Journal: Journal for immunotherapy of cancer

Article Title: Engineered oncolytic virus expressing B7H3-targeting BiTE enhances antitumor T-cell immune response.

doi: 10.1136/jitc-2024-009901

Figure Lengend Snippet: Figure 1 Expression of B7H3 in human malignancies and generation of an OV encoding B7H3 BiTE (OAd-B7H3-BiTE). (A) B7H3 (CD276) expression between tumor and normal samples analyzed by TIMER2.0 (http://timer.cistrome.org/). (B–C) Representative immunohistochemistry images (B) and quantification (C) of B7H3 staining in peritumoral and cancerous tissue from patients with lung adenocarcinoma (LUAD) (n=3), breast cancer (BC) (n=6) or colon adenocarcinoma (COAD) (n=2). Each pair of dots represents a patient. (D) Schematic of recombinant OAd-B7H3-BiTE genomes. (E) Cytotoxic effects of OAd-B7H3- BiTE. MGC-803 and U87 cells were infected with serial dilutions of OAd or OAd-B7H3-BiTE. Four days after infection, cell viability was examined. (F) Viral replication of OAd-B7H3-BiTE. MGC-803 and U87 cells were infected with OAd or OAd-B7H3- BiTE and then harvested at different time points. DNA extraction and quantitative PCR were performed to detect the copy number of the E1a gene. Fold change to 0 hour was calculated. (G) Serial volumes of anti-CD3 scFv were detected with anti– anti-CD3-scFv antibody by western blot. (H) HEK293 cells were infected with OAd or OAd-B7H3-BiTE at multiplicity of infection of 0.5. 48 hours after infection, supernatants were collected. B7H3 BiTE was detected with anti–anti-CD3-scFv antibody by western blot. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; OAd, oncolytic adenovirus; scFv, single- chain variable fragment.

Article Snippet: Deparaffinized and dehydrated sections were boiled for 10 min in 0.01 M citrate buffer and incubated with 3% hydrogen peroxide in methanol for 25 min to block endogenous peroxidase, incubated with B7H3 monoclonal antibody (1:3,000; #66481–1- Ig; Proteintech) and horseradish peroxidase (HRP)- conjugated goat anti- mouse IgG (H+L) (1:200; #GB23301; Servicebio) sequentially, and visualized with 0.05% 3,3- diaminobenzidine tetrachloride.

Techniques: Expressing, Immunohistochemistry, Staining, Recombinant, Infection, DNA Extraction, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal for immunotherapy of cancer

Article Title: Engineered oncolytic virus expressing B7H3-targeting BiTE enhances antitumor T-cell immune response.

doi: 10.1136/jitc-2024-009901

Figure Lengend Snippet: Figure 2 B7H3 expression in cell lines and the binding of delivered B7H3 BiTE to T and target cell. (A) B7H3 expression on MGC-803, SH-SY5Y, U87, SGC-7901 and Raji cells were detected using flow cytometry. (B) Binding of delivered B7H3 BiTE to target cells. Several cell lines were incubated with supernatants of the OAd-B7H3-BiTE-infected HEK293 cells. B7H3 BiTE binding was detected via flow cytometry with anti–anti-CD3-scFv antibody. (C) CD3 expression on Jurkat cells was detected using flow cytometry. (D) Binding of delivered B7H3 BiTE to T cells. PBMCs were incubated with supernatants of the OAd- B7H3-BiTE-infected HEK293 cells. B7H3 BiTE binding was detected via flow cytometry with anti–anti-CD3-scFv antibody. (E) Competition between delivered B7H3 BiTE and OKT3. PBMCs were incubated with supernatants of OAd or OAd-B7H3- BiTE-infected HEK293 cells. Then, cells were stained with fluorescein-conjugated OKT3 and examined throughflow cytometry. (F) Cell-bridging assay. A mixture of MGC-803/U87 and PBMCs was incubated with the supernatants of the OAd or OAd- B7H3-BiTE-infected HEK293 cells. The percentage of T/target cell pairs was quantified using flow cytometry. Statistical analysis was performed by unpaired Student’s t-test (n=3). NS: not significant; **p<0.01 and ****p<0.0001. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; OAd, oncolytic adenovirus; PBMC, peripheral blood mononuclear cell; scFv, single- chain variable fragment.

Article Snippet: Deparaffinized and dehydrated sections were boiled for 10 min in 0.01 M citrate buffer and incubated with 3% hydrogen peroxide in methanol for 25 min to block endogenous peroxidase, incubated with B7H3 monoclonal antibody (1:3,000; #66481–1- Ig; Proteintech) and horseradish peroxidase (HRP)- conjugated goat anti- mouse IgG (H+L) (1:200; #GB23301; Servicebio) sequentially, and visualized with 0.05% 3,3- diaminobenzidine tetrachloride.

Techniques: Expressing, Binding Assay, Flow Cytometry, Incubation, Infection, Staining, Cytometry

Journal: Journal for immunotherapy of cancer

Article Title: Engineered oncolytic virus expressing B7H3-targeting BiTE enhances antitumor T-cell immune response.

doi: 10.1136/jitc-2024-009901

Figure Lengend Snippet: Figure 3 T-cell activation mediated by oncolytic viruses-delivered B7H3 BiTE. (A–D) PBMCs were cultured alone or co- cultured with the indicated cell lines in the absence (Mock) or presence of the supernatants of OAd or OAd-B7H3-BiTE- infected cells for 48 hours. (A) Representative brightfield images are shown. (B) Representative flow cytometry histograms showing FSC-A of CD4+ and CD8+ T cells. (C) Cumulative data of flow cytometry showing CD69 or CD107a expression in CD4+ and CD8+ T cells. (D) The levels of IFN-γ and IL-2 in supernatants at 48 hours measured by ELISA. (E) T-cell proliferation assay. CFSE-stained PBMCs were co-cultured with MGC-803 cells and incubated with or without the supernatants of OAd or OAd-B7H3-BiTE-infected cells. Proliferation was determined after incubation for 4 days. Statistical analysis was performed by unpaired Student’s t-test (n=3). NS: not significant; **p<0.01, ***p<0.001, and ****p<0.0001. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; CFSE, 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester; IFN, interferon; IL, interleukin; OAd, oncolytic adenovirus; PBMC, peripheral blood mononuclear cell.

