Figure 2.
PVR and B7-H6 are essential for formation of robust immunological synapses between DOT cells and AML cells. (A) Summary of killing assays performed against the indicated KO HEL cell lines, compared with the mock-transfected cell line. Each dot represents a different DOT-cell donor. Data were generated in 3 independent experiments. (B) Killing assay performed against the mouse breast cancer cell line E0771 previously transfected to express human PVR, nectin-2, or B7-H6 compared with the mock-transfected cell line. DOT cells were selected for high expression of the respective counterreceptors (DNAM-1 or NKp30). Each dot represents a technical replicate. Data were generated in 2 independent experiments. (C) Representative flow cytometry plots of granzyme B (GzmB) and perforin (Prf) expression on DOT cells, when in contact with different cell lines or in the presence of interleukin-15 (IL-15) only. (D) Summary of the flow cytometry data depicted in panel C. Each dot represents a DOT-cell donor. Data were generated in 2 independent experiments. (E) Tumor load in the blood of a xenograft model of AML. Immunodeficient NSG-HuIL-15 mice were engrafted with control or PVR/B7-H6 double-KO HEL (luciferase positive) cell lines. Mice were treated with DOT cells intravenously or left untreated. Three weeks after tumor injection, mice were euthanized and tumor load in the blood was quantified by luminescence. Data were generated in 2 independent experiments. (F) Representative images of image-flow cytometry data of immunological synapses established between DOT cells and different HEL cell lines. (G) Quantification of filamentous actin (F-actin) signal within the area of interaction between the DOT cell and the tumor cell. Data are representative of 2 independent experiments. Statistical analysis was performed using 1-sample t test (hypothetical value: 100 for panel A, or 0 for panel B), 1-way analysis of variance followed by Šídák multiple comparisons test for panel E, or Kruskal-Wallis test followed by Dunn multiple comparisons test for panels D and G. WT, wild type.

PVR and B7-H6 are essential for formation of robust immunological synapses between DOT cells and AML cells. (A) Summary of killing assays performed against the indicated KO HEL cell lines, compared with the mock-transfected cell line. Each dot represents a different DOT-cell donor. Data were generated in 3 independent experiments. (B) Killing assay performed against the mouse breast cancer cell line E0771 previously transfected to express human PVR, nectin-2, or B7-H6 compared with the mock-transfected cell line. DOT cells were selected for high expression of the respective counterreceptors (DNAM-1 or NKp30). Each dot represents a technical replicate. Data were generated in 2 independent experiments. (C) Representative flow cytometry plots of granzyme B (GzmB) and perforin (Prf) expression on DOT cells, when in contact with different cell lines or in the presence of interleukin-15 (IL-15) only. (D) Summary of the flow cytometry data depicted in panel C. Each dot represents a DOT-cell donor. Data were generated in 2 independent experiments. (E) Tumor load in the blood of a xenograft model of AML. Immunodeficient NSG-HuIL-15 mice were engrafted with control or PVR/B7-H6 double-KO HEL (luciferase positive) cell lines. Mice were treated with DOT cells intravenously or left untreated. Three weeks after tumor injection, mice were euthanized and tumor load in the blood was quantified by luminescence. Data were generated in 2 independent experiments. (F) Representative images of image-flow cytometry data of immunological synapses established between DOT cells and different HEL cell lines. (G) Quantification of filamentous actin (F-actin) signal within the area of interaction between the DOT cell and the tumor cell. Data are representative of 2 independent experiments. Statistical analysis was performed using 1-sample t test (hypothetical value: 100 for panel A, or 0 for panel B), 1-way analysis of variance followed by Šídák multiple comparisons test for panel E, or Kruskal-Wallis test followed by Dunn multiple comparisons test for panels D and G. WT, wild type.

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