Journal: The Journal of Experimental Medicine

Article Title: Tumor-derived soluble CD155 inhibits DNAM-1–mediated antitumor activity of natural killer cells

doi: 10.1084/jem.20191290

Figure Lengend Snippet: Characterization of generated sCD155/BL6 and mock/BL6, and serum levels of sCD155. (A) Concentrations of mouse and human sCD155 in the culture supernatant of sCD155/BL6 ( n = 3), mock/BL6 ( n = 3), and HeLa ( n = 3) were analyzed 24 h after the start of the culture by CBA assay and ELISA, respectively. (B) Expression of membrane-bound CD155 on sCD155/BL6 and mock/BL6 was analyzed by using flow cytometry. (C) sCD155/BL6 ( n = 3) and mock/BL6 ( n = 3) were cultured (1.0 × 10 5 cells/well) in 96-well flat plates for 24 h, and then BrdU reagent was added to the cultures. BrdU incorporation was measured after culture for 12 h. (D) C57BL/6 WT mice were intravenously injected with different clones of sCD155/BL6 ( n = 4) and mock/BL6 ( n = 5) from those used in . Colony numbers in the lung were counted on day 17. (E) C57BL/6 WT mice were intravenously injected with sCD155/BL6 ( n = 5) or mock/BL6 ( n = 5) used in and , and analyzed for serum levels of sCD155 on days 0, 13, 17, and 21. (F) C57BL/6 WT mice were treated with mouse IgG2a, anti-NK1.1 antibody, rat IgG2a, or anti-CD8 antibody. Peripheral blood mononuclear cells on days 0, 4, and 7 were stained with antibodies against CD3, CD49b, and/or CD4. (G) C57BL/6 WT mice were intravenously injected with sCD155/BL6 or mock/BL6. Paraffin sections of lungs with colonized tumor and spleen on day 17 were stained as described in . Scale bars, 50 µm. Error bars indicate SD. Results were analyzed by using Student’s t test. For all analyses: *, P < 0.05; n.s., not significant.

Article Snippet: The reporter cells were incubated with 10 μg/ml of BSA or 2.5, 5.0, or 10 μg/ml of recombinant mouse CD155 protein (R&D Systems) at 37°C for 18 h. For positive control of GFP expression, plate-coated CD155-Fc protein (5 μg/ml) was used.

Techniques: Generated, Enzyme-linked Immunosorbent Assay, Expressing, Membrane, Flow Cytometry, Cell Culture, BrdU Incorporation Assay, Injection, Clone Assay, Staining

Journal: The Journal of Experimental Medicine

Article Title: Tumor-derived soluble CD155 inhibits DNAM-1–mediated antitumor activity of natural killer cells

doi: 10.1084/jem.20191290

Figure Lengend Snippet: sCD155 suppressed mouse and human NK cell activation in a DNAM-1–dependent manner. (A and C) IL-2–activated mouse NK cells derived from WT (A) and Cd226 −/− (C) mice were co-cultured with B16/BL6 (E:T = 1:1) in the presence of sCD155; mAbs against mouse DNAM-1, mouse TIGIT, or mouse CD96; or isotype control and analyzed for CD107a and IFN-γ expression. (D) Human NK cells isolated from PBMC in a healthy volunteer (HV) were co-cultured with COLO 205 (E:T = 1:1) in the presence of sCD155; mAbs against human DNAM-1, human TIGIT, or human CD96; or isotype control and analyzed for CD107a and IFN-γ expression. (B and E) IL-2–activated mouse NK cells (B) and human NK cells (E) were co-cultured with B16/BL6 (E:T = 1:1) and COLO 205 (E:T = 1:1) in the presence of mouse and human sCD155, respectively, or BSA (control) and analyzed for CD107a expression. (F) Mouse DNAM-1–GFP reporter cells were cultured in the presence of sCD155 or BSA (control) or stimulated with plate-coated mouse CD155-Fc fusion protein and analyzed for GFP expression by flow cytometry. Data are representative of two experiments (A, C, and D). Error bars indicate SD. Results were analyzed by using Student’s t test. For all analyses: *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.

Article Snippet: The reporter cells were incubated with 10 μg/ml of BSA or 2.5, 5.0, or 10 μg/ml of recombinant mouse CD155 protein (R&D Systems) at 37°C for 18 h. For positive control of GFP expression, plate-coated CD155-Fc protein (5 μg/ml) was used.

Techniques: Activation Assay, Derivative Assay, Cell Culture, Control, Expressing, Isolation, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Tumor-derived soluble CD155 inhibits DNAM-1–mediated antitumor activity of natural killer cells

doi: 10.1084/jem.20191290

Figure Lengend Snippet: sCD155 binds to activated CD8 + T cells. (A) DNAM-1, TIGIT, and CD96 expression on spleen CD8 + T cells stimulated with or without plate-coated anti-CD3 mAb and soluble anti-CD28 mAb in the presence of IL-2 was analyzed by using flow cytometry. The shaded histogram indicates isotype control, and the open histogram indicates each specific antibody. (B) sCD155 binding to CD8 + T cells. The naive (upper) or activated (bottom) CD8 + T cells were incubated with recombinant mouse CD155-His and then were stained with PE-conjugated anti-His mAb. The shaded histogram indicates isotype control, and the open histogram indicates anti-His mAb staining.

Article Snippet: The reporter cells were incubated with 10 μg/ml of BSA or 2.5, 5.0, or 10 μg/ml of recombinant mouse CD155 protein (R&D Systems) at 37°C for 18 h. For positive control of GFP expression, plate-coated CD155-Fc protein (5 μg/ml) was used.

Techniques: Expressing, Flow Cytometry, Control, Binding Assay, Incubation, Recombinant, Staining

Journal: The Journal of Experimental Medicine

Article Title: Tumor-derived soluble CD155 inhibits DNAM-1–mediated antitumor activity of natural killer cells

doi: 10.1084/jem.20191290

Figure Lengend Snippet: Steps used in the BLItz system affinity assay

Article Snippet: The reporter cells were incubated with 10 μg/ml of BSA or 2.5, 5.0, or 10 μg/ml of recombinant mouse CD155 protein (R&D Systems) at 37°C for 18 h. For positive control of GFP expression, plate-coated CD155-Fc protein (5 μg/ml) was used.

Techniques: Recombinant