Article Snippet: Deparaffinized and dehydrated sections were boiled for 10 min in 0.01 M citrate buffer and incubated with 3% hydrogen peroxide in methanol for 25 min to block endogenous peroxidase, incubated with B7H3 monoclonal antibody (1:3,000; #66481–1- Ig; Proteintech) and horseradish peroxidase (HRP)- conjugated goat anti- mouse IgG (H+L) (1:200; #GB23301; Servicebio) sequentially, and visualized with 0.05% 3,3- diaminobenzidine tetrachloride.

Techniques: Activation Assay, Cell Culture, Infection, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Staining, Incubation

Journal: Journal for immunotherapy of cancer

Article Title: Engineered oncolytic virus expressing B7H3-targeting BiTE enhances antitumor T-cell immune response.

doi: 10.1136/jitc-2024-009901

Figure Lengend Snippet: Figure 4 T-cell cytotoxicity mediated by OAd-B7H3-BiTE. (A–C) After labeling with CFSE, MGC-803 (A), U87 (B) or Raji (C) cells were infected with OAd or OAd-B7H3-BiTE; uninfected cells (Mock) were used as a control. After 24 hours, peripheral blood mononuclear cells were added and co-cultured for 48 hours. Representative flow cytometry plots (left) and cumulative data (right) are shown. Dead target cells are defined as CFSE+ Zombie Dye+. The percentage of dead target cells is calculated as follows: CFSE+ Zombie-Dye+/CFSE+. Statistical analysis was performed by unpaired Student’s t-test (n=3). **p<0.01, ***p<0.001, and ****p<0.0001. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; CFSE, 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester; OAd, oncolytic adenovirus.

Article Snippet: Deparaffinized and dehydrated sections were boiled for 10 min in 0.01 M citrate buffer and incubated with 3% hydrogen peroxide in methanol for 25 min to block endogenous peroxidase, incubated with B7H3 monoclonal antibody (1:3,000; #66481–1- Ig; Proteintech) and horseradish peroxidase (HRP)- conjugated goat anti- mouse IgG (H+L) (1:200; #GB23301; Servicebio) sequentially, and visualized with 0.05% 3,3- diaminobenzidine tetrachloride.

Techniques: Labeling, Infection, Control, Cell Culture, Flow Cytometry

Journal: Journal for immunotherapy of cancer

Article Title: Engineered oncolytic virus expressing B7H3-targeting BiTE enhances antitumor T-cell immune response.

doi: 10.1136/jitc-2024-009901

Figure Lengend Snippet: Figure 5 OAd-B7H3-BiTE treatment elicits potent therapeutic effects in vivo. (A) Schematic diagram of treatment schedule for MGC-803 bearing mice (B–G). (B) Representative flow cytometry plots showing human CD45+ CD3+ T cells in tumor and spleen of untreated mice. Flow cytometry plots gated on live-cell population. (C) Summary data for average tumor growth (left) and body weight changes (right) for all treatment groups. (D) Tumor pictures and weights for all treatment groups. (E–F) Primary tumors were collected and analyzed with reverse transcription-quantitative PCR to examine the expression of E1a (E) and B7H3 BiTE (F). (G) Representative H&E staining of the major organs for all treatment groups. Statistical analysis was performed by unpaired two-way analysis of variance (C) or unpaired Student’s t-test (D) (n=5). ***p<0.001, and ****p<0.0001. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; mRNA, messenger RNA; OAd, oncolytic adenovirus; PBS, phosphate-buffered saline; PBMC, peripheral blood mononuclear cell; s.c., subcutaneous injection.

Article Snippet: Deparaffinized and dehydrated sections were boiled for 10 min in 0.01 M citrate buffer and incubated with 3% hydrogen peroxide in methanol for 25 min to block endogenous peroxidase, incubated with B7H3 monoclonal antibody (1:3,000; #66481–1- Ig; Proteintech) and horseradish peroxidase (HRP)- conjugated goat anti- mouse IgG (H+L) (1:200; #GB23301; Servicebio) sequentially, and visualized with 0.05% 3,3- diaminobenzidine tetrachloride.

Techniques: In Vivo, Flow Cytometry, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Staining, Saline, Injection

Journal: Journal for immunotherapy of cancer

Article Title: Engineered oncolytic virus expressing B7H3-targeting BiTE enhances antitumor T-cell immune response.

doi: 10.1136/jitc-2024-009901

Figure Lengend Snippet: Figure 6 OAd-B7H3-BiTE increases T-cell infiltration and activation. Flow cytometry analysis of T cells in the tumor xenografts model treated as described in figure 5A. (A) Proportion of CD4+ and CD8+ TILs. (B–D) The expression of CD69 (B), Tim-3 (C) and PD-1 (D) on TILs. (E) Expression of CD69 on CD4+ and CD8+ T cells in the spleen. Statistical analysis was performed by unpaired Student’s t-test (n=5). NS: not significant; *p<0.05, **p<0.01. Data are presented as the mean±SD. BiTE, bispecific T-cell engager; OAd, oncolytic adenovirus; PD-1, programmed cell death-1; TILs, tumor-infiltrating lymphocytes; Tim-3, T cell immunoglobulin mucin-3.

