Journal: PLoS ONE

Article Title: Utility of Clostridium difficile Toxin B for Inducing Anti-Tumor Immunity

doi: 10.1371/journal.pone.0110826

Figure Lengend Snippet: CT26 cells were exposed to 500 ng/ml of TcdB for different time, and cell viability was measured by the PI staining as described in . Propidium iodide-positive cells were analyzed by Flow cytometry. The data shown represent one of three independent experiments. ***represents P<0.001 vs. control (unpaired two-tailed t- test). Error bars, SEM.

Article Snippet: Murine colon adenocarcinoma cell lines CT26 and CT26.CL25 (CT26 cells expressing the model antigen β-galactosidase) , the myeloma cell line p3x63Ag8.653, and the melanocytoma cell line B16-F10 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Staining, Flow Cytometry, Two Tailed Test

Journal: PLoS ONE

Article Title: Utility of Clostridium difficile Toxin B for Inducing Anti-Tumor Immunity

doi: 10.1371/journal.pone.0110826

Figure Lengend Snippet: Autologous splenocytes were co-cultured with bone marrow DCs (BMDCs) preloaded with live or TcdB-intoxicated CT26 cells for two weeks, and the supernatant was collected to measure IFN-γ production by ELISA. Splenocytes incubated with mere DCs or mere TcdB-treated tumor cells were set as controls. The data represent the mean of three independent determinations ± SEM. ***represents P<0.001 (unpaired two-tailed t- test).

Article Snippet: Murine colon adenocarcinoma cell lines CT26 and CT26.CL25 (CT26 cells expressing the model antigen β-galactosidase) , the myeloma cell line p3x63Ag8.653, and the melanocytoma cell line B16-F10 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Two Tailed Test

Journal: PLoS ONE

Article Title: Utility of Clostridium difficile Toxin B for Inducing Anti-Tumor Immunity

doi: 10.1371/journal.pone.0110826

Figure Lengend Snippet: Mice were subcutaneously immunized with PBS or TcdB-exposed CT26 cells (TcdB). In some experiments, mice were injected with lysate of TcdB-treated CT26 cells (TcdB-lysate). Mice were then challenged with 10 5 live CT26 cells on the opposite side of the groin and tumor growth was monitored. (A) Tumor volume was calculated using the formula: length×width 2 ×π/6. The data represent one of five independent experiments (n = 5∼8). **, P<0.01 vs. PBS; ***, P<0.001 vs. PBS (paired two-tailed t- test). Error bars, SEM. (B) The percentage of tumor-free mice was measured. The data in (B) represent a pool from five independent experiments (n = 5∼8 for each experiment).

Article Snippet: Murine colon adenocarcinoma cell lines CT26 and CT26.CL25 (CT26 cells expressing the model antigen β-galactosidase) , the myeloma cell line p3x63Ag8.653, and the melanocytoma cell line B16-F10 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Injection, Two Tailed Test

Journal: PLoS ONE

Article Title: Utility of Clostridium difficile Toxin B for Inducing Anti-Tumor Immunity

doi: 10.1371/journal.pone.0110826

Figure Lengend Snippet: Mice were immunized twice with PBS or TcdB-intoxicated CT26.CL25 (TcdB), and splenocytes were harvested 5 days after the second immunization. (A and B) splenocytes were restimulated with OVA, CT26.CL25 lysate, CT26 lysate, and β-galactosidase. (A) T-cell proliferation was determined by BrdU cell proliferation assay. (B) IL-2 production was measured by ELISA. The data in (A) and (B) represent the mean of three independent experiments ± SEM. *represents P<0.05 (one-way ANOVA). (C) Specific CTL induction. Splenocytes restimulated with CT26.CL25 lysate were tested for cytolytic activity against CT26.CL25 cells, parental CT26 cells, or myeloma p3x63Ag8.653 cells using cytotoxicity detection kit (LDH) assay. Representative data from one of three independent experiments are shown.

