Study on the Mechanisms of Allogeneic NK Cells Combined with Myeloid Cells in Treating Acute Myeloid Leukemia

Allogeneic hematopoietic stem cell microtransplantation (MST) has been shown in prior studies to improve outcomes compared to chemotherapy alone in acute myeloid leukemia, but its mechanism requires further investigation. In MST, donor peripheral blood mononuclear cells (GPBMCs) mobilized by granulocyte colony-stimulating factor (G-CSF), which contain a large number of NK cells, are infused after each chemotherapy cycle (typically 3 to 5 infusions total). This study was designed to investigate whether G-CSF-mobilized NK cells exert antitumor effects and the underlying mechanisms involved. NK cell phenotype, cytotoxicity, and cytokine production were compared before and after G-CSF mobilization. Seventy-two AML patients treated with MST were retrospectively analyzed. The effects of NK cell dose and KIR ligand mismatch on survival were studied. NK cells from 8 donors were isolated before and after G-CSF mobilization to assess phenotype, killing activity, and cytokine production. Gene expression and cell-cell communication were analyzed via single-cell RNA sequencing (scRNA-seq). In univariable analysis, higher NK cell doses were associated with improved survival; however, this association did not retain statistical significance after multivariable adjustment, whereas KIR ligand mismatch and donor activating receptor numbers were not. G-CSF mobilization increased the proportion of CD56^bright NK cells and upregulated the inhibitory receptor TIGIT, while downregulating NKG2D. Mobilized NK cells showed reduced cytotoxicity against K562 cells, lower perforin and granzyme B release, and decreased IFN-γ production. scRNA-seq revealed that mobilized NK cells upregulated genes associated with immune regulation, myeloid cell interaction, and chemokine signaling. Ligand-receptor pairing analysis suggested enhanced potential interactions with myeloid cells post-mobilization, particularly involving C5AR1 and CCL5 pathways. G-CSF mobilization alters the phenotype and reduces the direct cytotoxic function of NK cells, while enriching for subsets with immunomodulatory gene expression profiles. These findings generate hypotheses that mobilized NK cells may support antitumor immunity indirectly through crosstalk with myeloid cells. Further studies are needed to validate these interactions and their functional consequences in vivo.