itk  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: A . Top panel: Schematic representation of the protocol used to inhibit ITK activity. PSCA CAR-T cells were pre-treated for 2 hours with ITK inhibitors 1μM (BMS-509744, Ibrutinib or GNE-9822) or DMSO as control and then cocultured with HPAC WT cells for 0 or 10 minutes. Left panel: Representative Western blot of CD28 pY218 in CAR-T cells after stimulation with HPAC WT cells. Right panel: Normalized CD28 pY218 intensity with respect to CAR (CD3ζ). Significance was determined using one-way ANOVA. * = P<0.05. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). B. Top panel. Schematic representation of the experimental protocol. ITK expression was disrupted using CRISPR/Cas in Jurkat cells. WT and ITK-KO Jurkat cells were transduced to express a PSCA-specific CAR and cocultured with HPAC WT cells for 0 or 10 min. Left panel. Representative membrane of CD28 pY218 in CAR-T Jurkat cells after stimulation. Right panel. Normalized CD28 pY218 intensity quantified by densitometry (representative plot) and CD28 pY218 at 10 min post-stimulation. Significance was determined by paired t-test. ** = P<0.01. Each symbol represents one of 3 independent experiments, performed with different donor T cells.

Article Snippet: Primary antibodies: rabbit anti-CD28 pY218 (Abcam, ab138404), anti-CD28, rabbit ant-ZAP70 pY319 (Cell Signaling, #2717), rabbit anti-ZAP70 (Cell Signaling, #2705), rabbit anti-PLCγ pY783 (Abcam, ab76155), rabbit anti-PLCγ (Abcam, ab76155), rabbit anti-VAV1 pY174 (Abcam, ab76225), rabbit anti-VAV1 (Abcam, ab97574), rabbit anti-LCK (Abcam, ab32149), ITK (Cell Signaling, 77215), mouse anti-CD3ζ (Santa Cruz, sc-166275), mouse anti-B actin (Santa Cruz, sc-47778).

Techniques: Activity Assay, Control, Western Blot, Standard Deviation, Expressing, CRISPR, Membrane

Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: A . Schematic representation of WT and PYRP mutant CAR constructs. In the PYRP mutant, alanine (A) 217 and serine (S) 220 were substituted with prolines (P) to generate a proline-rich domain. B . PSCA-targeted WT and PYRP CAR-T cells were cocultured with HPAC WT cells for 0, 1, 10, 30, or 60 min. CAR molecules were immunoprecipitated using protein-L coated beads and co-immunoprecipitated proteins were analyzed by Western blot. Upper panel: Representative Western blot results showing ITK present in CAR immunoprecipitate. CD3ζ staining of the CAR molecule was used as a bait control. Lower panel: Intensity of ITK with respect a total CAR (CD3ζ) was analyzed by densitometry (representative plot). C. PSCA WT and PYRP CAR-T cells were stimulated with HPAC WT cells for 10 min and phosphorylation of Y218 was analyzed by mass spectrometry. Bar charts represent the intensity of phosphorylated Y218 normalized to the total CAR signal. Significance was determined by one-way Anova. * = P<0.05. Data is represented as the mean ± standard deviation (SD). D and F. PSCA WT and PYRP CAR-T cells were cocultured with HPAC WT cells. Supernatant was collected after 24 hours and cytokine production was measured by ELISA. Significance was determined by one-way ANOVA. * = P<0.05, ** = P<0.01. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). E. PSCA WT and PYRP CAR-T cells were cocultured with HPAC WT cells. RNA was extracted after 24 hours and IL-2 mRNA production was evaluated using quantitative Time PCR. CAR mRNA was used as normalization control. Significance was determined by one-way Anova. ** = P<0.01, *** = P<0.001. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). G. % of change in tumor volume (compared to baseline) is represented (n=5). Representative example of two independent experiments. Significance was determined by permutation two sample t-test. * = P<0.05.

Article Snippet: Primary antibodies: rabbit anti-CD28 pY218 (Abcam, ab138404), anti-CD28, rabbit ant-ZAP70 pY319 (Cell Signaling, #2717), rabbit anti-ZAP70 (Cell Signaling, #2705), rabbit anti-PLCγ pY783 (Abcam, ab76155), rabbit anti-PLCγ (Abcam, ab76155), rabbit anti-VAV1 pY174 (Abcam, ab76225), rabbit anti-VAV1 (Abcam, ab97574), rabbit anti-LCK (Abcam, ab32149), ITK (Cell Signaling, 77215), mouse anti-CD3ζ (Santa Cruz, sc-166275), mouse anti-B actin (Santa Cruz, sc-47778).

