Advancing NK cell immunotherapy in AML and overcoming cytokine-driven adaptive resistance
Ex vivo–expanded NK cells combined with monoclonal antibodies (mAbs) offer a promising immunotherapy for acute myeloid leukemia (AML). This approach aims to enhance NK cell–mediated cytotoxicity and persistence while leveraging antibody-based targeting. However, AML cells may develop adaptive resistance via cytokine-driven upregulation of NK cell inhibitory ligands, particularly IFN-γ–mediated HLA class I upregulation, reducing NK cell recognition. This study investigates how NK cell–exposed AML cells change their susceptibility to NK cells and whether this NK–AML interaction induces NK cell exhaustion.
First, NK cells from healthy donors were expanded using K562 feeder cells expressing 4-1BBL and membrane-bound IL-21, with IL-2 supplementation. These NK cells were tested against HL-60, MOLM-13 cells and primary AML cells with mAbs targeting CD33 at varying effector-to-target (E:T) ratios. Conditioned AML cells exposed to IFN-γ or to cytotoxicity supernatant (CS) from assays with HL-60 cells were analyzed using multiparameter flow cytometry (MPFC) and RNA sequencing. Expanded NK cells were cultured for 120h, with a restimulation after 72h with AML cells. Phenotypic and functional exhaustion were analyzed by multiparameter flow cytometry (MPFC).
Expanded NK cells retained CD16 expression, enabling combinatorial therapy with monoclonal antibodies (mAbs). NK cytotoxicity showed synergistic effects with mAbs against MOLM-13 cells (56.7% alone vs. 77.2% with αCD33) and primary AML cells (24.4% alone vs. 37.9% with αCD33). Cytotoxicity against HL-60 cells revealed, among other cytokines, notably high IFN-γ secretion (6.9 ng/mL; n = 5).
MOLM-13 cells exposed to cytotoxicity supernatant (CS) or IFN-γ showed reduced susceptibility to NK cell–mediated killing (56.7% untreated vs. 35.5% with CS vs. 33.5% with IFN-γ), as did primary AML cells (24.5% untreated vs. 16.7% with CS vs. 17.0% with IFN-γ). Mechanistically, this was linked to IFN-γ–induced HLA class I upregulation on MOLM-13 cells, confirmed by multiparameter flow cytometry (MPFC) and RNA sequencing. Anti-CD33 antibodies partially mitigated this reduction in cytotoxicity (MOLM-13: 33.5% vs. 64.2%; primary AML: 16.7% vs. 30.7%).
Neutralizing IFN-γ reduced HLA class I expression on MOLM-13 cells and improved NK-cell–mediated killing. Finally, NK cell exhaustion assays performed with HL-60 and MOLM-13 for 120h showed a marked decline in NK cytotoxicity (96% specific lysis vs. 31%) without notable upregulation of phenotypic exhaustion markers.
Ex vivo–expanded NK cells demonstrate strong therapeutic potential, particularly when combined with mAbs. However, AML cells exploit cytokine-driven resistance, such as IFN-γ–induced HLA class I upregulation, to evade NK cell cytotoxicity and induction of NK cell exhaustion. Future combinatorial strategies integrating NK cell–based therapies with antibody treatment may be further optimized by targeting these resistance mechanisms through NK cell genetic engineering.