T cell expansion

Following defined T cell differentiation in optimized conditions and CD8SP maturation as described above, CD8β⁺, CD4⁻, CD3⁺, TCRαβ⁺ were sorted using the MoFlo Astrios Cell Sorter (Beckman Coulter). The day before sorting, a plate coating solution was prepared using sterile PBS combined with anti-CD3 antibody (3 μg/ml; BioLegend, 317326) and RetroNectin (150 μg/ml; Takara Bio Inc., T100B). Tissue culture–treated 96-well plates (Fisher Scientific, 12-556-008) were precoated with 50 μl of coating solution overnight at 4°C. Coating solution was aspirated, and plates were washed with PBS immediately before use. The complete expansion medium was prepared by combining IMDM with GlutaMAX basal medium (Thermo Fisher Scientific, 31980030) with 4% B-27 supplement minus vitamin A (Thermo Fisher Scientific, 12587010), 0.50% (v/v) penicillin-streptomycin (Invitrogen, 15140122), 60 μM ascorbic acid (Sigma-Aldrich, A8960), 24 μM 2-mercaptoethanol, IL-7 (0.005 μg/ml; R&D Systems, 207-IL), IL-15 (0.005 μg/ml; R&D Systems, 247-ILB), IL-12 (0.5 μg/ml; R&D Systems, 219-IL), IL-18 (0.5 μg/ml; R&D Systems, 9124-IL), IL-21 (0.2 μg/ml; R&D Systems, 8879-IL), 100 μM Pan Caspase Inhibitor Z-VAD-FMK (R&D Systems, FMK001), and anti-CD28 antibody (3 μg/ml; BioLegend, 302934). Sorted cells were counted using a hemocytometer and seeded onto precoated plates at 75,000 cells per well in 100-μl expansion medium. Twenty-four hours after seeding, cells were harvested, pelleted by centrifugation for 300g for 5 min, and resuspended in fresh IMDM supplemented with GlutaMAX basal medium (Thermo Fisher Scientific, 31980030) with 4% B-27 supplement minus vitamin A (Thermo Fisher Scientific, 12587010), 0.50% (v/v) penicillin-streptomycin (Invitrogen, 15140122), 60 μM ascorbic acid (Sigma-Aldrich, A8960), 24 μM 2-mercaptoethanol, IL-7 (0.005 μg/ml; R&D Systems, 207-IL), and IL-15 (0.005 μg/ml; R&D Systems, 247-ILB). Cells were then reseeded onto plates coated with RetroNectin only. Cells were passaged on days 3 and 6. After 10 days, cells were restimulated following the same protocol for an additional 10 days.