Cytokine-stimulated and patient–derived primary human bystander-phenotype-positive CD8⁺ T cells are capable of killing leukemia and lymphoma cell lines. (A) Schematic of coculture cytotoxicity assay. Cells were sorted for bystander-phenotype-positive and -negative CD8⁺ T cells and cocultured with Nalm6, β2M knockout Nalm6, or Daudi cells at effector-to-target ratio of 1:1 for 16 hours. (B) Percentage killing of Nalm6 (n = 7; paired t test) and (C) β2M knockout Nalm6 cells by unstimulated (n = 3; unpaired t test), IL-15 (n = 5; paired t test), and IL-2 (n = 3; paired t test)–stimulated bystander-phenotype-positive and -negative CD8⁺ T cells. (D) Percentage killing of Daudi cells after coculture with IL15-stimulated T cells sorted for bystander-phenotype-positive and -negative CD8⁺ T cells (n = 3; paired t test). Experiments were performed in duplicates or triplicates for each donor. Individual donors are represented by the unique symbols in the figure. (E) Frequency of CAR⁺ T cells and CD8⁺, CD94⁺, CD160⁺, and NKG2D⁺ CAR-negative T cells (red cluster) were determined by flow cytometric analysis in PBMCs isolated from a healthy donor, as well as from Pt006 and Pt007, with blood drawn 6 to 9 days after CAR T-cell infusion. (F) Percentage killing of β2M knockout Nalm6 cells 16 hours after coculture with bystander-phenotype-positive and -negative CD8⁺ T cells. This experiment was done with technical triplicates, the mean of all experiments is shown as ± standard error of mean. Stim, stimulated; unstim, unstimulated; WT, wild-type.