Human ITK is required for optimal cytotoxicity in innate-like adaptive T lymphocytes. Innate-like adaptive T lymphocyte subsets were sorted from two to three healthy donors, expanded for 2 wk, and cocultured with CFSE-stained Raji cells with or without BiTE in the presence of DMSO or an ITK inhibitor (BMS509744). After 24 h of coculture, the supernatants were collected and analyzed with a LEGENDplex assay. The cells were acquired with an Attune NxT automated flow cytometry instrument to quantify the remaining CFSE⁺ Raji cells in equal volumes of sample. (A) Absolute numbers of Raji cells per well. (B) The ratio of the numbers of Raji cells in wells with and without BiTE. A higher ratio indicates more potent BiTE-induced Raji cell killing. (C) Secreted cytokines and cytotoxic effector molecules. Bars represent the mean and SEM. Statistical significance was determined for comparisons between samples with or without ITK inhibition. n.s., not significant. *, P < 0.05; **, P < 0.01, and two-tailed Wilcoxon’s rank sum tests with FDR adjustment. Representative results from two independent experiments are shown.