Journal: Cancer Immunology Research

Article Title: Cross-Presentation of the Oncofetal Tumor Antigen 5T4 from Irradiated Prostate Cancer Cells—A Key Role for Heat-Shock Protein 70 and Receptor CD91

doi: 10.1158/2326-6066.cir-14-0079

Figure Lengend Snippet: Figure 1. Radiation causes cell-cycle arrest, cell death, and changes to immunologically relevant molecules in DU145 cells. A, cell-cycle analysis of DU145 cells irradiated with increasing dose (x-axis) and incubated for 4, 24, or 48 hours, as indicated above the graphs. B, percentage of DU145 cells undergoing different types of cell death, as indicated, following 12-Gy irradiation and cultures up to 72 hours (x-axis). C, i, HMGB1 expression in fixed and permeabilized DU145 cells at different times after 12-Gy irradiation (x-axis) shown as mean fluorescence intensity (mfi); ii, soluble HMGB1, as detected by ELISA in the supernatant of DU145 cells as in (i). D, i, 5T4 expression in fixed and permeabilized DU145 cells 48 hours after irradiation; D, ii, representative FACS histograms. A–D, i, mean þ SEM of results from triplicate samples. E, Western blotting of 5T4 antigen in DU145 cell lysate with or without irradiation. Raw data (left), adjusted density (right). All experiments were repeated two to three times.

Article Snippet: The membrane was blocked with 5% nonfat dry milk and probed with a sheep polyclonal IgG 5T4 antibody (R&D Systems; AF4975) or a mouse monoclonal IgG1 anti-Hsp70 antibody (C92F3A-5) and with a mouse monoclonal antibody for GAPDH (0411; both from Santa Cruz Biotechnology) for 1 hour at room temperature followed by horseradish peroxidase–conjugated secondary antibody (Santa Cruz Biotechnology).

Techniques: Cell Cycle Assay, Irradiation, Incubation, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Cancer Immunology Research

Article Title: Cross-Presentation of the Oncofetal Tumor Antigen 5T4 from Irradiated Prostate Cancer Cells—A Key Role for Heat-Shock Protein 70 and Receptor CD91

doi: 10.1158/2326-6066.cir-14-0079

Figure Lengend Snippet: Figure 4. Partial effect of HMGB1–TLR4 pathway inhibitors on DC maturation and antigen cross-presentation. A, DCs were cocultured with 0 Gy or 12 Gy–treated DU145 cells in the presence or absence of 50 mmol/L glycyrrhizin (GA) for 72 hours. CD86 upregulation on DCs (i) and 5T4-specific T-cell stimulation (ii) were analyzed by flow cytometry. The columns show mean þ SEM of results from triplicate cultures. B, DCs were treated with LPS in the presence of inhibitory peptides targeting MyD88 (20 mmol/L) or TRIF (25 mmol/L). Control peptides (cell-permeable domain of the inhibitory peptide) were used at the same concentrations. Mean þ SEM of the percentage of TNFa-producing DCs are shown, as detected by cytokine flow cytometry. C, DCs were cultured in a cross-presentation assay in the presence of MyD88 and TRIF inhibitory peptides together or with control peptides (25 mmol/L each). Mean þ SEM of percentage of IFNgþ T cells from triplicate cultures are shown. The experiments were repeated two to three times.

Article Snippet: The membrane was blocked with 5% nonfat dry milk and probed with a sheep polyclonal IgG 5T4 antibody (R&D Systems; AF4975) or a mouse monoclonal IgG1 anti-Hsp70 antibody (C92F3A-5) and with a mouse monoclonal antibody for GAPDH (0411; both from Santa Cruz Biotechnology) for 1 hour at room temperature followed by horseradish peroxidase–conjugated secondary antibody (Santa Cruz Biotechnology).

Techniques: Cell Stimulation, Cytometry, Control, Cell Culture

Journal: Cancer Immunology Research

Article Title: Cross-Presentation of the Oncofetal Tumor Antigen 5T4 from Irradiated Prostate Cancer Cells—A Key Role for Heat-Shock Protein 70 and Receptor CD91

doi: 10.1158/2326-6066.cir-14-0079

Figure Lengend Snippet: Figure 3. DC maturation and antigen cross-presentation by irradiated tumor cells. A, representative dot plot showing uptake of CFSE-prelabeled DU145 cells (x- axis) after 0-Gy (i) or 12-Gy (ii) irradiation by DCs (HLA-DRþ cells). Phagocytic DCs are in the top right quadrant (Q2). iii, summary of results from 5 donors; each symbol represents the mean percrentage of DCs in Q2 from triplicate samples per donor. B, flow cytometry analysis of CD86 expression after 24-hour coculture of DCs without (Nil) or with 0-Gy or 12-Gy irradiated DU145 cells. Mean þ SEM of CD86 mean fluorescence intensity (mfi) from triplicate cultures is shown. C, flow cytometry of CD86 expression, analyzed on DCs gated as Q1 or Q2 DCs, respectively, after DCs coculture with DU145 cells. D, i, proliferation of CFSE-labeled 5T4- specific T cells 5 days after stimulation by autologous DCs cocultured with DU145 cells, as indicated. Mean þ SEM of CFSE (mfi) of T cells from triplicate cultures are shown; ii, representative histograms of CFSE dilution in T cells. The numbers represent the percentage of T cells that proliferated. E, 5T4-specific T cells were stimulated overnight with DCs (from 6 donors), cocultured with DU145 cells. Each symbol represents the mean

Article Snippet: The membrane was blocked with 5% nonfat dry milk and probed with a sheep polyclonal IgG 5T4 antibody (R&D Systems; AF4975) or a mouse monoclonal IgG1 anti-Hsp70 antibody (C92F3A-5) and with a mouse monoclonal antibody for GAPDH (0411; both from Santa Cruz Biotechnology) for 1 hour at room temperature followed by horseradish peroxidase–conjugated secondary antibody (Santa Cruz Biotechnology).

