Anti cd107a lamp 1

Anti-ULBP1, anti-ULBP2 and anti-ULBP3 antibodies were purchased from R&D systems (catalog numbers: MAB1380, 1248 and 1517 respectively) and used both for flow cytometry and for ELISA assays. Anti-ULBP1 antibody, (catalog number Sc33564, Santa Cruz Biotechnology) was used for western blotting of ULBP1. The W6/32 mAb was used for MHC-I staining. Anti-NKG2D antibody was purchased from R&D Systems (MAB139). The anti-CD99 (12E7) was used as an isotype control. Anti-CD107a (LAMP-1) was purchased from BioLegend (catalog number 328620). Anti-CD56 (Becton Dickinson) and anti-CD3 (BioLegend) antibodies were used to determine NK purity. Anti-VP2/3 and agnoprotein antibodies were produced in house as well as the rabbit polyclonal antibodies against VP1. Anti-T-Ag antibody was purchased from Abcam (Pab416). The commercial recombinant ULBP-1 Fc chimeric protein (R&D systems, catalog number 1380-UL) was used for the generation of ULBP1 standard curve.
CD16-Ig, NKp30-Ig and NKp46-Ig and NKG2D-Ig fusion proteins were generated in the human embryonic kidney 293T cells and were purified on a protein G column as described [38 (link)]. The fusion proteins used in this work were regularly assayed by SDS-PAGE protein gels, to ensure that the proteins were not degraded. Protein purity of all Ig fusion proteins used in this study was approximately 100%.

The expanded NK cells were incubated with or without C4-2 cells at a 1 : 1 ratio for 20 hours at 37°C. NK cells were collected and treated with a GolgiStop protein transport inhibitor (BD Bioscience) for 2 hours, followed by a single wash with Cyto-Fast Perm/wash 1x solution (BioLegend). Next, the cells were resuspended and Cyto-Fast Fix/Perm solution (BioLegend) was added and incubated for 20 min in the dark at room temperature. After being washed once, cells were immunostained with anti-CD107a (LAMP-1) (BioLegend) for 10 min. After being washed twice, the cells were resuspended in 200 μl PBS and the expression of CD107a was analyzed by flow cytometry (Merck, easyCyte HT).

NK cell degranulation was evaluated based on CD107a expression in NK cells and the supernatant’s granzyme B and perforin-1 levels. NK cells were cocultured with or without BIU-87/T24/UMUC-3/5637/SV-HUC-1 cells (E/T = 10:1) for 1 h. The GolgiStop protein transport inhibitor (BD Biosciences) was added at the beginning of the coculture. After coculture, NK cells were collected, stained with anti-CD107a (LAMP-1) (BioLegend, H4A3) for 15 min, and analyzed using flow cytometry. Perforin-1 and granzyme B concentrations in the supernatants were measured using human ELISA kits for PRF1/PFP (CSB-E09313h) and granzyme B (CUSABIO, CSB-E08718h), respectively, following the manufacturer’s instructions.