Cd57 percp cy5

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).

We isolated peripheral blood mononuclear cells (PBMC) from peripheral blood by Ficoll gradient and we stored them in liquid nitrogen for batched analysis. For multicolor flow cytometry analyses, we used the following monoclonal antibodies: from Becton Dickinson (BD, Franklin Lakes, NJ), CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CD71-PE, IgD-PerCP-Cy5.5, CD25-APC-Cy7, CD138-BV421, CCR4-PE, CD27-PE, CD95-BV421, CD28-BV421, TIM3-BV421, CCR6-BV421, CCR7-AF700, CXCR3-PE, IL-2-PE, IL-17-BV786, and IFN-γ-PE-Cy7; from Biolegend (San Diego, CA), CD19-BV510, CD56-FITC, CD27-APC, CD127-FITC, CD57-PerCp-Cy5.5, PD-1-APC-Cy7, CXCR5-FITC, LAG3-APC, IL-10-PerCP-Cy5.5, and TNF-α-FITC. From eBioscience (San Diego, CA), CD4-PE-Cy7, CD21-PE, and CD24-APC-Cy7 were used. From Miltenyi Biotec (Auburn, CA), CD25-APC and anti-KLRG1-PE were used. From Beckman Coulter (Indianapolis, IN), CD38-PE-Cy7 was used.
We performed intracellular staining for IL-2, IL-4, IL-17, IFN-g, and TNF-a together with extracellular markers for CD4⁺, CD8⁺, and CD19⁺. Cells were fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions.
Data were acquired (> 1 × 10⁶ events) on a 3-laser FACSLyric flow cytometer (BD Biosciences) and analyzed with FlowJo^® software.