Institute of Virology and Immunobiology, University of Würzburg, Versbacher Str. 7, 97078 Würzburg, Germany1
Institute of Pathology, University of Würzburg, Würzburg, Germany2
Centre for Peptide and Protein Engineering, Queens University of Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK3
Author for correspondence: Stefan Niewiesk. Fax +49 931 201 3934. e-mail niewiesk{at}vim.uni-wuerzburg.de
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Abstract |
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Introduction |
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In this paper, we have correlated the mortality of mice after intracerebral infection with virus spread and virus replication in the brain as well as with T cell activation. In addition, the contribution of primary as well as secondary CD4+ and CD8+ T cells for protection was evaluated in BALB/c, BaCF1 and C3H mice.
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Methods |
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Recombinant vaccinia viruses.
Recombinant vaccinia viruses (vvR) encoding the peptides LDRLVRLI (from MV Edmonston strain nucleocapsid protein aa 5259; vvL5259), VESPGQLI (MV nucleocapsid protein aa 8188; vvL8188) or YPALGLHEF (MV nucleocapsid protein aa 281289; vvL281289), bound to the HA-1 leader sequence to ensure peptide transport independent of TAP, have been described previously (Neumeister & Niewiesk, 1998 ).
Peptides.
The peptides S1 (Ac-ESPGQLIQRITDDPDVS-NH2; aa 8298) and S1v (Ac-ESPGQLIQRITDDP_VS-NH2; aa 8298 without aa 96) were synthesized by solid-phase methods using the Fmoc strategy on an automated peptide synthesizer (Fields & Noble, 1990 ). The peptides were purified by HPLC and the correct mass in each case was observed by fast atom bombardment mass spectroscopy.
Infection and immunization of mice.
Mice were infected intracerebrally with 2x104 TCID50 MV strain CAM/RBH in a 20 µl volume. Infected animals were weighed and monitored daily for clinical signs. For the generation of CD8+ T cells, C3H mice were infected intraperitoneally (i.p.) with 15x106 p.f.u. of either vvL5259 or vvL8188 with vvL281289 as a control. BALB/c mice were immunized i.p. with 5x106 p.f.u. of MV strain Edmonston (MV-Ed) and C3H mice were immunized i.p. with 5x105 p.f.u. MV-Ed. For peptide immunization, C3H mice were injected with 100 µg peptide emulsified in Freunds complete adjuvant.
Depletion of T cell subsets.
Supernatants from hybridomas YTS 191 (specific for mouse CD4) and YTS 169 (specific for mouse CD8) were purified by using a Sepharose-G column (Pharmacia) and dialysed against PBS. The amount of antibody was determined with the Bio-Rad protein assay and the effectiveness of the in vivo depletion of T cell subsets was confirmed by flow cytometry with monoclonal antibodies (MAbs) L3T4 (Pharmingen) for CD4+ T cells and MCA 609 G (Serotech) for CD8+ T cells. Mice were depleted by i.p. injection of 0·25 mg MAb 24 h prior to infection. Depletion was repeated every fourth day. This scheme of depletion was monitored by flow cytometry. Within the limit of detection (2%), no T cells of the respective T cell subset were found on days 1 and 7 after a single injection of MAb. In previous experiments, the injection of isotype-matched MAb of unrelated specificity did not influence the CD4+ and CD8+ T cell subsets.
Adoptive transfer.
For adoptive transfer, virus-specific T cells were purified by using nylon wool columns as described previously (Julius et al., 1973 ). Briefly, spleen cell suspensions were loaded onto nylon wool columns at a density of 5x107 cells/ml in Hanks balanced salt solution (HBSS) containing 5% FCS and incubated for 45 min at 37 °C. The columns were washed with two column volumes of warm (37 °C) HBSS/5% FCS and the cells in the effluent were pelleted at 300 g for 15 min at 4 °C. The efficiency of purification was determined by flow cytometry. The preparations always contained less than 4% Ig-bearing cells. One spleen equivalent of the pelleted cells was resuspended in 100 µl PBS/0·1% FCS and injected into the tail vein of the recipient animal. Immediately after transfer, the animals were depleted of CD8+ or CD4+ T cells.
Generation and culture of T cells and cytotoxicity and proliferation assays.
Generation and culture of CD4+ and CD8+ T cells as well as the cytolytic assay for CD8+ T cells have been described previously (Niewiesk et al., 1993 ; Neumeister & Niewiesk, 1998
). For direct ex vivo proliferation assays, single-cell suspensions of spleen cells were plated in triplicate at 5x105 cells per well into 96 well flat-bottom plates in RPMI 1640/1% mouse serum with or without 2·5 µg/ml gradient-purified, UV-inactivated MV. After 2 days, cultures were labelled with [3H]thymidine for 1620 h and harvested as described previously (Niewiesk et al., 1993
).
The stimulation index (SI) was calculated as the ratio of c.p.m. of MV-stimulated cells to that of medium controls.
Virus titration.
