Generation of Antigen-Specific CD8+ T Cells

Normal donor monocytes were plated on day 1 in 6-well plates at 5 × 10⁶ cells per well in RPMI-10 FBS supplemented with 10 ng ml⁻¹ IL-4 (Peprotech) and 800 IU ml⁻¹ GM-CSF (Peprotech) and incubated at 37 °C overnight. On day 2, fresh medium supplemented with 10 ng ml⁻¹ IL-4 and 1,600 IU ml⁻¹ GM-CSF was added to the monocytes and incubated at 37 °C for another 48 h. On day 4, non-adherent cells were removed, and immature dendritic cells washed and pulsed with 5 µM peptide in AIM-V-10% FBS supplemented with 10 ng ml⁻¹ IL-4, 800 IU ml⁻¹ GM-CSF, 10 ng ml⁻¹ lipopolysaccharide (Sigma-Aldrich), and 100 IU ml⁻¹ IFN-γ (Peprotech) at 37 °C overnight. Day 1 was repeated on days 4 and 8 to generate dendritic cells for the second and third stimulations on days 8 and 12, respectively.
On day 5, normal donor-matched CD8+ T cells were enriched using protein kinase inhibitor dasatinib (Sigma-Aldrich), dextramers, and anti-PE or anti-APC beads (Miltenyi Biotec) as previously described^{50 (link)}. Enriched T cells were co-incubated with the appropriate pulsed dendritic cells in AIM-V-10% FBS. The day 5 protocol was repeated on days 8 and 12 using dendritic cells generated on days 4 and 8 for the second and third stimulation, respectively. Expanded T cells were validated for antigen-specificity by staining with the appropriate dextramers and for activation marker 41BB/CD137 (BioLegend).