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.