Brains of infected mice were removed aseptically and passaged through steel sieves. Brain cell suspensions were serially diluted in MEM/5% FCS and incubated overnight on Vero cells in 48 well plates. After three washes with PBS/0·1% FCS, cells were cultured in MEM/5% FCS for 8 days before virus titres were determined. The titre was calculated according to the method of Spearman and Kärber (Kärber, 1931 ).
In situ hybridization.
MV-specific RNA was localized in brain sections with 35S-labelled RNA probes as described previously (Czub et al., 1996 ). 35S-labelled probes were obtained by in vitro transcription of the MV-specific pGEM-N clone (Cattaneo et al., 1987
) in the anti-mRNA orientation with T7 RNA polymerase (Boehringer).
Paraffin sections (5 µm) were refixed in PBS/5% paraformaldehyde (pH 7·4) for 15 min. Slides were acetylated in 0·1 M triethanolamineHCl buffer (pH 8·0) (Sigma) twice for 5 min and subsequently incubated in triethanolamine buffer/0·25% acetic anhydride. After washing in 2x SSC and dehydration, slides were denatured in deionized formamide/5% SSC (0·1x) at 65 °C for 15 min, drenched in cold 0·1x SSC and then subjected to prehybridization for 34 h. Prehybridization solution contained 2xSSC, 50% formamide and 1x Denhardts solution with 200 µg/ml tRNA as a carrier at 45 °C for 3 h.
Hybridization was performed at 45 °C for 16 h in a probe cocktail (0·3 M NaCl, 10 mM TrisHCl, pH 7·4, 1 mM EDTA, 1 mg/ml BSA, 0·02% Ficoll, 0·02% polyvinylpyrrolidone, 5 mM DTT, 50% formamide, 10% dextran sulphate with or without tRNA) containing MV nucleocapsid protein (N)-specific ssRNA (approximately 106 c.p.m. 35S-labelled riboprobe/µl). After hybridization, the slides were washed twice in 2xSSC (with 5 mM DTT) for 30 min followed by two washes for 30 min at 60 °C in 2xSSC (plus 0·1% Triton X-100, 1 mM EDTA and 5 mM DTT). Slides were treated for 40 min at 37 °C with 40 µg/ml RNase A and 10 U/ml RNase T1 in 10 mM TrisHCl (pH 7·5), 0·3 M NaCl and 5 mM DTT, followed by washing for 30 min at 60 °C in 2xSSC with 0·1% Triton X-100, 1 mM EDTA and 5 mM DTT. After dehydration, the slides were exposed to a photographic emulsion (Ilford K2 nuclear tract) for 24 weeks at 4 °C. After development, the slides were counterstained with haematoxylin and mounted permanently in xylene-soluble medium.
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Results |
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Discussion |
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Although CD8+ T cells play a dominant role in lymphocytic choriomeningitis virus and influenza A virus infection, they can be substituted for by CD4+ T cells (Muller et al., 1992 ; Eichelberger et al., 1991
). However, virus clearance by CD4+ T cells usually is less efficient, with delayed kinetics. In contrast, in MVE, CD4+ T cells are the most important T cell subset for protection. CD4+ T cells alone are protective in the resistant BALB/c mouse (Finke & Liebert, 1994
), whereas the primarily non-protective CD4+ T cell response in the C3H mouse can be rendered protective by peptide immunization. Although CD4+ T cells of BALB/c mice are highly lytic, experiments with mice unable to lyse target cells due to a mutation in the Fas molecule (gld mutant) demonstrate that Fas-dependent lysis does not contribute to protection (data not shown). Therefore, other factors seem to be involved in CD4+ T cell-mediated protection. It has been shown that antibody-mediated neutralization of IFN-
leads to breakdown of resistance in BALB/c mice (Finke et al., 1995
). As IFN-
has pleiotropic effects (e.g. direct antiviral activity, influence on antigen processing and presentation and migration), it is currently not clear which of these are important in overcoming MVE. In C3H and BaCF1 mice, CD4+ T cells are not protective alone but are able to provide help for a primary CD8+ T cell response (in BaCF1 mice). The lytic capacity of CD8+ T cells seems to be important for this T cell collaboration to succeed. Only Ld-restricted CD8+ T cells, which lyse MV-infected target cells well in vitro, confer protection after adoptive transfer (Niewiesk et al., 1993
; Neumeister & Niewiesk, 1998
). The induction of Kk-restricted CD8+ T cells, which do not lyse MV-infected target cells in vitro, does not lead to protection. Whether qualitative differences in T cell responses that depend on the immunogenetics of an individual generally play an important role in the outcome of virus diseases is not yet clear. A pattern that is probably similar to the observations made in the mouse MVE model is seen in human immunodeficiency virus infection. Patients that are able to control this infection for many years (long-term survivors) have been shown to generate strong CD8+ and CD4+ T cell responses, indicating that the balance between resistance and disease might depend on the composition of the respective T cell subsets (Rosenberg et al., 1999
; Kalams & Walker, 1998
).
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Acknowledgments |
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References |
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Received 19 April 2000;
accepted 8 August 2000.