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Preservation of motor function by inhibition of CD8+ virus peptide-specific T cells in Theiler’s virus infection ¹

AARON J. JOHNSON^*, JADEE UPSHAW^*, KEVIN D. PAVELKO^*,, MOSES RODRIGUEZ^*, and LARRY R. PEASE^*2

Departments of
^* Immunology and
Neurology, Mayo Graduate and Medical Schools, Rochester, Minnesota 55901, USA

²Correspondence: Department of Immunology, Mayo Graduate and Medical Schools, Mayo Clinic, 200 First St. SW, Rochester, MN 55905, USA. E-mail: pease.larry{at}mayo.edu

SPECIFIC AIMS

Central nervous system (CNS) infiltrating CD8+ T cells are potential mediators of neuropathology in models of multiple sclerosis induced by Theiler’s murine encephalomyelitis virus (TMEV) infection. We studied the effect of inhibiting a virus peptide-specific CD8+ T cell response in a mouse model of chronic progressive demyelination and motor dysfunction.

PRINCIPAL FINDINGS

1. The CD8+ T cell response toward the immunodominant D^b:VP2_121–130 epitope during TMEV infection can be inhibited
C57BL/6 mice mount a vigorous CTL response toward the TMEV peptide VP2_121–130 presented in the context of D^b. IFN-R-/- mice also develop a class I-restricted response dominated by this epitope. To evaluate the importance of the strong CTL response to TMEV in these animals, we inhibited the development of D^b:VP2_121–130 epitope-specific CTL during TMEV infection. Administration of the VP2_121–130 peptide 1 day before TMEV infection resulted in the complete inhibition of the immunodominant T cell response against the D^b:VP2_121–130 epitope normally observed among brain infiltrating CD8+ T cells 7 days postinfection (Fig. 1 A). Inhibition was peptide dependent, as 66% of CD8+ T cells stained positive with the D^b:VP2_121–130 peptide after treatment with E7 peptide (Fig. 1B ).

View larger version (24K): [in this window] [in a new window]   Figure 1. FACS analysis of inflammatory mononuclear cells isolated from the brains of TMEV infected IFN-R-/- mice pretreated with E7 or VP2121–130 peptides. The pooled brain infiltrating lymphocytes (n=3) of 7 day infected IFN-R-/- mice pretreated with A) VP2121–130 peptide or B) E7 peptide were isolated using a Percoll gradient and stained with Db/VP2 tetramer and anti CD8 antibody. Also shown is the staining of brain infiltrating lymphocytes from individual 45 day TMEV infected IFN-R-/- mice pretreated once weekly with C) VP2121–130 peptide and D) E7 peptide. The percentages of CD8+ T cells that stain positive and negative for Db:VP2121–130 tetramer staining are indicated.

IFN-R-/- mice display a rapid decline in motor function, becoming paralyzed and moribund by 7 wk after infection with TMEV. To deplete D^b:VP2_121–130 epitope-specific CD8+ T cells from the population of brain infiltrating CTL for the full duration of TMEV infection of IFN-R-/- mice, VP2_121–130 peptide was administered intravenously (i.v.) weekly. This regimen removed D^b:VP2_121–130 epitope-specific brain infiltrating CD8+ T cells from 45 day infected mice (Fig. 1C ). Long-term removal of D^b:VP2_121–130 epitope-specific CD8+ T cells was not achieved with administration of a control E7 peptide (Fig. 1D ).

2. Removal of D^b:VP2_121–130 epitope-specific CD8+ T cells does not result in greater virus load or a different pattern of infection
Removal of D^b:VP2_121–130 epitope-specific CD8+ T cells might be expected to increase in virus load during the course of infection. Using Northern blot analysis, we did observe a higher average virus load in animals treated i.v. with VP2 peptide 7, 14, 28, and 45 days postinfection vs. mock-treated control animals. This trend approached statistical significance (P=0.063). VP2_121–130 did not alter the pattern of disease. In both VP2_121–130 and E7 peptide treatment groups, TMEV is cleared from the gray matter but persists in the white matter on the periphery of demyelinated lesions. We can conclude that VP2 peptide treatment did not reduce virus load or change the pattern of TMEV infection in IFN-R-/- mice.

