Figure 4.

Degranulation of patient NK cells and cytolysis mediated by ch14.18/CHO. Patient PBMCs acquired post haploidentical HSCT were used to test the capacity of degranulation, measuring CD107a (LAMP-1) expression as a strong indicator of NK cell activation and cytolysis targeting the neuroblastoma cell lines LAN-1 and LS in a 6 h flow cytometry based kill assay (A–D). Besides testing the overall degranulation capacity, NK cell subsets distinguished by the major inhibitory killer cell immunoglobulin-like receptors (KIR) CD158a/KIR2DL1, CD159b/KIR2DL2 and CD158e/KIR3DL1 were analyzed for interpopulation differences in CD107a positivity and were evaluated in the context of the KIR receptor ligand model. Additionally, a 2 h-DELFIA-EuTDA release cytotoxicity assay was used to evaluate the cytolysis of tumor cells by patient PBMCs via ADCC and CDC (E, F). The testing conditions (E) comprised I) PBMCs versus tumor, II) PBMCs versus tumor and patient serum (after infusion of ch14.18/CHO), and III) PBMCs versus tumor and 1 µg/ml ch14.18/CHO. Moreover, cytolysis of the neuroblastoma cell line LAN-1 by PBMCs from healthy volunteer donors (HVDs) compared to patient PBMCs was assessed. (F) The comparison included the conditions I) [HVD PBMCs plus HVD serum] versus tumor, II) [HVD PBMCs plus HVD serum plus 1 µg/ml ch14.18/CHO] versus tumor, III) [patient PBMCs plus heat inactivated patient serum] versus tumor, and IV) [patient PBMCs plus patient serum] versus tumor. Data shown in (A–D) represent mean of (n = 12) independent experiments and different donors in triplicates, respectively. Data shown in (E) represent mean of (n_LAN-1 = 15; n_LS = 13) and (F) represent single values of (n_HVD = 5; n_patients = 10) independent experiments and different donors in triplicates, respectively. Statistical significance was determined by one-way ANOVA and Tukey post-hoc test. P-values below 0.05 were defined significant. * = <0.05, ** = <0.01, *** = <0.0001, **** = <0.0001. ns, not significant.