Expanding CCR7+ CD8+ T cells from PBMCs

From three healthy volunteers, PBMCs (1 x 10⁶ cells/mL) were prepared from heparinized blood (10cc) by Ficoll–Hypaque (GE Healthcare) density-gradient centrifugation. To expand CCR7⁺CD8⁺ T cells, isolated PBMCs were stimulated using anti-CD3, IL-15, IL-2, and retinoic acid. Cells were cultured as described previously. [11 (link)] We pooled cells for microarray as opposed to single cell RNA sequencing. CCR7⁺ CD8⁺ T cells were purified by CD8–APC (SK1, IgG1,κ; BD) and CCR7-strepavidin (3D12, IgG2a, κ). The cells were sorted using an FACS Aria device (Becton Dickinson) or a MoFlo cell sorter (Beckman Coulter) to isolate CCR7⁺ CD8⁺ and CCR7^- CD8⁺ T cells. We extracted mRNA from CCR7⁺CD8⁺ and CCR7^- CD8⁺ T cells using the ReliaPrep™ RNA Miniprep Systems (Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions. RNA purity and integrity were evaluated by ND-1000 Spectrophotometer (NanoDrop, Wilmington, DE, USA).