of June 13, 2013.
This information is current as
Localization to the Central Region of the
Signals and Sequences That Control CD28
Mariano Sanchez-Lockhart, Beth Graf and Jim Miller
2008; 181:7639-7648; ;
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Copyright © 2008 by The American Association of
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The Journal of Immunology
by guest on June 13, 2013
Signals and Sequences That Control CD28 Localization to the
Central Region of the Immunological Synapse1
Mariano Sanchez-Lockhart,* Beth Graf,* and Jim Miller2*†
During T cell interaction with APC, CD28 is recruited to the central region (cSMAC) of the immunological synapse. CD28-
mediated signaling through PI3K results in the recruitment of protein kinase C-? (PKC?) to the cSMAC, activation of NF-?B, and
up-regulation of IL-2 transcription. However, the mechanism that mediates CD28 localization to the cSMAC and the functional
consequences of CD28 localization to the cSMAC are not understood. In this report, we show that CD28 recruitment and
persistence at the immunological synapse requires TCR signals and CD80 engagement. Addition of mAb to either MHC class II
or CD80 results in the rapid displacement of CD28 from the immunological synapse. Ligand binding is not sufficient for CD28
localization to the immunological synapse, as truncation of the cytosolic tail of CD28 disrupts synapse localization without effecting
the ability of CD28 to bind CD80. Furthermore, a single point mutation in the CD28 cytosolic tail (tyrosine 188) interferes with
the ability of CD28 to preferentially accumulate at the cSMAC. PKC? distribution at the immunological synapse mirrors the
distribution of tyrosine 188-mutated CD28, indicating that CD28 drives the localization of PKC? even when CD28 is not localized
to the cSMAC. Mutation of tyrosine 188 also results in diminished activation of NF-?B, suggesting that CD28-mediated local-
ization of PKC? to the cSMAC is important for efficient signal transduction. These data reinforce the importance of the interplay
of signals between TCR and CD28 and suggest that CD28 signaling through PCK? may be mediated through localization to the
cSMAC region of the immunological synapse. The Journal of Immunology, 2008, 181: 7639–7648.
gands for the TCR as well as ligands for costimulatory receptors.
Many surface receptors can provide costimulation in conjunction
with the TCR, most notably among these is CD28. CD28 is the
most potent and best-described costimulatory molecule for T cells,
participating in many aspects of T cell activation, survival, and
differentiation (1–3). One of the best-characterized functions of
CD28 is its ability to enhance IL-2 expression through at least two
different mechanisms. CD28 promotes activation and nuclear
translocation of transcription factors (NF-?B and AP-1) that will
interact with the IL-2 enhancer and augment transcription (4). In
addition, CD28 increases IL-2 mRNA stability by a mechanism
that is not well characterized (1).
Despite the well-known functional importance of CD28, the bio-
chemical signaling pathways induced downstream of CD28 are not
clearly understood (2, 3). Part of the difficulty in identifying CD28-
mediated signals is that one major function of CD28 is to amplify
signals that can be initiated through the TCR. Profiling of protein
signaling intermediates as well as changes in gene expression have
suggested that CD28 costimulation functions primarily to modify
TCR-mediated signaling pathways (5–7). In support of this, Lck,
cell activation requires Ag-specific recognition in the con-
text of MHC on the surface of an APC. The interaction
with the APC provides the T cell with peptide-MHC li-
Itk, and PI3K, the kinases that can be recruited to the cytosolic tail
of CD28 and have been associated with CD28 costimulation, can
also be activated by TCR signaling in the absence of CD28 (8–14).
Point mutations in the Src homology domain (SH)32 and SH3
interaction motifs that recruit these signaling proteins to the CD28
cytosolic tail do not entirely reproduce the effects of CD28 defi-
ciency, suggesting that additional pathways and/or functional re-
dundancy within these pathways may be involved (15–20). For
example, we have shown that mutation of the PI3K interaction site
results in a failure of CD28 to enhance NF-?B activation and IL-2
transcription. However, this has little effect on IL-2 secretion, be-
cause this mutation does not inhibit the ability of CD28 to enhance
IL-2 mRNA stability (21).
