Noncytotoxic lytic granule-mediated CD8+ T cell inhibition of HSV-1 reactivation from neuronal latency

Abstract

Reactivation of herpes simplex virus type 1 (HSV-1) from neuronal latency is a common and potentially devastating cause of disease worldwide. CD8+ T cells can completely inhibit HSV reactivation in mice, with interferon-gamma affording a portion of this protection. We found that CD8+ T cell lytic granules are also required for the maintenance of neuronal latency both in vivo and in ex vivo ganglia cultures and that their directed release to the junction with neurons in latently infected ganglia did not induce neuronal apoptosis. Here, we describe a nonlethal mechanism of viral inactivation in which the lytic granule component, granzyme B, degrades the HSV-1 immediate early protein, ICP4, which is essential for further viral gene expression.

Figure 1. CD8⁺ T cells release lytic granules toward neurons within HSV-1 latently infected ganglia without activating neuronal caspases

(A) In situ confocal images of intact latently infected WT TG stained with antibodies to CD8α, GrB, and NeuN (neuronal nucleus). Top: CD8⁺ T cell with GrB polarized to the junction with a NeuN⁺ neuron. Bottom: Most CD8⁺ T cells have GrB dispersed throughout the cell. (B) Imaging of ex vivo cultures of latently infected WT TG. Top: CD8⁺ T cell in contact with two neurons polarizes TCR and GrB toward lower neuron only. Bottom: CD8⁺ T cells not contacting targets show no TCR or GrB polarization. (C–E) Imaging of ex vivo latently infected WT TG cultured 24–48 hrs with WT gB-CD8, which prevent reactivation from latency. (C) Two representative interactions between NeuN⁺ neurons and CD8⁺ T cells with CD107a polarized toward the junction with a neuron lacking activated caspases (VAD-FMK⁻). Interaction 2, bottom panel: the plane demarcated by the line between the cells (left) is shown en face (right) demonstrating an apparent secretory domain of an immunological synapse. (D) 10% ethanol induced neuronal caspase activation. (E) CD8⁺ T cells contacting NeuN⁻ non-infected fibroblast-like cells (left) or not contacting cells (right) lack surface CD107a expression. Data are representative of 11 TG from three separate experiments.

Figure 2. Pfn and GrB are required to maintain HSV-1 neuronal latency in vivo and in ex vivo ganglia cultures

(A) DNA was extracted from individual TG at designated times and HSV-1 genome copies was determined by quantitative real-time PCR (horizontal bar = mean). Data pooled from at least two independent experiments per time point. * p≤0.05 as calculated by ANOVA with Bonferroni post-test. (B) 34–41 dpi TG were dispersed and cultured at one-fifth TG equivalents per well. HSV-1 reactivation was indicated by the presence of infectious virus in serially sampled supernatants as assessed by plaque assay. Pooled data from three independent experiments presented as mean ± SEM. * p=0.0009; ** p=0.0002 as calculated by survival curve analysis (Log-rank test). (C) 14 dpi TG were dispersed and cultured at 1 TG per well in medium alone or in medium supplemented with 1000 U/ml recombinant IFN-γ (rIFN-γ), 100 μg/ml anti-CD8α monoclonal antibody (α-CD8α mAb), or 20 μg/ml anti-IFN-γ monoclonal antibody (α-IFN-γ mAb). Reactivation was assessed, analyzed, and presented as in (B). n=10 TG/condition. Data representative of two independent experiments. * p=0.0108; ** p=0.0049.

Figure 3. GrB cleaves the essential HSV-1 IE protein ICP4

(A&B) B6WT3 fibroblasts were infected with recombinant HSV-1 expressing EGFP from the ICP0 promoter for 1 hr, washed, and exposed to no, WT, Pfn^−/−, or GrB^−/− gB-CD8 for 5 hrs. (A) Histograms from flow analysis of recovered cells show total fibroblasts (CD8⁻) recovered from cultures (top left) and percent infected (EGFP⁺; top right). (B) Lysates of recovered cells were subjected to Western blot for ICP4 (Lane 1: Noninfected; Lanes 2–5: Infected with No, WT, Pfn^−/−, or GrB^−/− gB-CD8, respectively). Bar graphs show optical density (O.D.) readings that were not adjusted (middle) or adjusted for the number of infected fibroblasts recovered from each culture (right). (C) Lysates from 293T cells that were either non-transfected (lane 1) or transfected with an ICP4-expressing plasmid (lanes 2–5) and exposed to different concentrations of recombinant GrB (lanes 1–5: 0, 0, 25, 50, 100 nM GrB, respectively) for 1 hr at 37° C. Lysates from 293T cells transfected with an ICP4-expressing plasmid (D), lysates from B6WT3 cells infected with HSV-1 (E), or ICP4 immunoprecipitated from HSV-1-infected fibroblasts (F) were exposed to 0 (lanes 1–3, 5, 7) or 100 nM GrB (lanes 4, 6, 8) for varying times at 37° C (lanes 1–2: 0 hr; 3–4: 1 hr; 5–6: 2 hrs; 7–8: 3 hrs). Lane 1 contains non-transfected 293T cell lysate (D) or noninfected fibroblast lysate (E).