T Cell Receptor Signaling Can Directly Enhance the
Avidity of CD28 Ligand Binding
Mariano Sanchez-Lockhart1.¤, Ana V. Rojas2., Margaret M. Fettis1, Richard Bauserman3, Trissha R. Higa1,
Hongyu Miao2, Richard E. Waugh3, Jim Miller1*
1David H Smith Center for Vaccine Biology and Immunology and Department of Microbiology and Immunology, University of Rochester, Rochester, New York, United
States of America, 2Department of Biostatistics and Computational Biology, University of Rochester, Rochester, New York, United States of America, 3Department of
Biomedical Engineering, University of Rochester, Rochester, New York, United States of America
T cell activation takes place in the context of a spatial and kinetic reorganization of cell surface proteins and signaling
molecules at the contact site with an antigen presenting cell, termed the immunological synapse. Coordination of the
activation, recruitment, and signaling from T cell receptor (TCR) in conjunction with adhesion and costimulatory receptors
regulates both the initiation and duration of signaling that is required for T cell activation. The costimulatory receptor, CD28,
is an essential signaling molecule that determines the quality and quantity of T cell immune responses. Although the
functional consequences of CD28 engagement are well described, the molecular mechanisms that regulate CD28 function
are largely unknown. Using a micropipet adhesion frequency assay, we show that TCR signaling enhances the direct binding
between CD28 and its ligand, CD80. Although CD28 is expressed as a homodimer, soluble recombinant CD28 can only bind
ligand monovalently. Our data suggest that the increase in CD28-CD28 binding is mediated through a change in CD28
valency. Molecular dynamic simulations and in vitro mutagenesis indicate that mutations at the base of the CD28
homodimer interface, distal to the ligand-binding site, can induce a change in the orientation of the dimer that allows for
bivalent ligand binding. When expressed in T cells, this mutation allows for high avidity CD28–CD80 interactions without
TCR signaling. Molecular dynamic simulations also suggest that wild type CD28 can stably adopt a bivalent conformation.
These results support a model whereby inside-out signaling from the TCR can enhance CD28 ligand interactions by
inducing a change in the CD28 dimer interface to allow for bivalent ligand binding and ultimately the transduction of CD28
costimulatory signals that are required for T cell activation.
Citation: Sanchez-Lockhart M, Rojas AV, Fettis MM, Bauserman R, Higa TR, et al. (2014) T Cell Receptor Signaling Can Directly Enhance the Avidity of CD28 Ligand
Binding. PLoS ONE 9(2): e89263. doi:10.1371/journal.pone.0089263
Editor: Jon C.D. Houtman, University of Iowa, United States of America
Received November 12, 2013; Accepted January 17, 2014; Published February 24, 2014
Copyright: ? 2014 Sanchez-Lockhart et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the National Institute of Health (AI-83206, AI-090259, AI-105621, and PO1 HL018208). The funders had no role
in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤ Current address: Genomic Center, USAMRIID, Fort Detrick, Maryland, United States of America
. These authors contributed equally to this work.
Efficient T cell activation requires co-ligation of the T cell
antigen receptor (TCR) and costimulatory receptors. T cell
encounter with antigen in the absence of costimulation leads to
limited T cell activation and can induce anergy. The best
described costimulatory molecule is CD28. CD28 has been shown
to have a broad impact on many aspects of T cell function,
including T cell activation, elaboration of effector cytokine
expression, enhanced expansion and survival, upregulation of
metabolic activity, effector cell differentiation, memory responses,
and tolerance [1,2,3,4,5]. Interestingly, one of the key conse-
quences of the innate immune response to microbial challenge is
the upregulation on dendritic cells of CD80 and CD86, the ligands
for CD28. Thus, CD28 can be viewed as the T cell-associated
receptor for detection of pathogens.
CD28 functions as a modifier and amplifier of TCR-derived
signals . T cell activation takes place in the context of an
immunological synapse that is formed at the cell:cell contact
between a T cell and an antigen presenting cell (APC). T cell
signaling is initiated and sustained by the formation of TCR
microclusters that form at the periphery of the contact site and
move to and coalesce within the center of the immunological
synapse . TCR and CD28 are rapidly colocalized in these
microclusters . Although the precise mechanisms of CD28
costimulation are not fully understood, CD28 functions in part
through direct amplification of the TCR signal, for example
though activation of PI3K and Lck , and through unique
contributions, notably the recruitment of PKCh [9,10,11,12,13].
