Journal: Age and ageing

Article Title: Moderate physical activity associated with a higher naïve/memory T-cell ratio in healthy old individuals: potential role of IL15.

doi: 10.1093/ageing/afaa035

Figure Lengend Snippet: Figure 1. Impact of physical activity on T-cell subset distribution, thymic output and B-cell subset distribution. Percentage of (a) naïve CD4 T cells, (b) memory CD4 T cells in sedentary (n = 25) and physically active healthy older participants (n = 25). (c) Association between serum IL-15 levels and peripheral frequency of naïve CD4 T cells (n = 50). Percentage of (d) PTK7+ve

Article Snippet: For phenotypic characterisation of T-cell subset distribution, PBMCs (1 × 106/ml) were stained with a combination of fluorochrome-conjugated antibodies, anti-human CD3-PEcy7 (eBiosciences, clone UCHT1), anti-human CD4 Violet (eBiosciences, clone RPA-T4), anti-human CD8 PE (Immunotools, clone UCHT4), antihuman CCR7 FITC (R and D systems, clone 150,503), anti-human CD45RA APC (Biolegend, clone HI-100) and anti-human PTK7 (Miltenyi Biotech, clone 188B).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: ADAM10 tailors extracellular vesicles for content transfer rather than signaling by contact

doi: 10.64898/2026.02.12.705562

Figure Lengend Snippet: A. Summary table presenting the proteins identified and quantified by mass spectrometry analysis of ADAM10-KO cell lysates compared to control MCF7 cells and their corresponding secreted sEVs. B-C. sEVs secreted by MCF7 cells with knock-out of ADAM10 (A10 KO) or control cells (WT) were isolated by differential ultracentrifugation of conditioned culture media. Cell lysates and sEVs were analyzed by Western blot, testing for several proteins including EphrinB3, BCAM, E-cadherin and PTK7 ( B ), SRC ( C ). Histograms represent mean signal intensities normalized to controls ± SEM, calculated from n independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, n.s. non-significant (multiple unpaired t-test).

Article Snippet: Antibodies directed against SDC1 intracellular domain (D4Y7H) was from (cell signaling #12922, dilution 1/1000), GFP (A11122) from Thermofisher (dilution 1/1000), ADAM17 (abcam, #ab39162, 1/1000 or cell signaling #3976, 1/1000), ADAM10 (abcam, #ab1997, 1/1 000), GAPDH (Proteintech # 10494-1-AP), Flotillin-1 (BD Biosciences, #X11669), Ephrin B3 (santa cruz, # sc-514139), PTK7 (Proteintech, #17799), E-cadherin (R&D Systems, #AF748), BCAM (R&D Systems, #BAF148), STAT3 124H6 (cell signaling #9139), p-STAT3 (cell signaling #9131).

Techniques: Mass Spectrometry, Control, Knock-Out, Isolation, Western Blot

Journal: Cell reports. Medicine

Article Title: Identification and targeting of protein tyrosine kinase 7 (PTK7) as an immunotherapy candidate for neuroblastoma.

doi: 10.1016/j.xcrm.2023.101091

Figure Lengend Snippet: Figure 5. PTK7 CAR T cells have specific in vitro cytotoxicity (A) Twelve-hour flow cytometry cytotoxicity assay. Percentage cytotoxicity = the sum of 7AAD+, annexin V+, and 7AAD+ annexin V+ cells, gated on target cells only. n = 3 donors with n = 2 biological replicates. Four-hour cytotoxicity shown in Figure S5A. (B) Target cells and CAR T cells or mock T cell control were incubated with caspase-3/7 dye and live cell analysis was performed at the indicated time points. Processing definitions of caspase-3/7 activity (green object count) on target cells were determined. n = 1 donor. Error bars represent SD of 4 technical replicates. (legend continued on next page)

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Recombinant Human PTK7 Fc Chimera Protein R&D Systems 9799-TK-050 Recombinant Mouse PTK7 Fc Chimera Protein R&D Systems 10333-TK-050 Recombinant Human IL-2 PeproTech 200-02-50UG Recombinant Human IL-7 PeproTech 200-07-50UG CD3/CD28 Human T-Activator Dynabeads ThermoFisher Scientific 111.31D Polybrene EMD Millipore TR-1003 D-Luciferin PerkinElmer 122799 Accutase Stemcell technologies 7920 Biocytin hydrazide Biotium 90060 Sep-Pak tC18 vacuum cartridge Waters WAT054960 NeutrAvidin agarose resins Thermo Scientific 29201 TMT10plex Thermo Scientific Blasticidin CellGro 30-100-RB Puromycin Gemini 400-128P Doxorubicin Selleck Chemicals S1208 Melphalan Selleck Chemicals S8266 Topotecan Selleck Chemicals/McKesson Pharmaceuticals S9321/1109275 Critical commercial assays EasySep Human T cell Isolation Kit Stemcell technologies 17951 MycoAlert contamination kit Lonza LT07-318 Deposited data DepMap (DepMap, Broad (2022): DepMap 22Q2 Public. figshare.

