The DNAM-1 ligand, CD155/ PVR is required for DOT-cell targeting of AML cell lines. (A) Gene expression of ligands for DNAM-1, NKp30, NKp44, and NKG2D receptors in a panel of AML cell lines; results are normalized to the housekeeping genes GUSB and PSM6. (B) Protein expression of ligands for DNAM-1, NKp30, NKp44, and NKG2D receptors in a panel of AML cell lines; gray histograms represent isotype; black lines represent sample. (C) Summary of flow cytometry data depicted in panel B. Data represented as mean fluorescence intensity (MFI) increase relative to isotype control. (D) Representative flow cytometry plots of killing assay performed in the absence (basal) or presence of DOT cells that were previously incubated with blocking antibodies for the proteins indicated. (E) Summary of killing assays performed against HEL (left) and MOLM13 (right) cell lines. Results are normalized to the percentage of tumor cells targeted in the condition in which DOT cells are incubated with isotype control antibodies. Each dot represents a different DOT-cell donor. Data were generated in ≥3 independent experiments. (F) Flow cytometry plots depicting the phenotype of MOLM13 cell lines modified via CRISPR–CRISPR-associated protein 9 editing. (G) Summary of killing assays performed against the indicated KO cell lines, compared with the mock-transfected cell line. Each dot represents a different DOT-cell donor. Data were generated in >3 independent experiments. (H) Schematic representation of the experimental design of competitive killing assays. (I) Summary of killing assays performed against the indicated KO HEL cell lines in a competitive setting, compared with the mock-transfected cell line. Each dot represents a different DOT-cell donor. Data were generated in 3 independent experiments. Statistical analysis was performed using 1-sample t test (hypothetical value: 100). WT, wild type.