In addition to the well-described effects of CD28 on modulating
TCR signaling, recent studies indicate that TCR can also
modulate CD28 function. TCR signaling can induce a rapid
reorientation of the cytosolic tail domains within the CD28
homodimer as detected by a change in fluorescence resonance
energy transfer (FRET) . TCR signaling is also necessary for
sustained localization of CD28 to the immunological synapse
[11,13] and TCR signaling can induce CD28 polarization toward
CD80-positive cells . These results raise the possibility that
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Figure 1. TCR signaling can enhance the avidity of CD28 ligand binding. (A) Images from a cell adhesion assay. Top, a resting T cell and
CD80-coated bead brought into contact using micropipets. Middle, the same T cell 30s later after the cell and bead were separated. No adhesion was
observed. Bottom, image of a T cell bound to an anti-CD3 coated bead, which is out of the plane of focus, but clearly distal to the site of interaction of
the T cell with the CD80-bead. This image was taken shortly after the T cell was pulled away from the CD80-bead. In this case, the T cell-CD80-bead
adhesion has caused the T cell to dislodge from the micropipet. (B) Cell frequency adhesion of in vitro primed and resting WT DO11.10 T cells to
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TCR signaling may be able to enhance CD28 localization at the
immunological synapse by regulating the ability of CD28 to
interact with ligand. This process of inside-out signaling, whereby
intracellular signaling from one receptor can enhance ligand
binding of a second receptor, is well established for integrins
[15,16]. Inside-out signaling for integrins is initiated by a
conformational change in the cytosolic domains that is transduced
across the plasma membrane and results in a dramatic confor-
mational change in the multisegment extracellular domain. This
positions the integrin ligand binding domain in a membrane distal
position allowing for enhanced interaction with ligands. CD28
consists of a single immunoglobulin-like extracellular domain that
is unlikely to undergo large scale conformation changes, making
the finding that it can increase its affinity in response to
intracellular signaling all the more surprising.
In this report we have measured CD28 ligand binding using a
cell adhesion frequency assay and show that TCR signaling can
rapidly increase the avidity of CD28–CD80 interactions. Further-
more, our molecular dynamic (MD) simulation and site-directed
mutagenesis data support a model whereby the TCR-induced
increase in CD28 avidity results from a reorientation of the CD28
dimer to allow for bivalent ligand binding.
Materials and Methods
This study was carried out in strict accordance with the
recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health and with
the approval of the Animal Care and Use Committee at the
University of Rochester (Protocol Number 2002-159).
CD4-postive T cells were purified from WT or CD28-deficient
DO11.10 TCR transgenic mice or from CTLA-4-deficient, RAG-
deficient, 5C.C7 TCR transgenic mice (Taconic) and activated
in vitro with antigenic peptide and irradiated spleen cells. To limit
induction of CTLA4 expression, in some experiments WT and
CD28KO T cells were primed with CD80/CD86-negative
transfectants (ProAd-ICAM). T cells were used 7–10 days after
priming (14–21 days for CTLA-4-deficient T cells).
The K118I/K120P mutation was introduced into murine
CD28 by overlapping PCR and confirmed by DNA sequencing.
Both WT and K118I/K120P CD28 were fused at the C-terminus
to monomeric YFP with a 4 amino acid linker (RSTG) as
described  and cloned into the Murine Stem Cell Virus
retroviral vector, MIGR1 (which was deleted for the IRES-GFP).
The plasmid constructs were transiently transfected into the
Phoenix Ecotropic packaging cell line (provide by G. Nolan,
Stanford University, Palo Alto, CA) and virus in the supernatant
was concentrated by PEG precipitation (retro-X, Clontech).
CD28-deficient T cells were stimulated for 36 hours, isolated on
a ficoll gradient, and transduced with retrovirus in the presence of
4 mg/ml polybrene by centrifugation at 2000 rpm (Rcf 670) for 60
minutes. Expression of YFP and CD28 were determined by flow
cytometry 6–10 days after initial activation. WT and K118I/
K120P CD28-YFP-positive T cells were purified by flow
Cell Conjugation and Immunofluorescence Microscopy
B7-negative (ProAdICAM-1) and B7-positive (ProAdICAM-1/
B7-1) transfectants  were pre-incubated with or without
2.0 mg/ml of OVA peptide for 1 hour at 37uC, mixed with T cells
at a 1:1 ratio, and centrifuged at Rcf 2000 for 20 seconds at RT.
The cell pellet was incubated for 5 minutes at 37uC. T cells were
resuspended in 200 ml DMEM, plated on poly-L-lysine (Sigma, St.
Louis, MO) coated coverslips for 3 minutes at 37uC, and fixed in
3% (w/v) paraformaldehyde. Samples were imaged at room
temperature in Mowiol-DAPCO on a Zeiss Axiovert microscope
with a 6361.4 NA on a Plan-Apochromat oil immersion objective.
Images were collected utilizing a CoolSNAP HQ monochrome
CCD camera (Roper Scientific). Nearest-Neighbor deconvolution
and digital analysis were performed using SlideBook software
(Intelligent Imaging Innovations).
