Bv650 cd95

Samples from HLA-A2–positive subjects were dual stained with a mixture containing a pool of PE and APC tetramers, PBMCs (20 ×10^6) were stained simultaneously with 3 μl each in 200 μl of PBS supplemented with 2% BSA and incubated for 15 min at 37°C. Cells were then incubated with 30 μl each of anti-PE and anti-APC magnetic beads (Miltenyi Biotec, San Diego, CA, USA) and enriched on a MS-sized magnetic column according to the manufacturer’s instructions. After enrichment, flow-through cells were reserved for bulk memory and naive CD8⁺ T-cell sorting described in the following section. Both enriched and flowthrough cells samples were washed and then stained for 30 min at 4°C with 5 μl each:anti- CD8⁺ BV605(Biolegend), AF700 CD45RA(BD Bioscience), APC-Cy7 CCR7(Biolegend), PerCP- eFluor 710 TIGIT(eBioscience), PE-Cy7 PD-1(Biolegend), BV650 CD95(Biolegend), BV421 KLRG1(Biolegend), 2.5 μl each of anti-CD4/CD14/CD16/CD20/CD40-FITC (dump channel; eBioscience, Waltham, MA, USA), and 1:10,000 syTOX green viability dye(Thermo Fisher). CD8⁺ cells were sorted using a FACSAria II(BD Biosciences) into lysis buffer from an Allprep DNA RNA mini prep kit (Qiagen) and processed for DNA and RNA according to the manufacturer’s instructions.