Journal: EMBO Molecular Medicine

Article Title: MHC-I upregulation by macbecin II in the solid tumors potentiates the effect of active immunotherapy

doi: 10.1038/s44321-025-00213-7

Figure Lengend Snippet: ( A ) B16-Ova cells were treated with the indicated dose of macbecin II for 48 h and the cells were examined for H-2kb-Ova peptide presentation by FACS. One-way ANOVA with a Tukey post-hoc test was used for comparison ( n = 3/group, biological replicates). ( B ) BMDCs were isolated from the tibia of a C57BL/6 mouse. Dendritic cells (DC) were differentiated with GM-CSF treatment for 5 days. Loosely adherent cells were then isolated and treated with Ova (100 µg/mL) overnight. The next day, the cells were treated with the indicated dose of macbecin II for 48 h. Post-incubation, cells were washed and H-2Kb-Ova presentation was examined by FACS. Mean fluorescence intensity was calculated and compared by one-way ANOVA with a Tukey post-hoc test ( n = 3/group, biological replicates). ( C ) The OT-1 T cells were co-cultured with DC in the presence of Macbecin II or PBS, and they were examined for IFN-γ expression by FACS as described in ( B ). Statistical significance was analyzed by the one-way ANOVA with a Tukey post-hoc test ( n = 3/group, biological replicates). ( D ) B16-Ova cells (500 cells/well) were seeded into a 96-well plate and treated with the macbecin II (0.5 µM), OT-1 T cells (E:T ratio 10:1), and MHC-I blocking antibody (40 µg/mL) for 48 h. Dead cells and OT-1 T cells were washed off with PBS, and the remaining cancer cells were fixed with methanol and stained with crystal violet. The dye was then dissolved in 10% acetic acid, and the absorbance was measured at 590 nm. Statistical significance between groups was determined using the one-way ANOVA with a Tukey post-hoc test ( n = 3/group, biological replicates). Data are presented as mean +/−SEM. Data are represented as mean +/− SEM. .

Article Snippet: T Cell Activation/Expansion Kit, mouse , Miltenyi Biotec , 130-093-627.

Techniques: Comparison, Isolation, Incubation, Fluorescence, Cell Culture, Expressing, Blocking Assay, Staining

Journal: EMBO Molecular Medicine

Article Title: MHC-I upregulation by macbecin II in the solid tumors potentiates the effect of active immunotherapy

doi: 10.1038/s44321-025-00213-7

Figure Lengend Snippet: ( A ) BMDCs were treated with the indicated dose of macbecin II for 48 h. Post-incubation, cells were stained with an H-2Kb antibody and examined by FACS ( n = 3/group, biological replicates). Statistical inference was compared by one-way ANOVA with a Tukey post-hoc test. ( B ) OT-1 cells were co-cultured with B16-Ova cells and the expression of TNF-α in the medium was measured by ELISA. Statistical significance was determined using one-way ANOVA with a Tukey post-hoc test ( n = 3/group, biological replicates). ( C ) OT-1 T cells were co-cultured in the presence of DCs that were pulsed with Ova (DC-ova) with or without macbecin II, and then examined for CD69 surface expression by FACS at after three days. Statistical significance was determined using the one-way ANOVA with a Tukey post-hoc test ( n = 3/group, biological replicates). ( D ) E0771 cells transfected with the ova plasmid were treated with the indicated doses of macbecin II for 48 h. The cells were examined for the presentation of H-2Kb Ova peptide by FACS. One-way ANOVA with a Tukey post-hoc test was used for statistical analysis ( n = 3/group, biological replicates). ( E ) The BMDCs were isolated from the syngeneic mouse and pulsed with ova peptide with or without macbecin II. TNF-α levels were examined in T cells co-cultured with BMDCs ( n = 3/group, biological replicates). ( F ) E0771-Ova cells (500 cells/well) were seeded in a 96-well plate and treated with the indicated doses of macbecin II followed by co-culturing with T cells (E:T ratio 10:1) from ( E ) for 48 h. Dead cells were washed out with ice-cold PBS, and the cancer cells were fixed with methanol. and stained with crystal violet. The dye was then dissolved in 10% acetic acid, and readouts were obtained at 590 nm. Statistical significance between groups was determined using the one-way ANOVA with a Tukey post-hoc test ( n = 3/group, biological replicates). Data are presented as mean +/− SEM.

