Flow Cytometry
One sample of peripheral blood was obtained from each patient according to standard procedures. EDTA-treated blood samples were analyzed by 8-color flow-cytometry (Becton Dickinson Canto II Cytometer) using several combinations of fluorescence-labeled antibodies distributed in the following panels: Panel 1: CD3 FITC, CD4 APC/Cy7,  CD25 BV421, CD127 APC and FOXP3 PE; Panel 2: CD3  PerCP/Cy5, CD4 APC/Cy7, CD8 Alexa Fluor 488, HLA-DR PE, CD244 (2B4) APC,  CD279 (PD-1) BV421, CD38 BV510 and PE/Cy7 CD366 (Tim-3); Panel 3: CD3 PE/Cy7, CD4 PE/Cy5, CD8 BV510, CD27 APC/Cy7, CD57 APC, CD45RO FITC, T-bet BV421 and EOMES PE; Panel 4: CD3 APC, CD4 BV510, CD8 PE, CD45RA FITC, CD197 APC/Cy7. All antibodies were purchased from Biolegend Inc. (San Diego, CA) except EOMES PE (eBioscience). Briefly, 250 µL of blood was incubated with fluorochrome-conjugated antibodies for 30 min at room temperature prior to lysis with RBC Lysis Buffer (Biolegend Inc.) and fixation with 3% formaldehyde in PBS.
OneFlow ™ Setup Beads (BD Biosciences) were used to adjust instrument settings, set fluorescence compensation, and check instrument sensitivity. ‘Fluorescence minus one’ controls were used to determine positive and negative staining boundaries for each fluorochrome. Two hundred and fifty thousand events were recorded for each sample and analyzed with the FlowJo® software v.10.7.1 (FlowJo, LLC, Oregon, USA). FlowAI plugin was used to perform an automatic or interactive quality control on the .fcs data [10]. Lymphocyte population was identified in an SSC-A versus FSC-A plot, followed by doublets exclusion in a FSC-Height (FSC-H) by FSC-Area (FSC-A) scatter plot. Phenotypes used to define mayor and minor T cell populations are depicted in Table 3.