Lag 3 fitc

The following antibody-fluorochrome combinations were used: CD3-PE-Cy7, CD8-Horizon v450, CD4-Horizon v500, IFN-γ-FITC, PD-1-PE, CD45RO-BV650 (all from BD Biosciences, Oxford, UK), CD62L-APC-Cy7 (eBioscience, Hatfield, UK); LAG-3-FITC (R&D Systems, Abingdon, UK), and TIM-3-AF700 (eBioscience, Hatfield, UK). Ex vivo surface staining was performed on 1×10⁶ freshly isolated PBMC. Briefly, one microliter of a 1:50 dilution of each antibody was added to the cells and incubated for 30 minutes, at 4ºC, in the dark. Cells were washed twice with PBS/1% FCS and then resuspended in 200 μL PBS/1% FCS for data acquisition. Flow cytometry data acquisition was performed using a FACSCalibur. Propidium iodide (10 μg/mL) was added immediately prior to acquisition to discriminate dead cells from viable cells. Data analysis was performed using FlowJo (Treestar Inc., San Carlos, CA, version v10).

The following antibodies were used for T-cell phenotyping: CD8-BV650 (BD), CD4-BV605 (BD Biosciences), CD3-Alexa Fluor700 (BD Biosciences), CD95-BV711 (BD Biosciences), CD45RO-PerCPCy5.5 (BD Biosciences), CD25-APC-Cy7 (BD Biosciences), CD127-BV421 (BioLegend), CCR7-PE-Cy7 (BioLegend), CD45RA-ECD (Beckman Coulter), PD-1-BV785 (BioLegend), LAG3-FITC (R&D Systems), and TIM3-APC (R&D Systems). The dead cell exclusion stain (Live/Dead Aqua) was purchased from Invitrogen. To detect transduced NY-ESO-1^c259TCR-expressing cells, PE-conjugated pentamer reagents specific for the HLA-A*02:01 SLLMWITQC complex (ProImmune) were used at the manufacturer’s recommended concentrations.