Anti cd56 clone hcd56

PBMCs were thawed, stained with LIVE/DEAD Green Viability Dye (Invitrogen) for 15 minutes at 4°C and subsequently preincubated for 10 min with of FcR blocking reagent (Miltenyi). Afterward, cells were incubated for 25 minutes at 4°C with anti-CD3 (clone HIT3a; BioLegend), anti-CD14 (clone HCD14; BioLegend), anti-CD19 (clone HIB19; BioLegend), anti-CD56 (clone HCD56; BioLegend) and anti-CD16 (clone 3G8; BioLegend) antibodies. Cells were then sorted for CD56^dim CD16⁺ cNK cells and CD56^bright CD16^- NK cells on a BD FACSAria Fusion (BD Bioscience) at the Ragon Institute Imaging Core Facility.

Fluorochrome-conjugated peptide-HLA class I tetramers were produced as described previously (29 (link)). The specificities used in this study are detailed in Table II. Cells were stained 48 h after transfection with optimal titers of tetramer (0.2 μg with respect to the monomeric component in minimal residual volume) or anti-FLAG (clone M2; Sigma-Aldrich) mAb for 30 min at 4°C. Ba/F3 cells stably expressing LILRB1 were used as a positive control for tetramer binding. The LILRB1 receptor binds with equivalent affinity to a broad range of HLA molecules, and this binding is dependent on the correct folding of the HLA molecule and its association with β₂-microglobulin (30 (link)), but is independent of the presented peptide (31 (link)). Primary human PBMCs were stained similarly with 0.5 μg tetramer, anti-CD3 (clone HIT3a; BD Biosciences), and anti-CD56 (clone HCD56; BioLegend) mAbs. After RBC lysis in hypotonic buffer, cells were washed and analyzed using an LSRFortessa flow cytometer (BD Biosciences). For blocking experiments, cells were preincubated with the indicated mAb (10 μg/ml) for 15 min at 4°C.

Purified human CD3^-CD56⁺ dNK cells from uninfected, infected, and Tim-3-neutralized infected groups were air-dried onto Poly-L-lysine coated slides. After fixation in 4% paraformaldehyde for 30 min, slides were then blocked with goat serum for 1 h at room temperature. dNK cells were incubated overnight at 4°C with anti-Tim-3 (1/200, Abcam), anti-NKG2D (1/200, Abcam), anti-GranzymeA, anti-GranzymeB and anti-Perforin (1/200, all from Proteintech) or for 45 min with anti-CD56 (clone HCD56, Biolegend) and anti-KIR2DL4 (clone mAb33, Biolegend) antibody. After being washed three times with PBS, cells were then incubated with appropriate concentrations of secondary antibodies for 1 h at 37°C. Cy3 rabbit anti-goat IgM (1/500, Bioss) was used as the secondary antibody for anti-Tim-3 antibody, and Cy3 donkey anti-rabbit IgG (1/500, Bioss) was used as secondary antibody for anti-NKG2D, anti-GranzymeA, anti-GranzymeB and anti-Perforin. Subsequently, dNK cells were stained with the DAPI (nucleic acid stain 4’,6-diamidino-2-phenylindole) for 15 min and washed 3 times with PBS. Finally, confocal microscopy of cells was performed using Zeiss LSM880.