Article Snippet: T Cell Activation/Expansion Kit, mouse , Miltenyi Biotec , 130-093-627.

Techniques: Incubation, Staining, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Isolation

Journal: EMBO Molecular Medicine

Article Title: MHC-I upregulation by macbecin II in the solid tumors potentiates the effect of active immunotherapy

doi: 10.1038/s44321-025-00213-7

Figure Lengend Snippet: ( A ) Schematic of the experiment. HLA-matched PBMCs were treated with GM-CSF and IL-4 for 5 days to differentiate monocytes into dendritic cells (DC). The loosely adherent cells were isolated and treated with DCIS.com lysate, LPS (100 ng/mL), and C-diGMP (200 µM). Exosomes were then isolated by ultracentrifugation and co-cultured with HLA-matched PBMCs (20 µg/mL) for 5 days. The educated PBMCs were then expanded with CD3/CD28 microbeads and used for co-culturing with DCIS.com cells. ( B ) PBMCs from ( A ) were co-cultured with the DCIS.Com cells treated with or without macbecin II (0.5 µM). Followed by this, IFN-γ + CD8 T cell were examined by FACS. Statistical significance was analyzed by the one-way ANOVA with a Tukey post-hoc test ( n = 3/group, biological replicates). ( C ) DCIS.com cells (500 cells/well, n = 3/group, biological replicates) were seeded in a 96-well plate and co-cultured in the presence of educated PBMCs, the indicated dose of macbecin II, or 40 µg/mL CD8 blocking antibody for 48 h. Post-incubation, cells were washed with ice-cold PBS to dislodge the dead cells. The live cells were fixed with methanol at room temperature for 15 min and stained with crystal violet. The dye was dissolved in 10% acetic acid and readouts were obtained at 590 nm. Statistical significance was compared by one-way ANOVA with a Tukey post-hoc test. ( D ) DCIS.com cells (500 cells/well) were cultured in a 96-well plate and treated with (i) vehicle (ii) macbecin II (0.1 µM and 0.5 µM), (iii) PBMCs (1:10 and 1:20 ratio) alone or in combination for 48 h ( n = 3/group, biological replicates). After incubation, the cells were washed with ice-cold PBS to remove dead cells and PBMCs. Surviving cells were fixed in methanol and stained with crystal violet. The dye was dissolved in 10% acetic acid and readouts were obtained at 590 nm. ( E ) Combination index (CI) values were calculated by CompuSyn based on the inhibitory effect of macbecin II and PBMC treatment. A CI < 1 indicates synergy. ( F ) MCF10CA1a cells (1000 cells/well, n = 3/group, biological replicates) were seeded in a 96-well plate and treated with the indicated ratio of PBMCs for 48 h. Post-incubation, relative cell viability was calculated by the crystal violet assay method as mentioned in ( C ). Statistical significance was determined by one-way ANOVA with a Tukey post-hoc test. ( G , H ) MCF10CA1a cells (500 cells/well, n = 3/group, biological replicates) were seeded in a 96-well plate and co-cultured in the presence of educated PBMCs in combination with the indicated dose of macbecin II or 40 µg/mL CD8 antibody for 48 h. The co-culture experiment was performed as mentioned in ( C ). Combination index (CI) values were calculated by CompuSyn based on the inhibitory effect of macbecin II and PBMC treatment. A CI < 1 indicates synergy. ( H ) Data are represented as mean +/− SEM. .

Article Snippet: T Cell Activation/Expansion Kit, mouse , Miltenyi Biotec , 130-093-627.

