Fas ligand interactions contribute to CD8+ T-cell-mediated control of West Nile virus infection in the central nervous system

Abstract

West Nile virus (WNV) is a neurotropic flavivirus that causes encephalitis, most frequently in elderly and immunocompromised humans. Previous studies demonstrated that CD8+ T cells utilize perforin-dependent cytolytic mechanisms to limit WNV infection. Nonetheless, the phenotype of perforin-deficient CD8+ T cells was not as severe as that of an absence of CD8+ T cells, suggesting additional effector control mechanisms. In this study, we evaluated the contribution of Fas-Fas ligand (FasL) interactions to CD8+ T-cell-mediated control of WNV infection. Notably, the cell death receptor Fas was strongly upregulated on neurons in culture and in vivo after WNV infection. gld mice that were functionally deficient in FasL expression showed increased susceptibility to lethal WNV infection. Although antigen-specific priming of CD8+ T cells in peripheral lymphoid tissues was normal in gld mice, increased central nervous system (CNS) viral burdens and delayed clearance were observed. Moreover, the adoptive transfer of WNV-primed wild-type but not gld CD8+ T cells to recipient CD8(-/-) or gld mice efficiently limited infection in the CNS and enhanced survival rates. Overall, our data suggest that CD8+ T cells also utilize FasL effector mechanisms to contain WNV infection in Fas-expressing neurons in the CNS.

FIG. 1.

Fas expression in neurons after WNV infection. (A) Fas expression in WNV-infected cortical neurons. Cortical neurons were infected with WNV (MOI of 1), and 1 day later, neurons were stained for Fas (green) and WNV antigen (red). Uninfected neurons did not express Fas (upper panels a to d), whereas infected neurons did (lower panels e to h). p.i., postinfection. (B) Fas expression in brain sections. Brain tissue from wild-type mice was harvested on day 10 after infection with 10² PFU of WNV, sectioned, and stained for Fas expression. Neurons from uninfected mice were negative for Fas (upper panels i to l), whereas WNV-infected neurons stained positively for Fas expression (middle panels m to p). In WNV-infected mice, regions of the brain that stained negatively for WNV lacked the induction of Fas (lower panels q to t). Typical sections from cortex tissue selected after the review of more than five different brains are shown. WNV antigen and Fas expression are indicated by red and green fluorescence, respectively. DAPI was used as a nuclear stain.

FIG. 2.

Survival and viral titer analysis for wild-type and gld mice infected with WNV. (A) Kaplan-Meier survival curves. Wild-type (n = 50) and gld (n = 34) mice were infected with 10² PFU of WNV and monitored for mortality for 28 days. Survival differences as judged by the log rank test were statistically significant (P < 0.0001). (B to E) WNV tissue burdens in wild-type and gld mice. Infectious WNV levels in tissues from spleens (B), brains (C and E), and spinal cords (D and F) of wild-type and gld mice were measured using a viral plaque assay on BHK21 cells after tissues were harvested at the indicated time points. Data are expressed as log numbers of PFU per gram of tissue and reflect results for 10 to 12 mice per time point between days 2 and 10 and 3 to 8 mice per time point between days 14 and 35. For viral burden experiments, the dotted line represents the limit of sensitivity of viral detection and asterisks indicate statistically significant (P < 0.05) differences between wild-type and gld mice as judged by the Mann-Whitney test.

FIG. 3.

IFN-γ production by WNV-primed CD8⁺ T cells from wild-type (WT) and gld mice. Uninfected or WNV-infected splenocytes from wild-type or gld mice were harvested on day 7 and stimulated ex vivo with an immunodominant D^b-restricted NS4B peptide (SSVWNATTAI) or phorbol ester and ionomycin for 4 h. Cells were costained for CD8 and IFN-γ and analyzed by flow cytometry. (A) Representative flow cytometry profiles of intracellular IFN-γ staining in the absence of the peptide (upper panels) or in the presence of the peptide (middle panels) or phorbol ester (PMA) and ionomycin (bottom panels). The value in the top right corner indicates the percentage of CD8⁺ T cells that expressed IFN-γ after restimulation. (B and C) Total numbers of IFN-γ-producing CD8⁺ T cells after stimulation with NS4B peptides (B) or phorbol ester and ionomycin (C) were calculated. Each symbol represents data from an individual mouse, and lines indicate mean values. The numbers of IFN-γ⁺ CD8⁺ T cells in wild-type mice and those in gld mice after antigen-specific stimulation were not statistically different (P > 0.4), although there was a small increase (P = 0.01; asterisks) in the numbers of IFN-γ⁺ CD8⁺ T cells in gld mice after phorbol ester and ionomycin stimulation.

FIG. 4.

Survival rates and viral burdens after adoptive transfer of WNV-primed CD8⁺ T cells. CD8⁺ T cells were purified from naïve or WNV-primed wild-type and gld mice and transferred into either CD8^−/− or gld mice 1 day after infection. (A) Survival curves for CD8^−/− mice after the adoptive transfer of 10⁷ primed wild-type or gld CD8⁺ T cells. Differences in the survival curves for CD8^−/− mice that received no T cells and for CD8^−/− mice that received primed wild-type or gld cells were statistically significant (P ≤ 0.001). The number of mice in each group was 10 to 12. (B and C) Viral burden analysis of spleen, brain, and spinal cord tissues of recipient CD8^−/− mice on day 10 after the adoptive transfer of 10⁷ (B) or 3 × 10⁶ (C) naïve or primed wild-type or gld CD8⁺ T cells.

FIG. 5.

Control of WNV infection in primary neurons by primed CD8⁺ T cells. Cortical neurons were infected with WNV at an MOI of 0.001. After 1 h, purified naïve or WNV-primed CD8⁺ T cells from wild-type (WT) and gld mice were added at an E/T ratio of 50:1 or 10:1. After 48 h, supernatants were harvested and WNV production was measured by a plaque assay. Statistically significant reductions in WNV production compared to that in cultures of neurons without the addition of T cells are indicated by asterisks (**, P ≤ 0.005; *, P ≤ 0.05), and ns indicates that differences were not statistically significant.