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132 | Chapter 5 and 1000 U/ml IL-2 (Proleukin ® ; Chiron, München, Germany). Cultures were refreshed every 2-3 days and maintained till day 42. Five UCB-NK cultures were used for cytotoxicity assays and one UCB-NK culture for in vivo studies (both with a CD56 + cell purity of >95%). UCB- NK CD16 levels in matured UCB-NK cells were monitored using an antibody mix of human CD45VioGreen (1:11), CD56 APC-Vio770 (1:11) and CD16 APC (1:11). Similarly, UCB-NK CD16 expression in BRGS mice was monitored using an antibody mix of BV650 anti-mouse CD45 (clone 30-F11), Alexa Fluor® 700 anti-human CD45 (clone HI30), PE-CF594 anti-human CD56 (clone B159), all from BD, and APC-Vio770 anti-human CD56 (clone REA196) and APC CD16 (clone REA423) both from Miltenyi Biotec. NK cell cytotoxicity assays Flow cytometry was used for the read-out of cytotoxicity assays. Target cells (COLO320, SW480 and HT-29 were labelled with 5µM pacific blue succinimidyl ester (PBSE; Molecular Probes Europe, Leiden, The Netherlands) at a concentration of 1x10 7 cells per ml for 10 min at 37°C. The reaction was terminated by adding an equal volume of FCS, followed by incubation at room temperature for 5 min after which stained cells were washed twice and suspended in DMEM + 10% FCS to a final concentration of 5 x 10 5 /ml. Overnight activated PBNK cells and UCB-NK cells were washed with PBS and suspended in Glycostem Basal Growth Medium (GBGM) + 2% FCS to a final concentration of 5 x 10 5 /ml. Target cells were co-cultured with effector cells at an E:T ratio of 1:1 in a total volume of 250 µl in 96-well flat-bottom plates (5 x 10 4 targets in 100 µl of DMEM + 10% FCS incubated with 5 x 10 4 effectors in 100 µl of GBGM + 2% FCS, further supplemented with 25 µl of GBGM + 2% FCS and DMEM + 10% FCS medium). NK cells and target cells alone were plated out in triplicate as negative controls. Target cells were coated with 5µg/ml cetuximab (Merck, Darmstadt, Germany) for 1h at 4°C. To measure degranulation of NK cells, anti-CD107a PE (Miltenyi Biotech) was added in 1:20 dilution at the beginning of the assay. After incubation for 4hr at 37°C, cells were harvested and stained with CD56 APC Vio 770 (1:25) and CD16 APC (1:25) (Miltenyi Biotech) and 7AAD (1:500) (Sigma Aldrich). Degranulation of NK cells was measured by detecting cell surface expression of CD107a. In vivo studies The EGFR + RAS mut SW480 cell line and EGFR +++ RAS wt A431 cell line were stably transduced with Gaussia Luciferase (Gluc) for in vivo studies. Lentiviral (LV) supernatant of Cerulean Fluorescent Protein (CFP) positive Gluc virus (LV-CFP-Gluc) was kindly provided by Dr. Tom Würdinger 26 . SW480 and A431 cells with Gluc expression of 95% were used for mouse studies. Immunodeficient BRGS mice (BALB/c Rag2 tm1Fwa Il2rg tm1Cgn Sirpa NOD ) were used in this study. Twenty-four adult mice (male, 8 weeks old) received an intravenous (i.v) tail vein injection