Antigen presenting cells (APCs) form the critical link between innate and adaptive immune responses in mammals. The mechanisms of lymphocyte activation in lower vertebrates, however, are largely unknown. Our preliminary studies suggest that the cellular constituents responsible for antigen presentation are remarkably conserved from zebrafish to mammals. In mammals, dendritic cells (DCs) are the most potent cells in priming an adaptive response through direct activation of lymphocytes. We have identified DC-like cells in the lymphoid organs of zebrafish and initiated studies to enable their prospective isolation and functional testing. We will begin our proposed studies in the adult to determine the cellular effectors of antigen presentation. We will develop functional assays to determine which cell types can traffic foreign antigen to secondary lymphoid organs and stimulate proliferation of antigen-specific T lymphocytes. To complement these functional approaches, we will identify markers capable of enriching APCs from hematolymphoid tissues. These basic studies will set the stage for an assessment of the ontogeny of immunity in the zebrafish embryo. We will make use of novel and existing transgenic lines to fluorescently label APCs and the major branches of the hematolymphoid system, and leverage the unique attributes of zebrafish to image the immune response to pathogen in real time in living animals. These studies will help us determine the predominant sites of immune cell interactions. With the ability to specifically identify each of the APC subsets, we will utilize this information to determine the cellular architecture of secondary lymphoid organs under steady-state conditions. We will subsequently ascertain the changes that result in these tissues following immune challenge. Finally, we will ablate APCs using a conditional transgenic toxin to determine the effects upon lymphoid architecture and the overall immune response. Taken together, these studies will provide a new level of precision in the study of the zebrafish immune response, and will lead to a better understanding of the role of APCs in bridging the ancient and acquired arms of the vertebrate immune system. In addition, these efforts will provide unprecedented, real-time study of APC behavior in living animals. Ultimately, this work should lead to new discoveries regarding antigen presenting cells by utilizing the unique imaging and genetic advantages of the zebrafish.
We will utilize the unique advantages of zebrafish to develop the tools and assays to ultimately study antigen presenting cells directly through microscopic observation in translucent animals. We have discovered that zebrafish possess dendritic cells, by far the most efficient antigen presenting cells in mammals, which should allow us to better understand how the innate immune system communicates with lymphocytes of the adaptive immune system. The conservation of immune cell types and functions between zebrafish and mammals, ability to directly visualize interactions between immune cell types and between host and pathogen, and ability to discover new gene functions through forward genetic approaches should enable new discoveries in the zebrafish to eventually be translated to the clinic.
