T-cell triggering thresholds are modulated by
the number of antigen within individual
T-cell receptor clusters
Boryana N. Manza,b, Bryan L. Jacksona,c, Rebecca S. Petita,c, Michael L. Dustind, and Jay Grovesa,b,c,1
aHoward Hughes Medical Institute, Department of Chemistry, and
cPhysical Biosciences and Materials Sciences Divisions, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and
Pathogenesis, Skirball Institute of Biomolecular Medicine and Department of Pathology, New York University School of Medicine, New York, NY 10016
bBiophysics Graduate Group, University of California, Berkeley, CA 94720;
dProgram in Molecular
Edited* by Ronald D. Vale, University of California, San Francisco, CA, and approved April 15, 2011 (received for review January 10, 2011)
T cells react to extremely small numbers of activating agonist
peptides. Spatial organization of T-cell receptors (TCR) and their
peptide-major histocompatibility complex (pMHC) ligands into
microclusters is correlated with T-cell activation. Here we have
designed an experimental strategy that enables control over the
number of agonist peptides per TCR cluster, without altering the
total number engaged by the cell. Supported membranes, parti-
tionedwith gridsofbarriersto lateralmobility, provideaneffective
way of limiting the total number of pMHC ligands that may be
assembled within a single TCR cluster. Observations directly reveal
that restriction of pMHC content within individual TCR clusters
can decrease T-cell sensitivity for triggering initial calcium flux at
fixed total pMHC density. Further analysis suggests that triggering
thresholds are determined by the number of activating ligands
available to individual TCR clusters, not by the total number en-
countered by the cell. Results from a series of experiments in which
the overall agonist density and the maximum number of agonist
per TCR cluster are independently varied in primary Tcells indicate
that the most probable minimal triggering unit for calcium signal-
ing is at least four pMHC in a single cluster for this system. This
threshold is unchanged by inclusion of coagonist pMHC, but costi-
mulation of CD28 by CD80 can modulate the threshold lower.
cell biophysics ∣ cell patterning ∣ immune synapse
teins on the surface of antigen presenting cells. Some studies have
suggested that Tcells may react to even a few individual agonist
peptide molecules (1–3). Moreover, Tcells maintain this extreme
sensitivity without spontaneous triggering in the absence of
agonist, which could lead to autoimmune disorders (4). Various
mechanisms for how high sensitivity to agonist with apparent
immunity to stochastic noise have been proposed. Some include
T-cell receptors (TCR) monomer triggering by coreceptor or self-
peptide-major histocompatibility complex (pMHC) heterodimer-
ization (5, 6) or force-induced conformational changes (7).
Cooperativity among multiple receptors within the TCR-pMHC
clusters is also implicated (8), which may result from conforma-
tion-induced clustering (9), kinetic segregation (10), or lipid-
mediated assembly (11). However, the specific physical mechan-
isms by which these remarkable capabilities are achieved remain
unresolved, due largely to experimental limitations. Key to ulti-
mately determining, and experimentally verifying, the molecular
mechanism of TCR triggering is the ability to manipulate TCR
cluster assembly in living T cells.
In the following, we present results from experiments that
directly probe the consequences of differentially partitioning ago-
nist, coagonist, and null pMHC among TCR clusters in primary T
cells. The strategy is based on the hybrid live cell–supported
membrane configuration, in which a solid supported lipid bilayer
takes the place of the antigen presenting cell (12–14) (Fig. 1).
Histidine-tagged variants of MHC class II, intercellular adhesion
cells exhibit an exquisite ability to recognize extremely low
densities of agonist peptide antigen displayed in MHC pro-
molecule-1 (ICAM-1), and CD80 for costimulation experiments
are linked to the membrane through Ni2þ-chelating lipid groups
(14, 15). The lipids and proteins diffuse freely and as monomers
on the supported membrane (16) (Fig. S1). Peptide composition
and pMHC density are under direct experimental control. TCR
clustering is controlled through grid patterns of metal lines,
which have been prefabricated onto the underlying solid sub-
strate. These structures, known as diffusion or mobility barriers
(12, 16), block lateral transport of lipids and supported mem-
brane associated proteins. Molecules diffuse freely within each
corral, but are unable to hop between separate corrals. As the
TCR engage antigen pMHC at high antigen densities, clustering
ensues until all pMHC within a single corral of the grid-parti-
tioned supported membrane are coalesced into a single cluster
with their cognate TCR receptors (12) (Fig. 1D and Fig. S2).
The number of pMHC within each supported membrane corral
determines the maximum pMHC content of the corresponding
TCR cluster that may assemble on the T cell. Thus adjusting
the grid size at constant pMHC density titrates the maximum
number of pMHC per TCR cluster without changing the number
of antigens engaged by the Tcell. The total number of TCR and
other signaling molecules within clusters is not limited by the
substrate partitions. We refer to this physical manipulation of
molecular organization within living cells as a spatial mutation
(12, 17, 18). In the present application, T cells differing only in
the peptide agonist distribution among TCR clusters are gener-
ated and compared side-by-side.