Techniques: Irradiation, Cytometry, Expressing, Labeling

Journal: Cancer Immunology Research

Article Title: Cross-Presentation of the Oncofetal Tumor Antigen 5T4 from Irradiated Prostate Cancer Cells—A Key Role for Heat-Shock Protein 70 and Receptor CD91

doi: 10.1158/2326-6066.cir-14-0079

Figure Lengend Snippet: Figure 5. TLR4 polymorphism does not affect tumor antigen cross-presentation. A, i, TLR4 expression on monocytes from 5 donors with the Asp299 (299A) and 4 donors with the polymorphic Gly299 (299G) allele; ii, TLR4 expression on day 5 DCs. iii, day 5 DCs were stimulated with LPS and TNFa production was measured by flow cytometry. Symbols represent percentage of positive cells from individual donors. The boxes show the 25% and 75% percentiles of the combined data, and the lines represent the medians. B, DC phenotyping from donors as in A. DCs were cocultured with 0 Gy or 12 Gy–irradiated DU145 cells. Each line represents an individual donor. C, stimulation of 5T4-specific T cells with DCs, derived from 3 donors in each group (as in A), loaded with untreated, 12 Gy–irradiated, or oxaliplatin-treated DU145 cells, respectively. Mean þ SEM of percentage of IFNgþ T cells are shown from triplicate samples. NS, not statistically significant.

Article Snippet: The membrane was blocked with 5% nonfat dry milk and probed with a sheep polyclonal IgG 5T4 antibody (R&D Systems; AF4975) or a mouse monoclonal IgG1 anti-Hsp70 antibody (C92F3A-5) and with a mouse monoclonal antibody for GAPDH (0411; both from Santa Cruz Biotechnology) for 1 hour at room temperature followed by horseradish peroxidase–conjugated secondary antibody (Santa Cruz Biotechnology).

Techniques: Expressing, Cytometry, Irradiation, Derivative Assay

Journal: Cancer Immunology Research

Article Title: Cross-Presentation of the Oncofetal Tumor Antigen 5T4 from Irradiated Prostate Cancer Cells—A Key Role for Heat-Shock Protein 70 and Receptor CD91

doi: 10.1158/2326-6066.cir-14-0079

Figure Lengend Snippet: Figure 6. Hsp70 inhibition abolishes antigen cross-presentation. A, the effect of VER155008 on DU145 cell numbers after 72-hour culture. B, different types of cell death as detected by Annexin/PI staining in the absence or presence of VER155008. C, surface expression of Hsp70 (gray) versus isotype (black) in the absence or presence of VER155008: (i) summary from triplicates; (ii) representative histograms. D, effect of VER155008- treated or untreated DU145 cells on CD86 expression of DCs following a 24-hour coculture: (i) representative histograms; (ii) summary from triplicates. E, stimulation of 5T4- specific T cells in a cross-presentation experiment with DCs loaded with VER155008 or PES-treated or untreated DU145 cells. This experiment was carried out with DCs derived from 2 donors. A–E, mean þ SEM of results from triplicate samples are shown.

Article Snippet: The membrane was blocked with 5% nonfat dry milk and probed with a sheep polyclonal IgG 5T4 antibody (R&D Systems; AF4975) or a mouse monoclonal IgG1 anti-Hsp70 antibody (C92F3A-5) and with a mouse monoclonal antibody for GAPDH (0411; both from Santa Cruz Biotechnology) for 1 hour at room temperature followed by horseradish peroxidase–conjugated secondary antibody (Santa Cruz Biotechnology).

Techniques: Inhibition, Staining, Expressing, Derivative Assay

Journal: Cell Host & Microbe

Article Title: Identification of Human Single-Domain Antibodies against SARS-CoV-2

doi: 10.1016/j.chom.2020.04.023

Figure Lengend Snippet:

Article Snippet: The biotinylated human Tim-3, human IL-33 and human 5T4 were purchased from ACROBiosystems Inc.

Techniques: Virus, Recombinant, Saline, Luciferase, Expressing, Plasmid Preparation, Software

Journal: Cell Host & Microbe

Article Title: Identification of Human Single-Domain Antibodies against SARS-CoV-2

doi: 10.1016/j.chom.2020.04.023

Figure Lengend Snippet:

Article Snippet: Human TPBG / 5T4 , ACROBiosystems , Cat# TPG-H52E5.

Techniques: Virus, Recombinant, Saline, Luciferase, Expressing, Plasmid Preparation, Software