T cell recognition of Ag is associated with the formation of a
tight cell-cell contact with APC, termed the immunological syn-
apse (IS) (22, 23). Distinct membrane domains (supramolecular
activation clusters or SMAC) are defined according to the distri-
bution of the proteins recruited to and sorted within the IS. The
most central cluster (cSMAC) is defined by the presence of TCR,
a peripheral cluster (pSMAC) is defined by the distribution of the
integrin LFA-1, and a more distal cluster is defined by the local-
ization of CD45 (24–26). Because many of the molecules associ-
ated with the cSMAC are involved in proximal T cell activation, it
was initially thought that this region was the signaling compart-
ment of the IS. However, the cSMAC is now thought to be the site
for TCR down-regulation (27, 28) and sustained TCR signaling is
thought to be mediated by the constant recruitment of new TCR
complexes that form along the outside of the pSMAC (29–32).
According to this model, these TCR microclusters signal as they
*The David H. Smith Center for Vaccine Biology and Immunology and the†Depart-
ment of Microbiology and Immunology, University of Rochester, Rochester, NY
Received for publication August 5, 2008. Accepted for publication October 1, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Grant R01-AI063418 from the National Institutes of
Health (to J. M.). M.S.L. was supported by National Institutes of Health Training
2Address correspondence and reprint requests to Dr. Jim Miller, Center for Vaccine
Biology and Immunology, University of Rochester, Box 609, 601 Elmwood Avenue,
Rochester, NY 14642-8609. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: SH, Src homology domain; SMAC, supramolecu-
lar activation cluster; cSMAC, central SMAC; pSMAC, peripheral SMAC; PKC?,
protein kinase C-?; IS, immunological synapse; WT, wild type; IRES, internal ribo-
somal entry site; CT, cytosolic tail; DIC, differential interference contrast; CFP, cyan
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
by guest on June 13, 2013
migrate through the pSMAC toward the cSMAC. TCR microclus-
ters ultimately accumulate at the cSMAC, where they stop signal-
ing, and are targeted for down-regulation. In contrast, CD28 sig-
naling has been indirectly associated with cSMAC localization.
CD28 has been shown to be recruited to the IS and to accumulate
at the cSMAC along with TCR (21, 33–35). CD28 is required for
recruitment of protein kinase C-? (PKC?) to the cSMAC region
(21, 34). In CD28-deficient T cells or T cells expressing a mutation
in the PI3K interaction site, PKC? is still recruited to the IS, but it
is not focused into the cSMAC region. This results in a loss in
PKC?-dependent activation of NF-?B and up-regulation of IL-2
transcription, suggesting that CD28-dependent activation of PI3K
within the IS contributes to CD28 costimulation (21). CD28 en-
gagement outside the IS (in trans) can still enhance IL-2 secretion,
but PKC? is not recruited to either the TCR or CD28 and IL-2 gene
transcription is not up-regulated. Rather, CD28 in trans enhances
IL-2 secretion through the induction of IL-2 mRNA stabilization
(36). These observations suggest that CD28 localization at the IS
may be an important component for the transduction of some, but
not all, aspects of CD28-mediated T cell costimulation.
Little is known about the molecular mechanisms involved in the
recruitment of CD28 to the IS. CD28 distributes around the plasma
membrane of nonpolarized T cells and, within minutes of T cell-
APC interaction, CD28 accumulates to the contact area, concen-
trating into the cSMAC. It has been shown that ligand interaction
with CD80 or CD86 is important to promote CD28 recruitment to
the IS (33, 35). But the signals that drive CD28 targeting to the
cSMAC and the intracellular sorting motifs within CD28 that me-
diate this localization are not understood. In this report, we have
addressed both of these issues. We show that sustained signaling
from both TCR and CD28 is required to maintain CD28 within the
IS. Furthermore, we have identified a single point mutation at
tyrosine 188 (Y188F) that diminishes CD28 localization to the
cSMAC. The mislocalization of Y188F CD28 was mirrored by
corresponding mislocalization of PKC? and resulted in a reduction
in NF-?B nuclear translocation. These observations support the
model that CD28 costimulation may be controlled in part by lo-
calization of CD28 to the cSMAC region of the IS.
