Rhil 18

NK cell degranulation was measured as described previously 26 (link). Briefly, PBMCs were incubated with K562 cells [5:1 effector : target (E : T) ratio] for 3 h at 37°C following overnight stimulation with a combination of 50 ng/ml recombinant human (rh)IL-12 and rhIL-18 (Miltenyi Biotec, R&D Systems). CD107a-PE antibody (BD Biosciences) was added at the time of stimulation with target cells along with 1 mM monensin prior to staining and acquisition.

To assess proliferation and further characterisation of the differentiation of NK cells, PBMC were permeabilised and stained with anti-Ki67-PE (eBioscience, Hatfield, UK), Granzyme-B-FITC and Perforin-Cy5.5/PerCP (Biolegend, London, UK) directly ex-vivo. For intracellular staining for IFNγ production; PBMC were incubated with rhIL12 and rhIL15 (10ng/ml) (R&D systems, Abingdon, UK), for 19 hours at 37°C. 1mM monensin (Sigma-Aldrich, Gillingham, UK) was added for the final 3 hours. Cells were then stained with anti-CD3-Cy5.5/PerCP or CD3/Pe-Cy7, CD16-APCy7, CD56-FITC, and subsequently fixed and permeabilised, followed by intracellular staining for IFNγ-v450 (BD Biosciences, Oxford, UK). Dead cells were excluded by fixable live dead stain.
For degranulation, PBMCs were incubated with K562 cells (5:1 E:T ratio) for 3 hours following overnight stimulation with a combination of 50ng/ml rhIL12 and rhIL18 (Miltenyi Biotech). Anti-CD107a-PE mAb (BD Biociences, Oxford, UK) was added at the time of stimulation with target-cells and monensin (1mM) added during the last 2 hours of incubation prior to staining and acquisition.