A protocol for obtaining cytotoxic T cell responses to the flavivirus West Nile (WNV) has been developed . CBA/H (H-2) mice were immunized with 10 p.f.u. WNV intravenously and their spleen cells used directly in cytotoxic assays. This method reliably produced WNV-immune Tc cells which showed WNV-specific cytotoxic activity on infected L929 (H-2) target cells. There was inadequate lysis of infected targets by WNV-immune spleen cells when the m.o.i. was less than 100 p.f.u. WNV, or when tertiary mouse embryo fibroblasts, resident peritoneal macrophages or thioglycollate-induced peritoneal macrophages were used as targets. Only L929 cells infected for 16 h with WNV at a m.o.i. of 100 were suitable targets. Cytotoxic activity against WNV-infected target cells was first detected 4 days after immunization, peaked on day 5 and declined rapidly after day 7. An immunizing dose of 10 p.f.u. of WNV was adequate for significant cytotoxicity to be detected; however, the cytotoxic response increased with increasing immunizing doses to plateau levels when 10 p.f.u. WNV were used. The cells responsible for lytic activity were H-2-restricted, Thy-1, Lyt-2, L3T4 and virus-specific with respect to WNV and influenza virus.


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