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Background: As tumor antigen-specific CD4+ T cells can mediate strong therapeutic anti-tumor responses in melanoma patients we set out to establish a comprehensive screening strategy for the identification of tumor-specific CD4+ T cell epitopes suitable for detection, isolation and expansion of tumor-reactive T cells from patients. Methods and Findings: To scan the human melanoma differentiation antigens TRP-1 and TRP-2 for HLA-DRB1*0301-restricted CD4+ T cell epitopes we applied the following methodology: Splenocytes of HLA-DRB1*0301-transgenic mice immunized with recombinant adenovirus encoding TRP-1 (Ad5.TRP-1) or TRP-2 (Ad5.TRP-2) were tested for their T cell reactivity against combinatorial TRP-1- and TRP-2-specific peptide libraries. CD4+ T cell epitopes thus identified were validated in the human system by stimulation of peripheral blood mononuclear cells (PBMC) from healthy donors and melanoma patients. Using this strategy we observed that recombinant Ad5 induced strong CD4+ T cell responses against the heterologous tumor antigens. In Ad5.TRP-2-immunized mice CD4+ T cell reactivity was detected against the known HLA-DRB1*0301-restricted TRP-260-74 epitope and against the new epitope TRP-2149-163. Importantly, human T cells specifically recognizing target cells loaded with the TRP-2149-163-containing library peptide or infected with Ad5.TRP-2 were obtained from healthy individuals, and short termin vitro stimulation of PBMC revealed the presence of epitope-reactive CD4+ T cells in melanoma patients. Similarly, immunization of mice with Ad5.TRP-1 induced CD4+ T cell responses against TRP-1-derived peptides that turned out to be recognized also by human T cells, resulting in the identification of TRP-1284-298 as a new HLA-DRB1*0301-restricted CD4+ T cell epitope. Conclusions: Our screening approach identified new HLA-DRB1*0301-restricted CD4+ T cell epitopes derived from melanoma antigens. This strategy is generally applicable to target antigens of other tumor entities and to different HLA class II molecules even without prior characterization of their peptide binding motives. [ABSTRACT FROM AUTHOR]