Journal: Molecular therapy. Methods & clinical development

Article Title: Dasatinib is a potent enhancer for CAR T cell generation by CD3-targeted lentiviral vectors.

doi: 10.1016/j.omtm.2022.12.002

Figure Lengend Snippet: Figure 1. Dasatinib enhances CD3-targeted gene transfer into activated PBMCs in a time- and dose-dependent manner Freshly thawed primary human PBMCs were activated for 3 days with aCD3 and aCD28 antibodies in presence of IL-7/15 prior to transduction with CD3-LV. (A) Cells were incubated in 50 nM dasatinib-containing medium for 5 h with increasing particle numbers. Fold increase in transduction compared with transduction in absence of dasatinib is shown for four vector stocks. Each data point is an average value referring to a particular vector stock tested on one to four donors. *p < .05, by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. (B) Cells were incubated in 0.39–100 nM dasatinib-containing medium for 7 h in presence of vector particles. Dose- dependent increase in GFP-expression 3 days after transduction with 2.5 1010 CD3-LV particles is shown for four to five donors. (C and D) Cells were incubated without (w/ o), 25 nM, 50 nM, or 100 nM dasatinib-containing medium for 3 h, 5 h, or 7 h in presence of vector particles. (C) Representative dot plots of GFP-expression 3 days after transduction. (D) Superimposed symbols for different concentrations connected by line show GFP-expression 3 days after transduction. Data are based on three donors, and values are shown as mean ± standard error of the mean (SEM).

Article Snippet: For T cell activation prior to transduction, plates were coated with 1 mg/mL anti-human CD3 (aCD3; clone OKT3; Miltenyi Biotec), and freshly isolated or thawed PBMCs were cultivated with 3 mg/mL anti-human CD28 (aCD28; clone 15E8; Miltenyi Biotec)-containing medium for 3 days.

Techniques: Transduction, Incubation, Plasmid Preparation, Expressing

Journal: Journal of Biological Chemistry

Article Title: Alternative Antigen Receptor (TCR) Signaling in T Cells Derived from ZAP-70-deficient Patients Expressing High Levels of Syk

doi: 10.1074/jbc.m908568199

Figure Lengend Snippet: FIG. 2. Syk is highly expressed in proliferating ZAP-70-defi- cient T cells and is phosphorylated in response to TCR ligation. The level of Syk expression in freshly obtained (A) and proliferating (B) T cells (1 3 106 cell equivalents) isolated from ZAP-70–/– patients and control donors was monitored using an aSyk mAb. Immunoblots were then reprobed with an aErk2 mAb. C, Syk was immunoprecipitated from proliferating ZAP-70–/– T cells from the two patients and identi- cally treated control CD41 T cells (5 3 106 cell equivalents) following no stimulation (–) or stimulation (1) with an aCD3 mAb. Immunoprecipi- tates (IP) were immunoblotted (IB) with an a-Tyr(P) (a-PTyr) mAb to assess the phosphorylation status of Syk (upper panel). The position of Syk is indicated by an arrow. The blot was then reprobed with an aSyk mAb (lower panel).

Article Snippet: The aCD3 OKT3 hybridoma line was purchased from the ATCC, and the aCD3 UCHT1 mAb was from Pharmingen (San Diego, CA).

Techniques: Ligation, Expressing, Isolation, Control, Western Blot, Immunoprecipitation, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Alternative Antigen Receptor (TCR) Signaling in T Cells Derived from ZAP-70-deficient Patients Expressing High Levels of Syk

doi: 10.1074/jbc.m908568199

Figure Lengend Snippet: FIG. 3. LAT is tyrosine-phosphorylated and associates with PLC-g1 and SLP76 in CD3-activated Sykhi/ZAP-702 T cells. A, LAT was immunoprecipitated (IP) from control and Sykhi/ZAP-702 T cells (1 3 107 cell equivalents) following either no stimulation (–) or stimulation with a cross-linked aCD3 mAb (1). Immunoprecipitates were immunoblotted (IB) with an a-Tyr(P) (a-PTyr) mAb to assess the phosphorylation status of LAT (upper panel) and then reprobed with an aLAT pAb (bottom panel). B, lysates from nonstimulated (–) and CD3- stimulated (1) control and Sykhi/ZAP-702 T cells were immunoprecipi- tated with aPLC-g1 Ab, and the presence of tyrosine-phosphorylated PLC-g1 was assessed by immunoblotting with an a-Tyr(P) mAb (upper panel). Blots were reprobed with an aPLC-g1 Ab (bottom panel). C, similarly, lysates were immunoprecipitated with an aSLP76 Ab, and the presence of tyrosine-phosphorylated proteins was monitored.

