Role of CD4+ and CD8+ T cells in the prevention of measles virus-induced encephalitis in mice

Gerald Weidinger1, Stefanie Czub2, Claudia Neumeister1, Pat Harriott3, Volker ter Meulen1 and Stefan Niewiesk1

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, Queen’s 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


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Depending on their major histocompatibility complex (MHC) haplotype, inbred mouse strains are either resistant (H2-d, BALB/c), susceptible (H2-k, C3H) or partially resistant (H2-dxk, BaCF1) to intracerebral infection with the neurotropic rodent-adapted measles virus (MV) strain CAM/RBH. Here, mortality is demonstrated to be correlated directly with virus spread and virus replication in the CNS and to be inversely correlated with the activation of MV-specific T cells. Previously, it has been shown that primary CD4+ T cells alone are protective in the resistant background. In the susceptible background, CD4+ T cells acquire protective capacity after immunization with a newly defined CD4+ T cell epitope peptide. In the partially resistant mice, CD4+ T cells provide help for CD8+ T cells and protect in cooperation with them. It seems that the lytic capacity of CD8+ T cells is crucial in providing protection, as MV-specific Ld-restricted CD8+ T cells, which are highly lytic in vitro after transfer, protect naive animals against MV-induced encephalitis (MVE). In contrast, Kk-restricted CD8+ T cells with low lytic capacity do not protect. In the MVE model, CD4+ T cells are able to protect either alone (resistant mice), through cooperation with CD8+ T cells (intermediate susceptible) or after immunization as secondary T cells (susceptible mice). CD8+ T cells are able to protect alone after immunization if they are cytolytic. Thus, susceptibility and resistance depend upon the functional composition of CD4+ and CD8+ T cells governed by the MHC haplotype.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
In the mouse model of measles virus (MV)-induced encephalitis (MVE), susceptibility and resistance depend on the major histocompatibility complex (MHC). Mice with the H2-d haplotype (e.g. BALB/c mice) are resistant, mice with the H2-k haplotype (e.g. C3H mice) are susceptible and animals of the F1 generation (BaCF1) are partially resistant. BALB/c mice generate a rigorous cytotoxic T cell response against MV-infected target cells whereas C3H mice do not (Niewiesk et al., 1993 ). In contrast, CD8+ T cells from both mouse strains recognize target cells infected with a recombinant vaccinia virus expressing the MV nucleocapsid protein (vvN) (Neumeister & Niewiesk, 1998 ). The difference in recognition is due to the fact that Ld (the restriction element of BALB/c mice) binds MV epitope peptide 100 times better than does Kk, the restriction element of C3H mice (Neumeister & Niewiesk, 1998 ). The low binding ability of Kk-restricted epitope peptides explains the difference in recognition between MV- versus vvN-infected target cells. Whereas MV replicates abortively in mouse cell lines, vvN produces large amounts of the nucleocapsid protein and consequently epitope peptide, permitting lysis by Kk-restricted CD8+ T cells. In vivo, however, depletion of CD8+ T cells by monoclonal antibody does not affect resistance in BALB/c mice (Finke & Liebert, 1994 ). In contrast, depletion of CD4+ T cells leads to breakdown of resistance. This indicates that CD4+ T cells act as effector T cells in eliminating MV, probably through their lytic capacity (Niewiesk et al., 1993 ). Apparently, CD4+ T cells from C3H mice lack the effector function, as they do not protect against MVE. However, as the state of resistance comprises three modes (resistant, partially resistant and susceptible), this cannot simply be explained by the function of CD4+ T cells alone.

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.


   Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Mice.
BALB/cOlaHsd (BALB/c), with the MHC haplotype H2-d, BALB/kOlaHsd (BALB/k) and C3H/HeNHsd (C3H) mice (both H2-k), bought from Harlan Winkelmann (Borchem, Germany), were specific pathogen free (specification according to the breeder). The F1 generation of BALB/c and C3H mice (BaCF1), with the MHC haplotype H2-dxk, was bred at the Institute of Virology and Immunobiology. Every 3–4 months, animals were checked for pathogens by serological examination. Animals were kept in a barrier system with light negative pressure (150 MPa) and a 12 h day (artificial light) and were fed and watered ad libitum. Room temperature (21±2 °C) and humidity (55±5%) were regulated by air conditioning. Mice were used between the ages of 6 and 18 weeks.

