Mart 1 dextramer

DCs were generated by isolating CD14⁺ cells from PBMCs (Discovery Life Sciences Inc.) using the CD14 enrichment kit (Miltenyi) according to the manufacturer’s instructions and cultured in Dendritic Cell media (Cellgro) containing 1% human AB serum supplemented with rhIL-4 (100 ng/ml) and rhGM-CSF (100 ng/ml, Peprotech). The media was removed on day 4 of culture and replaced with fresh media containing 1% human AB serum supplemented with rhIL-4, rhGM-CSF, and hIFN-γ (10 ng/ml, Peprotech), lipopolysaccharide (10 ng/ml, MilliporeSigma), and MART-1 peptide (2.5 μg/ml, Anaspec). On day 5, the mature DCs were harvested and irradiated at 30 Gy. Naive CD8⁺ T cells were isolated using the Naive CD8 Isolation kit (StemCell) and cultured with irradiated DCs in media containing 5% human AB serum supplemented with rhIL-21 (30 ng/ml), MART-1 peptide, rhIL-7 (5 ng/ml), and rhIL-15 (5 ng/ml). Fresh media supplemented with rhIL-7 and rhIL-15 was added on day 3, 5, and 7. Cells were harvested on day 10 and antigen specificity was assessed by staining with MART-1 Dextramer (Immudex) and analyzing by flow cytometry.

The following antibodies were used for the analysis of surface and intracellular markers: anti-CD73 (2C5) antibody was discovered at AstraZeneca (18 (link)) and conjugated to fluorochrome in house; anti-CD73 (TY/11.8) was purchased from BioLegend; anti-Ki-67 (SolA15), anti-granzyme B (GB11), and anti-CD8 (OKT8) were purchased from eBioscience; and FcR blocking reagent was purchased from Miltenyi Biotec. Intranuclear staining was performed using Fixation/Permeabilization buffer solution (eBioscience) according to the manufacturer’s instructions. MART-1 dextramer was purchased from Immudex, and the staining was performed according to the manufacturer’s instructions. In some experiments, cells were treated with EGF (50 ng/ml), EGFR inhibitors (osimertinib, 1 μM, AstraZeneca), NF-κB inhibitor (IKK inhibitor VII, 1 μM, MilliporeSigma), ERK inhibitor (LY3214996, 1 μM, Selleckchem), or PKC inhibitor (Go 6983, 10 μM, MilliporeSigma) for 48 hours before analysis. Stained cells were analyzed on a LSRFortessa (BD Biosciences), and data were analyzed with FlowJo software.