In vitro antigen stimulation assay. DC were generated from healthy donor monocytes using GM-CSF and IL-4. On Day 7, DC were loaded with 2 μg/mL PSA and various concentrations of either a NS IgG1 or the anti-PSA IgG1. DC were also loaded with equimolar amounts of the anti-PSA IgE antibody. Four hours after loading, the DC were matured with TNF-α and IFN-α. Autologous T cells were added 24 hours later. Cells were incubated for 7 days and restimulated with freshly generated and pulsed DC weekly. The cells were stimulated for a total of 3 rounds. One day after the third stimulation in the presence of Brefeldin A, T cells were harvested and analyzed for activation of CD4 and CD8 IFN-γ producing T cells by intracellular cytokine staining and flow cytometry. CD3+/CD8- (CD4, left quadrants) and CD3+/CD8+ (CD8, right quadrants) that stain positively for IFN-γ are a measure of specific activation.
Daniels-Wells et al. BMC Cancer 2013 13:195 doi:10.1186/1471-2407-13-195