{blacksquare} Recombinant vaccinia viruses.
Recombinant vaccinia viruses (vvR) encoding the peptides LDRLVRLI (from MV Edmonston strain nucleocapsid protein aa 52–59; vvL52–59), VESPGQLI (MV nucleocapsid protein aa 81–88; vvL81–88) or YPALGLHEF (MV nucleocapsid protein aa 281–289; vvL281–289), bound to the HA-1 leader sequence to ensure peptide transport independent of TAP, have been described previously (Neumeister & Niewiesk, 1998 ).

{blacksquare} Peptides.
The peptides S1 (Ac-ESPGQLIQRITDDPDVS-NH2; aa 82–98) and S1v (Ac-ESPGQLIQRITDDP_VS-NH2; aa 82–98 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.

{blacksquare} 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 1–5x106 p.f.u. of either vvL52–59 or vvL81–88 with vvL281–289 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 Freund’s complete adjuvant.

{blacksquare} 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.

{blacksquare} 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.

{blacksquare} 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 16–20 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.

{blacksquare} 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 ).

{blacksquare} 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 triethanolamine–HCl 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 3–4 h. Prehybridization solution contained 2xSSC, 50% formamide and 1x Denhardt’s 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 Tris–HCl, 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 Tris–HCl (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 2–4 weeks at 4 °C. After development, the slides were counterstained with haematoxylin and mounted permanently in xylene-soluble medium.


   Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Mortality after intracerebral MV infection correlates with virus spread, virus replication and T cell activation
After intracerebral infection with the neurotropic MV strain CAM/RBH, susceptible mice succumb to infection between day 5 and day 9 (Niewiesk et al., 1993 ). To investigate virus spread within the CNS, brains from resistant BALB/c mice (H2-d) and susceptible BALB/k mice (H2-k) were investigated for the presence of virus by in situ hybridization for MV nucleocapsid mRNA at 5, 6 and 7 days post-infection. In resistant mice, virus was found close to the site of injection in the cortex and hippocampus (Fig. 1b, d) and single neurons were shown to be infected. In contrast, in susceptible mice, virus was present throughout the brain in cortex, hippocampus and thalamus of both hemispheres (Fig. 1a, c) and more neurons were infected. Virus titres obtained by virus reisolation from the brains of infected animals from day 3 to day 12 confirmed these data. Virus was reisolated from brain tissue of susceptible C3H mice from day 4 to day 8 (17 of 24 animals) with titres between 102 and 104 TCID50. No virus was detectable on day 10 or day 12, after the acute infection. Virus was found in brain tissue of only two of 17 resistant BALB/c mice at titres lower than 103 TCID50 (Fig. 2a). From day 4 to day 7, a strong MV-specific CD4+ T cell proliferation (SI between 4 and 7) was found in spleen cells of resistant BALB/c mice, whereas the T cell response from spleen cells of susceptible C3H mice was below the threshold of detection (Fig. 2b). To confirm the causal relationship between virus replication and T cell activation, C3H mice were immunized before challenge. One week after immunization with MV, C3H mice were protected against intracerebral challenge with the neurotropic MV strain CAM/RBH and all 10 animals survived. No virus could be reisolated from immunized animals on days 5 or 7 (Fig. 2a) and a strong MV-specific T cell proliferation was observed from spleen cells of immunized C3H mice on day 4 (Fig. 2b). These data suggest that mortality correlates with virus spread and replication in brain tissue of infected mice and is inversely correlated with CD4+ T cell activation.



