This renewal application is focused on the investigation of afferent immune cells that make the first in vivo contact with tumor cells. Tumor cells have been gene modified to increase their in vivo immunogenicity and to serve as gene delivery devices for the in vivo transduction of pre-immune cells. The feasibility of this method has been demonstrated in the preliminary result section by the generation and injection of retroviral vector packaging tumor cell lines capable of in vivo transducing cells in syngeneic mice. Transduced cells subsequently accumulate in the regional lymph node. In vivo transduction of tumor-specific T-cells by retroviral vector packaging tumor cells will be used to pursue three goals. First, in vivo transduced and thereby gene marked, tumor specific T-cells will be analyzed. The investigator expects that this novel procedure of marking tumor-specific pre-effector cells will significantly contribute to our understanding of the afferent limb of immunity as it occurs in vivo. In particular, the factors influencing the phenotype of transduced cells and their survival are of interest. The influence of the microenvironment provided by the tumor, e.g. by expression of B7 or production of cytokines, on the nature of transduced cells will be determined. The influence of the macroenvironment provided by the host, e.g. Fas, Fas-ligand or perforin deficiency, on transduced T-cell survival will be evaluated. Second, a novel method will be tested to control gene activity of genes previously transduced into tumor-specific T-cells by pharmacological means. The transduced gene product, to be activated by drug administration in vivo is the intracellular domain of CD28 fused to the drug responsive gene product, gyrase B or FK506 binding protein 12. The pharmacological drugs to be used to induce CD28 signaling are coumermycin or the dimer of FK506, FK1012. Third, in vivo activation of CD28 in combination with a CTLA-4 blocker or with apoptotic protease blocking genes expressed on a dicistronic transcript will be used to test the hypothesis that apoptosis is the rate limiting event in tumor immunity in vivo.
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