Generation of Ag-specific cytotoxic T lymphocytes by DC transfected with in vitro transcribed influenza virus matrix protein (M1) mRNA.

Abstract

Application of DC transfected with tumor Ag RNA is promising for DC-based tumor immunotherapy. In this study, Ag-specific cytotoxic T lymphocytes (CTL) were generated by priming lymphocytes with DC transfected with in vitro transcribed (IVT) influenza virus matrix protein M1 (M1) mRNA. Human UC blood-CD34+ cell-derived DC were transfected with IVT mRNA encoding either the enhanced green fluorescence protein (EGFP), or M1 by square-wave electroporation. DC were confirmed to have typical morphology and phenotype. DC transfected with IVT EGFP mRNA were analyzed with the FACScan flow cytometer, to confirm the efficiency of this transfection method. On Days 7, 14, 21 and 28 after the start of DC culture, DC were harvested and electroporated with M1 mRNA. The transfected DC were co-cultured with autologous UC blood CD34- cells. One week after the fourth priming of autologous CD34 negative cells with M1 mRNA electroporated DC, Ag-specific CTL activity was evaluated. To prepare target cells, M1 mRNA was added to autologous DC 48 h prior to CTL assays. Our CTL assays results indicate that UC blood CD34+ cell-derived DC transfected with M1 mRNA by electroporation stimulated Ag-specific CTL responses that are capable of recognizing and lysing autologous DC loaded with M1 mRNA. M1 mRNA transfected DC-primed CTL showed a significant cytotoxic activity against M1 mRNA loaded autologous DC, while nearly baseline cytotoxic activity was recorded for the M1 mRNA unloaded DC. Our results showed that mRNA-transfected DC are potent stimulators of T-cell immunity in vitro. In addition, mRNA-loaded DC can function as targets in CTL cytotoxicity assays, which offer a practical substitute for tumor cells in assays to test the immunological effects of specific Ags.