NK Cell Immunophenotyping and IFN-γ Analysis

REAfinity^TM Recombinant Antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany) were used for NK cell monitoring. Anti-CD56-FITC, CD3-PE-Vio770, CD45-VioBlue, CD16-PE were used to identify CD56dim and CD56 bright NK cells. The four stages were discriminated by using CD11b-APC-Vio770 and CD27-APC. ABCC3 expression was assessed before and after each vaccination as previously described [17 (link)], by using a primary antibody anti-ABCC3 (Thermo Fisher Scientific, Waltham, MA, USA) and a secondary anti-rabbit Alexa Fluor488 antibody (Abcam) according to manufacturer’s instructions. PBLs were then fixed and permeabilized using the Cytofix/Cytoperm solution (BD Biosciences, Franlin Lakes, NJ, USA) and intracellularly stained with an anti-IFN-γ (Miltenyi Biotec) antibody. NK cells were gated and then analyzed by flow cytometry for IFN-γ assessment. Acquisition of stained samples was performed using a MACSQuant (Miltenyi Biotec) flow cytometer, and data were analyzed using Flowlogic software (version 7.2, Miltenyi Biotec).