Figure 5.

Treatment with 5-aza-2'-deoxycytidine or TNF-α upregulates ICAM-1 expression and improves trastuzumab-mediated NK cell cytotoxicity. (A, D) HEK293 cells were treated with 1 µM 5-aza-2'-deoxycytidine (5AZA) for 72 hours (A) or with 100 ng/mL TNF-α for 48 hours (D). Expression of ICAM-1 and ErbB2 was analyzed by flow cytometry. Representative histograms are shown. (B, E) haNK cells combined with trastuzumab, or CAR-engineered NK-92/5.28.z cells were incubated for 2 hours with HEK293 cells pretreated with 5AZA (B) or TNF-α (E). Specific cytotoxicity was measured using a Europium-based cytotoxicity assay. (C, F) Specific lysis is depicted as fold-change in comparison to cytotoxicity of the NK cells against HEK293 control cells without 5AZA or TNF-α pretreatment. Data were pooled from three independent experiments. Mean values±SEM are shown. CAR, chimeric antigen receptors; haNK, high-affinity FcγRIIIa-modified NK-92 cells; NK, natural killer; TNF-α, tumor necrosis factor alpha.