cDNAs encoding functional T cell receptor (TCR) alpha and beta chains from a CD4+ T cell line (SG6) generated by repeated stimulation of a melanoma patient's peripheral blood mononuclear cells with HLA-DP4-restricted, NY-ESO-1-specific peptide p161-180 were cloned using a 5'rapid amplification of cDNA end method. Three different TCR alpha chains and 7 TCR beta chains were found among the 84 alpha and 162 beta cDNA clones tested. By screening different combination of the alpha/beta chains using RNA electroporation, TRAV9-1 (Valpha22.1) and TRBV20-1 (Vbeta2) were found to be the functional pair in line SG6. Antibody blocking experiments confirmed that the specificity of TRAV9-1/TRBV20-1 mRNA-transfected T cells were CD4 dependent and HLA-DP4 restricted. A retroviral vector expressing both TRAV9-1 and TRBV20-1 was constructed and used for transduction of OKT3-stimulated peripheral blood lymphocytes from melanoma patients. TCR-transduced CD4 T cells were capable of recognizing peptide-pulsed antigen-presenting cells (Epstein-Barr virus transformed B-cells, dendritic cells, and peripheral blood mononuclear cells), and protein-pulsed dendritic cells. Transduced cells were also capable of proliferation upon peptide stimulation and recognized peptide concentrations that were recognized by the parental line (0.2 microM). In contrast to SG6, which could not recognize human tumors, TCR-transduced CD4 T cells could specifically recognize NY-ESO-1/HLA-DP4-expressing melanoma cells. Major histocompatibility complex class II TCR-transduced CD4 T cells provides an alternative source of tumor antigen-specific T cells for adoptive immunotherapy of cancer patients.