Materials and Methods
Cells and reagents
6132 Pro cell transfectants expressing class II (I-Ad) alone (ProAd) or in
combination with CD80 (ProAd-B7), ICAM-1 (ProAd-ICAM), or both
(ProAd-ICAM-B7) and purification of CD4-positive T cells from DO11.10
TCR-transgenic BALB/c mice have been previously described (21). All
cell lines were maintained in DMEM (Invitrogen Life Technologies) sup-
plemented with 10% FCS, 2 mM glutamine, 0.1 mM nonessential amino
acids, and 50 ?M 2-ME. CD4?T cells (either from CD28?/?or CD28-
deficient mice) were stimulated with 0.2 ?g/ml OVA peptide (323–339)
presented by irradiated syngeneic spleen cells and 20 U/ml human rIL-2.
When noted, CD28-deficient T cells were activated and 1 day later trans-
duced with retroviruses containing wild-type (WT) or mutated murine
CD28 (16) or WT murine CD28 fused to cyan fluorescent protein (CFP)
(CD28-CFP) (35). Additional mutations in CD28, where portions of the
cytosolic tail were placed by alanine (ala scan) or where the entire cytosolic
tail was deleted (?CT), were constructed by overlapping PCR, confirmed
by DNA sequencing, and subcloned into the MIGR retroviral vector. All of
the viruses used (but not CD28-CFP) contain GFP expressed from an in-
ternal ribosome entry site (IRES) as a marker for transduced cells. Infection
efficiency ranged from 10 to 60%, and cells were sorted to match the level of
CD28 expression before each experiment. Abs against PKC? (C-18) and
NF-?B p65 (A) were purchased from Santa Cruz Biotechnology; Abs against
BD Pharmingen and fluorescently labeled; species-specific, secondary Abs
were obtained from Jackson ImmunoResearch Laboratories. Abs against
CD28 (37.51), CD80 (1G10), and MHC class II (M5/114) were also purified
from hybridoma supernatants by protein A-Sepharose. Soluble CD80 (rmB7-
1/hFc) was purchased from R&D Systems.
Peptide-pulsed APC (2 ?g/ml OVA peptide) were centrifuged with T cells
for 20 s at a relative centrifugal force of 2000 ? g. The cell pellet was
incubated for 5 min at 37°C, resuspended in complete medium, and either
incubated for various times or immediately plated on poly-L-lysine-coated
coverslips. After plating, conjugates were incubated for 5 min at 37°C to
allow cell binding to the poly-L-lysine. Cells were fixed in 3% (w/v) para-
formaldehyde, permeabilized in 0.3% (v/v) Triton X-100, and stained. For
NF-?B localization, the incubation time before plating on poly-L-lysine-
coated coverslips was increased to 35 min, and nuclei were labeled with
Hoechst stain after fixation and permeabilization. Samples were analyzed
on a Zeiss Axiovert microscope controlled by SlideBook software (Intel-
ligent Imaging Innovations). Nearest-Neighbor deconvolution, digital anal-
ysis, and three-dimensional rendering were accomplished using SlideBook
software. The threshold for the images was set using the APC (CD28 and
PKC?) or the T cell (MHC class II) fluorescent intensity as nonspecific
staining since these cells should not express the corresponding proteins.
CD28 and PKC? distribution within the IS was calculated on a midplane
image by measuring the length of the cell-cell contact that contained CD28
or PKC? and dividing it by the length of the entire interaction site between
the T cell and the APC. NF-?B localization to the nucleus was quantified
by defining a nuclear mask (Hoechst staining) and calculating the fluores-
cence intensity of NF-?B staining within the mask area.
Live cell microscopy
ProAd-ICAM-B7 were plated on Delta T culture dishes (Bioptechs) and
incubated overnight at 37°C. Ag (2 ?g/ml) was loaded for 2 h and the dish
was mounted on a heated stage maintained at 37°C. CD28-CFP-expressing
T cells were added and allowed to interact with the APC. Conjugates were
selected based on sustained recruitment of CD28 toward the APC. Images
were collected every 21 s for 5 min, 2 ?g/ml control (anti-MHC class I) or
blocking (either anti-CD80 or anti-MHC class II) mAbs were added and
imaging was continued for an additional 5–20 min.
