Journal: Cell Reports Medicine

Article Title: Selective refueling of CAR T cells using ADA1 and CD26 boosts antitumor immunity

doi: 10.1016/j.xcrm.2024.101530

Figure Lengend Snippet:

Article Snippet: PE adenosine deaminase Antibody , Santa Cruz , Cat# sc-28346; RRID: AB_626634.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, LDH Cytotoxicity WST Assay, Chemotaxis Assay, cDNA Synthesis, Modification, Membrane, Software

Journal: Cell Reports Medicine

Article Title: Selective refueling of CAR T cells using ADA1 and CD26 boosts antitumor immunity

doi: 10.1016/j.xcrm.2024.101530

Figure Lengend Snippet: Rational design and implementation of ADA1 in CAR T cells (A) Four different versions of ADA1 were designed for use in CAR T cell therapy. (B) Human PBMCs ( n = 3) were stimulated with 100 μM ADO, 5 μg/mL coated caveolin-1 protein (CD26), 1 μg/mL coated OKT3 mAb (CD3), combined CD3/ADO, or combined CD3/CD26. After 48 h, ecto-ADA1 was measured by ADA activity assay. (C) HER2-MR-CAR T cells ( n = 3) were stimulated with 1 μg/mL coated OKT3 mAb (CD3), 10 μg/mL coated mAb 134-2C2 (CD26), or combined CD3/CD26. After 48 h, cell culture medium was subjected to ADA1 ELISA. p = 0.0352 for CD3/CD26 vs. CD3/CD28. (D) HER2-MR-CAR T cells ( n = 3), HER2-CAR T cells, or NT cells were cultured at indicated density for 24 h. After incubation, IFN-γ was measured using ELISA. The experiments were conducted in triplicate. ∗ p value for HER2-MR-CAR T cells + A549 vs. HER2-CAR T cells + A549. (E) mRNA sequencing ( n = 3) was used to analyze inflammatory cytokines, granzyme A, and granzyme B. (F) RT-qPCR was used to measure IFN-γ ( n = 3). (G) mA.26.HER2 ( n = 3) or NT cells were cultured either with or without A549 tumor cells, while mA.26.GPC3 or NT cells were cultured either with or without Huh7 cells. 24 h later, IFN-γ were measured using ELISA. p = 0.0000008 and p = 0.000236 for mA.26.HER2 and mA.26.GPC3 vs. NT. p = 0.0000003 for mA.26.HER2 vs. NT (A549). p = 0.0000001 for mA.26.GPC3 vs. NT (Huh7). Error bars represent SEM. p values were determined by two-tailed t test.

Article Snippet: The antibodies used for T cell phenotyping and cytokine production analyses are: CD3-BV785 (BioLegend, 317329), CD8-Pacific Blue (BioLegend, 344717), CCR2-BUV395 (BD, 747854), CCR5-FITC (BioLegend, 313705), CD26-FITC (BD, 555436), ADA1-PE (Santa Cruz, sc-28346), PD-1-Alexa flour 647 (BD, 566851), PD-1-APC (BioLegend, 329908), LAG-3-Pacific Blue (BioLegend, 369341), TIM-3-PE(BD Biosciences, 565570), TIGIT-AF647(R&D Systems, FAB7898N), IL2-BV711 (BD #563946), IFN gama-PE-CF594 (BD, 562392), GZMB-APC (BioLegend, 372203), Perforin-BV711 (BioLegend, 08129), T-bet-BV785 (BioLegend, 644835), IFN-γ-BV570 (BioLegend, 502534) and GZMB-PE (BioLegend, 372208).

Techniques: Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Sequencing, Quantitative RT-PCR, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Selective refueling of CAR T cells using ADA1 and CD26 boosts antitumor immunity

doi: 10.1016/j.xcrm.2024.101530

Figure Lengend Snippet: Overexpression of CD26 resisted TGF-β1 suppression and promoted CAR T cell mobility and proliferation (A) Rv-CD26-transduced T cells were cultured in the presence of TGF-β1 for 48 h, and CD26 expression was detected by flow cytometry. The experiments were conducted in duplicate. (B and C) CCR2 and CCR5 expression was determined by flow cytometry. The experiments were conducted in duplicate. (D) The heatmap shows the expression levels of chemokine receptor genes. Triplicate samples ( n = 3) were used for each group and are represented on the x axis. (E and F) Rv-CD26-transduced T cells ( n = 3) were subjected to a fluorescent migration assay and Transwell migration assay. (G) PBMCs ( N = 2) were transduced with retroviral vectors expressing HER2-CAR (HER2-CAR), ADA1.CD3scFv and HER2-CAR (ADA1.CD3scFv-HER2-CAR), CD26 and HER2-CAR (CD26-HER2-CAR), or ADA1.CD3scFv/CD26 and HER2-CAR (HER2-MRCAR) and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). The T cells were then cultured for 4 days and subjected to flow analysis. (H) GPC3-MR-CAR T cells and GPC3-CAR T cells were expanded in vitro , and the cell numbers were determined at different time points using a hemocytometer. The results are presented as a growth curve. The figure indicates the p values for GPC3-MR-CAR vs. GPC3-CAR. The experiments were conducted in triplicate. Error bars represent SEM. p values were determined by two-tailed t test.

