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

M Axmann, GJ Schütz, JB Huppa

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)

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.

Original languageEnglish
Article numbere53157
Pages (from-to)e53157
JournalJournal of visualized experiments : JoVE
Volume2015
Issue number104
DOIs
Publication statusPublished - 30 Oct 2015

Keywords

  • Bioengineering
  • Calcium flux measurement
  • Förster resonance energy transfer
  • Immunological synapse
  • Issue 104
  • Receptor-ligand interaction kinetics
  • Single molecule microscopy
  • T-cell antigen recognition
  • Amino Acid Sequence
  • T-Lymphocytes/immunology
  • Humans
  • Molecular Sequence Data
  • Major Histocompatibility Complex/immunology
  • Fluorescence Resonance Energy Transfer/methods
  • Antigens/immunology
  • Antigen-Presenting Cells/immunology
  • Lipid Bilayers
  • Receptors, Antigen, T-Cell/immunology
  • Protein Binding
  • Microscopy, Fluorescence/methods

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