Article Snippet: Murine colon adenocarcinoma cell lines CT26 and CT26.CL25 (CT26 cells expressing the model antigen β-galactosidase) , the myeloma cell line p3x63Ag8.653, and the melanocytoma cell line B16-F10 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: BrdU Cell Proliferation Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Lactate Dehydrogenase Assay

Journal: PLoS ONE

Article Title: Utility of Clostridium difficile Toxin B for Inducing Anti-Tumor Immunity

doi: 10.1371/journal.pone.0110826

Figure Lengend Snippet: (A and B) Protection against pre-injected tumors. Four hours after lethal CT26 tumor cell challenge, mice were injected with PBS, TcdB-exposed CT26 cells (TcdB), or CT26 lysate (TcdB-lysate). (A) Tumor volume was measured. The data represent one of four independent experiments (n = 5∼8). ***represents P<0.0001 vs. PBS (paired two-tailed t- test). Error bars, SEM. (B) The percentage of tumor-free mice was determined. The data presented is a pool from four independent experiments (n = 5∼8 for each experiment). (C and D) Mice vaccinated with TcdB intoxicated CT26 cells are not protected against myeloma p3x63 cells. Mice were challenged with lethal myeloma p3x63 cells and then immunized with TcdB-intoxicated CT26 cells (TcdB) or vehicle control (PBS) at a different site 4 h later. (C) Mouse tumor volume was measured (n = 8). (D) The percentage of tumor-free mice was measured (n = 8). (E and F) The anti-tumor immunity induced by TcdB-intoxicated tumor cells is long lasting. Mice surviving the first challenge with CT26 cells after either prophylactic or therapeutic vaccination with TcdB-treated tumor cells were rechallenged with 10 6 (10 times the LD100) CT26 cells 3 months after the first challenge. The age-matched naive mice were challenged with 10 6 CT26 cells as control. Tumor volume (E) and the percentage of tumor-free mice (F) were evaluated. The data shown represent one of three independent experiments. ** in e, P<0.01 between prophylactic group or therapeutic group vs. PBS (paired two-tailed t- test); *** in f, P<0.0001 vs. PBS (Log-rank test). Error bars, SEM.

Article Snippet: Murine colon adenocarcinoma cell lines CT26 and CT26.CL25 (CT26 cells expressing the model antigen β-galactosidase) , the myeloma cell line p3x63Ag8.653, and the melanocytoma cell line B16-F10 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Injection, Two Tailed Test

Journal: Oncoimmunology

Article Title: Interleukin-21 combined with PD-1 or CTLA-4 blockade enhances antitumor immunity in mouse tumor models

doi: 10.1080/2162402X.2017.1377873

Figure Lengend Snippet: Addition of mIL-21 improves antitumor activity of mCTLA-4 mAb in the CT26 colon carcinoma model. Antitumor activity of mIL-21 (50 μg/mouse) and anti-mCTLA-4 mAb (clone UC10-4F10; 400 μg/mouse) when administered alone or in combination on the days indicated in the table. Median tumor volumes ( left hand panel ) and individual tumor volumes ( right hand panels ) are shown. CR = complete regression. Asterisks (*, **) indicate p < 0.05 or p < 0.01, respectively, for differences between the mCTLA-4 mAb group and either the control group or mIL-21 group (each comparison is p < 0.05), or the mCTLA-4 + mIL-21 combination group and either the control group or mIL-21 group (each comparison is p < 0.01) for ‘treatment effect’ by 2-way repeated measures ANOVA. Data are representative of results from two separate studies.

Article Snippet: CT26 cells (CRL-2639, ATCC) were cultured in RPMI 1640 media (SH30027, HyClone) supplemented with 10% FBS and GlutaMAX.

Techniques: Activity Assay

Journal: Oncoimmunology

Article Title: Interleukin-21 combined with PD-1 or CTLA-4 blockade enhances antitumor immunity in mouse tumor models

doi: 10.1080/2162402X.2017.1377873

Figure Lengend Snippet: Effect of immune cell subset depletion on mIL-21 + CTLA-4 or PD-1 blockade in the MC38 and CT26 models. (A) Mice implanted SC with (A) MC38 or (B) CT26 tumor cells were injected IP with PBS ( upper left ), or depleting mAbs directed against CD4+ ( upper right ), CD8+ ( lower left ), or NK ( lower right ) cells on days 3, 9, and 16 post-tumor implant. Two days after initiating depleting mAb treatment, the mice were administered PBS ( x's ), mCTLA-4 mAb (9D9-mIgG2b, 200 μg/mouse; filled circles ), mIL-21 (50 μg/mouse; filled diamonds ), a combination of mIL-21 and mCTLA-4 mAb ( open circles ), mPD-1 mAb (4H2-mIgG1, 200 μg/mouse; filled squares ), or a combination of mIL-21 and mPD-1 mAb ( open squares ). Median tumor volumes (mm 3 ) are plotted vs. days post tumor cell implant. Data are representative of results from two separate studies. See Supplemental Table 1 for statistical analyses.

Article Snippet: CT26 cells (CRL-2639, ATCC) were cultured in RPMI 1640 media (SH30027, HyClone) supplemented with 10% FBS and GlutaMAX.

