CD40-CD40 ligand interactions promote trafficking of CD8+ T cells into the brain and protection against West Nile virus encephalitis

Abstract

Recent studies have established a protective role for T cells during primary West Nile virus (WNV) infection. Binding of CD40 by CD40 ligand (CD40L) on activated CD4+ T cells provides an important costimulatory signal for immunoglobulin class switching, antibody affinity maturation, and priming of CD8+ T-cell responses. We examined here the function of CD40-dependent interactions in limiting primary WNV infection. Compared to congenic wild-type mice, CD40(-/-) mice uniformly succumbed to WNV infection. Although CD40(-/-) mice produced low levels of WNV-specific immunoglobulin M (IgM) and IgG, viral clearance from the spleen and serum was not altered, and CD8+ T-cell priming in peripheral lymphoid tissues was normal. Unexpectedly, CD8+ T-cell trafficking to the central nervous system (CNS) was markedly impaired in CD40(-/-) mice, and this correlated with elevated WNV titers in the CNS and death. In the brains of CD40(-/-) mice, T cells were retained in the perivascular space and did not migrate into the parenchyma, the predominant site of WNV infection. In contrast, in wild-type mice, T cells trafficked to the site of infection in neurons. Beside its role in maturation of antibody responses, our experiments suggest a novel function of CD40-CD40L interactions: to facilitate T-cell migration across the blood-brain barrier to control WNV infection.

FIG. 1.

Survival data and WNV infection in wild-type and CD40^−/− mice. (A) Wild-type (n = 45) and CD40^−/− (n = 25) C57BL/6 mice were infected with 10² PFU WNV via a subcutaneous route in several independent experiments. A significant difference in survival between the two groups of mice was observed (P < 0.0001). (B to E) WNV burden in wild-type and CD40^−/− mice at days 2, 4, 6, 8, and 10 after infection. (B) Serum WNV RNA levels were determined by quantitative RT-PCR and expressed as genomic equivalents of WNV per ml of serum. (C to E) WNV was measured from the spleen (C), brain (D), and spinal cord (E) by viral plaque assay. Samples were obtained from 4 to 10 mice per time point per group. The dotted line represents the limit of sensitivity of the assay. Asterisks indicate time points at which differences are statistically significant compared to wild-type mice.

FIG. 2.

WNV antigen staining in the brain. The brains of infected (left) CD40^−/− and (right) wild-type mice were harvested 10 days after infection with WNV, sectioned, and stained with anti-WNV MAbs. Arrows indicate examples of infected cells. Typical sections from the cerebellum, cortex, hippocampus, and medulla at ×40 magnification are shown after staining samples from at least five mice per group.

FIG. 3.

WNV-specific antibody responses. Serum samples from wild-type or CD40^−/− mice were collected at the indicated time points. The development of WNV-specific IgM (A) or IgG (B) was determined after incubating serum with purified WNV E protein. (C) Neutralizing activity of serum samples from wild-type or CD40^−/− mice at the indicated days was determined by a plaque reduction neutralization titer assay. Asterisks indicate significant differences between wild-type and CD40^−/− mice (P < 0.05). Experiments represent 4 to 10 mice per time point, and individual experiments were performed in duplicate.

FIG. 4.

T-cell activation and leukocyte trafficking after WNV infection. (A and B) Wild-type or CD40^−/− mice were mock treated or infected with WNV. Seven days later, splenocytes were harvested and assayed for activation. The graphs show the percentage of CD4⁺ IFN-γ⁺ and CD8⁺ IFN-γ⁺ cells after ex vivo restimulation of mock- or WNV-infected splenocytes with phorbol ester and ionomycin (A) or a D^b-restricted WNV NS4B peptide (B). The data are an average of at least two independent experiments and reflect 7 to 10 mice per group. Asterisks indicate significant differences (P ≤ 0.05) from mock-treated mice. (C and D) On day 9 after WNV infection of wild-type or CD40^−/− mice, brain leukocytes were recovered by Percoll gradient purification and phenotyped with FITC-conjugated anti-CD3 antibody and APC-conjugated anti-CD8 antibody (C) or FITC-conjugated anti-CD45 antibody (D). The data are an average of the total number of CD3⁺ CD4⁺, CD3⁺ CD8⁺, or CD45⁺ cells and represent three independent experiments with a total of nine mice. Asterisks indicate significant differences (P ≤ 0.05) between wild-type and CD40^−/− mice.

