There is accumulating evidence that CD4+ T cells or ?helper? T cells play critical roles in the induction and maintenance of anti-tumor responses. Few class II-restricted melanoma epitopes recognized by CD4+ T cells have been thus far reported and no immunotherapeutic approach has yet been developed successfully in order to generate and stimulate anti-tumor CD4+ T cells in humans. CD4+ T cells recognize tumor epitopes presented by class II molecules at the surface of the melanoma cells or antigen presenting cells. The definition of such epitopes is of major importance because it will allow the development of new vaccine trials designed to induce anti-tumor responses, which are measurable, by precise assays in vivo. We have recently been successful in identifying novel class II-restricted melanoma epitopes and have established experimental strategies that will allow us in the near future to identify other novel epitopes. Our long-term goal is to develop the knowledge required for the optimization of peptide vaccines in order to induce antitumor T cell responses in vivo. This application focuses on determining the best peptide sequences capable of stimulating specific high-avidity melanoma-reactive CD4+ T cells. The specific Aims are (1) To identify MHC Class II-restricted epitopes encoded by the NY-ESO-1 gene and recognized by Th1- type CD4+ T cells generated from peripheral blood lymphocytes (PBL) of patients with melanoma; (2) To isolate and characterize the Th2 and Th0-type CD4+ T cells that recognize the NY-ESO-1 derived epitopes (3) To identify peptide analogues based on amino acid (aa) substitutions of the original sequence in order to enhance antigen recognition, and dissect the induction of tumor-reactive CD4+ T cell activation events; Using these tools, we will be prepared to prospectively analyze the CD4+ T cell immune responses of patients with melanoma or other NY-ESO-1-expressmg tumors and to use the epitopes to be identified as vaccine components in inimunotherapeutic protocols. Taken together, these data will allow us to better understand the role of ?helper? T cells in the anti-tumor responses in vivo and to design more effective immunotherapeutic clinical protocols based on the modulation of patients responses to such epitopes in vivo.