Nkg2c alexa fluor 700

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).

Antibodies for cell surface staining were CD3-APC, CD56-FITC, CD16-PercP, CD8-PercP (CD8α, clone SK1), KIR2DL5 (CD158f)-BV421, and NKG2D-PECy7 (BD Biosciences, San Jose, CA, USA). Antibody TCRγδ-PE was obtained from BioLegend (San Diego, CA, USA) and antibodies NKG2A-PE and NKG2C-AlexaFluor700 were obtained from R&D Systems (Minneapolis, MN, USA). CD8+ T cells were analyzed within the T lymphocyte gate. Both CD8+ and CD8-T cells were considered for the analysis of TCRγδ expression. CD56+ cells were analyzed within CD3 ± gate to differentiate between NK and NKT cells.
For intracellular staining of interferon gamma (IFNγ), tumor necrosis factor alfa (TNFα) and granzyme B (GZB) from NK cells, PBMCs were treated for 4h at 37 °C with Hsp70 peptide 1 μgr/mL (Abcam, Cambridge, UK) and brefeldin-A (BD Biosciences, San Jose, CA, USA). Cells were then stained with antibodies against CD3, CD56 and CD16. After fixation and permeabilization with IntraPrep Reagent (Beckman Coulter, Indianapolis, IN, USA), cells were stained with antibodies against IFNγ-PE (Beckman Coulter, Indianapolis, IN, USA), TNFα-PE (Beckman Coulter, Indianapolis, IN, USA) or GZB-PE (BD Biosciences).
Data acquisition was performed in a BD LSRFortessa X-20 flow cytometer using FACS Diva software (BD Biosciences). Data analysis was performed with FlowJo_V10 software (TreeStar, Ashland, OR, USA).

Conjugated antibodies for surface staining CD3-APC, CD4-PercP, CD8-APC-H7, CD16-PercP, CD56-FITC, CD107a-PE-Cy7, NKp44-BUV395, and NKp46-BUV650 were purchased from BD Biosciences (San Jose, CA), whereas PD1-BUV650, NKG2A-PE, and NKG2C-Alexa Fluor700 were purchased from R&D Systems (Minneapolis, MN). Tregs cells were characterized by staining with CD4-PercP, CD25-PE-Cy5 and CD127-FITC (R&D Systems). CD4+ and CD8+ T cell subpopulations were determined by staining with CCR7-FITC and CD45RA-PE-Cy7 (Biosciences) as follows: naïve (CD45RA+CCR7+), central memory (TCM) (CD45RA-CCR7+), effector memory (TEM) (CD45RA-CCR7-) and terminally differentiated effector memory (TEMRA) (CD45RA+CCR7-) cells.
For intracellular staining of granzyme B (GZB) from NK and NKT cells, PBMCs were treated for 4 h at 37°C with Hsp70 peptide 1 µgr/ml (Abcam, Cambridge, UK) to stimulate cytolytic activity of NK cells, in the presence of brefeldin A (BD Biosciences). Cells were then stained with antibodies against CD3, CD56 and CD16 conjugated to APC, FITC and PercP, respectively. After fixation and permeabilization with IntraPrep Permeabilization Reagent (Beckman Coulter), cells were stained with an antibody against GZB-PE (BD Biosciences) and then acquired and analyzed in a BD LSRFortessa X-20 flow cytometer (BD Biosciences) using FACS Diva (BD Biosciences) and FlowJo_V10 software (TreeStar).