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TU2218 inhibits immunosuppression induced by TGFβ or coculture with cancer cells or deficient costimulatory signal. a CD4 + IFNγ + or CD8 + IFNγ + of CD3 (%) in PBMCs with indicated treatment condition. Human PBMCs were treated with indicated condition for 24 h and analyzed by flow cytometry. (One-way ANOVA, Dunnett vs. anti-CD3 + TGFβ) b IFNγ secretion by indicated treatment condition. The amount of IFNγ in PBMC culture supernatant was measured with ELISA. Human PBMCs were treated with indicated condition for 72 h. c The population of NKG2D + cells on CD56 dim or CD56 bright NK cells in human PBMCs compared to vehicle. NK2GD + cells (%) were assessed by flow cytometry. d The population of NKG2D + cells on NK92 cell lines (left) compared to vehicle and cellular cytotoxicity of K562 in NK92/K562 (E/T ratio = 2:1) coculture system (right). e Recovery of IFNγ secretion by indicated treatment condition. The amount of IFNγ in PBMC culture supernatant was measured with ELISA and calculated as relative ratio to anti-CD3/CD28 group. f Population of IFNγ-producing CD4 or CD8 T lymphocyte subsets in PBMC with indicated treatment condition analyzed by flow cytometry. Noted aCD3 indicates anti-CD3 stimulation, TU indicates TU2218, and V indicates vactosertib. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (One-way ANOVA, Dunnett or Tukey, vs. anti-CD3 + TGFβ or anti-CD3/CD28 + TGFβ or TGFβ)

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: ALK5/VEGFR2 dual inhibitor TU2218 alone or in combination with immune checkpoint inhibitors enhances immune-mediated antitumor effects

doi: 10.1007/s00262-024-03777-4

Figure Lengend Snippet: TU2218 inhibits immunosuppression induced by TGFβ or coculture with cancer cells or deficient costimulatory signal. a CD4 + IFNγ + or CD8 + IFNγ + of CD3 (%) in PBMCs with indicated treatment condition. Human PBMCs were treated with indicated condition for 24 h and analyzed by flow cytometry. (One-way ANOVA, Dunnett vs. anti-CD3 + TGFβ) b IFNγ secretion by indicated treatment condition. The amount of IFNγ in PBMC culture supernatant was measured with ELISA. Human PBMCs were treated with indicated condition for 72 h. c The population of NKG2D + cells on CD56 dim or CD56 bright NK cells in human PBMCs compared to vehicle. NK2GD + cells (%) were assessed by flow cytometry. d The population of NKG2D + cells on NK92 cell lines (left) compared to vehicle and cellular cytotoxicity of K562 in NK92/K562 (E/T ratio = 2:1) coculture system (right). e Recovery of IFNγ secretion by indicated treatment condition. The amount of IFNγ in PBMC culture supernatant was measured with ELISA and calculated as relative ratio to anti-CD3/CD28 group. f Population of IFNγ-producing CD4 or CD8 T lymphocyte subsets in PBMC with indicated treatment condition analyzed by flow cytometry. Noted aCD3 indicates anti-CD3 stimulation, TU indicates TU2218, and V indicates vactosertib. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (One-way ANOVA, Dunnett or Tukey, vs. anti-CD3 + TGFβ or anti-CD3/CD28 + TGFβ or TGFβ)

Article Snippet: Erythrocytes were lysed and single-cell suspensions were incubated with Fc block and stained with the following antibodies: anti-mouse CD31-FITC (clone 390, eBioscience), anti-mouse VCAM-1-APC (clone 429, Biolegend) anti-mouse CD45-BV510 (clone 30-F11, BD Bioscience), anti-mouse CD3-APC-Cy7 (clone 145-2C11, Invitrogen), anti-mouse CD8-BB515 (clone 53–6.7, BD Bioscience), and anti-mouse IFNγ-APC (clone XMG1.2, BD Bioscience).

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay

TU2218 blocks suppressive activity of regulatory T cell for proliferation of TCR-elicited effector T lymphocytes. a Activity of Treg suppression on proliferation of T effector cell (Teff). Histograms and % proliferation showing division of CFSE-labeled CD4 + CD25 − Teff purified from pooled eight donor-derived PBMCs, cultured with anti-CD3 and anti-CD28 at Treg/Teff ratios of 3:1 for 5 days. Purified Teff cells without Treg cells (0:1) were used as a positive control of Teff proliferation. b Percentage of Treg (CD25 + CFSE-) after Treg and Teff coculture for 5 days. Noted TU indicates TU2218 and V indicates vactosertib and anti-TGFβ indicates anti-TGFβ neutralizing antibody

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: ALK5/VEGFR2 dual inhibitor TU2218 alone or in combination with immune checkpoint inhibitors enhances immune-mediated antitumor effects

doi: 10.1007/s00262-024-03777-4

Figure Lengend Snippet: TU2218 blocks suppressive activity of regulatory T cell for proliferation of TCR-elicited effector T lymphocytes. a Activity of Treg suppression on proliferation of T effector cell (Teff). Histograms and % proliferation showing division of CFSE-labeled CD4 + CD25 − Teff purified from pooled eight donor-derived PBMCs, cultured with anti-CD3 and anti-CD28 at Treg/Teff ratios of 3:1 for 5 days. Purified Teff cells without Treg cells (0:1) were used as a positive control of Teff proliferation. b Percentage of Treg (CD25 + CFSE-) after Treg and Teff coculture for 5 days. Noted TU indicates TU2218 and V indicates vactosertib and anti-TGFβ indicates anti-TGFβ neutralizing antibody

Article Snippet: Erythrocytes were lysed and single-cell suspensions were incubated with Fc block and stained with the following antibodies: anti-mouse CD31-FITC (clone 390, eBioscience), anti-mouse VCAM-1-APC (clone 429, Biolegend) anti-mouse CD45-BV510 (clone 30-F11, BD Bioscience), anti-mouse CD3-APC-Cy7 (clone 145-2C11, Invitrogen), anti-mouse CD8-BB515 (clone 53–6.7, BD Bioscience), and anti-mouse IFNγ-APC (clone XMG1.2, BD Bioscience).

Techniques: Activity Assay, Labeling, Purification, Derivative Assay, Cell Culture, Positive Control