Advanced Quantification of Receptor-Ligand Interaction Lifetimes via Single-Molecule FRET Microscopy.

Area of Science:

• Immunology
• Biophysics
• Computational Biology

Background:

• Receptor-ligand interactions, particularly T cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) binding, are fundamental to T cell activation and immune responses.
• The duration of TCR-pMHC interactions (dwell time) is critical for signaling efficacy, distinguishing between agonist strengths.
• Existing methods like surface plasmon resonance (SPR) lack cellular context, while single-molecule Förster resonance energy transfer (smFRET) offers a cellular perspective but requires robust analysis.

Purpose of the Study:

• To introduce a novel mathematical model for determining receptor-ligand interaction lifetimes using survival analysis.
• To develop and validate a comprehensive computational pipeline for extracting TCR-pMHC interaction kinetics from microscopy data.
• To provide an automated, robust, and unbiased methodology for studying T cell activation mechanisms.

Main Methods:

• Development of a survival analysis-based mathematical model to quantify exponentially distributed interaction lifetimes.
• Creation of an image analysis pipeline to process raw microscopy data and extract kinetic parameters.
• Validation of the model and pipeline using simulated data and multiple TCR-pMHC interaction pairs.

Main Results:

• The mathematical model accurately determines receptor-ligand interaction lifetimes, verified with simulated data.
• The analysis pipeline successfully extracts kinetic data from microscopy images, demonstrating robustness across various TCR-pMHC pairs.
• The automated software suite enhances data processing throughput and minimizes experimental bias.

Conclusions:

• This methodology offers a refined tool for precise measurement of TCR-pMHC interaction dwell times in a cellular context.
• The findings provide deeper insights into the mechanisms governing T cell activation and immune response modulation.
• The developed software facilitates high-throughput, unbiased analysis of immune cell interactions.