Journal: British Journal of Cancer

Article Title: Epigenetic remodelling of gene expression profiles of neoplastic and normal tissues: immunotherapeutic implications

doi: 10.1038/bjc.2012.361

Figure Lengend Snippet: Phenotypic and functional analyses of changes induced by in vitro 5-AZA-CdR. ( A ) TS/A cells either untreated (—), or treated with 1 μ ℳ 5-AZA-CdR (- - - -), were incubated with the FITC-conjugated anti-MHC class I, anti-H-2D d , or anti- H-2L d mAbs. Cells were then analysed by flow cytometry. ( B ) Cytolytic activity of anti-P1A H-2L d -restricted CTL was measured by chromium release assay against 51 Cr-labelled TS/A cells untreated (—) or treated (- - - -) with 1 μ ℳ 5-AZA-CdR every 12 h for 2 days, at effector/target ( E / T ) ratios of 50 : 1, 17 : 1, 6 : 1, and 2 : 1. For peptide pulsing, 51 Cr-labelled target cells (10 6 per ml) were incubated with the MHC class I L d -restricted peptide corresponding to amino acids 35–43 of P1A protein, at 1 μ ℳ final concentration, for 30 min at 37 °C, and then washed twice before use (crossed squares and triangle points). Cytotoxicity was expressed as per cent of lysis. Data reported are representative of three independent experiments.

Article Snippet: The FITC-conjugated anti-MHC class I monoclonal antibody (mAb) MCA2189F was purchased from Serotec (Dusseldorf, Germany), the FITC-conjugated anti-MHC class I H-2D d from Abcam (Cambridge, UK), and the FITC-conjugated anti-MHC class I H-2 L d mAb from Acris Antibodies GmbH (Herford, Germany).

Techniques: Functional Assay, In Vitro, Incubation, Flow Cytometry, Activity Assay, Release Assay, Concentration Assay, Lysis

Journal: Journal for Immunotherapy of Cancer

Article Title: Antigen mimicry as an effective strategy to induce CSPG4-targeted immunity in dogs with oral melanoma: a veterinary trial

doi: 10.1136/jitc-2021-004007

Figure Lengend Snippet: HuDo-CSPG4 vaccination is effective in inducing an anti-CSPG4 cellular immune response in dogs. (A) Flow cytometry analysis of the frequency of circulating B cells, CD4 + and CD8 + T cells, and MDSC collected from canine melanoma patients before (Pre-Vax) and after the fourth HuDo-CSPG4 vaccination (Post-Vax). Graphs show the percentage of CD21 + B cells (gated on live cells), of CD4 + and CD8 + T cells (gated on CD5 + cells) and of MHC-II – CD14 – (gated on CD11b + cells) cells. The numbers of dogs in which a difference in the frequency (fold change >1.1 or fold change <1.1) of a cell population was observed comparing Pre-Vax and Post-Vax PBMC are indicated above in each graph. Student’s t-test, *p=0.0151. (B) Cytotoxic assays to quantify the ability of Pre-Vax and Post-Vax PBMC to kill CSPG4-positive CMM-12 cells. Representative dot plots of one dog analyzed, showing the percentage of 7-AAD + dead cells among CFSE + cells (upper panels) are shown. Results are shown as the fold change between the percentage of CMM-12 cells lysed after incubation with Post-Vax and Pre-Vax PBMC for each dog analyzed (lower, left panel, Student’s t-test, *p=0.0260), and as the percentage of dogs of which PBMC induced an increased CMM-12 cell lysis (responders) or not (non responders) (lower, right panel). (C) and (D) Kaplan-Meier curves comparing the overall survival (C) and the disease-free-interval (DFI, (D), in days, of vaccinated dogs who develop (responders, continuous blue line) or not (non responders, dotted blue line) a cytotoxic response against the canine CMM-12 cell line. The median survival times (MST) in days for each group has been reported in the overall survival graph. Log-rank test, p=0.2819. 7-AAD, 7-Amino-ActinomycinD; CSPG4, chondroitin sulfate proteoglycan 4; Do, dog; Hu, human; MDSC, myeloid derived suppressor cells; PBMC, peripheral blood mononuclear cells; FITC, fluorescein isothiocyanate; MHC, major histocompatibility complex; CFSE, carboxyfluorescein succinimidyl ester.

Article Snippet: Thawed PBMC were incubated with human IgG to block the Fc receptor and then stained with the following mAbs: rat anti-dog CD5-fluorescein isothiocyanate (FITC), rat anti-dog CD4-RPE-cy7, rat anti-dog CD8-Pacific Blue, mouse anti-dog B cells-Alexa Fluor 647, purified mouse anti-dog CD11b, rat anti-dog major histocompatibility complex (MHC) II-FITC and mouse anti-human CD14-Alexa fluor 647 (all from AbD Serotec).