View larger version (169K):
[in this window]
[in a new window]
 
Fig. 1. Comparison of virus spread in brain tissue from resistant and susceptible mice. Brain tissue from infected resistant BALB/c (H2-d) mice (b, d) and susceptible BALB/k (H2-k) mice (a, c) on day 7 after infection was subjected to in situ hybridization with an MV nucleocapsid protein-specific probe. Part of the left hemisphere (a, b) with infected neurons (arrows) and (in higher magnification of the same area) infected neurons within the cortex (c, d) are shown. Magnification x25 (a, b), x100 (c, d).

 


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2. Virus replication in CNS and MV-specific T cell activation. After intracerebral infection of naive BALB/c and C3H mice and MV-immune C3H mice, virus titres (a) and T cell proliferation (b) were determined. The threshold of detection for the titration assay is 102 TCID50 and an SI of >2 signifies MV-specific T cell proliferation. In (a), symbols represent C3H mice ({triangleup}), BALB/c mice ({bullet}) and immunized C3H mice ({diamondsuit}). In (b), because UV-inactivated MV was used, T cell proliferation reflects the growth of CD4+ T cells. Bars (light shaded, BALB/c; filled, C3H; heavy shaded, immunized C3H) represent the means (±SD) of five mice per group and time-point. nd, Not done.

 
Secondary CD4+ T cells are able to protect C3H mice against MVE
Immunization of C3H mice with MV leads to activation of CD4+ T cells as well as CD8+ T cells and antibodies. To demonstrate that CD4+ T cells alone are able to protect C3H mice, MV-specific stimulation with a CD4+ T cell epitope was attempted. Amino acids 77–98 of the MV nucleocapsid protein have been reported previously to contain a CD4+ T cell epitope (Giraudon et al., 1992 ). In proliferation studies with overlapping peptides, we found that CD4+ but not CD8+ T cell proliferation was stimulated by peptide S1 (aa 82–98) (data not shown). After immunization with peptide S1, C3H mice were protected against MVE (Table 1). To confirm the specificity of protection, mice were immunized with a variant S1 peptide lacking aa 96 (S1v). Immunization with S1v did not protect against MVE. The efficiency of immunization with S1 was compared to adoptive transfer of secondary MV-specific CD4+ T cells. The same level of protection was achieved after transfer of CD4+ T cells and immunization with S1 peptide.


View this table:
[in this window]
[in a new window]
 
Table 1. Peptide immunization with a CD4+ T cell epitope peptide protects C3H mice against MVE

 
Primary CD8+ T cells require CD4+ T cell help to overcome CNS infection with MV
Whereas BALB/c mice are resistant and C3H mice are susceptible to MVE, the F1 generation is intermediately susceptible. As an intermediate phenotype cannot be explained by the function of CD4+ T cells alone, the contributions of CD4+ and CD8+ T cells for protection against MVE were analysed. The depletion of either CD4+ or CD8+ T cells rendered BaCF1 mice susceptible to MVE (Fig. 3). This demonstrated that CD4+ T cells alone from BaCF1 mice cannot protect against MVE. However, they seemed to be able to provide T cell help, as they were able to confer (partial) protection in conjunction with CD8+ T cells.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3. CD4+ as well as CD8+ T cell responses are required to protect BaCF1 mice partially. BaCF1 mice were infected and left untreated ({square}) or depleted of CD8+ ({bullet}) or CD4+ ({triangleup}) T cells by MAb treatment. The numbers signify surviving/total animals. BaCF1 mice were significantly more susceptible than control animals (Fisher’s exact test) after depletion of CD4+ (P<0·002) and CD8+ (P<0·002) T cells.