FIG. 5.

Chemokine and chemokine receptor levels. (A) Quantitative RT-PCR analysis of chemokine mRNA levels in the brains of wild-type and CD40^−/− mice were measured 9 days after infection with 10² PFU of WNV. Total RNA was analyzed for the expression of CCL2, CCL5, CXCL9, and CXCL10. The data are expressed as copies of chemokine mRNA per copy of glyceraldehyde-3-phosphate dehydrogenase (control) and were obtained from nine mice per group. (B) Chemokine receptor levels on splenic CD8⁺ T cells. Wild-type or CD40^−/− mice were infected with WNV. On day 7, splenocytes were harvested and stained with antibodies against CCR2, CXCR3, and CCR5. Cells were analyzed by flow cytometry. The data are the average of three independent experiments, and none of the differences were statistically significant.

FIG. 6.

Lymphocyte trafficking patterns in the brain parenchyma after WNV infection. The brains of wild-type and CD40^−/− mice were harvested 9 days after infection with WNV, sectioned, and costained for CD31 (green) and CD3 (red); cell nuclei were stained with ToPro3 (blue) (A and B) or stained with hematoxylin and eosin (C and D). Yellow arrows indicate examples of leukocytes associated with meninges or blood vessels, and white arrows indicate CD3⁺ T cells that have migrated into the parenchyma. Representative images are shown after staining four mice per group in three independent experiments. The insets show a higher-power image and denote the increased density of cells associated with the meninges in CD40^−/− mice. (E) Quantitation of colocalization of CD3⁺ cells with CD31⁺ cells. Ten different fields were counted at ×400 magnification to determine the percentage of CD3⁺ cells associated with CD31⁺ endothelium or in the parenchyma. The data are presented as the percentage of CD31-associated or parenchyma-associated cells in a ×400 field and are derived from experiments from three different mice per group.

FIG. 7.

Adoptive transfer of CD45.1⁺ CD8⁺ T cells into CD45.2⁺ wild-type and CD40^−/− mice. WNV-primed CD45.1⁺ CD8⁺ T cells from wild-type mice were adoptively transferred into CD45.2⁺ wild-type or CD40^−/− mice 4 days after WNV infection. Leukocytes were isolated from brains 9 days after infection (5 days after transfer), stained with antibodies to CD45.1 and CD8, and analyzed by flow cytometry. (A) Representative flow cytometry profiles showing CD45.1⁺ CD8⁺ T cells in the brain after adoptive transfer into CD40^−/− (left) and wild-type (right) mice. (B) Total number of CD45.1⁺ CD8⁺ T cells that migrated into the brains of CD40^−/− and wild-type mice. The difference in the levels of CD45.1 CD8⁺ T cells in the brains of wild-type and CD40^−/− mice was statistically significant (P < 0.05). (C) The brains of recipient CD40^−/− (a and c) and wild-type (b and d) C45.2 mice were harvested 9 days after WNV infection and 5 days after adoptive transfer of WNV-primed CD45.1⁺CD8⁺ T cells from wild-type mice (a and c). Brains were sectioned, and costained with anti-CD31 (green) (a, b, and d), anti-CD45.1 (red) (a, b, and d), or isotype control antibodies (c). Cell nuclei were stained with ToPro3 (blue). Panel d is from a WNV-infected wild-type C45.2 control mouse that did not receive CD45.1 cells. Representative images are shown after staining brains from several mice.