Techniques: Isolation, Cell Culture, Blocking Assay, Incubation, Staining, Crystal Violet Assay, Co-Culture Assay

Journal: EMBO Molecular Medicine

Article Title: MHC-I upregulation by macbecin II in the solid tumors potentiates the effect of active immunotherapy

doi: 10.1038/s44321-025-00213-7

Figure Lengend Snippet: ( A ) Extracellular vesicles were isolated from dendritic cells that were pulsed with DCIS.com lysate. They were examined by electron microscopy. Representative image is shown. ( B ) The size distribution of p13nsEV was examined by Nanoparticle tracking analysis (NTA). The median size of p13nsEV was 148.5 nm. ( C ) The exosomes were isolated from the BMDCs pulsed with B16-F1 lysate by ultracentrifugation (UCF) or with the use of ExoQuick kit. The pallet or the supernatant fraction isolated from the UCF was then incubated with the T cells isolated from the spleen. Similarly, exosome fraction or exosome-depleted fraction from ExoQuick preparation was incubated with the T cells. The T cells were then co-cultured with the B16-F1 cells and TNF-α was examined ( n = 3/group, biological replicates). The result was analyzed by the unpaired two-tailed Student’s t-test ( n = 3/group, biological replicates). ( D ) The T cells treated with the supernatant and pallet fraction were co-cultured with the B16-F1 cells (E:T ratio 15:1) for 48 h and relative cell viability was examined. After the incubation, the cells were washed with ice-cold PBS to remove dead cells. The live cells were fixed with methanol at room temperature for 15 min and then stained with crystal violet. The dye was dissolved in 10% acetic acid, and readouts were obtained at 590 nm. The result was analyzed by the unpaired two-tailed Student’s t-test ( n = 3/group, biological replicates). ( E ) DCIS.com cells (500 cells/well, n = 3/group, biological replicates) were seeded in a 96-well plate and co-cultured in the presence of PBMCs, macbecin II (0.1 µM), or 40 µg/mL MHC-I blocking antibody for 48 h. The IL2-ep13nsEV were prepared from the dendritic cells that were pulsed with DCIS.com lysate and they were used to educate T cells in the PBMC. After the incubation, the cells were washed with ice-cold PBS to remove dead cells. The live cells were fixed with methanol at room temperature for 15 min and then stained with crystal violet. The dye was dissolved in 10% acetic acid, and readouts were obtained at 590 nm. Statistical significance was determined using the one-way ANOVA with a Tukey post-hoc test. ( F ) Schematic of the experiment. BMDC were treated with GM-CSF for 5 days to differentiate monocytes into dendritic cells (DCs). The DCs were treated with ova peptide, LPS (100 ng/ml), C-diGMP (200 µM), and macbecin II followed by IL2-ep13nsEV isolation. The T cells were isolated from the spleen of syngeneic mice that were subcutaneously injected with ova peptide (5 µg for 10 days), with or without macbecin II, and they were activated in the presence of IL2-ep13nsEV. ( G ) Activated and expanded spleenocytes from ( E ) were examined for antigen-specific priming by tetramer assay. Unpaired Student’s t-test was used for data analysis ( n = 3/group/ biological replicates). ( H ) B16-F1 cells were seeded in a 96-well plate. Bone marrow-derived dendritic cells (BMDCs) isolated from syngeneic mice were pulsed with B16-F1 lysate and treated with or without macbecin II. Subsequently, T cells isolated from the spleen of syngeneic mouse were co-cultured with the BMDCs for antigenic priming. T cells co-cultured with macbecin II-treated, lysate-pulsed BMDCs were designated as T+, while those co-cultured with macbecin II-untreated BMDCs were designated as T−. The primed T cells (E:T ratio 10:1) were then co-cultured with B16-F1 cells treated with or without macbecin II for 48 h. After incubation, dead cells and T cells were washed off with PBS, and the remaining cancer cells were fixed with methanol and stained with crystal violet. The dye was dissolved in 10% acetic acid, and absorbance was measured at 590 nm. Statistical significance between groups was determined using one-way ANOVA with Tukey’s post-hoc test ( n = 3/group, biological replicates). ( I ) MDA-MB-231 cells (1000 cells/well, n = 3/group, biological replicates) were cultured in the presence of in vitro educated PBMCs from Fig. for 48 h. Post-incubation, cells were washed with ice-cold PBS to dislodge the dead cells and PBMCs. The live cells were fixed in methanol at room temperature for 15 min and stained with crystal violet. The dye was dissolved in 10% acetic acid and readouts were obtained at 590 nm. ( J ) E0771 cells (500 cells/well) were cultured in a 96-well plate and treated with (i) vehicle, (ii) macbecin II (0.1 µM and 0.5 µM), (iii) CD8 T cells isolated from the spleen of an E0771-bearing syngeneic mouse and activated with exosomes isolated from E0771 lysate-loaded BMDCs (50 µg/mL) for 5 days (E:T ratio 5:1 and 10:1) alone or in combination for 48 h ( n = 3/group, biological replicates). After incubation, the cells were washed with ice-cold PBS to remove dead cells and T cells. The surviving cells were fixed in methanol and stained with crystal violet. The dye was dissolved in 10% acetic acid and readouts were obtained at 590 nm. ( K ) Combination index (CI) values were calculated by CompuSyn based on the inhibitory effect of macbecin II and T cell treatment. CI < 1 indicates synergy. ( L ) TNF-α expressions were measured for PBMCs that were treated with macbecin II. ( M ) Relative cell viability was assessed using the MTS assay in PBMCs that were treated with macbecin II. ( n = 3/group, biological replicates). Data are represented as mean +/− SEM ( n = 5/group, biological replicates).