3. CD4+ T cells and NK cells do not increase with the removal of D^b:VP2_121–130 epitope-specific CD8+ T cells
The removal of D^b:VP2_121–130 epitope-specific CD8+ T cells could alter the brain infiltrating T cell response. Therefore, we assessed the relative frequency of brain infiltrating NK cells, CD4+T cells, D^b:VP2_121–130-specific CD8+ T cells, and non-D^b:VP2_121–130-specific CD8+ T cells after treatment with VP2_121–130 or E7 peptides. Analysis of E7 and VP2_121–130 peptide-treated groups excluding D^b:VP2_121–130 epitope-specific CD8+ T cells showed that the proportions of CD4+, NK, and CD8+ T cells not bearing receptors specific for the D^b:VP2_121–130 epitope remained unchanged. This demonstrates that inhibiting the expansion of D^b:VP2_121–130- specific CD8+ T cells through VP2_121–130 peptide pretreatment does not affect the expansion of other lymphocyte compartments. Nor did we detect a compensatory increase in CD8+ T cells that could be specific for other subdominant epitopes after VP2_121–130 peptide treatment by CTL analysis.

4. The influence of D^b:VP2_121–130 epitope-specific CNS infiltrating CD8+ T cells on the extent of demyelination in IFN-R-/- mice
To address the contribution of antiviral CD8+ T cells in the demyelination of axons, the animals depleted of D^b:VP2_121–130 epitope-specific T cells were analyzed histologically for demyelinating lesions. Two sets of experimental conditions were assessed: one 6 wk after infection and one 4 wk after infection. In animals evaluated 6 wk after TMEV infection, the E7-treated mice (n=12) averaged 18.3% demyelination whereas the VP2-treated mice (n=11) averaged 15.1% demyelination (P=0.49). There was not a significantly different amount of lymphocyte inflammation in the meninges (P=0.73) or gray matter (P=0.16) when sections from the two treatment groups were compared. In a second experiment, differences in demyelination approached but did not reach statistical significance 4 wk after virus infection (P=0.056). Mice pretreated with the irrelevant E7 peptide (n=8) developed average demyelination covering 16.9% of the white matter in the cord and those pretreated with VP2_121–130 peptide developed 9.8% demyelination. We concluded from these studies that the removal of D^b:VP2_121–130 epitope-specific CD8+ T cells from chronically infected IFN-R-/- mice did not strongly influence the extent of demyelination, although the animals lacking CD8⁺ T cells specific for the VP2_121–130 epitope tended to have somewhat less demyelination than those treated with the irrelevant peptide.

5. Inhibition of the development of D^b:VP2_121–130 epitope-specific CD8+ T cells preserves the motor ability in IFN-R-/- mice
To address whether virus-specific CD8+ T cells contribute to developing motor dysfunction, we compared the ability of IFN-R-/- mice that received E7 peptide treatment to the ability of IFN-R-/- mice that received VP2 peptide treatment to negotiate trials on a rotarod. Mice placed on the rotarod try to remain on the surface of the slowly spinning rod as it accelerates from 5 to 40 rpm over a 7 min period. In the first set of trials, uninfected IFN-R-/- mice were able to stay on the rotarod for 154 ± 29 s. Forty-five days after TMEV infection, animals depleted of anti-viral D^b:VP2_121–130 epitope-specific CD8+ T cells remained on the rotarod significantly longer (76±20 s) than E7 peptide-treated animals (34±10 s) (P=0.012) (Table 1 A). In a second independent experiment, VP2 peptide-treated animals also remained on the rotarod significantly longer (61±7.4 s) than E7 peptide-treated animals (27±8.0 s) (P=0.007) (Table 1B) . This functional analysis demonstrates that VP2 peptide-treated mice, though generally not as mobile as uninfected controls, have significantly better motor ability than E7 peptide-treated mice. These data provide clear evidence that the VP2_121–130 peptide-specific CD8⁺ T cells play a substantial role in developing motor dysfunction that is a hallmark of this murine model of multiple sclerosis.

