Anti nkg2d

FACS staining was performed using standard procedures. Staining was performed with the following conjugated antibodies: anti CD56, anti CD3, anti CD16 (all from Biolegend), anti 2B4, anti NKp46, anti CD69 (BD), anti NKp30, anti NKp44, anti NKG2D, anti DNAM1, anti NKG2C (R&D), anti CEACAM1 (R&D), anti TIGIT (eBioscience), anti NKG2A (R&D). Antibodies against chemokine receptors that were used included anti CCR1, anti CCR2, anti CCR3, anti CCR5, anti CCR7, anti CCR10, anti CXCR1, anti CXCR2, anti CXCR3, anti CXCR4, and anti CX3CR1. Other antibodies used were: anti CD57 and antibodies against the killer cell immunoglobulin-like receptors-KIR2DL1/DS1, anti KIR2DL2/DL3, and anti KIR3DL1/KIR3DS1. Unless otherwise noted, all antibodies in this study were purchased from Biolegend. In all FACS results shown in this paper, dead cells, neutrophils, and monocytes were excluded from analysis. We used the FCS Express version 4 program for FACS analysis. We performed correction for the MFI of different unrelated FACS staining, by dividing the individual staining MFI (of blood and SFs NK cells) by the background isotype control of the same staining (normalized MFI).

The purification and activation of NK cells was previously described (27 (link)). To assess NK cell degranulation, 5 x 10⁴ NK cells were co-incubated with 3 x 10⁴ uninfected HSB-2 cells or HSB-2 cells 72 hours after HHV-6A infection, or alternatively, with U20-J-Jhan, U21-J-Jhan or control transfectants. Where indicated, NK cell receptors were blocked using 0.25 µg anti-NKG2D (1.25µg/mL; R&D Systems) antibody. Alternatively, NK cells were incubated with 0.25 µg of a control antibody targeting CD99 (1.25µg/mL). NK cells were discriminated in FACS using CD56-PE (0.5µl/100µl, BD Biosciences) and degranulation was assessed using CD107a-APC (0.5µl/100µl, Biolegend). Both conjugated antibodies were added after intermixture of NK and target cells. The experiment was performed for two hours and analyzed on a FACSCalibur machine. For analysis of degranulation, NK cells were gated according to their appearance in the forward/side scatter and for CD56 expression. CD107a positive percentage was determined on the gated NK cell population.

Freshly isolated PBMCs were stained with antibodies specific to the cell surface markers of T cells, B cells, and the NK lymphocyte lineage, including anti-CD3, CD16, anti-CD19, CD56, CD4, CD8, γδ TCR (BD Biosciences, San Jose, CA, USA), anti-NKG2D, and anti-DNAM-1 (R&D Systems, Minneapolis, MN, USA). The cells were also stained with NIR zombie dye (BioLegend, San Diego, CA, USA) to exclude dead cells from the analysis. The proportion of circulating regulatory T cells (cTreg) was assessed in cryopreserved PBMCs. The stained cells were analyzed using a BD Canto instrument (BD Biosciences), and the data were analyzed using the FlowJo software package (version 10.1, Tree Star, Ashland, OR, USA).

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).