Article Snippet: Deparaffinized and dehydrated sections were boiled for 10 min in 0.01 M citrate buffer and incubated with 3% hydrogen peroxide in methanol for 25 min to block endogenous peroxidase, incubated with B7H3 monoclonal antibody (1:3,000; #66481–1- Ig; Proteintech) and horseradish peroxidase (HRP)- conjugated goat anti- mouse IgG (H+L) (1:200; #GB23301; Servicebio) sequentially, and visualized with 0.05% 3,3- diaminobenzidine tetrachloride.

Techniques: Activation Assay, Flow Cytometry, Expressing

Journal: Cell

Article Title: Therapeutic potential of co-signaling receptor modulation in hepatitis B

doi: 10.1016/j.cell.2024.05.038

Figure Lengend Snippet:

Article Snippet: InVivoMab anti-mouse L-Selectin (CD62L) , Bio X Cell , Cat# BE0021; RRID: AB_1107665.

Techniques: Purification, Virus, Recombinant, Staining, Saline, Fluorsave, Sequencing, DNA Labeling, Cell Isolation, Sample Prep, Reverse Transcription, Software, Microscopy

Journal: eLife

Article Title: Antibody escape by polyomavirus capsid mutation facilitates neurovirulence

doi: 10.7554/eLife.61056

Figure Lengend Snippet:

Article Snippet: Antibody , ChromPure Rat IgG , Jackson ImmunoResearch , Cat#012-000-003 , See Materials and methods for concentrations.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Recombinant, Virus, Purification, Reverse Transcription, Transfection, Avidin-Biotin Assay, Blocking Assay, SYBR Green Assay, DNA Purification, Mutagenesis, Software

Journal: Cancer science

Article Title: Enhancer of zeste homolog 2 downregulates E-cadherin by mediating histone H3 methylation in gastric cancer cells.

doi: 10.1111/j.1349-7006.2008.00743.x

Figure Lengend Snippet: Fig. 1. Profiling of enhancer of zeste homolog 2 (EZH2) mRNA expression in gastric cancer cell lines, gastric cancer tissue, and non-cancerous mucosa. (a) EZH2 mRNA from cancer cells including gastric, prostate, and breast cancer cell lines was determined by quantitative real-time reverse transcription–polymerase chain reaction (RT-PCR). After normalizing with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the ratio of each cell line (mean ± SD) carried out in duplicate from two different experiments, was calculated based on the standard curve. (b) Western blot analysis showed expression of EZH2 protein in 12 gastric cancer cell lines. (c) The level of E-cadherin mRNA expression in 12 gastric cancer cell lines was confirmed by quantitative real-time RT-PCR. (d) EZH2 mRNA from 22 pairs of gastric cancer and non-cancerous gastric mucosa was determined by quantitative real-time RT-PCR. The values from each tissue sample were calculated as above. The significant difference between gastric cancer tissue and non-cancerous mucosa was determined by Mann–Whitney U-test.

Article Snippet: Probing antibodies included EZH2 (1:1000), E-cadherin (1:2500), β-catenin (1:500) (all supplied by BD Transduction Laboratories), and β-actin (1:50; Santa Cruz Biotechnology).

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, MANN-WHITNEY

Journal: Cancer science

Article Title: Enhancer of zeste homolog 2 downregulates E-cadherin by mediating histone H3 methylation in gastric cancer cells.

doi: 10.1111/j.1349-7006.2008.00743.x

Figure Lengend Snippet: Fig. 2. Restoration of E-cadherin mRNA in MKN1 gastric cancer cell line after knockdown of enhancer of zeste homolog 2 (EZH2) accompanied by chromatin remodeling without DNA methylation in the promoter region of the E-cadherin gene. (a) Restoration of E-cadherin mRNA analyzed by reverse transcription–polymerase chain reaction (RT-PCR) in MKN1 cells was visualized with 6% polyacrylamide gel electrophoresis. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siRNA, short interfering RNA. (b) The restored expression level of E-cadherin mRNA was quantified by real-time RT-PCR. (c) Chromatin immunoprecipitation (ChIP) assay was carried out using DNA–protein complex isolated from MKN1 cells transfected with EZH2 siRNA for 96 h and immunoprecipitated (IP) with various antibodies. The PCR product of each IP DNA and input DNA was visualized with 6% polyacrylamide gel electrophoresis. The number under each gel is the ratio of IP DNA versus input DNA quantified. A, antibody; B, no antibody; C, input. Individual ChIP assays were repeated at least twice to confirm the reproducibility of the PCR-based experiment. The results of ChIP assays are shown as a diagram for each antibody and statistical analyses (student’s t-test) were carried out (H3K27me3; H3-Lys-27 trimethylation, H3K9me2; H3-Lys-9 dimethylation). (d) Methylation-specific polymerase chain reaction analyses from the DNA of MKN1 cells transfected with EZH2 siRNA (e) or negative control siRNA (c), using primer sets that specifically amplify either unmethylated (U) or methylated (M). Control templates from human genomic placenta DNA, treated with SssI methylase (S) or untreated (P), are shown. Ma, 100 bp DNA ladder marker. (e) Combined bisulfite restriction analysis (COBRA) from the DNA of MKN1 and MKN28 cells transfected with EZH2 siRNA (E) or negative control siRNA (C), using primer sets for the E-cadherin gene. MKN28 was a positive control for E-cadherin expression. PCR products were digested with specific restriction enzymes including SnaBI or TaqI. (f) Bisulfite sequencing of the E-cadherin CpG island. Top, map of the CpG island. Twenty- two individual CpG dinucleotides are indicated as vertical lines. Primer, location of bisulfite sequencing primers. Numbers indicate positions relative to transcription start. TS, transcriptional start. Bottom, each circle indicates a CpG site in the primary sequence, and each line of circles represents analysis of a single cloned allele. Black circles, methylated CpG sites; white circles, unmethylated CpG sites.

