Role of CD8+ T cells in control of West Nile virus infection - PubMed (original) (raw)

Role of CD8+ T cells in control of West Nile virus infection

Bimmi Shrestha et al. J Virol. 2004 Aug.

Abstract

Infection with West Nile virus (WNV) causes fatal encephalitis more frequently in immunocompromised humans than in those with a healthy immune system. Although a complete understanding of this increased risk remains unclear, experiments with mice have begun to define how different components of the adaptive and innate immune response function to limit infection. Previously, we demonstrated that components of humoral immunity, particularly immunoglobulin M (IgM) and IgG, have critical roles in preventing dissemination of WNV infection to the central nervous system. In this study, we addressed the function of CD8(+) T cells in controlling WNV infection. Mice that lacked CD8(+) T cells or classical class Ia major histocompatibility complex (MHC) antigens had higher central nervous system viral burdens and increased mortality rates after infection with a low-passage-number WNV isolate. In contrast, an absence of CD8(+) T cells had no effect on the qualitative or quantitative antibody response and did not alter the kinetics or magnitude of viremia. In the subset of CD8(+)-T-cell-deficient mice that survived initial WNV challenge, infectious virus was recovered from central nervous system compartments for several weeks. Primary or memory CD8(+) T cells that were generated in vivo efficiently killed target cells that displayed WNV antigens in a class I MHC-restricted manner. Collectively, our experiments suggest that, while specific antibody is responsible for terminating viremia, CD8(+) T cells have an important function in clearing infection from tissues and preventing viral persistence.

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Figures

FIG. 1.

FIG. 1.

Survival data and viral burden data for wild-type (WT), CD8−/−, and MHC class Ia−/− C57BL/6J mice inoculated with WNV. (A) Wild-type, CD8−/−, and MHC class Ia−/− mice were inoculated via footpad with 102 PFU of WNV and monitored for 28 days. The survival curves were constructed using data from three to five independent experiments. The numbers of animals were n = 50 for wild-type, n = 43 for CD8−/−, and n = 32 for MHC class Ia−/− mice. Survival differences between wild-type and CD8−/− or MHC class Ia−/− mice were statistically significant (P < 0.0001). (B) Levels of viral RNA in serum. Viral RNA levels were determined from serum of wild-type or CD8−/− mice after WNV infection at the indicated days by a real-time fluorogenic RT-PCR assay. Data are expressed as genomic equivalents of WNV RNA per milliliter of serum and reflect the average of five independent mice per time point. The dashed line represents the limit of sensitivity of the assay. (C to E) Infectious virus levels in tissues. Virus levels were measured from the spleen (C), upper (USC) and lower (LSC) halves of the spinal cord (D), and brain (E) of wild-type and CD8−/− mice by a viral plaque assay in BHK21 cells after tissues were harvested at the indicated days after inoculation. Data are shown as the average PFU per gram of tissue or milliliter of serum and reflect five mice per time point for either wild-type or CD8−/− mice. The dashed line represents the limit of sensitivity of the assay.

FIG. 1.

FIG. 1.

Survival data and viral burden data for wild-type (WT), CD8−/−, and MHC class Ia−/− C57BL/6J mice inoculated with WNV. (A) Wild-type, CD8−/−, and MHC class Ia−/− mice were inoculated via footpad with 102 PFU of WNV and monitored for 28 days. The survival curves were constructed using data from three to five independent experiments. The numbers of animals were n = 50 for wild-type, n = 43 for CD8−/−, and n = 32 for MHC class Ia−/− mice. Survival differences between wild-type and CD8−/− or MHC class Ia−/− mice were statistically significant (P < 0.0001). (B) Levels of viral RNA in serum. Viral RNA levels were determined from serum of wild-type or CD8−/− mice after WNV infection at the indicated days by a real-time fluorogenic RT-PCR assay. Data are expressed as genomic equivalents of WNV RNA per milliliter of serum and reflect the average of five independent mice per time point. The dashed line represents the limit of sensitivity of the assay. (C to E) Infectious virus levels in tissues. Virus levels were measured from the spleen (C), upper (USC) and lower (LSC) halves of the spinal cord (D), and brain (E) of wild-type and CD8−/− mice by a viral plaque assay in BHK21 cells after tissues were harvested at the indicated days after inoculation. Data are shown as the average PFU per gram of tissue or milliliter of serum and reflect five mice per time point for either wild-type or CD8−/− mice. The dashed line represents the limit of sensitivity of the assay.

