NY-ESO-1–specific T cells are of high avidity, recognize the naturally processed antigen, and have cytolytic potential in a patient with MM after alloSCT. We analyzed the peptide specificity of the CD4⁺ T-cell responses against NY-ESO-1_51-70 and NY-ESO-1_121-140 and of the CD8⁺ response against NY-ESO-1_51-70, performing an ELISPOT in combination with peptide titration experiments (A). Following a single cycle of antigen-specific stimulation with peptides NY-ESO-1_51-70 or NY-ESO-1_121-140, patient BMT47's T cells were able, especially in the case of the NY-ESO-1_51-62–specific cell lines, to recognize the antigen in nanomolar concentrations and in a highly specific fashion. Irrelevant peptides are shown as an empty circle (NY-ESO-1_51-70–specific CD4⁺), triangle (NY-ESO-1_51-70–specific CD8⁺), and diamond (NY-ESO-1_121-140–specific CD4⁺) and were used at a concentration of 10 μmol/mL. (A) When we analyzed whether the patient's NY-ESO-1_51-62–specific CTLs would demonstrate cytolytic potential in a granzyme B ELISPOT, we observed that the patient's CD8⁺ T cells indeed secreted this cytoloytic molecule upon exposure to autologous EBV-B cells pulsed with NY-ESO-1 peptide but not the irrelevant control (B). Furthermore, CD4⁺ as well as CD8⁺ T cells specific for NY-ESO-1_51-62 not only recognized this peptide but also produced IFN-γ in response to the naturally processed antigen in form of autologous EBV-B cells infected with vaccinia virus recombinant for full-length NY-ESO-1 (VV NY-ESO-1) (C). Irrelevant VV recombinant for influenza nucleoprotein (VV NP) was used as a control. Bars show the mean spot number of duplicate ELISPOT experiments, with error bars indicating SEM.