Journal: Frontiers in oncology

Article Title: Chimeric Antigen Receptor T Cells Targeting Integrin αvβ6 Expressed on Cholangiocarcinoma Cells.

doi: 10.3389/fonc.2021.657868

Figure Lengend Snippet: FIGURE 1 | Integrin αvβ6 was upregulated in cholangiocarcinoma (CCA) tissues. Immunohistochemical (IHC) staining was performed to examine integrin αvβ6 expression in CCA tissues obtained from liver fluke-associated CCA patients. (A) Negative staining of normal bile duct epithelial cells and hepatocytes surrounding the tumor area was observed. Positive staining of the integrin αvβ6 protein was detected in the cytoplasm and membrane of tumor cells with (B) low and (C) high expression levels. Scale bars represent 50 µm. (D) Expression levels of integrin αvβ6 in CCA tissues were summarized as H-scores. (E) Cumulative overall survival curves of CCA patients with different expression levels of integrin αvβ6. The cumulative survival time of patients who had high expression levels of integrin αvβ6 was significantly shorter than that of the patients who had low/negative expression levels of integrin αvβ6 (p=0.043).

Article Snippet: After blocking of nonspecific binding with 5% skim milk, the tissue sections were incubated overnight with an anti-αvβ6 monoclonal antibody (dilution 1:100, Clone 6.2A1; Biogen Inc., Cambridge, MA, USA) that was generously provided by Dr. Shelia M. Violette.

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, Negative Staining, Staining, Membrane

Journal: Frontiers in oncology

Article Title: Chimeric Antigen Receptor T Cells Targeting Integrin αvβ6 Expressed on Cholangiocarcinoma Cells.

doi: 10.3389/fonc.2021.657868

Figure Lengend Snippet: FIGURE 2 | Expression of integrin αvβ6 on cholangiocarcinoma (CCA) cell lines. (A) Integrin αvβ6 was stained with anti-integrin αvβ6 mAb in the indicated CCA cell lines and analyzed by flow cytometry. A representative result of flow cytometry analysis demonstrates CCA cell lines stained with isotype-control antibody (gray peaks), and CCA cell lines stained with anti-integrin αvβ6 mAb (white peaks). (B) A quantification bar-graph summarizes the data from 3 independent experiments (***p<0.001). The data with error bar represent mean ± standard error of mean (SEM). Differences between the group of β6-negative A375.puro cells and those of cell lines with positive expression of integrin αvβ6 were analyzed by one-way analysis of variance (ANOVA). (C) Immunofluorescence images of CCA cell lines show membranous and granular expression of integrin β6 stained with anti-β6 polyclonal antibody (green) and nuclei staining (Hoechst 33342; blue), and A375.β6 was used as a positive control. Scale bars represent 10 µm.

Article Snippet: After blocking of nonspecific binding with 5% skim milk, the tissue sections were incubated overnight with an anti-αvβ6 monoclonal antibody (dilution 1:100, Clone 6.2A1; Biogen Inc., Cambridge, MA, USA) that was generously provided by Dr. Shelia M. Violette.

Techniques: Expressing, Staining, Cytometry, Control, Positive Control

Journal: Frontiers in oncology

Article Title: Chimeric Antigen Receptor T Cells Targeting Integrin αvβ6 Expressed on Cholangiocarcinoma Cells.

doi: 10.3389/fonc.2021.657868

Figure Lengend Snippet: FIGURE 3 | Generation of chimeric antigen receptor (CAR) T cells targeting integrin αvβ6. (A) The schematic map demonstrates the A20-2G and A20-4G CAR constructs. The sequence of A20 (integrin αvβ6-targeting ligand) was cloned in-frame into lentiviral vectors linked with the sequences of CD8 hinge, CD28 transmembrane domain (TM), costimulatory domain of CD28/CD3ζ (A20-2G), or CD28/4-1BB/CD27/CD3ζ (A20-4G). (B) Expression of A20-2G CAR and A20-4G CAR proteins in Lenti-X 293T cells was detected by immunoblotting analysis using anti-CD3ζ antibody. The data shown represent the mean of three experiments, all of which showed similar results. (C) Representative histogram shows expression of A20-2G and A20-4G CAR on the surface of T cells as examined by anti-cMyc FITC-conjugated antibody and flow cytometric analysis. (D) Expression of A20-2G or A20-4G CAR on transduced T cells was examined and summarized from eight healthy donors as mean ± standard deviation (SD). Non-transduced (NT) T cells were used as control. (E) Representative flow cytometric gating of CD3+CD56−T cell population (upper) and summarized data (lower). The phenotypes of CAR T cells were cytotoxic CD8+ T cells and helper CD4+ T cells. Flow cytometric gating was based on cells stained with isotype-match control antibody. (F) The subgroups of A20-2G or A20-4G CAR T cells consisted of CD45RA−CD62L+ central memory (TCM) and CD45RA+CD62L+ naïve T cells. These data were derived from four healthy donors (ns, non-significant, *p<0.05, **p<0.01, ***p<0.001).