The primary goal of the present study is to determine how ago-
nist distribution among TCR clusters governs T-cell activation.
We perform a two parameter titration experiment in which the
overall antigen peptide surface density as well as its partitioning
among TCR clusters are independently controlled in living
Tcells. The results indicate that the threshold antigen densities
for triggering Ca2þflux (in terms of the number of antigens per
cell) are dependent on agonist partitioning among TCR clusters.
Most significantly, when antigen dose-response functions ob-
tained on different grid partition sizes are analyzed in terms of
the maximum number of antigens per TCR cluster (determined
by pMHC content within individual membrane corrals), they col-
lapse onto a single curve.
We observe that T cells trigger at an average agonist density
of approximately two per TCR cluster, irrespective of the total
number of agonist engaged by the cell. The term triggering
threshold is used here to describe the average density at which
Author contributions: B.N.M., M.L.D., and J.T.G. designed research; B.N.M. performed
research; B.N.M., B.L.J., R.S.P., and M.L.D. contributed new reagents/analytic tools;
B.N.M. analyzed data; and B.N.M., M.L.D., and J.T.G. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence should be addressed. E-mail: JTGroves@lbl.gov.
This article contains supporting information online at www.pnas.org/lookup/suppl/
www.pnas.org/cgi/doi/10.1073/pnas.1018771108PNAS ∣ May 31, 2011 ∣ vol. 108 ∣ no. 22 ∣ 9089–9094
supported membrane system may be less physiological than live
APCs, it is far better controlled and therefore offers more quan-
titative information on the behavior of the TCR signaling system
under specific conditions.
In conclusion, the TCR cluster size titration experiments
described here arguably constitute the most quantitative analysis
of TCR triggering thresholds to date. The results are generally
consistent with the extreme, near-single-molecule sensitivity of
T cells to antigen long thought to exist. Cluster size titration
further reveals that spatial organization into TCR clusters is cri-
tical to achieve this sensitivity. The universal observation that
Ca2þflux triggering thresholds were determined on a per TCR
cluster basis, rather than a per T-cell basis, raises interesting ques-
tions concerning large-scale cooperativity in receptor signaling
clusters and apparent lack of cooperativity between clusters.
Materials and Methods
Mice and T cells. AND CD4+ T-cell blasts were prepared by in vitro stimulation
of spleen and lymph node cells from F1 cross of ANDx B10.Br mice (Jackson
Laboratory) with 1–2 μM MCC peptide. Cells were maintained in IL-2 every
48 hr and used on days 5 and 7.
Patterned Supported Proteolipid Bilayers. Glass slides were patterned by
e-beam lithography at University of California, Berkeley Microfabrication
Lab and Molecular Foundry, Lawrence Berkeley National Laboratory.
Liposomes with 97.5 mol % 1,2-dioleoyl-sn-glycero-3-phosphocholine,
2 mol % 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiace-
tic acid)succinyl] (nickel salt) and 0.5 mol% Texas Red® 1,2-dihexadecanoyl-
sn-glycero-3-phosphoethanolamine, triethylammonium salt, prepared by
sonication or extrusion, were injected over substrate in Biophtech FCS2 flow
chamber. His-tagged proteins were expressed in Hi5 cells and purified by
Ni-nitrilotriacetic acid affinity (14, 22). Surface density of MCC–MHC was
measured with AlexaFluor conjugated MCC peptide and calibrated with
supported bilayers (43).
Fluorescence Imaging. For calcium flux, cells were loaded with 1 μM fura-2-
acetoxymethyl and imaged at 340 and 380 nm with emission at 510 nm
via 40xS Fluor objective on Quantix 57 or CoolSnap K4 cameras. Fields of view
with patterns were monitored every 7 or 15 s for 20 min. Cell motion and
fluorescence intensity were tracked and analyzed semiautomatically in
Imaris, Metamorph, Matlab, and Excel. TCR, actin, and CD45 were imaged
via 100x N.A.1.3 objective on CoolSnapHQ camera. Total internal reflection
fluorescence (TIRF) was done via 100x 1.45 N.A. TIRF objective on a Cascade
512B EM CCD camera.
ACKNOWLEDGMENTS. The authors acknowledge Andrew DeMond, Nina
Hartman, Joseph Hickey, and Jeff Nye for experimental reagents, and the
Molecular Foundry, Lawrence Berkeley National Laboratory for substrate
preparation. Work at the Molecular Foundry was supported by the Office
of Science, Office of Basic Energy Sciences, of the US Department of Energy
under Contract DE-AC02-05CH11231. J.T.G. acknowledges support from the
Chemical Sciences, Geosciences and Biosciences Division, Office of Basic
Energy Sciences, of the US Department of Energy under Contract DE-AC03-
76SF00098. M.L.D. was supported by National Institutes of Health R37
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