To explore the similarities and differences in the biological phenotypes and cytotoxicity of resident and expanded natural killer cells derived from mononuclear cells of human bone marrow between healthy donors (HD) and patients with B-cell acute lymphoblastic leukemia (B-ALL) .

Bone marrow blood of HD and B-ALL was collected, and mononuclear cells were enriched by density gradient centrifugation. Flow cytometry was used to analyze the phenotypes of resident NK cells (HD-NK and BALL-NK) in the two types of blood. Then, with the aid of our well-established 14-day "3ILs"-based (short for IL-2, IL-15, IL-18) culture system for in vitro high-efficient activation and expansion of NK cells, we conducted cell counting, flow cytometry analysis and in vitro co-culture killing tests to compare the phenotypes and cytotoxicity of expanded HD-NK cells (eHD-NK) and expanded BALL-NK cells (eBALL-NK) in bone marrow blood. Comparisons between two groups were performed using independent samples t-test. Comparisons among the four groups (HD-NK, eHD-NK, BALL-NK, and eBALL-NK) were conducted using two-way repeated measures ANOVA, followed by Bonferroni's post hoc test for pairwise comparisons.

Compared with the healthy donor (HD) group, the proportion of total CD3^-CD56⁺ NK cells in the non-activated, expanded resident NK cell population from patients with B-cell acute lymphoblastic leukemia (B-ALL) was significantly lower [ (4.21 ± 0.92) % vs (14.25 ± 1.15) %]. In addition, the frequencies of activated NK cell subsets were reduced, including CD16⁺ NK cells [ (46.82 ± 2.96) % vs (67.87 ± 2.12) %], NKG2D⁺ NK cells [ (22.45 ± 2.12) %vs (50.82 ± 5.65) %], and NKp46⁺ NK cells [ (7.66 ± 1.73) % vs (17.27 ± 1.75) %]. Following in vitro activation and expansion, the proportion of total NK cells was significantly increased in both the expanded HD-NK (eHD-NK) group and the expanded B-ALL-derived NK (eBALL-NK) group [ (61.52 ± 3.18) % vs (14.25 ± 1.15) %]; (24.63 ± 2.07) % vs (4.21 ± 0.92) %]. Moreover, the eHD-NK group exhibited higher proportions of total NK cells [ (61.52 ± 3.18) % vs (24.63 ± 2.07) %] and multiple activated NK cell subsets than the eBALL-NK group, including NKG2D⁺ NK cells [ (80.63 ± 2.03) % vs (57.83 ± 8.55) %], CD25⁺ NK cells [ (37.45 ± 3.21) %vs (20.90 ± 5.15) %], and NKp46⁺ NK cells [ (27.23 ± 2.30) % vs (9.51 ± 0.98) %]. Cell viability analysis demonstrated that the efficiency of in vitro activation and expansion of total NK cells was higher in the HD group than in the B-ALL group. Compared with the eHD-NK group, the eBALL-NK group showed significantly higher proportions of apoptotic cells, including Annexin V⁺7-AAD- cells [ (4.50 ± 0.35) % vs (2.72 ± 0.43) %] and Annexin V⁺ cells [ (5.13 ± 0.62) % vs (3.29 ± 0.58) %], while no significant differences in cell cycle distribution were observed between the two groups. In vitro cytotoxicity co-culture assays revealed that eHD-NK cells exhibited stronger cytotoxic activity against the tumor cell lines Nalm6 and U937 compared with eBALL-NK cells.

Compared with HDs, bone marrow–derived resident and activated NK cells from B-ALL patients exhibited reduced proportions, diminished activated subpopulation content, impaired in vitro activation capacity, decreased cellular viability, and attenuated cytotoxicity. This study provides valuable reference for subsequent investigations into the underlying pathogenic mechanisms and the development of clinical therapeutic strategies.