View this table: [in this window] [in a new window]   Table 1. Motor function in peptide-pretreated, chronically infected IFN-R-/- micea

CONCLUSION

The mechanisms underlying neuropathology in the TMEV model of MS have not been defined. Direct virus pathology and immune-mediated injury are suspected causes of tissue damage in the CNS. Here we demonstrate that TMEV-specific CD8+ T cells mediate neurological injury in a model where their presence or absence does not significantly decrease virus load during infection. Furthermore, motor deficits develop in these animals in a manner that is not tightly related to the extent of demyelination that develops in the chronic phase of the disease. In animals infected for 6 wk, we characterized mice with similar levels of demyelination and found them to have very different levels of motor function.

Administration of the viral VP2_121–130 peptide before virus infection inhibited the development of the brain infiltrating CD8+ T cells that are specific for the D^b:VP2_121–130 viral epitope. The treatment resulted in a major change in the TMEV-induced disease process, as there was significantly reduced motor dysfunction in VP2-treated IFN-R-/- mice when compared with the severe debilitation seen in mice treated with an irrelevant peptide. The behavioral differences between mice in these two treatment groups demonstrate that antiviral CD8+ T cells can contribute to neurological injury in a virus-based model where neurons are not actively infected and where expression of class I molecules on neurons and axons is largely absent. However, as these VP2 peptide-treated mice do not retain full motor function, antiviral CD8+ T cells are not the exclusive mechanism causing motor dysfunction. Also emerging from these studies is the conclusion that direct action by the virus on CNS tissues is not the exclusive basis of neurological injury, as the VP2_121–130-treated mice had comparable or higher virus loads but less neurological deficit than the E7 peptide-treated control animals.

Our finding that inhibition of brain-infiltrating CD8+ D^b:VP2_121–130-specific T cells results in decreased neurological injury raises questions regarding what these cells are doing. Earlier work from our lab, as well as studies using the mouse hepatitis virus (MHV) model, have implicated CD8+ T cells in the development of demyelinated lesions. However, we had found that extensive demyelination can occur in the absence of significant motor deficits; in the current studies, deficits in motor function were not strongly associated with demyelination. Perhaps the intensity of the CD8+ T cell response (reflected in the rate of demyelination) is a key factor in determining the extent of motor deficit. The experiments reported here suggest that differences in demyelination rate may exist between animals containing TMEV-specific CD8+ brain infiltrating lymphocytes and those that do not. At 4 wk postinfection, animals lacking these T cells had an average demyelination of 9.8%; mice that had the brain infiltrate developed 16.9% demyelination. By 6 wk after infection, animals in these treatment groups had 15.1% and 18.3% demyelination respectively. One interpretation of these findings is that virus-specific, CNS infiltrating T cells incite an intense response that leads to rapid demyelination and damage to neurons (Fig. 2 ), whereas animals lacking these virus-specific T cells undergo a more gradual demyelination in which oligodendrocytes are damaged but neurons are spared. Both groups eventually plateau at comparable levels of demyelination, but one group develops more neuronal damage. Our current findings highlight the role of CD8+ T cells in this process.

View larger version (26K): [in this window] [in a new window]   Figure 2. Schematic diagram. Antiviral CD8+ T cells contribute to motor dysfunction during TMEV infection. 1) Antiviral CD8+ T cells kill TMEV infected glial cells including oligodendrocytes. This results in demyelinated axons. 2) Uninfected neurons, which lack expression of class I molecules, are spared by antiviral CD8+ T cells. However, 3) exposed axons are susceptible to secondary injury through cytokines or death receptors expressed by CD8+ T cells or other immune cells infiltrating the CNS.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0373fje; to cite this article, use FASEB J. (October 15, 2001) 10.1096/fj.01-0373fje

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