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: Cancer Immunology, Immunotherapy

Article Title: Preclinical development of T-cell receptor-engineered T-cell therapy targeting the 5T4 tumor antigen on renal cell carcinoma

doi: 10.1007/s00262-019-02419-4

Figure Lengend Snippet: High throughput bulk sequencing of the T-cell receptor β chain for 5T4 p17 -specific CD8 + T-cell clones isolated from healthy or kidney cancer donors

Article Snippet: To confirm 5T4 expression on surface after MVA-5T4 infection, target cells were stained with a 5T4-specific mAb (clone 524744; R&D systems, Minneapolis, MN) at 1 μg/mL followed by a 1:50 dilution of a secondary PE-labeled anti-mouse IgG1 mAb (clone A85-1; BD Biosciences) for analysis by flow cytometry.

Techniques: High Throughput Screening Assay, Sequencing, Clone Assay, Isolation

Journal: Cancer Immunology, Immunotherapy

Article Title: Preclinical development of T-cell receptor-engineered T-cell therapy targeting the 5T4 tumor antigen on renal cell carcinoma

doi: 10.1007/s00262-019-02419-4

Figure Lengend Snippet: Characteristics of 5T4 p17 -specific CDR3 regions of TRA and TRB . IMGT junction analysis is shown for a TRA -CDR3 regions and b TRB -CDR3 regions. Red boxes indicate N nucleotides and/or D regions ( TRB ) between V gene and J gene encoded sequence. c V- and J-gene usage for TRA and TRB are shown. Color shading identifies common gene use between TCRs. d The net charge for CDR1, 2, and 3 amino acid sequences from 5T4 17–25 -specific TCRs are shown

Article Snippet: To confirm 5T4 expression on surface after MVA-5T4 infection, target cells were stained with a 5T4-specific mAb (clone 524744; R&D systems, Minneapolis, MN) at 1 μg/mL followed by a 1:50 dilution of a secondary PE-labeled anti-mouse IgG1 mAb (clone A85-1; BD Biosciences) for analysis by flow cytometry.

Techniques: Sequencing

Journal: Cancer Immunology, Immunotherapy

Article Title: Preclinical development of T-cell receptor-engineered T-cell therapy targeting the 5T4 tumor antigen on renal cell carcinoma

doi: 10.1007/s00262-019-02419-4

Figure Lengend Snippet: 5T4 p17 -specific TCRs expression on CD8 + T-cells from healthy donors and redirected effector functions against peptide-pulsed T2 targets. a Vector structure of 5T4 p17 -specific TCR assembly: TRB and TRA are transcribed under control of the MSCV promotor; P2A cleaves the primary transcripts to equal molar quantity of TRA and TRB in the cytosol. The enlarged graph indicates the gene segments and CDR3s, contributing to TRA and TRB sequence assembly. The relative location for the introduced transmembrane cysteines in TRAC and TRBC are indicated. b Cell surface expression of 5T4 p17 -specific TCRs stained by 5T4 p17 /HLA-A2 tetramer at day 7 post-transduction is shown in comparison to tetramer staining of the native T-cell clone expressing the corresponding TCR (upper panels). Cell surface expression of 5T4 p17 -specific TCRs on healthy donor T-cells is shown after sorting for 5T4 p17 /HLA-A2 tetramer + cells and 12 days expansion (lower panels). CD8 + T-cells expressing 5T4 p17 -specific TCRs were tested for recognition of c T2 cells pulsed with 10 nM of 5T4 p17 or control HLA-A2-binding peptides DDX3Y 428–436 or UTY 148–156 in a 4-h cytotoxicity assay. The effector: target ratio (E:T) was 10:1. d TNF-α release was measured by ELISA in culture supernatants harvested after 18 h co-culture of effector T-cells with T2 cells pulsed with 10 nM of 5T4 p17 or the control peptide DDX3Y 428–436 at 10:1 E:T. e Effector T-cells were tested for recognition of T2 cells pulsed with 5T4p17 peptide in a 4-h cytotoxicity assay at 10:1 E:T. f TNF-α release was measured by ELISA in culture supernatants harvested after 18 h co-culture of effector T-cells with T2 cells pulsed with 5T4 p17 peptide at 10:1 E:T

Article Snippet: To confirm 5T4 expression on surface after MVA-5T4 infection, target cells were stained with a 5T4-specific mAb (clone 524744; R&D systems, Minneapolis, MN) at 1 μg/mL followed by a 1:50 dilution of a secondary PE-labeled anti-mouse IgG1 mAb (clone A85-1; BD Biosciences) for analysis by flow cytometry.

