Gamma interferon transduced 9L gliosarcoma. Cytokine gene therapy and its relevance to cellular therapy with alloreactive cytotoxic T lymphocytes.

Abstract

In earlier studies, we demonstrated that intratumoral infusions of alloreactive cytotoxic T lymphocytes (aCTL), sensitized to the major histocompatibility complex (MHC) antigens of the host, effectively retarded the intracranial growth of Fischer 9L gliosarcoma. We further demonstrated that continuous in vitro exposure to gamma-interferon (gammaIFN) upregulates MHC on 9L gliosarcoma cells and that they were better targets of anti-Fischer aCTL. We hypothesized that the efficacy of cellular therapy with aCTL could be further improved by in situ transduction of the tumor with retroviral vectors coding for gammaIFN, which would generate continuous secretion of the cytokine and maintain upregulated MHC expression by the tumor cells. 9L gliosarcoma and Herpes simplex virus thymidine kinase (tk) transductants of those cells were transduced with a retrovirus carrying the murine gammaIFN gene. By limiting dilution, clones of these cells, designated 9Lgamma 7, 9Lgamma tk8, and 9Lgamma tk10, which produced similar levels of gammaIFN (383-411 ng gammaIFN/10(6) cells/24 h) were isolated. The production of gammaIFN by one clone, 9Lgamma 7, was stable when monitored over 6 weeks in vitro. The clones also demonstrated upregulated MHC class I expression, and the tk-transduced clones maintained their sensitivity to ganciclovir. Compared to the wildtype cells, 9Lgamma 7 had approximate 6- and 1.5-fold increases in the relative antigen densities of MHC I and II, respectively. Addition of exogenous gammaIFN to 9Lgamma 7 cultures did not significantly increase the MHC expression. In cytotoxicity assays, 9Lgamma 7 cells, or 9Lgamma 7 incubated with exogenous gammaIFN, were better targets of aCTL than the parental 9L cells. The growth rate of 9Lgamma-transduced cells was decreased compared to the wildtype cells both in vitro and in vivo. Proliferation studies with transwell plated 9L, 9Lgamma 7, and 9Lgamma tk10 cells in various combinations revealed that the secreted cytokine itself caused a decrease in proliferation. However, the transduced cells exhibited a much reduced growth rate, which likely was a consequence of redirected metabolic activity of the cells. In vivo growth of the 9L and 9Lgamma 7 tumors in rat brains given identical inoculums similarly demonstrated significantly reduced 9Lgamma 7 tumor volumes at various timepoints, indicative of slower growth of the gammaIFN-producing tumors.