Detection of gp100 specific immunity in 4 vaccinated melanoma patients. Reactivity against gp10044–59 in uncultured PBMCs or in 10–20 day IVS cultures was determined by IFN-γ ELISPOT assays. 20 μM WT or Q56A peptide was used to stimulate CD4+ T cells in both IVS cultures and ELISPOT assays. Data from uncultured PBMCs from patient #3 are not shown, because all values were < 20 spots/106 cells. CDC27, an unrelated HLA-DR4-restricted control peptide. IVS, in vitro stimulation. Pre, pre-vaccine. P2, P4, P8, PBMCs collected from patients post 2, 4 or 8 vaccinations. SFC, spot forming colonies, mean± SEM. WT, wild type gp10044–59. * P<0.02, one-tailed t test compared to CDC27.