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ATCC b16f0 mouse melanoma cell line

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 <t>B16F0-hCD19-mCherry</t> 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.

B16f0 Mouse Melanoma Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 1354 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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b16f0 mouse melanoma cell line - by Bioz Stars, 2026-06

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1) Product Images from "ATG5-mediated inducible autophagy sustains CAR-T cell durability under solid tumor stress"

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

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2026.1720544

Figure Legend 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.

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

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 <xref ref-type=

Supplementary Figure 2 . " title="... for assessing oxidative stress and effector functional characteristics. B16F0-hCD19 target cells (1 × 10 5 cells) were ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend 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 .

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

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. .

Figure Legend 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. .

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

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 <xref ref-type=

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 . " title="... TIL analysis. C57BL/6 mice were subcutaneously inoculated with B16F0-hCD19 tumor cells (3 × 10 5 cells). In ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend 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 .

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

Image Search Results

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

Supplementary Figure 2 . " width="100%" height="100%">

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 .

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. .

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 .

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

Efficiency of lentiviral transduction with il12, il15 and il15Rα or il18 genes and the level of differentiation of bone marrow-derived dendritic cells (DCs) stimulated with B16 F0 tumor antigens (TAg). Concentration of overexpressed cytokines IL-12 (A) , IL-15 (B) , IL-18 (C) in supernatants collected after 48 hours of DCs cultures measured using ELISA. Percentage of CD11c + cells on the 10 th day of DCs cultured (D) . Expression of CD40 (E) , CD80 (F) , CD86 (G) and MHC II (H) molecules on the surface of CD11c + cells (MFI) (bar plots and representative overlay histograms). Results are presented as mean+SD calculated for 5–6 samples per group. Differences between groups were estimated using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons post-hoc test (A–D, F–H) or the parametric Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test (E) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the DC/TAg control cells; crosses (X) indicate a statistically significant difference between the given group and the DC/Vctrl/TAg control cells, asterisks (*) under the line indicate statistically significant differences between the given groups – (*/ x p<0.05; **/ xx p<0.01; ***/ xxx p<0.001; ****/ xxxx p<0.0001). IC – isotype control , MFI – mean fluorescence intensity .

Journal: Frontiers in Immunology

Article Title: Co-delivery of IL-12/IL-15/IL-18 engineered DC vaccines with anti-IL-10R and nanoconjugated methotrexate in melanoma

doi: 10.3389/fimmu.2026.1773836

Figure Lengend Snippet: Efficiency of lentiviral transduction with il12, il15 and il15Rα or il18 genes and the level of differentiation of bone marrow-derived dendritic cells (DCs) stimulated with B16 F0 tumor antigens (TAg). Concentration of overexpressed cytokines IL-12 (A) , IL-15 (B) , IL-18 (C) in supernatants collected after 48 hours of DCs cultures measured using ELISA. Percentage of CD11c + cells on the 10 th day of DCs cultured (D) . Expression of CD40 (E) , CD80 (F) , CD86 (G) and MHC II (H) molecules on the surface of CD11c + cells (MFI) (bar plots and representative overlay histograms). Results are presented as mean+SD calculated for 5–6 samples per group. Differences between groups were estimated using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons post-hoc test (A–D, F–H) or the parametric Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test (E) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the DC/TAg control cells; crosses (X) indicate a statistically significant difference between the given group and the DC/Vctrl/TAg control cells, asterisks (*) under the line indicate statistically significant differences between the given groups – (*/ x p<0.05; **/ xx p<0.01; ***/ xxx p<0.001; ****/ xxxx p<0.0001). IC – isotype control , MFI – mean fluorescence intensity .

Article Snippet: The non-metastatic murine melanoma B16 F0 cell line (ECACC 92101204) was cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM, ATCC) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS; Sigma−Aldrich).