Techniques: Mutagenesis, Construct, Immunoprecipitation, Western Blot, Staining, Control, Phospho-proteomics, Mass Spectrometry, Standard Deviation, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: A . Top panel: Schematic representation of the protocol used to inhibit ITK activity. PSCA CAR-T cells were pre-treated for 2 hours with ITK inhibitors 1μM (BMS-509744, Ibrutinib or GNE-9822) or DMSO as control and then cocultured with HPAC WT cells for 0 or 10 minutes. Left panel: Representative Western blot of CD28 pY218 in CAR-T cells after stimulation with HPAC WT cells. Right panel: Normalized CD28 pY218 intensity with respect to CAR (CD3ζ). Significance was determined using one-way ANOVA. * = P<0.05. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). B. Top panel. Schematic representation of the experimental protocol. ITK expression was disrupted using CRISPR/Cas in Jurkat cells. WT and ITK-KO Jurkat cells were transduced to express a PSCA-specific CAR and cocultured with HPAC WT cells for 0 or 10 min. Left panel. Representative membrane of CD28 pY218 in CAR-T Jurkat cells after stimulation. Right panel. Normalized CD28 pY218 intensity quantified by densitometry (representative plot) and CD28 pY218 at 10 min post-stimulation. Significance was determined by paired t-test. ** = P<0.01. Each symbol represents one of 3 independent experiments, performed with different donor T cells.

Article Snippet: The following antibodies were used for immune and cancer cell phenotyping: G4S Linker AF488 (Cell signaling, 50515), CD3 BV711(biolegend, 317328), CD4 PE-Cy7(Biolegend, 300512), CD8 BUV395 (BD, 563795), rabbit ITK (Cell Signaling, 77215), anti-rabbit AF488(Cell Signaling 4412S), PSCA PE (Santa Cruz, sc-80654).

Techniques: Activity Assay, Control, Western Blot, Standard Deviation, Expressing, CRISPR, Membrane

Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: A . Schematic representation of WT and PYRP mutant CAR constructs. In the PYRP mutant, alanine (A) 217 and serine (S) 220 were substituted with prolines (P) to generate a proline-rich domain. B . PSCA-targeted WT and PYRP CAR-T cells were cocultured with HPAC WT cells for 0, 1, 10, 30, or 60 min. CAR molecules were immunoprecipitated using protein-L coated beads and co-immunoprecipitated proteins were analyzed by Western blot. Upper panel: Representative Western blot results showing ITK present in CAR immunoprecipitate. CD3ζ staining of the CAR molecule was used as a bait control. Lower panel: Intensity of ITK with respect a total CAR (CD3ζ) was analyzed by densitometry (representative plot). C. PSCA WT and PYRP CAR-T cells were stimulated with HPAC WT cells for 10 min and phosphorylation of Y218 was analyzed by mass spectrometry. Bar charts represent the intensity of phosphorylated Y218 normalized to the total CAR signal. Significance was determined by one-way Anova. * = P<0.05. Data is represented as the mean ± standard deviation (SD). D and F. PSCA WT and PYRP CAR-T cells were cocultured with HPAC WT cells. Supernatant was collected after 24 hours and cytokine production was measured by ELISA. Significance was determined by one-way ANOVA. * = P<0.05, ** = P<0.01. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). E. PSCA WT and PYRP CAR-T cells were cocultured with HPAC WT cells. RNA was extracted after 24 hours and IL-2 mRNA production was evaluated using quantitative Time PCR. CAR mRNA was used as normalization control. Significance was determined by one-way Anova. ** = P<0.01, *** = P<0.001. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). G. % of change in tumor volume (compared to baseline) is represented (n=5). Representative example of two independent experiments. Significance was determined by permutation two sample t-test. * = P<0.05.

Article Snippet: The following antibodies were used for immune and cancer cell phenotyping: G4S Linker AF488 (Cell signaling, 50515), CD3 BV711(biolegend, 317328), CD4 PE-Cy7(Biolegend, 300512), CD8 BUV395 (BD, 563795), rabbit ITK (Cell Signaling, 77215), anti-rabbit AF488(Cell Signaling 4412S), PSCA PE (Santa Cruz, sc-80654).

Techniques: Mutagenesis, Construct, Immunoprecipitation, Western Blot, Staining, Control, Phospho-proteomics, Mass Spectrometry, Standard Deviation, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Inherited human ITK deficiency impairs IFN-γ immunity and underlies tuberculosis

doi: 10.1084/jem.20220484

Figure Lengend Snippet: AR ITK deficiency with TB. (A) Pedigree of the kindreds. Black symbols indicate affected individuals. Arrows indicate the probands. Genotypes for ITK are also shown. M, mutated. (B) Cranial T2-weighted MRI for P1. The high-intensity lesion in the right temporal lobe disappeared after 9 mo of anti-TB therapy (post-tx). (C) Surgical biopsy of an enlarged mesenteric lymph node of P1 showing central caseation on H&E staining and acid-fast bacilli (arrows) on Ziehl–Neelsen staining. (D) A thoracic CT scan of the lung of P3 showing tree-in-bud signs. Radiological improvement was observed after 4 mo of anti-TB therapy. (E) Variants detected by WES. (F) Sanger sequencing of the ITK variants. (G) Population genetics of ITK . The minor allele frequency (MAF) and CADD scores for all non-synonymous variants reported in the gnomAD database are shown. Three homozygous variants found in our private cohort are also shown. The CADD score of 35.0 for the c.496C>T (R166*) allele is well above the MSC of 20.7 ( ; ; horizontal dotted line). (H) Gene-level negative selection. Like other genes with mutations underlying AR IEI, ITK is not under negative selection, as shown by CoNeS . (I) Schematic representation of the ITK protein. PH, pleckstrin homology domain; SH, Src homology domain. Previously reported homozygous or compound heterozygous mutations are also shown.