Techniques: Expressing, Plasmid Preparation, Control, Sequencing, Staining, Transduction, Comparison, Binding Assay, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Co-Culture Assay

Journal: Cancer Immunology, Immunotherapy

Article Title: Preclinical development of T-cell receptor-engineered T-cell therapy targeting the 5T4 tumor antigen on renal cell carcinoma

doi: 10.1007/s00262-019-02419-4

Figure Lengend Snippet: Cytotoxicity of 5T4 p17 -specific TCR transduced CD8 + T-cells against T2 pulsed with alanine-substituted 5T4 p17 peptides. a Binding of alanine-substituted 5T4 p17 peptides to HLA-A2 was determined by stabilization of HLA-A2 on the surface of peptide-pulsed T2 cells. CMV pp65 is an HLA-A2 + T-cell epitope from CMV and serves as a positive control. Cell surface HLA-A2 was assessed by immunostaining and flow cytometry. b CD8 + T-cells expressing 5T4 p17 -specific TCRs were tested for recognition of T2 cells pulsed with the progenitor 5T4 p17 peptide or HLA-A2 binding alanine-substituted variants in a 4-h cytotoxicity assay with a 10:1 E:T

Article Snippet: To confirm 5T4 expression on surface after MVA-5T4 infection, target cells were stained with a 5T4-specific mAb (clone 524744; R&D systems, Minneapolis, MN) at 1 μg/mL followed by a 1:50 dilution of a secondary PE-labeled anti-mouse IgG1 mAb (clone A85-1; BD Biosciences) for analysis by flow cytometry.

Techniques: Binding Assay, Positive Control, Immunostaining, Flow Cytometry, Expressing, Cytotoxicity Assay

Journal: Cancer Immunology, Immunotherapy

Article Title: Preclinical development of T-cell receptor-engineered T-cell therapy targeting the 5T4 tumor antigen on renal cell carcinoma

doi: 10.1007/s00262-019-02419-4

Figure Lengend Snippet: Cytotoxicity of 5T4 p17 -specific TCR-transduced CD8 + T-cells against 5T4-expressing tumor targets. CD8 + T-cells expressing 5T4 17–25 -specific TCRs were tested for recognition of tumor target lines in a 4-h cytotoxicity assay with 10:1 E:T. For each target cell-line, 5T4 17–25 -specific TCR transduced effector CD8 + T-cells were plotted in the following order: HD_A-2, HD_A-15, HD_B-19, HD_B-21, HD_C-3, HD_C-17, KCD_D-6, and untransduced. a Targets are 5T4 + /HLA-A2 + RCC cell lines (A498, BB65, TREP, DOBSKI; red bars), the 5T4 + /HLA-A2 − RCC line (SST548; open bars) and the 5T4 − /HLA-A2 + LCL (BB65-LCL; gray shading). b Targets are 5T4 + /HLA-A2 + breast cancer line (MDA231; blue bars), a 5T4 + /HLA-A2 − breast cancer line (BT20; open bars) and 5T4 + /HLA-A2 + colon cancer line (SW480, green bars). CD8 + T-cells with (gTRAC_KO) or without (gCtrl) native TRA disruption expressing 5T4 p17 -specific TCRs were tested for recognition of c 5T4 + /HLA-A2 + RCC cell-lines (A498, BB65, DOBSKI), the 5T4 + /HLA-A2 − RCC line (SST548), the 5T4 − /HLA-A2 + BB65-LCL, and d paired primary 5T4 + /HLA-A2 + RCC and autologous PTEC cell-lines from four RCC patients

Article Snippet: To confirm 5T4 expression on surface after MVA-5T4 infection, target cells were stained with a 5T4-specific mAb (clone 524744; R&D systems, Minneapolis, MN) at 1 μg/mL followed by a 1:50 dilution of a secondary PE-labeled anti-mouse IgG1 mAb (clone A85-1; BD Biosciences) for analysis by flow cytometry.

Techniques: Expressing, Cytotoxicity Assay, Disruption

Journal: Cancer Immunology, Immunotherapy

Article Title: Preclinical development of T-cell receptor-engineered T-cell therapy targeting the 5T4 tumor antigen on renal cell carcinoma

doi: 10.1007/s00262-019-02419-4

Figure Lengend Snippet: Cytotoxicity of 5T4 p17 -specific TCR-transduced CD8 + T-cells against MVA-5T4 infected targets. a Schematic of human 5T4 protein, p17–25 is in the signal sequence region. Trans: transmembrane region, Cyto: cytoplasmic region. b T2 cells and BB65-LCL were infected with MVA-WT or MVA-5T4, and cell surface expression of 5T4 was analyzed by immunostaining and flow cytometry 24 h after infection. The fraction of cells positive for cell surface 5T4 after MVA-5T4 infection is indicated. CD8 + T-cells expressing 5T4 p17 -specific TCRs were tested for recognition of c T2 and d BB65-LCL cells infected by MVA-WT, MVA-5T4, or uninfected cells pulsed with 10 nM 5T4 p17 peptide in a 6-h cytotoxicity assay at a 10:1 E:T

Article Snippet: To confirm 5T4 expression on surface after MVA-5T4 infection, target cells were stained with a 5T4-specific mAb (clone 524744; R&D systems, Minneapolis, MN) at 1 μg/mL followed by a 1:50 dilution of a secondary PE-labeled anti-mouse IgG1 mAb (clone A85-1; BD Biosciences) for analysis by flow cytometry.

Techniques: Infection, Sequencing, Expressing, Immunostaining, Flow Cytometry, Cytotoxicity Assay