Article Snippet: Probing antibodies included EZH2 (1:1000), E-cadherin (1:2500), β-catenin (1:500) (all supplied by BD Transduction Laboratories), and β-actin (1:50; Santa Cruz Biotechnology).

Techniques: Knockdown, DNA Methylation Assay, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Small Interfering RNA, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Isolation, Transfection, Immunoprecipitation, Methylation, Negative Control, Control, Marker, Combined Bisulfite Restriction Analysis Assay, Positive Control, Methylation Sequencing, Sequencing, Clone Assay

Journal: Cancer science

Article Title: Enhancer of zeste homolog 2 downregulates E-cadherin by mediating histone H3 methylation in gastric cancer cells.

doi: 10.1111/j.1349-7006.2008.00743.x

Figure Lengend Snippet: Fig. 3. Knockdown of enhancer of zeste homolog 2 (EZH2) restores E-cadherin expression and induces the membranous translocation of β-catenin from the nucleus. (a) The re-expression of E-cadherin was also detected by Western blot analysis, without changing β-catenin expression after EZH2 knockdown. (b) Human gastric cancer MKN1 cells transfected with EZH2 short interfering RNA (siRNA) show the restoration and membranous distribution of E-cadherin (E-cadherin, red; EZH2, green). (c) MKN1 cells transfected with EZH2 siRNA showed the translocation of β-catenin from the nucleus to the membrane (β-catenin, red; EZH2, green).

Article Snippet: Probing antibodies included EZH2 (1:1000), E-cadherin (1:2500), β-catenin (1:500) (all supplied by BD Transduction Laboratories), and β-actin (1:50; Santa Cruz Biotechnology).

Techniques: Knockdown, Expressing, Translocation Assay, Western Blot, Transfection, Small Interfering RNA, Membrane

Journal: Cancer science

Article Title: Enhancer of zeste homolog 2 downregulates E-cadherin by mediating histone H3 methylation in gastric cancer cells.

doi: 10.1111/j.1349-7006.2008.00743.x

Figure Lengend Snippet: Fig. 4. Effect of E-cadherin restored by the knockdown of enhancer of zeste homolog 2 (EZH2) on the invasive capacity of human gastric cancer MKN1 cells. (a) The invading cells were quantitated by dissolving stained cells with colorimetric reading of the optical density (OD) at 560 nm. (b) Forty-eight hours after transfection with EZH2 short interfering RNA (siRNA) or control siRNA, cells were re-seeded in new dishes at a concentration of 1.0 × 105 cells, then enumerated. Bars, SD; points, mean of three independent experiments.

Article Snippet: Probing antibodies included EZH2 (1:1000), E-cadherin (1:2500), β-catenin (1:500) (all supplied by BD Transduction Laboratories), and β-actin (1:50; Santa Cruz Biotechnology).

Techniques: Knockdown, Staining, Transfection, Small Interfering RNA, Control, Concentration Assay

Journal: Cancer science

Article Title: Enhancer of zeste homolog 2 downregulates E-cadherin by mediating histone H3 methylation in gastric cancer cells.

doi: 10.1111/j.1349-7006.2008.00743.x

Figure Lengend Snippet: Fig. 5. Correlation between enhancer of zeste homolog 2 (EZH2) expression, expression of E-cadherin, and the localization of β-catenin in gastric cancer tissues. The expression of these proteins was evaluated by immunohistochemistry using 114 human gastric adenocarcinomas. (a) EZH2 is normally expressed in the neck region, the proliferative zone for gastric mucosa. HE, hematoylin–eosin. (b) High expression of EZH2 correlated with loss of E-cadherin expression and nuclear and cytoplasmic localization of β-catenin. (c) No expression of EZH2 correlated with membranous localization of E-cadherin or β-catenin. Lymphoblasts served as appropriate positive controls for EZH2. (d) The relationship between EZH2 expression and negative or membranous localization of E-cadherin expression in 114 gastric cancer tissues (χ2-test, P < 0.0001). Bar, 100 μm (a–c).

Article Snippet: Probing antibodies included EZH2 (1:1000), E-cadherin (1:2500), β-catenin (1:500) (all supplied by BD Transduction Laboratories), and β-actin (1:50; Santa Cruz Biotechnology).

Techniques: Expressing, Immunohistochemistry

Journal:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: Expression of murine CD40L by recombinant canarypox vector. Recombinant canarypox viruses vCPmCD40L and vCPmSP-D-CD40L were generated as described in Materials and Methods. (A) RT-PCR. CEF cells were infected by vCPmCD40L, vCPmSP-D-CD40L or its parental control ALVAC II at 5 MOI for 3 days. Total RNA was isolated from infected CEF cells using Trizol Reagent and subjected to RT-PCR with specific primers amplifying the coding sequence of murine CD40L or SP-D-CD40L. The identity of the amplified product was further verified by DNA sequencing (not shown). (B) Flow cytometry. Human PBMCs were infected by vCPmCD40L, vCPmSP-D-CD40L or ALVAC II at 5 MOI for 24 hrs and stained with PE-labeled anti-mouse CD40L mAb. (C) Western blot. Hela cells were infected by vCPmCD40L, vCPmSP-D-CD40L, or ALVAC II at 10 MOI for 24–48hrs. Cell lysates or magnetic beads incubated with supernatant of infected cells were subjected to Western blot and detected with a goat anti-mouse CD40L or anti-mouse SP-D antibody. As expected, the membrane CD40L was about 35–40kD and soluble multimeric form of CD40L was about 70kD.

