Sample Collection and Flow Cytometry

Heparinized peripheral blood samples were collected during study visits. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation (Biochrom GmbH, Germany) according to the manufacturer’s instructions as described previously [56], and were then cryopreserved in liquid nitrogen for later analysis. All samples of the same patient (different time points) were processed and analyzed simultaneously.

For the characterization of NK cell subpopulations, defrozen PBMCs were washed and incubated with human Fc fragments (Miltenyi Biotec, Germany) to block unspecific antibody binding, followed by the incubation with anti-CCR7-BV421, anti-NKG2C-AlexaFlour488, anti-DNAM-1/CD226-PerCP/Cy5.5, anti-CD158a/h-PE, anti-CD56-PE/Dazzle594, anti-NKG2A/CD159a-PE/Cy7, anti-CD94-APC, anti-Lin( CD3, CD19, CD14, CD20)-Alexa700, anti-CD16-APCfire750, anti-NKG2D-BV510, anti-CX₃CR1-BV605, anti-NKp46-BV650 and anti-CD127-BV785. Viable cells were identified with “LIVE/DEAD™ Fixable Dead Cell Stain Kits “ (Thermofisher). Data were acquired at Cytoflex LX flow cytometer (Beckman Coulter).