Figure 1.
7C6 inhibits the in vivo outgrowth of AML in 2 mouse models. (A-F) C1498-MICB and AML model in immunocompetent C57BL/6 mice. (G-J) WEHI-3-MICB and AML model in immunocompetent Balb/c mice. (A) 7C6-mIgG2b inhibits the shedding of MICB by C1498 cells. The C1498-MICB cell line was cultured for 24 hours with the indicated antibodies at the indicated doses, followed by analysis of soluble MICB shed into supernatants with a sandwich enzyme-linked immunosorbent assay (ELISA) and surface MICB on cells via flow cytometry. MFI, mean fluorescence intensity. (B-E) C57BL/6 mice were inoculated IV with 2 × 106 C1498-MICB cells, which corresponds to day 0. Mice were treated via IP injections with 200 μg of the indicated antibodies on days 5 and 6; on day 14, mice were treated with just 100 μg of the indicated antibodies. Euthanasia and analysis of blood and bone marrow were performed on day 21. (B) Illustration of the procedure and identification of ZsGreen+ C1498-MICB in the blood by flow cytometry. (C-E) 7C6-mIgG2a reduces the frequency (C) and absolute numbers (D) of C1498-MICB in the blood and bone marrow of mice. (E) Similar results were found via histopathology. (C) The frequency of C1498-MICB was measured after staining blood cells with an antigen-presenting cell-conjugated CD45 antibody followed by identification of ZsGreen+ C1498-MICB by flow cytometry. (D) The absolute numbers of C1498-MICB were determined after multiplying the C1498-MICB frequency by the concentration of total blood cells and bone marrow cells from femur. Wright-Giemsa staining (top) and hematoxylin-eosin staining (bottom) of blood smears and femurs, respectively. (E) The arrows indicate leukemia-like cells. (F) 7C6-mIgG2a prolongs the survival of leukemia-bearing mice. C57BL/6 mice were inoculated IV with 1 × 106 C1498-MICB cells and treated with the indicated antibodies on days 5, 6, and 14 (same antibody treatment regimen of experiment shown in panels C-E). Mice were euthanized when they showed physical signs of disease (weight loss, limb paralysis, moribund state, and/or curved posture). (G) 7C6-mIgG2a inhibits the MICB shedding by WEHI-3-MICB and stabilizes the surface protein. WEHI-3-MICB was treated for 24 hours with the indicated concentrations of the indicated antibodies, followed by analyses of soluble MICB shed into supernatants by sandwich ELISA and surface MICB on cells by flow cytometry. (H-I) 7C6 inhibits WEHI-3-MICB in vivo. Balb/c mice were inoculated IV with 2 × 106 WEHI-3-MICB and, on days 5 and 6, treated with 0.2 mg of 7C6-mIgG2a or isotype. On day 14, only 0.1 mg of antibodies were administered. Euthanasia and analyses of leukemia by flow cytometry were done on day 20. (J) 7C6 prolongs the survival of mice in the WEHI-3-MICB model. Balb/c mice were inoculated IV with 5 × 105 WEHI-3-MICB and treated with 0.2 mg of 7C6-mIgG2a or isotype on days 1, 4, and 8. As in panel F, mice were euthanized when they showed physical signs of disease. Data representative of 3 (A,G) and pooled of 2 (C-D,F,H-J) independent experiments. Data in panel E are representative of 5 mice per antibody and 2 naïve mice. (C-D,H-I) **P < .01; ***P < .001, (Mann-Whitney test). In panels F and J, the P values were calculated by Mantel-Cox test. In panels A and G, 3 wells per antibody concentration were used. (C-D,H-I) Each dot indicates 1 mouse. Data are (A,G) mean ± standard error or (C-D,H-I) median ± interquartile range.

7C6 inhibits the in vivo outgrowth of AML in 2 mouse models. (A-F) C1498-MICB and AML model in immunocompetent C57BL/6 mice. (G-J) WEHI-3-MICB and AML model in immunocompetent Balb/c mice. (A) 7C6-mIgG2b inhibits the shedding of MICB by C1498 cells. The C1498-MICB cell line was cultured for 24 hours with the indicated antibodies at the indicated doses, followed by analysis of soluble MICB shed into supernatants with a sandwich enzyme-linked immunosorbent assay (ELISA) and surface MICB on cells via flow cytometry. MFI, mean fluorescence intensity. (B-E) C57BL/6 mice were inoculated IV with 2 × 106 C1498-MICB cells, which corresponds to day 0. Mice were treated via IP injections with 200 μg of the indicated antibodies on days 5 and 6; on day 14, mice were treated with just 100 μg of the indicated antibodies. Euthanasia and analysis of blood and bone marrow were performed on day 21. (B) Illustration of the procedure and identification of ZsGreen+ C1498-MICB in the blood by flow cytometry. (C-E) 7C6-mIgG2a reduces the frequency (C) and absolute numbers (D) of C1498-MICB in the blood and bone marrow of mice. (E) Similar results were found via histopathology. (C) The frequency of C1498-MICB was measured after staining blood cells with an antigen-presenting cell-conjugated CD45 antibody followed by identification of ZsGreen+ C1498-MICB by flow cytometry. (D) The absolute numbers of C1498-MICB were determined after multiplying the C1498-MICB frequency by the concentration of total blood cells and bone marrow cells from femur. Wright-Giemsa staining (top) and hematoxylin-eosin staining (bottom) of blood smears and femurs, respectively. (E) The arrows indicate leukemia-like cells. (F) 7C6-mIgG2a prolongs the survival of leukemia-bearing mice. C57BL/6 mice were inoculated IV with 1 × 106 C1498-MICB cells and treated with the indicated antibodies on days 5, 6, and 14 (same antibody treatment regimen of experiment shown in panels C-E). Mice were euthanized when they showed physical signs of disease (weight loss, limb paralysis, moribund state, and/or curved posture). (G) 7C6-mIgG2a inhibits the MICB shedding by WEHI-3-MICB and stabilizes the surface protein. WEHI-3-MICB was treated for 24 hours with the indicated concentrations of the indicated antibodies, followed by analyses of soluble MICB shed into supernatants by sandwich ELISA and surface MICB on cells by flow cytometry. (H-I) 7C6 inhibits WEHI-3-MICB in vivo. Balb/c mice were inoculated IV with 2 × 106 WEHI-3-MICB and, on days 5 and 6, treated with 0.2 mg of 7C6-mIgG2a or isotype. On day 14, only 0.1 mg of antibodies were administered. Euthanasia and analyses of leukemia by flow cytometry were done on day 20. (J) 7C6 prolongs the survival of mice in the WEHI-3-MICB model. Balb/c mice were inoculated IV with 5 × 105 WEHI-3-MICB and treated with 0.2 mg of 7C6-mIgG2a or isotype on days 1, 4, and 8. As in panel F, mice were euthanized when they showed physical signs of disease. Data representative of 3 (A,G) and pooled of 2 (C-D,F,H-J) independent experiments. Data in panel E are representative of 5 mice per antibody and 2 naïve mice. (C-D,H-I) **P < .01; ***P < .001, (Mann-Whitney test). In panels F and J, the P values were calculated by Mantel-Cox test. In panels A and G, 3 wells per antibody concentration were used. (C-D,H-I) Each dot indicates 1 mouse. Data are (A,G) mean ± standard error or (C-D,H-I) median ± interquartile range.

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