Search

Cytotoxic T (Tc) cells were generated in mice of five H-2 haplotypes against the flaviviruses Kunjin and West Nile (WNV). A panel of recombinant vaccinia viruses which between them expressed cDNA of the entire Kunjin virus genome were used to infect targets. Anti-Kunjin virus responses to determinants derived from non-structural proteins, especially NS3, NS4A and NS4B, were dominant in most mouse strains; usually only one class I major histocompatibility complex (MHC) restriction element was involved. WNV-immune Tc cells showed similar but not identical patterns of antigen recognition to Kunjin virus-immune Tc cells. The extent to which WNV-immune Tc cells recognized Kunjin virus-encoded determinants varied considerably between mice of different MHC haplotypes.

A series of recombinant vaccinia viruses expressing various parts of the entire Kunjin virus (KUN) coding region was used to analyse the cytotoxic T (Tc) cell responses to KUN. CBA/H mice inoculated with KUN or West Nile virus were shown to develop responses to KUN or various vaccinia virus expression constructs in either primary cytotoxic assays, or after secondary stimulation of the Tc cells in vitro with KUN antigens. Tc cells from CBA mice showed the strongest response to target cells infected with recombinant vaccinia viruses expressing parts of the KUN NS3 and NS4A proteins, and only a weak response to the other structural or non-structural proteins. Further analysis of deleted versions of the NS3-NS4A region showed that the main epitope recognized was derived from a sequence of 99 amino acids spanning parts of NS3 and NS4A. No other major epitopes were detected by Tc cells from CBA mice in the remaining 3333 amino acids of the KUN polypeptide.

Murine cytomegalovirus (MCMV)-immune lymph node (LN) cells were generated following 4 days culture of draining popliteal LN cells removed after bilateral hind footpad inoculation of mice with 4 X 10(2) p.f.u. MCMV. This cell population expressed both cytotoxic activity against MCMV-infected mouse embryo fibroblasts (MEF) and the capacity to release lymphokine upon stimulation with MCMV-infected MEF. Cytotoxicity and lymphokine release were Class I, H-2-restricted, virus-specific and dependent upon Thy1. 2+, Lyt2+ cells. Mixing of 5 X 10(5) MCMV-immune T cells with an excess of syngeneic MCMV-infected MEF stimulators produced detectable levels of the lymphokine interleukin-3 by 1 h, with a maximum rate of its release between 1 and 6 h and plateau levels by 14 h. The capacity to stimulate virus-specific MCMV-immune T cells was acquired by MEF at 1 h after MCMV infection with maximum stimulator ability attained by 8 h. In the presence of a fixed number of MCMV-immune T cells, and titration to excess of syngeneic 3 h or 18 h MCMV-infected MEF, lower interleukin-3 plateau levels were obtained with 3 h than with 18 h MCMV-infected stimulators. This suggested that fewer T cells responded to 3 h than to 18 h MCMV-infected MEF.

A cytotoxic response to murine cytomegalovirus (MCMV) was obtained by the culture of lymph node cells from mice inoculated with MCMV into both hind footpads 7 days previously. The cytotoxicity was mediated by Thy1.2+, Lyt2+, H-2-restricted effector cells and was virus-specific. Investigation of the in vitro conditions established that T cell proliferation was necessary for optimal generation of cytotoxicity, that proliferation was dependent upon Thy1.2+, Lyt2+ cell populations and that supernatants from concanavalin A-activated spleen cells enhanced the levels of cytotoxicity obtained.

Inflammatory cells were isolated from West Nile virus (WNV)-infected CBA/H (H-2k) mouse brains, and their function and cell surface markers were studied. Two populations of cytolytic lymphocytes were detected. One population, which lysed WNV-infected and uninfected L929 (H-2k), YAC-1 (H-2a), P815 (H-2d), OH (H-2KdDk) and 2R (H-2KkDb) target cells, had a phenotype of L3T4- Lyt2- Thy1 +/- asialo GM1+ and hence were natural killer (NK) cells. The other, which lysed WNV-infected cells to a greater extent than uninfected L929 cells, had a phenotype of L3T4- Lyt2+ Thy1+ asialo GM1- and were cytotoxic T cells. In addition, the presence of the latter population was demonstrated by the specific lysis of syngeneic WNV-infected astrocytes, a cell type which is resistant to NK cell lysis. The cell surface markers of isolated inflammatory cells were determined by two colour flow cytometry. This showed that 15 to 40% of the cells were Thy1+, among which about 3% were Lyt2+. No L3T4+ cells were detected.

Combined in vivo and in vitro protocols for the generation of anti-murine cytomegalovirus (anti-MCMV) cytotoxic responses were investigated using BALB/c mice and syngeneic mouse embryo fibroblast target cells. Injections of doses of MCMV from 10(2.6) up to 10(6.1) p.f.u. into the hind footpad, harvest of draining popliteal lymph node cells after 6 to 8 days, followed by 4 days culture of these cells gave similar and optimal cytotoxic activity against MCMV-infected target cells. After injection of 10(4.6) p.f.u. into the hind footpad, MCMV was detectable at about 10(3) to 10(4) p.f.u. per popliteal node after 3 days, but became undetectable in most animals by 6 days. Cell numbers in the draining popliteal lymph node increased following MCMV inoculation into the hind footpad, but the extent of the increase was inversely related to virus dose and bore no relationship to the anti-MCMV cytotoxic potential of the cells. Lymph node cells applied directly to target cells upon harvest from MCMV-infected mice never gave detectable anti-MCMV cytotoxic activity at 2, 4, 6, 8, 10, 12, 17 or 19 days post-infection, but lysis of uninfected syngeneic targets was obtained 4 to 8 days post-infection.

A protocol for obtaining cytotoxic T cell responses to the flavivirus West Nile (WNV) has been developed in vivo. CBA/H (H-2k) mice were immunized with 10(6) 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-2k) 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(3) 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(6) 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.