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| Item Type: | Article |
|---|---|
| Title: | Measuring TCR-pMHC binding in situ using a FRET-based microscopy assay |
| Creators Name: | Axmann, M., Schütz, G.J. and Huppa, J.B. |
| 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. |
| Keywords: | Amino Acid Sequence, Antigen-Presenting Cells, Antigens, Fluorescence Resonance Energy Transfer, Lipid Bilayers, Major Histocompatibility Complex, Fluorescence Microscopy, Molecular Sequence Data, Protein Binding, T-Cell Antigen Receptors, T-Lymphocytes / Immunology |
| Source: | Journal of Visualized Experiments |
| ISSN: | 1940-087X |
| Publisher: | JoVE |
| Number: | 105 |
| Page Range: | e53157 |
| Date: | 30 October 2015 |
| Official Publication: | https://doi.org/10.3791/53157 |
| PubMed: | View item in PubMed |
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