The activation of T cells has been shown to require at least two signals via molecules present on professional antigen-presenting cells: signal 1 via a peptide/MHC complex, and signal 2 via a costimulatory molecule. Studies are being conducted on the impact of hyperexpression of multiple costimulatory molecules in the activation of T cells. Pox virus vectors (vaccinia and avipox) are being used because of their ability to efficiently express multiple transgenes. Murine tumor cells provided with signal 1 and infected with either recombinant vaccinia or avipox vectors containing a TRIad of COstimulatory Molecules (B7-1/ICAM-1/LFA-3, designated TRICOM) induce the activation of T cells to a far greater extent than cells infected with any one or two costimulatory molecules. Despite this T-cell hyperstimulation using TRICOM vectors, no evidence of apoptosis above that seen using the B7-1 vector was observed. Experiments using a four-gene construct have shown that TRICOM recombinants can enhance antigen-specific T-cell responses in vivo. Studies are also ongoing to determine if dendritic cells infected with these TRICOM vectors are rendered more efficient in enhancing T-cell responses. Peptide-pulsed dendritic cells infected with rF-TRICOM or rV-TRICOM induced cytotoxic T-lymphocyte activity in vitro and in vivo to a markedly greater extent than peptide-pulsed dendritic cells. These studies thus demonstrate the ability of vectors to introduce three costimulatory molecules into cells, thereby activating T-cell populations to levels far greater than those achieved with the use of one or two costimulatory molecules. This new threshold of T-cell activation in vaccine design is currently being studied.