Figure 8. Depletion of ILKAP promotes antigen-specific CTL-mediated tumor cell killing.

(A) Cell survival of antigen loaded and untreated HCT 116 WT or ILKAP KO cells using 3 sgRNAs after 3 days of co-culturing with different ratios of PBMCs containing antigen specific CTLs. Bar graphs show normalized mean ± SD of triplicate representative of three independent experiments. (B) Cell survival of antigen loaded and untreated Panc-1 WT or ILKAP KO cells using 2 sgRNAs after 3 days of co-culturing with different ratios PBMCs containing antigen-specific CTLs. Bar graphs show normalized mean ± SD of triplicate representative of three independent experiments (C) ILKAP protein levels normalized to β-actin determined by Simple Western system. Bar graphs show normalized mean ± SD (n=3). (n.d. – not detectable). (D) Cell death of HCT 116 WT or ILKAP KO cells untreated or treated with 100 ng/mL IFN-γ or 40 ng/mL TNF-α determined with live/dead staining (FVS780) using flow cytometry. Bar graphs show mean ± s.e.m (n=3). (E) Fold change of HLA-A2, ICAM-1 and PD-L-1 cell surface levels after treatment with 100 ng/mL IFN-γ or 40 ng/mL TNF-α of WT or HCT 116 ILKAP KO cells. Bar graphs show mean ± s.e.m (n=3). (F) Mean fluorescence intensities (MFI) of HLA-A2, ICAM-1 and PD-L-1 of HCT 116 WT or HCT 116 ILKAP KO cells. Bar graphs show mean ± s.e.m (n=3). (G) ILKAP protein levels of Panc-1 cells transiently transfected with ILKAP or ILKAP H154D assessed by western blot. (H) Level of mICAM-1 measured with flow cytometry and (I) secreted sICAM-1 levels determined by ELISA of transiently transfected Panc-1 cells. For (A) and (B), two-way ANOVA corrected for multiple comparison according to Dunnett was used to determine statistical significance (*p<0.05, **p<0.01, ***p<0.001****, p<0.0001). Two-tailed t tests with adjustments for multiple comparisons were performed (D and E). For (C and F) unpaired two-tailed t test was used to determine statistical significance (*p<0.05, **p<0.01).