Nkp44

To determine the involvement of specific CLRs as putative attachment factors for HBsAg on DCs, we performed neutralisation experiments by blocking single CLR using specific functional grade anti‐human CLR antibodies. Briefly, PBMCs were seeded at 2 × 10⁶ cells mL⁻¹ in 48‐well plates (Corning) and pre‐incubated for 30 min at 37°C in presence or not of blocking anti‐human DCIR, FcɣRIIA, MMR, FcεRIα, DECTIN1, BDCA2 (R&D systems), NKp44 (Invitrogen and Biolegend), ILT7 (Invitrogen) antibodies (single or in combination) or specific isotype controls. After 30 min of CLR blockade, 25 µg mL⁻¹ of FL‐HBsAg was added in the culture for an additional 2 h at 37°C. Subsequently, PBMCs were thoroughly washed with cold PBS2% FBS to remove solution‐free anti‐human CLR antibodies or FL‐HBsAg. Hence, cells were stained with anti‐CD11c, HLA‐DR, Lineage cocktail, CLEC9A, CD123, (BD), CD45 (Biolegend) and BDCA1 (Beckman Coulter) antibodies and fixed with FACS lysing solution (BD). FL‐HBsAg‐positive cDC1s, cDC2s and pDCs in each condition were then analysed using LSRII Flow Cytometer and FACSDiva software (BD).

Peripheral blood mononuclear cells were seeded at 1 × 10⁶ cells mL⁻¹ in 96‐well plates (Corning) and cultured for 30 min at 37°C in presence or not of blocking anti‐human FcɣRIIA, FcεRIα, DCIR, BDCA2 antibody, DECTIN1, MMR (R&D systems), NKp44 (Invitrogen and Biolegend), ILT7 (Invitrogen) functional grade antibodies or specific isotype controls. Native or deglycosylated rec‐HBsAg (from Pichia or Human) were added or not in the culture and the cells were subsequently cultured for 5 h in presence or not of a mixture of TLR ligands comprising polyI:C (30 µg mL^–1), Imiquimod (R848, 1 µg mL⁻¹), and Class‐A CpG oligonucleotide ODN‐2336 (CpGA, 1 µm) (Invivogen). 1 µg mL⁻¹ of Brefeldin A (BD) was added for the last 4 h. Later on, cells were stained for surface markers allowing to define cDC1s, cDC2s and pDCs (CD11c, HLA‐DR [BD], Lin, CD45 [Biolegend], cDC1/BDCA1 [Beckman], BDCA2 and BDCA3 [Miltenyi]) and then fixed and permeabilised according to manufacturer' instructions (BD Biosciences). Intracellular cytokine staining was then performed using the fluorochrome‐labelled anti‐human TNF⍺, IL‐12p40/70 (BD), IFN⍺ (Miltenyi) antibodies and anti‐human IFNλ1 (Novus, Abingdon, UK) antibody stained with mix‐n‐stain CF488 (Biotium, Fremont, CA, USA). Analyses were done by flow cytometry using LSRII Flow Cytometer and FACSDiva software v.8.