Techniques: In Vitro, Cytometry, Cytotoxicity Assay, Control, Incubation, Cell Analysis, Activity Assay

Journal: Frontiers in Immunology

Article Title: PTK7-Targeting CAR T-Cells for the Treatment of Lung Cancer and Other Malignancies

doi: 10.3389/fimmu.2021.665970

Figure Lengend Snippet: PTK7-CAR generation, cell-surface expression, and transduction of human T cells. (A) PTK7-CAR was generated by fusing PTK7-specific scFv to the co-stimulatory signaling domain of the 4-1BB (BB-ICD) and activating signaling domain of CD3ζ (CD3ζ-ICD), a T2A ribosomal skipping sequence, and tEGFR was included for the detection of CAR-modified T cells. (B) 293T cells transfected with control or PTK7-CAR constructs express both CAR and the marker gene tEGFR. (C) PTK7-CAR expression on transduced human CD4 + and CD8 + T cells was approximately 40–60% and 20–30%, respectively, as determined by tEGFR and CAR co-staining.

Article Snippet: In some assays, it was conducted in the presence of soluble PTK7 protein (OriGene).

Techniques: Expressing, Transduction, Generated, Sequencing, Modification, Transfection, Control, Construct, Marker, Staining

Journal: Frontiers in Immunology

Article Title: PTK7-Targeting CAR T-Cells for the Treatment of Lung Cancer and Other Malignancies

doi: 10.3389/fimmu.2021.665970

Figure Lengend Snippet: PTK7 is overexpressed on several tumor cell lines. CHO and PTK7-CHO cells served as negative and positive controls, respectively. PTK7 overexpression was observed on NSCLC (H520, H1975, H1299), SCLC (H446, H69), MDA-DB-468 breast cancer (BC), BxPC3 pancreatic cancer (PC), OVCAR3 ovarian cancer (OC) cells. Black and red lines denote the control (secondary antibody alone) and PTK7 staining, respectively.

Article Snippet: In some assays, it was conducted in the presence of soluble PTK7 protein (OriGene).

Techniques: Over Expression, Control, Staining

Journal: Frontiers in Immunology

Article Title: PTK7-Targeting CAR T-Cells for the Treatment of Lung Cancer and Other Malignancies

doi: 10.3389/fimmu.2021.665970

Figure Lengend Snippet: PTK7-CAR T cells release IFN-γ and IL-2 and proliferate in response to PTK7-positive target cells. (A) Control or PTK7-CAR T cells from healthy donors (n = 3) were co-cultured with CHO and PTK7-CHO and various PTK7-expressing tumor cell lines for 24 h before performing IFN-γ and IL-2 ELISA. Mean and SEM are shown. (B) T cells were labeled with CFSE and co-cultured for 3 days with CHO, PTK7-CHO, H520, H1975, or H1299 cells in the absence of exogenous IL-2, and CFSE dilution was analyzed by flow cytometry. A representative histogram from three independent assays is shown.

Article Snippet: In some assays, it was conducted in the presence of soluble PTK7 protein (OriGene).

Techniques: Control, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Labeling, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: PTK7-Targeting CAR T-Cells for the Treatment of Lung Cancer and Other Malignancies

doi: 10.3389/fimmu.2021.665970

Figure Lengend Snippet: PTK7-CAR T cells kill PTK7-positive tumor cell lines. GL-expressing tumor target cells were co-cultured with control or PTK7-CAR T cells at the varying effector-to-target ratios in triplicate wells of white 96-well plates. Target cell viability was monitored 18 h later by using Bright-Glo™ Luciferase Assay System according to the manufacturer’s instructions. The percent lysis (%) was calculated by using the following equation: 1-[bioluminescence value in sample well (target cells + CAR T cells)/maximum bioluminescence value (target cells alone)]. Shown are means ± SEM of % cell killing in triplicate wells.

Article Snippet: In some assays, it was conducted in the presence of soluble PTK7 protein (OriGene).

Techniques: Expressing, Cell Culture, Control, Luciferase, Lysis

Journal: Frontiers in Immunology

Article Title: PTK7-Targeting CAR T-Cells for the Treatment of Lung Cancer and Other Malignancies

doi: 10.3389/fimmu.2021.665970

Figure Lengend Snippet: PTK7-CAR2 T cells retain effector function upon recursive target exposure. (A) Schematics of the long-term cytotoxicity assay. (B) Counts of H520 and H446 target cells after each round of recursive co-culture (rounds 1–3, R1–R3) with control or PTK7-CAR T cells. (C) Counts of control or PTK7-CAR T cells after each round of recursive co-culture with target cells. (D) Intracellular staining for granzyme B of control or PTK7-CAR T cells at the end of round 1 and 3 co-culture with H520 tumor cells. (E) PD-1 expression in control or PTK7-CAR T cells after rounds 1 and 3 of recursive co-culture with H520 tumor cells. (F) Percentage of PD-1 + TIM-3 + cells in control or PTK7-CAR T cells after rounds 1 and 3 of recursive co-culture with H520 tumor cells. Data are shown as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, determined by repeated-measures two-way ANOVA with Tukey’s post hoc test.

