Augmenting immunogenicity by genetically modifying tumor cells to express costimulatory molecules has proven to be a promising therapeutic strategy in murine tumor models and is currently under investigation in human clinical trials for metastatic cancer. However, there are significant technical and logistic problems associated with implementing strategies requiring direct gene modification of primary tumor cells. In an effort to circumvent these problems, we are developing a strategy in which the costimulatory signal required for tumor-specific T lymphocyte activation is provided by a genetically modified human fibroblast (trans-costimulation). We have evaluated the efficiency of CD80- and CD86-mediated trans-costimulation in the activation of human CD8+ and CD4+ T lymphocytes in MHC class I- and class II-restricted lymphoproliferation reactions. Our studies demonstrate that the efficiency of CD80- or CD86-mediated trans-costimulation of purified human CD8+ and CD4+ T lymphocytes is comparable to cis-costimulation under defined conditions. Moreover, a dose-response relationship consistent with the predicted two-hit kinetics of the reaction was evident in trans-costimulation reactions in which the ratio of target cells expressing either signal 1 or signal 2 was varied incrementally from 1:10 to 10:1. Importantly, the level of cell-surface CD86 required for trans-costimulation is equivalent to that constitutively expressed by human peripheral blood monocytes. These results may have significant implications for the clinical implementation of this type of cancer immunotherapy and also raise questions about the possibility of trans-costimulating autoreactive T lymphocytes in vivo.