Our laboratory discovered a novel antigen presenting pathway in which some professional antigen presenting cells (APCs) could present antigens from the extracellular fluids on class I molecules. This project's overall theme is an analysis of this pathway with a major emphasis on in vivo studies which should provide important information about its physiological relevance. Another goal is to elucidate its underlying cellular and molecular mechanisms. The importance of these goals is that this pathway is likely to play a major role in the generation of CTL responses to several pathogens including viruses and cancers. Furthermore, this pathway has the potential to be exploited for vaccine delivery. We have 3 specific Aims. The goal of Aim 1 is to elucidate the various underlying mechanisms of the pathway. We have previously defined a novel process in which exogenous antigens are transferred from phagosomes to the cytosol by unknown mechanism. In one set of experiments we will seek to elucidate this mechanism. There is also a second pathway which does not involve transfer of antigen to the cytoplasm. We will critically examine three distinct hypothesis for how this pathway operates. The goal of Aim 2 is to determine what APCs and what mechanisms operate to elicit CTL responses to exogenous antigens in in vivo situations. Our underlying hypotheses are that in vivo dendritic cells are the key. APCs for initiating CTL responses of exogenous antigens and that the physiologically important mechanism is the phagosome-to-cytosol pathway. Our experimental approach will be to use animals that genetically lack key component of the cytosolic pathway or specific APCs and also to use antigens that are dependent on cytosolic versus vacuolar processing. We will also test the ability of different APCs to reconstitute the pathway. The goal of Aim 3 is to examine the role of the exogenous pathway in the generation of CTL responses. We hypothesize that it is the major pathway for initiating primary and secondary CTL responses to viruses that do not infect professional APCs and to somatic cells of transplants which lack endogenous APCs. In addition, we hypothesize that this pathway allows immune surveillance against pathogens that chronically infect phagocytes. Our experimental approach will examine whether CTLs are stimulated in vivo when professional APCs cannot synthesize the antigen (e.g. cannot be infected with a virus) and when animals lack the exogenous pathway of presentation. We will also examine the importance of MHC class II presentation and costimulation in the generating responses in these settings.
