Journal: Molecular therapy. Methods & clinical development

Article Title: Autologous antigen-presenting cells efficiently expand piggyBac transposon CAR-T cells with predominant memory phenotype.

doi: 10.1016/j.omtm.2021.03.011

Figure Lengend Snippet: Figure 4. PB-HER2-CAR-T cells have robust and sustained antitumor activity against HER2-positive tumors in vitro (A) An impedance-based tumor-cell-killing assay was used to evaluate PB-HER2-CAR-T cells. Tumor cells (U- 2OS) were plated, and 24 h later, CAR-T cells were added at different effector-to-target (E:T) ratios. Real-time impedance traces of tumor cells without treatment (green) and with co-culture of CAR-T cells (magenta, blue, and red) were shown. Each experiment was duplicated, and mean ± SD of the normalized cell index at each time point is shown in the graph. (B) Antitumor efficacy of PB-HER2- CAR-T cells against HER2-positive tumor cell lines. Real- time impedance traces of tumor cells without treatment (green) and tumor cells co-cultured with PB-CD19-CAR-T cells (blue) or PB-HER2-CAR-T cells (red) at an E:T ratio of 1:1 were acquired for 72 h. (C) Sequential killing assay of PB-HER2-CAR-T cells. Real-time impedance traces of tumor cells without treatment (green) and tumor cells co- cultured with PB-HER2-CAR-T cells (red) were acquired for 72 h in each round. Mean ± SD of normalized cell index at 72 h in each round were also evaluated (n = 3). (D) The expression of HER2-CAR molecules on T cells during sequential co-culture. Representative dot plots are shown. (E) Mean fluorescent Index of HER2-CAR expression on T cells during sequential co-culture. (F) The expression of PD-1, TIM-3, and LAG3 on HER2-CAR-T cells during sequential co-culture.

Article Snippet: Cell-surface expression of the HER2-CAR molecules on PB-HER2CAR-T cells or CD19-CAR molecules on PB-CD19-CAR-T cells was determined using the flow cytometry using recombinant human ErbB2/HER2-Fc chimera protein or recombinant human CD19 Fc chimera protein, respectively (R&D Systems, Minneapolis, MN, USA), followed by a goat anti-human IgG Fc fragment-specific antibody conjugated to fluorescein isothiocyanate (FITC; Merck Millipore, Burlington, MA, USA).

Techniques: Activity Assay, In Vitro, Co-Culture Assay, Cell Culture, Expressing

Journal: Molecular therapy. Methods & clinical development

Article Title: Autologous antigen-presenting cells efficiently expand piggyBac transposon CAR-T cells with predominant memory phenotype.

doi: 10.1016/j.omtm.2021.03.011

Figure Lengend Snippet: Figure 5. PB-HER2-CAR-T cells efficiently control HER2-positive tumor in vivo (A) Schematics of in vivo study and tumor re-challenge experiments in the HER2-positive tumor xenograft murine model. SJCRH30-FFluc was subcutaneously injected at the dorsal wall of the mice to form a tumor. One week after tumor engraftment, mice intravenously received either vehicle, approximately 6 106 of PB-CD19-CAR-T cells, or PB-HER2-CAR-T cells. (B) Tumor growth was measured as bioluminescence signal intensity (BLI) and expressed as total flux (p/s). The BLI images of each mouse and the tumor volumes measured as total flux (p/s) are shown. The PB-HER2-CAR-T cells group demon- strated a statistically significant tumor reduction measured as the mean total flux at day 28 (*), compared to the PB- CD19-CAR-T cells group (Mann-Whitney U test, p < 0.01). The SJCRH30-FFLuc tumor cells were re-inocu- lated into the corresponding contralateral thoracic wall of the mice on day 28 after the primary tumor inoculation. BLI images on days 7 and 14 after tumor re-challenges are indicated by arrowheads. (C) Blood samples were collected from each mouse on day 42 after primary tumor inoculation, and the presence of PB-HER2-CAR-T cells was assessed using flow cytometry. HER2CAR+/human CD3 population could be explained as caused by the non-specific binding of anti-human IgG-Fc antibody to mouse PBMCs, as shown in Figure S11.