Article Snippet: T Cell Activation/Expansion Kit, mouse , Miltenyi Biotec , 130-093-627.

Techniques: Isolation, Electron Microscopy, Incubation, Cell Culture, Two Tailed Test, Staining, Blocking Assay, Injection, Derivative Assay, In Vitro, MTS Assay

Journal: EMBO Molecular Medicine

Article Title: MHC-I upregulation by macbecin II in the solid tumors potentiates the effect of active immunotherapy

doi: 10.1038/s44321-025-00213-7

Figure Lengend Snippet: ( A ) Schematic of the experiment. ( B ) E0771 cells (200 K cells) were implanted in syngeneic mice by intraductal injection. Animals were injected with the vaccine alone or in combination with macbecin II at the indicated time and dose. ( C ) Representative tumor pictures were taken at the endpoint (left). Tumor growth was monitored and compared by two-way ANOVA ( n = 10/group, biological replicates). Scale bar, 1 cm. ( D ) Tumor weight was measured at the endpoint and compared by one-way ANOVA with a Tukey post-hoc test ( n = 10/group, biological replicates). ( E ) Representative HE images showing lung micro-metastases from each group (up). Lung metastases were measured (down) and compared by one-way ANOVA ( n = 5/group, biological replicates). Scale bar, 100 µm. ( F , G ) CD8 ( F ) and CD4 ( G ) TILs among the CD3+ cells were measured in the dissociated tumor by FACS and compared by one-way ANOVA with a Tukey post-hoc test ( n = 4/group, biological replicates). ( H , I ) IFN-γ positive CD4 ( H ) and CD8 ( I ) TILs were measured in the dissociated tumor by FACS and compared by one-way ANOVA with a Tukey post-hoc test ( n = 4/group, biological replicates). ( J , K ) Schematic of the experiment. B16-F1 cells (2 × 10 5 cells) with or without ectopic MHC-I expression were implanted in syngeneic mice by subcutaneous injection. Animals were treated with the IL2-ep13nsEV alone or in combination with macbecin II at the indicated time and dose. ( L ) Representative tumor images were taken at the endpoint (left). Tumor growth was monitored, and the result was analyzed by the two-way ANOVA ( n = 10/group, biological replicates) test. Scale bar, 1 cm. ( M ) Tumor weight was measured at the endpoint and the result was analyzed by the one-way ANOVA with a Tukey post-hoc test ( n = 10/group, biological replicates). ( N ) Representative HE images showing lung micro-metastases from each group (left). Lung metastases were measured (right) and the result was analyzed by the one-way ANOVA (n = 5/group, biological replicates) test. Scale bar, 100 µm. ( O , P ) CD8 ( O ) and CD4 ( P ) TILs among the CD3+ cells were measured in the dissociated tumor by FACS, and the result was analyzed by the one-way ANOVA with a Tukey post-hoc test ( n = 4/group, biological replicates). ( Q , R ) IFN-γ positive CD4 ( Q ) and CD8 ( R ) TILs were measured in the dissociated tumor by FACS, and the result was analyzed by the one-way ANOVA with a Tukey post-hoc test ( n = 4/group, biological replicates). ( S , T ) MHC-I expression was examined in tumor ( S ) and DC ( T ) cells in dissociated tumor by FACS and the result was analyzed by the one-way ANOVA with a Tukey post-hoc test ( n = 3/group, biological replicates). Data are presented as mean +/−SEM. .

