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

M Axmann, GJ Schütz, JB Huppa

Publikation: Beitrag in FachzeitschriftArtikelBegutachtung

5 Zitate (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.

OriginalspracheEnglisch
Aufsatznummere53157
Seiten (von - bis)e53157
FachzeitschriftJournal of visualized experiments : JoVE
Jahrgang2015
Ausgabenummer104
DOIs
PublikationsstatusVeröffentlicht - 30 Okt 2015

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