b16f0 mouse melanoma cell line  (ATCC)

Journal: Frontiers in Immunology

Article Title: ATG5-mediated inducible autophagy sustains CAR-T cell durability under solid tumor stress

doi: 10.3389/fimmu.2026.1720544

Figure Lengend Snippet: In vitro cytotoxicity of ATG5 or LC3b OE mCAR-T cells under an immunosuppressive TME–mimicking condition. (A) Schematic overview of the in vitro long-term cytotoxicity assay. mCAR-T cells were co-cultured with B16F0-hCD19-mCherry tumor cells at various effector-to-target (E:T) ratios (0.5:1 to 4:1) for 96 h in the presence of TGF-β (5 ng/mL) to mimic an immunosuppressive TME. Tumor cell confluency was quantified based on mCherry fluorescence intensity using a CellCyte™ live-cell imaging system. (B) Cytotoxicity kinetics of each mCAR-T cell group over time across different E:T ratios. Data represent mean ± SEM (n = 3). Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test to compare the main effects among pMIG, ATG5 OE, and LC3b OE CAR-T cells within each E:T ratio. *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: The B16F0 (mouse melanoma) cell line was purchased from the American Type Culture Collection (ATCC), and the Plat-E (ecotropic retroviral packaging) cell line was obtained from Cell Biolabs (San Diego, CA, USA).

Techniques: In Vitro, Cytotoxicity Assay, Cell Culture, Fluorescence, Live Cell Imaging

Journal: Frontiers in Immunology

Article Title: ATG5-mediated inducible autophagy sustains CAR-T cell durability under solid tumor stress

doi: 10.3389/fimmu.2026.1720544

Figure Lengend Snippet: ATG5 OE reduces oxidative stress while enhancing effector function of mCAR-T cells under TGF-β–mediated immunosuppressive conditions. (A) Schematic illustration of the experimental design for assessing oxidative stress and effector functional characteristics. B16F0-hCD19 target cells (1 × 10 5 cells) were co-cultured with mCAR-T cells at an effector-to-target (E:T) ratio of 1:1 in the presence of TGF-β (5 ng/mL) and IL-2 (100 IU/mL). Intracellular cytokine staining (ICS) assays were performed in separate wells supplemented with GolgiPlug™, whereas ROS measurements and phenotypic analyses were conducted in parallel wells without GolgiPlug™. After 24 h of co-culture, cells were harvested and subjected to flow cytometric analysis. (B–E) Assessment of intracellular and mitochondrial ROS in mCAR-T cells co-cultured with B16F0-hCD19 tumor cells in the presence of TGF-β (5 ng/mL). (B) Representative histograms of total cellular ROS measured by CellROX™ Deep Red staining. (C) Quantification of cellular ROS levels expressed as mean fluorescence intensity (MFI ×10³). (D) Representative histograms of mitochondrial superoxide measured by MitoSOX™ Red staining. (E) Quantification of mitochondrial ROS levels expressed as MFI (×10²). In panels (C, E) , gray dotted lines indicate the basal status without tumor co-culture. (F–I) Functional characterization of mCAR-T cells following co-culture with B16F0-hCD19 tumor cells under TGF-β–mediated immunosuppressive conditions. (F) Representative histograms of intracellular perforin staining. (G) Quantification of perforin expression expressed as MFI (×10³). (H) Representative flow cytometry plots showing IFN-γ and TNF-α expression in pMIG, ATG5 OE, and LC3b OE mCAR-T cells. (I) Quantification of TNF-α + IFN-γ + cells among OE mCAR-T cells. (J, K) Analysis of exhaustion marker expression. (J) Representative flow cytometry plots showing PD-1 and TIM-3 expression in OE mCAR-T cells. (K) Quantification of PD-1 + TIM-3 + cells among OE mCAR-T cells. Individual replicates are shown as dots, and bars indicate mean ± SEM (n = 3). Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Supplementary Figure 2 .

Article Snippet: The B16F0 (mouse melanoma) cell line was purchased from the American Type Culture Collection (ATCC), and the Plat-E (ecotropic retroviral packaging) cell line was obtained from Cell Biolabs (San Diego, CA, USA).