Article Snippet: Cell-surface expression of the HER2-CAR molecules on PB-HER2CAR-T cells or CD19-CAR molecules on PB-CD19-CAR-T cells was determined using the flow cytometry using recombinant human ErbB2/HER2-Fc chimera protein or recombinant human CD19 Fc chimera protein, respectively (R&D Systems, Minneapolis, MN, USA), followed by a goat anti-human IgG Fc fragment-specific antibody conjugated to fluorescein isothiocyanate (FITC; Merck Millipore, Burlington, MA, USA).

Techniques: Control, In Vivo, Injection, MANN-WHITNEY, Cytometry, Binding Assay

Journal: Nature Communications

Article Title: RNA polymerase II pausing factor NELF in CD8 + T cells promotes antitumor immunity

doi: 10.1038/s41467-022-29869-2

Figure Lengend Snippet: a , b Percentages of CD62L + CD45RA + cells within CD4 + ( a ) and CD8 + ( b ) populations of 28z or 28z-hNELFB CAR-T cells after 10 days of in vitro expansion, n = 4/group. c Survival curves of Raji tumor-bearing mice receiving PBS or mock T cells, CD19 CAR-28z T cells, or CD19 CAR-28z-hNELFB T cells. d Model of enhancer–promoter looping mediated by NELFB, Pol II, and TCF1. Mean differences were compared using Student’s t -test. Log-rank (Mantel–Cox) tests were used for survival analyses. Source data are provided as a Source Data file.

Article Snippet: In vitro expanded cells were stained by FITC-labeled human CD19 (ACROBiosystems, CD9HF2H225UG) and subsequently purified by Anti-FITC MicroBeads (Miltenyi Biotec, 130-048-701) before the detection of NELFB overexpression by western blot or CAR expression by FITC-labeled human CD19.

Techniques: In Vitro

Journal: Immuno-Oncology and Technology

Article Title: Overcoming antigen loss in CAR T therapy with Vγ9Vδ2 CAR T-cells

doi: 10.1016/j.iotech.2025.101053

Figure Lengend Snippet: Antigen-specific expansion of Vγ9Vδ2 and αβ T-cells transduced with a CD19-specific CAR. (A) Contour plots of a representative donor showing the percentages of CAR-positive cells in Vγ9Vδ2 T-cells expanded with either IL-2 (black) or IL-2/IL-15 (red) and αβ T-cell (blue) cultures before the Ag-specific expansion started, and after 7 and 14 days in a CD19-coated plate. (B) Percentages of CAR-positive T-cells in all cultures through the 14-day Ag-specific expansion. Bars represent the mean value, and error bars show the standard deviation ( n = 6, three independent experiments). Ag, antigen; CAR, chimeric antigen receptor; IL, interleukin.

Article Snippet: After 1 week, the cells were transferred to a plate coated with a recombinant human CD19 protein (1 μg/ml, R&D Systems, Minneapolis, MN) for 2 weeks to specifically expand the engineered CAR T-cells.

Techniques: Transduction, Standard Deviation

Journal: Immuno-Oncology and Technology

Article Title: Overcoming antigen loss in CAR T therapy with Vγ9Vδ2 CAR T-cells

doi: 10.1016/j.iotech.2025.101053

Figure Lengend Snippet: Antitumor effect of Vγ9Vδ2 and αβ CAR T-cells against CD19-positive tumor cell lines. (A) Intracellular staining analysis was used to determine the levels of the degranulation marker CD107a and the cytokines TNF-α and IFN-γ in Vγ9Vδ2 T-cells expanded with either IL-2 (black) or IL-2/IL-15 (red) and αβ T-cell (blue) cultures after a 5-h co-culture with the CD19-positive tumor cell lines K562-CD19, Daudi and Nalm-6 ( n = 4, three independent experiments). (B) Representative plot for long-term cytotoxicity of Vγ9Vδ2 T-cells expanded with either IL-2 or IL-2/IL-15 and αβ T-cell cultures against the CD19-positive cell lines K562-CD16 (left plot), Daudi (middle plot) and Nalm-6 (right plot). Data are presented as the average of triplicates. One-way analysis of variance (ANOVA) with Tukey multiple comparisons test was used to compare the different curves. (C) Time to kill 50% of the tumor cells (KT50) was compared between Vγ9Vδ2 T-cells expanded with either IL-2 or IL-2/IL-15 and αβ T-cell cultures. Each donor is represented with a different symbol, and lines represent the average of all ( n = 4, three independent experiments). Two-way ANOVA with Tukey multiple comparisons test was used to compare the different cultures. (D) Proliferation of Vγ9Vδ2 T-cells expanded with either IL-2 or IL-2/IL-15 and αβ T cell upon encountering the cell lines Daudi or Nalm-6. Stacked bar graphs represent the percentage of cells in each generation after a 5-day co-culture with the tumor cells. Bars represent the mean value, and error bars show the standard deviation ( n = 4, three independent experiments). CAR, chimeric antigen receptor; IFN, interferon; IL, interleukin; TNF-α, tumor necrosis factor-α. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: After 1 week, the cells were transferred to a plate coated with a recombinant human CD19 protein (1 μg/ml, R&D Systems, Minneapolis, MN) for 2 weeks to specifically expand the engineered CAR T-cells.

