FRET system to monitor TCR-pMHC binding in situ

Shown is the FRET (Förster Resonnance Energy Transfer) system, which we apply to measure interactions between the T cell receptor (in blue) on the T cell and peptide-loaded MHC molecules (in yellow and red) on the antigen presenting cell as they occur within the immunological synapse. To do so reproducibly, we typically attach the corresponding fluorescent dyes Alexa Fluor 555 (green) and Alexa Fluor 647 (red) to the interaction partners in a site-specific manner. As of now there are no methods available to label cell-bound receptors such as the TCR site-specifically with organic dyes. Hence we modified a TCR-reactive monoclonal antibody to serve as a monovalent probe (shown here in light brown).This probe does not interfere with the TCR-pMHC interaction but can readily be labeled in a site-specific manner. When TCR and pMHC are in the bound state, the corresponding FRET dyes are as close as ~ 41 Å, which supports a FRET-efficiency of more than 50%. Such strong FRET signals allow  us to quantify TCR-pMHC interactions within the immune synapse both on a bulk and single molecule level.

The displayed model is a composite of a TCR-pMHC structure and the TCR-H57 Fab stucture.



Single molecule FRET events within the Immunological Synapse


The movie above shows the FRET channel of a microscopy experiment in which we image the Immunological Synapse between a T cell and a glass-supported lipid bilayer. As explained in the illustration on top, cell-associated TCRs are decorated with the H57 scFv-Cy3 as FRET donor. Bilayer-embedded pMHCs are labeled in low abundance with Cy5 and serve as FRET acceptor. The duration of single molecule FRET events, which appear temporarily as flashes of light and which represent single TCR-pMHC interactions, can be easily monitored. The outline of the Cy3-labeled T cell bleeds through in this raw FRET movie .

time frame: 165ms/ image