Techniques: Flow Cytometry, Incubation, Lysis, Derivative Assay, Immunopeptidomics

Journal: bioRxiv

Article Title: Morphologic characterization and cytokine response of chicken bone-marrow derived dendritic cells to infection with high and low pathogenic avian influenza virus

doi: 10.1101/2024.02.06.579192

Figure Lengend Snippet: Comparative analysis of surface markers on immature and mature ckBM-DCs. Cells were cultured in the presence of 50 ng/ml GM-CSF + 50 ng/ml IL-4 for 6 days, and then stimulated with 500 ng/ml LPS for 30 hours. (A) Immature cells are on the left (A1,B1,C1) and mature cells are on the right (A2,B2,C2). Immunofluorescence analysis was performed using a FITC labeled mouse-anti-chicken MHC-II antibody (A1, A2). Cells were also stained with mouse anti-chicken CD11c (B1,B2) and mouse anti-chicken CD40 (C1,C2) followed by a goat-anti-mouse secondary. A representative image is shown for each at 100x magnification. (B) Cellular RNA was extracted to measure expression levels of surface markers in mature ckBM-DCs. RNA was normalized using the Ck 28S house-keeping gene. The data is expressed as the fold change in mRNA levels between immature (negative control) and mature ckBM-DCs for MHC-II, CD11c, CD40, CD80, CD83, and CD86. The data shown is a representative of three independent experiments. Error bars represent the standard deviation.

Article Snippet: Immunofluorescence labelling was performed to analyze the DC markers using FITC labeled mouse-anti-chicken MHC-II in combination with mouse anti-chicken CD40 (Bio-Rad, Hercules, CA), mouse anti-chicken CD11c (8F2, IgG2a) followed by incubation with a goat anti-mouse-Ig secondary Ab (Thermo-fisher Scientific, Waltham, MA).

Techniques: Cell Culture, Immunofluorescence, Labeling, Staining, Expressing, Negative Control, Standard Deviation

Journal: bioRxiv

Article Title: Morphologic characterization and cytokine response of chicken bone-marrow derived dendritic cells to infection with high and low pathogenic avian influenza virus

doi: 10.1101/2024.02.06.579192

Figure Lengend Snippet: Functionality of immature ckBM-DCs. Cells were cultured in the presence of 50 ng/ml GM-CSF + 50 ng/ml IL-4 for 6 days. (A) ckBM-DCs were incubated with 0.5-um carboxylate modified fluorescent red latex beads or (B) FITC labeled-inactivated H5N9 avian influenza virus for 4 hours. Following incubation cells were counterstained with DAPI, washed 5x with PBS, and visualized with immunofluorescence microscopy. A representative image is shown for each at 100x magnification.

Article Snippet: Immunofluorescence labelling was performed to analyze the DC markers using FITC labeled mouse-anti-chicken MHC-II in combination with mouse anti-chicken CD40 (Bio-Rad, Hercules, CA), mouse anti-chicken CD11c (8F2, IgG2a) followed by incubation with a goat anti-mouse-Ig secondary Ab (Thermo-fisher Scientific, Waltham, MA).

Techniques: Cell Culture, Incubation, Modification, Labeling, Virus, Immunofluorescence, Microscopy

Journal: bioRxiv

Article Title: Morphologic characterization and cytokine response of chicken bone-marrow derived dendritic cells to infection with high and low pathogenic avian influenza virus

doi: 10.1101/2024.02.06.579192

Figure Lengend Snippet: Distribution of sialic acid receptors on ckBM-DCs and susceptibility to pandemic H1N1 and H5N9 viruses. Immature ckBM-DCs (A1, B1) were stained with FITC-labeled MAA (SA-α2,3-Gal) (A2) or TRITC-labeled SNA (SA-α2,6-Gal) (B2), counter stained with DAPI, and visualized by immunofluorescence microscopy. CkBM-DCs were infected at an MOI of 1 with A/turkey/Virginia/SEP-4/2009 H1N1 (SA-α2,6-Gal preference) and A/turkey/Wisconsin/68 H5N9 (SA-α2,3-Gal preference). At 20 HPI, viral-infected cells, H1N1 (C1) and H5N9 (C2), were washed 2x with PBS, fixed with methanol, and observed by microscopy. Viral NP proteins, H1N1 (D1) and H5N9 (D2), were detected using a mouse-anti-NP antibody followed by a FITC-conjugated anti-mouse IgG secondary (D1,D2). A representative image is shown for each at 100x (A1,A2,B1,B2) and 200x (C1,C2,D1,D2) magnification.

Article Snippet: Immunofluorescence labelling was performed to analyze the DC markers using FITC labeled mouse-anti-chicken MHC-II in combination with mouse anti-chicken CD40 (Bio-Rad, Hercules, CA), mouse anti-chicken CD11c (8F2, IgG2a) followed by incubation with a goat anti-mouse-Ig secondary Ab (Thermo-fisher Scientific, Waltham, MA).

Techniques: Staining, Labeling, Immunofluorescence, Microscopy, Infection