Micropipet Cell Adhesion Assay
Streptavidin-coupled dynabeads (Invitrogen) were coated with
125 ng/ml biotinylated mouse-anti-human IgG1 (BD Phamingen)
and used to capture 50–250 ng/ml mouse CD80-hIgG1Fc fusion
protein (R&D). The beads were blocked with 1.0 mg/ml mouse
uPAR-hIgG1Fc fusion protein (R&D) and the level of CD80
bound (100–500 molecules/mm2) was determined by staining with
anti-CD80-PE and comparison to QuantiBrite beads (BD
Pharmingen). For TCR stimulation, 1.0 mg/ml anti-CD3 (2C11)
was bound to latex beads. The adhesion frequency of T cells to
CD80-beads was determined at room temperature essentially as
described [17,18,19]. Briefly, a previously activated, resting T cell
was brought into contact with a CD80-coated bead using
micromanipulators for 2 sec and then separated. Adhesion was
scored by visual distortion of the T cell membrane upon
dissociation. Contact was repeated 25 times for each individual
T cell. The T cell was then brought into contact with an anti-CD3
coated bead and another 25 impingements with anti-CD28 beads
Model Building and MD Simulations
The extracellular domain of CD28 is connected to the
(GKHLCPSPLFP), which was not included in the crystal structure
of CD28 (PDB ID 1YJD). We manually added this segment to the
crystal structure using the Build tool in pymol  and oriented
this segment to generate a disulfide-linked dimer using the editing
tool in pymol. MD simulations were carried out with the AMBER
force field . Three independent trajectories were carried out
for each system to improve MD sampling and provide a way of
control beads (None) or beads coated with CD80 (CD80-Fc) in the absence (Unstim, empty bars) and after (Anti-CD3; filled bars) stimulation. No
increase in adhesion to CD80 was detected when cells were stimulated with beads coated with anti-MHC class I (not shown). (C and D) WT and CD28-
deficient T cells were primed with CD80/CD86-negative APC to reduce upregulation of CTLA-4. (C) Flow cytometry of CTLA-4 expression on WT (thin
line) and CD28-deficient (thick line) T cells after in vitro priming. Isotype control (filled) and WT T cells primed in the presence of CD28 costimulation
(dashed line) are included as negative and positive controls. (D) Cell frequency adhesion of WT and CD28-deficient (CD28KO) T cells that were primed
with CD80/CD86-negative cells, to CD80-beads in the absence (Unstim) and after (Anti-CD3) stimulation. (E) Adhesion frequency of WT and CTLA-4-
deficient (CTLA-4KO) T cells to control beads (None) or beads coated with increasing concentrations of CD80 in the absence (Unstim) and after (Anti-
CD3) stimulation. Cell adhesion data are presented as mean 6 SD of the adhesion frequency for individual cells (25 impingements each; n=9–10,
except for 62.5 ng/ml CD80-Fc in panel E, n=6; p values for t tests between samples are shown; ns, not significant).
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assessing errors . The molecules were neutralized by adding
Cl-atoms and solvated in a truncated octahedron box. The size of
the box was set to assure an 8 A˚distance between the molecule
and the edge of the box. Initial conformations were minimized as
follows: 1) the protein was held fixed, while the solvent was
minimized by 500 steps of steepest descent followed by another
500 steps of conjugate gradient; 2) the whole system was
minimized by 1,000 steps of steepest descent followed by another
1,000 steps of conjugate gradient. After minimization, MD
simulations were carried out using the ff99SB  force field
and the TIP3P water model  in the NPT ensemble. The
temperature was held constant at 300 K using the Langevin
thermostat with a collision frequency g=1.0ps21. Hydrogen
bonds were constrained using the SHAKE algorithm, [25,26]
which allowed a time step of dt=2 fs. We used periodic boundary
conditions with the Particle Mesh Ewald method  with a 9 A˚
cutoff to calculate long range electrostatic forces. The system was
initially allowed to equilibrate at constant temperature for 50 ps
without pressure coupling. After this, the pressure was kept
constant at 1 atm by means of the Berendsen barostat .
Trajectories were analyzed using the cpptraj program from the
AMBER suite. This includes calculations of distances, RMSD’s
and buried surface areas. When calculating the RMSD with
respect to the initial conformation, the stalk region was not
Binding of CD80 to WT CD28, K118I/K120P CD28 was
modeled based on the binding mode predicted for CD80 and
CTLA-4 . To test whether this binding mode was also stable
for CD28, A 41 ns MD simulation of a CD28 monomer bound to
a CD80 monomer was carried out. The simulation was stable with
RMSD values below 5A˚(data not shown). To dock the CD80
molecules onto the simulated conformations, we used pymol’s
TCR Signaling Can Enhance the Avidity of CD28–CD80
To determine whether TCR could enhance CD28–CD80
interactions we used a cell frequency adhesion assay [17,18,19,30].
In this assay T cells are captured on a micropipet and repeatedly
brought into contact with beads coated with CD80 (Figure 1A).
Adhesion is detected by visual distortion of the T cell membrane
during separation of the T cell-bead contact. The frequency of
adhesion events is determined by the concentration of CD28 on
the T cell surface, the concentration of CD80 on the bead, and the
avidity of the CD28–CD80 interaction. A base line of adhesion
was first established in the absence of TCR signaling, the T cell
was then brought into contact with an anti-CD3 coated bead and
the adhesion frequency to CD80-beads was measured. As shown
in the bottom panel of Figure 1A, the T cell interaction site with
the anti-CD3-bead is distal from the interaction site with the
CD80-coated bead. Thus, any impact of TCR engagement is not
mediated by local effects of CD3 cross-linking in the plasma
membrane. Wild type (WT) T cells show a clear increase in
adhesion frequency after stimulation with anti-CD3 (Figure 1B).
To confirm that this enhanced adhesion was mediated through
CD28, we compared WT and CD28-deficient T cells. To limit
any contribution of CTLA-4 to the adhesion assay, WT and
CD28-deficient T cells were primed in the absence of CD28
costimulation to limit the induction of CTLA-4 expression
(Figure 1C) . Enhanced adhesion to CD80-beads after TCR
signaling was only detected in WT cells, indicating that this
adhesion was mediated by CD28 (Figure 1D). To further exclude a
potential contribution of CTLA-4, we found that TCR signaling of
CTLA4-deficient T cells could enhance binding to CD80-beads
(Figure 1E). Taken together, these data indicate that TCR
signaling can increase the avidity of CD28–CD80 interactions.
