Characterization of immune responses of patients that completed the treatment. a. Lymphoproliferation assay of PBMCs collected at indicated time points and stimulated with different melanoma-associated antigens (NY-ESO-1, tyrosinase, gp100, MART-1, survivin, MAGE-A1, and MAGE-A3). Proliferative activity was reported only for antigen-positive culture. Pre: prevaccination time; m: months. b. Intracellular cytokine staining (ICS) performed on T cell lines derived from in vitro expansion of PBMCs collected at different time points during treatment, with the tyrosinase, NY-ESO-1 and gp100 peptide pools. Histograms represent percentages of IFN-γ positive cells assessed within the CD3 + CD8+ gate after 6 h stimulation with IFN-DCs pulsed with the indicated peptides pools. Unpulsed IFN-DC were used as a control at each time point and for each antigen resulting in almost undetectable or very low levels of IFN-γ positive cells (% ranging from 0.02 to 0.08) for all samples, except in the case of both NY-ESO-1-specific samples (0.2%) from Pt5. Cells were analyzed by flow cytometry using a FACS DIVA and FlowJo software (version 10; TreeStar).