The dominant mechanism for cytotoxic CD8+ T-cell-mediated viral clearance and killing of virally infected cells is the release of cytolytic granules containing the pore-forming protein perforin, the proteoglycan serglycin, and a family of serine proteases known as the granzymes. Granzymes A and B are the most abundant granzymes in cytolytic granules, and granzyme B is a mediator of caspase-dependent apoptosis. Granzymes appear to be packaged within the cytolytic granule in complex with serglycin. Approximately 30 to 50 granzyme molecules bind to each molecule of ser-glycin, forming a large granzyme particle that ranges in size from 40 to 200 nm in diameter. After T-cell receptor recognition of the appropriate peptide/MHC class I complex on target cells and the formation of the immune synapse, the granzyme/serglycin complex and perforin are exocytosed by the cytotoxic T-cell (Lieberman, 2003). Some confusion currently exists regarding the role of perforin in the steps subsequent to cytolytic granule exocytosis, but the current model suggests that granzymes, either free or in complex with serglycin, bind to the surface of the target cell and are endocytosed. This event appears to occur in the absence of perforin (Froelich et al., 1996; Pinkoski et al., 1998; Shi et al., 1997). Once endocytosed, however, perforin may function to facilitate the transfer of granzymes from target cell endosomes to the cytosol via endosomolysis, apparently without plasma membrane pore formation (Browne et al., 1999; Froelich et al., 1996; Metkar et al., 2002). Regardless of the precise mechanism of action, perforin is clearly required for the full killing function of cytotoxic T-cells, and it was therefore logical to assess the role of perforin in T-cell-mediated axon killing.

Our laboratory has demonstrated, using Theiler's virus infection of C57BL/6 H-2b mice, that perforin may be a critical component in the induction of neurological deficits once demyelination is established (Murray et al., 1998). We found that perforin deficiency broke viral resistance, resulting in viral persistence and consequent demyelination (Fig. 15). Despite significant demyelination throughout the spinal cord, however, perforin-deficient C57BL/6 H-2b mice failed to develop the significant functional deficits associated with demyelination in susceptible strains of mice. Perforin-defi-cient mice did not acquire hind-limb paralysis, did not show a decrease in spontaneous activity, and did not develop signfi-cant changes in hind-limb stride, suggesting that neurological deficits associated with demyelination are dependent on perforin, and, by extension, on functional cytotoxic T-cells (Murray et al., 1998). Furthermore, we have recently observed that the number of large axons in the spinal cord does not differ between infected wild-type C57BL/6 mice and perforin-deficient C57BL/6 mice, strengthening the argument that neurological function in these animals is preserved as a result of axon preservation. Likewise, perforin contributes to neurological deficit and axon dropout in susceptible B10.Q H-2q mice. In these experiments, we found that wild-type B10.Q mice developed significant demyelination, a dramatic decrease in the frequency of large axons in the spinal cord, and significant neurological dysfunction, as measured by rotarod performance, but that large axon frequency and neurological function were relatively preserved in perforin-defi-cient B10.Q mice, despite marked demyelination.

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