Memory B cells, but not long-lived plasma cells, possess antigen specificities for viral escape mutants

Memory B cells have the unique capacity to recognize variants of West Nile virus, likely providing protection against mutant viruses that escape antibody neutralization

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

Tumor Necrosis Factor Alpha Protects against Lethal West Nile Virus Infection by Promoting Trafficking of Mononuclear Leukocytes into the Central Nervous System ▿

West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis in humans, especially in immunocompromised individuals. Previous studies have shown essential protective roles for antiviral cytokines (e.g., alpha interferon [IFN-α] and IFN-γ) against WNV in mice. However, studies using cell culture offer conflicting answers regarding whether tumor necrosis factor alpha (TNF-α) has an anti-WNV function. To test the biological significance of TNF-α against WNV in vivo, experiments were performed with TNF receptor-1 (TNF-R1)-deficient and TNF-α-depleted C57BL/6 mice. TNF-R1−/− mice had enhanced mortality and decreased survival time after WNV infection compared to congenic wild-type mice. Consistent with this, administration of a neutralizing anti-TNF-α monoclonal antibody also decreased survival after WNV infection. Relatively small differences in viral burdens in peripheral tissues of TNF-R1−/− mice were observed, and this occurrence correlated with a modest antiviral effect of TNF-α on primary macrophages but not dendritic cells. In contrast, the viral titers detected in the central nervous systems of TNF-R1−/− mice were significantly increased compared to those of wild-type mice, although TNF-α did not have a direct antiviral effect in primary neuron cultures. Whereas no defect in priming of adaptive B- and T-cell responses in TNF-R1−/− mice was observed, there were significant reductions in accumulations of CD8+ T cells and macrophages in the brain. Our data are most consistent with a model in which interaction of TNF-α with TNF-R1 protects against WNV infection by regulating migration of protective inflammatory cells into the brain during acute infection

Activation of procathepsin H.

<p><b>A</b>. Coomassie gel of proCTSH incubated for 3 hours in sodium acetate(NaAC) buffer at various pHs (4.5, 5.0, 5.5), MES buffer at various pHs (5.5, 6.0, 6.5), thermolysin activated CTSH (TH), human liver purified CTSH (HS), and protein ladder (LD). Arrow indicates mature CTSH product. <b>B.</b> Assessment of CTSH enzymatic activity from samples in (A) and thermolysin alone (T). Data shown is relative fluorescence units (RFUs) from excitation emission readings at 360/480nm. <b>C</b>. Coomassie gel of procathepsin H (PH), proCTSH activated with CTSL for various minutes (5, 20, 60), thermolysin activated CTSH(TH) and protein ladder (LD). Arrow indicates mature CTSH product. <b>D</b>. Assessment of R-AMC (cathepsin H substrate) cleavage from proCathepsinH (proCTSH), CTSL, thermolysin (Therm), proCTSH activated with CTSL (CTSL CTSH), and thermolysin activated CTSH (Therm CTSH). Data shown is relative fluorescence units (RFUs) from excitation emission readings at 360/480nm.</p

Assessment of cathepsin H point mutants.

<p>(A) Measurement of cathepsin H aminopeptidase activity in HEK293T cell lines overexpressing the indicated forms of cathepsin H. Activity is expressed as RFUs (relative fluorescent units) detected after cleavage of cathepsin H substrate. (B) Western blot analysis of cathepsin H and actin in HEK293T cell lines expressing the indicated forms of cathepsin H. Arrow indicates pro form and mature form of cathepsin H.</p

Crystal structure of procathepsin H.

<p>(A) Overall structure of procathepsin H. Left, the prodomain and the mature domain are colored in violet and cyan respectively. The mini-chain is colored in blue. The prodomain contains two conserved sequence motifs: ERFNIN motif (orange) and GNFD motif (green). The four disulfide bonds in the structure are highlighted in yellow.Right, the 2<i>F</i>_<i>o</i>-<i>F</i>_<i>c</i> electron density map (top) and <i>F</i>_<i>o</i>-<i>F</i>_<i>c</i> omit map (bottom) of the mini-chain are displayed as the grey mesh at a contour level of 1.2<i>σ</i> and 3<i>σ</i> respectively. (B) Superposition of procathepsin H (cyan) and procathepsin L (violet). (C) Superposition of human procathepsin H (cyan) and mature porcine cathepsin H (wheat). The mini-chain of human procathepsin H is shown in blue and that of mature porcine cathepsin H is shown in orange. (D) Hydrogen bonding interactions between the β strand from prodomain (violet) and β strand from right subdomain (cyan). Backbones of the residues involved are shown as sticks. The distances between atoms are indicated by dashes. (E) Interactions between hydrophobic residues from prodomain (violet) and the mature domain (cyan). Sidechains of the residues involved are shown as sticks. (F) Hydrogen bonding interactions between the C-terminal linker of prodomain (violet) and core enzyme (cyan). Backbones and sidechains of the residues involved are shown as sticks. The distances between atoms are indicated by dashes.</p

The stability of the mini-chain in cathepsin H and procathepsin H.

<p>The snapshots of the mini-chain and mature domain complexes before simulations (<i>left</i>) and the overlay of the mini-chain conformations during the simulations (<i>right</i>) in systems (A) 8pch_1glyc, (B) 6czk_1glyc, and (C) 6czk_2glyc. The cathepsin H (orange) and the procathepsin H (blue) systems are distinguished by their colors. The glycans present are shown in stick representation. (D) The per-residue RMSF of the mainchain atoms in the mini-chain calculated from the simulations and the B-factors from the crystal structures averaged by residue. The three systems 8pch_1glyc (filled orange circle), 6czk_1glyc (empty blue circle), and 6czk_2glyc (filled blue circle) are shown in different styles. The B-factors for 8PCH (orange) and 6CZK (blue) are shown as lines. (E) The distribution of native (left) and total (right) contact numbers computed from the simulations. Two residues are identified as contacting residues when any of the heavy atoms from the two are within 4.5 Å. The total contact number is evaluated for all residue pairs between the mini-chain and the mature domain. The native contacts are the ones from the total contacts that are also present in the native crystal structure. Results from the three systems 8pch_1glyc (solid orange line), 6czk_1glyc (dashed blue line), and 6czk_2glyc (solid blue line) are shown.</p

Crystallographic data and refinement statistics.

<p>Crystallographic data and refinement statistics.</p

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