Nkg2c apc

In brief, healthy donors and patient’s total PBMC were stained for surface markers with the following fluorochrome conjugated antibodies: CD19-BUV737, CD107a-BUV395, CD3-BV786, CD33-BV711 & CD14-BV650 (for AML study), CD15-BV650 & CD30-BV605 (for HL study), PD1-FITC, CD62L-PerCP-Cy5.5, CD57-PE-CF594, CD16-AF700 from Beckton Dickinson, CD69-PE from Beckman Coulter, CD45RO-VioGreen, CD7-VioBlue, CD56-PE-Vio770, CD45RA-APC-Vio770, and NKG2C-APC from Miltenyi Biotec. Cell viability was determined using DAPI (BD Biosciences). Cells were stained with the corresponding antibodies in FACS buffer (PBS, 2% FBS) on ice for 25–30 min and washed twice with the same FACS buffer and acquired on BD LSR-Fortessa instrument (Blue-Yellow/Green-Red-Violet-Ultraviolet) (BD Bioscience). Flow Cytometry Standard (FCS) files were analyzed using FlowJo software v10.6.1 (Becton, Dickinson and Company, Ashland, OR). The gating strategy for conventional flow cytometry and the determination of the “percentage” of selected cells in the figures is described in the Supplemental Figure S1.

Frozen PBMCs from three AFB (CMV⁺) and six EUB (three CMV⁺, three CMV^-) donors were thawed and allowed to rest overnight, as previously described. For each donor, 10⁶ cells were resuspended in phosphate-buffered saline supplemented with 2% FBS and incubated with human Fc blocking solution (BD Biosciences) for 10 min at 4 °C. Cells were then stained with the following antibodies for 30 min at 4 °C: CD3 VioGreen (BW264/56, Miltenyi Biotec), CD14 V500 (M5E2, BD Biosciences), CD57 Pacific Blue (HNK-1, BioLegend), NKp46 PE (9E2/NKp46, BD Biosciences), CD16 PerCP-Cy5.5 (3G8, BD Biosciences), CD56 APC-Vio770 (REA196, Miltenyi Biotec), NKG2A FITC (REA110, Miltenyi Biotec) and NKG2C APC (REA205, Miltenyi Biotec). The cells were then washed and acquired on the MACSQuant cytometer (Miltenyi Biotec), and the data were analysed using FlowJo (v.10.7.1)⁷² .

Paired PBMCs and perfusate MNCs were resuspended in PBS/0.1% BSA to create a 2x cell solution. This was resuspended in Carboxyfluorecin succinimidyl ester (CFSE) staining solution (CellTrace™ CFSE Cell Proliferation Kit) (Life Technologies™, Paisely, UK) to make a final CFSE concentration of 5 μM and incubated for 10 min, 37°C. Staining was quenched with 5 volumes ice‐cold Roswell Park Memorial Institute Medium (RPMI) 1640 + Glutamax (Gibco®, Life Technologies™) supplemented with 10% fetal bovine serum (Hyclone®, Thermoscienticic, Northumberland, UK), penicillin, streptomycin and glutamine (Gibco®, Life Technologies™) (R‐10) and incubated for 5 min, 4°C. Cells were washed three times in R‐10 then recounted. PBMCs and liver MNCs were incubated in R‐10 supplemented with 5% HS (Sigma) in addition to Recombinant Human IL‐2 100 U/ml (PeproTech, London, UK), IL‐12 10 ng/ml (PeproTech), IL‐15 25 ng/ml (R&D Systems, Oxford, UK), IL‐18 100 ng/ml (Medical and Biological Laboratories, Japan), or a cocktail of all four for 6 days. Media and cytokines were changed every 2–3 days. A CFSE FMO was included. On day 0 and 6 PBMCs and liver MNCs underwent staining with Zombie Violet™ Fixable Viability Kit (Biolegend®), CD3‐BV510 (Biolegend®), CD56‐PECy7 (Biolegend®), NKG2C‐APC (Miltenyi Biotec), CXCR6‐PerCP/Cy5.5 (Biolegend®), and CD49a‐PE (BD Biosciences).