Early B-Cell Activation after West Nile Virus Infection Requires Alpha/Beta Interferon but Not Antigen Receptor Signaling▿

The B-cell response against West Nile virus (WNV), an encephalitic Flavivirus of global concern, is critical to controlling central nervous system dissemination and neurological sequelae, including death. Here, using a well-characterized mouse model of WNV infection, we examine the factors that govern early B-cell activation. Subcutaneous inoculation with a low dose of replicating WNV results in extensive B-cell activation in the draining lymph node (LN) within days of infection as judged by upregulation of the surface markers CD69, class II major histocompatibility complex, and CD86 on CD19+ cells. B-cell activation in the LN but not the spleen was dependent on signals through the type I alpha/beta interferon (IFN-α/β) receptor. Despite significant activation in the draining LN at day 3 after infection, WNV-specific B cells were not detected by immunoglobulin M enzyme-linked immunospot analysis until day 7. Liposome depletion experiments demonstrate that B-cell activation after WNV infection was not affected by the loss of F4/80+ or CD169+ subcapsular macrophages. Nonetheless, LN myeloid cells were essential for control of viral replication and survival from infection. Overall, our data suggest that the massive, early polyclonal B-cell activation occurring in the draining LN after WNV infection is immunoglobulin receptor and macrophage independent but requires sustained signals through the type I IFN-α/β receptor

Tolerance is established in polyclonal CD4(+) T cells by distinct mechanisms, according to self-peptide expression patterns.

Studies of repertoires of mouse monoclonal CD4(+) T cells have revealed several mechanisms of self-tolerance; however, which mechanisms operate in normal repertoires is unclear. Here we studied polyclonal CD4(+) T cells specific for green fluorescent protein expressed in various organs, which allowed us to determine the effects of specific expression patterns on the same epitope-specific T cells. Peptides presented uniformly by thymic antigen-presenting cells were tolerated by clonal deletion, whereas peptides excluded from the thymus were ignored. Peptides with limited thymic expression induced partial clonal deletion and impaired effector T cell potential but enhanced regulatory T cell potential. These mechanisms were also active for T cell populations specific for endogenously expressed self antigens. Thus, the immunotolerance of polyclonal CD4(+) T cells was maintained by distinct mechanisms, according to self-peptide expression patterns

Tregs control the development of symptomatic West Nile virus infection in humans and mice

West Nile virus (WNV) causes asymptomatic infection in most humans, but for undefined reasons, approximately 20% of immunocompetent individuals develop West Nile fever, a potentially debilitating febrile illness, and approximately 1% develop neuroinvasive disease syndromes. Notably, since its emergence in 1999, WNV has become the leading cause of epidemic viral encephalitis in North America. We hypothesized that CD4+ Tregs might be differentially regulated in subjects with symptomatic compared with those with asymptomatic WNV infection. Here, we show that in 32 blood donors with acute WNV infection, Tregs expanded significantly in the 3 months after index (RNA+) donations in all subjects. Symptomatic donors exhibited lower Treg frequencies from 2 weeks through 1 year after index donation yet did not show differences in systemic T cell or generalized inflammatory responses. In parallel prospective experimental studies, symptomatic WNV-infected mice also developed lower Treg frequencies compared with asymptomatic mice at 2 weeks after infection. Moreover, Treg-deficient mice developed lethal WNV infection at a higher rate than controls. Together, these results suggest that higher levels of peripheral Tregs after infection protect against severe WNV disease in immunocompetent animals and humans

Epitope discovery in West Nile virus infection: Identification and immune recognition of viral epitopes

Cytotoxic T lymphocytes (CTL) play an important role in the control and elimination of infection by West Nile virus (WNV), yet the class I human leukocyte antigen (HLA)-presented peptide epitopes that enable CTL recognition of WNV-infected cells remain uncharacterized. The goals of this work were first to discover the peptide epitopes that distinguish the class I HLA of WNV-infected cells and then to test the T cell reactivity of newly discovered WNV epitopes. To discover WNV-immune epitopes, class I HLA was harvested from WNV (NY99 strain)-infected and uninfected HeLa cells. Then peptide epitopes were eluted from affinity-purified HLA, and peptide epitopes from infected and uninfected cells were comparatively mapped by mass spectroscopy. Six virus-derived peptides from five different viral proteins (E, NS2b, NS3, NS4b, and NS5) were discovered as unique to HLA-A*0201 of infected cells, demonstrating that the peptides sampled by class I HLA are distributed widely throughout the WNV proteome. When tested with CTL from infected individuals, one dominant WNV target was apparent, two epitopes were subdominant, and three demonstrated little CTL reactivity. Finally, a sequence comparison of these epitopes with the hundreds of viral isolates shows that HLA-A*0201 presents epitopes derived from conserved regions of the virus. Detection and recovery from WNV infection are therefore functions of the ability of class I HLA molecules to reveal conserved WNV epitopes to an intact cellular immune system that subsequently recognizes infected cells

Pivotal Role of Antibody and Subsidiary Contribution of CD8+ T Cells to Recovery from Infection in a Murine Model of Japanese Encephalitis▿

The immunological correlates for recovery from primary Japanese encephalitis virus (JEV) infection in humans and experimental animals remain poorly defined. To investigate the relative importance of the adaptive immune responses, we have established a mouse model for Japanese encephalitis in which a low-dose virus inoculum was administered into the footpads of adult C57BL/6 mice. In this model, ∼60% of the mice developed a fatal encephalitis and a virus burden in the central nervous system (CNS). Using mice lacking B cells (μMT−/− mice) and immune B cell transfer to wild-type mice, we show a critically important role for humoral immunity in preventing virus spread to the CNS. T cell help played an essential part in the maintenance of an effective antibody response necessary to combat the infection, since mice lacking major histocompatibility complex class II showed truncated IgM and blunted IgG responses and uniformly high lethality. JEV infection resulted in extensive CD8+ T cell activation, judged by upregulation of surface markers CD69 and CD25 and cytokine production after stimulation with a JEV NS4B protein-derived H-2Db-binding peptide and trafficking of virus-immune CD8+ T cells into the CNS. However, no significant effect of CD8+ T cells on the survival phenotype was found, which was corroborated in knockout mice lacking key effector molecules (Fas receptor, perforin, or granzymes) of cytolytic pathways triggered by T lymphocytes. Accordingly, CD8+ T cells are mostly dispensable for recovery from infection with JEV. This finding highlights the conflicting role that CD8+ T cells play in the pathogenesis of JEV and closely related encephalitic flaviviruses such as West Nile virus