Maintenance of antigen specific T cells in vitro requires periodic stimulation with specific antigen presented by appropriate APC, and the presence of IL-2, T cell growth factor. An early result of T cell antigen receptor (TCR) occupation by antigen is the rapid hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate, liberating the second messengers inositol trisphosphate and sn- 1,2-diacylglycerol. Diacylglycerol, in synergy with the increase in intracellular calcium induced by inositol triphosphate, activates the serine-threonine-specific protein kinase C (PKC). We have previously shown that constitutive expression and activation of PKCgamma in fibroblasts results in increased proliferation rate in vitro, increased saturation density and lowered requirement for growth factors. This prompted us to constitutively express PKC in antigen specific T cell clones in order to test its growth promoting effect on these cells, and to explore the possibility that T cells might behave as if their antigen receptors were perpetually stimulated by antigen. T cells which constitutively expressed PKC had stable high level expression of IL-2 receptors, increased proliferation rate, increased longevity in vitro, and complete independence of antigen, while maintaining strict antigen specificity and effector function. In the studies proposed here we plan to: I. Use newly constructed amphotropic retroviral vectors to transduce PKCgamma, as well as alpha and beta genes, into human anti-tumor effector T cells and evaluate in vitro what effect constitutive overexpression of these genes has on proliferation, longevity and anti-tumor effector function of these cells. II. Use PKC transduction to facilitate isolation of tumor specific T cell clones from tumor patients peripheral blood, lymph nodes or tumor samples. III. Use a well established animal tumor model of adoptive immunotherapy to test the effect of PKC transduction into T cell lines and clones used to mediate immunotherapy. IV. Construct new retroviral vectors which contain a suicide gene capable of destroying the transfected effector cells, once they have accomplished their immunotherapeutic function, for future application of this method in human clinical trials of adoptive immunotherapy of cancer.
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