Techniques: Transduction, Derivative Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Expressing, Control, Fluorescence

The ability of DCs genetically modified to produce IL-12, IL-15/Il-15Rα or IL-18 and stimulated with B16 F0 tumor antigens to prime splenocytes. Percentage of CD8 + cells (A) , CD4 + cells (B) and NK cells (C) among splenocytes obtained after 5-day coculture with DCs. Percentage of effector cells (CD107a + ) among CD8 + (D) , CD4 + (E) and NK cells (F) after 2-hour incubation with B16 F0 cells. Concentration of IFN-γ (G) and IL-10 (H) in supernatants collected after 5 days of cocultured DCs and spleen cells. Ratio of IFN-γ and IL-10 concentration (I) . Cytotoxic activity of splenocytes presented as a percentage of dead B16 F0 tumor cells after 4-hour incubation with effector cells in a ratio of 10:1 (effector:target) (J) . Results are presented as mean+SD calculated for 6–12 samples per group. Differences between groups were estimated using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons post-hoc test (A–D, G–I) , the parametric one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (E, J) or the parametric Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test (F) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the DC/TAg control cells; crosses (X) indicate a statistically significant difference between the given group and the DC/Vctrl/TAg control cells, asterisks (*) under the line indicate statistically significant differences between the given groups – (*/ x p<0.05; **/ xx p<0.01; ***/ xxx p<0.001; ****/ xxxx p<0.0001).

Journal: Frontiers in Immunology

Article Title: Co-delivery of IL-12/IL-15/IL-18 engineered DC vaccines with anti-IL-10R and nanoconjugated methotrexate in melanoma

doi: 10.3389/fimmu.2026.1773836

Figure Lengend Snippet: The ability of DCs genetically modified to produce IL-12, IL-15/Il-15Rα or IL-18 and stimulated with B16 F0 tumor antigens to prime splenocytes. Percentage of CD8 + cells (A) , CD4 + cells (B) and NK cells (C) among splenocytes obtained after 5-day coculture with DCs. Percentage of effector cells (CD107a + ) among CD8 + (D) , CD4 + (E) and NK cells (F) after 2-hour incubation with B16 F0 cells. Concentration of IFN-γ (G) and IL-10 (H) in supernatants collected after 5 days of cocultured DCs and spleen cells. Ratio of IFN-γ and IL-10 concentration (I) . Cytotoxic activity of splenocytes presented as a percentage of dead B16 F0 tumor cells after 4-hour incubation with effector cells in a ratio of 10:1 (effector:target) (J) . Results are presented as mean+SD calculated for 6–12 samples per group. Differences between groups were estimated using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons post-hoc test (A–D, G–I) , the parametric one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (E, J) or the parametric Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test (F) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the DC/TAg control cells; crosses (X) indicate a statistically significant difference between the given group and the DC/Vctrl/TAg control cells, asterisks (*) under the line indicate statistically significant differences between the given groups – (*/ x p<0.05; **/ xx p<0.01; ***/ xxx p<0.001; ****/ xxxx p<0.0001).

Techniques: Genetically Modified, Incubation, Concentration Assay, Activity Assay, Control

Growth of B16 F0 tumors in mice after immunotherapy and chemoimmunotherapy composed of a chemotherapeutic agent, anti-IL-10R antibody, and genetically modified DC-based vaccines stimulated with B16 F0 tumor antigens. Scheme of immunotherapy (A) and chemoimmunotherapy (E) treatment created with BioRender.com . Growth kinetics of B16 F0 tumor in mice treated with immunotherapy (B) or chemoimmunotherapy (F) . Violin plot presenting individual tumor volume and designated median tumor volume for each group, calculated on the 19 th day of the immunotherapy (C) or chemoimmunotherapy (G) experiment. Results are presented as median for 3–10 mice per group. Table presenting B16 F0 tumor growth inhibition (TGI) calculated on the 19 th day of the experiment in relation to the non-treated group (nt) (D, H) . Differences between groups were estimated using the two−way ANOVA followed by Tukey’s multiple comparisons post−hoc test (B, F) or non-parametric Kruskal−Wallis test followed by Dunn’s multiple comparisons test (C, G) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the non-treated control group (nt); hashtags (#) above a bar indicate a statistically significant difference between the given group and the HES-MTX treated group (H-M) – (*p<0.05; **/ ## p<0.01; ***p<0.001; ****p<0.0001).

