The role of antigen cross-presentation in the vaccine-induced activation of cytotoxic T-lymphocytes

Abstract

Antigen delivery systems that are efficient in inducing CD8+ T-cell mediated immune responses are required for the development of novel vaccines against intracellular pathogens and cancer. Although many new approaches were already investigated in mice and also humans, low immunogenicity of tested vaccines is still a major challenge in the field. Therefore it is important to study the molecular and cellular function of particular vaccines, in order to develop strategies that overcome this limitation. Beside “directpresentation” of endogenous proteins, antigen “cross-presentation” by professional antigen presenting cells (APCs) is an essential pathway for the major histocompatibility complex (MHC) class I presentation of exogenous antigens. It becomes more and more evident that cross-presentation is not only involved in physiological responses to various pathogens and malignancies, but also mediates specific cytotoxicity in response to vaccines. This thesis aimed on analyzing the molecular and cellular requirements for antigen cross-presentation, especially in response to selected vaccines. In chapter I we characterized the cell types involved in the cross-presentation of antigens that are encapsulated into a particulate biodegradable vaccine. These poly(lactic-co-glycolic) acid (PLGA) microspheres are approved as drug delivery system in humans and display promising properties to serve as therapeutic vaccine against cancer. In the murine system, we could show that cross-presentation of PLGA MS-encapsulated antigen can be performed by both dendritic cells (DCs) and macrophages. In contrast to the current idea that CD8+ DCs are the important cell type in cross-presentation we provide evidence for a dominant role of CD8- DCs and macrophages. In chapter II we investigated the intracellular fate of PLGA MS after phagocytosis by APCs. Based on the encapsulation of inorganic nanocrystals, we introduce a novel method to label PLGA MS for electron and fluorescent microscopy, as well as magnetic cell sorting. Using these tools, we were able to show that PLGA MS do not enter the cytosol after uptake, but are stored in lysosomal vesicles for at least 72h. Since cross-presentation of encapsulated antigen already occurred at much earlier time points, our data have implications on the intracellular cross-presentation pathway. In chapter III we studied the molecular requirements concerning antigen-stability on the efficiency of DNA vaccines and immune responses induced by recombinant vaccinia virus (VV). We could show that targeting antigens for rapid degradation increases direct-presentation in vitro, but in contrast inhibits in vivo responses to DNA vaccination and recombinant VV. The results shown here argue for a dominant role of cross-presentation in the systems analyzed. In chapter IV we investigated the role of heat-shock proteins (HSPs) and other cellular factors on the cross-presentation of cell-associated antigen. We provide evidence that the stable full-length protein, but not antigenic peptides, is the source of antigen transfer to APCs. At the same time we exclude several HSPs as mediators for cross-presentation in this system. Finally we show biochemical strategies to find interaction partners of a viral antigen that can mediate the transfer to APCs. Taken together the data accumulated in this thesis contribute to the development of novel strategies to enhance immune responses following vaccination.