The CD8 TCell Hypothesis

Using the Theiler's virus model of MS, we have investigated the role of MHC class I and class II in the progression of demyelination and the development of functional neurological deficits. We found that C57BL/6 x 129/J mice (H-2b haplotype) were resistant to demyelination induced by Theiler's virus and never developed clinical disease. In contrast, mice of the identical haplotype that were deficient in expression of MHC class I (C57BL/6 x 129/J P2-microglob-ulin-/-) developed extensive demyelination (Fig. 9). These animals failed to develop significant clinical signs of functional deficit as measured by hind-limb motor-evoked potentials, spontaneous vertical and horizontal movement assessment, rotarod performance, and activity wheel behavior (Fiette et al., 1993; Pullen et al., 1993; Rivera-Quinones et al., 1998; Rodriguez et al., 1993; Ure and Rodriguez, 2002). On the other hand, mice of the same haplotype that were deficient in expression of MHC class II (C57BL/6 x 129/J Ab0) developed not only extensive demyelination but also severe neurological impairment, and were, in fact, frequently moribund by 120 days postinfection (Njenga et al., 1996; Rivera-Quinones et al., 1998). Our findings indicate that physiological function was likely preserved secondary to the upregulation and redistribution of sodium channels along demyelinated axons. In fact, whereas chronically infected mice of a susceptible haplotype (SJL/J mice) showed a severe loss of sodium channel density and intensity along axons, chronically infected ß2-microglobulin-/-mice showed a dramatic upregulation and redistribution of axonal sodium channels, even though overall levels of myelin loss and distribution of demyelinated lesions were similiar between the two strains (Rivera-Quinones et al., 1998) (Fig. 10). Furthermore, axons, as marked by either Bielschowski staining or antineurofilament staining, were preserved in chronically infected ß2-microglobulin-/- mice, even in regions of significant cellular infiltrate and demyeli-nation (Fig. 11). In contrast, there was significant disruption and degeneration of axons in chronically infected SJL/J mice (Rivera-Quinones et al., 1998). These findings indicate

Figure 9 Three-dimensional reconstruction of demyelinated lesions in chronically infected mice.

Infected P2m+/+ mice do not exhibit demyelination, whereas the extent and distribution of demyelinated lesions is qualitatively similar in class I-deficient mice (P2m--), SJL/J mice, and class Il-deficient mice (Ab0). For each pair of reconstructions, the left profile shows gray matter (green) with an overlay of demyelinated areas (red); the right profile shows normal white matter (white) and demyelinated lesions (red).

Figure 9 Three-dimensional reconstruction of demyelinated lesions in chronically infected mice.

Infected P2m+/+ mice do not exhibit demyelination, whereas the extent and distribution of demyelinated lesions is qualitatively similar in class I-deficient mice (P2m--), SJL/J mice, and class Il-deficient mice (Ab0). For each pair of reconstructions, the left profile shows gray matter (green) with an overlay of demyelinated areas (red); the right profile shows normal white matter (white) and demyelinated lesions (red).

Figure 10 Saxitoxin binding associated with voltage-dependent sodium channels in mouse spinal cord. Normal axons have a distribution of sodium channels corresponding to the spacing of the nodes of Ranvier, and this distribution is integral to the normal saltatory conduction of the axon potential along myelinated axons. After demyelination, sodium channels are redistributed to a more uniform pattern, while axon loss results in loss of sodium channel labeling. (A) Spinal cord from infected class I-deficient mice (P2m-/-) showing evidence of sodium channel redistribution but intense channel labeling. (B) In contrast, spinal cord sections from chronically infected SJL/J mice show substantial loss of sodium channel labeling, consistent with a loss of axons.