Article Snippet: The membrane was then blocked and probed with goat anti-mouse CD40L or goat anti-human CD40L antibody (R&D Systems, Inc., Minneapolis, MN), followed by horseradish peroxidase-conjugated anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Expressing, Recombinant, Plasmid Preparation, Generated, Reverse Transcription Polymerase Chain Reaction, Infection, Control, Isolation, Sequencing, Amplification, DNA Sequencing, Flow Cytometry, Staining, Labeling, Western Blot, Magnetic Beads, Incubation, Membrane

Journal:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: CD40L expressed by recombinant ALVAC virus boosts the immunogenicity of an HIV-1 canarypox vaccine in mice. Female Balb/c mice of 6–8 wks age were immunized 3 times with an HIV-1 canarypox vaccine, vCP1452, with either of vCPmCD40L or vCPmSP-D-CD40L or ALVAC II. Six weeks after the last immunization, mice were sacrificed and spleens were taken and used for preparation of splenocytes. (A) IFN-γ ELISpot. Splenocytes were stimulated with P815 cells pulsed with an H-2Kd restricted HIV-1 Gag peptide, AMQMLKETI, for 16 hrs. Cells producing IFN-γ were detected and counted as described in Materials and Methods. (B) and (C) MHC-I tetramer analysis. Splenocytes were stained with anti-mouse CD8 mAb and AMQMLKETI/H-2Kd tetramer. (B) shows representative flow cytometry results for each group of mice. Numbers are percentage of tetramer positive cells in CD8+ cells. (C) shows summary of the tetramer data for each group of mice. (D) to (G) Polyfunctional CD8+ T cells analyzed by flow cytometry. Splenocytes were stained with anti-mouse IFN-γ and anti-mouse TNF-α mAbs and co-expressing cells are measured in (D) and (E) or anti-mouse IFN-γ and anti-mouse CD107a mAbs and co-expressing cells are measured in (F) and (G). (D) and (F) show representative flow cytometry results and (E) and (G) show summary of the polyfunctional CD8+ T cell data for each group of mice. (H) CD8+ T cells producing IFN-γ analyzed by flow cytometry. Splenocytes were stained with anti-mouse CD8 and anti-mouse IFN-γ mAbs. (I) and (J) Memory HIV-1-Gag specific CD8+ cells analyzed by flow cytometry. Splenocytes were stained with anti-mouse CD8, anti-mouse CD127 (IL-7Rα) mAbs, and AMQMLKETI/H-2Kd tetramer. In (I) CD8+/Tetramer+ cells are gated and measured by CD127 (y-axis). CD8+ T cells from naïve mice unstained for CD127 was used as control for CD127 gating. (J) shows summary data of CD127 expression in tetramer+ cells. (K) Lymphocyte proliferation. Splenocytes were stimulated with HIV-1 p24 Gag for 6 days and [3H]-thymidine incorporation was measured. (L) Cytokine production. Splenocytes were stimulated with HIV-1 p24 Gag for 5 days. IFN-γ and IL-4 in the supernatant were measured by ELISA. (M) Serum anti-Gag antibody. HIV-1 p24 Gag was used as antigen, and mice serum anti-Gag antibodies were measured by ELISA. Data shown are mean±SEM. n=4–10. *: P<0.05. **: P<0.01.

Article Snippet: The membrane was then blocked and probed with goat anti-mouse CD40L or goat anti-human CD40L antibody (R&D Systems, Inc., Minneapolis, MN), followed by horseradish peroxidase-conjugated anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Recombinant, Virus, Immunopeptidomics, Enzyme-linked Immunospot, Staining, Flow Cytometry, Expressing, Control, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: CD40L expressed by recombinant ALVAC virus enhances TNF-α and IL-12 production of human MDDCs. Human immature MDDCs were infected with recombinant ALVAC virus expressing human CD40L called vA3131-2, an ALVAC-HIV vaccine called vCP205, or a parental control, ALVAC II at an MOI of 10, or incubated with medium alone, or with CD40L trimer (2 μg/ml) as a positive control at 37°C for 24 h. The production of TNF-α and IL-12 was determined by intracellular staining flow cytometry. (A) Data are taken from the HIV-1-infected participant #1 and are representative of experiments with MDDCs derived from two HIV-1-infected and two HIV-1-uninfected individuals. The numbers in each gate represent the percentage of TNF-α or IL-12-positive MDDCs in total MDDCs. (B) Pooled data from all participants are shown. Data shown are mean±SEM. **: P<0.01.