Article Snippet: T Cell Activation/Expansion Kit, mouse , Miltenyi Biotec , 130-093-627.

Techniques: Injection, Expressing

Journal: EMBO Molecular Medicine

Article Title: MHC-I upregulation by macbecin II in the solid tumors potentiates the effect of active immunotherapy

doi: 10.1038/s44321-025-00213-7

Figure Lengend Snippet: ( A ) Flow cytometry analysis for INF-γ-CD8 + T cells in the four groups. ( B ) Granzyme B expression was examined by immunohistochemistry (left) and quantified (right) in the four groups. The staining intensity was analyzed by the unpaired two-tailed Student’s t-test ( n = 4/group, biological replicates). ( C ) LAMP1 expression was examined through immunohistochemistry (left) and quantified (right) in the four groups. The staining intensity was measured and analyzed by the one-way ANOVA with a Tukey post-hoc test ( n = 4/group, biological replicates). ( D , E ) MHC-I expression was examined in EPCAM+ tumor cells and CD11c+ dendritic cells in the dissociated tumor by FACS. Statistical analysis was performed using the one-way ANOVA with a Tukey post-hoc test ( n = 3/group, biological replicates). ( F , G ) Animal weight ( F ) and AST activity ( G ) in the blood of mice were measured at the end-point. Data are represented as mean +/− SEM ( n = 5/group, biological replicates). ( H ) B16-Ova cells (500 cells/well, n = 3/group, biological replicates, were seeded in a 96-well plate and treated with a combination of macbecin II (0.1 µM) and OT-1 T cells (E:T ratio 5:1) with or without ectopic MHC-I expression for 48 h. The T cells were activated with IL2-ep13nsEV that were isolated from B16-Ova lysate-pulsed BMDCs (E:T ratio 5:1). After the incubation, cells were washed with ice-cold PBS to remove dead cells. The live cells were fixed with methanol at room temperature for 15 min and stained with crystal violet. The dye was dissolved in 10% acetic acid and measured at 590 nm. ( I ) E0771 cells (500 cells/well, n = 3/group, biological replicates) were seeded in a 96-well plate and treated with a combination of T cells (E:T ratio 5:1) and macbecin II (0.1 µM) with or without ectopic MHC-I expression for 48 h. The T cells were isolated from the spleen of E0771-bearing syngeneic mouse and they were activated with IL2-ep13nsEV that were isolated from E0771 lysate-pulsed BMDCs. After the incubation, cells were processed as described in ( H ). Statistical significance was determined by the one-way ANOVA with a Tukey post-hoc test. ( J ) MHC-I was ectopically expressed in B16-F1 cells, and the MHC-I expression was confirmed by FACS. The result was analyzed by the unpaired two-tailed Student’s t-test ( n = 3/group, biological replicates). ( K ) Cell viability was examined by MTS assay for B16-F1 with or without MHC-I over expression (OE) ( n = 3/group, biological replicates). ( L , M ) Animal weight ( L ) and AST activity ( M ) were measured at the end-point ( n = 5/group, biological replicates). Data are presented as mean +/− SEM.