Journal: Frontiers in Immunology

Article Title: Co-delivery of IL-12/IL-15/IL-18 engineered DC vaccines with anti-IL-10R and nanoconjugated methotrexate in melanoma

doi: 10.3389/fimmu.2026.1773836

Figure Lengend Snippet: Growth of B16 F0 tumors in mice after immunotherapy and chemoimmunotherapy composed of a chemotherapeutic agent, anti-IL-10R antibody, and genetically modified DC-based vaccines stimulated with B16 F0 tumor antigens. Scheme of immunotherapy (A) and chemoimmunotherapy (E) treatment created with BioRender.com . Growth kinetics of B16 F0 tumor in mice treated with immunotherapy (B) or chemoimmunotherapy (F) . Violin plot presenting individual tumor volume and designated median tumor volume for each group, calculated on the 19 th day of the immunotherapy (C) or chemoimmunotherapy (G) experiment. Results are presented as median for 3–10 mice per group. Table presenting B16 F0 tumor growth inhibition (TGI) calculated on the 19 th day of the experiment in relation to the non-treated group (nt) (D, H) . Differences between groups were estimated using the two−way ANOVA followed by Tukey’s multiple comparisons post−hoc test (B, F) or non-parametric Kruskal−Wallis test followed by Dunn’s multiple comparisons test (C, G) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the non-treated control group (nt); hashtags (#) above a bar indicate a statistically significant difference between the given group and the HES-MTX treated group (H-M) – (*p<0.05; **/ ## p<0.01; ***p<0.001; ****p<0.0001).

Techniques: Genetically Modified, Vaccines, Inhibition, Control

Evaluation of leukocyte subpopulations infiltrating B16 F0 tumor tissue after administration of immunotherapy or chemoimmunotherapy. Scheme of the multiparameter flow cytometry analysis of myeloid and lymphoid cells infiltrating B16 F0 tumor nodules (prepared for one representative sample from group IX in the chemoimmunotherapy experiment) (A) . Percentage of live CD45 + cells in tumor (B, H) . Percentage of each leukocyte’s population among CD45 + cells (C, I) . Percentage of CD8 + (D, J) , CD4 + (E, K) , Treg (F, L) and NK cells (G, M) infiltrating tumor tissue. Results are presented as mean+SD calculated for 3–6 mice per group. Differences between groups were estimated using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons post-hoc test (B, G, J, M) , the parametric one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (D) or the parametric Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test (K, L) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the non-treated control group (nt); hashtags (#) above a bar indicate a statistically significant difference between the given group and the HES-MTX treated group (H-M) ; crosses (X) indicate a statistically significant difference between the given group and the DC/Vctrl/TAg treated group; asterisks (*) under the line indicate statistically significant differences between the given groups – (*/ # / x p<0.05; **/ xx p<0.01; ***/ xxx p<0.001; ****p<0.0001).

Journal: Frontiers in Immunology

Article Title: Co-delivery of IL-12/IL-15/IL-18 engineered DC vaccines with anti-IL-10R and nanoconjugated methotrexate in melanoma

doi: 10.3389/fimmu.2026.1773836

Figure Lengend Snippet: Evaluation of leukocyte subpopulations infiltrating B16 F0 tumor tissue after administration of immunotherapy or chemoimmunotherapy. Scheme of the multiparameter flow cytometry analysis of myeloid and lymphoid cells infiltrating B16 F0 tumor nodules (prepared for one representative sample from group IX in the chemoimmunotherapy experiment) (A) . Percentage of live CD45 + cells in tumor (B, H) . Percentage of each leukocyte’s population among CD45 + cells (C, I) . Percentage of CD8 + (D, J) , CD4 + (E, K) , Treg (F, L) and NK cells (G, M) infiltrating tumor tissue. Results are presented as mean+SD calculated for 3–6 mice per group. Differences between groups were estimated using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons post-hoc test (B, G, J, M) , the parametric one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (D) or the parametric Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test (K, L) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the non-treated control group (nt); hashtags (#) above a bar indicate a statistically significant difference between the given group and the HES-MTX treated group (H-M) ; crosses (X) indicate a statistically significant difference between the given group and the DC/Vctrl/TAg treated group; asterisks (*) under the line indicate statistically significant differences between the given groups – (*/ # / x p<0.05; **/ xx p<0.01; ***/ xxx p<0.001; ****p<0.0001).

