Detection of spontaneous CD4+ T-Cell responses in melanoma patients against a tyrosinase-related protein-2-derived epitope identified in HLA-DRB1*0301 transgenic mice

Purpose: The frequently expressed differentiation antigen tyrosinase-related protein-2 (TRP-2) has repeatedly been described as a target of spontaneous cytotoxic T-cell responses in melanoma patients, suggesting that it might be an ideal candidate antigen for T cell -> based immunotherapy. As a prerequisite for immunization, T-cell epitopes have to be identified. Whereas a number of HLA class I-presented TRP-2-derived epitopes are known, information about HILA class 11 presented antigenic ligands recognized by CD4(+) T helper (Th) cells is limited. Experimental Design: The search for TRP-2-derived Th epitopes was carried out by competitive in vitro peptide binding studies with predicted HLA-DRB1 *0301 ligands in combination with peptide and protein immunizations of HLA-DRB1 *0301 transgenic mice. In vivo selected candidate epitopes were subsequently verified for their immunogenicity in human T-cell cultures. Results: This strategy led to the characterization of TRP-2(60-74) as an HLA-DRB1 *0301-restricted Th epitope. Importantly, TRP-2(60-74)- reactive human CD4+ Th cell lines, specifically recognizing target cells loaded with recombinant TRP-2 protein, could be established by repeated peptide stimulation of peripheral blood lymphocytes from several HLA-DRB1 *03(+) melanoma patients. Even short-term peptide stimulation of patients' peripheral blood lymphocytes showed the presence of TRP-260-74- reactive T cells, suggesting that these T cells were already activated in vivo. Conclusion: Peptide TRP-2(60-74) might be a useful tool for the improvement of immunotherapy and immune monitoring of melanoma patients.