Pd 1 bv711 clone eh12.2h7

Cryopreserved PBMCs were thawed and 1x10⁶ cells per sample washed with PBS containing 2% FCS, 2 mM EDTA, and 0.01% sodium azide at room temperature. After Fc receptor blocking with Gamunex (human immunoglobulin; GRIFOLS) and labeling of dead cells with ethidium monoazide (EMA; Biotinum), the cells were stained with the following titrated monoclonal antibodies: CD3-A700 (clone UCHT1, Biolegend), CD56-FITC (clone HCD56, Biolegend), CD4-PerCP (clone SK3, BD Bioscience), CD8-APC-Cy7 (clone SK1, Biolegend) and PD-1-BV711 (clone EH12.2H7, Biolegend). Lastly, after additional washing the samples were measured with a LSRII cytometer (BD) and processed with BD FACSDiva software 6.1.3. Single color controls were used for automatically calculated compensation.

The following antibodies were used for flow cytometry analysis: huCD45-PB (clone HI30; Sony Biotechnology), pan-γδTCR-PE (clone IMMU510; Beckman-Coulter), mCD45-APC (clone 30-F11, Sony Biotechnology), αβTCR-FITC (clone IP26; Biolegend), CD4-PeCy7 (clone RPA-T4, Biolegend), CD8-PerCPCy5.5 (clone RPA-T8, Biolegend), PD-1-BV711 (clone EH12.2H7, Biolegend), and TIM3-BV650 (clone F38-2E2, Biolegend). To exclude non-viable cells from the analysis, Fixable Viability Dye eFluor506 was used (eBioscience). All samples were analyzed on a BD LSRFortessa using FACSDiva Software (BD Biosciences).