Techniques: Flow Cytometry, Control

Estimation of TAM and MDSC subpopulation in B16 F0 tumor tissue after applied therapy. Percentage of TAM (A, D) , TAMs MHC II high /TAMs MHC II low (B, E) and MDSC (C, F) among CD45 + cells in tumors. Results are presented as mean+SD calculated for 3–6 mice per group. Differences between groups were estimated using the parametric one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (A, D) or the parametric Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test (F) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the non-treated control group (nt); hashtags (#) above a bar indicate a statistically significant difference between the given group and the HES-MTX treated group (H-M); crosses (X) indicate a statistically significant difference between the given group and the DC/Vctrl/TAg treated group; asterisks (*) under the line indicate statistically significant differences between the given groups – (*/ # p<0.05; **/ ## / xx p<0.01; ***/ xxx p<0.001; ****/ #### p<0.0001).

Journal: Frontiers in Immunology

Article Title: Co-delivery of IL-12/IL-15/IL-18 engineered DC vaccines with anti-IL-10R and nanoconjugated methotrexate in melanoma

doi: 10.3389/fimmu.2026.1773836

Figure Lengend Snippet: Estimation of TAM and MDSC subpopulation in B16 F0 tumor tissue after applied therapy. Percentage of TAM (A, D) , TAMs MHC II high /TAMs MHC II low (B, E) and MDSC (C, F) among CD45 + cells in tumors. Results are presented as mean+SD calculated for 3–6 mice per group. Differences between groups were estimated using the parametric one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (A, D) or the parametric Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test (F) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the non-treated control group (nt); hashtags (#) above a bar indicate a statistically significant difference between the given group and the HES-MTX treated group (H-M); crosses (X) indicate a statistically significant difference between the given group and the DC/Vctrl/TAg treated group; asterisks (*) under the line indicate statistically significant differences between the given groups – (*/ # p<0.05; **/ ## / xx p<0.01; ***/ xxx p<0.001; ****/ #### p<0.0001).

Techniques: Control

Impact of applied immunotherapy and chemoimmunotherapy on the induction of systemic antitumor in B16 F0 melanoma model. Scheme of the flow cytometry analysis of restimulated splenocytes (A) . Percentage of CD8 + (B, H) , CD4 + (C, I) , and NK cells (D, J) restimulated splenocytes. Percentage of CD107a + cells among CD8 + (E, K) , CD4 + (F, L) and NK cells (G, M) . Results are presented as mean+SD calculated for 3–6 mice per group. Differences between groups were estimated using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons post-hoc test (E, H, K) , the parametric one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (B, F, G, I) or the parametric Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test (C, D, J, L, M) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the non-treated control group (nt); hashtags (#) above a bar indicate a statistically significant difference between the given group and the HES-MTX treated group (H-M) ; crosses (X) indicate a statistically significant difference between the given group and the DC/Vctrl/TAg treated group; asterisks (*) under the line indicate statistically significant differences between the given groups – (*/ # p<0.05; **/ ## / xx p<0.01; ***/ ### p<0.001; ****/ #### p<0.0001).

