Journal: Journal of the National Cancer Institute

Article Title: Response of established human breast tumors to vaccination with mammaglobin-A cDNA.

doi: 10.1093/jnci/djh261

Figure Lengend Snippet: Fig. 1. Membrane stabilization of the HLA-A2 molecule by mammaglobin-A– derived candidate epitopes. Computer-assisted predicted HLA-A2–binding af- finities of the candidate epitopes were confirmed by membrane stabilization of the HLA-A2 molecule in T2 cells, a human T-B lymphoblastoid hybrid cell line deficient in transporter associated with antigen-processing proteins. T2 cells were incubated in the presence of each peptide in complete medium (see “Subjects and Methods”). After 24 hours, HLA-A2 expression was determined with flow cytometric analysis using the BB7.2 anti–HLA-A2 monoclonal anti- body (solid bars). T2 cells incubated in the presence of the GILGFVFTL influenza–derived epitope (Flu) were used as a positive control, and T2 cells incubated in the presence of HLA-A3–restricted PLLENVISK (Mam-A3.1) mammaglobin-A–derived epitope were used as negative control. Results are expressed as the mean fluorescence shift, which represents the difference be- tween the mean fluorescence obtained with T2 cells cultured in the presence of peptides and the mean fluorescence obtained with T2 cells cultured in the absence of peptides. Results are presented as the mean of four independent experiments performed in quadruplicate. Error bars represent upper 95% con- fidence intervals. Fig. 2. Epitope immunodominance of CD8 cytotoxic T lymphocytes (CTLs) from mammaglobin-A cDNA–vaccinated HLA-A2 /hCD8 mice. Five HLA- A2 /hCD8 mice were each vaccinated with either mammaglobin-A cDNA (solid bars) or empty vector (open bars). A) Immunodominance of the CD8

Article Snippet: The HLA-A2–binding ability of the peptides was confirmed by cell membrane stabilization of the HLA-A*0201 molecule in transporter associated with antigen processing (TAP)–deficient T2 cells, a human T-B lymphoblastoid hybrid cell line obtained from the American Type Culture Collection.

Techniques: Membrane, Derivative Assay, Binding Assay, Incubation, Expressing, Positive Control, Negative Control, Cell Culture, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Fine analysis of spontaneous MAGE-C1/CT7-specific immunity in melanoma patients

doi: 10.1073/pnas.1002155107

Figure Lengend Snippet: CT7-specific CD4+ T-cell clones recognize naturally processed antigen and exert cytotoxicity. CT7-specific CD4+ T-cell clones from ZH-616, ZH-793, and ZH-522 were incubated with autologous mature dendritic cells that were pulsed with 10 μg/mL recombinant CT7 protein or irrelevant RAB38 protein for 16 h followed by IFN-γ ICS. (A) Peptide titration assay for CT7/616/8 (Left), CT7/793/3 (Center), and CT7/522/6 (Right) was measured by IFN-γ ICS assay. HLA-matched tumor cell lines were pulsed with the relevant short peptide for 1 h and incubated with the individual CD4+ T-cell clones. CT7/616/8 was incubated with aa 451–458 loaded B-LCL NW2412, CT7/793/3 with aa 136–148 loaded B-LCL NW2412, and CT7/522/6 with aa 779–787 loaded melanoma cell line ESTAB 007. (B) Using targets and effectors as in B, perforin secretion was measured by ELISPOT. An HLA-A*0201/NY-ESO-1157–165-specific CD8+ T-cell clone and an HLA-DP*0402/TetanusToxoid947–963-specific CD4+ T-cell clone stimulated with relevant peptides and APC were used as positive and negative control, respectively. Assay was performed in triplicate. Error bars depict ±3× SD. (C) Cytotoxicity of aforementioned clones from three respective patients (ZH-616, ZH-522, ZH-793) was measured in a chromium release assay using melanoma cell line ESTDAB007 pulsed with either relevant or mock peptides as targets in the presence or absence of blocking antibodies for HLA-DR or HLA-DQ. (D) One representative experiment of two is shown for all four figures.

Article Snippet: NW2412 B-LCL cell line was obtained as a generous gift from Elke Jäger (Krankenhaus Nordwest, Frankfurt), and melanoma cell line SK-MEL-37 was obtained as generous gift of L. J.