Lysines at the Base of the CD28 Dimer Interface Control
the Valency of CD28 Ligand Binding
One possible mechanism that could account for increased
CD28 ligand binding is a change in valency. CD28 is a
homodimer that contains two identical functional ligand-binding
sites. Yet, soluble recombinant CD28 can only interact with ligand
monovalently . Evans et al.  obtained a crystal structure of
monomeric CD28 (PDB ID 1YJD ) in complex with the Fab
fragment from the 5.11A1 mitogenic antibody . Although the
CD28-Fab complex did not dimerize, a model for the CD28 dimer
was proposed based on the lattice contacts in the crystal. The
model is shown in Figure 2A. The structure of the monomer is
very similar to that of CTLA-4, [29,35,36] and the CD28 dimer
presents the same v-type topology of CTLA-4. However,
superposition of the two homodimers (Figure 2B) shows that
CD28 adopts a more compact conformation. This difference in
orientation between the subunits in the CD28 and CTLA-4
dimers is significant because it is thought to control the valency of
ligand binding. Although both ligand-binding sites are open in
CD28, the CD28 ligands, CD80 and CD86, are elongated
structures and steric interference distal to the ligand-binding site is
thought to preclude bivalent binding . In contrast, the dimer
interface in CTLA-4 creates a greater angle between the ligand
binding sites eliminating the steric interference that prevents
bivalent binding in CD28. This difference in valency between
these structurally related molecules raises the possibility that in the
context of the cell membrane, TCR signaling could induce a
Figure 2. K118I/K120P CD28 rapidly adopts a conformation that would allow bivalent binding in molecular dynamic simulations.
(A) Structural model of the extracellular domains of the CD28 homodimer illustrating the location of the ligand binding sites (red) and K118/K120
(yellow) at the base of the dimer interface. The K118/K120 residues are only visible on the left hand side monomeric unit in this view. (B) The
structural model of CD28 (red) is superimposed on that of CTLA-4 (blue). The illustration shows that the monomeric units are structurally similar, but
when forming the dimer, their relative orientations are different. Additional residues including the interchain di-sulfide bond were added at the end
of the CD28 structural model shown (see methods for details). (C and D) Three independent trajectories were simulated of WT CD28 (C) or K118I/
K120P CD28 (D) with the crystal structure of CD28 as the initial conformation and the RMSD with respect to the initial conformation are shown over
time. For simulations of K118I/K120P CD28 (D), there is considerable rearrangement of the subunits at the beginning of the simulations, reflected by
an increase in the RMSD. For trajectories 2 and 3, RMSD values start to stabilize towards the end of the simulation. (E and F) CD80 molecules were
docked onto the simulated WT CD28 dimers (E) and K118I/K120P CD28 dimers (F), to obtain the corresponding CD28+ CD80 complexes. The surface
area buried between carboxy-terminal domains of the docked ligands was calculated at various times. A value of zero indicates no buried surface and
thus no contact between the ligands, which would allow for bivalent ligand binding. The fraction of bivalent-competent conformations along each of
the three independent trajectories is indicated and the average over all three trajectories was 26% for WT CD28 and 81% for K118I/K120P CD28. (G) A
representative conformation from simulations of WT CD28 (red) showing docked CD80 ligands (cyan). It can be seen that the orientation of the ligand
binding sites precludes bivalent binding due to steric interference at the distal end of CD80. (H) A representative conformation from simulations of
K118I/K120P CD28 (purple) showing bivalently-bound CD80 ligands (cyan).
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Figure 3. K118I/K120P CD28 is polarized toward CD80-positive cells in the absence of TCR signaling. (A and B) CD28-deficient, DO11.10
CD4 T cells were retrovirally transduced with WT or K118I/K120P CD28 fused to YFP, stained with anti-CD28 and analyzed by flow cytometry. Two
color display (A) shows total transduced protein expression as detected by YFP (FL1, x-axis) and cell surface expression of CD28 as detected by anti-
CD28 staining (FL3, y-axis). Single color display (B) shows relative cell surface expression of WT CD28 (thick line) and K118I/K120P (KK/IP) CD28 (thin
line). (C and D) Representative images of WT and K118I/K120P CD28-YFP localization in cell:cell conjugates with CD80-negative (C) and CD80-positive
(D) APC in the presence (+Ag) and absence (-Ag) of TCR signaling. DIC (digital image correlation) and fluorescent images are shown. (E) Individual
conjugates were visually scored for CD28 polarization toward the interacting APC and the percentage of conjugates displaying polarized CD28 is
shown (n=25–28). (F) The efficiency of CD28 recruitment to CD80-positive APC was calculated by determining the ratio of YFP fluorescence within
the T cell:APC contact site to the YFP fluorescence in the T cell plasma membrane distal to the contact site. Values for individual cells, population
medians and statistical analysis (non-parametric Kruskal-Wallis ANOVA with Dunn’s multiple comparison) are shown (ns, not significant; * p,0.05;
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change in the orientation of the CD28 monomers within the
homodimer, that would allow for bivalent binding. This change in
valency could then result in a functionally significant increase in
CD28 avidity that would regulate CD28 ligand binding.
