Analysis of the effect of type I interferon on the response of human DC subsets to influenza virus

Dendritic cells (DCs) are a key component in both the innate and adaptive immune responses. Upon virus infection, DCs are activated causing the cells to produce pro-inflammatory chemokines and cytokines, such as type I interferon (IFN). Activated DCs migrate to the lymph nodes and present antigen to B and T cells. Influenza virus encodes a potent immune antagonist, the NS1 protein, which blocks DC activation and the production of IFN from infected cells. As a result, influenza virus is a poor activator of both mouse and human DCs in vitro. However, in vivo a strong immune response to influenza virus infection is generated in both species suggesting that other factors may contribute to the activation of DCs. We hypothesized that the environment in which a DC encounters a virus in vivo will affect the DC response to infection. Specifically, we are interested in how the pro-inflammatory molecule, type I IFN, a critical component of the viral immune response, will affect DC activation.;We have examined the activation profiles of the major human DC subsets: conventional DCs, both cultured and directly isolated from blood (cDCs and mDCs), plasmacytoid DCs and monocytes. Our data show that the activation of all cell types in response to influenza virus infection is enhanced when pre-exposed to IFN. However, the degree of priming and kinetics of activation are different in the pDCs relative to the other cell types.;Human experimentation is often restricted by the availability of patient samples. Even when samples are obtainable, it is often difficult to study cells of low abundance and find adequate controls. In this thesis two approaches were tried to overcome the limitations associated with human research. First, we developed a comprehensive computational model for cDC activation following IFN pretreatment and virus infection. Our model provides insight into the kinetics of IFN induction in pretreated DCs and identifies extracellular IFN level as the key regulator of the overall dynamics of activation. Furthermore, the model predicts that IFN mRNA induction in vivo will occur at a faster rate than in vitro, and is determined by the level of IRF7 mRNA present in the cell at the early stages of influenza virus infection.;In order to overcome the limitation that results from a scarcity of circulating cells in the blood, we developed and validated a new technique for studying the activation profile of rare cell subsets. With this new method, we are now able to pursue research in human subjects with significantly reduced patient material. This technique is applicable to all studies where cell numbers are limiting.