Article Snippet: In some assays, it was conducted in the presence of soluble PTK7 protein (OriGene).

Techniques: Cytotoxicity Assay, Co-Culture Assay, Control, Staining, Expressing

Journal: Frontiers in Immunology

Article Title: PTK7-Targeting CAR T-Cells for the Treatment of Lung Cancer and Other Malignancies

doi: 10.3389/fimmu.2021.665970

Figure Lengend Snippet: Systemic treatment with PTK7-CAR2 T cells leads to tumor growth control and increased survival of mice in both human tumor xenograft models. (A) NSG mice were s.c. implanted with H520 or H69 tumor cells, after 7 days, received two intravenous infusion of control or PTK7-CAR2 T cells (5 × 10 6 cells in 100 µl PBS) week apart and tumor growth quantified by measuring tumor size. Data are shown as mean ± SEM (n = 5 mice per group). *P < 0.05, ***P < 0.001, and ****P < 0.0001, determined by repeated-measures two-way ANOVA with Tukey’s post hoc test. (B) Kaplan–Meier survival curves summarizing three independent experiments (n = 11 mice per group). ***P < 0.001, and ****P < 0.0001 determined by log-rank test. (C) Frequency of human CD3 + tEGFR + CAR T cells in the peripheral blood collected 10 days after T cell infusion or at the end of experiment. Data are shown as mean ± SEM (n = 4 mice per group). **P < 0.01 and ***P < 0.001, determined by repeated-measures two-way ANOVA with Tukey’s post hoc test. (D) Representative IHC images and quantification of T-cell infiltration in tumor tissues (n = 3) from treated mice harvested at the end of experiment. Scale bars, 100 µm. Data are shown as mean ± SEM (n = 3 mice per group). ***P < 0.001, determined by repeated-measures two-way ANOVA with Tukey’s post hoc test.

Article Snippet: In some assays, it was conducted in the presence of soluble PTK7 protein (OriGene).

Techniques: Control

Journal: Frontiers in Immunology

Article Title: PTK7-Targeting CAR T-Cells for the Treatment of Lung Cancer and Other Malignancies

doi: 10.3389/fimmu.2021.665970

Figure Lengend Snippet: PTK7-CAR2 T cells do not mediate detectable on-target off-tumor toxicity. Control or PTK7-CAR T cells were tested reactivity against a panel of primary human normal epithelial cells or HUVECs in the cytotoxicity assays at the indicated effector-to-target ratios. Shown are mean ± SEM of % cell killing in triplicate wells. **P < 0.01, determined by repeated-measures two-way ANOVA with Tukey’s post hoc test.

Article Snippet: In some assays, it was conducted in the presence of soluble PTK7 protein (OriGene).

Techniques: Control

Journal: Cell reports. Medicine

Article Title: Identification and targeting of protein tyrosine kinase 7 (PTK7) as an immunotherapy candidate for neuroblastoma.

doi: 10.1016/j.xcrm.2023.101091

Figure Lengend Snippet: Figure 5. PTK7 CAR T cells have specific in vitro cytotoxicity (A) Twelve-hour flow cytometry cytotoxicity assay. Percentage cytotoxicity = the sum of 7AAD+, annexin V+, and 7AAD+ annexin V+ cells, gated on target cells only. n = 3 donors with n = 2 biological replicates. Four-hour cytotoxicity shown in Figure S5A. (B) Target cells and CAR T cells or mock T cell control were incubated with caspase-3/7 dye and live cell analysis was performed at the indicated time points. Processing definitions of caspase-3/7 activity (green object count) on target cells were determined. n = 1 donor. Error bars represent SD of 4 technical replicates. (legend continued on next page)

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Recombinant Human PTK7 Fc Chimera Protein R&D Systems 9799-TK-050 Recombinant Mouse PTK7 Fc Chimera Protein R&D Systems 10333-TK-050 Recombinant Human IL-2 PeproTech 200-02-50UG Recombinant Human IL-7 PeproTech 200-07-50UG CD3/CD28 Human T-Activator Dynabeads ThermoFisher Scientific 111.31D Polybrene EMD Millipore TR-1003 D-Luciferin PerkinElmer 122799 Accutase Stemcell technologies 7920 Biocytin hydrazide Biotium 90060 Sep-Pak tC18 vacuum cartridge Waters WAT054960 NeutrAvidin agarose resins Thermo Scientific 29201 TMT10plex Thermo Scientific Blasticidin CellGro 30-100-RB Puromycin Gemini 400-128P Doxorubicin Selleck Chemicals S1208 Melphalan Selleck Chemicals S8266 Topotecan Selleck Chemicals/McKesson Pharmaceuticals S9321/1109275 Critical commercial assays EasySep Human T cell Isolation Kit Stemcell technologies 17951 MycoAlert contamination kit Lonza LT07-318 Deposited data DepMap (DepMap, Broad (2022): DepMap 22Q2 Public. figshare.

Techniques: In Vitro, Cytometry, Cytotoxicity Assay, Control, Incubation, Cell Analysis, Activity Assay