Fitc mouse anti human cd3

We performed cell surface staining to characterize the cell populations in each sample. Briefly, cells were incubated with specific fluorescence-labeled monoclonal antibodies at 4 °C for 30 min. CD14 and CD16 were used to identify the monocyte subpopulation. CD3, CD56, BDCA2, and CD123 were used for T cells, NK cells, NKT cells, and plasmacytoid dendritic cells. Events were acquired with a FACS Canto II (Becton Dickinson, USA) and analyzed with FlowJo software v.10.3 (Tree Star Inc., USA) (https://www.flowjo.com/solutions/flowjo). The following antibodies were used for cell surface staining: PE-Cy7 Mouse Anti-Human CD14 (BD Pharmingen), APC-Cy7 Mouse Anti-Human CD16 (BD Pharmingen), FITC Mouse Anti-Human CD3 (BD Pharmingen), PE-Cy7 Mouse Anti-Human CD56 (BD Pharmingen), APC anti-human CD303 (BDCA-2) Antibody (BioLegend), and PerCP/Cyanine5.5 anti-human CD123 Antibody (BioLegend).

Cell sorting was performed to separate CD3+CD4+ and CD3+CD8+ lymphocyte populations. The absolute numbers of the PBMCs were determined using DXH 500 (Beckman Coulter, CA, USA) equipment in order to adjust the volume of each antibody. FITC mouse anti-human CD3, PE mouse anti-human CD4, and PerCP-Cy 5.5 mouse anti-human CD8 antibodies (BD Pharmingen, CA, USA) were used according to the manufacturer’s instructions and incubated for 15 min at room temperature, in the dark. Cells were further washed in PBS, centrifuged at 450× g for 5 min, and pelleted cells resuspended in PBS until sorting using a FACSAria III^TM from Becton Dickinson, CA, USA. About 10,000 events were acquired to define the gating strategy using morphologic and fluorescence parameters. Using a dot-plot from FSC (forward light scatter) vs. SSC (Side scatter), lymphocytes were gated and, after excluding doublet, lymphocytes subsets were identified by gating CD3+CD4+ and CD3+CD8+, and non-CD3+. Monocytes were initially selected by morphologic parameters, doublets were excluded, and monocytes were identified by CD4 positivity since we did not use a specific monocyte marker. No major differences were observed in the percentage of lymphocyte subpopulations and monocytes obtained from the different groups (Figure S1).

Cells were stained with membrane antibodies diluted in PBS containing 2% FBS and fixed. For in vitro experiments, cell viability was evaluated by the LIVE/DEAD® Fixable Near-IR Dead Stain Kit (Invitrogen). Membrane antibodies FITC mouse anti-human CD3, PE mouse anti-human iNKT cell (clone 6B11) and PerCP/Cy5.5 mouse anti-human CD69 were provided by BD Biosciences (San Jose, CA). Appropriate isotype control was used for each marker. Cells were analysed by FACS Canto II™ and FacsDiva™ software (BD, Franklin Lakes, NJ) and data were analysed using FlowJo™ software (version 7.5.5 Treestar, Ashland, OR). CD69 expression was analysed gating on CD3(+)6B11(+) cells, defined as iNKT cells.