Article Snippet: After blocking of nonspecific binding with 5% skim milk, the tissue sections were incubated overnight with an anti-αvβ6 monoclonal antibody (dilution 1:100, Clone 6.2A1; Biogen Inc., Cambridge, MA, USA) that was generously provided by Dr. Shelia M. Violette.

Techniques: Construct, Sequencing, Clone Assay, Expressing, Western Blot, Standard Deviation, Control, Staining, Derivative Assay

Journal: Frontiers in oncology

Article Title: Chimeric Antigen Receptor T Cells Targeting Integrin αvβ6 Expressed on Cholangiocarcinoma Cells.

doi: 10.3389/fonc.2021.657868

Figure Lengend Snippet: FIGURE 4 | Cytotoxic effects of A20-2G and A20-4G chimeric antigen receptor (CAR)-T cells on integrin αvβ6-negative A375.puro cells, integrin αvβ6-positive A375.β6 cells, and three integrin αvβ6-positive cholangiocarcinoma (CCA) cell lines, including KKU055, KKU100, and KKU213A. The effector CAR T cells were co-cultured with target cells at the indicated effector to target (E:T) ratios (1.25:1, 2.5:1, and 5:1) for 24 h (solid line) or 48 h (dashed line). (A) Cytotoxicity of target cells after co-culturing with effector cells quantified by crystal violet staining assay. Integrin αvβ6-negative A375.puro cell line served as negative control while integrin αvβ6-positive A375.β6 cells was served as positive control. (B) Cytotoxic effects of A20-2G and A20-4G CAR T cells on integrin αvβ6-negative A375.puro cells with non-transduced (NT) T cells used as a control for comparison. (C) Cytotoxic effects of A20-2G and A20-4G CAR T cells on integrin αvβ6-positive A375.β6 cells with NT T cells used as a control for comparison. Cytotoxic effects of A20-2G and A20-4G CAR T cells on integrin αvβ6-positive KKU055 (D), KKU100 (E), and KKU213A (F) cells as compared to the killing activity of NT T cells on the same target cells. Percentage of cytotoxicity relative to the total number of tumor cells alone (set at 0% cytotoxicity) from three independent experiments presented as mean ± standard error of the mean (SEM) (*p<0.05, **p<0.01).

Article Snippet: After blocking of nonspecific binding with 5% skim milk, the tissue sections were incubated overnight with an anti-αvβ6 monoclonal antibody (dilution 1:100, Clone 6.2A1; Biogen Inc., Cambridge, MA, USA) that was generously provided by Dr. Shelia M. Violette.

Techniques: Cell Culture, Staining, Negative Control, Positive Control, Control, Comparison, Activity Assay

Journal: Frontiers in oncology

Article Title: Chimeric Antigen Receptor T Cells Targeting Integrin αvβ6 Expressed on Cholangiocarcinoma Cells.

doi: 10.3389/fonc.2021.657868

Figure Lengend Snippet: FIGURE 6 | Production of interferon-γ (IFN-γ) and cell proliferation non-transduced (NT) T cells, A20-2G CAR T cells, and A20-4G CAR T cells after co-culture with integrin αvβ6-expressing target cells. (A) IFN-γ production was examined by intracellular cytokine staining and flow cytometry analysis in NT T cells, A20-2G CAR T cells, and A20-4G CAR T cells after co-culturing with integrin αvβ6-negative A375.puro or integrin αvβ6-positive A375.β6 cells. (B) IFN-γ expression from three individual healthy donors. Data are presented as mean ± standard error of the mean (SEM). (C) Proliferation of NT cells, A20-2G CAR T cells, and A20-4G CAR T cells was examined after co-culturing for 3 days with integrin αvβ6-negative A375.puro or integrin αvβ6-positive A375.β6 cells at an effector to target (E:T) ratio of 5:1 without addition of exogenous cytokine. (D) Summary data of T cell proliferation derived from 3 individual healthy donors. (*p<0.05, **p<0.01, ***p<0.001).

Article Snippet: After blocking of nonspecific binding with 5% skim milk, the tissue sections were incubated overnight with an anti-αvβ6 monoclonal antibody (dilution 1:100, Clone 6.2A1; Biogen Inc., Cambridge, MA, USA) that was generously provided by Dr. Shelia M. Violette.

Techniques: Co-Culture Assay, Expressing, Staining, Cytometry, Derivative Assay