To determine whether enhanced CD28 ligand binding could be
mediated through a reorientation of the monomers to allow for
bivalent binding, we modified the dimer interface. Based on the
crystal structure of CD28, it was proposed that the presence of two
charged residues (K118 and K120, see Figure 2A) at the base of
the dimer interface prevents CD28 from adopting a CTLA-4-like
orientation . Charge repulsion between these residues is
thought to drive the base of the interface apart and, consequently,
position the membrane distal ligand binding sites closer together.
In CTLA4 the corresponding residues are hydrophobic (isoleucine
and proline) and are thought to stabilize the base of the dimer
interface, allowing the ligand binding sites to separate. To
determine the impact of these two lysine residues in the orientation
of the CD28 monomeric units, we used molecular dynamic (MD)
simulations to study the effect of mutating these lysine residues to
the corresponding hydrophobic residues in CTLA-4. Three
independent simulations were carried out for the WT CD28
dimer and for the K118I/K120P mutant, with the crystal
structure of CD28 as the initial conformation. For WT CD28
molecules, the dimer model is stable and the molecules largely
remain in compact structures and the root mean square deviation
(RMSD) with respect to the initial conformation remains below 5
A˚(Figure 2C). In contrast, K118I/K120P CD28 does not remain
in the initial conformation, as evidenced by a large initial increase
in RMSD (Figure 2D). The structures eventually stabilize,
although continued fluctuations in RMSD in the trajectories
indicate that a final stable structure was not reached in the
simulations. During the simulations, K118I/K120P CD28 mole-
cules quickly undergo conformational changes that bring the 118–
120 regions of the two monomers together, disrupting the initial
orientation, causing the ligand binding regions to separate.
To assess whether the molecules populate conformations that
would allow bivalent binding, CD80 molecules were docked onto
the CD28 dimers, based on the ligand interface in the CD80-
CTLA-4 co-crystals . The buried surface between the
carboxy-terminal domains of CD80 was calculated to measure
the extent of steric interference that would preclude bivalent
binding. The WT CD28 simulations generate large buried
surfaces indicating that the ligands would often physically collide,
indicative of monovalent conformations (Figure 2E). An illustra-
tion of this steric interference that precludes bivalent binding is
shown in Figure 2G. In contrast, 81% of K118I/K120P CD28
conformations result in no buried surface between the CD80
molecules (Figure 2F), indicating that these conformations could
bind ligand bivalently. An example of such conformation is shown
in Figure 2H. This gain-in-function mutation is distal to the
ligand-binding site and MD simulations do not predict any
changes at the binding site itself that would suggest a change in
affinity. Rather the MD simulations predict that K118I/K120P
CD28 adopts a stable conformation that would accommodate
bivalent binding. These data suggest that a change in valency is
sufficient to provide a functionally relevant increase in ligand
K118I/K120P CD28 Can Bind CD80 in the Absence of TCR
To assess the impact of potential bivalent binding on CD28
ligand interactions, the K118I/K120P mutations were introduced
into CD28 by site-directed mutagenesis. WT and mutated CD28
were fused to YFP at the C terminus and retrovirally transduced
into CD28-deficient T cells. Both WT and K118I/K120P CD28
were expressed at equivalent levels at the cell surface (Figure 3 A
and B). Importantly, the relative ratio of cell surface CD28 (as
measured by antibody staining) to total CD28 (as measured by
YFP fluorescence) was equivalent, indicating that the mutations
did not impact the efficiency of CD28 expression at the plasma
membrane (Figure 3A). To assess the ability of CD28 to interact
with ligand, cell:cell conjugates were formed between T cells and
APCs, and the localization of CD28 was determined by
fluorescence microscopy (Figure 3 C–F). Neither WT nor
K118I/K120P CD28 was polarized toward CD80-negative APC
in the presence or absence of TCR signaling (Figure 3 C and E),
indicating that CD28 polarization to the immunological synapse
was dependent on ligand binding. For WT CD28, CD28-ligand
binding requires concurrent TCR signaling  and WT CD28 is
polarized toward CD80-positve APC only in the presence of
antigen (Ag) (Figure 3 D–F). In contrast, K118I/K120P CD28 is
polarized toward cells expressing CD80 in the presence and
absence of Ag (Figure 3 D–F). Thus, mutation of the two lysine
residues bypasses the apparent need for activation of ligand
binding, allowing K118I/K1210P CD28 to be efficiently recruited
towards the immunological synapse in the absence of TCR
To confirm that the TCR-independent polarization of K118I/
K120P CD28 to CD80-positve cells is mediated through enhanced
ligand binding, we used the cell adhesion frequency assay
(Figure 4). CD28-deficient T cells transduced with WT CD28
showed the same dependence on TCR signaling for adhesion to
CD80-beads as seen in Figure 1 for T cells expressing endogenous
CD28. In contrast, CD28-deficient T cells transduced with
K118I/K120P CD28 showed significant binding to CD80-beads
even in the absence of TCR signaling (Figure 4). These results,
together with the simulations showing that K118I/K120P CD28
Figure 4. K118I/K120P CD28 binds CD80 with high avidity in
the absence of TCR signaling. Cell frequency adhesion of CD28-
deficient, DO11.10 T cells retrovirally transduced with WT CD28-YFP or
K118I/K120P CD28-YFP to control beads (None) or beads coated with
CD80 in the absence (Unstim) and after (Anti-CD3) stimulation. Cell
adhesion data are presented as mean 6 SD of the adhesion frequency
for individual cells (25 impingements each; n=6–7 for control beads
and 12–14 for CD80-Fc beads; p values for t tests between samples are
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adopts bivalent conformations support the model that CD28
ligand interactions can be regulated by a change in valency.
