Defined stimulation of T cells

The goal of this proposal is to explore the precise requirements for cell activation via specific cell surface receptors. This intent will be achieved by developing cellular assays allowing online measurement of activation parameters and by combining two established high-end methods of single-molecule spectroscopy, namely atomic force microscopy (AFM) and wide-field fluorescence microscopy (WFM) for temporally and quantitatively defined triggering of receptors and determination of the resulting response. The development of this microscope will be the subject of a separate project proposed by Dr. Enderlein from the Institute for Biophysics, Johannes Kepler University of Linz (see enclosure). The specific aim of this project that is interdependent and closely linked to the technical one, is the design and establishment of the required cellular assays. As target cells we will use T cells, which play a key role in the immune system of higher organisms. A central element of their functioning is their highly specific recognition and, after activation, destruction of pathogens. The crucial molecule for the initial activation of T cells is the T cell receptor (TCR) specifically recognizing a peptide derived from an antigenic protein of the pathogen. Although the TCR is one of the best-studied cell surface receptors, there are still unknown the precise requirements for efficient and productive T cell activation. The variables include number of TCRs triggered, valency of the ligand(s), affinity, the kinetic of the binding, and the influence of coreceptors. The employment of the microscopic techniques as proposed here are supposed to give an answer to this fundamental question in immunology by allowing precise triggering of the TCR and its co-receptors. Together with the developed cellular assays, T cell activation will not only be investigated from individual, combined or cumulative TCRlcoreceptor triggering events but also monitored online providing a clear picture of the T cell activation mechanism. The knowledge of this central immunological process is instrumental to design novel therapeutic regiments to specifically direct the outcome of the immune response either to the signs of infection or to induction of immmunological tolerance. The possibility to therapeutically switch the immune system between activation and tolerization is the basis to develop both new vaccine strategies as well as specific treatments for various immunological diseases including allergy (asthma, atopic dermatitis, rhinitis) and autoimmune disorders (rheumatoid arthritis, arteriosclerosis, multiple sclerosis), which are caused by a break of immunological tolerance to harmless or self substances.

During this project we were aiming at visualizing the spatio-temporal reorganization and action of key molecules of the signaling pathways of T cells upon recognition of pathogens using an ultra-sensitive single molecule microscopy technique, called single dye tracing (SDT). Indeed, this novel technique has provided new vistas into the function of the tested molecules that allow redefining several key steps in the signaling pathway of T cells. Knowledge of this central signaling process of the immune system is instrumental to design novel therapeutic regiments to specifically direct the outcome of the immune response either to the signs of infection or to induction of immunological tolerance. The possibility to therapeutically switch the immune system between activation and tolerization is the basis to develop both new vaccine strategies as well as specific treatments for various immunological diseases including allergy and autoimmune disorders. SDT, established by our partners in Linz and controlled only by a few laboratories worldwide, is unique in that it allows tracking the movement and function of single molecules in living cells in real time. All other techniques available so far allow only ensemble measurements of large numbers of molecules determined by the sensitivity level of the respective method. It is this sensitivity of single molecules, which allows by SDT for the first time to analyze how individual molecules act time- and space-resolved in the cell. Therefore, single molecule analysis was ranked by the "Science" journal within the ten breakthroughs of the year 2003. The specific aim of this project was the preparation of biological tools for the SDT technique. We followed two alternative routes: (i) Monovalent antibody fragments to key signaling molecules were established and labeled with fluorescent dyes. Using these reagents we recorded and analyzed for the first time the trajectories of receptors on the surface of T cells. The results obtained provide a novel picture of the architecture of the plasma membrane of T cells and its reorganization upon recognition of pathogens. (ii) On living cells, antibodies can only be used to label surface molecules. In order to reach the interior of cells, we generated genetic fusions of signaling molecules with variants of green fluorescent protein GFP. Using these reagents our main achievement was the visualization of the molecular folding processes in Lck, the central molecule in T cell signaling whose structure is strongly influencing its activity, in living T cells. In another part of the project we could identify an important marker for development of regulatory T cells, which play a key role in concerting immune responses and preventing autoimmune reactions. Additionally we have established a system, which allows to stimulate T cells in a spatio-temporally defined way using a miniaturized needle with antibodies bound to its tip.