A polyclonal anti-vaccine CD4 T cell response detected with HLA-DP4 multimers in a melanoma patient vaccinated with MAGE-3.DP4-peptide-pulsed dendritic cells



During the last few years, HLA class I tetramers have been successfully used to demonstrate anti-vaccine CD8 CTL proliferation in cancer patients vaccinated with tumor antigens. Frequencies of CTL as low as 10–6 among CD8 cells were observed even in patients showing tumor regression. Little is known about the role of tumor-antigen-specific CD4 T cells in the context of these anti-vaccine responses. Therefore, we developed a very sensitive approach using fluorescent class-II–peptide multimers to detect antigen-specific CD4 T cells in vaccinated cancer patients. We produced HLA-DP4 multimers loaded with the MAGE-3243–258 peptide and used them to stain ex vivo PBL from melanoma patients injected with dendritic cells pulsed with several class I and class II tumor antigenic peptides, including the MAGE-3243–258 peptide. The multimer+ CD4 T cells were sorted and amplified in clonal conditions; specificity was assessed by their ability to secrete IFN-γ upon contact with the MAGE-3 antigen. We detected frequencies of about 1×10–6 anti-MAGE-3.DP4 cells among CD4 cells. A detailed analysis of one patient showed an anti-MAGE-3.DP4 CD4 T cell amplification of at least 3000-fold upon immunization. TCR analysis of the clones from this patient demonstrated a polyclonal response against the MAGE-3 peptide.