CD8+ T cells require perforin to clear West Nile virus from infected neurons - PubMed (original) (raw)

CD8+ T cells require perforin to clear West Nile virus from infected neurons

Bimmi Shrestha et al. J Virol. 2006 Jan.

Abstract

Injury to neurons after West Nile virus (WNV) infection is believed to occur because of viral and host immune-mediated effects. Previously, we demonstrated that CD8+ T cells are required for the resolution of WNV infection in the central nervous system (CNS). CD8+ T cells can control infection by producing antiviral cytokines (e.g., gamma interferon or tumor necrosis factor alpha) or by triggering death of infected cells through perforin- or Fas ligand-dependent pathways. Here, we directly evaluated the role of perforin in controlling infection of a lineage I New York isolate of WNV in mice. A genetic deficiency of perforin molecules resulted in higher viral burden in the CNS and increased mortality after WNV infection. In the few perforin-deficient mice that survived initial challenge, viral persistence was observed in the CNS for several weeks. CD8+ T cells required perforin to control WNV infection as adoptive transfer of WNV-primed wild-type but not perforin-deficient CD8+ T cells greatly reduced infection in the brain and spinal cord and enhanced survival of CD8-deficient mice. Analogous results were obtained when wild-type or perforin-deficient CD8+ T cells were added to congenic primary cortical neuron cultures. Taken together, our data suggest that despite the risk of immunopathogenesis, CD8+ T cells use a perforin-dependent mechanism to clear WNV from infected neurons.

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Figures

FIG. 1.

Survival and viral load analysis for wild-type and perforin-deficient C57BL/6 mice inoculated with 102 PFU of WNV. (A) Wild-type and perforin-deficient mice were inoculated with WNV and monitored for morbidity and mortality for 28 days. The numbers of animals were n = 50 for wild-type mice and n = 41 for perforin-deficient mice. Survival differences were statistically significant (P < 0.0001). (B to E) WNV tissue burden. Infectious WNV levels were measured from the (B) spleen, (C) spinal cord, and (D) brain of wild-type and perforin-deficient mice using a viral plaque assay in BHK21 cells after tissues were harvested at the indicated days. Data are shown as the average PFU per gram of tissue and reflect 5 to 10 mice per time point for either wild-type or perforin-deficient mice. (E) Persistent WNV infection in the brain of surviving wild-type and perforin-deficient mice as determined by plaque assay. For all viral burden experiments, the dotted line represents the limit of sensitivity of viral detection and asterisks denote statistically significant (P < 0.05) differences between wild-type and perforin-deficient mice.

FIG. 2.

Detection of WNV antigen in the brains of infected wild-type and perforin-deficient mice by immunohistochemistry. Brains from equivalently moribund wild-type (a to d) and perforin-deficient (e to h) mice were harvested at day 10 after infection with 102 PFU of WNV, sectioned, and stained for WNV antigen. Examples of infected cells or injured neurons are indicated with black and red arrows, respectively. Typical sections are shown from the cerebellum (a and e), brain stem (b and f), cerebral cortex (c and g), and spinal cord (d and h) after review of more than 10 independent brains from either wild-type or perforin-deficient mice.

FIG. 3.

Characterization of perforin-deficient CD8+ T cells. (A)Levels of CD45+ inflammatory cells in the brains of wild-type and perforin-deficient mice. Brain sections from wild-type and perforin-deficient mice at day 10 after infection were stained with rat anti-mouse CD45 antibody. The number of CD45+ cells per high-power field was counted from 10 independent fields from each mouse. No significant difference was observed (P > 0.1). (B) Migration of CD8+ and CD4+ T cells into the brains of WNV-infected mice. Wild-type and perforin-deficient mice (n = 9 per group) were infected with 102 PFU of WNV. At day 10, brain leukocytes were isolated by Percoll gradient centrifugation and phenotyped with phycoerythrin-conjugated anti-CD8 or anti-CD4 antibodies. The data are expressed as average numbers of CD8+ or CD4+ T cells and reflect the total number of brain leukocytes recovered after Percoll gradient centrifugation multiplied by the percentage that expressed CD8α (Ly-2) chain or CD4 antigen as measured by flow cytometry. No significant difference was observed (P = 0.9). (C) Intracellular staining of IFN-γ production by CD8+ T cells in the spleens of WNV-infected mice. Wild-type and perforin-deficient mice (n = 8 per group) were infected with 102 PFU of WNV. At day 7 after infection, splenocytes were harvested and left untreated (Mock) or stimulated ex vivo with phorbol ester and ionomycin for 4 h. Cells were then stained with FITC-conjugated anti-CD8 antibody and Alexa 647-conjugated anti IFN-γ antibody and analyzed by flow cytometry. The data are expressed as average numbers of CD8+ T cells producing IFN-γ. Asterisks indicate differences that are significantly different compared to mock-treated cells (P ≤ 0.05). No difference in IFN-γ expression was observed between wild-type and perforin-deficient CD8+ T cells (P = 0.7).

