The major population of mature monocytic JMML cells are similar to HD peripheral blood monocytes in NK cell ligand expression and are relatively resistant to NK cell lysis. (A) NK cells were cocultured with target cells at different E:T ratios (10:1, 5:1, 2.5:1, and 1.25:1). JMML cells (n = 2 patient samples) were used as targets in a standard cytotoxicity assay. K562 cells were used as a positive control for NK cell cytotoxicity. (B) PBMCs of 5 HDs and 5 patients with JMML were stained with CD14-FITC, CD33-APC, and PE-labeled anti-MIC A/B, CD112, CD155, HLA-ABC, or HLA-E Abs (all m-a-h IgG1), or isotype control, washed and analyzed immediately by flow cytometry. Data were acquired using a fluorescence-activated cell sorting (FACSCalibur) cytometer. Data were preanalyzed using FlowJo to gate live cells based on side and forward scatter and 7-aminoactinomycin D exclusion. Gated events were exported into a new FCS3 file, totaling 60 files. FCS3 files were uploaded into SPADE and analyzed collectively. A minimum spanning tree was constructed based on forward and side scatter and expression of CD33 and CD14. CD14⁺CD33⁺ nodes were identified as monocytes. (C) MIC A/B, Nectin2, PVR, HLA-ABC, and HLA-E expression levels on CD14⁺CD33⁺ monocytes were analyzed from HDs and patients with JMML. AU, arbitrary units; HD, healthy donor; MIC, Major histocompatibility complex class I-related chain A and B; PVR, poliovirus receptor.