Ccr7 bv605

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.

For immunofluorescence staining of whole blood and PBMC, antibodies included CD45-ECD (Beckman Coulter, Indianapolis IN), CD3-e780, CD4-e450, CD8-AF700, CD15-FITC (eBioscience, San Diego, CA), CD45-FITC, CD4-PerCP-Cy5-5, CD14-PECy7, HLA-DR-APC, HLA-DR-BV421, CD8-APC-H7, CCR7-BV605, CD25-BV605, CD45RA-PECy7, CD45RO-APC-H7, CCR4-PECy7, CD38-APC, CD127-BV650 (BD Bioscience, San Jose, CA), CD14-AF700, and CD3-AF700 (Biolegend, San Diego, CA). For phosphoSTAT staining, antibodies included CD3-BV785 (Biolegend), CD4-BV605, CD8-BV510, CD14-PECy7, CD19-BV421, CD16-BV650, pSTAT3-647, pSTAT5-PE (BD Biosciences), and pSTAT1-488 (Cell Signaling Technologies, Danvers, MA). Cell types were identified according to the criteria of the Human Immunology Project [19 (link)], and a full list of antibodies used to identify individual cell types including those not reported here can be found in Additional file 2: Table S1. Hematological toxicity was graded according to NCI CTCAE v4.0 guidelines and is reported in Additional files 3 and 4: Tables S2 and S3.