Mouse tumor cells transfected with syngeneic MHC class II and costimulatory molecule genes are therapeutic vaccines in mice, provided they do not coexpress the class II-associated invariant chain (Ii). We demonstrated previously that the vaccine cells present tumor peptides via the endogenous antigen presentation pathway to activate CD4(+) and CD8(+) T cells. Because of their efficacy in mice, we are translating this vaccine strategy for clinical use. To obtain MHC class II(+)CD80(+)Ii(-) human tumor cells, we developed retroviruses encoding HLA-DR and CD80. The HLA-DR virus encodes the DRalpha and DRbeta0101 chains using an internal ribosomal entry site to coordinate expression. SUM159PT mammary carcinoma and Mel 202 ocular melanoma cells transduced with the retroviruses DRB1/CD80 express high levels of DRB0101 and CD80 on the cell surface in the absence of Ii. Irradiated SUM159PT/DR1/CD80 vaccines stimulate proliferation of non-HLA-DRB0101 peripheral blood mononuclear cells and present an exogenous DR1-restricted tetanus toxoid (TT) peptide, indicating that the transduced DRB0101 is functional. SUM159PT/DR1/CD80 vaccines were further transduced with a retrovirus encoding the TT fragment C gene, as a model tumor antigen. These cells stimulate IFN-gamma release from TT-primed human DRB0101 peripheral blood mononuclear cells, demonstrating their ability to present "endogenous" tumor antigen. Depletion and antibody blocking experiments confirm that MHC class II-restricted, endogenously synthesized epitopes are presented to CD4(+) T cells. Therefore, the MHC class II vaccines are efficient antigen-presenting cells that activate tumor-specific MHC class II-restricted, CD4(+) T lymphocytes, and they are a novel and potential immunotherapeutic for metastatic cancers.