Fig 2: In vivo persistence and safety analysis of ErbB2CAR-T cells. (A) The mice were injected with 2 × 106 cells IP NAR-LUC cells. After 20 days, they were treated with ErbB2CAR (2.5 × 106 IP or 10 × 106 IP or 10 × 106 IV cells), UNT, or were left untreated. They were euthanized after 45 days. Cells from the spleen, ovary, and blood were collected and stained with anti-human CD3 and analyzed by flow cytometry. Each group included an average of 8–11 mice ± SEM. Data were analyzed by one-way ANOVA test * p < 0.05. (B) The leftmost pie diagram depicts the percentage of CCR7 and/or CD45RA expression out of CD3/ErbB2CAR-GFP-positive T cells (average of four independent transductions). The mice were then euthanized 45 days post-NAR-LUC injection. Splenic single-cell suspension was prepared and stained with anti-human CD3/CCR7/CD45RA and analyzed by flow cytometry. The three right pies depict the percentage of CCR7 and/or CD45RA expression out of CD3-positive cells. EM—effector memory cells, CM—central memory, TN—naïve T cells, and EFF—effector T cells. Each group included an average of 5–11 mice. (C) The mice were euthanized 45 days post-NAR-LUC inoculation, and RNA levels from the splenic cells were measured for PD-1 and TIM-3. The relative expressions were normalized to the housekeeping gene GAPDH, and UNT-treated mice were considered as 1. Each group included an average of 4–5 mice ± SEM. Data were analyzed by 1-way ANOVA test * p < 0.05. (D) Primary cells from different tissues of healthy human donors were co-cultured with lymphocyte T cells transduced for 24 h with ErbB2CAR at an E:T ratio of 2:1. Supernatants were collected and secreted IFNγ was measured by ELISA. The tested target cells included: SKOV3 and OVCAR8 cells (positive control), KEpC—Kidney Epithelial Cells, LEpC—Lung Epithelial Cells, PC—Pancreatic Cells, CMEnC—Cardiac Microvascular Endothelial Cells, RBC—Red Blood Cells, and NT—No Target.

Fig 3: Impaired production of IL-9 by ITK-deficient T lymphocytes. (A) T-blasts from P1 and P2 were stimulated for 2 h with various reagents. Secreted cytokines were determined with the LEGENDplex assay. Technical duplicates were prepared for the unstimulated and anti-CD3 mAb-conjugated bead-stimulated samples. (B) Expression of IRF4. T-blasts from P2, her mother, and local controls were either left unstimulated or were stimulated with an anti-CD2/CD3/CD28 mAb cocktail for 24 h and analyzed by flow cytometry. For pharmacological ITK inhibition, T-blasts from controls were cultured in the presence of DMSO or an ITK inhibitor (BMS509744). Bars represent the mean and SEM. Statistical significance was determined for differences between the two ITK-deficient patients on the one hand, and local and familial controls on the other. *, P < 0.05, two-tailed Wilcoxon’s rank sum tests with FDR adjustment. Representative results from two independent experiments are shown.

Fig 4: Impaired production of IFN-γ and TNF by ITK-deficient T lymphocytes. (A and B) PBMCs from P1 and P2 (obtained at the ages of 19 and 18 yr, respectively) and their father were stimulated for (A) 18 h or (B) 3 or 5 d with IL-2. Secreted cytokine levels were determined in LEGENDplex assays. Technical duplicates were prepared for (A) P1 or (B) P1, P2, and their father (except P2 for 3 d). (C and D) Expanded T cell blasts (T-blasts) from P1 and P2 were stimulated for 2 h with various reagents and then for an additional 5 h in the presence of a secretion inhibitor. Secreted cytokines were determined with the LEGENDplex assay, and intracellular cytokine levels were determined by flow cytometry. Technical duplicates were prepared for the unstimulated and anti-CD3 mAb-conjugated bead-stimulated samples. (E) Lentiviral rescue. P2’s T-blasts underwent lentiviral transduction with the EV or the WT ITK CDS. Cells were selected on puromycin. Cells were either left unstimulated or stimulated with anti-CD3 mAb-conjugated beads for 5 h. Cytokine production was assessed by flow cytometry. (F) Pharmacological ITK inhibition. Magnetically enriched CD4+ T-blasts from four healthy donors were incubated for 1 h with DMSO or an ITK inhibitor (BMS509744) and were then stimulated for 6 h. Secreted cytokines were determined in LEGENDplex assays. In A–D and F, bars represent the mean and SEM. In A–D, statistical significance was determined for differences between the two ITK-deficient patients on the one hand, and local and familial controls on the other. *, P < 0.05; **, P < 0.01; ***, P < 0.001, and two-tailed Wilcoxon’s rank sum tests with FDR adjustment.

Fig 5: Phosphorylation of ZAP70. Total T lymphocytes from P1, P2, and two healthy controls (Ctrl) were analyzed by immunoblotting for phosphorylated ZAP70 (Y319) after 5 min of stimulation with anti-CD3 mAb. A representative result from two independent experiments is shown. Source data are available for this figure: SourceData FS3.