α cd3 apc

For the T cell activation assay, HLA-A2 positive human DCs were adhered for 1 h at 37 °C in X-vivo medium (Lonza) containing 2% human serum in 96 wells plates. Cells were then washed and incubated with gp100 short (10 μM gp100 (residues 280–288); GenScript) or long (100 μM gp100 (residues 272–300); JPT) peptide with T cell receptor stimulation mix (4 μg/ml R-848 and poly(I:C); Enzo Life Sciences) for 4 h. After incubation, cells were washed and resuspended in X-vivo medium containing Jurkat E6.1 cells (ratio 1 DC: 2 T cells) and co-cultured for 18 h at 37 °C. T cells were stained with αCD69-FITC (1:10; BD biosciences), αCD3-APC (1:10; eBiosciences) and Propidium Iodide (PI; 0.5 μg/ml) and analyzed with a FACS Calibur (BD biosciences). CD69 surface levels as a measure of T cell activation were analyzed using FlowJo software and Prism 5. Briefly, cells were gated on viability (PI), followed by gating for CD3-positive cells, and the CD69 signals were determined. T cell activation was calculated as the ratio of CD69 positive cells stimulated with long over stimulation with short peptide.