The importance of several lymphocyte populations for the ability of 7C6 in inhibiting AML. (A-B) Antibody-mediated depletion of NK cells does not abrogate the 7C6’s efficacy against AML. C57BL/6 mice were inoculated IV with 2 × 10⁶ C1498-MICB (day 0) and treated via IP injections with 200 μg 7C6-mIgG2a or isotype on days 5 and 6. On day 14, mice were treated with just 100 μg of 7C6-mIgG2a or isotype. Furthermore, mice were treated with 100 μg control IgG (cIgG) or α-NK1.1 on days −1, 0, 7, and 14. Analysis of leukemia cells in the blood and bone marrow were performed on day 20 by flow cytometry. (A) Frequency of C1498-MICB in the blood and bone marrow. (B) Absolute numbers of C1498-MICB in the blood and bone marrow. (C) Validation of NK cell depletion. A healthy mouse was bled via submandibular puncture and treated with 0.1 mg of α-NK1.1. Two days later, the mouse was euthanized and its blood collected for analyses. NK cells are the CD49b⁺ and CD3ε⁻ cells. The gatings of lymphocytes, doublets discrimination, and identification of viable CD45.2⁺ cells (not shown) antecede the plots shown in panel C. (D-E) Antibody-mediated depletion/blockade of T cells and NKG2D. C57BL/6 mice were treated with the indicated depleting/blocking antibodies on days −1, 0, 7, and 14. The mice were also inoculated IV with 2 × 10⁶ C1498-MICB (day 0) and treated with 0.2 mg 7C6-mIgG2a or isotype control on days 5 and 6. On day 14, mice received only 0.1 mg of 7C6-mIgG2a or isotype. Analyses of leukemia cells (D) in the blood and (E) bone marrow were done by flow cytometry 3 weeks after leukemia cell inoculation. Data pooled from (A-B,D-E) 2 independent experiments or (C) representative of 2 mice and 2 independent experiments. *P < .05; **P < .01; ***P < .001, as calculated (A-B,D-E) with Mann-Whitney tests comparing 2 treatment groups at a time. (A-B,D-E) Median ± interquartile range.