Browsing by Subject "Vaccinia virus"

Natural killer (NK) cells are lymphocytes of the innate immune response with well-demonstrated activities against viral infections and tumors. Because of these abilities, we sought to glean insights into the mechanisms of NK cell activation so that they may be applied toward the design of new therapies.

NK cells are particularly critical for the control of poxviral infections. Vaccinia virus (VV) is the most-studied member of the poxviral family. It is robustly immunogenic and functions as the live vaccine responsible for the successful elimination of smallpox. VV infection provides a useful model for studying NK cell activation: NK cells play an important role in its clearance and the virus efficiently activates NK cells and recruits them to the site of infection. We had previously used this model to identify Toll-like receptor (TLR)-dependent and -independent mechanisms of NK cell activation to VV. One method of TLR-independent activation to VV requires the activation receptor NKG2D, which recognizes host ligands expressed upon viral infection by accessory cells such as dendritic cells (DCs) and macrophages.

In the first aim of this thesis, we sought to determine how the ligands for the NKG2D activation receptor become upregulated in the context of VV infection. Specifically, we asked whether interleukin-18 (IL-18), known to play a role in the innate immune response, could boost the expression of NKG2D ligands on DCs in response to viral infection. Using an in vivo infection model with IL-18R-deficient mice, our results confirmed an important role for IL-18 in NK cell activation to VV and viral control. We then made use of an NK-DC co-culture to show that IL-18 signaling on DCs, in addition to NK cells, is necessary to achieve efficient NK cell activation to viral infection. We further demonstrated in a cell-transfer experiment that cell-extrinsic IL-18 signaling is critical for NK cell activation in vivo. DC ablation via a mouse model designed to specifically ablate CD11c+ cells showed that DCs are also required for NK cell activation to VV in vivo. We finally showed how IL-18 can act on DCs in vivo and in vitro to boost the expression of Rae-1, an NKG2D ligand. Collectively, our data uncover a novel mechanism whereby NK cells become activated by IL-18 control of NKG2D ligand expression on DCs.

In the second aim of this project, we detailed how IL-18 signaling results in the upregulation of the NKG2D ligand Rae-1. Using an in vitro macrophage model, we showed how recombinant IL-18 was sufficient to upregulate Rae-1 expression. We compared IL-18 control of Rae-1 expression to LPS, a TLR ligand that also signals through the common adaptor MyD88 to govern Rae-1 expression. Using chemical inhibitors to cell signaling molecules, we then identified the importance of MyD88 signaling through PI3K. We then revealed that glycogen synthase kinase 3 (GSK-3) can act as a negative regulator of Rae-1 expression downstream of IL-18/TLR signaling. Specifically, we have shown that during inflammatory signaling, PI3K (acting downstream of MyD88) can inhibit GSK-3 to relieve its tonic suppression of Rae-1 expression and upregulate the NKG2D ligand. Finally, we showed that PI3K and GSK-3 signaling are also important to Rae-1 expression on DCs - the accessory cell where IL-18 signals to control Rae-1 expression to boost NK cell activation against VV.

In its entirety, this work seeks to address how NK cells become activated in the context of VV infection in order to identify new ways NK cells may be harnessed therapeutically.

Orthopoxviruses encode many genes that are not essential for viral replication, which often account for differences in pathogenesis among otherwise closely related orthopoxviruses. Although dendritic cells (DCs) are essential to the generation of an effective anti-viral immune response, the effects of different orthopoxviruses on DC function is poorly understood. The objective of these studies was to determine the effect of different orthopoxviruses on DCs. Cowpox virus (CPXV) is ideally suited to this purpose because it encodes the largest and most representative set of accessory genes among orthopoxviruses, it is endemic in mouse populations, and can infect humans.