Article Snippet: The membrane was then blocked and probed with goat anti-mouse CD40L or goat anti-human CD40L antibody (R&D Systems, Inc., Minneapolis, MN), followed by horseradish peroxidase-conjugated anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Recombinant, Virus, Infection, Expressing, Control, Incubation, Positive Control, Staining, Flow Cytometry, Derivative Assay

Journal:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: CD40L expressed by recombinant ALVAC virus promotes human MDDCs maturation independent of TNF-α secretion. Immature MDDCs were infected with recombinant ALVAC virus expressing human CD40L, vA3131-2; an ALVAC-HIV vaccine, vCP205; or a parental control, ALVAC II at an MOI of 10, or incubated with medium alone at 37°C for 48 h. Expression of surface molecules was analyzed by flow cytometry. (A) Numbers in CD83 row represent percentage of CD83 positive cells in total MDDCs and numbers in CD86 and CD80 rows represent the mean fluorescence intensity of specific staining (histogram with solid line) subtracted from the value of background staining with matched isotype control mouse mAbs (dotted histogram). Data shown are from HIV-1-infected participant #1 and representative of six experiments performed with MDDCs obtained from three HIV-1-uninfected blood donors and three HIV-1-infected individuals. (B) Pooled data from all six participants studied are shown. Data shown are mean±SEM. **: P<0.01. (C) Immature human MDDCs were infected or not (medium) with vA3131-2 (CD40L expressing), vCP205, or ALVAC II at an MOI of 10 and then incubated in the presence of a blocking anti-human TNF-α Ab or the control isotype immunoglobulin (20 μg/mL). After 24 h incubation, MDDCs were collected and stained for CD83 expression to monitor DC maturation. Numbers in each dot plot represent percentage of CD83 positive cells in total MDDCs. Expression of CD83 on MDDCs treated with isotype immunoglobulin is similar to that on MDDCs treated with medium. Increasing the concentration of blocking anti-human TNF-α Ab up to 100 μg/mL did not further reduce the CD83 expression in all conditions (data not shown). The results represent one of five experiments.

Article Snippet: The membrane was then blocked and probed with goat anti-mouse CD40L or goat anti-human CD40L antibody (R&D Systems, Inc., Minneapolis, MN), followed by horseradish peroxidase-conjugated anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Recombinant, Virus, Infection, Expressing, Control, Incubation, Flow Cytometry, Fluorescence, Staining, Blocking Assay, Concentration Assay

Journal:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: Effects of ALVAC recombinant vA3131-2 expressing human CD40L on apoptosis of DCs. Immature MDDCs were infected or not (medium control) with either parental ALVAC II, ALVAC recombinant vA3131-2 expressing human CD40L, or an HIV canarypox vaccine vCP205 at an MOI of 10 at 37°C for 48 h. Infected cells were harvested and early apoptotic marker caspase-3 was determined by intracellular staining and flow cytometry using FITC- or PE-conjugated mAb against human caspase-3. (A) MDDCs were intracellularly stained for caspase-3 expression to detect early apoptotic MDDCs. The level of caspase-3 expression (x axis) is shown for each of the conditions. Background staining with FITC-conjugated isotype control Ab is shown by a solid gray histogram; medium-treated MDDCs are shown by a histogram with a dotted line, MDDCs infected with vA3131-2 are represented with a histogram with a dark line, and MDDCs infected with parental ALVAC II are represented with a histogram with a light line. The results represent one of five experiments from two HIV-1-uninfected and three HIV-1-infected donors. (B) Pooled data from all participants are shown. Data shown are mean±SEM. **: P<0.01.

Article Snippet: The membrane was then blocked and probed with goat anti-mouse CD40L or goat anti-human CD40L antibody (R&D Systems, Inc., Minneapolis, MN), followed by horseradish peroxidase-conjugated anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Recombinant, Expressing, Infection, Control, Marker, Staining, Flow Cytometry

Journal:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: CD40L expressed by recombinant ALVAC virus enhances specific antiviral CTL activity. CD4+ T cell-containing or -depleted PBMCs from four HIV-1-infected individuals and three HIV-1-uninfected individuals were cocultured with autologous MDDCs that were either pulsed or not with HLA-restricted epitopes of HIV-1 proteins (for HIV-1 infected individuals) or EBV proteins (for HIV-1-uninfected individuals). MDDCs were previously infected or not (medium) with parental ALVAC II or ALVAC recombinant vA3131-2 expressing human CD40L at an MOI of 10 for 48 h. On day 10, specific CTL activity was assessed by intracellular flow cytometric analysis of IFN-γproducing CD8+ T cells and 51Cr release assay. (A) Representative intracellular IFN-γ flow cytometric data obtained from HIV-1-positive participant #1. (B) Summary data from intracellular IFN-γ flow cytometry of HIV-1-positive participant #2–#4 are graphically depicted. Open bars represent CD4+ T cell-containing condition and dark bar represent CD4+ T cell -depleted conditions. (C) A representative 51Cr release assay result from HIV-1-positive participant #1 is shown. Similar results were obtained with HIV-1-positive participant #2 (data not shown). (D) Summary data from intracellular IFN-γ flow cytometric analysis of EBV-positive participant #5–#7 are graphically depicted. Open bars represent CD4+ T cell -containing condition and dark bars represent CD4+ T cell -depleted conditions. (E) A representative 51Cr release assay result from EBV-positive participant #6 is shown. Similar results were obtained with EBV-positive participant #5 (data not shown). DC, MDDCs not pulsed with peptide; DCp, MDDCs pulsed with peptide; vA3131-2/DCp, vA3131-2-infected MDDCs pulsed with peptide; ALVAC/DCp, parental ALVAC II-infected MDDCs pulsed with peptide.

