Anti nkp46

24-well plates were coated overnight at 4 °C with 20 μg/ml anti-NK1.1 (PK136, eBiosciences), anti-Ly49D (4E5, BD Pharmingen), anti-NKp46 (polyclonal, R&D systems). The day after, splenocytes were prepared and NK cells were enriched using negative selection kit (Invitrogen). NK cells (4 × 10⁵ to 5 × 10⁵ cells) were stimulated for 6 hours at 37 °C. CD107a (BD Pharmingen) was added (1:300 dilution) to the culture during the stimulation. After the first hour, GolgiPlug™ Protein Transport Inhibitor (BD Pharmigen) was dispensed into the culture. 1 μg/ml of phorbol-12-myristate-13-acetate (PMA) plus 0.25 μg/ml of ionomycin was used for positive control and PBS alone for negative control. For cytokine stimulation, IL-18 (1 ng/ml) plus IL-12 (1 ng/ml,) was used. After 6 hours, cells were stained with Fc block and surface antibodies as described above. For the intracellular staining, cells were fixed and then permeabilized using Cytofix/Cytoperm kit (BD Pharmingen), followed by staining with anti-IFN-γ (XMG1.2).

NK cells and PBMCs were pre-incubated at 37°C for 24hrs with anti-NKp46 (10 ug/mL; R&D Systems) or isotype control (10 ug/mL; R&D Systems). Next, PBMCs from high viral load CHB patients and healthy controls were incubated with K562 cells at an E:T of 10:1. Human hepatoblastoma derived HepG2 and HepG2.215 cells were cultured as previously described [24 (link)]. Healthy control NK cells were incubated with the hepatocellular carcinoma cell lines (HepG2 and HepG2.2.15 respectively) at E:Ts of 1:1, 5:1, 10:1 and 20:1. NK cell killing activity was analyzed using a lactate dehydrogenase (LDH) cytotoxicity assay kit (Promega, Madison, WI, USA) according to manufacturer’s instructions [25 (link)].

Optimal cutting temperature (OCT) compound embedded liver biopsy cryosections and paraffin-embedded sections (both 5 μm) were incubated with anti-NKp46 (Clone 195314, R&D Systems), anti-SMA-α (Rabbit polyclonal, Abcam) or TGF-β (Rabbit polyclonal, Abcam), respectively, according to our previously described protocols26 (link)38 (link). Double staining was performed using mouse anti-NKp46 and anti-SMA-α to evaluate the co-location of hepatic NK cells and HSCs. High-powered fields (hpf, 400×) were used to count positive cells. Positive cells were counted in three different fields by two independent observers.

The expansion of isolated pNK cell was performed with a GMP-compliant method at CHA Biotech (Seongnam, Korea). The cells were seeded on a γ-globulin (Greencross, Yongin, Korea) and anti-NKp46 (R&D Systems, Minneapolis, MN, USA)-coated flask and cultured in Alys505NK serum-free medium (CSTI, Sendai, Japan) supplemented with 1000 IU/mL recombinant human IL-2 (Novartis, Basel, Switzerland), 50 ng/mL recombinant human IL-18 (R&D system, Minneapolis, MN, USA), and 5% heat-inactivated autologous plasma. Fresh culture medium was added every 1 to 3 days depending on the cell density (2 × 10⁶ cells/mL). On Day 6, the cells were transferred to a culture bag (NIPRO, Osaka, Japan), and cultured for 14 days. The pNK cells were cryopreserved with Cryostor CS5 (BioLife Solutions, Bothell, WA, USA).

Immunostaining was performed on whole mount corneas as previously described [54 (link)]. Briefly, corneas were dissected from whole eyes and radial cuts were made to flatten the cornea. Corneas were fixed with buffered 2% paraformaldehyde (EMS, Hatfield, PA) for 1h, washed 3 x 5 min with PBS, and then permeabilized and blocked with PBS containing 0.1% Triton X-100 and 2% bovine serum albumin for 30 min. Corneas were incubated with fluorescent-labeled antibodies overnight at 4°C and then washed with PBS before being mounted in Airvol 205 (Sigma Aldrich, St. Louis, MO). Image analysis and quantification were performed using a DeltaVision microscope (Applied Precision, Carlsbad, CA). Corneal epithelial nuclei, and those nuclei showing a mitotic figure, were visualized using DAPI (Sigma Aldrich, St. Louis, MO); corneal nerves and NK cells were stained with anti-beta tubulin III (Tuj-1) and anti-Nkp46, respectively (R&D Systems, Inc., Minneapolis, MN); blood vessels, platelets and γδ T cells were stained with anti-CD31 (MEC13.3), anti-CD41 (MWReg30) and anti-γδ T cell receptor (GL3), respectively (BD Biosciences, San Jose, CA); and neutrophils with anti-LY6G (Invitrogen, Carlsbad, CA). Limbal venule diameter measurements and counts of platelets, neutrophils, γδ T cells, NK cells and dividing epithelial cells were made from images captured with a 20X objective.

Natural killer cell-mediated lysis of DLD-1 cells was assessed by xCELLigence System (Roche), a label-free real-time monitoring assay of adherent cell lysis (26 (link)). It measures electrical impedance across interdigitated micro-electrodes integrated on the bottom of tissue culture E-Plates. The electrode impedance, which is recorded as a dimensionless parameter denominated cell index (CI) value, provides quantitative information about the status of the adherent cells, including cell number, viability, and morphology. DLD-1 targets (15,000 cells) were seeded into the wells of 96 E-Plates in 100 μl of RPMI 1640 medium plus 10% human serum and their adhesion was monitored for 5 h. IL-2-activated purified NK cells were added in a volume of 50 μl/well at 1:1 E:T ratio. Cocultures were assessed by the system with a measurement of CI every 15 min for up to 300 min. Results expressed as CI, were normalized with RTCA Software (Roche, Applied Science, Mannheim, Germany) (nCI) and are expressed as % specific lysis:% lysis = (nCI (no effector) − nCI (effector))/nCi (no effector) × 100. In some cocultures, NK cells were pretreated with anti-NKp46, anti-NKp30, anti-NKG2D, or anti-DNAM-1 mAbs (R&D System).

For in situ immunofluorescence of NK cells, femurs from the indicated mice were fixed in paraformaldehyde-lysine-periodate fixative for 8 h, and decalcified in decalcifying buffer (10% EDTA in PBS (w/v), pH 7.4). Then femurs were rehydrated in 30% sucrose solution for 24 h and frozen in OCT for sectioning. BM sections were rehydrated in PBS and blocked in 10% donkey serum. Next, the sections were incubated with primary antibodies (anti-NKp46 (R&D), DX5 (eBioscience) and anti-B220 (BD), and anti-activated caspase3 (Cell Signaling)) for 2 h at room temperature followed by washing in 0.1% Tween20/PBS for three times. Sections were then incubated with Alexa488-, Alexa594- or Alexa405-conjugated secondary antibodies (Invitrogen). After washing for three times, DAPI was added and subjected to dehydrating in EtOH gradient: 70, 85, 95, 100%, 3 min each. Mounted sections were analysed by confocal microscopy (Olympus FV1000). For cell immunofluorescence, NK cells at different stages were isolated by flow cytometry, and fixed on 0.01% poly-L-Lysine treated coverslips with paraformaldehyde (Sigma-Aldrich) for 20 min at RT. Then cells were permeabilized, and stained with primary and secondary antibodies.