CART cells.⁷ In vitro expansion of T cells from patients with CLL (n = 8) cultured with idelalisib (C) and duvelisib (D), with the 50% inhibitory concentration (IC₅₀) for PI3Kδ and PI3Kγ shown as vertical dashed lines. Flow cytometric analysis of the frequency of CD8⁺ T cells (E) and CD4/CD8 T-cell ratios (F) in duvelisib-containing and control T-cell cultures. (G) Expression of the immune checkpoint molecules TIM3, LAG3, and PD1 on the CD8⁺ T cells. (H) Frequency of TIM3 on the CD4⁺ T-cell subset. LAG3 and PD1 yielded no significant changes and are not shown on the CD4⁺ subset. (I) Distribution of T cells according to naive and memory phenotypes in control and duvelisib-containing cultures. T-cell populations were defined as follows: naive and T_SCM (CD45RA⁺CD45RO⁻CCR7⁺), central memory (CD45RA⁻CD45RO⁺CCR7⁺), T-memory and effector memory (CD45RA⁻CD45RO⁺CCR7⁻), and terminal effector cells (CD45RA⁺CD45RO⁻CCR7⁻). *P < .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001.