Article Snippet: The membrane was then blocked and probed with goat anti-mouse CD40L or goat anti-human CD40L antibody (R&D Systems, Inc., Minneapolis, MN), followed by horseradish peroxidase-conjugated anti-goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Recombinant, Virus, Activity Assay, Infection, Expressing, Release Assay, Flow Cytometry

Journal: Cell reports

Article Title: Inactivation of p53 in Human Keratinocytes Leads to Squamous Differentiation and Shedding via Replication Stress and Mitotic Slippage.

doi: 10.1016/j.celrep.2014.10.012

Figure Lengend Snippet: Figure 2. p53 Restrains MYC-Induced Epidermal Differentiation (A) Expression of GFP (green) and p53 (red) by immunofluorescence of HKMYCER cells infected with control vector CTGFP (CT) or with shP53 as indicated after activation of MYC by addition of OHT for 3 days. Note that within the mixed cul- tures, p53 is undetected in cells expressing shP53 (green cells, broken line). (B) Detection of p53 and its target p21 by western blot in CT or shP53 cells treated with OHT for 3 days as indicated. Bar histograms represent quantitation of the western blots normalized to GAPDH (GDH). (C) Expression of p53 as measured by RT-PCR in CT or shP53 cells after activation of MYC by OHT for 3 days. (D) Typical phase contrast and green fluorescence (GFP) of live shP53 cells 4 days postinfection with or without OHT as indicated. (E) Percent of CT or shP53 GFP cells expressing the terminal differentiation marker keratin K1 after addition of OHT for 5 days, as determined by immunofluorescence. (F) Immunofluorescence for involucrin (green) of CT or shP53 cells, 5 days after addition of OHT. Nuclear DNA by DAPI (blue). (G) Percent of CT or shP53 cells expressing in- volucrin (Invol) after addition of OHT for 5 days, as determined by flow-cytometry. Error bars are SEM of duplicate samples of representative experiments. *p < 0.05. Scale bar, 50 mm.

Article Snippet: Lentiviral constructs were control plKO1 (CT; Sigma-Aldrich), control GFP pLVTHM (CTGFP), and three constructs expressing shRNA specific against p53: the GFP expressing vector pLVUH-shp53 (shP53) and two non-GFP constructs pLKO1-p53-shRNA-427 (shP53-427; Addgene; Kim et al., 2007) and pLKO1-p53-shRNA-941 (shP53-941; Addgene; Kim et al., 2007).

Techniques: Expressing, Infection, Control, Plasmid Preparation, Activation Assay, Western Blot, Quantitation Assay, Reverse Transcription Polymerase Chain Reaction, Marker, Cytometry

Journal: Cell reports

Article Title: Inactivation of p53 in Human Keratinocytes Leads to Squamous Differentiation and Shedding via Replication Stress and Mitotic Slippage.

doi: 10.1016/j.celrep.2014.10.012

Figure Lengend Snippet: Figure 3. Disruption of Endogenous p53 in Human Keratinocytes Causes Replication Stress (A) Expression of p53, its target p21, and p63 in HK infected with CTGFP (CT) or with shP53 as indicated, untreated or treated with the genotoxic drug doxorubicin (DOXO) for 24 hr. Bar histogram represents quantitation of western blots normalized to GAPDH (GDH). (B) Western blot for the cell cycle regulators indicated in CT or shP53 cells, 2 or 3 days postinfection; Cyc, cyclin. See also Figure S2A. (C) Bar histogram shows expression of p53 in CT or shP53 cells as measured by RT-PCR 2 days postinfection. See also Figure S2A. (D) Double immunostaining for p53 (red) and gH2AX (green) 3 days post- infection. Note that cells with low p53 tend to accumulate DNA damage (arrows) and vice versa. Nuclear DNA by DAPI (blue). See also Figure S5. Scale bar, 50 mm. (E) Distribution histogram shows number of foci of gH2AX per nucleus, as scored by immunofluorescence as in Figure 3D and image analyses. Left bar

Article Snippet: Lentiviral constructs were control plKO1 (CT; Sigma-Aldrich), control GFP pLVTHM (CTGFP), and three constructs expressing shRNA specific against p53: the GFP expressing vector pLVUH-shp53 (shP53) and two non-GFP constructs pLKO1-p53-shRNA-427 (shP53-427; Addgene; Kim et al., 2007) and pLKO1-p53-shRNA-941 (shP53-941; Addgene; Kim et al., 2007).

Techniques: Disruption, Expressing, Infection, Quantitation Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Double Immunostaining

Journal: Cell reports

Article Title: Inactivation of p53 in Human Keratinocytes Leads to Squamous Differentiation and Shedding via Replication Stress and Mitotic Slippage.

doi: 10.1016/j.celrep.2014.10.012

Figure Lengend Snippet: Figure 4. Disruption of Endogenous p53 in Human Keratinocytes Causes Mitotic Slip- page and Loss of Clonogenic Potential (A) Percent of total or polyploid (polyp) HK infected with shP53 undergoing DNA synthesis (BrdU+) for the periods of time postinfection indicated, as quantitated by flow cytometry and normalized to HK infected with CTGFP (CT). See also Figure S2B. (B) Percent of cells in the G1/S (2N), G2/M (4N), or polyp phases of the cell cycle in CT (gray) or shP53 (light red) cells 3 days postinfection, as determined by flow cytometry. Red arrow shows the shift of shP53 cells toward polyploidy, a consequence of mitotic slippage. See also Fig- ures S2B and S2C. (C) Double immunostaining for K1 (green, arrow heads) and mitotic cyclin B (CycB, red; arrows). Nuclear DNA by DAPI (blue). shP53 cells dis- played frequent accumulation of cytoplasmic cy- clin B (cytopl+), and scoring is shown in the bar histogram. See also Movie S2. (D) Clonogenic capacity of CT or shP53 cells (10,000 cells plated 3 days postinfection). Bar and circle histograms show the number (bars) and percent (circles) of actively shP53 growing col- onies (light gray) versus small abortive, differenti- ated colonies (dark gray). Error bars are SEM of duplicate (A–C) or triplicate (D) samples of representative experiments. *p < 0.05.

