Cd56 fitc

For surface marker staining of γδTc, the following anti-human antibodies from BioLegend, unless otherwise indicated, were employed: TCRγδ PE (clone B1, 1:25); TCR Vδ1 FITC (Miltenyi, clone REA173, 1:10); TCR Vδ2 PerCP (clone B6, 1:25); NKG2D APC (BD Biosciences, Mississauga, ON, Canada, 1:25); CD56 FITC (clone MEM-188, 1:5); CD69 AF700 (clone FN50, 1:4); CD94 FITC (clone DX22, 1:5); CD95 APC (clone DX2, 1:100); HLA ABC PE (clone W6/32, 1:10); FasL PE (clone NOK-1, 1:5); and CD40L APC (clone 24–31, 1:5).
Anti-human MICA/B PE (BioLegend, clone 6D4, 0.1 µg) was used to stain breast cancer cell lines.

After stimulation with 20 ng/ml phorbol myristate acetate (PMA) (Sigma, USA) plus 1 µg/ml ionomycin (Sigma) for 1 hour at 37°C in 5% CO₂, cells were cultured for 4 hours in the presence of 2 µM/L monensin (Sigma). Then, cells were stained with fluorochrome-conjugated antibodies to CD3-PerCP/cy5.5, CD56-FITC, CD8-FITC and CD4-FITC (Biolegend, San Diego, CA) at 4°C in the dark. Cells were then fixed and permeabilized, followed by intracellular staining for IFN-γ-PE, TNF-α-PE and isotype matched controls (Biolegend, San Diego, CA) at 4°C in the dark. Finally, cells were detected by flow cytometry.

A3.01 and ACH-2 cells (transformed CD4⁺ T cells, National Institutes of Health (NIH) AIDS Reagents Program, catalog nos. ARP-166 and ARP-349, respectively) were plated in BCM with 10 ng ml⁻¹ of TNF and incubated at 37 °C for 18 h. The cells were washed in BCM to remove TNF. NK cells were isolated from uninfected participant PBMC and cocultured with either the reactivated A3.01 or ACH-2 cells at a 1:3 E:T ratio in BCM at 37 °C for 6 h in the presence of the scDbs or the parental IgG molecules (at the indicated concentrations), monensin (BD Bioscience) and CD107a-BV421 (BioLegend, catalog no. 328625). The supernatants were collected and analyzed by Human CD8/NK Panel Legendplex (BioLegend) per the manufacturer’s protocol. The cells were washed in PBS and stained with the following antibodies (1:50 dilution)/dye (1:1,000 dilution) at 4 °C for 45 min: CD3-BV605 and CD56-FITC (BioLegend; catalog nos. 317321 and 304603, respectively) and Fixable Viability Dye-eFluor780. The cells were washed, and samples were acquired on an Intellicyt iQue Screener Plus. Data were analyzed with FlowJo (see Supplementary Fig. 6 for gating example). NK cells are defined as live CD3⁻CD56⁺ lymphocytes. All conditions were tested in triplicate, mean and s.d. shown.