Techniques: Clone Assay, Incubation, Recombinant, Titration, Enzyme-linked Immunospot, Negative Control, Release Assay, Blocking Assay

Journal: Frontiers in Immunology

Article Title: Interleukin-18 Amplifies Macrophage Polarization and Morphological Alteration, Leading to Excessive Angiogenesis

doi: 10.3389/fimmu.2018.00334

Figure Lengend Snippet: Schematic of the proposed mechanism by which interleukin (IL)-18 amplifies macrophage (Mφ) M2 polarization and its morphological alteration, leading to excessive angiogenesis. IL-18 amplifies IL-10-induced increases in the production of osteopontin (OPN) and thrombin as soluble mediators derived from Mφ, yielding the generation of thrombin-cleaved form of OPN (Thr-OPN). Subsequently, Thr-OPN binds to integrins α4/α9 receptors on Mφ, which in turn augments M2 polarization of Mφ with higher expression of CD163 and its morphological alteration. Furthermore, CD163 may be responsible for mediating the direct cell–cell interaction between these Mφs and endothelial cells, ultimately resulting in the excessive angiogenesis.

Article Snippet: The mouse leukemic monocyte Mφ cell line, RAW264.7 cells (DS Pharma Biomedical), and the mouse endothelial cell line, b.End5 cells (DS Pharma Biomedical), were cultured in Dulbecco’s modified Eagle’s medium supplemented with 2 mM l -glutamine and 10% heat-inactivated fetal bovine serum.

Techniques: Derivative Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Interleukin-18 Amplifies Macrophage Polarization and Morphological Alteration, Leading to Excessive Angiogenesis

doi: 10.3389/fimmu.2018.00334

Figure Lengend Snippet: Characteristic behavior of macrophages (Mφs) during angiogenesis. (A) Representative series of time-lapse images at 4 h intervals from 0 to 16 h extracted from Video S1 which shows live-cell imaging of Matrigel tube formation assay where endothelial cells (green) and Mφs [interleukin (IL)-10 + IL-18] (red) were cocultured. Scale bar represents 50 µm. (B) Higher magnification images of white rectangle region in panel (A) were reconstructed from 4 h 00 min to 7 h 00 min in Video S5 in Supplementary Material. The time elapsed after starting the movie is indicated in hours:minutes in the bottom left of each panel. White arrowheads highlight the characteristic behavior of Mφ (IL-10 + IL-18) as well as the cell–cell interaction with endothelium in respective image. Scale bar represents 10 µm.

Article Snippet: The mouse leukemic monocyte Mφ cell line, RAW264.7 cells (DS Pharma Biomedical), and the mouse endothelial cell line, b.End5 cells (DS Pharma Biomedical), were cultured in Dulbecco’s modified Eagle’s medium supplemented with 2 mM l -glutamine and 10% heat-inactivated fetal bovine serum.

Techniques: Live Cell Imaging, Tube Formation Assay

Journal: Frontiers in Immunology

Article Title: Interleukin-18 Amplifies Macrophage Polarization and Morphological Alteration, Leading to Excessive Angiogenesis

doi: 10.3389/fimmu.2018.00334

Figure Lengend Snippet: Ultrastructural analysis of cell–cell interaction between macrophages (Mφs) [interleukin (IL)-10 + IL-18] and endothelia. SEM images were obtained at 4 h after coculture of b.End5 with Mφs (IL-10 + IL-18) on Matrigel. Lower images are magnified regions from red rectangles in the corresponding upper panels. Magnification and scale bars: (A) upper, ×1.0 K, 50 µm; Lower, ×5.0 K, 10 µm; (B) upper, ×1.0 K, 50 µm; lower, ×2.0 K, 20 µm; (C) upper, ×1.0 K, 50 µm; lower, ×8.0 K, 5 µm; (D) upper, ×1.0 K, 50 µm; lower, ×3.0 K, 10 µm; (E) upper, ×1.0 K, 50 µm; lower, ×5.0 K, 10 µm; (F) upper, ×1.0 K, 50 µm; lower, ×4.0 K, 10 µm; (G) upper, ×1.0 K, 50 µm; lower, ×2.5 K, 20 µm; (H) upper, ×1.0 K, 50 µm; lower, ×2.5 K, 20 µm; (I) upper, ×1.0 K, 50 µm; lower, ×3.5 K, 10 µm; respectively. Black or white arrows indicate Mφs or endothelial cells, respectively. Red arrowheads indicate pseudopodia of Mφs interacting with endothelial cells.

Article Snippet: The mouse leukemic monocyte Mφ cell line, RAW264.7 cells (DS Pharma Biomedical), and the mouse endothelial cell line, b.End5 cells (DS Pharma Biomedical), were cultured in Dulbecco’s modified Eagle’s medium supplemented with 2 mM l -glutamine and 10% heat-inactivated fetal bovine serum.

Techniques: