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J. E. Knickelbein, R. L. Hendricks; Lytic Granule-Mediated, Noncytolytic CD8 T Cell Inhibition of HSV-1 Reactivation From Neuronal Latency. Invest. Ophthalmol. Vis. Sci. 2008;49(13):821.
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Herpes simplex virus type 1 (HSV-1) establishes lifelong latent infections in sensory neurons. Reactivation of latent HSV-1 in neurons of the trigeminal ganglion (TG) can lead to recurrent ocular infections and development of herpes stromal keratitis, a leading infectious cause of blindness worldwide. Understanding and exploiting mechanisms responsible for maintaining HSV-1 in a latent state might represent the most promising approach to preventing this devastating disease. The purpose of our studies is to identify and characterize the mechanisms employed by HSV-specific CD8 T cells (HSV-CD8) to block HSV-1 reactivation from latency in sensory ganglia, thereby potentially limiting recurrent herpetic disease.
Wild-type, perforin (Pfn) knock-out, and granzyme B (GrB) knock-out mice were used in a murine model of HSV-1 corneal infection. Flow cytometry was employed to quantify and phenotype CD8 T cells from dispersed latently infected TG. Standard viral plaque assays and PCR analysis for viral genome copies were used to quantify viral reactivation from latency. Interactions between HSV-CD8 and latently infected neurons or susceptible fibroblasts were imaged by live- and fixed-cell epifluorescence microscopy.
HSV-CD8 can secrete IFN-γ to inhibit reactivation in some, but not all neurons, suggesting the involvement of additional effector mechanism(s). We now show that HSV-CD8 contain functional GrB-containing lytic granules and that the lytic granule components Pfn and GrB are necessary for optimal protection from HSV-1 reactivation in sensory neurons both in vivo and in ex vivo TG cultures. We further establish that HSV-CD8 exhibit vectorial release of lytic granules into immunologic synapses formed with both latently infected neurons and susceptible fibroblasts in ex vivo TG cultures. Importantly, while lytic granule release activates the caspase system of fibroblasts leading to morphologic signs of apoptosis, similar lytic granule release does not result in caspase activation or morphologic signs of apoptosis in neurons. Together our findings establish that directed lytic granule release by HSV-CD8 is required to block neuronal HSV-1 reactivation, that lytic granules block HSV-1 reactivation in neurons without activating caspases or inducing apoptosis, and that GrB is one, but apparently not the only, lytic granule component required to block HSV-1 reactivation in neurons.
A therapeutic vaccine designed to induce a potent CD8 T cell response to latent HSV-1 may provide protection from reactivation without eliminating non-regenerating sensory neurons.
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