Calcium imaging of T cell:APC conjugates was done essentially as previ-
ously described (37). ProAd-ICAM-B7 cells were preincubated with 2.0
?g/ml OVA peptide for 2 h at 37°C and labeled with Alexa Fluor 633
(Molecular Probes). T cells were loaded with 1 ?M Indo-1AM (Molecular
Probes) for 30 min at 37°C. T cells and APC were mixed at a 1:1 ratio in
solution and run on an LSRII flow cytometer (BD Bioscience) to establish
a baseline for intracellular calcium levels in the T cells. The T cell:APC
mixture was then centrifuged for 15 s at a relative centrifugal force of
2000 ? g at room temperature in a microfuge. The cell pellets were re-
suspended in warm medium and run on the flow cytometer at 37°C. T
cell:APC conjugates were identified by dual Indo-1/Alexa Fluor 633 flu-
orescence, and intracellular calcium levels were determined by ratiometric
analysis of Indo-1-Blue to Indo-1-Violet fluorescence using FlowJo soft-
ware (Tree Star).
CD28-ligand interactions are required to recruit and sustain
CD28 at the IS
CD28 colocalizes with TCR at the cSMAC of the IS in a ligand-
dependent manner (33, 35). The requirement for CD28 engage-
ment with CD80 to recruit CD28 to the IS can be shown by in-
teraction of T cells with APC that do and do not express CD80.
Endogenous CD28, detected by anti-CD28 staining, is only re-
cruited to the IS in conjunction with CD80-positive APC (Fig. 1,
A and B). Retroviral transduction of CD28-deficient T cells with a
CD28-CFP fusion protein also demonstrates efficient recruitment
of CD28 to the IS only with CD80-positive APC (Fig. 1, A and B).
Similarly, CD28 recruitment to the IS was disrupted when ligand
binding was blocked by the preincubation of APC with anti-CD80
mAb, while no effect was observed by the addition of a control
anti-class I mAb (Fig. 1, C and D).
To determine whether continuous engagement with ligand is
required to sustain CD28 at the IS, we allowed CD28 to be re-
cruited at the IS and then added anti-CD80 mAb to block CD80-
CD28 interactions (Fig. 2A). Within 5 min of anti-CD80 addition,
7640CD28 LOCALIZATION TO THE IMMUNOLOGICAL SYNAPSE
by guest on June 13, 2013
the majority of conjugates lack CD28 localization at the IS. Ad-
dition of a control anti-class I mAb has no effect on CD28 accu-
mulation at the IS. TCR signals remained intact when CD80-CD28
interactions were blocked, because this treatment does not inhibit
calcium influx on T cells (data not shown). The rapid displacement
of CD28 from the IS after anti-CD80 mAb addition can be visu-
alized in real-time using T cells expressing the CD28-CFP chimera
(Fig. 2B and video 1 in supplemental data4). CD28-CFP-positive T
cell:APC were imaged for 5 min to confirm that CD28 was stably
recruited to the IS. Blocking anti-CD80 mAb was added and the
conjugates were followed for an additional 15–20 min. Approxi-
mately 5 min after mAb addition, CD28-CFP began to diffuse
away from the APC and by 10 min was distributed evenly around
the T cell surface. In contrast, CD28-CFP remained polarized to-
ward the APC the entire period of time when anti-class I mAb
were added (Fig. 2C and video 2 in supplemental data). These
results demonstrated the crucial requirement of CD28-CD80 in-
teractions not only to initially recruit, but also to maintain, CD28
within the IS.
CD28 requires constant TCR signal to persist at the IS
To determine whether TCR engagement is required to sustain
CD28 at the IS, TCR-peptide-class II interactions were blocked
with the addition of anti-class II-blocking mAb. It has been shown
that this treatment leads to the rapid inhibition of calcium influx,
prevention of new TCR microcluster formation, and cessation of
sustained TCR signaling (32, 38, 39). To confirm the efficacy of
anti-class II blocking, conjugates between Indo-1-loaded CD4?T
cells and Alexa Fluor 633-labeled ProAd-ICAM-B7 were formed
and the magnitude of the T cell calcium flux in T cell:APC con-
jugates was determined on the flow cytometer (Fig. 3, A–C).
Blocking amounts of anti-class II mAb were added and conjugates
were analyzed to simultaneously measure the effect of anti-class II
on intracellular calcium flux and on T cell:APC conjugate stability
over time. After a lag of several minutes, calcium influx was rap-
idly inhibited in the entire population of T cells (Fig. 3, B and C).
