Host NK cell differentiation, maturation, and function are severely impaired in CML chimeric mice. (A) Schematic of experimental setup. Bone marrow cells from C57BL/6 CD45.2 transgenic BCR::ABL1 (CML) mice are transplanted into sublethally irradiated B6.SJL-Ptprca Pepcb/BoyJ mice. Nonmalignant CD45.2 cells were used to establish the control group. Splenic, blood, and bone marrow NK cells were collected at the end point (∼8-10 weeks after treatment) for further analysis. (B) The frequencies in total host cells (left) and absolute numbers (right) of bone marrow and splenic NK cells determined by flow cytometry. (C) Simple linear regression analysis showing a negative correlation between NK cell frequencies of total CD45.1⁺ cells in the spleen., and CML burden defined as frequencies of CML Gr1⁺CD11b⁺ myeloid cells of total CD45 splenic cells. Frequencies (D) and absolute numbers (E) of main NK cell maturation subsets in the spleen and bone marrow. (F) Frequencies of KLRG1⁺ cells in total host NK cells in control and CML mice. (G) NK cell maturation score (arbitrary units) in control and CML mice. Median with a range, unpaired 2-tailed t test. (H) Frequencies of Ki-67⁺ NK cells. (I) Frequencies (left Y-scale) and absolute counts (right Y-scale) of CLP (left plot) and NKP (right plot) in CML mice compared with control, mean ± standard deviation (SD). (J) Frequencies of NKp46⁺ cells in total host NK cells in the spleen. (K) Altered expression of inhibitory receptors on CML-exposed NK cells from spleen. (L) Left: target-specific degranulation in NK cells from control and leukemic mice. NK cells were sorted from the spleen and incubated with Yac-1 cells at an effector-to-target (E:T) ratio of 1:2 or alone for 4 hours. Degranulation was assessed by the presence of surface CD107a on NK cells measured by flow cytometry. Right: frequencies of IFN-γ⁺ and granzyme B⁺ NK cells after a 4-hour PMA/ionomycin stimulation. For panels B, D-F, H-L: mean ± SD, unpaired 2-tailed t test. For all experiments, ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001; n = 3 to 12 mice per group. CLP, common lymphoid progenitors; GzmB, granzyme B; Imm, immature; Lag-3, lymphocyte activating gene 3; M1, intermediate; M2, mature NK cell subset; NKP, NK cell progenitors; TIGIT, T-cell immunoreceptor with immunoglobulin and tyrosine-based inhibitory motif domains.