Techniques: Injection

Journal: Oncoimmunology

Article Title: Interleukin-21 combined with PD-1 or CTLA-4 blockade enhances antitumor immunity in mouse tumor models

doi: 10.1080/2162402X.2017.1377873

Figure Lengend Snippet: TIL immunophenotypes in CT26 tumors from mice treated with mIL-21 +/− mPD-1 or mCTLA-4 mAbs. TILs from mice implanted SC with CT26 tumor cells and treated with control mIgG, mIL-21, mCTLA-4 mAb (9D9-mIgG2b), mPD-1 mAb (4H2-mIgG1), or mIL-21 + mPD-1 or mCTLA-4 mAbs were isolated on study day 16, stained with various markers of immune cell subsets, and evaluated by flow cytometry. The (A) %CD8+ of live CD45+, (B) %CD25+FoxP3+ of CD4+, (C) %CD335+ of live CD45+, and (D) %PD-1+CD69- of CD8+ cells, are shown for each treatment group, indicated on the x-axes. The (E) ratios of the % CD8+ T cells of live CD45+ (Teff) to the % CD4+CD25+ of live CD45+ (Tregs) are plotted. Each symbol represents data from one mouse in the group and mean values are indicated with horizontal lines. Asterisks (*, **, ***, ****) indicate p < 0.05, p < 0.01, p < 0.001 or p < 0.0001, respectively, for differences between the groups indicated by 1-way ANOVA. Study was conducted one time.

Article Snippet: CT26 cells (CRL-2639, ATCC) were cultured in RPMI 1640 media (SH30027, HyClone) supplemented with 10% FBS and GlutaMAX.

Techniques: Isolation, Staining, Flow Cytometry

Journal: Oncoimmunology

Article Title: Interleukin-21 combined with PD-1 or CTLA-4 blockade enhances antitumor immunity in mouse tumor models

doi: 10.1080/2162402X.2017.1377873

Figure Lengend Snippet: Summary of antitumor efficacy of mIL-21+mCTLA-4 mAb or mPD-1 mAb.

Article Snippet: CT26 cells (CRL-2639, ATCC) were cultured in RPMI 1640 media (SH30027, HyClone) supplemented with 10% FBS and GlutaMAX.

Techniques: Activity Assay

Journal: Oncoimmunology

Article Title: Interleukin-21 combined with PD-1 or CTLA-4 blockade enhances antitumor immunity in mouse tumor models

doi: 10.1080/2162402X.2017.1377873

Figure Lengend Snippet: Experimental Design.

Article Snippet: CT26 cells (CRL-2639, ATCC) were cultured in RPMI 1640 media (SH30027, HyClone) supplemented with 10% FBS and GlutaMAX.

Techniques: Injection

Journal: iScience

Article Title: Cellular resistance to an oncolytic virus is driven by chronic activation of innate immunity

doi: 10.1016/j.isci.2022.105749

Figure Lengend Snippet: Characterization and analysis of VSV-resistant CT26-derived clones (A) Infection spread tracking by real-time live-cell fluorescence microscopy in naïve CT26 cells and resistant clones C1 and C4 after infection with VSV-D51 (MOI = 0.1 PFU/cell). (B) Viral production progeny after 24hpi. Significant differences by t -test are marked with asterisks (∗∗∗ p< 0.001; ∗∗ p< 0.01). (C) RT-qPCR analysis of viral RNA (RNA-L) produced in cells inoculated at low MOI (0.1 PFU/cell) after 8 hpi. Data are presented as mean ± SEM. (D) Principal component analysis of global gene expression in resistant C1, C4, and naïve CT26 cells. (E) Correlation between differential gene expression values in the resistant C1 clone and each of the three B16 resistant clones (R1, R2 and R5). (F) Gene ontology analysis of the 10 highest enrichment processes comparing C1 and naïve CT26 cells. Green: observed ratio, Blue: expected ratio.

Article Snippet: Mouse: CT26 , ATCC , ATCC Cat# CRL-2639; RRID: CVCL_7255.

Techniques: Derivative Assay, Clone Assay, Infection, Fluorescence, Microscopy, Quantitative RT-PCR, Produced, Expressing

Journal: iScience

Article Title: Cellular resistance to an oncolytic virus is driven by chronic activation of innate immunity

doi: 10.1016/j.isci.2022.105749

Figure Lengend Snippet:

Article Snippet: Mouse: CT26 , ATCC , ATCC Cat# CRL-2639; RRID: CVCL_7255.

Techniques: Recombinant, SYBR Green Assay, Expressing, Sequencing, Software