Ying Kai Chan

THE ROLE OF THE CHEMOKINE CXCL10 AND ITS RECEPTOR CXCR3 IN WEST NILE ENCEPHALITIS VIRUS. Ying Kai Chan¹, Robyn S. Klein². Department of Biology, Washington University, St. Louis, MO¹; Departments of Anatomy and Neurobiology, Pathology and Immunology, Washington University School of Medicine, St. Louis, MO².

Introduction. West Nile Virus (WNV) is a neurotropic flavivirus that is transmitted by mosquitoes and can cause West Nile encephalitis in humans, animals and birds. It is endemic in parts of Africa, Europe, the Middle East and Asia, with outbreaks occurring annually in North America too. People with impaired immune systems are at greatest risk for severe neurological disease. Previously, the chemokine CXCL10 was demonstrated to be secreted by neurons in order to recruit CD8+ T-cells through its receptor CXCR3. In this study, we attempted to understand how CXCR3 affects the trafficking of lymphocytes into the murine brain and how its ligand CXCL10 affects the level of activated CD8+ T-cells in the spleen during WNV infection.

Methods. Wild type and CXCR3^–/– mice were bred until 7 weeks old and infected with 100 pfu of West Nile Virus. On day 9, they were perfused and their brains were harvested. They were stained with anti-CD8, anti-CD4, anti-CD3 and anti-NK antibodies and then analyzed by flow cytometry. Subsequently, wild type and CXCL10^–/– mice were infected similarly and their spleens were harvested on day 7. The splenocytes were stimulated with a WNV peptide and then stained for CD8. Cells were then permeabilized with saponin buffer and stained for intracellular IFN-. Finally, samples were analyzed by flow cytometry.

Results. More CD8+ T-cells were demonstrated to traffick into the CNS in wild type mice, as compared to the CXCR3^–/–mice. Using a standard t test, p was 0.013 and the results were significant. Subsequently, CXCL10^–/– mice showed a higher level of CD8+ IFN-+ splenocytes, as compared to wild type mice. Using a standard t test, p was 0.02 and the results were significant.

Conclusion. Here, we demonstrate that the expression of CXCR3 on CD8+ T-cells is essential for their recruitment into the CNS for viral clearance. However, CXCL10^–/– mice had a larger proportion of splenocytes expressing IFN-g when restimulated with WNV peptide in vitro, compared to wild type mice. This suggests that while loss of CXCL10 does not inhibit the ability of splenocytes to respond to WNV peptides, signaling via its receptor, CXCR3, is required for the trafficking of CD8+ cells into the CNS. Taken collectively, we have demonstrated the important regulatory role of the chemokine CXCL10 and its receptor CXCR3 in the trafficking of lymphocytes into the CNS and the level of activation of T-cells in the periphery.