 
Secondary CD8+ T cells restricted by Ld, but not by Kk, protect against MVE
As primary CD8+ T cells are able to protect against MVE with the help of CD4+ T cells, the protective potential of secondary CD8+ T cells was evaluated. The main difference between CD8+ T cells from the resistant BALB/c mouse and the susceptible C3H mouse is the amount of epitope peptide required to lyse MV-infected target cells in vitro (Neumeister & Niewiesk, 1998 ). To investigate whether the poor recognition of MV-infected target cells in vitro was important for protection in vivo, C3H mice were immunized with two vaccinia virus recombinants (vvL52–59 and vvL81–88) expressing the two Kk-restricted epitopes of the nucleocapsid protein (aa 52–59 and 81–88). These epitopes were bound to a signal peptide enabling TAP-independent transport into the endoplasmic reticulum in order to induce good CTL responses (Neumeister & Niewiesk, 1998 ). Immunization yielded CD8+ T cells that did not recognize MV-infected targets in vitro but did recognize target cells expressing the MV nucleocapsid via a recombinant vaccinia virus (Fig. 4a). After immunization, mice were not protected and morbidity (as seen by weight loss) and mortality did not differ between mice immunized with a control vaccinia virus and the recombinant vaccinia viruses expressing the epitopes (Table 2). This indicates that, in C3H mice, secondary CD8+ T cells do not protect and that the lack of in vitro recognition of MV-infected target cells results in a lack of protection in vivo. In contrast, Ld-restricted CD8+ T cells from BALB/c mice lysed MV-infected target cells well (Fig. 4b) and protected against MVE after transfer into naive, CD4+ T cell-depleted BALB/c mice (Table 2).



View larger version (9K):
[in this window]
[in a new window]
 
Fig. 4. MV-specific lytic activity of CD8+ T cells correlates with protection against MVE. C3H mice (a) and BALB/c mice (b) were immunized with 5x106 p.f.u. vvL52–59. Four weeks later, spleen cells were stimulated in vitro with peptide 52–59 and the cytolytic capacity of T cells was tested on L929 target cells infected with vvR expressing the nucleoprotein of influenza A virus ({circ}), MV strain Edmonston ({blacktriangleup}) or vvR expressing the nucleocapsid protein of MV strain Edmonston ({blacksquare}). Similar results were obtained after immunization with vvL81–88. E:T, Effector:target.

 

View this table:
[in this window]
[in a new window]
 
Table 2. Lack of protein in mice by Kk-restricted cytotoxic T cells

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
In various mouse models of virus encephalitis, CD8+ T cells play a dominant role in combating infection. In the absence of CD4+ T cells, CD8+ T cells are often generated efficiently and clear virus from the CNS (Nash et al., 1987 ; Leist et al., 1987 ; Moskophidis et al., 1987 ). In the mouse model of MVE, however, depletion of CD4+ T cells leads to the breakdown of resistance in the resistant BALB/c mouse, indicating that CD8+ T cells alone are not protective. However, primary CD8+ T cells are able to confer partial protection against MVE with CD4+ T cell help (in BaCF1 mice). Secondary MV-specific CD8+ T cells alone are able to protect after adoptive transfer in CD4+ T cell-depleted BALB/c mice. The requirement for CD4+ T cell help during the primary, but not the secondary, CD8+ T cell response might be due to the fact that MV replicates poorly in the CNS and abortively in peripheral organs of mice (data not shown). It has been shown that the primary clonal burst of CD8+ T cells after abortive virus infection is reduced in the absence of CD4+ T cell help, although the number of memory cells is not affected (Zimmermann et al., 1997 ). In addition, in CNS infection with mouse hepatitis virus, CD8+ T cells without CD4+ T cell help become apoptotic and are not able to clear the virus (Stohlman et al., 1998 ).

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-{gamma} leads to breakdown of resistance in BALB/c mice (Finke et al., 1995 ). As IFN-{gamma} 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 ).


   Acknowledgments
 
This work was supported in part by Deutsche Forschungsgemeinschaft, Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, Pfleger-Stiftung and the World Health Organization.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Cattaneo, R., Rebmann, G., Baczko, K., ter Meulen, V. & Billeter, M. A.(1987). Altered ratios of measles virus transcripts in diseased human brains.Virology160, 523-526.[Medline]

Czub, S., Müller, J. G., Czub, M. & Müller-Hermelink, H. K.(1996). Nature and sequence of simian immunodeficiency virus-induced central nervous system lesions: a kinetic study.Acta Neuropathologica92, 487-498.[Medline]

