Macsquant analyser 10

PBMC were thawed, and washed with RPMI 1640 supplemented with 10% fetal calf serum (Lonza, Walkersville, MD, USA). For flowcytometry, 500 000 viable PBMC were stained with anti‐CD8‐FITC(RPA‐T8); anti‐CD3‐Amcyan(SK7); anti‐CD161‐eFluor450(HP‐3G10); Anti‐TCR Vα7.2‐PE(3C10); anti‐CD56‐APC(N901); anti‐CD56‐APC‐eFluor780(CMSSB); anti‐CD38‐PerCp‐eFluor710(HB7); or anti‐HLA‐DR‐PerCP‐Cyanine5.5(LN3) for 20 minutes at 4°C. Marker expression was detected by flowcytometry (MACSQuant Analyser 10 [Miltenyi Biotec, Cologne, Belgium]). MAIT‐cells were defined as CD3⁺CD161⁺Vα7.2⁺ cells within the lymphocyte gate and NK‐cells were defined as CD3⁻CD56⁺ cells within the lymphocyte gate.
Liver aspirates and paired blood were stained with anti‐CD3‐Alexa‐Fluor700(OKT‐3); anti‐CD56‐APC‐eFluor780(CMSSB); anti‐CD45‐PE‐eFluor610(HI30); anti‐CD235a‐FITC(HIR2) or anti‐CD4‐FITC(13B8.2); anti‐CD3‐eVolve605(RPA‐T8); anti‐TCR Vα7.2‐PE(3C10); anti‐CD161‐eFluor450(HP‐3G10); and Live/Dead Aqua (Life Technologies, Carlsbad, CA, USA). Cells were analysed using a FACS ARIA cell sorter (BD‐Biosciences), San Jose, CA, USA and MACSQuant Analyser 10. All antibodies were purchased from eBioscience (San Diego, CA, USA), Beckman (Brae, CA, USA) or Biolegend (London, UK).

Neutralising capacity of antibodies from ZIKV patients was determined via flow cytometry.54 Briefly, pooled patient and healthy plasma samples at 1:500, 1:1000 and 1:2000 dilutions were incubated with ZIKV or DENV‐3 at MOI 10 for 2 h at 37°C with gentle agitation (350 rpm). Virus–antibody suspensions were then added in duplicates to HEK293T cells (ATCC) at 37°C. After 2 h, media were removed and Dulbecco's modified Eagle's medium (DMEM; GE Healthcare Life Sciences, Pittsburgh, PA, USA) with 10% foetal bovine serum (FBS; GE Healthcare Life Sciences) was added. After 48 h, cells were harvested and stained as described,54 using ZIKV NS3 protein‐specific rabbit polyclonal antibody29 or DENV human monoclonal antibody 1B,25 and counter‐stained with fluorophore‐tagged goat anti‐rabbit or anti‐human IgG (H+L) (Thermo Fisher Scientific). Cells were acquired with MACSQuant Analyser 10 (Miltenyi Biotec, Bergisch Gladbach, Germany). The assay was carried out in duplicates with two independent experiments. Flow cytometry results were analysed with FlowJo (version 10.4.1; Tree Star Inc. Ashland, OR, USA). Data of patient and pooled healthy neutralisation assays were normalised using the respective untreated infections and calculated as a percentage of virus‐only control infection.