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80 | Chapter 3 in combination with CET upon exposure to cervical cancer cell lines expressing low levels of EGFR (C33a, HeLa and SiHa: denoted in Figure 2c by triangles). In contrast, degranulation levels in UCB-NK were generally high. PBNK, PBNK + CET and UCB-NK cytotoxicity levels per histological subtype and HPV type of cervical cancer cell lines are shown in Supplementary Figure 3. It shows that irrespective of HPV or histological tumor type, highest cytotoxicity was consistently achieved by UCB-NK. Expression of NK activating receptors and their ligands and their contribution to mediating cytotoxicity To investigate the involvement of activating receptors in mediating the cytotoxic activity of PBNK and UCB-NK, the expression of the two major NK activating receptors DNAM-1 and NKG2D on the NK cells described to be involved in the recognition of cervical cancer cells, and their respective ligands, i.e. PVR and MICA/B, ULBPs, on the tested cervical cancer cell lines, were assessed. Similarly, high levels of DNAM-1 and NKG2D were observed on both PBNK and UCB-NK (Figure 3a). The cell lines showed differential expression of the NK activating ligands, but all were positive for PVR, the DNAM-1 ligand, and at least one of the NKG2D ligands (Figure 3b). From the panel of cell lines, SiHa (with highest expression levels of PVR and ULBP-2/5/6) and C33A (with lowest expression levels of PVR and ULBP- 2/5/6) were selected as target cells in functional blocking studies. The relatively low ligand expression levels on C33A required combined blocking of DNAM-1 and NKG2D to achieve a significant reduction in either PBNK- or UCB-NK-mediated cytotoxicity (Figure 3b). In contrast, blocking either DNAM-1 or NKG2D already led to significant reductions of cytotoxicity in SiHa cells (Figure 3c). These data show dependence (at least in part) of both PBNK and UCB-NK on DNAM-1 and NKG2D for their cytotoxic potency. Differential expression of NK inhibitory receptors and their ligands in relation to level of cytolysis To investigate the effect of NK inhibitory receptors on the observed cytotoxic efficacy, the expression levels of KIR2D and NKG2A on the NK cells, and of their respective ligands, i.e. HLA-ABC/-G and HLA-E 39 , on the cervical cancer cell lines, were assessed (Figure 4a, b). Irrespective of overnight activationwith IL-2/IL-15, PBNK expressed high levels of both KIR2D and NKG2A, whereas UCB-NK only expressed equivalent levels of NKG2A, but no KIR2D. All classical and non-classical HLA molecules were expressed on all ten cervical cancer cell lines, but in widely varying degrees (Figure 4b). Correlation analyses showed a relationship only between HLA-ABC expression levels and levels of cytotoxicity achieved by PBNK, with lower HLA-ABC levels allowing for higher levels of cytotoxicity (P = 0.036, Figure 4c). In contrast, PBNK + CET (Figure 4d) and UCB-NK cytotoxicity was totally independent of HLA- ABC expression levels (Figure 4e). No other correlations were found between cytotoxicity levels and HLA-E or HLA-G expression levels on cervical cancer cell lines (data not shown).