We take advantage of FRET to measure in situ the synaptic interactions between cell-bound TCRs and their cognate ligands, pMHC, embedded in a planar lipid bilayer. We engineered a site-specifically labelled TCR-reactive scFv to tag cell-surface-located TCRs. When the TCR is bound to its ligand, its associated scFv brings its label (FRET donor) close enough to the FRET acceptor dye attached to the MHC-embedded peptide to give rise to a FRET signal. We recorded smFRET to measure the t1/2 values of synaptic TCR–pMHC interactions. We combined smFRET and bulk FRET measurements to determine synaptic Kd values and kon values. We applied antibody-mediated blockade of CD4 to assess its contribution to TCR–pMHC binding.
Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature .
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Quantifying TCR-pMHC Interactions via FRET
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Corresponding Organization :
Other organizations : Stanford Medicine, Johannes Kepler University of Linz, Agilent Technologies (United States), Howard Hughes Medical Institute
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Variable analysis
independent variables
- Antibody-mediated blockade of CD4
- FRET signal (to measure the t1/2 values of synaptic TCR–pMHC interactions)
- Synaptic Kd values
- Synaptic kon values
- Site-specifically labelled TCR-reactive scFv to tag cell-surface-located TCRs
- FRET acceptor dye attached to the MHC-embedded peptide
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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