Imaging the Molecular Dynamics of Immune Recognition
T-cell antigen recognition, immunological synapse, membrane proximal signaling, biophotonics, protein engineering
The Huppa lab applies a blend of biochemical and advanced imaging methods to identify cellular parameters underlying the phenomenal sensitivity of T-cells towards antigen. Given the moderate T-cell receptor (TCR) affinity for antigenic peptide/MHC complexes (pMHCs) and the unique three-dimensional constraints within the immunological synapse, conventional biochemical approaches are alone insufficient to inform how T-cells manage to detect the presence of even a single peptide/MHC antigen (pMHC) among thousands of structurally highly related yet non-stimulatory pMHCs on the surface of and antigen presenting cells (APCs). To this end Dr. Huppa and his coworkers devise and customize (single molecule) imaging modalities, which allow them to track synaptic key players in the course of T-cell recognition, synchronize their interactions with synaptic ligands through photo-uncaging and visualize them with millisecond temporal resolution via (single molecule) Förster Resonance Energy Transfer (FRET) measurements. Superresolution methodologies are applied to resolve synaptic conditions for receptor-ligand interactions below the diffraction limit of visible light, and to relate TCR engagement to downstream signaling and effector functions. Individual APC-associated parameters are reconstituted with the use of defined functionalized glass-supported planar lipid bilayers and assayed for their role in T-cell recognition. A central goal is to understand how cellular parameters are controlled in T-cell/APC development as a means to tune T-cell responsiveness in health and disease.
Collaborating research groups where PhD Students can perform their research stay
Mark M. Davis, Howard Hughes Medical Institute & Stanford School of Medicine, CA, USA
Tobias P. Dick, German Cancer Research Center, Heidelberg, Germany
Volker Haucke, Leibnitz Institute for Molecular Pharmacology, Berlin, Germany
Michael Hudecek, Center for Internal Medicine, Würzburg, Germany
Hidde L. Ploegh, Massachusetts Institute of Technology and Whitehead Institute of Biomedical Research, Cambridge, MA, USA
Lawren C. Wu, Amgen Inc., San Francisco, CA 9, USA
Know-how and infrastructure of the research group
Dr. Huppa is an expert in the field of T-cell antigen recognition, synaptic receptor-ligand interactions, T-cell signaling, protein engineering for the generation of imaging (e.g. site-specifically fluorescence-labeled T-cell ligands and monomeric single chain FV fragments) and antigen-specific T-cell isolation probes (e.g. pMHC-tetramers and streptamers), and advanced molecular imaging modalities. His lab has access to standard equipment required for molecular biology and tissue culture, specialized equipment to cultivate and process insect and E.coli cells in large quantities, a 4°C cold room (for protein refolding and dialysis), high speed and ultra centrifuges (for processing cell suspensions, media supernatant), to concentration devices (capacities ranging from 100 ml to 5 l), to a state-of-the-art FPLC (for protein purification) and HPLC-system (for reverse phase peptide purification) and a high-powered bath sonication device (for the generation of small unilamellar vesicles, which are required for manufacturing functionalized planar supported lipid bilayers used for T-cell stimulation and TIRF imaging). Furthermore, lab members have access to a radioisotope lab, flow cytometers and FACsorters. Together with his lab members Dr. Huppa has custom-built a temperature-controlled single molecule imaging platform (with customized excitation and emission beam paths), which is equipped with six lasers of different wavelengths (405 nm, 488 nm, 515 nm, 532 nm, 561nm, 647 nm) to conduct noise-attenuated multi-channel total internal reflection (TIRF) microscopy, stochastical superresolution microscopy and photo-uncaging studies. An integrated Xenon-excitation lamp allows for simultaneous measurements of intracellular calcium levels. A motorized stage supports precise sample alignment in X, Y and Z, and a motorized dichroic filter turret and a fast emission filter wheel enable optional switching between different excitation/emission wavelength settings.