CD4+ T-cell responses are required for clearance of West Nile virus from the central nervous system - PubMed (original) (raw)

Comparative Study

. 2006 Dec;80(24):12060-9.

doi: 10.1128/JVI.01650-06. Epub 2006 Oct 11.

Affiliations

Comparative Study

CD4+ T-cell responses are required for clearance of West Nile virus from the central nervous system

Elizabeth M Sitati et al. J Virol. 2006 Dec.

Abstract

Although studies have established that innate and adaptive immune responses are important in controlling West Nile virus (WNV) infection, the function of CD4(+) T lymphocytes in modulating viral pathogenesis is less well characterized. Using a mouse model, we examined the role of CD4(+) T cells in coordinating protection against WNV infection. A genetic or acquired deficiency of CD4(+) T cells resulted in a protracted WNV infection in the central nervous system (CNS) that culminated in uniform lethality by 50 days after infection. Mice surviving past day 10 had high-level persistent WNV infection in the CNS compared to wild-type mice, even 45 days following infection. The absence of CD4(+) T-cell help did not affect the kinetics of WNV infection in the spleen and serum, suggesting a role for CD4-independent clearance mechanisms in peripheral tissues. WNV-specific immunoglobulin M (IgM) levels were similar to those of wild-type mice in CD4-deficient mice early during infection but dropped approximately 20-fold at day 15 postinfection, whereas IgG levels in CD4-deficient mice were approximately 100- to 1,000-fold lower than in wild-type mice throughout the course of infection. WNV-specific CD8(+) T-cell activation and trafficking to the CNS were unaffected by the absence of CD4(+) T cells at day 9 postinfection but were markedly compromised at day 15. Our experiments suggest that the dominant protective role of CD4(+) T cells during primary WNV infection is to provide help for antibody responses and sustain WNV-specific CD8(+) T-cell responses in the CNS that enable viral clearance.

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Figures

FIG. 1.

FIG. 1.

Survival of CD4-deficient mice after WNV infection. Wild-type (n = 30), isotype control antibody-treated (n = 20), MHC class II−/− (n = 29), CD4 antibody-depleted (n = 20), and CD4−/− (n = 37) C57BL/6 mice were infected with 102 PFU of WNV in at least two independent experiments. Significant decreases in survival compared to that of wild-type mice were as follows: MHC class II−/− mice, P = 0.0003; CD4 antibody-depleted mice, P = 0.0004; CD4−/− mice, P < 0.0001.

FIG. 2.

FIG. 2.

WNV burdens in tissues. WNV burdens in the spleens (A), brains (B), and spinal cords (C) of wild-type (WT), isotype control antibody-treated, MHC class II−/−, and CD4 antibody-depleted mice were determined by viral plaque assay of samples from 7 to 10 mice per time point per group at days 5, 10, and 15 after infection. (D) WNV levels in the CNSs of individual MHC class II−/− mice at later times after infection as determined by viral plaque assay. The dotted line indicates the limit of sensitivity of the assay. Asterisks indicate time points at which differences were statistically significant (P ≤ 0.05).

FIG. 3.

FIG. 3.

WNV antigen staining in the brains of wild-type (WT) and CD4-depleted mice. The brains of wild-type and CD4 antibody-depleted mice were harvested 20 days after WNV infection, sectioned, and stained for WNV antigen. Examples of infected cells are indicated by arrows. Representative images from the cerebellum, cortex, hippocampus, and medulla are shown after a review of brains from at least three independent wild-type or CD4 antibody-depleted mice.

FIG. 4.

FIG. 4.

Antibody responses against WNV. Serum samples from wild-type (WT), MHC class II−/−, and CD4 antibody-depleted mice were collected at the indicated time points. The development of WNV-specific IgM (A) or IgG (B) antibodies was determined after incubation of serum with adsorbed control or purified WNV E protein. Neutralizing activity of serum samples (C) from wild-type and CD4-deficient mice on days 5 and 10 after infection was determined by a flow cytometry-based neutralization assay. Asterisks indicate significant differences between wild-type and CD4-deficient mice (P < 0.05). Data are an average of at least three independent experiments performed in duplicate and reflect 5 to 10 mice per group. (D) Passive administration of immune or naive serum from wild-type mice to MHC class II−/− mice (_n_ = 5) 10 days after WNV infection. The arrow indicates the time at which immune or naive serum was administered. The difference in survival was not statistically significant (_P_ > 0.2).

FIG. 5.

FIG. 5.

CD8+ T-cell activation after WNV infection. Mock- or WNV-infected splenocytes from wild-type (WT) and MHC class II−/− mice were harvested and stimulated ex vivo with a Db-restricted NS4B peptide. Cells were stained with antibodies against CD8 and IFN-γ and analyzed by flow cytometry on days 7 and 15. (A) Representative flow cytometry profiles showing intracellular IFN-γ staining of splenic CD8+ T cells after WNV infection in wild-type or MHC class II−/− mice. The percentage of CD8+ IFN-γ+ cells divided by the total number of CD8+ T cells is indicated in the top right corner. Percentages (B) and total numbers (C) of CD8+ IFN-γ+ splenocytes are also shown. Data are an average of at least three independent experiments and reflect 5 to 10 mice per group. Asterisks indicate statistically significant differences from mock-infected mice (*, P < 0.05) or between days 7 and 15 (**, P < 0.005).

FIG. 6.

FIG. 6.

Infiltrating brain leukocytes. Brains were harvested from WNV-infected wild-type (WT) and MHC class II−/− mice on days 9 and 15. Leukocytes were isolated by Percoll gradient centrifugation and double stained for CD3 and CD8. (A) Representative flow cytometry profiles showing CD3+ CD8+ T cells in the brain on day 9 after WNV infection in wild-type and MHC class II−/− mice. (B) Total number of CD3+ CD8+ leukocytes on day 9 postinfection. (C) Percent CD8+ IFN-γ+ staining of brain CD8+ leukocytes on day 9 after ex vivo restimulation with a Db-restricted WNV NS4B peptide. Significant differences from mock-infected mice are noted by asterisks. (D) CD3+ CD8+ leukocyte numbers in the brain on day 15 postinfection. Significant differences in the number of leukocytes between wild-type and MHC class II−/− mice are noted by asterisks (P < 0.05). Data are an average of at least three independent experiments and reflect five to eight mice per group.

FIG. 7.

FIG. 7.

Effector CD4+ T-cell responses. CD4+ T cells were purified from WNV-primed wild-type (WT), IFN-γ−/−, perforin−/−, or Fas ligand-deficient mice or uninfected wild-type mice and transferred 24 h after infection into CD4−/− mice (n = 9 to 18 mice for each CD4+ T-cell transfer). Significant changes were noted for mice receiving wild-type primed CD4+ T cells compared to naive CD4+ T cells (P < 0.0001). No significant difference was observed after adoptive transfer of primed wild-type or primed effector molecule-deficient CD4+ T cells (_P_ > 0.1).

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