CT antigen NY-ESO-1 elicits a strong allogeneic CD4⁺ and CD8⁺ T-cell response in a patient with MM who received allogeneic stem cell transplantation. ELISPOT assays for the determination of NY-ESO-1–specific CD4⁺ and CD8⁺ T cells in MM patient BMT47 were performed after a single cycle of antigen-specific stimulation using a series of overlapping 20mer peptides spanning the full sequence of NY-ESO-1. Stimulating CD4⁺ as well as CD8⁺ T cells with a pool of peptides covering NY-ESO-1 1-90, a CD8⁺ (A) and a CD4⁺ (B) response against NY-ESO-1 region 51-50 were detected. Using T-APCs pulsed with single 20mer peptides as targets, we found that both responses were directed against NY-ESO-1_51-70 (A, B). Using 12mer peptides overlapping by a single AA and spanning NY-ESO-1 region 51-70, we were able to further characterize the CD8⁺ and CD4⁺ responses as both being directed against NY-ESO-1_51-62 (A, B). Using pooled 20mer peptides covering NY-ESO-1 80-180, a second CD4⁺ response directed against NY-ESO-1 region 111-150 was detected (C). This CD4⁺ response was further defined as being specific for NY-ESO-1_121-140 (C). MAGEA3 peptide 271-279 and NY-CO-58 peptide 151-180 served as control peptides (control) for analyses of NY-ESO-1–specific CD8⁺ and CD4⁺ responses, respectively. Bars show the mean spot number of duplicate ELISPOT experiments, with error bars indicating SEM.