WT CD28 can Adopt Conformations that Allow for
If TCR inside-out signaling is regulating CD28 ligand binding
through a change in valency, then WT CD28 must be able to
adopt a dimer orientation that would accommodate bivalent
ligand binding. To assess this possibility, we carried out MD
simulations of CD28 with the dimer orientation of CTLA-4 (see
Figure 2B) as the initial conformation. We used the CD28 crystal
structure  to build the monomers, but they were superimposed
on the model of a CTLA-4 dimer (PDB ID 3OSK) , resulting
in a molecule with a greater distance between the ligand binding
sites. The initial conformation used in these simulations is not
stable and there is considerable rearrangement in the orientation
of the monomers at the beginning of the simulation, as evidenced
by a increase in the RMSD (Figure 5A). However, it should be
noted that no restraints have been imposed on the C-termini
connecting the extracellular and transmembrane domains. Such a
restraint could contribute to stabilization of bivalent conforma-
tions. Nevertheless, in all trajectories, the molecule primarily
populates bivalent conformations. When we docked CD80
molecules on the CD28 conformations we found that, on average,
79% of conformations can accommodate two ligands (Figure 5B).
These conformations did not resemble that of CTLA-4, where the
orientation of the subunits is such that the ligands lie parallel to
each other. Instead, in our simulations, bivalency was achieved by
a slight rotation of the CD28 monomers around the dimer
interface. When the CD80 ligands were docked onto CD28, the
CD80 molecules formed an angle with each other placing their
distal ends far enough apart to allow for bivalent binding
Therefore, we designed a new initial conformation in which the
subunits of CD28 were rotated with respect to their orientation in
Figure 5. WT CD28 adopts a bivalent binding conformation when starting from the CTLA-4 dimer orientation. MD simulations were
run with WT CD28 starting from a CTLA-4 dimer orientation. (A) The RMSD of three independent trajectories with respect to the initial conformation
are shown over time. There is considerable rearrangement of the subunits at the beginning of the simulations, reflected by an increase in the RMSD.
The conformational fluctuations stabilize after the first 100 ns of simulation, although the trajectories adopt different conformations. This instability
was not inherent to this dimer orientation, as the RMSD of MD simulations of CTLA-4 remained below 4A˚(data not shown). (B) CD80 molecules were
docked onto the simulated CD28 dimers to obtain the corresponding CD28-CD80 complexes. The surface area buried between the docked ligands
was calculated at various times along each of the three independent trajectories to estimate the fraction of bivalent-competent conformations; the
average over all three trajectories was 79%. (C) A representative conformation of CD28 (green) showing docked CD80 ligands (cyan) illustrates the
potential for bivalent ligand binding.
Regulation of CD28 Avidity
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the crystal structure in the fashion observed in the simulations
starting from the CTLA-4 dimer orientation. Care was taken that,
despite the rotation in the initial conformation, the hydrophobic
residues that formed the dimer interface in the crystal structure
were still part of the interface. This new initial conformation was
stable, as indicated by RMSDs below 5A˚for three independent
simulations (Figure 6A). The small fluctuations observed during
the simulations produced only bivalent conformations (Figure 6B),
as in the example shown Figure 6C. Interestingly, because of the
rotation of the dimer interface the CD80 molecules come off at a
more oblique angle as seen in the rotated view in Figure 6D. These
results suggest that in the presence of an appropriate external
force, that might be initiated by a TCR-induced reorganization of
the CD28 cytosolic domains, the WT CD28 dimer can form stable
dimer interfaces that position the ligand binding sites in a spatial
orientation that allows for bivalent interactions with CD80.
In this report we show that TCR signaling can increase the
avidity of CD28 ligand binding. These results are consistent with
previous studies showing that TCR signaling can initiate CD28
localization at the immunological synapse  and that sustained
TCR signaling is required to maintain CD28 at the immunolog-
ical synapse [11,13]. Taken together, the results indicate that the
ability of TCR signaling to increase the 2-dimensional avidity of
CD28 ligand interactions would drive the effective recruitment of
CD28 to the immunological synapse and ultimately the transmis-
sion of functional costimulatory signals from CD28 that are
required for T cell activation. Traditionally we think of CD28 as a
regulator of TCR signaling and most models of TCR/CD28
signal integration take place during downstream signaling events.
Our results create a new paradigm for this process indicating that
TCR can actually modify CD28 function and that integration of
TCR and CD28 signaling may be initiated through regulation of
ligand binding events at the plasma membrane. These results
imply that TCR signaling may regulate the ability of both the
primary costimulatory molecule, CD28, and the primary adhesion
molecule, LFA-1, to engage their respective ligands. Thus, initial
TCR signaling can coordinate LFA-1 and CD28 ligand engage-
ment during immunological synapse formation, providing new
insights into how coincidence detection of antigen recognition in
the context of costimulation regulates the initiation of T cell
immune responses and determines the fate of T cells following
initial encounter with antigen.
