Journal: International immunopharmacology

Article Title: Dominant negative TGFβ receptor II and truncated TIM3 enhance the antitumor efficacy of CAR-T-cell therapy in prostate cancer.

doi: 10.1016/j.intimp.2023.110807

Figure Lengend Snippet: Fig. 1. Expression of TIM3 and TGFβRII and their ligands in prostate cancer: (A) Expression of GAL9 in prostate cancer and normal prostate tissues in the GEPIA 2 database; (B) Expression of TGF-β in prostate cancer and normal prostate tissues in the GEPIA 2 database; (C) Correlation between the expression of TIM3 and TGFβRII, GAL9, and TGF-β in the GEPIA 2 database; (D) IHC of GAL9 in prostate cancer in the HPA database; (E) IHC of TGF-β in prostate cancer in the HPA database; (F) The left panel shows unsupervised graph-based clustering of all samples visualized by UMAP delineated by cell type, and the right panel shows the expression of TIM3 and TGFβRII in different cell populations in the single-cell sequencing results; (G) Expression of TIM3 and TGFβRII in T cells in the single-cell sequencing results (the left panel is a scatter plot, and the right panel is a violin plot); (H) Coexpression of TIM3 and TGFβRII in T cells in the single-cell sequencing results, where 1 (blue) represents T cells that coexpress TIM3 and TGFβRII, and 0 (gray) represents other T cells.

Article Snippet: Untransduced (UTD) T cells, PSMA-CAR-T cells, and DT-PSMA-CAR- L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 T cells were co-cultured with prostate cancer cells at ratios of 1:1, 5:1, and 10:1 for 24 h.The T-cell culture medium was supplemented with appropriate concentrations (0 ng/ml, 5 ng/ml, 20 ng/ml, and 50 ng/ml) of TGF-β (P01137, Suzhou Novoprotein Scientific) and/or (0 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) TIM3 activating monoclonal antibodies (MAB23651, R&D Systems) from the day before coculture to the end of coculture.

Techniques: Expressing, Sequencing

Journal: International immunopharmacology

Article Title: Dominant negative TGFβ receptor II and truncated TIM3 enhance the antitumor efficacy of CAR-T-cell therapy in prostate cancer.

doi: 10.1016/j.intimp.2023.110807

Figure Lengend Snippet: Fig. 2. Preparation and phenotypic characterization of DT-PSMA-CAR-T cells: (A) Gene structure of PSMA-CAR and DT-PSMA-CAR; (B) Expansion of the UTD group, PSMA-CAR-T group, and DT-PSMA-CAR-T group T cells 14 days after electroporation; (C) The positivity rate of CAR in the PSMA-CAR-T group and DT-PSMA-CAR-T group, after magnetic bead sorting, assessed by flow cytometry; (D) Expression of TIM3 and TGFβRII on the three groups of T cells assessed by flow cytometry before performing in vitro killing assays; (E) Expression of CD4 and CD8 on the three groups of T cells assessed by flow cytometry before performing in vitro killing assays; (F) Expression of CD45RO on the three groups of T cells assessed by flow cytometry before performing in vitro killing assays.

Article Snippet: Untransduced (UTD) T cells, PSMA-CAR-T cells, and DT-PSMA-CAR- L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 T cells were co-cultured with prostate cancer cells at ratios of 1:1, 5:1, and 10:1 for 24 h.The T-cell culture medium was supplemented with appropriate concentrations (0 ng/ml, 5 ng/ml, 20 ng/ml, and 50 ng/ml) of TGF-β (P01137, Suzhou Novoprotein Scientific) and/or (0 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) TIM3 activating monoclonal antibodies (MAB23651, R&D Systems) from the day before coculture to the end of coculture.

Techniques: Electroporation, Flow Cytometry, Expressing, In Vitro

Journal: International immunopharmacology

Article Title: Dominant negative TGFβ receptor II and truncated TIM3 enhance the antitumor efficacy of CAR-T-cell therapy in prostate cancer.

doi: 10.1016/j.intimp.2023.110807

Figure Lengend Snippet: Fig. 4. Analysis of the resistance of DT-PSMA-CAR-T cells to immune suppression caused by TIM3 activation in vitro was performed by tumor lysis assays: (A and B) UTD group, PSMA-CAR-T group, and DT-PSMA-CAR-T group T cells were cocultured with LNCAP cells in the presence of 10 μg/ml TIM3 activating monoclonal antibody; (C and D) Under an effector-to-target ratio of 5:1, the three groups of T cells were cocultured with LNCAP cells in the presence of different concentrations of TIM3 activating monoclonal antibody; (E) Cytokine secretion was detected in the supernatant of all experimental groups in (C).

Article Snippet: Untransduced (UTD) T cells, PSMA-CAR-T cells, and DT-PSMA-CAR- L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 T cells were co-cultured with prostate cancer cells at ratios of 1:1, 5:1, and 10:1 for 24 h.The T-cell culture medium was supplemented with appropriate concentrations (0 ng/ml, 5 ng/ml, 20 ng/ml, and 50 ng/ml) of TGF-β (P01137, Suzhou Novoprotein Scientific) and/or (0 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) TIM3 activating monoclonal antibodies (MAB23651, R&D Systems) from the day before coculture to the end of coculture.