Article Snippet: Lentiviral constructs were control plKO1 (CT; Sigma-Aldrich), control GFP pLVTHM (CTGFP), and three constructs expressing shRNA specific against p53: the GFP expressing vector pLVUH-shp53 (shP53) and two non-GFP constructs pLKO1-p53-shRNA-427 (shP53-427; Addgene; Kim et al., 2007) and pLKO1-p53-shRNA-941 (shP53-941; Addgene; Kim et al., 2007).

Techniques: Disruption, Infection, DNA Synthesis, Cytometry, Double Immunostaining

Journal: Cell reports

Article Title: Inactivation of p53 in Human Keratinocytes Leads to Squamous Differentiation and Shedding via Replication Stress and Mitotic Slippage.

doi: 10.1016/j.celrep.2014.10.012

Figure Lengend Snippet: Figure 5. Loss of Endogenous p53 in Human Keratinocytes Triggers Terminal Differenti- ation, Stratification, and Shedding (A) Dot plots show flow-cytometry analyses for GFP (green) versus light scatter (SSC-A; left) or involucrin (invol, right) of HK infected with shP53. Bar histograms show percent of differentiating (high light scatter; diff. in dot plot) or Invol positive (i+ in dot plot) cells in the GFP (+) or non-GFP () populations, as indicated. CT, cells infected with control vector. See also Figure S4A. (B) Double immunostaining for GFP (green) and Invol (red) 5 days postinfection of HK with CTGFP (CT) or shP53 as indicated; nuclear DNA by DAPI (blue). Bar histograms show percentage of cells positive for Invol and K1 (flow cytometry), or K10 (immunofluorescence) in shP53 cells relative to CT cells. Scale bar, 50 mm. See also Figure S4. (C) Expression of differentiation markers K1, ker- atin K10, filaggrin (Fil), and Invol, as determined by RT-PCR in shP53 cells relative to CT cells 2 days postinfections. (D) Snap frames from Movies S4 and S6 showing the accumulation of shP53 cells in mitosis (arrows) 5 days postinfection. Nuclear DNA by NucBlue (blue). Amplified insets show a cell blocked in metaphase (bottom panel: DNA only). See also Movies S4, S5, and S6. Bar histogram shows percent of shP53 cells shed into the medium, relative to control cells. Error bars are SEM of duplicate samples of repre- sentative experiments. *p < 0.05. Scale bars repre- sent 50 mm (B and D) or 10 mm (small insets in D).

Article Snippet: Lentiviral constructs were control plKO1 (CT; Sigma-Aldrich), control GFP pLVTHM (CTGFP), and three constructs expressing shRNA specific against p53: the GFP expressing vector pLVUH-shp53 (shP53) and two non-GFP constructs pLKO1-p53-shRNA-427 (shP53-427; Addgene; Kim et al., 2007) and pLKO1-p53-shRNA-941 (shP53-941; Addgene; Kim et al., 2007).

Techniques: Cytometry, Infection, Control, Plasmid Preparation, Double Immunostaining, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Cell reports

Article Title: Inactivation of p53 in Human Keratinocytes Leads to Squamous Differentiation and Shedding via Replication Stress and Mitotic Slippage.

doi: 10.1016/j.celrep.2014.10.012

Figure Lengend Snippet: Figure 6. A Mutant Inactive Conformation of p53 Induces Keratinocyte Differentiation and Reduces the Great Proliferative Poten- tial Cell Compartment (A) Detection of p53 by western blotting in kerati- nocytes expressing control vector (CT) or the temperature-sensitive mutant p53 (p53ts) 48 hr after the temperature switch to 32C or 39C as indicated. Bar histogram show quantitation of p53 western blots normalized to GAPDH (GDH). Note that p53 accumulates at 39C (inactive conformation). (B) Top: immunofluorescence for p53 (green) on p53ts cells as in (A); Bottom: double immunofluo- rescence for involucrin (Invol, green) and p53 (red) on p53ts cells 4 days after the temperature switch. Note the binucleate differentiating p53ts cells that accumulate p53 at 39C (arrows). Nuclear DNA by DAPI (blue). See also Figure S7A. Scale bar, 50 mm. (C) Representative flow cytometry for the expres- sion of Invol (top) or the hyperproliferative marker keratin K16 (bottom) in p53ts cells 6 days after the temperature switch, as indicated. Bar histograms show percent of morphologically differentiated (high scatter) cells or cells positive for Invol or K16 as indicated, normalized to CT cells; quantitation of positive cells by the gates indicated on the histo- grams (+), as determined by a negative isotype antibody control (dotted line; CD8). See also Figure S7B. (D) Clonogenic capacity of p53ts cells plated 5 days after the 39C temperature switch. A total of 2,500 proliferative cells were plated. Bar and circle histogram show number (bars) or percent (circles) of actively shP53 growing colonies (light gray) versus small abortive, differentiated colonies (dark gray). Error bars are SEM of duplicate (C) or triplicate (D) samples of representative experiments. *p < 0.05.

Article Snippet: Lentiviral constructs were control plKO1 (CT; Sigma-Aldrich), control GFP pLVTHM (CTGFP), and three constructs expressing shRNA specific against p53: the GFP expressing vector pLVUH-shp53 (shP53) and two non-GFP constructs pLKO1-p53-shRNA-427 (shP53-427; Addgene; Kim et al., 2007) and pLKO1-p53-shRNA-941 (shP53-941; Addgene; Kim et al., 2007).

Techniques: Mutagenesis, Western Blot, Expressing, Control, Plasmid Preparation, Quantitation Assay, Cytometry, Marker