Techniques: Functional Assay, Cell Culture, Staining, Co-Culture Assay, Fluorescence, Expressing, Flow Cytometry, Marker

Journal: Frontiers in Immunology

Article Title: ATG5-mediated inducible autophagy sustains CAR-T cell durability under solid tumor stress

doi: 10.3389/fimmu.2026.1720544

Figure Lengend Snippet: ATG5 OE mCAR-T cells exhibit enhanced in vivo antitumor efficacy with irradiation preconditioning. (A) Schematic overview of the in vivo experimental design. C57BL/6 mice were subcutaneously inoculated with B16F0-hCD19 tumor cells (3 × 10 5 cells). In the IR group, whole-body irradiation (5 Gy) was administered on day 6, followed by adoptive transfer of hCD19 mCAR-T cells (5–7.5 × 10 6 cells) on day 7. In the Non-IR group, mCAR-T cells were adoptively transferred without irradiation. Tumor size was monitored at the indicated time points. (B) Kaplan–Meier survival analysis. Survival curves of tumor-bearing mice treated with untransduced CD8 + T cells (UTD), pMIG control, or ATG5 OE mCAR-T cells under Non-IR (left, dashed lines) or IR (right, solid lines) conditions. A tumor size ≥ 225 mm² was used as a surrogate endpoint for survival analysis for humane reasons. Statistical significance was assessed using the log-rank (Mantel-Cox) test, followed by Holm–Šídák’s multiple comparisons test for group comparisons. (C) Tumor growth kinetics. Mean tumor growth curves under Non-IR (left) and IR (right) conditions. Data are presented as mean ± SEM. Statistical significance was determined using two-way ANOVA with Tukey’s post hoc test. (D) Individual tumor growth trajectories. Tumor growth curves for individual mice treated with UTD, pMIG, or ATG5 OE mCAR-T cells under Non-IR (upper panels) and IR (lower panels) conditions. Group sizes were as follows: Non-IR, n = 15, 10, and 11; IR, n = 16, 10, and 12 for UTD CD8 + T cells, pMIG, and ATG5 OE mCAR-T cells, respectively. (E) Comparison of tumor sizes at defined time points. Tumor sizes measured on day 15 (Non-IR) and day 17 (IR). Each dot represents an individual mouse, and horizontal lines indicate mean values. Data are presented as mean ± SEM. Group sizes were as follows: Non-IR, n = 12, 8, and 10; IR, n = 11, 10, and 12 for UTD CD8 + T cells, pMIG, and ATG5 OE mCAR-T cells, respectively. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, *** p < 0.0001. .

Article Snippet: The B16F0 (mouse melanoma) cell line was purchased from the American Type Culture Collection (ATCC), and the Plat-E (ecotropic retroviral packaging) cell line was obtained from Cell Biolabs (San Diego, CA, USA).

Techniques: In Vivo, Irradiation, Adoptive Transfer Assay, Control, Comparison

Journal: Frontiers in Immunology

Article Title: ATG5-mediated inducible autophagy sustains CAR-T cell durability under solid tumor stress

doi: 10.3389/fimmu.2026.1720544

Figure Lengend Snippet: ATG5 OE enhances the functional activity of mCAR-T cells in vivo under IR conditions. (A) Schematic overview of the in vivo tumor model and TIL analysis. C57BL/6 mice were subcutaneously inoculated with B16F0-hCD19 tumor cells (3 × 10 5 cells). In the IR group, mice received IR (5 Gy) on day 11, followed by adoptive transfer of anti-hCD19 mCAR-T cells (5 × 10 6 cells, i.v.) on day 12. In the Non-IR group, mCAR-T cells were transferred without IR. Tumors were harvested on day 20 for TIL analysis. (B, C) Tumor infiltration of CAR-T cells under Non-IR and IR conditions. (B) Representative flow cytometry plots showing CD8α and Thy1.1 expression among TILs under Non-IR (upper) and IR (lower) conditions. (C) Quantification of CD8 + CAR-T cells among total TILs. (D, E) Cytokine-producing capacity of CD8 + OE mCAR-TILs. The gating strategy is shown in Supplementary Figure 3A . (D) Representative flow cytometry plots showing IFN-γ and TNF-α expression. (E) Quantification of TNF-α + IFN-γ + cells among CD8 + OE mCAR-TILs. (F, G) Degranulation capacity of CD8 + OE mCAR-TILs. (F) Representative histograms of CD107a expression (IgG shown as a control). (G) Quantification of CD107a + cells among CD8 + OE mCAR-TILs. (H, I) Expression of exhaustion markers on CD8 + OE mCAR-TILs. (H) Representative flow cytometry plots showing PD-1 and TIM-3 expression. (I) Quantification of PD-1 + TIM-3 + cells among CD8 + OE mCAR-TILs. (J, K) Proliferative status of CD8 + OE mCAR-TILs. (J) Representative histograms of Ki67 expression (IgG shown as a control). (K) Quantification of Ki67 + cells among CD8 + OE mCAR-TILs. Individual replicates are shown as dots, and bars indicate mean ± SEM. Open dots represent the Non-IR group, and filled dots represent the IR group. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Supplementary Figure 3 .

Article Snippet: The B16F0 (mouse melanoma) cell line was purchased from the American Type Culture Collection (ATCC), and the Plat-E (ecotropic retroviral packaging) cell line was obtained from Cell Biolabs (San Diego, CA, USA).

Techniques: Functional Assay, Activity Assay, In Vivo, Adoptive Transfer Assay, Flow Cytometry, Expressing, Control