NK cytotoxicity assays were performed as previously described by Soland et al.16 (link) Briefly, MSC, nvMSC-HLA-G1 48 hours after transfection, and vMSC-HLA-G1 were plated in a flat-bottomed 96-well microplate (BD Falcon, Franklin Lakes, NJ) (1 × 105 cells/ml; 50 μl/well), and incubated in triplicate with different concentrations of NK-92 MI cells (ATCC, Rockville, MD) (20 × 105 cells/ml; 10 × 105 cells/ml; 5 × 105 cells/ml; 1 × 105 cells/ml; 2 × 104 cells/ml; 1 × 104 cells/ml; 50 μl/well) in α-MEM complete medium without phenol red (Gibco). NK-92MI cell line was used due to its robust proliferation rate in cell culture, and strong cytolytic activity towards several cells lines and primary cells without IL-2 supplementation. Furthermore, the NK-92 MI are also similar to activated NK cells with respect to their surface receptor expression.45 (link) The cytotoxicity tests were run at 20:1, 10:1, 5:1, 1:1, 0.2:1, and 0.1:1 E:T ratios following the guidelines of the CytoTox 96H Non-Radioactive Cytotoxicity Assay (Promega).