Article Snippet: The antibodies used for T cell phenotyping and cytokine production analyses are: CD3-BV785 (BioLegend, 317329), CD8-Pacific Blue (BioLegend, 344717), CCR2-BUV395 (BD, 747854), CCR5-FITC (BioLegend, 313705), CD26-FITC (BD, 555436), ADA1-PE (Santa Cruz, sc-28346), PD-1-Alexa flour 647 (BD, 566851), PD-1-APC (BioLegend, 329908), LAG-3-Pacific Blue (BioLegend, 369341), TIM-3-PE(BD Biosciences, 565570), TIGIT-AF647(R&D Systems, FAB7898N), IL2-BV711 (BD #563946), IFN gama-PE-CF594 (BD, 562392), GZMB-APC (BioLegend, 372203), Perforin-BV711 (BioLegend, 08129), T-bet-BV785 (BioLegend, 644835), IFN-γ-BV570 (BioLegend, 502534) and GZMB-PE (BioLegend, 372208).

Techniques: Over Expression, Cell Culture, Expressing, Flow Cytometry, Migration, Transwell Migration Assay, Transduction, Retroviral, Labeling, In Vitro, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Selective refueling of CAR T cells using ADA1 and CD26 boosts antitumor immunity

doi: 10.1016/j.xcrm.2024.101530

Figure Lengend Snippet: ADA1.CD3scFv enhances CAR T cell expansion preferentially without impacting tumor cells (A and B) Luciferase reporter Jurkat-Dual or Jurkat-NFAT cells either expressing CD26 or lacking CD26 were transduced with overexpressing vectors of ADA1 or ADA1.CD3scFv and cultured for 24 h. After incubation, the cells ( n = 3) were subjected to an ADA activity assay. CD26-positive Jurkat T cells transduced with ADA1.CD3scFv overexpressing vector were used as the maximum value to calculate the percentage. The data are presented as the percentage of ADA1 or ADA1.CD3scFv binding with the Jurkat T cells. The figure indicates the p values for the binding of ADA1 with CD26 + Jurkat T cells vs. the binding of ADA1.CD3scFv with CD26 + Jurkat T cells. (C) Jurkat T cells ( n = 3) were transduced with retroviral vector expressing ADA1, ADA1.CD3scFv, CD26, ADA1, and CD26 or ADA1.CD3scFv and CD26, respectively. Jurkat T cells were labeled with CFSE, and cell proliferation was measured by flow analysis. The experiments were conducted in duplicate. (D) The conditioned medium collected from the culture of HER2-MR-CAR T cells, HER2-CAR T cells, or NT human T cells was added to A549 cell cultures ( n = 3) and incubated for 24 or 48 h. The numbers of tumor cells were quantified daily using a hemocytometer. p = 0.000002 for ADA1 vs. ADA1.CD3scFv at both 24 and 48 h. (E and F) HER2-MR-CAR T cells or HER2-CAR T cells were co-cultured with A549 tumor cells in a Transwell plate for 72 h. Similarly, GPC3-MR-CAR T cells or GPC3-CAR T cells were co-cultured with Huh7 tumor cells. The CAR T cells and tumor cells were counted using a hemocytometer and are presented. (G) 293T cells were transduced with retroviral vectors expressing either ADA1 or ADA1.CD3scFv. After 48 h of culture, the cell culture medium was collected and added to the culture of CD26-negative or CD26-positive Jurkat-NFAT T cells for 24 h. The Jurkat-NFAT cells were then subjected to luciferase activity assay. p = 0.0034 for ADA1 vs. ADA1.CD3scFv in CD26-negative Jurkat T cells. p = 0.0000003 for ADA1.CD3scFv in CD26-positive Jurkat T cells vs. CD26-negative Jurkat T cells. The experiments were conducted in triplicate. Error bars represent SEM. p values were determined by two-tailed t test.

Article Snippet: The antibodies used for T cell phenotyping and cytokine production analyses are: CD3-BV785 (BioLegend, 317329), CD8-Pacific Blue (BioLegend, 344717), CCR2-BUV395 (BD, 747854), CCR5-FITC (BioLegend, 313705), CD26-FITC (BD, 555436), ADA1-PE (Santa Cruz, sc-28346), PD-1-Alexa flour 647 (BD, 566851), PD-1-APC (BioLegend, 329908), LAG-3-Pacific Blue (BioLegend, 369341), TIM-3-PE(BD Biosciences, 565570), TIGIT-AF647(R&D Systems, FAB7898N), IL2-BV711 (BD #563946), IFN gama-PE-CF594 (BD, 562392), GZMB-APC (BioLegend, 372203), Perforin-BV711 (BioLegend, 08129), T-bet-BV785 (BioLegend, 644835), IFN-γ-BV570 (BioLegend, 502534) and GZMB-PE (BioLegend, 372208).

