1. Protein kinase C and cytokine regulation of CD3-AK-response induced by alphaCD3: The CD3-AK model is used to determine the biological function of PKC isozymes in T cell activation. After screening the effect of a panel of different PKC antagonists on the alphaCD3-induced proliferation (PR) and cytotoxic response (PR), they were shown to give differential effect on the PR and CR, suggesting that different PKC isozymes are inhibited by these different antagonists. To further determine the production of PKC isozymes by western blot and the expression of PKC mRNA by northern blot will allow us to determine which isozyme(s) is responsible for regulating proliferation or generation of killer cells. 2. Glutathione regulation of IL-4 dependent activated killer cells: Glutathione (GSH) regulates the activation and differentiation of IL-4 dependent CD3-AK cells. BSO, a GSH synthetase inhibitor, decreases both the proliferation and cytolytic activity of the killer cells. BSO does not affect the production and exocytosis of the cytolytic granules which mediate the lytic reactions. These results suggest that the reduction of intracellular GSH which regulates the thiol enzyme activity will impair the ubiquitination pathway whose function is critically dependent on thiol enzymes. Thus reduced cellular GSH leads to disruption of normal cell cycle and production of defective cytolytic granules, which results in reduced proliferation and reduced killer cell activity. 3. Tumor cell-induced immunosuppression: Tumor cell/cell free products suppress CD3-AK response. IL-4 or PKC antagonists alone partially restores the response and the combination of the two may fully restore the response. These studies should help to devise a strategy to override the tumor-induced immunosuppression.
