Journal: Cancer Research Communications

Article Title: Epigenetic Priming by Hypomethylation Enhances the Immunogenic Potential of Tolinapant in T-cell Lymphoma

doi: 10.1158/2767-9764.CRC-23-0415

Figure Lengend Snippet: Effect of necrosome protein expression in TCL cell lines on tolinapant-induced cell death. A, Western blots of parental BW5147, RIPK3 − / − BW5147 and MLKL − / − BW5147 cell lysates (untreated). B, Cytotox-NIR signal captured by real-time microscopy (IncuCyte), detecting membrane permeabilization during lytic cell death after treatment of parental BW5147, RIPK3 − / − BW5147 and MLKL − / − BW5147 cells with tolinapant in the absence or presence of zVAD. C, Western blots of Karpas-299 cells transduced with control gRNA (KARPAS-299-CTR gRNA) or with RIPK3 gRNA (KARPAS-299-RIPK3 gRNA) constructs treated with tolinapant ± zVAD for 0, 2, or 6 hours. D, Real-time microscopy (IncuCyte) measurement of membrane permeabilization by measuring Cytotox-NIR signal after treatment of KARPAS-299-CTR gRNA or KARPAS-299-RIPK3 gRNA cells with tolinapant. E, Western blots of EL4-PAR and CASP8 KO EL4 (EL4-C8KO) cell lysates after 24-hour treatment with tolinapant. F, Real-time microscopy (IncuCyte) measurement of membrane permeabilization by measuring Cytotox-NIR signal after treatment of EL4-PAR or EL4-C8KO cells with tolinapant.

Article Snippet: EL4 (catalog no. 85023105, RRID:CVCL_0255) and Karpass-299 (catalog no. 06072604, RRID:CVCL_1324) cells were obtained from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK).

Techniques: Expressing, Western Blot, Microscopy, Membrane, Transduction, Control, Construct

Journal: Cancer Research Communications

Article Title: Epigenetic Priming by Hypomethylation Enhances the Immunogenic Potential of Tolinapant in T-cell Lymphoma

doi: 10.1158/2767-9764.CRC-23-0415

Figure Lengend Snippet: Dosing with a combination of tolinapant plus decitabine drives increased efficacy in necroptosis-model of TCL (EL4-C8KO model). Volumes of EL4-PAR ( A ) and C8KO ( B ) EL4 tumors in wild-type mice treated with decitabine (0.3 mg/kg i.p., every day × 3 doses) and tolinapant dosing (25 mg/kg orally, every day × up to 46 doses) as single agents or in combination. C, Kaplan–Meier plot showing survival (tumors reaching 1,000 mm 3 as endpoint) of mice bearing EL4-C8KO (C8KO) tumors.

Article Snippet: EL4 (catalog no. 85023105, RRID:CVCL_0255) and Karpass-299 (catalog no. 06072604, RRID:CVCL_1324) cells were obtained from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Small Molecule Inhibitors Targeting Nuclear Factor κB Activation Markedly Reduce Expression of Interleukin-2, but Not Interferon-γ, Induced by Phorbol Esters and Calcium Ionophores

doi: 10.3390/ijms222313098

Figure Lengend Snippet: Eomes promoted the expression of IFN-γ mRNA in EL4 cells. ( A , B ) Whole cell lysates were prepared from Control #1, Control #2, Eomes #1, and Eomes #2 EL4 transfectants and used for Western blotting using anti-FLAG, anti-RelA, and anti-NFATc2 antibodies. Blots were representative of five independent experiments ( A ). The amount of FLAG-Eomes, RelA, and NFATc2 was normalized to that of γ1-actin. The amount of the FLAG-Eomes protein in the Eomes #2 EL4 transfectant was set to 1-fold. The amounts of the RelA protein and NFATc2 protein in the Control #1 EL4 transfectant were set to 1-fold. The FLAG-Eomes protein (fold), RelA protein (fold), and NFATc2 protein (fold) are shown as the mean ± S.E. of five independent experiments. ( C , D ) Control #1, Control #2, Eomes #1, and Eomes #2 EL4 transfectants were treated with (+) or without (−) PMA (10 nM) and IM (1 µM) for 6 h. IFN-γ mRNA ( C ) and IL-2 mRNA ( D ) were measured by quantitative PCR. mRNA levels without PMA and IM in each EL4 transfectant were set to 1-fold. Data are shown as the mean ± S.E. of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Mouse lymphoma EL4 cells (RCB1641) were obtained from the RIKEN BioResource Research Center (Tsukuba, Japan).