Techniques: Staining, Marker, Co-Culture Assay, Standard Deviation

Journal: Immuno-Oncology and Technology

Article Title: Overcoming antigen loss in CAR T therapy with Vγ9Vδ2 CAR T-cells

doi: 10.1016/j.iotech.2025.101053

Figure Lengend Snippet: Antitumor effect of Vγ9Vδ2 and αβ CAR T-cells against CD19-negative tumor cell lines. (A) Intracellular staining analysis was used to determine the levels of the degranulation marker CD107a and the cytokines TNF-α and IFN-γ in Vγ9Vδ2 T-cells expanded with either IL-2 (black) or IL-2/IL-15 (red) and αβ T-cell (blue) cultures after a 5-h co-culture with the CD19-negative tumor cell lines K562 and Molm-14 ( n = 4, three independent experiments). (B) Representative plot for long-term cytotoxicity of Vγ9Vδ2 T-cells expanded with either IL-2 or IL-2/IL-15 and αβ T-cell cultures against the CD19-negative cell lines K562 (left plot) and Molm-14 (right plot). Data are presented as the average of triplicates. One-way analysis of variance (ANOVA) with Tukey multiple comparisons test was used to compare the different curves. (C) Time to kill 20% of the tumor cells (KT20) was compared between Vγ9Vδ2 T-cells expanded with either IL-2 or IL-2/IL-15 and αβ T-cell cultures. Each donor is represented with a different symbol, and lines represent the average of all ( n = 4, three independent experiments). One-way ANOVA with Tukey multiple comparisons test was used to compare the different curves. (D) Proliferation of Vγ9Vδ2 T-cells expanded with either IL-2 or IL-2/IL-15 and αβ T-cell upon encountering the cell lines K562 and Molm-14. Stacked bar graphs represent the percentage of cells in each generation after a 5-day co-culture with the tumor cells. Bars represent the mean value, and error bars show the standard deviation ( n = 4, three independent experiments). CAR, chimeric antigen receptor; IFN, interferon; IL, interleukin; TNF-α, tumor necrosis factor-α. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: After 1 week, the cells were transferred to a plate coated with a recombinant human CD19 protein (1 μg/ml, R&D Systems, Minneapolis, MN) for 2 weeks to specifically expand the engineered CAR T-cells.

Techniques: Staining, Marker, Co-Culture Assay, Standard Deviation

Journal: Immuno-Oncology and Technology

Article Title: Overcoming antigen loss in CAR T therapy with Vγ9Vδ2 CAR T-cells

doi: 10.1016/j.iotech.2025.101053

Figure Lengend Snippet: Antitumor effect of Vγ9Vδ2 and αβ CAR T-cells during antigen loss. (A) Long-term cytotoxicity of Vγ9Vδ2 T-cells expanded with either IL-2 or IL-2/IL-15 and αβ T-cell cultures against mixed tumor population of the CD19-negative cell line K562 and the CD19-positive cell line K562-CD19. Data are presented as the average of duplicates. One-way analysis of variance with Tukey multiple comparisons test was used to compare the different curves. (B) Time to kill 20% of the tumor cells (KT20) was compared between Vγ9Vδ2 T-cells expanded with either IL-2 or IL-2/IL-15 and αβ T-cell cultures. Dots represent average, and error bars represent the range ( n = 3, two independent experiments). CAR, chimeric antigen receptor; IL, interleukin. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: After 1 week, the cells were transferred to a plate coated with a recombinant human CD19 protein (1 μg/ml, R&D Systems, Minneapolis, MN) for 2 weeks to specifically expand the engineered CAR T-cells.

Techniques: