Cd8 alexa fluor 700

The cultured cells or isolated PBMCs were stimulated with 0.5 μg/ml concentration of the peptide pool. After 1 h of culture (37°C, 5% CO₂), the cells were supplemented with brefeldin A solution (BioLegend) and cultured for 4 h. The samples stimulated with the peptide solvent alone (20% DMSO in PBS) were used as unstimulated controls. The cells were transferred to a V‐bottom 96‐well plate (Nalgene) and stained as described¹⁴ with live/dead fixable stain and the following antibodies: CD4‐PE‐Cy7 and CD8‐Alexa Fluor 700 (Exbio), CD3‐PerCP‐Cy5.5, TNF‐α‐APC, IFNγ‐PE (Becton Dickinson). For T cell phenotype analyses, the cells were stained with live/dead fixable stain and the following antibodies: CD4‐PE‐Cy7 and CD8‐Alexa Fluor 700 (Exbio), CD3‐PerCP‐Cy5.5 and TNF‐α‐APC (Becton Dickinson), and CD62L‐FITC and CD45RO‐PE (Exbio). The cells were analyzed by FACSAria II (Becton Dickinson) and the data were processed by FlowJo software (Tree Star). The frequency of reactive T cells was calculated as the difference between the frequency of the cytokine‐producing T cells of the vehicle‐stimulated sample and the peptide pool‐stimulated sample of the same donor.

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.