Techniques: Expressing, Control, Western Blot, Transfection, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Small Molecule Inhibitors Targeting Nuclear Factor κB Activation Markedly Reduce Expression of Interleukin-2, but Not Interferon-γ, Induced by Phorbol Esters and Calcium Ionophores

doi: 10.3390/ijms222313098

Figure Lengend Snippet: Binding of FLAG-Eomes, RelA, and NFATc2 to the IFN-γ promoter, CNS−22, and CNS+30 in EL4 transfectants. ( A – C ) The Control #2 EL4 transfectant (Control) and Eomes #2 EL4 transfectant (Eomes) were treated with (+) or without (−) PMA (10 nM) and IM (1 µM) for 2 h. ChIP assays were performed for FLAG-Eomes ( A ), RelA ( B ), and NFATc2 ( C ). Quantitative PCR was used to measure the amounts of fifteen different DNA regions. The IFN-γ promoter (−0.1 kb), CNS−22, and CNS+30 are shown in this figure. Other regions for FLAG-Eomes, RelA, and NFATc2 are shown in , respectively. Immunoprecipitated (IP) DNA (% input) is shown as the mean ± S.E. of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Mouse lymphoma EL4 cells (RCB1641) were obtained from the RIKEN BioResource Research Center (Tsukuba, Japan).

Techniques: Binding Assay, Control, Transfection, Real-time Polymerase Chain Reaction, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Small Molecule Inhibitors Targeting Nuclear Factor κB Activation Markedly Reduce Expression of Interleukin-2, but Not Interferon-γ, Induced by Phorbol Esters and Calcium Ionophores

doi: 10.3390/ijms222313098

Figure Lengend Snippet: Effects of TPCA-1 on IFN-γ and IL-2 mRNA expression in EL4 transfectants. ( A – C ) Control #1, Control #2, Eomes #1, and Eomes #2 EL4 transfectants were pretreated with TPCA-1 for 1 h and then treated with (+) or without (−) PMA (10 nM) and IM (1 µM) for 2 h in the presence or absence of TPCA-1 at the indicated final concentrations. Cell viability (%) is shown as the mean ± S.E. of three independent experiments ( A ). IFN-γ mRNA ( B ) and IL-2 mRNA ( C ) were measured by quantitative PCR. mRNA levels with PMA and IM in each EL4 transfectant were set to 100%. IFN-γ mRNA (%) and IL-2 mRNA (%) are shown as the mean ± S.E. of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Mouse lymphoma EL4 cells (RCB1641) were obtained from the RIKEN BioResource Research Center (Tsukuba, Japan).

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Small Molecule Inhibitors Targeting Nuclear Factor κB Activation Markedly Reduce Expression of Interleukin-2, but Not Interferon-γ, Induced by Phorbol Esters and Calcium Ionophores

doi: 10.3390/ijms222313098

Figure Lengend Snippet: Effects of IKK-16 on IFN-γ and IL-2 mRNA expression in EL4 transfectants. ( A – C ) Control #1, Control #2, Eomes #1, and Eomes #2 EL4 transfectants were pretreated with IKK-16 for 1 h and then treated with (+) or without (−) PMA (10 nM) and IM (1 µM) for 2 h in the presence or absence of IKK-16 at the indicated final concentrations. Cell viability (%) is shown as the mean ± S.E. of three independent experiments ( A ). IFN-γ mRNA ( B ) and IL-2 mRNA ( C ) were measured by quantitative PCR. mRNA levels with PMA and IM in each EL4 transfectant were set to 100%. IFN-γ mRNA (%) and IL-2 mRNA (%) are shown as the mean ± S.E. of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Mouse lymphoma EL4 cells (RCB1641) were obtained from the RIKEN BioResource Research Center (Tsukuba, Japan).

