Cd16 ecd

Peripheral blood mononuclear cells (PBMC) from CLL patients and HC were isolated and cryopreserved as described earlier.^{25 (link)} To enrich for NK cells, samples from CLL patients were CD19-depleted using CD19 immunomagnetic microbeads (Miltenyi Biotec, Bergish Gladbach, Germany). Afterwards, PBMC were washed with ice-cold phosphate-buffered saline containing 0.5% bovine serum albumin (PBA) and stained for 30 minutes at 4°C with saturating amounts of CD56 APC-Alexa Fluor 750, CD16 ECD, CD158 (a,h) APC, CD158 (e1,e2) APC, CD158 (b1, b2, j) APC, p75 PE, NKG2A PE-Cy7, BTLA PC7, CD160 PE, ILT2 APC (Beckman Coulter, Brea, CA, USA), KLRG1 Alexa Fluor 488, NKG2D Pe-Cy7 (ThermoFisher Scientific), CD3 FITC, NKp30 APC, CD57 PerCP-Cy5.5 (Biolegend, San Diego, CA, USA), CD3 PE (BD Biosciences, Franklin Lakes, NJ, USA) and NKG2C Alexa Fluor 700 (R&D systems, Minneapolis, MN, USA). Cells were washed twice with PBA, resuspended, and analyzed on a BD FACSCanto flow cytometer. Data analysis was performed using Flowjo Mac Version 10. Gating strategy can be found in supplemental Figure 4 (Supplemental Digital Content).

NK3.3-LTV cells were analyzed for changes in cell surface marker expression compared to non-immortalized NK3.3 cells. All cells (1 x 10⁵ cells per antibody) were centrifuged at 300 g x 5 min, media aspirated, and 1.5 µL single antibody + 20 µL staining buffer (phosphate buffered saline (PBS) + 1% bovine serum albumin + 1% sodium azide) were added to cells. Sample tubes were incubated for 30 min on ice, followed by addition of 200 µL staining buffer and incubated for another 10 min on ice. Samples were analyzed using LSRFortessa Cell Analyzer (BD Biosciences, Franklin Lakes, NJ). Data were analyzed using FlowJo software. Antibodies used include: NKp46 PE/Cy7 (#331915, BioLegend, San Diego, CA); NKp30 PE (#325207, BioLegend); NKp44 PE (#IM3710, Beckman Coulter, Brea, CA); CD94 PE (#130-098-974, Miltenyi Biotec, Auburn, CA); NKG2D APC (#120-003-706, Miltenyi Biotec); CD161 APC-Alexa Fluor750 (#B30630, Beckman Coulter); CD158e1 BV421 (#312713, BioLegend); CD158a,h PE/Cy7 (#A66899, Beckman Coulter); CD244 PerCp Cy5.5 (#B21171, Beckman Coulter); CD16 ECD (#A33098, Beckman Coulter); CD133 APC (#130-098-829, Miltenyi Biotec); CD57 BV421 (#563896, BD Biosciences); CD158 FITC (#339503, BioLegend).