title: Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay.
abstract: T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (µM range) to antigen when binding is measured in vitro(1). In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) (2). Measurements are based on Förster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.
authors.affiliation: Institute for Applied Physics - Biophysics, Vienna University of Technology.
authors.forename: Markus
authors.initials: M
authors.lastname: Axmann
authors.affiliation: Institute for Applied Physics - Biophysics, Vienna University of Technology.
authors.forename: Gerhard J
authors.initials: GJ
authors.lastname: Schütz
authors.affiliation: Institute for Hygiene and Applied Immunology, Medical University of Vienna; Johannes.Huppa@meduniwien.ac.at.
authors.forename: Johannes B
authors.initials: JB
authors.lastname: Huppa
mesh_terms: D000595:Amino Acid Sequence; D000938:Antigen-Presenting Cells / Q000276:immunology; D000941:Antigens / Q000276:immunology*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008051:Lipid Bilayers; D008285:Major Histocompatibility Complex / Q000276:immunology*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D000941:Antigens; D008051:Lipid Bilayers; D011948:Receptors, Antigen, T-Cell