In contrast, the majority of T cell:APC conjugates were stable for
at least 15 min after the inhibition of calcium signaling (Fig. 3D).
This provides a kinetic window where we could evaluate the re-
quirement for sustained TCR signaling in CD28 localization to the IS
without disrupting T cell:APC interactions. To determine whether
CD28 remains recruited to the IS when TCR signals were blocked,
T cells:ProAd-ICAM-B7 conjugates were formed in the presence
of Ag and incubated for 10 min to allow IS formation and CD28
recruitment toward the APC. Blocking Abs for class II were added
and conjugates were fixed 5 or 10 min later (Fig. 3E). As a neg-
ative control, conjugates were treated with anti-class I mAb. CD28
remained recruited to the APC contact area in most of the conju-
gates 5 min after the addition of anti-class II mAb. However, after
10 min of TCR blocking, localization of CD28 to the IS was lost
in most of the conjugates.
To visualize CD28 displacement from the APC when TCR sig-
nals are blocked, we performed a live cell-imaging analysis.
CD28-CFP-expressing T cells were allowed to interact with Ag-
pulsed APC for 10 min. Conjugates were selected for CD28 lo-
calization to the IS, blocking class II mAb was added, and CD28
localization was followed for another 20 min in the presence of the
blocking mAb (Fig. 3F and video 3 in supplemental data). CD28
remained polarized toward the APC for ?8 min and then rapidly
diffused away, adopting a nonpolarized distribution. Nevertheless,
even after complete loss of CD28 polarization within the IS, T
cells remained in close contact with the APC for at least 20 min
after MHC class II-blocking Ab was added. This indicates that
cessation of TCR signaling results in displacement of CD28, well
4The online version of this article contains supplemental material.
retrovirally transduced CD28KO T cells expressing a CD28-CFP fusion protein (CD28-CFP), and Ag-pulsed APC were analyzed based on their ability to
recruit CD28 to the IS. CD80-APC are ProAd-ICAM cells that express MHC class II and ICAM-1, but not CD80 or CD86; while CD80?APC are
ProAd-ICAM-B7 cells that express MHC class II, ICAM-1, and CD80. CD28?/?T cells were stained with anti-CD28 to label endogenous CD28, while
localization of CD28-CFP was determined by CFP fluorescence. Representative images (A) and percentage of conjugates that recruit CD28 to the IS (B;
n ? 50–65 conjugates) from one experiment representative of two (CD28-CFP) or three (CD28?/?) are shown. C and D, T cell conjugates with Ag-pulsed
ProAd-ICAM-B7 APC were formed in the presence of 2 ?g/ml mAb against CD80 (anti-CD80) or MHC class I (anti-MHC I) and analyzed for recruitment
of CD28 to the IS. Representative images (C) and percentage of conjugates that recruit CD28 to the IS (D; n ? 50–65 conjugates) from one experiment
representative of three is shown. In all of the images, the T cell is oriented toward the top in each panel. ?, p ? 0.0001 by two-population proportion Z-Student
CD28 recruitment to the IS requires ligand binding. A and B, Conjugates between DO11.10 TCR-transgenic, CD4?T cells (CD28?/?), or
7641The Journal of Immunology
by guest on June 13, 2013
before complete dissolution of the IS. Taken together, these data
indicate that steady-state localization of CD28 at the IS depends on
continuous CD28 ligand binding and TCR signaling.
