Figure 1
Monitoring of CTL spiking by WB technology. CD8+ T cells from an HLA-A0201 restricted gp100280–288 specific CTL clone were added to 300 μl WB from an unrelated donor in the presence of the specific or a control (Melan-A/MART-127–35) peptide at a 10 μg/ml concentration. Following 5 hour incubation at 37°C, RNAlater was added to the samples and total cellular RNA was extracted, reverse transcribed and amplified in the presence of primers and probes specific for IL-2, IFN-γ. The expression of the indicated genes from triplicate samples was analyzed by using, as reference, the expression of β-actin house keeping gene (y axes). Standard deviations, never exceeding 5% of the reported values were omitted. A threshold of 2-fold increase in specific gene expression over control values was considered as cut-off for the definition of positive responses. Numbers of CTL spiked into WB were reported on x axes. (triangles = IFN-γ gene; squares = IL-2 gene; filled symbols = specific peptide stimulation; empty symbols = control peptide stimulation).