Based on our previous work supporting the common presence of anti-EphA2 and anti-MAGE6 CD4+ T cells in the peripheral blood of HLA-DR4+ patients with melanoma or RCC based on cytokine-based ELISPOT readout assays (17, 22), we now analyzed similar patients for the status of antigen-specific CD4+ T cells at the single cell level by flow cytometry by implementing fluorescently labeled MHC Class II-peptide tetramers. Here, CD4 is linked to melanoma.