Biology Reference
In-Depth Information
INTRODUCTION
T-cell antigen receptor (TCR) clustering was long thought to be dependent on bind-
ing of its ligand, a major histocompatibility complex (MHC) molecule presenting a
pathogen-derived peptide. Early confocal microscopy studies showed that upon rec-
ognition of its antigen, the TCR concentrated in the contact area between the T cell
and the cognate peptide/MHC (pMHC)-expressing antigen-presenting cell (APC),
forming the immunologic synapse (IS) (
Grakoui et al., 1999; Monks, Freiberg,
Kupfer, Sciaky, & Kupfer, 1998
). With the advance in confocal imaging techniques
and the development of supported lipid bilayers incorporating pMHC molecules and
adhesion molecules, it was observed that IS formation is preceded by ligand-
dependent formation of microclusters, 0.5-1
m large structures containing TCRs
and downstream signaling molecules, that move and coalesce in the IS (
Varma,
Campi, Yokosuka, Saito, & Dustin, 2006; Yokosuka et al., 2005
). However, in
the last decade, it has become clear that clusters of the TCR can be detected at
the cell surface of T cells in absence of any ligand. These clusters, with size ranging
from tens to a few hundred nanometers and therefore referred to as TCR nanoclus-
ters, were first shown via electron microscopic analysis of cell surface replicas of
intact cells (
Kumar et al., 2011; Schamel et al., 2005
) and of T-cell membranes
(
Lillemeier et al., 2010
) and more recently by superresolution light microscopy tech-
niques (
Lillemeier et al., 2010; Sherman et al., 2011
).
The relevance of determining the clustering state of a receptor before ligand bind-
ing lies in the fact that preclustering can give rise to synergy in ligand binding and/or
receptor output, which would result in receptor properties that cannot be explained
by considering the receptor as a monomer. Theoretical and experimental studies on
the chemotactic receptors in
E. coli
have shown that clustering of these receptors is
essential for permitting the exquisitely sensitive chemotactic response of these bac-
teria (
Bray, Levin, &Morton-Firth, 1998
). Similar mechanisms appear to play a role
in TCR-dependent signaling. Even though monomeric TCRs have a low affinity for
their cognate pMHC ligand, T cells only need to detect a few pMHC molecules on
the APC in order to become activated (
Demotz, Grey, & Sette, 1990; Harding &
Unanue, 1990; Irvine, Purbhoo, Krogsgaard, & Davis, 2002
). Our own studies indi-
cate that TCR complexes present in nanoclusters become more activated than mo-
nomeric TCRs under conditions of weak stimulation (
Schamel et al., 2005
) and
that they play a direct role in the increase in T-cell sensitivity accompanying the tran-
sition from naive to memory T cells (
Kumar et al., 2011; Molnar, Deswal, &
Schamel, 2010; Molnar et al., 2012; Schamel & Alarcon, 2013
). Knowing whether
a receptor is present at the cell surface as a cluster before ligand binding should not
only help to explain the biology of that receptor but also open new possibilities for
interfering with its function for therapeutic purposes.
The technique consists of fixing cells with paraformaldehyde (PFA) to avoid any
protein movements at later steps, followed by labeling with antibodies against the
protein of interest and colloidal gold-coupled secondary reagents. Cells are adhered
m
