Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

Area of Science:

• Immunology
• Cell Biology
• Biophysics

Background:

• T-cells recognize peptide-MHC (pMHC) complexes on antigen-presenting cells (APCs) via T-cell receptors (TCRs).
• TCRs typically exhibit moderate affinity for antigens in vitro, yet mediate highly sensitive T-cell responses in vivo.
• Understanding the in situ binding dynamics of TCR-pMHC interactions is crucial for elucidating T-cell activation.

Purpose of the Study:

• To develop and validate a microscopy-based methodology for monitoring TCR-pMHC binding in situ within the T-cell synapse.
• To quantify the affinity and kinetics of TCR-pMHC interactions at the single-molecule level.
• To establish a versatile platform for studying other receptor-ligand interactions in a native cellular environment.

Main Methods:

• Utilized a microscopy approach employing Förster Resonance Energy Transfer (FRET) between fluorescently labeled TCR and pMHC.
• Developed an artificial lipid bilayer system mimicking APC membranes for in situ binding assays.
• Measured FRET efficiency to determine inter-dye distance and infer binding kinetics.

Main Results:

• Successfully visualized and quantified TCR-pMHC binding within the T-cell synapse in situ.
• Detected single-molecule FRET events, enabling determination of TCR-pMHC binding affinity and off-rate.
• Provided a method to interpolate the on-rate of TCR-pMHC interactions.

Conclusions:

• The developed microscopy technique offers a sensitive platform for studying T-cell antigen recognition in real-time and in situ.
• This approach allows for precise characterization of TCR-pMHC binding kinetics, crucial for understanding T-cell sensitivity.
• The methodology is adaptable for investigating diverse receptor-ligand interactions within their native cellular contexts.