Journal: Frontiers in Immunology

Article Title: Co-delivery of IL-12/IL-15/IL-18 engineered DC vaccines with anti-IL-10R and nanoconjugated methotrexate in melanoma

doi: 10.3389/fimmu.2026.1773836

Figure Lengend Snippet: Impact of applied immunotherapy and chemoimmunotherapy on the induction of systemic antitumor in B16 F0 melanoma model. Scheme of the flow cytometry analysis of restimulated splenocytes (A) . Percentage of CD8 + (B, H) , CD4 + (C, I) , and NK cells (D, J) restimulated splenocytes. Percentage of CD107a + cells among CD8 + (E, K) , CD4 + (F, L) and NK cells (G, M) . Results are presented as mean+SD calculated for 3–6 mice per group. Differences between groups were estimated using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons post-hoc test (E, H, K) , the parametric one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (B, F, G, I) or the parametric Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test (C, D, J, L, M) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the non-treated control group (nt); hashtags (#) above a bar indicate a statistically significant difference between the given group and the HES-MTX treated group (H-M) ; crosses (X) indicate a statistically significant difference between the given group and the DC/Vctrl/TAg treated group; asterisks (*) under the line indicate statistically significant differences between the given groups – (*/ # p<0.05; **/ ## / xx p<0.01; ***/ ### p<0.001; ****/ #### p<0.0001).

Techniques: Flow Cytometry, Control

Activity of restimulated splenocytes obtained from B16 F0 melanoma-bearing mice after immunotherapy and chemoimmunotherapy. Concentration of IFN-γ (A, D) , IL-10 (B, E) and IL-4 (C, F) in supernatants after restimulation of spleen cells with B16 F0 cells, measured using ELISA assay. Results are presented as mean+SD calculated for 3–6 mice per group. Differences between groups were estimated using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons post-hoc test (A, C, F) or the parametric Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test (B, D, E) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the non-treated control group (nt); hashtags (#) above a bar indicate a statistically significant difference between the given group and the HES-MTX treated group (H-M); crosses (X) indicate a statistically significant difference between the given group and the DC/Vctrl/TAg treated group; asterisks (*) under the line indicate statistically significant differences between the given groups – (*/ # p<0.05; **/ ## p<0.01; ***/ ### p<0.001; ****/ #### p<0.0001).

Journal: Frontiers in Immunology

Article Title: Co-delivery of IL-12/IL-15/IL-18 engineered DC vaccines with anti-IL-10R and nanoconjugated methotrexate in melanoma

doi: 10.3389/fimmu.2026.1773836

Figure Lengend Snippet: Activity of restimulated splenocytes obtained from B16 F0 melanoma-bearing mice after immunotherapy and chemoimmunotherapy. Concentration of IFN-γ (A, D) , IL-10 (B, E) and IL-4 (C, F) in supernatants after restimulation of spleen cells with B16 F0 cells, measured using ELISA assay. Results are presented as mean+SD calculated for 3–6 mice per group. Differences between groups were estimated using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons post-hoc test (A, C, F) or the parametric Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test (B, D, E) . The asterisks (*) presented in the graphs indicate statistically significant differences between the given groups and the non-treated control group (nt); hashtags (#) above a bar indicate a statistically significant difference between the given group and the HES-MTX treated group (H-M); crosses (X) indicate a statistically significant difference between the given group and the DC/Vctrl/TAg treated group; asterisks (*) under the line indicate statistically significant differences between the given groups – (*/ # p<0.05; **/ ## p<0.01; ***/ ### p<0.001; ****/ #### p<0.0001).

Techniques: Activity Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control

The effect of chemoimmunotherapy with DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg cell vaccine on the inhibition of B16 F0 tumor growth.

Journal: Frontiers in Immunology

Article Title: Co-delivery of IL-12/IL-15/IL-18 engineered DC vaccines with anti-IL-10R and nanoconjugated methotrexate in melanoma

doi: 10.3389/fimmu.2026.1773836

Figure Lengend Snippet: The effect of chemoimmunotherapy with DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg cell vaccine on the inhibition of B16 F0 tumor growth.