FIG. 4.

WNV infection after adoptive transfer of naïve or WNV-primed CD8+ T cells. CD8+ T cells were purified from naïve or WNV-primed wild-type, IFN-γ-deficient, or perforin-deficient mice and transferred 24 h after infection into CD8-deficient (A, B, C, D, E, and G) or K b × D b class I MHC-deficient (F) mice. (A) Viral burden at day 10 after adoptive transfer into CD8-deficient mice of 10 × 106 CD8+ T cells from naïve and WNV-primed wild-type mice. (B) Viral burden at day 10 after adoptive transfer into CD8-deficient mice of 10 × 106 CD8+ T cells from WNV-primed wild-type or perforin-deficient mice. (C) Survival curve of CD8-deficient mice after adoptive transfer of WNV-primed wild-type or perforin-deficient CD8+ T cells at 24 h postinfection. The number of mice was 12 to 14 for each arm, and the survival difference between adoptive transfer of primed wild-type and perforin-deficient CD8+ T cells was statistically significant (P = 0.01). (D) Effect of depletion of CD8+ T cells with an anti-CD8 or isotype control antibody after adoptive transfer of CD8+ T cells to CD8-deficient mice. (E) Viral burden at day 10 after adoptive transfer of 3 × 106 WNV-primed wild-type or perforin-deficient CD8+ T cells into CD8-deficient mice. (F) Viral burden at day 10 after adoptive transfer of 10 × 106 WNV-primed wild-type or perforin-deficient CD8+ T cells into K b × D b class I MHC-deficient mice. (G) Viral burden at day 10 after adoptive transfer of 10 × 106 WNV-primed wild-type or IFN-γ-deficient CD8+ T cells into CD8-deficient mice.

FIG. 5.

Survival of wild-type mice with WNV infection after depletion of natural killer cells. Natural killer cells were depleted from wild-type mice after treatment with anti-NK1.1 antibody 2 days before and after infection with WNV. (A) Depletion of NK cells was confirmed by flow cytometry after staining with FITC-conjugated anti-CD49b. (B) WNV infection of NK cell-depleted mice. Twenty mice were treated with either anti-NK1.1 or the isotype control (anti-SARS ORF7a) antibody and monitored for survival. No statistically significant differences in mortality were observed (P > 0.1).

FIG. 6.

CD8+ T-cell-mediated control of WNV infection in primary cortical neurons. (A) Cortical neurons were infected with an MOI of 0.001 and 48 h later costained for neuronal and WNV antigen infection using naïve or WNV immune serum and an anti-MAP2 antibody. Purity of neuron cultures was assessed by comparing the MAP2 (a) and DAPI (b) staining. Infection of cells was evaluated after staining with WNV-immune (c) or WNV-naïve (d) rat serum. Infection of neurons was confirmed by merging the MAP2, anti-WNV, and DAPI images (e and f). (B) Clearance of WNV infection from neurons by CD8+ T cells. One hour after WNV infection, purified naive or WNV-primed CD8+ T cells from wild-type (WT) and perforin-deficient mice were added at an 80:1 or 10:1 E:T ratio. Supernatants were harvested 48 h later, and the reduction of WNV production was measured by plaque assay. Asterisks denote differences that are statistically significant (P < 0.05) compared to the addition of no CD8+ T cells.

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CD8+ T cells require perforin to clear West Nile virus from infected neurons - PubMed (original) (raw)