Eichelberger, M., Allan, W., Zijlstra, M., Jaenisch, R. & Doherty, P. C.(1991). Clearance of influenza virus respiratory infection in mice lacking class I major histocompatibility complex-restricted CD8+ T cells.Journal of Experimental Medicine174, 875-880.[Abstract]

Fields, G. B. & Noble, R. L.(1990). Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids.International Journal of Peptide and Protein Research35, 161-214.[Medline]

Finke, D. & Liebert, U. G.(1994). CD4+ T cells are essential in overcoming experimental murine measles encephalitis.Immunology83, 184-189.[Medline]

Finke, D., Brinckmann, U. G., ter Meulen, V. & Liebert, U. G.(1995). Gamma interferon is a major mediator of antiviral defense in experimental measles virus-induced encephalitis.Journal of Virology69, 5469-5474.[Abstract]

Giraudon, P., Buckland, R. & Wild, T. F.(1992). The immune response to measles virus in mice. T-helper response to the nucleoprotein and mapping of the T-helper epitopes.Virus Research22, 41-54.[Medline]

Julius, M. H., Simpson, E. & Herzenberg, L. A.(1973). A rapid method for the isolation of functional thymus-derived murine lymphocytes.European Journal of Immunology3, 645-649.[Medline]

Kalams, S. A. & Walker, B. D.(1998). The critical need for CD4 help in maintaining effective cytotoxic T lymphocyte responses.Journal of Experimental Medicine188, 2199-2204.[Free Full Text]

Kärber, G.(1931). Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche.Archiv für Experimentelle Pathologie und Pharmakologie162, 480-487.

Leist, T. P., Cobbold, S. P., Waldmann, H., Aguet, M. & Zinkernagel, R. M.(1987). Functional analysis of T lymphocyte subsets in antiviral host defense.Journal of Immunology138, 2278-2281.[Abstract/Free Full Text]

Moskophidis, D., Cobbold, S. P., Waldmann, H. & Lehmann-Grube, F.(1987). Mechanism of recovery from acute virus infection: treatment of lymphocytic choriomeningitis virus-infected mice with monoclonal antibodies reveals that Lyt-2+ T lymphocytes mediate clearance of virus and regulate the antiviral antibody response.Journal of Virology61, 1867-1874.[Medline]

Muller, D., Koller, B. H., Whitton, J. L., LaPan, K. E., Brigman, K. K. & Frelinger, J. A.(1992). LCMV-specific, class II-restricted cytotoxic T cells in {beta}2-microglobulin-deficient mice.Science255, 1576-1578.[Medline]

Nash, A. A., Jayasuriya, A., Phelan, J., Cobbold, S. P., Waldmann, H. & Prospero, T.(1987). Different roles for L3T4+ and Lyt 2+ T cell subsets in the control of an acute herpes simplex virus infection of the skin and nervous system.Journal of General Virology68, 825-833.[Abstract]

Neumeister, C. & Niewiesk, S.(1998). Recognition of measles virus-infected cells by CD8+ T cells depends on the H-2 molecule.Journal of General Virology79, 2583-2591.[Abstract]

Niewiesk, S., Brinckmann, U., Bankamp, B., Sirak, S., Liebert, U. G. & ter Meulen, V.(1993). Susceptibility to measles virus-induced encephalitis in mice correlates with impaired antigen presentation to cytotoxic T lymphocytes.Journal of Virology67, 75-81.[Abstract]

Rosenberg, E. S., LaRosa, L., Flynn, T., Robbins, G. & Walker, B. D.(1999). Characterization of HIV-1-specific T-helper cells in acute and chronic infection.Immunology Letters66, 89-93.[Medline]

Stohlman, S. A., Bergmann, C. C., Lin, M. T., Cua, D. J. & Hinton, D. R.(1998). CTL effector function within the central nervous system requires CD4+ T cells.Journal of Immunology160, 2896-2904.[Abstract/Free Full Text]

Zimmermann, C., Seiler, P., Lane, P. & Zinkernagel, R. M.(1997). Antiviral immune responses in CTLA4 transgenic mice.Journal of Virology71, 1802-1807.[Abstract]

Received 19 April 2000; accepted 8 August 2000.