Regulation of receptor-ligand interactions through this inside-
out receptor cross-talk provides an interesting paradigm for the
sequential receptor activation events that control complex cellular
processes. This pathway has been extensively characterized for
integrins, including signaling from the activating receptors,
Figure 6. WT CD28 can adopt a stable conformation that would allow bivalent binding. MD simulations were run with WT CD28 starting
from a rotated dimer orientation predicted from the simulations starting from the CTLA-4 dimer orientation. (A) The RMSD with respect to the initial
conformation for three independent trajectories indicate that these dimer conformers are very stable. (B) When CD80 molecules were docked onto
the conformations, there was no surface buried between the ligands, indicating that all conformations were bivalent. A representative bivalent
conformation of CD28 (green) with docked CD80 molecules (cyan) is shown to illustrate the CD28 dimer (C) and in a rotated view to show the
orientation of the docked CD80 molecules (D).
Regulation of CD28 Avidity
PLOS ONE | www.plosone.org9 February 2014 | Volume 9 | Issue 2 | e89263
modifications and structural changes within the cytosolic domains,
association with regulatory proteins, and the conformational
changes that occur within the extracellular domain to enhance
integrin ligand binding [15,16]. Inside-out signaling has also been
implicated in regulating ligand binding for cadherin  and L1
 adhesion receptors and differential reactivity to monoclonal
antibodies supports a change in the orientation of the multiple
extracellular domains. Cytokine signaling has been shown to
enhance ligand binding to the IgA and IgG Fc receptors [40,41].
The signaling pathways that mediate this effect have been
identified [42,43], but the changes to the extracellular domains
to allow for enhanced ligand binding remain unknown. Interesting
for CEACAM1 , interactions between GXXXG transmem-
brane motifs appears to stabilize cis-dimers in the inactive form.
Calmodulin binding to the cytosolic domains disrupts the dimers,
allow the monomeric CEACAM1 molecules to form trans-
interactions and cell adhesion with neighboring cells. Our results
indicate that CD28 is also regulated through inside-out signaling
supporting the possibility that inside-out regulation of receptor-
ligand interactions may be a general mechanism for receptor
cross-talk at the cell surface.
In our micropipet adhesion experiments we have isolated TCR
and CD28 ligands on separate surfaces (anti-CD3-coated and
CD80-coated beads, respectively). Because these beads contact the
T cell at discrete sites on the plasma membrane, it is unlikely that
the ability of TCR signaling to enhance CD28 ligand binding is
mediated by local changes within the plasma membrane (such as
actin polymerization, lipid membrane domains, or receptor
capping). Rather, it is more likely to be mediated by activation
of specific downstream signaling pathways that can function at a
distance. During T cell activation by antigen presenting cells,
TCR and CD28 are colocalized within both microclusters and
within the cSMAC of the mature immunological synapse. So it is
possible that additional local effects of TCR engagement, or
contributions of signaling from additional receptors, could further
impact on the activation (or inactivation) of CD28-ligand binding.
Both CD28 and CTLA-4 can interact with the ligands, CD80
and CD86. The receptor-ligand binding interface is conserved and
both ligands can form functional interactions with both receptors
[33,45]. However, the interactions between these four receptor-
ligand pairs are not identical. CTLA-4 binds to both CD80 and
CD86 with a higher affinity than CD28 and CD80 binds to both
CTLA-4 and CD28 with a high affinity than CD86 (the hierarchy
CD28:CD80. CD28:CD86) . The relative difference in
affinity of CTLA-4 to CD28 for binding to CD80 is 20-fold,
whereas the difference in binding to CD86 is only 8-fold . In a
competitive environment (such as after CTLA-4 is induced on T
cells), CTLA-4 could then out compete CD28 for binding to
CD80 to a greater degree than binding to CD86. This predicted
preference for binding in a competitive environment has been
confirmed experimentally . The kinetics of expression of these
receptor-ligand pairs and ligand-specific blocking experiments
have suggested that CD86 functions to promote T cell activation
(consistent with the requirement for CD28 costimulation for T cell
activation), while CD80 suppresses T cell activation (consistent
with CTLA-4 mediated suppression). CD28 is constitutively
expressed on naı ¨ve T cells, whereas CTLA-4 is only induced
following activation . Likewise, CD86 is constitutively
expressed on APC and rapidly upregulated upon activation,
whereas expression of CD80 on APC is kinetically delayed
[47,48]. Thus, during initial T cell priming when CD28
costimulation is required for activation, CD86 is the primary
ligand available on APC. Later, in the response, when the
magnitude of T cell activation needs to be tempered, the inhibitory
receptor, CTLA-4, is induced on the T cells, coincident with the
induction of CD80 expression on the APC. In vivo blocking
experiments with CD80 or CD86-specific antibodies or with
CD80/CD86 selective knockout models generally support this
association of CD28 with CD86 and CTLA4 with CD80 (for
example see [49,50,51,52,53,54]). However, these findings remain
controversial with other groups reporting role for CD80 in T cell
activation and CD86 in immunosuppression (for example see
[55,56,57]). Whether these differences in CD80 and CD86
function in vivo are related to preferential interactions with CD28
and CTLA-4 or whether they are also related to the selective
activation of T effector subsets or T regulatory cells and/or to
restricted expression of CD80 or CD86 within specific target
tissues is not clear [45,58]. In our studies we have shown that TCR
can increase CD28 ligand binding to CD80 in the presence and
absence of CTLA-4 expression. Given the very rapid off-rate of
monomeric CD28 from CD86 and the predicted 100-fold increase
in the stability of bivalent versus monovalent binding in solution
, we would predict that impact of TCR-mediated activation of
CD28 ligand binding would be more significant in interactions
with CD86 than CD80.