Techniques: Inhibition

Selective, dose-dependent binding and uptake of murine CD137 aptamer by CD137-expressing cells. A total of 2 × 10 5 A20 or A20-CD137 cells prefixed with 1% paraformaldehyde were treated with fluorescein maleimide (FAM)-labeled murine CD137 aptamer at different concentrations (50, 100, and 500 nM). (a) The size of murine aptamer (222 nt) and the successful labeling of the aptamer with fluorescein maleimide (FAM) were detected using agarose gel electrophoresis. (b) CD137 expression on A20 and A20-CD137 cells. (c) Histograms showing the binding of CD137 aptamer to A20 (upper panel) or A20-CD137 (bottom panel) cells when given at different concentrations. (d) Comparisons of CD137 aptamer binding across different concentrations in A20 or A20-CD137 cells. (e) Comparisons of the % population between A20 and A20-CD137 cells that had bound to the CD137 aptamer at a given concentration. A total of 5 × 10 5 CD137-expressing (A20-CD137 or B16-CD137) or control (A20 or B16) cell lines were incubated with murine CD137 aptamer (0.5, 1, or 2 μg) for 2 h at 37°C. Comparisons between the amount of CD137 aptamer internalized by (f) A20 and A20-CD137 or (g) B16 and B16-CD137 cells. Comparisons of CD137 aptamer uptake when given at different amounts for (h) A20-CD137 and (i) B16-CD137 cells. The extent of CD137 aptamer internalization was expressed as fold change relative to control cells treated with 0.5 μg aptamer, or as absolute copy number per 1 μg total RNA. Dashed lines at a ratio of 1 represent the normalization reference. Data are shown as means ± SEM. Numbers above the brackets indicate P -values. * P < .05, ** P < .01, and *** P < .001 using unpaired Students’ t -test for (e), (f), and (g), while using one-way ANOVA with Bonferroni’s multiple comparison test for (d), (h), and (i).

Journal: Immunotherapy Advances

Article Title: Targeting intratumoral regulatory T cells by CD137 aptamer-shRNA chimeras

doi: 10.1093/immadv/ltaf037

Figure Lengend Snippet: Selective, dose-dependent binding and uptake of murine CD137 aptamer by CD137-expressing cells. A total of 2 × 10 5 A20 or A20-CD137 cells prefixed with 1% paraformaldehyde were treated with fluorescein maleimide (FAM)-labeled murine CD137 aptamer at different concentrations (50, 100, and 500 nM). (a) The size of murine aptamer (222 nt) and the successful labeling of the aptamer with fluorescein maleimide (FAM) were detected using agarose gel electrophoresis. (b) CD137 expression on A20 and A20-CD137 cells. (c) Histograms showing the binding of CD137 aptamer to A20 (upper panel) or A20-CD137 (bottom panel) cells when given at different concentrations. (d) Comparisons of CD137 aptamer binding across different concentrations in A20 or A20-CD137 cells. (e) Comparisons of the % population between A20 and A20-CD137 cells that had bound to the CD137 aptamer at a given concentration. A total of 5 × 10 5 CD137-expressing (A20-CD137 or B16-CD137) or control (A20 or B16) cell lines were incubated with murine CD137 aptamer (0.5, 1, or 2 μg) for 2 h at 37°C. Comparisons between the amount of CD137 aptamer internalized by (f) A20 and A20-CD137 or (g) B16 and B16-CD137 cells. Comparisons of CD137 aptamer uptake when given at different amounts for (h) A20-CD137 and (i) B16-CD137 cells. The extent of CD137 aptamer internalization was expressed as fold change relative to control cells treated with 0.5 μg aptamer, or as absolute copy number per 1 μg total RNA. Dashed lines at a ratio of 1 represent the normalization reference. Data are shown as means ± SEM. Numbers above the brackets indicate P -values. * P < .05, ** P < .01, and *** P < .001 using unpaired Students’ t -test for (e), (f), and (g), while using one-way ANOVA with Bonferroni’s multiple comparison test for (d), (h), and (i).

Article Snippet: Murine B lymphoma cell line A20, melanoma cell line B16, and hepatoma cell line Hepa1-6 were purchased from American Type Cell Culture (ATCC; Manassas, VA, USA).

Techniques: Binding Assay, Expressing, Labeling, Agarose Gel Electrophoresis, Concentration Assay, Control, Incubation, Comparison

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