NKRLs are variably expressed by AML blasts. The expression of 6 activating (MICA, MICB, CD155, CD112, ULBP1, and ULBP2/5/6) and 3 inhibitory (PD-L1, PD-L2, and HLA class I) NKRLs was detected by flow cytometry on cryopreserved bone marrow blasts harvested from 66 patients with a new diagnosis of AML. Side scatter (SSC-A) and CD45 were used to gate the blast population. CD33 and CD34 were used as additional markers of disease. The RFI was calculated for each ligand as follows: mean fluorescence intensity (MFI) of the ligand/MFI of the unstained control. (A) NKRL RFIs calculated and plotted for all 66 patients. Each replicate is shown; black horizontal line corresponds to median values. (B) Representative plots of a patient expressing only inhibitory ligands (HLA-I and PD-L2). (C) Representative plots of a patient with high expression of both activating and inhibitory NKRLs. (D) Representative plots of a patient with a double blast population (CD33^posCD34^pos and CD33^posCD34^dim), where the expression of ULBP1 was strikingly different: the CD33^posCD34^pos blasts expressed low levels of ULBP1 (RFI = 5.5), whereas the activating ligand was highly expressed on CD33^posCD34^dim cells (RFI = 69.5). Blue histograms correspond to unstained control MFI; red histograms correspond to NKRL MFI. (E) An RFI score ranging from 0 to 2 was calculated for each ligand as follows: (1) samples with RFI <2 (ie, less than twofold increase of median fluorescence above background) were considered negative and assigned score 0; (2) Samples with an RFI value between 2 and the III quartile were assigned score 1; (3) Samples with RFI equal to or above the III quartile were assigned score 2. Panel E represents the distribution of patients according to the RFI score for each NKRL.