Article Snippet: The membrane was probed with the following primary antibodies: anti-ITK mAb for the N-terminal epitope (Cat: ab32039; Abcam, Clone: Y401, 1:1,000, 4 °C overnight), anti-ITK mAb for the C-terminal epitope (Cat: 77215; Cell Signaling Technology, Clone: E4X7M, 1:1,000, 4 °C overnight), anti-phospho-PLCγ1 (Y783) rabbit polyclonal antibody (Cat: 2821, 1:1,000; Cell Signaling Technology, 4 °C overnight), HRP-conjugated anti-DDK mAb (Cat: 637311, Clone: L5, 1:2,000; BioLegend, room temperature for 1 h), and HRP-conjugated anti-vinculin mAb (Cat: sc-73614, Clone: 7F9, 1:1,000; Santa Cruz, 4 °C overnight).

Techniques: Staining, Computed Tomography, Sequencing, Selection

Journal: The Journal of Experimental Medicine

Article Title: Inherited human ITK deficiency impairs IFN-γ immunity and underlies tuberculosis

doi: 10.1084/jem.20220484

Figure Lengend Snippet: 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 (Ca 2+ ) 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 Ca 2+ concentration was determined with Indo-1. Representative results from at least two independent experiments are shown. Source data are available for this figure: .

Article Snippet: The membrane was probed with the following primary antibodies: anti-ITK mAb for the N-terminal epitope (Cat: ab32039; Abcam, Clone: Y401, 1:1,000, 4 °C overnight), anti-ITK mAb for the C-terminal epitope (Cat: 77215; Cell Signaling Technology, Clone: E4X7M, 1:1,000, 4 °C overnight), anti-phospho-PLCγ1 (Y783) rabbit polyclonal antibody (Cat: 2821, 1:1,000; Cell Signaling Technology, 4 °C overnight), HRP-conjugated anti-DDK mAb (Cat: 637311, Clone: L5, 1:2,000; BioLegend, room temperature for 1 h), and HRP-conjugated anti-vinculin mAb (Cat: sc-73614, Clone: 7F9, 1:1,000; Santa Cruz, 4 °C overnight).

Techniques: Expressing, Over Expression, Western Blot, Phospho-proteomics, Control, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: Inherited human ITK deficiency impairs IFN-γ immunity and underlies tuberculosis

doi: 10.1084/jem.20220484

Figure Lengend Snippet: 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.

Article Snippet: The membrane was probed with the following primary antibodies: anti-ITK mAb for the N-terminal epitope (Cat: ab32039; Abcam, Clone: Y401, 1:1,000, 4 °C overnight), anti-ITK mAb for the C-terminal epitope (Cat: 77215; Cell Signaling Technology, Clone: E4X7M, 1:1,000, 4 °C overnight), anti-phospho-PLCγ1 (Y783) rabbit polyclonal antibody (Cat: 2821, 1:1,000; Cell Signaling Technology, 4 °C overnight), HRP-conjugated anti-DDK mAb (Cat: 637311, Clone: L5, 1:2,000; BioLegend, room temperature for 1 h), and HRP-conjugated anti-vinculin mAb (Cat: sc-73614, Clone: 7F9, 1:1,000; Santa Cruz, 4 °C overnight).

Techniques: Flow Cytometry, Transduction, Inhibition, Incubation, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Inherited human ITK deficiency impairs IFN-γ immunity and underlies tuberculosis

doi: 10.1084/jem.20220484

Figure Lengend Snippet: 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.

Article Snippet: The membrane was probed with the following primary antibodies: anti-ITK mAb for the N-terminal epitope (Cat: ab32039; Abcam, Clone: Y401, 1:1,000, 4 °C overnight), anti-ITK mAb for the C-terminal epitope (Cat: 77215; Cell Signaling Technology, Clone: E4X7M, 1:1,000, 4 °C overnight), anti-phospho-PLCγ1 (Y783) rabbit polyclonal antibody (Cat: 2821, 1:1,000; Cell Signaling Technology, 4 °C overnight), HRP-conjugated anti-DDK mAb (Cat: 637311, Clone: L5, 1:2,000; BioLegend, room temperature for 1 h), and HRP-conjugated anti-vinculin mAb (Cat: sc-73614, Clone: 7F9, 1:1,000; Santa Cruz, 4 °C overnight).

Techniques: Expressing, Flow Cytometry, Inhibition, Cell Culture, Two Tailed Test