FIG. 2.

FIG. 2.

Histopathology and immunohistochemistry after infection with WNV (A) Histopathology of CNS tissue from wild-type and CD8−/− mice. CNS tissues from equivalently moribund wild-type (a to d) and CD8−/− (e to h) mice were harvested at day 10 after infection with 102 PFU of WNV, sectioned, and stained with hematoxylin and eosin. Typical sections from the cerebellum, hippocampus, and spinal cord are shown after review of more than 10 independent brains. In samples from wild-type infected mice (thick arrows) the Purkinje neurons of the cerebellum, the CA1 neurons of the hippocampus, and the anterior horn motor neurons are identified with blue, red, and green arrows, respectively. In samples from CD8−/− mice (thin arrows) these neurons are again delineated; however, significantly more neuronal degeneration is observed. (B) Detection of WNV infection in the CNS by immunohistochemistry in wild-type and CD8−/− mice. The brains of wild-type (i to l) and CD8−/− (m to p) mice were harvested 10 days after infection with WNV, sectioned, and stained with rat anti-WNV polyclonal serum or a control negative polyclonal rat serum. Typical sections are shown from the cerebellum (i and m), brain stem (j and n), cerebral cortex (k and o), and hippocampus (l and p) after review of more than 10 independent brains from either wild-type or CD8−/− mice.

FIG. 3.

FIG. 3.

Trafficking of CD8+ T cells into the brain of WNV-infected mice. Groups of wild-type mice were infected with 102 PFU of WNV subcutaneously. At the indicated days, brain leukocytes were recovered by Percoll gradient centrifugation and phenotyped with phycoerythrin-conjugated anti-CD8 antibodies. The data are expressed as a scatter plot and reflect the total number of brain CD45+ leukocytes recovered after Percoll gradient centrifugation of individual brains multiplied by the percentage that expressed CD8α (Ly-2) chain antigen as detected by flow cytometry.

FIG. 4.

FIG. 4.

Development of specific antibodies to WNV in wild-type and CD8−/− mice. Serum was collected from wild-type (WT) or CD8−/− mice at the indicated days after infection with 102 PFU of WNV. The development of specific IgM or IgG antibodies to WNV was determined after incubating serum with adsorbed control or purified WNV E protein. Data are the averages of 5 to 10 mouse experiments per time point performed in duplicate.

FIG. 5.

FIG. 5.

Delayed clearance of WNV from the brains of surviving CD8−/− and MHC-Ia−/− mice. Wild-type (WT), CD8−/−, and MHC class Ia−/− mice were infected with 102 PFU of WNV. Surviving animals were euthanized at 28 or 35 days after infection, and brains were analyzed for infectious virus by plaque assay as described in the Fig. 2 legend. Data are shown as the average PFU per gram of tissue and reflect three to five mice per time point.

FIG. 6.

FIG. 6.

Primary CD8+-T-cell-mediated killing of WNV targets. (A) Splenocytes were harvested from mice that were infected with 106 PFU of WNV at 4, 6, and 8 days after infection. CD8+ T cells were purified by negative antibody selection and mixed with calcein AM-labeled MC57GL target cells (MC57GLWNV-E or MC57GLvector) at various E/T ratios. Target cell killing was measured by assessing the release of calcein AM with a 96-well-plate fluorimeter. Specific lysis was determined after subtracting the amount of calcein AM release for target cells that were incubated without CD8+ T cells. One representative experiment of two is shown. (B) Class I-restricted killing of MC57GLWNV-E target cells. MAbs to class I MHC molecules (Kb + Db) were added to target cells prior to the addition of effector CD8+ T cells. (Inset) Flow cytometry profile demonstrating the purity of CD8+ T cells after negative selection by antibody-coated magnetic beads.

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