Techniques: Luciferase, Expressing, Transduction, Cell Culture, Incubation, Activity Assay, Plasmid Preparation, Binding Assay, Retroviral, Labeling, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Selective refueling of CAR T cells using ADA1 and CD26 boosts antitumor immunity

doi: 10.1016/j.xcrm.2024.101530

Figure Lengend Snippet: MR-CAR T cells demonstrated antitumor activity in xenograft mouse models (A–C) A549 tumor-bearing mice ( n = 5) were treated with a single dose of either 5 × 10 6 or 1 × 10 7 or two doses of 2 × 10 6 (at 1 week intervals) HER2-MR-CAR T cells, HER2-CAR T cells, or PBS. Tumor size and mouse body weight were monitored every 2–3 days. Data represent mean ± SD ( n = 5). (D–F) At week 0, mice ( n = 10) were intercostally injected with 2 × 10 6 A549-luc tumor cells. One week after tumor implantation, the mice were administered either 2 × 10 6 T cells or PBS through the tail vein. Tumor development was monitored weekly using bioluminescence in vivo imaging (D). Mean photon count with SDs of mice groups is shown at the indicated time points (E). p = 0.04513, 0.01437, or 0.002137 for MR-CAR vs. ADA1.CD3scFv-HER2-CAR at weeks 9, 10, or 11 individually. Mouse survival was monitored (F). p = 0.0177 for HER2-MR-CAR vs. HER2-CAR in survival. (G and H) A murine HCC xenograft model was established in NSG mice ( n = 5) by subcutaneous inoculation of 2 × 10 6 Huh7 tumor cells on the right flank. When the average tumor size reached 4–6 mm in diameter, experimental mice were treated with a single dose of 2 × 10 6 GPC3-MR-CAR T cells, 2 × 10 6 GPC3-CAR T cells, or PBS. Tumor size and mouse body weight were monitored every 2–3 days. Data represent mean ± SD ( n = 5). p = 0.0209 and p = 0.00191 for GPC3-MR-CAR T cells vs. GPC3-CAR T cells on days 9 and 13 respectively. No difference in body weight was observed among groups. Error bars represent SEM. p values were determined by two-tailed t test.

Article Snippet: The antibodies used for T cell phenotyping and cytokine production analyses are: CD3-BV785 (BioLegend, 317329), CD8-Pacific Blue (BioLegend, 344717), CCR2-BUV395 (BD, 747854), CCR5-FITC (BioLegend, 313705), CD26-FITC (BD, 555436), ADA1-PE (Santa Cruz, sc-28346), PD-1-Alexa flour 647 (BD, 566851), PD-1-APC (BioLegend, 329908), LAG-3-Pacific Blue (BioLegend, 369341), TIM-3-PE(BD Biosciences, 565570), TIGIT-AF647(R&D Systems, FAB7898N), IL2-BV711 (BD #563946), IFN gama-PE-CF594 (BD, 562392), GZMB-APC (BioLegend, 372203), Perforin-BV711 (BioLegend, 08129), T-bet-BV785 (BioLegend, 644835), IFN-γ-BV570 (BioLegend, 502534) and GZMB-PE (BioLegend, 372208).

Techniques: Activity Assay, Injection, Tumor Implantation, In Vivo Imaging, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Selective refueling of CAR T cells using ADA1 and CD26 boosts antitumor immunity

doi: 10.1016/j.xcrm.2024.101530

Figure Lengend Snippet:

Article Snippet: The antibodies used for T cell phenotyping and cytokine production analyses are: CD3-BV785 (BioLegend, 317329), CD8-Pacific Blue (BioLegend, 344717), CCR2-BUV395 (BD, 747854), CCR5-FITC (BioLegend, 313705), CD26-FITC (BD, 555436), ADA1-PE (Santa Cruz, sc-28346), PD-1-Alexa flour 647 (BD, 566851), PD-1-APC (BioLegend, 329908), LAG-3-Pacific Blue (BioLegend, 369341), TIM-3-PE(BD Biosciences, 565570), TIGIT-AF647(R&D Systems, FAB7898N), IL2-BV711 (BD #563946), IFN gama-PE-CF594 (BD, 562392), GZMB-APC (BioLegend, 372203), Perforin-BV711 (BioLegend, 08129), T-bet-BV785 (BioLegend, 644835), IFN-γ-BV570 (BioLegend, 502534) and GZMB-PE (BioLegend, 372208).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, LDH Cytotoxicity WST Assay, Chemotaxis Assay, cDNA Synthesis, Modification, Membrane, Software