Cd56 bv510

The expression of ectonucleotidases (CD39 and CD73) on αβDNT and activated Treg cells was evaluated by flow cytometry. The gating strategy for activated Treg cells was performed as previously described (5 (link)). PBMCs were stained with directly conjugated antibodies for 30 min at 4°C in the dark. The cells were washed before flow cytometry analysis. Antibodies used included anti-human CD3-BV785 (clone SK7), CD4-APC-fire750 (clone SK3), αβTCR-BV421 (clone IP26), CD56-BV510 (clone HCD56), CCR7 (CD197)-PE-cy7 (clone G043H7), HLA-DR-AF700 (clone L243), CD73-PE (clone AD2), CD39-BV605 (clone A1, BioLegend, San Diego, CA, USA), CD45RA-BV711 (clone HI100), CD38-BUV737 (clone HB7), CD25-PE-CF594 (clone M-A251), CD8-FITC (clone SK1), CD8-BUV395 (clone RPA-T8, BD Biosciences, San Diego, CA, USA), and the corresponding isotype controls. Data were acquired on a BD LSR Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

Lymphocytes 5 × 10⁶/mL were stimulated with 1 μg/mL PepMix in a CTL medium for 2 h and then for an additional 12 h in the presence of 1:1000 diluted Golgi plug (BD Biosciences, Franklin Lakes, NJ, USA). After incubation, cells were harvested with PBS and stained with the LIVE/DEAD Blue Dead Cell Staining Kit (Thermofisher) and antibodies to CD45RA-BB515, CD27-BV650, CD45RO-BV786, PD1-PECF594, CCR7-BV605 (BD Horizon, BD Biosciences), CD56-BV510, TIGIT (VSTM3)-PE/Cy7 (Biolegend, San Diego, CA, USA), CD8-AlexaFluor700 (Exbio, Prague, Czech Republic) and CD57—APC (BD Pharmingen). The cells were then washed with PBS, fixed using IC fixation and permeabilisation Buffer (eBioscience, San Diego, CA, USA) for 20 min and stained intracellularly with antibodies against IFN-gamma-PE, CD3-APCCy7 (Biolegend, San Diego, CA, USA) and CD4-PacificBlue (Exbio) in a permeabilisation buffer. The cells were washed and resuspended in a FACS buffer (FB-PBS containing 0.09% sodium azide, 1% BSA). The cells were measured using the BD LSR Fortessa 5 L flow cytometer (BD Biosciences). The obtained data were analysed by the FlowJo 10.5 software (TreeStar, Ashland, OR, USA). The gating strategy is shown in Figure 1.

RNK001 potency was determined by challenging them with K562 cells. Briefly, 150.000 RNK001 cells were co-cultured with 100.000 K562 cells in the presence of CD107a-PECy7 (Biolegend, #328618) at 37 °C for 4 h. After 1 h, Brefeldin A (BD Bioscience, #5550229) was added. Next, cells were harvested and stained with CD56-BV510 (Biolegend, # 318340) and eFluor780 (eBiosciences, #65-0865-14) at 4 °C for 30 min. Cells were then fixed and permeabilized using eBiosciences Fix/Perm buffer (#00-5123-43, #005223-56 and #00-8333-56). Finally, cells were stained intracellularly for IFN-γ-FITC (BD bioscience, #554700) and Perforin-PE (Biolegend, #308106), washed, acquired on the Gallios flowcytometer (Beckmann Coulter) and analyzed using Kaluza V2.1.3.