Isolation and Characterization of CMV-Specific CD8+ T Cells

PE-conjugated HLA-A2 tetramers refolded around the CMV pp65_495-503 epitope NLVPMVATV (NLV) were generated as described previously^{48 (link),49 (link)} and used at a final concentration of 10 μg/mL. Directly conjugated mAbs were obtained from commercial suppliers: (i) anti-CD3-APC-H7, anti-CD8-BV786, anti-CD14-BV510, anti-CD19-BV510, anti-CD45-RA-PerCP-Cy5.5, anti-CD57-APC, anti-CD127-BV421, anti-CD160-PerCP-Cy5.5, anti-CCR7-PE-Cy7, anti-CTLA-4-PE-Cy7, anti-BTLA-APC, anti-2B4-FITC, anti-2B4-PE, and anti-Tim-3-AF700 (BD Biosciences); (ii) anti-CD27-BV605 and anti-PD-1-PE/Dazzle 594 (BioLegend); and (iii) a panel of anti-TCR-Vβ-FITC and anti-TCR-Vβ-PE mAbs (Beckman Coulter). The acute effects of dasatinib administration were monitored using an IOTest Beta Mark TCR Repertoire Kit (BD Biosciences). Non-viable cells were excluded from the analysis using LIVE/DEAD Fixable Aqua (Life Technologies). Data were acquired using a Fortessa X-20 flow cytometer (BD Biosciences) and analyzed with FlowJo software version 10.0.7 (Tree Star). Viable CD3⁺ CD8⁺ TCR-Vβ⁺ cells were sorted using an Influx^TM Cell Sorter (BD Biosciences).