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G. Schütz, J. Huppa:
"Förster resonance energy transfer to study TCR-pMHC interactions in the immunological synapse";
The Immune Synapse: Methods and Protocols, 158 (2017), 207 - 229.

T-cell antigen recognition is remarkably efficient: when scanning the surface of antigen-presenting cells
(APCs), T-cells can detect the presence of just a few single antigenic peptide/MHCs (pMHCs), which are
often vastly outnumbered by structurally similar non-stimulatory endogenous pMHCs (Irvine et al.,
Nature 419(6909):845-849, 2002; Purbhoo et al., Nat Immunol 5(5):524-530, 2004; Huang et al.,
Immunity 39(5):846-857, 2013). How T-cells achieve this is still enigmatic, in particular in view of the
rather moderate affinity that TCRs typically exert for antigenic pMHCs, at least when measured in vitro
(Davis et al., Ann Rev Immunol 16:523-544, 1998). To shed light on this in a comprehensive manner, we
have developed a microscopy-based assay, which allows us to quantitate TCR-pMHC interactions in situ,
i.e., within the special confines of the nascent immunological synapse of a T-cell contacting a planar-supported
lipid bilayer functionalized with the costimulatory molecule B7-1, the adhesion molecule
ICAM-1, and pMHCs (Huppa et al., Nature 463(7283):963-967, 2010) (Fig. 1). Binding measurements
are based on Förster resonance energy transfer (FRET) between site-specifically labeled pMHCs and
TCRs, which are decorated with recombinant site-specifically labeled single-chain antibody fragments
(scFV) derived from the TCRβ-reactive H57-597 antibody (Huppa et al., Nature 463(7283):963-967,
2010). FRET, a quantum-mechanical phenomenon, involves the non-radiative coupling of dipole moments
of two adjacent fluorophores, a donor molecule and an acceptor molecule. FRET efficiency is inversely
proportional to the sixth power of the inter-dye distance. Hence, it can be employed as a molecular ruler
(Stryer and Haugland, Proc Natl Acad Sci, USA 58(2):719-726, 1967) or, as is the case here, to score for
interactions of appropriately labeled molecules. To facilitate both quantitative and single-molecule readout,
it is important to conjugate donor and acceptor dyes in a site-specific manner.
While SLBs mimic some but certainly not all properties of a plasma membrane of a living cell, their
use features a number of operational advantages: SLBs can be prepared in a fluid state, thereby facilitating
the spatial rearrangements that accompany the formation of an immunological synapse (Grakoui
et al., Science 285(5425):221-227, 1999). The imaging of a three-dimensional binding process is
reduced to two dimensions, which saves time and fluorophore-emitted photons and allows for fast measurements.
Furthermore, images can be acquired in noise-attenuated total internal reflection (TIR)
mode, so far a necessity for single-molecule detection within the immunological synapse. Importantly,
the stimulatory potency of pMHCs is very well preserved compared to cell surface-embedded pMHCs.
Hence, while in principle artificial, SLBs are still a good approximation of the physiologic scenario a
T-cell encounters when approaching an APC. Vice versa, the reconstitutive approach offers unique
opportunities to interrogate the influence of accessory molecules on T-cell antigen recognition in a
highly quantitative manner.