Philadelphia University + Thomas Jefferson University

Research Projects

Research Projects

Anti-viral Protection by Type I Interferon (Type I IFN) & NFκB

The mechanism whereby Type I IFNs and NFκB protect from viral infections and how viruses evade their function in vivo remain incompletely understood. ECTV encodes a Type I IFN decoy receptor (EVM166) but its role in pathogenesis was unknown. Using a deletion mutant virus, we showed that EVM166 is essential for ECTV virulence and that mice can be protected from mousepox by vaccinating with recombinant EVM166. We also demonstrated that Type I IFN act local in tissues and not systemically and that Type I IFN signaling can be restored in the liver and mice cured from mousepox if treated with a mAb that neutralizes EVM166 (Figure 1). Moreover, we showed that the transcription factor NFκB is essential for resistance to mousepox and that an ECTV mutant lacking an NFκB inhibitor activates NFκB more effectively in vivo. Notably, NFκB activation compensates for defects in the Type I IFN pathway, such as a deficiency in the transcription factor IRF7. Thus, the overlap between the Type I IFN and NFκB pathways allows the host to overcome genetic or pathogen-induced deficiencies in Type I IFN. These findings may also explain why some pathogens target both pathways to cause disease.

Treatment with blocking 10G7 but not with non-blocking 10F3 monoclonal antibody decreases overall virus loads as determined by whole body imaging.

Figure 1. Treatment with blocking 10G7 but not with non-blocking 10F3 monoclonal antibody decreases overall virus loads as determined by whole body imaging. BALB/c mice were infected in the footpad with ECTV expressing luciferase. At 5 dpi, the mice were treated with the indicated antibodies and imaged 2 days later for light emission. The mouse on the top-left is an uninfected control. The bar graph (right) shows the mean ± SEM quantitative luminescence intensity of the two groups (From Xu et al. PLoS Pathog. 2012 Jan;8(1):e1002475 ).

More recently, we have found that the main producers of Type I in the draining lymph node are inflammatory monocytes which sense infection via a mechanism that involves the detection of intracellular virus, the signaling adapter STING, and the transcription factors IRF7 and NFκB. The inflammatory monocytes are recruited to the lymph node by chemokines produced by dendritic cells, which recognize infection via the pathogen recognition receptor TLR9, the adapter MyD88 and IRF7.


Anti-viral Protection by Memory CD8 T-Cells

Memory CD8 T-cells control viral replication in the draining lymph node.

Figure 2. Memory CD8 T-cells control viral replication in the draining lymph node. B6 mice were adoptively transferred with naïve (N-GFP) or VACV immune (M-GFP) CD8 T-cells from B6 mice expressing GFP under the β-actin promoter and/or infected in the footpad with ECTV expressing the red fluorescence protein mCherry (ECTV-mCherry). The draining lymph nodes were collected at 5 dpi and 5 µm sections of the whole organ were imaged by confocal microscopy at 10X (from Remakus et al. Memory CD8+ T cells can outsource IFN-γ production but not cytolytic killing for antiviral protection ( from Cell Host Microbe. 2013 May 15;13(5):546-57).

Work in my laboratory has shown that memory CD8 T-cells maintained in the absence of antigen protect mice from mousepox. Moreover, we showed that a major mechanism of memory CD8 T-cell protection is to restrict virus spread from the draining lymph node (Figure 2). Further, we demonstrated that both dominant and subdominant epitopes can be protective and that protection by memory CD8 T cells required IFNγ and perforin. However, while the CD8 T cells must express perforin autonomously, IFNγ can be outsourced to other cells.

 

 

 

 

 

 


Anti-viral Protection by Natural Killer Cells

NK cells control virus spread in the Lymph node

Figure 3. NK cells control virus spread in the Lymph node. The figure shows the lymph node of a mouse with green fluorescent NK cells (B6-NKp46GFP) 2.5 days after infection with ectromelia virus expressing the red fluorescent protein mCherry.

Until recently, most of our knowledge about NK cells in ant-viral protection had been learned from studies that used mouse cytomegalovirus. While it had been known for some time that NK cells also played a role in the natural resistance of C57BL/6 (B6) mice to mousepox, the mechanisms remained elusive. We showed that B6 mice resistance to mousepox requires the direct cytolytic function of NK cells. Furthermore, we showed that the activating receptor NKG2D is required for optimal NK cell-mediated resistance to lethal mouse pox. Moreover, we showed that similar to memory CD8 T-cells, NK cells protect, at least in part, by restricting virus spread from the draining lymph node (Figures 3 and 4). We also demonstrated that natural resistance of B6 mice to mousepox requires CD94 on NK cells, and that B6 mice deficient in Qa1b, the ligand for CD94, are also susceptible. In addition, we found that B6 mice lose their resistance to mousepox as they age, and that this is due to NK cell defects in maturation and migration to the draining lymph node. This established a new mechanism that can explain the increased susceptibility of the aged to viral diseases.


Anti-viral Protection by cytolytic CD4 T-cells (CD4 CTL)

NK-infected cell interaction

Figure 4. NK-infected cell interaction. A Natural Killer cell (red, stained with anti-NKp46) interacting with ectromelia virus-infected cells (green, ectromelia-GFP) in the lymph node of a mouse that has been infected 2.5 days earlier with ectromelia virus expressing green fluorescent protein.

CD4 T-cells are generally regarded as helpers and regulators of the immune response. Although cytolytic CD4 T-cells have been described, whether those generated during the course of a viral infection play a role in virus control remained unknown. We showed that during acute infection with ectromelia virus, the mouse homolog of the human virus of smallpox, large numbers of CD4 T-cells in the draining lymph node and liver of resistant mice have a CTL phenotype. We also show that these cells kill targets in vivo in a perforin-dependent manner and that mice with specific deficiency of perforin in CD4 T-cells have impaired virus control. Thus, our work demonstrated for the first time that CD4 CTL killing of infected cells is an important mechanism of antiviral defense.