Cd8 pb clone sk1

Peripheral blood mononuclear cells (PBMC) from healthy subjects were isolated from heparinized whole blood by density gradient centrifugation and 1x10⁶ PBMCs were stained with viable and dead cells LIVE/DEAD™ Fixable Aqua dead cell stain kit (Life Technologies) for 15 min followed by an extracellular staining. First CCR7 PerCp Cy 5.5 was stained (clone G043H7, Biolegend) for 15 min at 37 °C followed by CD3 AF700 (clone OKT3, Biolegend), CD4 BV605 (clone RPA-T4, BD Biosciences), CD8 PB (clone SK1, Biolegend), CD45 RA FITC (clone HI100, Biolegend), CD56 APC-Cy7 (clone HCD56, Biolegend), CD19 PE-Cy7 (clone HIB19, Biolegend), CD26 APC (clone BA5b, Biolegend) for 15 min at 4°C. Non-specific binding of Fc-receptors was blocked by 2% polyclonal IgG (Flebogamma). CD26 surface expression was measured with CytoFLEX LX (Beckman Coulter) and data was analyzed with FlowJo software 10.0.08. Gating strategies are shown in the Supplementary Information.

Cells were stained with Vβ4-fluorescein isothiocyanate (FITC) (TRBV29-1, clone WJF24; Beckman Coulter, Brea, CA, USA), αβTCR-phycoerythrin (PE) (clone BW242/412; Miltentyi Biotec, Bergisch Gladbach, Germany), CD3-Pacific Blue (PB) (clone UCHT1; Becton Dickinson [BD]), CD4-PeCy7 (clone RPA-T4; eBioscience, Thermo Fisher Scientific, Breda, the Netherlands), CD8-allophycocyanin (APC) (clone RPA-T8; BD), CD8-PB (clone SK1; BioLegend, San Diego, CA, USA), or RPE-conjugated NY-ESO-1_157–165 HLA∗02:01 (SLLMWITQV) pentamer (ProImmune, Oxford, UK). Samples were fixed using 1% paraformaldehyde (PFA) in PBS, measured on a FACSCanto II flow cytometer (BD, Eysins, Switzerland), and analyzed using FACSDiva (BD, Eysins, Switzerland) or FlowJo (BD, Eysins, Switzerland) software.