Ch l pe

Healthy donor PBMCs were stimulated overnight with IL-15 (1ng/ml) and then co-incubated with target cells at 5:1 effector:target ratio for 4 hours. Golgistop (BD Biosciences) was added 1 hour after co-incubation and cells were then stained for surface markers REA147-FITC (Miltenyi Biotech), CH-L-PE (BD Biosciences), CD56-PE/Cy7 (Biolegend) and CD3-PerCP (Biolegend). Cells were then fixed and permeabilized, stained for IFNγ-BV421 (Biolegend) and TNFα-AF700 (Biolegend) and analysed by flow cytometry on a BD FACS Aria. Data was analysed by FlowJo_V10 software.

Healthy PBMC or isolated NK cells (average purity of 96% NK cells achieved using the Miltenyi Biotec NK isolation kit) were blocked with 10% AB serum for 20 minutes at 4°C then washed and stained with antibodies for 30 minutes at 4°C in FACs buffer (PBS, BSA 1%, Sodium Azide 0.05%). Surface antibodies used were REA147-FITC (Miltenyi Biotech), CH-L-PE (BD Biosciences), CD56-PE/Cy7 (Biolegend) CD3-PerCP (Biolegend) and CD16-APC (Biolegend). Cells were then washed in FACs buffer and analysed by flow cytometry using a BD FACs Aria. Analysis was performed using FlowJo_V10 software. For HLA-C staining, B-cell lines were incubated with DT9 antibody for 30 minutes in FACs buffer then washed in FACs buffer and stained with anti-mouse secondary antibody-AF647. Cells were then analysed by flow cytometry using a BD Accuri and data analysed by FlowJo_V10.

Healthy donor PBMCs or isolated NK cells were stimulated overnight with IL-15 (1ng/ml; R&D Systems) and co-incubated with target cells at 10:1, 5:1 or 1:1 effector:target ratios as indicated for 4 hours with anti-CD107a-AF647. For assays using primary CLL cells as targets, CLL cells were isolated using a B-CLL isolation kit (Miltenyi Biotech) before co-incubation with healthy donor PBMC. For assays in combination with anti-CD20 antibodies, target cells were incubated with indicated antibody (1μg/ml; in-house) for 20 minutes at 4°C prior to addition of PBMC or purified NK cells (Miltenyi Biotech NK isolation kit) at a 10:1 effector:target ratio. Excess anti-CD20 antibody in the medium was removed by two washes with RPMI media prior to addition of PBMC. Golgistop (BD Biosciences) was added 1 hour after co-incubation and cells were then stained with REA147-FITC (Miltenyi Biotech), CH-L-PE (BD Biosciences), CD56-PE/Cy7 (Biolegend) CD3-PerCP (Biolegend) and analysed by flow cytometry on a BD FACS Aria. Data was analysed by FlowJo_V10 software.