We hypothesized that CPXV would have novel mechanisms of evading the immune response that other orthopoxviruses lack, which may exert maximal effect in the context of antigen presenting cells such as DCs, allowing for discovery of novel viral strategies of immune evasion. To test this, CPXV was used to infected mouse bone marrow-derived DCs (BMDCs), and the effect of the virus on DC survival, expression of T-cell costimulatory molecules and cytokine production was determined. The effects of vaccinia virus strain Western Reserve (VV), the prototype of the species, and modified vaccinia virus strain Ankara (MVA), a promising vaccine vector, on mouse BMDCs were also determined. Confirming the hypothesis that CPXV would have different effects on mouse BMDCs from other orthopoxviruses, BMDCs infected with CPXV survived longer in culture than those infected with MVA or VV. In addition, CPXV specifically downregulated MHC I, MHC II, CD40, and CD86, and induced production of significant levels of IL-6 and IL-10.

Because IL-10 has many suppressive effects on the immune system, inducing IL-10 may provide a selective advantage to CPXV in vivo. To examine the role of IL-10 in a CPXV infection, wild type and IL-10 deficient mice were infected intranasally with CPXV. The effect of CPXV infection on disease morbidity, viral loads, inflammation and the protective immune response was determined. As expected, IL-10 was important in controlling inflammation during CPXV infection, but there was no effect on viral replication or clearance. Surprisingly, IL-10 was important in generation of a protective memory response to CPXV, which may reflect a novel role for IL-10 in the immune response.

Immune responses against viral infections are mediated through a complex process by diverse populations of cells, that can be harnessed for tumor immunotherapies and vaccinations. Vaccinia virus (VV) is the most studied member of the poxvirus family and is responsible for the successful elimination of smallpox worldwide. It is unique among well-studied viruses in that it replicates solely in the host cytoplasm and is able to elicit one of the longest lasting immunity in recorded human history. Its success in vaccination has led to the development of adjuvants with VV epitopes and recombinant VV vectors for other infectious diseases and cancer immunotherapy. However, the mechanism behind how VV elicits such a strong immune response from the immune system remains insufficiently understood.

Previous studies have shown that although activation of NK cells is critical for the initial control of VV infection, efficient activation of CD8+ T cell response is required for the eradication of VV infection. It has also been demonstrated that gammadelta T cells play an important part in the immune response against VV infection. However, both processes remain relatively undefined. What promotes CD8+ T cell activation and subsequent generation of CD8+ memory T cells in response to VV infection is still not very well dissected, and the mechanisms that govern gammadelta T cells response to VV are relatively unknown. This thesis examines these questions through three main aims: 1) influence of gammadelta T cells on CD8+ T cell activation, 2) gammadelta T cell direct cytotoxicity against VV infection, and 3) mechanisms that govern CD8+ memory T cell formation. The overall goal of this thesis is to understand the mechanisms behind gammadelta T and CD8+ T cells responses against VV infection.

We found that gammadelta T cells play an important role in promoting CD8+ T cell response to VV infection. We showed that gammadelta T cells serve not only as antigen presenting cells to CD8+ T cell activation, but also as mediators of other signals of CD8+ T cell response in vivo. We further demonstrated that cell-intrinsic MyD88 signaling in gammadelta T cells is required for activation of CD8+ T cells. Contrary to conventional expectations, we found that NKG2D expression in both NK and CD8+ T cells only have partial effect on the elimination of VV post-infection. Instead, we found that NKG2D is an important activator of gammadelta T cell cytotoxicity for VV clearance. Lastly, we demonstrated that Notch1, but not Notch2, deficiency increases the formation of CD8+ memory T cells, through modulating the expression of TCF1/Tcf7. We discovered that cleaved Notch1 intracellular domain binds upstream of Tcf7 and controls the expression of Tcf7 for CD8+ memory T cell formation.

These results demonstrated a critical role for gammadelta T cells in viral clearance and the regulation of adaptive T cell response, with insights into the formation of CD8+ memory T cells. Collectively, this dissertation seeks to better understand how gammadelta T and CD8+ T cells respond to VV infection, with the hopes of shedding additional light on the design of more effective vaccine strategies based on the precise manipulation of immune cell populations for infectious diseases and cancer immunotherapy.