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86 | Chapter 3 with 20 ng/mL IL-15 (CellGenix). During the first 14 days of culture, low molecular weight heparin (LMWH) (Clivarin ® ; Abbott, Wiesbaden, Germany) in a final concentration of 20 µg/ ml and a low-dose cytokine cocktail consisting of 10 pg/ml GM-CSF (Neupogen), 250 pg/ ml G-CSF and 50 pg/ml IL-6 (CellGenix) were added to the expansion cultures. Cells were refreshed with new medium twice a week and maintained at 37°C, 5% CO 2 . On day 14, the NK cell differentiation process was initiated by addition of NK cell differentiation medium consisting of the same basal medium with 2% human serum but with high-dose cytokine cocktail consisting of 20 ng/ml of IL-7, SCF, IL-15 (CellGenix) and 1000 U/ml IL-2 (Proleukin ® ; Chiron, München, Germany). Cultures were refreshed every 2-3 days and maintained till day 35. For cytotoxicity assays, UCB-NK was used with CD56 + cells >85% purity. In vitro NK cytotoxicity assays Cervical cancer cell lines (target cells) were labelled with 5 µM pacific blue succimidyl ester (PBSE; Molecular Probes Europe, Leiden, The Netherlands) in a concentration of 1x10 7 cells/mL for 15 min at 37°C. After incubation, cells were washed and resuspended in DMEM culture medium to a final concentration of 1x10 6 /mL. PBNK and UCB-NK were washed with PBS and also resuspended in GBGM medium to a final concentration of 1x10 6 /mL. Target cells were co-cultured in triplicate with effector cells (PBNK or UCB-NK), with or without 5 µg/ml CET at an E:T ratio of 1:1 in a total volume of 100 µl in FACs tubes (5 x 10 4 targets in 50 µl of DMEM culture medium incubated with 5 x 10 4 effectors in 50 µl of GBGMmedium). PBNK, UCB-NK and target cells alone were cultured in triplicate as controls. To measure degranulation by PBNK and UCB-NK, anti-CD107a PE (Miltenyi Biotech, Germany) was added at the beginning of the assay. After incubation for 4h at 37°C, cells were harvested and stained with 7AAD, CD56 (labeled with APC-Vio770) and CD16 (labeled with APC) (all from Miltenyi Biotech, Germany). For NK flow cytometry and blocking experiments NKG2D PE (clone ON72, Beckman Coulter) and DNAM-1 FITC (clone DX11, BD Pharmingen™) were used at 10 µg/ml. Further, Killer-cell immunoglobulin-like receptor 2D (PanKIR2D) FITC (clone NKVFS1) and CD94/NKG2A PE-Vio770 (Clone REA110) (both from Miltenyi Biotec) were used to screen inhibitory receptor expression on PBNK and UCB-NK. BD LSR Fortessa™ was used for read-out of the cytotoxicity assays. Data were analyzed using Kaluza software (Beckman coulter). Percentages of specific NK degranulation were calculated as ∆CD107a + NK cells (i.e. [target cells + NK cells] minus [NK cells only]) and percentages of cytotoxicity as ∆7AAD + target cells (i.e. [target cells + NK cells] minus [target cells only]). See Supplementary Figure 1 for a representative gating example. Statistical analysis Statistical analysis was performed using Graph Pad Prism software. Statistical significance of differences between conditions were determined using a parametric paired t test, unpaired