PURPOSE: Recombinant poxvirus vaccines have been explored as tumor vaccines. The immunogenicity of these vaccines can be enhanced by co-expressing costimulatory molecules and tumor-associated antigens. While the B7-CD28 interaction has been most comprehensively investigated, other costimulatory molecules utilize different signaling pathways and might provide further cooperation in T cell priming and survival. 4-1BB (CD137) is a TNF family member and is critical for activation and long-term maintenance of primed T cells. This study was conducted to determine if a poxvirus expressing the ligand for 4-1BB (4-1BBL) could further improve the immune and therapeutic responses of a previously reported poxvirus vaccine expressing a triad of costimulatory molecules (B7.1, ICAM-1, and LFA-3). EXPERIMENTAL DESIGN: A recombinant vaccinia virus expressing 4-1BBL was generated and characterized in an in vitro infection system. This vaccine was then used alone or in combination with a vaccinia virus expressing CEA, B7.1, ICAM-1, and LFA-3 in CEA-transgenic mice bearing established MC38 tumors. Tumor growth and immune responses against CEA and other tumor-associated antigens were determined. The level of anti-apoptotic proteins in responding T cells was determined by flow cytometry on tetramer selected T cells. RESULTS: The combination of 4-1BBL with B7.1-based poxvirus vaccination resulted in significantly enhanced therapeutic effects against CEA-expressing tumors in a CEA-transgenic mouse model. This was associated with an increased level of CEA-specific CD4(+) and CD8(+) T cell responses, induction of antigen spreading to p53 and gp70, increased accumulation of CEA-specific T cells in the tumor microenvironment, and increased expression of bcl-X(L) and bcl-2 in CD4(+) and CD8(+) T cells in vaccinated mice. CONCLUSION: 4-1BBL cooperates with B7 in enhancing anti-tumor and immunologic responses in a recombinant poxvirus vaccine model. The inclusion of costimulatory molecules targeting distinct T cell signaling pathways provides a mechanism for enhancing the therapeutic effectiveness of tumor vaccines.