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Small Molecule Inhibitors Targeting Nuclear Factor κB Activation Markedly Reduce Expression of Interleukin-2, but Not Interferon-γ, Induced by Phorbol Esters and Calcium Ionophores

doi: 10.3390/ijms222313098

Figure Lengend Snippet: Effects of TPCA-1 on the binding of RelA, FLAG-Eomes, and NFATc2 to the IFN-γ promoter, CNS−22, and CNS+30 in EL4 transfectants. ( A – C ) Eomes #2 EL4 transfectants were pretreated with (+) or without (−) TPCA-1 for 1 h and then treated with (+) or without (−) PMA (10 nM) and IM (1 µM) for 2 h in the presence or absence of TPCA-1 (20 µM). ChIP assays were performed for FLAG-Eomes ( A ), RelA ( B ), and NFATc2 ( C ) using control IgG (light color bars) or specific antibodies (dark color bars). Quantitative PCR was used to measure the amounts of DNA regions. Immunoprecipitated (IP) DNA (% input) is shown as the mean ± S.E. of four ( A ) and three ( B , C ) independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Mouse lymphoma EL4 cells (RCB1641) were obtained from the RIKEN BioResource Research Center (Tsukuba, Japan).

Techniques: Binding Assay, Control, Real-time Polymerase Chain Reaction, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: Glycogen Synthase Kinase 3 Inactivation Compensates for the Lack of CD28 in the Priming of CD8 + Cytotoxic T-Cells: Implications for anti-PD-1 Immunotherapy

doi: 10.3389/fimmu.2017.01653

Figure Lengend Snippet: T-cell activation with anti-CD28 enhances cytolytic T-cell (CTL) killing of antigen specific target cells through glycogen synthase kinase 3 (GSK-3). (A) OT-1 CD8 + CTLs were activated with OVA peptide incubated in the presence (right panel) or absence (left panel) of SB415286 with or without anti-CD28 or blocking CD28 (CTLA-4 IgG fusion protein). After 5 days, CTLs were washed and counted before incubation with target (OVA-EL4) cells at the ratios shown for 4 h. Lactate dehydrogenase release was measured as an indication of target cell killing. Histogram depicts measurements normalized for background non-specific killing. OVA alone: light gray bars; anti-CD28: light blue bars; CTLA-4 IgG: dark blue bars (error bars based on triplicate values in individual experiments, data shown representative of four independent experiments). (B) Histogram showing MFI values of programmed cell death 1 (PD-1) expression as measured by flow cytometry. (C) Flow cytometry profiles of GZMB and Lamp-1 in the presence and absence of SB415286 alone, combined with anti-CD28 or CTLA-4 IgG. Error bars based on triplicate values in individual experiments; data shown representative of three independent experiments.

Article Snippet: EL4 cells were incubated with 10 nM OVA 257–264 peptide (Bachem) for 1 h at 37°C and treated with mitomycin C (Sigma-Aldrich, St. Louis, MO, USA) (final concentration of 10 μg/mL) prior to mixing with primary T-cells by coculturing at a ratio of 1:5 of EL4 and T-cells to generate cytotoxic T-cells.

Techniques: Activation Assay, Incubation, Blocking Assay, Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Glycogen Synthase Kinase 3 Inactivation Compensates for the Lack of CD28 in the Priming of CD8 + Cytotoxic T-Cells: Implications for anti-PD-1 Immunotherapy

doi: 10.3389/fimmu.2017.01653

Figure Lengend Snippet: CD28 activation is comparable to glycogen synthase kinase 3 (GSK-3) inactivation enhancing cytolytic function. (A) OT-1 CD8 + cytolytic T-cells (CTLs) were transfected with scrambled (left panel) or GSK-3 (right panel) small-interfering RNA (siRNA) prior to activation with OVA peptide and incubated with or without anti-CD28 or blocking CD28 (CTLA-4 IgG fusion protein). After 5 days CTLs were washed and counted before incubation with target (OVA-EL4) cells at the ratios shown for 4 h. Lactate dehydrogenase release was measured as an indication of target cell killing. Histogram depicts measurements normalized for background non-specific killing. OVA alone: light gray bars; anti-CD28: light blue bars; CTLA-4 IgG: dark blue bars (error bars based on triplicate values in individual experiments, data shown representative of four independent experiments). (B) Histogram showing MFI values of programmed cell death 1 (PD-1) expression as measured by flow cytometry. (C) Flow cytometry profiles of GZMB and Lamp-1 in either scrambled or GSK-3 siRNA transfected cells stimulated with Ova alone, or combined with anti-CD28 or CTLA-4 IgG. Error bars based on triplicate values in individual experiments; data shown representative of three independent experiments.

