Isolation of HLA-A^negHEK293. (A) Loss of HLA-A2 and HLA-A3 protein expression. Flow cytometry analysis of HLA-A2 and HLA-A3 expression on parental HEK293 and 3 derived genetically modified clones with loss of HLA-A (numbered 18.1, 8.18, and 83). Dotted lines represent isotype (HLA-A2) or SA-PE (HLA-A3) controls; solid line represents HLA-A expression without IFN-γ and TNF-α, and filled lines represent HLA-A expression after culturing with 600 IU/mL IFN-γ and 10 ng/mL TNF-α for 48 hours. Dashed lines in the parental column represent HLA-A2 or HLA-A3 expression on EBV-LCL. (B) Resistance to CTL-mediated lysis. Parental HEK293 and derived HLA-A^neg clones were cultured with IFN-γ and TNF-α for 48 hours and pulsed with serial dilutions of the cognate HLA-A3 peptide RVWDLPGVLK (derived from PANE1 and recognized by CTL clone 7A7)²⁷  or the HLA-A2 peptide CIPPDSLLFPA (derived from C19ORF48/A2 and recognized by CTL clone GAS2B3-5)²⁸  and evaluated for recognition by CTL clones in a 4-hour ⁵¹Cr release assay at an effector-to-target ratio of 20:1.HLA-A2⁺ LCL (hatched bar) that expresses PANE1mHAg (not peptide-loaded) were used as a positive control.