Cells and flow cytometry
Viable PBMCs were isolated and cryopreserved according to Center for Human Immunology (CHI) protocols (Staining was performed using dedicated lyophilized antibody plates for each of compensation, fluorescence-minus-one controls, and study sample staining (all BD Biosciences). Sample staining plates included up to 10 study samples in addition to control PBMCs from a healthy donor, and staining was preceded by an additional incubation with LIVE/DEAD Fixable Blue Dead Cell Stain (Thermo Fisher Scientific). Acquisition was performed with a Becton Dickinson LSRFortessa, using DIVA 8 software, acquiring 250,000 cells for each sample. Subsequent analysis to determine population frequencies used FlowJo version 9.6.2. Compensation performed with unstained cells and Becton Dickinson compensation beads was used to aid acquisition monitoring. Subsequently a final compensation matrix was calculated using FlowJo during postacquisition analysis.