Natural Killer (NK) Cell-Mediated Immune Evasion Plays a Key Role in Clonal Persistence, Refractoriness, and Relapse in Acute Myeloid Leukemia (AML). Growing evidence indicates that alterations in the expression of innate immune recognition ligands by leukemic blasts contribute to the inhibition of NK cell effector responses, thereby favoring disease maintenance. However, the immunophenotypic mechanisms underlying this process remain poorly understood.

In this study, we investigated the expression profile of NK cell ligands on leukemic blasts and their association with clinical outcomes and measurable residual disease (MRD).

A total of 127 bone marrow samples collected at diagnosis from adult AML patients were analyzed. Samples were processed within 24 hours, lysed using ammonium chloride solution, and stained with monoclonal antibodies targeting CD45, CD34, CD38, CD123, CD112, CD155, and HLA-E. Flow cytometry analysis was performed using a FACSCanto II cytometer (BD Biosciences), applying validated panels for both diagnosis and MRD assessment. At least 500,000 viable events were acquired per sample, and analysis was standardized using internal controls and healthy donor bone marrow samples. Patients were treated according to standard AML therapeutic protocols, including induction with cytarabine and anthracyclines (7+3 regimen), followed by high-dose cytarabine (HiDAC) consolidation, according to clinical and biological eligibility. Leukemic stem cell (LSC)-enriched subpopulations were defined as CD45dim/CD34⁺/CD38⁻/CD123⁺, along with CD34⁻ blasts. Activating (CD112, CD155) and inhibitory (HLA-E) NK ligands were assessed by both the frequency of positive cells and median fluorescence intensity (MFI). MRD was measured in post-induction samples with a minimum sensitivity of 10⁻⁴, and clinical outcomes (remission, relapse, refractoriness, early death) were obtained from electronic medical records, with a minimum follow-up of 12 months.

Patients with unfavorable clinical outcomes showed, at diagnosis, a significant reduction in CD112 (p = 0.004) and CD155 (p = 0.013) expression, along with increased HLA-E levels (p = 0.009), when compared to patients in remission and healthy controls. MRD positivity after induction was associated with higher HLA-E expression (p = 0.027) and a trend toward decreased CD112 (p = 0.078) and CD155 (p = 0.065), particularly within the CD34⁺ subpopulation. Among CD34⁺ blasts, higher HLA-E expression (p = 0.006) and lower CD155 levels (p = 0.021) were observed compared to CD34⁻ cells, suggesting enhanced immune evasion capacity in the LSC-enriched fraction.

These findings support the hypothesis that phenotypic alterations in NK cell recognition ligands contribute to immune escape and MRD persistence. The consolidation of the inhibitory HLA-E/NKG2A axis as a key mechanism of immune suppression in AML highlights its potential as a therapeutic target. Altogether, our results support the development of immunomodulatory strategies aimed at restoring innate immune surveillance and preventing LSC persistence, with the potential to reduce relapse rates and improve clinical control of AML.