Ligand binding is not sufficient to recruit CD28 to the IS
To identify the cis-elements in CD28 that mediate localization to
the IS, we first generated a truncation of CD28 that lacks the cy-
tosolic tail (?CT) (for schematic, see Fig. 5A). The mutation does
not effect cell surface expression of CD28. WT and ?CT CD28 are
expressed at equivalent levels at the cells surface following retro-
viral transduction into CD28-deficient T cells (Fig. 4A). To deter-
mine whether the CD28 cytosolic tail is required for recruitment to
the IS, conjugates between retrovirally transduced T cells express-
ing either WT or ?CT CD28 and Ag-pulsed ProAd-ICAM-B7
were analyzed by microscopy (Fig. 4, C and D). ?CT CD28-ex-
pressing T cells showed a significantly reduced ability to recruit
CD28 molecules toward the APC. Furthermore, even in those
?CT-expressing T cells where CD28 was polarized toward the
APC, significant CD28 was detected outside of the IS, a morphol-
ogy that was not detected with cells expressing WT CD28
To confirm that the failure to recruit ?CT CD28 to the IS was
not secondary to a defect in ligand binding, we tested the ability of
?CT CD28 to bind CD80. WT or ?CT CD28-expressing T cells
were labeled with a soluble CD80/Ig fusion protein and analyzed
by flow cytometry (Fig. 4A). Equivalent levels of CD28 were de-
tected on WT and ?CT-expressing T cells by labeling either with
anti-CD28 mAb or with the soluble CD80/Ig fusion protein. Dou-
ble staining with CD28 and CD80/Ig was not feasible, because the
CD28 mAb used blocks the CD80 interaction site. Therefore, we
used the IRES-GFP reporter cassette in the retroviral vector to
normalize for levels of retroviral gene expression and then com-
pared the cell surface labeling with anti-CD28 mAb and with the
CD80/Ig fusion protein for a given level of GFP expression (Fig.
4B). WT and ?CT CD28 bound equivalent levels of CD80/Ig fu-
sion protein for any given amount of CD28 expression level. These
results confirmed that the failure of ?CT CD28 to localize at the IS
was not due to expression levels or inability to interact with CD80,
but was a direct result of the cytosolic tail deletion. Furthermore,
CD28 localization to the IS is not mediated passively by binding to
ligand expressed on the surface of the APC. Taken together these
data indicate that CD28 ligand binding is necessary, but not suf-
ficient, to target CD28 to the IS and additional signals from the
TCR and cis-elements in the CD28 cytosolic tail are also required.
Mutation of tyrosine at position 188 disrupts the ability of
CD28 to focus at the IS
To identify the specific motif within the cytosolic tail of CD28 that
mediates IS localization, we first analyzed a series of amino acid
point mutations in suspected protein interaction motifs. We tar-
geted the YMNM motif at positions 170–173, that upon tyrosine
phosphorylation generates a SH2 binding site that has been shown
to recruit PI3K and Grb-2 (8, 9, 40). We also targeted prolines at
178 and 187/190, that have been implicated in Itk and Lck SH3
domain interactions, respectively (11–14) and two additional ty-
rosine residues at 188 and 197 (Fig. 5A). Conjugates between
CD28-deficient T cells expressing different point mutations and
Ag-pulsed ProAd-ICAM-B7 were formed and analyzed by micros-
copy. Strikingly, only one of these mutations showed a significant
defect in its ability to focus CD28 into the IS, a point mutation of
the tyrosine at position 188 (Fig. 5B). To test the role of additional
segments of the cytosolic tail, a series of clustered alanine replace-
ments were generated (Fig. 5A). Replacement of aa 179–182 and
183–186 with alanines had no effect on CD28 localization to the
IS. In contrast, alanine substitutions of residues 187–190 (which
contains Y188) and of residues 192–194 inhibited CD28 localiza-
tion to the IS. These results indicate that residues 187–194, and
especially Y188, are required for CD28 focusing within the IS.
To more carefully address the role of the cytosolic tail on CD28
localization in the IS, we focused our analyses on the single amino
acid mutation Y188F (Fig. 6). T cells expressing Y188F CD28
showed a delay in the kinetics of CD28 accumulation at the IS
compared with WT CD28 (Fig. 6B). At 15 min, there was a sig-
nificant difference in the ability of WT and Y188F CD28 to be
recruited at the IS, but by 45 min both WT and Y188F CD28 were
recruited to the IS with similar frequency. However, even when
Y188F was recruited to the IS, it was often incomplete and CD28
A, WT DO11.10 T cell conjugates with Ag-pulsed ProAd-ICAM-B7 APC
were incubated for 10 min to allow IS formation, 2 ?g/ml mAb against
MHC class I or CD80 was added, and the cells were cultured for 5 or 10
min before fixation and microscopic analysis. Percentage of conjugates that
recruit CD28 to the IS (n ? 60 conjugates) is shown. ?, p ? 0.0001 by
two-population proportion Z-Student statistical test. B and C, Conjugates
between CD28-CFP-expressing T cells and Ag-pulsed ProAd-ICAM-B7
APC were selected based on CD28 recruitment at the IS. Live cell imaging
was done for 5 min before addition of 2 ?g/ml anti-CD80 (B) or anti-MHC
class I (C) and continued for 15 min after mAb addition. Representative
images at various time points are shown; complete movies are shown in
videos 1 and 2 in supplemental data. The apparent change in morphology
in the T cell following anti-class I addition reflects the T cell crawling onto
the top of the APC (see video 2 in supplemental data). DIC, Differential
CD28 ligand binding is required to sustain CD28 at the IS.
