Recently, we have demonstrated that tumor-specific CD4+ Th cell responses can be rapidly induced in advanced melanoma patients by vaccination with peptide-loaded monocyte-derived dendritic cells. Most patients showed a T cell reactivity against a melanoma Ag 3 (MAGE-3) peptide (MAGE-3(243-258)), which has been previously found to be presented by HLA-DP4 molecules. To analyze the functional and specificity profile of this in vivo T cell response in detail, peptide-specific CD4+ T cell clones were established from postvaccination blood samples of two HLA-DP4 patients. These T cell clones recognized not only peptide-loaded stimulator cells but also dendritic cells loaded with a recombinant MAGE-3 protein, demonstrating that these T cells were directed against a naturally processed MAGE-3 epitope. The isolated CD4+ Th cells showed a typical Th1 cytokine profile upon stimulation. From the first patient several CD4+ T cell clones recognizing the antigenic peptide used for vaccination in the context of HLA-DP4 were obtained, whereas we have isolated from the second patient CD4+ T cell clones which were restricted by HLA-DQB1*0604. Analyzing a panel of truncated peptides revealed that the CD4+ T cell clones recognized different core epitopes within the original peptide used for vaccination. Importantly, a DP4-restricted T cell clone was stimulated by dendritic cells loaded with apoptotic or necrotic tumor cells and even directly recognized HLA class II- and MAGE-3-expressing tumor cells. Moreover, these T cells exhibited cytolytic activity involving Fas-Fas ligand interactions. These findings support that vaccination-induced CD4+ Th cells might play an important functional role in antitumor immunity.