Autoimmunity

Our ability to manipulate and/or target the T-cell compartment for therapeutic benefit will hinge upon our deeper understanding of the genes regulating immune development and autoimmunity. Self-tolerance is maintained at two levels: “Central tolerance” is achieved in the thymus where autoreactive T cells are either deleted or anergized, a process for which the transcription factor AIRE is indispensable. The second level of tolerance, or “peripheral tolerance”, is maintained by regulatory T cells. Regulatory T cells have a specific phenotype (CD4+CD25+), and their generation is dependent upon expression of the forkhead protein Foxp3. To date over ten single genes have been identified (including AIRE and Foxp3) that, when mutated, cause autoimmune manifestations. However, many autoimmune diseases are of unknown genetic origin, as for example insulin dependent diabetes mellitus (IDDM, or Type I diabetes). IDDM is an autoimmune disorder in which T cells attack pancreatic islet cells from self. Zebrafish and higher vertebrates show a high level of conservation in pancreatic development, with very similar endocrine and exocrine compartments. T cell development is also highly conserved, and zebrafish have all the necessary components (including T cell receptor, antigen presenting cells, MHC class I and II molecules) to mount an adaptive immune response. In addition, we have cloned the zebrafish orthologs of AIRE and Foxp3, and have shown that Foxp3 is, as expected, only expressed in mature T cells, but not in immature T cells or B cells.

The goal of this project is twofold. First, we are in the process of inactivating the zebrafish Foxp3 gene using TILLING. Once an inactivating mutation is identified, we will breed the Foxp3-/- individuals onto the lck-promoter-GFP transgenic background. In this way we will be able to monitor autoimmune manifestations by in-vivo fluorescent imaging. An example of a potential outcome is shown in the Figure, which shows either discrete, localized accumulation of fluorescent T cells, or diffuse infiltration (pictures are taken from a leukemia transplantation model). If successful, this model will represent a paradigm for autoimmunity in zebrafish. The second goal is to establish the zebrafish as a vertebrate model system to identify and characterize genes involved in the development of autoimmune diseases, including IDDM. In order to achieve this goal we plan to use the lck promoter–GFP transgenic line in a forward genetic mutagenesis screen to visualize T-cell infiltration of organs such as the pancreas, heart muscle, and the central nervous system, as an indicator of autoimmune activation. We are using multispectral in-vivo imaging to visualize T-cell migration in-vivo in wildtype and mutagenized adult zebrafish. Mutants with aberrant infiltration of T cells into organs will be subjected to more detailed analysis, with the aim of positional cloning of interesting candidates.