7642CD28 LOCALIZATION TO THE IMMUNOLOGICAL SYNAPSE
by guest on June 13, 2013
was detected outside of the IS (Fig. 6A). These results indicate that
Y188 is required for efficient polarization of CD28 to the IS. A
further defect in Y188 was detected when the morphology of
CD28 localization at the IS was compared. WT CD28 was signif-
icantly more focused into the center of the IS (indicative of
cSMAC localization) compared with Y188F CD28 at both time
points evaluated (Fig. 6, C and D). Mutation of Y188F did not
interfere with the ability of CD28 to interact with CD80, as
633-labeled ProAd-ICAM-B7 APC. The cells were analyzed by flow cytometry to established a baseline of Indo-1 fluorescence and then briefly pelleted
to induce T cell:APC conjugate formation (thin arrow on the left in A–C). The cells were resuspended, rapidly returned to the flow cytometer, and
maintained at 37°C. T cell:APC conjugates were gated based on coincident Indo-1 and Alexa Fluor 633 fluorescence and the change in the ratio of violet
to blue fluorescence of Indo-1, indicative of an increase in intracellular calcium, was monitored over time. Data are presented as dot plots of individual
conjugates (A and B) and as median calcium response of the population (C). After 7 min, anti-MHC class II was added to one sample (double arrow in
B and C), which resulted in a rapid decline in calcium signaling within all of the T cell-APC conjugates. Ionomycin was added at the end of the assay (thin
arrow on the right in A–C) to assure that all T cells were effectively loaded with Indo-1. D, The samples in A and B were analyzed for the percentage of
T cells that were in conjugates with APC over time in the presence and absence of anti-class II mAb. E, T cell:ProAd-ICAM-B7 conjugates were incubated
for 10 min to allow IS formation, 2 ?g/ml mAb against MHC class I or class II was added, and the cells were cultured for 5 or 10 min before fixation and
microscopic analysis. Percentage of conjugates that recruit CD28 to the IS (n ? 50 conjugates) is shown. This is one experiment representative of three.
?, p ? 0.0001 by two-population proportion Z-Student statistical test. F, Conjugates between CD28-CFP-expressing T cells and Ag-pulsed ProAd-
ICAM-B7 APC were selected based on CD28 recruitment at the IS. Live cell imaging was done for 2 min before addition of 2 ?g/ml anti-MHC class II
and continued for 20 min after mAb addition. Representative images at various time points are shown; the complete movie is shown in video 3 in
CD28 requires constant TCR signal to persist at the IS. A–C, T cells were loaded with Indo-1-AM and mixed with Ag-pulsed, Alexa Fluor
CD28 to the IS. CD28-deficient T cells were transduced
with a retrovirus expressing WT CD28 or a truncated
CD28 that lacks the cytosolic tail (?CT) in conjunction
with an IRES-GFP cassette. A and B, T cells were sorted
for GFP expression and then labeled with isotype con-
trol mAb, anti-CD28 mAb, or CD80/Ig fusion protein
and levels of staining were compared with GFP expres-
sion. A, Dot plots of flow cytometric analyses are
shown. B, Relative mean fluorescent intensity (MFI) of
anti-CD28 and CD80/Ig binding, normalized to GFP
shows that for any given level of CD28 expression, WT
and ?CT CD28 binds equivalent levels of CD80/Ig. C
and D, WT or ?CT CD28-expressing T cell:APC con-
jugates were incubated for 15 min and analyzed by mi-
croscopy. Percentage of conjugates that recruit CD28 to
the IS (n ? 50) is shown (C). This is one experiment
representative of two. ?, p ? 0.0001 by two-population
proportion Z-Student statistical test. Images that repre-
sent the different phenotype observed between WT and
?CT CD28-expressing T cells are shown (D). The T
cell is oriented toward the top in each panel. Note that
even in the ?CT CD28-expressing T cells that were
scored as CD28 recruitment to the IS, CD28 recruitment
was often incomplete (image on right).