Our results support a model whereby TCR signal regulates
CD28 ligand binding through a change in valency within the
CD28 dimer. Although CD28 is expressed as a disulfide linked
dimer, recombinant soluble CD28 has been shown to only bind
ligand monovalently . In the crystallographic model, mono-
valency is determine by the orientation of the dimer interface. In
contrast, the homologous receptor, CTLA-4, presents a different
dimer interface and can bind bivalently [29,32,35,36]. Therefore,
we considered the possibility that TCR-mediated inside-out
signaling would enhance CD28 ligand binding through a change
in valency. Like integrins, CD28 activation would be initiated by a
change in the orientation of the cytosolic tails in the dimer ,
but rather than inducing large scale conformational change in the
lumenal domains like integrins, the rigid body immunoglobulin
domains of CD28 would simply change their orientation in respect
to one another, allowing for bivalent ligand binding and increased
avidity. Consistent with this hypothesis, we have shown that
disruption of a single ligand-binding site within the CD28 dimer,
results in a significant decrease in recruitment to the immunolog-
ical synapse, indicating that both ligand binding sites within a
CD28 dimer are important for efficient ligand interaction .
To test this model more directly, we show here that introduction
of two CTLA-4 residues at the base of the dimer interface of CD28
allows for enhanced ligand binding in the absence of TCR
signaling. This gain-in-function mutation is distal to the ligand
binding site and MD simulations do not predict any changes at the
binding site itself that would suggest a change in affinity. Rather
the MD simulations predict that K118I/K120P CD28 adopts a
stable conformation that would accommodate bivalent binding.
These data suggest that a change in valency is sufficient to provide
a functionally relevant increase in ligand interactions. Although
mutation of K118/K120 bypasses the need for TCR signaling for
ligand binding, TCR signaling does result in increased localization
of K118I/K120P CD28 to the immunological synapse (Figure 3F).
It is possible that the K118I/K120P mutation does not fully mimic
the dimer reorientation induced by TCR signaling or that
additional factors can contribute to the organization of the mature
immunological synapse. Nevertheless, our data do show that an
extracellular conformational change in CD28 leads to increased
avidity of the molecule and capping behavior commensurate with
the natural activation response.
Regulation of CD28 Avidity
PLOS ONE | www.plosone.org10February 2014 | Volume 9 | Issue 2 | e89263
To determine whether WT CD28 could stably adopt a bivalent
conformation we used MD simulations. We do not yet understand
how TCR signals are transduced through the cytosolic domain to
induce high avidity CD28 conformers, so we could not simulate
this process precisely. Rather, we started the simulations with WT
CD28 sequences in the dimer interface orientation of CTLA-4.
This orientation was not stable, but WT CD28 did mostly reside in
conformations that could interact with CD80 bivalently. Interest-
ingly, the CD28 conformers that could bind bivalently adopted a
rotated dimer orientation. When we started from this conforma-
tion, the MD simulations predicted a very stable orientation that
remained bivalent. These data suggest that WT CD28 can form
stable dimer interfaces that position the ligand binding sites in a
spatial orientation that allows for bivalent interactions with CD80.
We do not know what other constraints might be imposed by
TCR-mediated inside-out signaling. Nevertheless, this result
suggests that in the presence of an appropriate external force,
that could be initiated by a TCR-induced reorganization of the
CD28 cytosolic domains, the CD28 dimer can more favorably
adopt a bivalent-binding conformation.
In recognition of the biological importance of CD28, a number
of immunotherapeutics have been designed to target CD28. The
most effective of these has been a soluble form of CTLA-4 (CTLA-
4-Ig; Abatacept), which has been FDA approved to treat RA and
may be useful in suppressing other autoimmune diseases
[59,60,61]. Small molecule mimics of the CTLA-4 ligand-binding
site have also been developed [62,63,64,65], but both these and
CTLA4-Ig target the CD80 and CD86 ligands and indiscrimi-
nately inhibit both CD28 and CTLA-4 binding and function. Our
identification of a novel step in the regulation of CD28 activity
provides a new conceptual framework on how signaling through
this receptor is controlled. The proposed structurally distinct high
and low avidity isoforms of CD28 could provide a new platform
for isolation of CD28-specific immunotherapeutics.
We thank Deb Fowell, Alan Grossfield, Scott Leddon, and Tim Mosmann
for comments on the manuscript. The Center for Integrated Research
Computing at the University of Rochester provided the necessary
computing systems (BlueHive and BlueGene clusters) to run the MD
Conceived and designed the experiments: MSL AVR REW JM.
Performed the experiments: MLS AVR MMF TRH RB. Analyzed the
data: MSL AVR MMF RB JM. Contributed reagents/materials/analysis
tools: MSL AVR MMF TRH HM REW. Wrote the paper: AVR JM.
Provided support and technical advice: HM REW.
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