Nkp46 v450

For phenotypical analysis, PBMCs isolated from children with CHB and uninfected controls were stained with fluorochrome-conjugated antibodies to CD3-phycoerythrin (PE)-cyanin 7 (Cy7), NK group 2D (NKG2D)-PE (eBioscience, Hatfield, UK), CD56-energy-coupled dye (ECD) (Beckman Coulter, High Wycombe, UK), tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-PE, CD16-allophycocyanin (APC)-Cy7, CD94-fluorescein isothiocyanate (FITC), human leucocyte antigen D-related (HLA-DR)-V500, NKp46-V450 (BD Biosciences, Oxford, UK), NKG2A-Alexa700, NKG2C-PerCP (R&D Systems, Abingdon, UK) and NKp30-APC (Miltenyi Biotec, Surrey, UK) or isotype-matched controls, in the presence of fixable live/dead stain (Invitrogen, Paisley, Scotland, UK). Cells were acquired on a fluorescence activated cell sorter (FACS) LSRII multi-colour flow cytometer and analysed using FlowJo analysis software (Tree Star, Ashland, OR, USA).

For phenotypic analysis of NK cells, PBMC were stained with the following fluorochrome conjugated antibodies or isotype matched controls: CD3-Cy5.5/PerCP or CD3/Pe-Cy7, (eBioscience, Hatfield, UK), CD56-PE Texas Red (Beckman Coulter, High Wycombe, UK), CD56-FITC, CD16-APC Cy7, HLA-DR V500, CXCR3-Cy.5./PerCP, CD57-BV605, NKp46-V450, TRAIL-PE, (BD Biosciences, Oxford, UK), TRAIL-BV421, CD62L-AF700, CXCR6-PE, CD69-APC, CCR7-BV421, KLRG1-FITC, (Biolegend, London, UK), NKG2A-APC, NKG2C-Cy5.5/PerCP, NKG2D-PE (R&D systems, Abingdon, UK), NKp30-APC, NKp44-PE (Miltenyi Biotec, Surrey, UK), in the presence of fixable live/dead stain (Invitrogen, Paisley, Scotland). For phenotypic analysis of T cells PBMC were stained with the fluorochrome conjugated antibodies or isotype matched controls: CD3/Pe-Cy7, CD8-AF700, CD4-APC Cy7 (eBioscience, Hatfield, UK), CD38-PE Texas Red, CD14-V500, CD19-V500 (Biolegend, London, UK). Flourescence minus-one (FMOs) were used for gating purposes for all flourochromes; an example of the gating strategy is shown in S1A Fig. Cells were acquired on a FACS LSRII multicolour flow cytometer (Beckton Dickinson) and analysed using Flow Jo analysis software (Tree star, Ashland, OR, USA). In addition to percentage, absolute numbers of NK cell subsets were calculated by multiplying their percent by total lymphocyte count.