Analysis of the immunophenotype
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood before drug administration (Day 0) and in different time‐points during treatment (Day 90 and Day 180). PBMCs were obtained by density centrifugation on Lympholyte (Cedarlane, Burlington, Canada) following manufacturer instruction, washed in PBS and frozen in standard FBS/10% DMSO freezing medium. For analysis, cells were thawed in RPMI‐1640 medium and washed in PBS (Lonza); the staining was performed using 1 million cells for each time point. The cells were firstly stained with LIVE/Dead™ Fixable Aqua—Dead Cell Stain Kit (Thermo Fisher) and incubated 10′ in the dark. After a PBS wash, cells were stained with a mixture of antibodies (see Table List of antibodies employed for Lymphocyte immunophenotyping