Figure 3.
7C6 inhibits AML by triggering antibody-dependent phagocytosis. (A) Identification of macrophages and validation of their depletion in the spleen and bone marrow. C57BL/6 mice were inoculated IV with 0.2 mL of control or clodronate liposomes on days −8 and −1. On day 0, spleens and femoral bone marrows were collected after euthanasia, and CD11b+ F4/80+ macrophages, which are also negative for CD49b, CD3ε, B220, and Ly6G (supplemental Figure 6A-B), were analyzed by flow cytometry. Data representative of 2 mice per experiment and 2 independent experiments. (B-C) Characterization of spleen and bone marrow macrophages from leukemic mice. C57BL/6 mice were inoculated IV with 2 × 106 C1498-MICB and euthanized on day 20 for analyses. Naïve mice did not receive the leukemia cells and were euthanized together with the leukemic mice. Macrophages were identified by flow cytometry as in panel A. Spleen and bone marrow macrophages (B) acquire the ZsGreen fluorescence and (C) express high levels of Fc activating receptors in mice that were inoculated with C1498-MICB. (C) Red = isotype, Blue = anti-CD16/32 or anti-CD64. (D) Macrophages are required by 7C6 to inhibit AML. C57BL/6 mice were inoculated IV with 0.2 mL of clodronate or control liposomes on days −1, 5, and 14, and with 2 × 106 C1498-MICB on day 0. Antibody treatments were performed on days 5, 6, and 14, and euthanasia for flow cytometry-based analyses of leukemia burdens on day 19. (E) 7C6-mIgG2a triggers antibody-dependent phagocytosis. C57BL/6 mice’s bone marrow cells were differentiated to macrophages via 7 to 14 days culture with 10 ng/mL macrophage colony-stimulating factor. Before assay, C1498-MICB cells were treated with 20 μg/mL of 7C6-mIgG2a, 7C6-DANA, or isotype. Subsequently, these leukemia cells were labeled with CFSE and cocultured at 37°C or just incubated at 4°C with macrophages for 1 hour. After this, macrophages were detached with 10 mmol/L EDTA, labeled with F4/80 antibody, and analyzed by flow cytometry. (F) Fc receptor recognition is required by 7C6 to inhibit AML. Frequency (left) and absolute numbers (right) of leukemia cells are shown. C57BL/6 mice were inoculated IV with 2 × 106 C1498-MICB and treated with antibodies on days 5, 6, and 14. Euthanasia and flow cytometry-based analyzes of leukemia cells in the blood and bone marrow were done on day 19. Data are (B,D,F) median ± interquartile range or (E) mean ± SD, and are representative of (A) 2 or (E) 3, or (B,D,F) pooled of 2 independent experiments. *P < .05; **P < .01; ***P < .001, Mann-Whitney test comparing (B,D,F) 2 groups or (E) 2-way ANOVA with Bonferroni’s post hoc test.

7C6 inhibits AML by triggering antibody-dependent phagocytosis. (A) Identification of macrophages and validation of their depletion in the spleen and bone marrow. C57BL/6 mice were inoculated IV with 0.2 mL of control or clodronate liposomes on days −8 and −1. On day 0, spleens and femoral bone marrows were collected after euthanasia, and CD11b+ F4/80+ macrophages, which are also negative for CD49b, CD3ε, B220, and Ly6G (supplemental Figure 6A-B), were analyzed by flow cytometry. Data representative of 2 mice per experiment and 2 independent experiments. (B-C) Characterization of spleen and bone marrow macrophages from leukemic mice. C57BL/6 mice were inoculated IV with 2 × 106 C1498-MICB and euthanized on day 20 for analyses. Naïve mice did not receive the leukemia cells and were euthanized together with the leukemic mice. Macrophages were identified by flow cytometry as in panel A. Spleen and bone marrow macrophages (B) acquire the ZsGreen fluorescence and (C) express high levels of Fc activating receptors in mice that were inoculated with C1498-MICB. (C) Red = isotype, Blue = anti-CD16/32 or anti-CD64. (D) Macrophages are required by 7C6 to inhibit AML. C57BL/6 mice were inoculated IV with 0.2 mL of clodronate or control liposomes on days −1, 5, and 14, and with 2 × 106 C1498-MICB on day 0. Antibody treatments were performed on days 5, 6, and 14, and euthanasia for flow cytometry-based analyses of leukemia burdens on day 19. (E) 7C6-mIgG2a triggers antibody-dependent phagocytosis. C57BL/6 mice’s bone marrow cells were differentiated to macrophages via 7 to 14 days culture with 10 ng/mL macrophage colony-stimulating factor. Before assay, C1498-MICB cells were treated with 20 μg/mL of 7C6-mIgG2a, 7C6-DANA, or isotype. Subsequently, these leukemia cells were labeled with CFSE and cocultured at 37°C or just incubated at 4°C with macrophages for 1 hour. After this, macrophages were detached with 10 mmol/L EDTA, labeled with F4/80 antibody, and analyzed by flow cytometry. (F) Fc receptor recognition is required by 7C6 to inhibit AML. Frequency (left) and absolute numbers (right) of leukemia cells are shown. C57BL/6 mice were inoculated IV with 2 × 106 C1498-MICB and treated with antibodies on days 5, 6, and 14. Euthanasia and flow cytometry-based analyzes of leukemia cells in the blood and bone marrow were done on day 19. Data are (B,D,F) median ± interquartile range or (E) mean ± SD, and are representative of (A) 2 or (E) 3, or (B,D,F) pooled of 2 independent experiments. *P < .05; **P < .01; ***P < .001, Mann-Whitney test comparing (B,D,F) 2 groups or (E) 2-way ANOVA with Bonferroni’s post hoc test.

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