Article Snippet: EL4 cells were incubated with 10 nM OVA 257–264 peptide (Bachem) for 1 h at 37°C and treated with mitomycin C (Sigma-Aldrich, St. Louis, MO, USA) (final concentration of 10 μg/mL) prior to mixing with primary T-cells by coculturing at a ratio of 1:5 of EL4 and T-cells to generate cytotoxic T-cells.

Techniques: Activation Assay, Transfection, Small Interfering RNA, Incubation, Blocking Assay, Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Glycogen Synthase Kinase 3 Inactivation Compensates for the Lack of CD28 in the Priming of CD8 + Cytotoxic T-Cells: Implications for anti-PD-1 Immunotherapy

doi: 10.3389/fimmu.2017.01653

Figure Lengend Snippet: Anti-CD28 enhances cytolytic T-cell (CTL) killing of antigen-specific target cells to similar extent as glycogen synthase kinase 3 (GSK-3) inhibitors. OT-1 CD8 + CTLs were activated with OVA peptide incubated in the presence or absence of one of four small molecule inhibitors (from top to bottom; SB415286, CHIR99021, L803mts, SB216763) with or without anti-CD28 or blocking CD28 (CTLA-4 IgG fusion protein). After 5 days, CTLs were washed and counted before incubation with target (OVA-EL4) cells at the ratios shown for 4 h. Lactate dehydrogenase release was measured as an indication of target cell killing. Dark blue bars on left panel depicts background non-specific killing (non-pulsed target cell death). Error bars based on triplicate values in individual experiments; data shown representative of three independent experiments.

Article Snippet: EL4 cells were incubated with 10 nM OVA 257–264 peptide (Bachem) for 1 h at 37°C and treated with mitomycin C (Sigma-Aldrich, St. Louis, MO, USA) (final concentration of 10 μg/mL) prior to mixing with primary T-cells by coculturing at a ratio of 1:5 of EL4 and T-cells to generate cytotoxic T-cells.

Techniques: Incubation, Blocking Assay

Journal: Frontiers in Immunology

Article Title: Glycogen Synthase Kinase 3 Inactivation Compensates for the Lack of CD28 in the Priming of CD8 + Cytotoxic T-Cells: Implications for anti-PD-1 Immunotherapy

doi: 10.3389/fimmu.2017.01653

Figure Lengend Snippet: Cytolytic potential of cytolytic T-cells (CTLs) primed in vivo can be enhanced in vitro with anti-CD28 antibody. OT-1 mice were injected intravenously with ova peptide alone (A) or in combination with SB415286 (B) . Spleens were extracted on day 7 . Ex vivo purified T-cells were then subjected to further stimulation with OVA peptide in the presence (right panel) or absence (left panel) of SB415286 with or without anti-CD28 or blocking CD28 (CTLA-4 IgG fusion protein). After 5 days, CTLs were washed and counted before incubation with target (OVA-EL4) cells at the ratios shown for 4 h. Lactate dehydrogenase release was measured as an indication of target cell killing. Histogram depicts measurements normalized for background non-specific killing. (Right panels) Flow cytometry profiles of GZMB, Lamp-1, and PD-1 in the presence and absence of SB415286, combined with anti-CD28 or CTLA-4 IgG (mean and SD of six mice per group). Error bars based on triplicate values in individual experiments; data shown representative of two independent experiments.

Article Snippet: EL4 cells were incubated with 10 nM OVA 257–264 peptide (Bachem) for 1 h at 37°C and treated with mitomycin C (Sigma-Aldrich, St. Louis, MO, USA) (final concentration of 10 μg/mL) prior to mixing with primary T-cells by coculturing at a ratio of 1:5 of EL4 and T-cells to generate cytotoxic T-cells.

Techniques: In Vivo, In Vitro, Injection, Ex Vivo, Purification, Blocking Assay, Incubation, Flow Cytometry