Facsfortessa instrument

Transfected or infected vFcγR expressing cells were harvested using Accutase (Sigma-Aldrich) to retain surface molecules upon detachment. Harvested cells were washed in PBS, equilibrated in staining buffer (PBS, 3% FCS) and sedimented at 1000 g and 10°C for 3 min. Cells were then incubated with staining buffer containing rituximab, Cytotect, or herceptin. Cells were then incubated in an adequate volume of staining buffer containing either mAbs, Fcγ fragment, or FcγR ectodomains pre-incubated with an αHis-PE antibody (30 min, 4°C). Human His-tagged FcγR ectodomains were used at a final concentration of 5 µg/ml (1:50 dilution from reconstituted 0.25 mg/ml stock solution; Sino Biological, 10389-H08H1). Pre-incubation with Protein G (Rockland, Biotin conjugated) was performed prior to incubation with FcγR ectodomains (diluted 1:100). Further incubation steps were carried out at 4°C for 1 hr and followed by three washing steps in staining buffer. Dead cells were excluded via DAPI stain. Analysis was performed on a FACS Fortessa instrument (BD Bioscience).

Peripheral blood mononuclear cells (PBMCs) were separated from fresh blood samples by Ficoll-Hypaque gradients (Sigma-Aldrich, St. Louis, MO, USA) and processed within 4 h. The reconstitution of NK cells after HSCT was determined by eight-color multiparameter flow cytometry using the following mAbs: anti-CD45-FITC (clone; HI30, BD bioscience, San Diego, CA, USA), anti-CD3-V450 (clone; UCHT1, BD bioscience, San Diego, CA, USA), anti-CD16-V500 (clone 3G8; BD bioscience, San Diego, CA, USA), anti-CD56-PE-Cy7 (clone; B159, BD bioscience, San Diego, CA, USA), anti-NKGD-APC (clone; 1D11, BD bioscience, San Diego, CA, USA), anti-NKG2A-PE (clone; REA110, Miltenyi Biotec, Bergisch Gladbach, Germany), anti-NKG2C-Alexa700 (clone 134591, R&D Systems Inc., Minneapolis, MN, USA), and anti-CD57-V450 (clone; TB01, ebioscience, San Diego, CA, USA). PBMCs were also intracellularly stained with anti-CD3ζ-PE (clone; 6B10.2, BD bioscience, San Diego, CA, USA) and anti-FcεRIγ-FITC (FcRγ) (Millipore, Merck Millipore, Burlington, MA, USA) for the analysis of FcεRIγ-deficient NK cells (g-NK cells). NK cell gating and analyses of the NK subpopulation are shown in Figure A1. Flow cytometry data were collected on an FACS Fortessa instrument (BD bioscience, San Jose, CA, USA), using FlowJo version 10.0.6 software (Tree Star, Ashland, OR, USA).

PBMCs were purified from healthy donor blood by centrifugation via Lymphoprep Medium according to the supplier instructions (Anprotec). HCMV-infected MRC-5 or HFF cells (MOI = 3, 72hpi) were incubated with titrated amounts of Cytotect at 37°C for 1 hr in a 5% CO₂ atmosphere. After washing 2× with medium, 5 × 10⁵ PBMCs were incubated on opsonized infected cells for 6 hr (100 µl per well in RPMI/10% FCS) in the presence of αCD107a-, αCD56-BV650, and Golgi-Plug/Golgi-Stop (according to supplier, BD). After incubation, PBMCs were harvested, washed in staining buffer (PBS/3% FCS), and incubated with αCD3-FITC 30 min at 4°C. After two final washing steps in staining buffer, analysis was performed on a FACS Fortessa instrument (BD Bioscience).

Isolated liver leukocytes or PBMCs were blocked with 5 μg/mL anti-mouse CD16/CD32 antibodies (BD Bioscience) and stained with fluorochrome-conjugated antibodies for 30 min at 4 °C in the dark. The cells were washed twice with PBS containing 3% FBS and fixed in 0.5 mL of Fluorofix buffer (BioLegend) for 30 min. The fixed cells were washed with PBS containing 3% FBS once and then analyzed on a FACSFortessa instrument (BD Bioscience). The fluorochrome-conjugated antibodies used in this study included FITC-conjugated anti-CD3, FITC-conjugated anti-CD11b, phycoerythrin-conjugated anti-CD45, APC-conjugated anti-CD8, APC-conjugated anti-CD115, APC/Cy7-conjugated anti-CD45, APC/Cy7-conjugated anti-Ly6G, BV605-conjugated anti-CD4, BV605-conjugated anti-Ly6G, BV421-conjugated anti-Ly6C, PerCP/Cy5.5-conjugated anti-CD19, PerCP/Cy5.5-conjugated anti-F4/80 (all purchased from BioLegend) and BV421-conjugated anti-CD11a (BD Bioscience). The compensation settings of the flow cytometer were determined with single-stained Ultracomp eBeads (Thermo Fisher Scientific). The frequency of each cell subpopulation obtained by flow cytometric analysis was further multiplied by the total isolated cell count to obtain the cell number for each subpopulation.

In total, 0.5–1 × 10⁶ cells were harvested from the 16-h or 10-day stimulation cultures, washed, and incubated with Live/Dead Aqua V510 for 15 min on ice. The cells were then washed again and surface-stained for 30 min on ice with the following antibodies: anti-CD3-FITC (BioLegend, clone UCHT1, Cat# 561802), anti-CD4-BV421 (BioLegend, clone OKT4, Cat# 317434), anti-CD8-PerCPCy5.5 (BD Pharmingen™, clone RPA-T8, Cat# 560662), anti-CD27-PE (BD Pharmingen™, clone M-T271, Cat# 555441), and anti-CD45RA-APC-H7 (BD Pharmingen™, clone HI100, Cat# 560674). After fixation and permeabilization with Cytofix and Perm (BD Bioscience, Cat# 554714) on ice for 15 min, intracellular staining was performed on ice for 30 min with anti-TNF-PE-Cy7 (BD, clone MAb11, Cat # 557647) and anti-IFNγ-APC (BD Pharmingen™, clone B27, Cat# 554702). After the final wash, the cells were resuspended in 200 μL FACS buffer. A FACSFortessa instrument (BD Bioscience) was used to acquire data, which were analyzed using FlowJo software (Treestar).