Fig 2: Expansion of the γδ and DN αβ T cell populations in Itk-deficient mice. Five WT and Itk-deficient mice (C57BL/6 background) were studied in two batches of experiments. (A) Frequencies of αβ and γδ T cells in the thymus (Thy) and spleen (Spl). (B) Phenotypes of γδ T cells. CD24 staining was performed for only three of the five mice. (C) αβ T cell subsets. (D) Phenotypes of DN αβ T cells. Bars represent the mean and SEM. Statistical significance was determined for differences between WT and Itk-deficient mice. n.s., not significant. *, P < 0.05, two-tailed Wilcoxon’s rank sum tests with FDR adjustment.

Fig 3: Atypical phenotypes of ITK-deficient DN αβ and γδ T lymphocytes. PBMCs from P1 (obtained at the ages of 18 and 19 yr), P2 (obtained at the age of 16 yr, free from EBV viremia), P3 (at the age of 4 yr), heterozygous members of their families, one patient with a homozygous LOF (R29C) mutation of ITK with EBV viremia but no history of TB, and one FAS-deficient patient were studied by flow cytometric immunophenotyping. (A) A representative plot of CD38 and T-bet expression. (B and C) Phenotypes of (B) DN αβ T and (C) Vδ2− γδ T lymphocytes. Results from three batches of experiments are compiled. (D–G) FlowSOM-guided clustering analysis. (D and E) UMAP plots of (D) all DN αβ T lymphocytes and (E) 2,500 cells per group. Equal numbers of cells were randomly sampled from each individual in each group. (F) Heatmap of scaled median expression levels. (G) Cluster abundance. In B, C, and G, the bars represent the mean and SEM. In B and C, statistical significance was determined for differences between ITK-deficient patients and adult, pediatric, and familial controls combined. ***, P < 0.001, two-tailed Wilcoxon’s rank sum tests with FDR adjustment.

Fig 4: Analysis of ITK expression and function. (A–C) Studies of ITK alleles in an overexpression system. (A and B) Immunoblotting of the ITK protein with two different mAbs. (C) Phosphorylation of phospholipase C-γ1. (D and E) Immunoblotting for endogenous ITK protein with two different antibodies, on total T lymphocytes from P1, P2, their father, and one healthy control (Ctrl). (F) TCR signaling assay. Total T lymphocytes from P1, P2, their father, and one healthy control were analyzed by immunoblotting for general tyrosine phosphorylation (pY) and for the phosphorylation of phospholipase C-γ1 without stimulation or after 2 or 5 min of stimulation with anti-CD3 mAb. (G) Calcium (Ca2+) influx assay. Total T lymphocytes from P1, P2, their father, and one healthy control were stimulated by TCR crosslinking (with anti-CD3 and anti-IgG mAbs) or ionomycin. Intracellular Ca2+ concentration was determined with Indo-1. Representative results from at least two independent experiments are shown. Source data are available for this figure: SourceData F2.

Fig 5: 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.