Figure 10 Saxitoxin binding associated with voltage-dependent sodium channels in mouse spinal cord. Normal axons have a distribution of sodium channels corresponding to the spacing of the nodes of Ranvier, and this distribution is integral to the normal saltatory conduction of the axon potential along myelinated axons. After demyelination, sodium channels are redistributed to a more uniform pattern, while axon loss results in loss of sodium channel labeling. (A) Spinal cord from infected class I-deficient mice (P2m-/-) showing evidence of sodium channel redistribution but intense channel labeling. (B) In contrast, spinal cord sections from chronically infected SJL/J mice show substantial loss of sodium channel labeling, consistent with a loss of axons.

that MHC class I-restricted CD8+ T-cells are likely to be critical mediators of axon damage associated with demyelina-tion in susceptible strains of mice (Fig. 12).

Further evidence in support of this CD8+ T-cell hypothesis is provided by our recent studies assessing axon transport in chronically infected mice. Using retrograde labeling of spinal axon tracts, we have demonstrated a failure of retrograde axon transport in mice with demyelination and functional deficits (Ure and Rodriguez, 2000) (Fig. 13). However, we found that retrograde axonal transport was preserved in demyelinated mice with deletion of MHC class I (Ure and Rodriguez, 2002), suggesting that the lack of CD8+ cytotoxic T-cells protected axonal integrity while not affecting demyelinated lesion load (Ure and Rodriguez, 2002). A specific CD8+ T-cell-mediated response is further supported by our experiments showing that depletion of antigen-specific cytotoxic T-cells restricted to a viral peptide (VP2121-130) resulted in preservation of neurological function, as measured by rotarod performance (Johnson et al., 2001). Therefore, specific presentation of a Theiler's viral epitope within the context of H-2Db is necessary for at least a component of the neurological dysfunction associated with infection, although VP2 peptide depletion has no effect on the extent of viral infection or the level of demyelination induced by infection (Johnson et al., 2001). Finally, ongoing experiments in our laboratory indicate that VP2 peptide depletion of the MHC class I cytotoxic T-cell response to Theiler's virus infection preserves spinal axons, as measured by neurofilament staining, axon counting, and retrograde labeling. Thus, antiviral CD8+ cytotoxic T-cells are clearly important for the loss of axons and development of neurological dysfunction associated with chronic demyelination in the Theiler's virus model of MS.

The hypothesis that CD8+ T-cells are critical in the pathogenesis of Theiler's virus infection is relevant specifically to mice of the H-2b haplotype. Observations regarding the role of MHC class I in resistance vs. susceptibility to viral persistance and demyelination were made in recombinant inbred strains of the C57BL background (Rodriguez and David, 1985; Rodriguez et al., 1986), and the immunodominant VP2121-130 peptide response was observed only in H-2b mice (Fig. 14). The hypothesis probably does not explain the mechanism of demyelination and neurological deficit in susceptible strains such as SJL/J mice (H-2s haplotype). For example, disruption of MHC class I function by deletion of P2-microglobulin in SJL/J mice results in more demyelina-tion and increased neurological deficit (Begolka et al., 2001), and in the SJL/J strain it has been suggested that MHC class II-restricted T-cell response (Miller et al., 1987), and epitope-spreading to myelin antigens may be the mechanism of demyelination and neurological deficit (Croxford et al., 2002; Miller et al., 1997). However, CD8+ MHC class I-restricted cytotoxic T-cells have definitely been implicated in human MS, and it is likely that demyelination and neurological dysfunction in humans are a combination of both CD8+- and CD4+-mediated mechanisms (Neumann, 2003). Recent pathological studies have shown that CD8+ T-cells may be the most common subset of T-cells in the MS brain and appear to be clonally expanded in MS lesions (Babbe et al., 2000). Recent experiments in collaboration with Hans Lassmann have demonstrated intense expression of MHC class I in oligodendrocytes, neurons, axons, and astrocytes in the MS lesion (Hoftberger et al., 2003), and autopsy studies have demonstrated that CD8+ T-cells are statistically associated with axonal injury in MS (Bitsch et al., 2000). In addition, CD8+ T-cells have been shown to injure neurons and transect axons in vitro (Medana et al., 2000, 2001), and imaging studies have indicated that axonal loss in MS is a direct correlate for disability (Narayanan et al., 1997; Truyen et al., 1996; van Waesberghe et al., 1999). These studies suggest that CD8+ T-cells may be the primary effectors for axonal injury and neurological deficits in MS.

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