Article Snippet: T Cell Activation/Expansion Kit, mouse , Miltenyi Biotec , 130-093-627.

Techniques: Flow Cytometry, Expressing, Immunohistochemistry, Staining, Two Tailed Test, Activity Assay, Isolation, Incubation, MTS Assay, Over Expression

Journal: EMBO Molecular Medicine

Article Title: MHC-I upregulation by macbecin II in the solid tumors potentiates the effect of active immunotherapy

doi: 10.1038/s44321-025-00213-7

Figure Lengend Snippet: ( A ) E0771 cells (500 cells/well) were cultured in a 96-well plate in the presence of (i) vehicle, (ii) anti-PD-1 (20 µg and 40 µg), and (iii) Macbecin II (0.1 µM and 0.5 µM). CD8 T cells were isolated from the spleen of an E0771-bearing syngeneic mouse and activated with CD3/CD28 beads. The activated CD8 T cells were co-cultured with the E0771 cells ( n = 3/group, biological replicates 5:1 E:T ratio). After 48 h, the cells were washed with ice-cold PBS to remove dead cells and T cells. The surviving cells were fixed in methanol and stained with crystal violet. The dye was dissolved in 10% acetic acid and readouts were obtained at 590 nm. ( B ) Combination index (CI) values were calculated by CompuSyn based on the inhibitory effect of anti-PD-1 and macbecin II treatment. CI < 1 indicates synergy. ( C , D ) Animal weight ( A ) and AST activity ( B ) were measured at the end-point ( n = 5/group, biological replicates). Data are presented as mean +/− SEM.

Article Snippet: T Cell Activation/Expansion Kit, mouse , Miltenyi Biotec , 130-093-627.

Techniques: Cell Culture, Isolation, Staining, Activity Assay

Journal: EMBO Molecular Medicine

Article Title: MHC-I upregulation by macbecin II in the solid tumors potentiates the effect of active immunotherapy

doi: 10.1038/s44321-025-00213-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: T Cell Activation/Expansion Kit, mouse , Miltenyi Biotec , 130-093-627.

Techniques: Recombinant, Over Expression, Plasmid Preparation, Sequencing, Proliferation Assay, Red Blood Cell Lysis, Staining, Activation Assay, Isolation, Cell Isolation, cDNA Synthesis, Bradford Protein Assay, AST Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Frontiers in Immunology

Article Title: Reinforcement of cell-mediated immunity driven by tumor-associated Epstein-Barr virus (EBV)-specific T cells during targeted B-cell therapy with rituximab

doi: 10.3389/fimmu.2023.878953

Figure Lengend Snippet: AC EBV -mediated long-term proliferation of EBV-specific memory T cells. Frequency of (A) CTV low T cells (CD3, CD8, CD4) examined after seven days of stimulation with APCs loaded with the EBV antigen cocktail (AC EBV , E:T 10:1) and visualized after subtraction from those stimulated with unloaded APCs. Results of four independent experiments are expressed as mean ± SD. (B) Frequency of A02LMP2A + T cells in HLA-A*02:01 + , EBV-seropositive donors (n=4, grey dots) determined by A02LMP2A dextramer staining before and after stimulation (day 0/7). As control, unloaded APCs were co-cultured with autologous CD3 + T cells (Ø). Frequencies measured using a nonsense dextramer (background control) were subtracted. Results of four independent experiments are displayed as contiguous grey dots for individual donors and as mean values in response to unloaded and AC EBV -loaded. Statistical analysis was performed using the non-parametric Wilcoxon test. CTV, CellTrace Violet.

Article Snippet: To determine the proliferative capacity of EBV-specific memory T cells, freshly isolated CD3 + T cells were labeled with CellTrace Violet (CTV) Proliferation Kit (2 mM, Thermo Fisher Scientifics, Waltham MA) according to the manufacturer’s instructions.

Techniques: Staining, Control, Cell Culture