Article Snippet: The aCD3 OKT3 hybridoma line was purchased from the ATCC, and the aCD3 UCHT1 mAb was from Pharmingen (San Diego, CA).

Techniques: Immunoprecipitation, Control, Phospho-proteomics, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Alternative Antigen Receptor (TCR) Signaling in T Cells Derived from ZAP-70-deficient Patients Expressing High Levels of Syk

doi: 10.1074/jbc.m908568199

Figure Lengend Snippet: FIG. 4. Calcium mobilization is increased in CD3-cross-linked Sykhi/ZAP-702 T cells. Sykhi/ZAP-702 T cells and control T cells derived from two donors (CTRL 1 and CTRL 2) were loaded with the calcium-sensitive dye Indo-1 and analyzed for increases in [Ca21]i fol- lowing stimulation with 10 mg/ml of the UCHT1 aCD3 mAb (A). Equiv- alent experiments were performed following stimulation with 1–5 mg/ml of the UCHT1 aCD3 mAb (B). Changes in [Ca21]i were monitored by fluorometry as a measure of the ratio of emission at 405/485 nm (ordinate axis) using a Photon Technologies spectrofluorometer for the indicated time period.

Article Snippet: The aCD3 OKT3 hybridoma line was purchased from the ATCC, and the aCD3 UCHT1 mAb was from Pharmingen (San Diego, CA).

Techniques: Control, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: Alternative Antigen Receptor (TCR) Signaling in T Cells Derived from ZAP-70-deficient Patients Expressing High Levels of Syk

doi: 10.1074/jbc.m908568199

Figure Lengend Snippet: FIG. 5. Decreased Erk activation in Sykhi/ZAP-702 T Cells stim- ulated via the TCRzCD3 complex. A, cell lysates (1 3 106 cell equiv- alents) were obtained from Sykhi/ZAP-702 and control T cells (CTRL) that were either incubated in medium (–) or stimulated with an aCD3 mAb (1) for 3 min at 37 °C. Lysates were processed as described under “Experimental Procedures” and immunoblotted with a polyclonal Ab that recognizes the doubly phosphorylated forms of Erk1 and Erk2. B, the blot was stripped and reprobed with an aErk2 mAb. C, Erk1 and Erk2 were immunoprecipitated from cell lysates (5 3 106 cell equiva- lents) using aErk1/aErk2 pAbs, and kinase activity was determined by an immune complex kinase assay using MBP as a substrate.

Article Snippet: The aCD3 OKT3 hybridoma line was purchased from the ATCC, and the aCD3 UCHT1 mAb was from Pharmingen (San Diego, CA).

Techniques: Activation Assay, Control, Incubation, Immunoprecipitation, Activity Assay, Immune Complex Kinase Assay

Journal: Journal of Biological Chemistry

Article Title: Alternative Antigen Receptor (TCR) Signaling in T Cells Derived from ZAP-70-deficient Patients Expressing High Levels of Syk

doi: 10.1074/jbc.m908568199

Figure Lengend Snippet: FIG. 7. Defective TCRzCD3-induced proliferation in Sykhi/ZAP- 702 cells. Sykhi/ZAP-702 T cells derived from both ZAP-70-deficient patients and control T cells were stimulated with either immobilized aCD3 mAb (1 mg/ml) or a combination of exogenous IL-2 (100 U/ml) and aCD3 mAbs. After 3 days of culture, cells were pulsed with 1 mCi of [3H]thymidine and harvested 18 h later. Values shown are the mean 6 SD of data obtained in one of three representative experiments. The stimulation index was determined as described under “Experimental Procedures.”

Article Snippet: The aCD3 OKT3 hybridoma line was purchased from the ATCC, and the aCD3 UCHT1 mAb was from Pharmingen (San Diego, CA).

Techniques: Derivative Assay, Control