Techniques: Activation Assay, In Vitro, Lysis

Journal: International immunopharmacology

Article Title: Dominant negative TGFβ receptor II and truncated TIM3 enhance the antitumor efficacy of CAR-T-cell therapy in prostate cancer.

doi: 10.1016/j.intimp.2023.110807

Figure Lengend Snippet: Fig. 5. Analysis of the resistance of DT-PSMA-CAR-T cells to immune suppression caused by TIM3 and TGFβRII activation in vitro was performed by tumor lysis assays: (A and B) UTD group, PSMA-CAR-T group, and DT-PSMA-CAR-T group T cells were cocultured with LNCAP cells in the presence of 20 ng/ml TGF-β and 10 μg/ml TIM3 activating monoclonal antibody; (C and D) Under an effector-to-target ratio of 5:1, the three groups of T cells were cocultured with LNCAP cells in the presence of different combinations of TGF-β and TIM3 activating monoclonal antibody; (E) Cytokine secretion was detected in the supernatant of all experimental groups in (C).

Article Snippet: Untransduced (UTD) T cells, PSMA-CAR-T cells, and DT-PSMA-CAR- L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 T cells were co-cultured with prostate cancer cells at ratios of 1:1, 5:1, and 10:1 for 24 h.The T-cell culture medium was supplemented with appropriate concentrations (0 ng/ml, 5 ng/ml, 20 ng/ml, and 50 ng/ml) of TGF-β (P01137, Suzhou Novoprotein Scientific) and/or (0 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) TIM3 activating monoclonal antibodies (MAB23651, R&D Systems) from the day before coculture to the end of coculture.

Techniques: Activation Assay, In Vitro, Lysis

Journal: International immunopharmacology

Article Title: Dominant negative TGFβ receptor II and truncated TIM3 enhance the antitumor efficacy of CAR-T-cell therapy in prostate cancer.

doi: 10.1016/j.intimp.2023.110807

Figure Lengend Snippet: Fig. 6. DT-PSMA-CAR-T cells can inhibit the growth of NSG mouse xe nografts in an environment activated by TIM3 and TGFβRII: (A) The timeline of animal experiments: 1 × 106 GAL9- PSMA-PC3 cells were subcutaneously injected; two weeks later, 5 × 106 UTD T cells, PSMA-CAR-T cells, or DT- PSMA-CAR-T cells were infused per mouse via the tail vein; and biolumi nescence imaging was performed weekly; (B) The location and tumor burden of the xenografts displayed by bioluminescence imaging; (C) Quanti tative results of tumor burden obtained by Aniview100.

Article Snippet: Untransduced (UTD) T cells, PSMA-CAR-T cells, and DT-PSMA-CAR- L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 T cells were co-cultured with prostate cancer cells at ratios of 1:1, 5:1, and 10:1 for 24 h.The T-cell culture medium was supplemented with appropriate concentrations (0 ng/ml, 5 ng/ml, 20 ng/ml, and 50 ng/ml) of TGF-β (P01137, Suzhou Novoprotein Scientific) and/or (0 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) TIM3 activating monoclonal antibodies (MAB23651, R&D Systems) from the day before coculture to the end of coculture.

Techniques: Injection, Imaging

Journal: Genome Biology

Article Title: Comprehensive analyses of tumor immunity: implications for cancer immunotherapy

doi: 10.1186/s13059-016-1028-7

Figure Lengend Snippet: Varied levels of CD8 T-cell infiltration in tumors highly expressing inhibitory receptors. a , b High CTLA4/TIM3-expressing tumors in melanoma/KIRC show different CD8 T-cell infiltration levels. Dashed lines in both panels are the hypothetical high CTLA4 or TIM3 cutoff. Tumor purity is indicated by color. Arrows in b point to selected TCGA samples for immunohistochemistry (IHC) analysis. c Sample with low TIM3 expression and CD8 T-cell infiltration used as a negative control. TIM3- or CD8-expressing cells are brown in color. Selected samples with (1) high TIM3 expression and (2) low ( d ) or high ( e ) CD8 T-cell infiltration showed the existence of two KIRC sample groups. TIM3 expression in d is twice as high as in e according to RNA-seq data. d Image represents about 15 % TCGA KIRC samples while e represents 5 %. The upper and lower panels were synchronized. TIM3 was expressed in cancer cells ( d , e ) as well as in lymphocytes ( e ). High magnification insets are presented in d and e to illustrate TIM3 expression in different cell types. Yellow boxes indicate lymphocytes; red boxes indicate tumor cells

Article Snippet: Sections were next incubated for 1 h at RT with the goat polyclonal anti-TIM3 antibody (1/400, AF2365, R&D Systems) diluted in Da Vinci Green Diluent (Biocare Medical).

Techniques: Expressing, Immunohistochemistry, Negative Control, RNA Sequencing