Ligand binding is not sufficient to recruit
7643 The Journal of Immunology
by guest on June 13, 2013
measured by CD80/Ig binding by flow cytometry (data not shown),
suggesting that mislocalization within the IS is a direct conse-
quence of the mutation of the tyrosine at position 188.
CD28 determines PKC? localization within the IS
To evaluate signals downstream of CD28, we analyzed the local-
ization of PKC?. Costimulation through either LFA-1 or CD28 can
recruit PKC? to the IS, but CD28 costimulation is required to
specifically localize PKC? to the cSMAC and induce PKC?-de-
pendent activation of NF-?B (21). To determine how mislocaliza-
tion of Y188F CD28 impacts on PKC?, conjugates between
CD28-deficient T cells expressing either WT or Y188F CD28
and Ag-pulsed APC were analyzed. To avoid LFA-1-mediated
recruitment of PKC? to the IS, the APC used did not express
ICAM-1 and were either CD80 positive (ProAd-B7) or CD80 neg-
ative (ProAd). Y188F CD28 effectively retained it ability to recruit
PKC? to the IS with no significant differences with WT CD28-
expressing T cells (Fig. 7, A and B). However, similar to CD28,
PKC? recruitment was delayed (data not shown) and PKC? dis-
tribution at the IS was more diffuse in T cells expressing Y188F
compared with WT CD28 (Fig. 7C).
To determine whether PKC? localization at the IS correlates
with CD28 localization, the distribution of both molecules was
measured at the IS in each conjugate and these values were directly
compared (Fig. 7D). In the majority of T cells expressing WT
CD28, both CD28 and PKC? were focused within the IS, while in
the majority of T cells expressing Y188F, both CD28 and PKC?
were broadly distributed across the IS. Strikingly, the data sets are
partially overlapping and the linear regression lines for these two
populations are essentially identical, suggesting that the localiza-
tion of CD28 is the primary determinant for the localization of
PKC?. To confirm that CD28 and PKC? focusing in the IS rep-
resented localization to the cSMAC, T cell:APC conjugates were
focusing to the IS. A, The sequence of the WT CD28 cytosolic tail is shown
on the top line. The localization and amino acid substitution of single
amino acid point mutations are shown above and below, respectively. Only
the amino acid change is shown and all of the individual mutations are
included on a single line. The clustered alanine replacements (Ala-A
through Ala-D) are shown on lines 3–6. Only the amino acid alterations are
shown; all other positions are WT. The sequence of the cytosolic tail trun-
cation (?CT) is shown on the bottom. In this case, only the sequence
remaining in the truncated construct is shown. The remainder of the cyto-
solic tail (indicated by dots) is deleted. B and C, CD28-deficient T cells
were retrovirally transduced with the mutants shown in A and T cell:
ProAd-ICAM-B7 conjugates were screened for CD28 focusing at the IS.
Focusing was defined as CD28 concentration within ?60% of the IS area.
Data are the percentage of conjugates that focus CD28 to the IS (n ? 50).
Mutation scan identifies a single site that controls CD28
CD28 to focus at the IS. Conjugates between WT or Y188F CD28-ex-
pressing T cells and Ag-pulsed ProAd-ICAM-B7 were formed and ana-
lyzed for CD28 localization. A, Representative images of conjugates are
shown. The T cell is oriented toward the top in each panel. For WT CD28,
the majority of conjugates display efficient recruitment of CD28 to the IS
and the majority of CD28 is focused into the cSMAC (as shown in the two
examples). In contrast, in Y188F-expressing T cells, even when CD28 is
recruited to the IS (the two images on the left), CD28 recruitment was often
incomplete. B and C, Percentage of conjugates that recruit CD28 to the IS
(B, n ? 60) or that focus CD28 within the IS (C, n ? 50) at 15 or 45 min
after conjugate formation are shown. #, p ? 0.001 by two-population
proportion Z-Student statistical test. D, The distribution of CD28 within
the IS was determined by the percentage of the IS that contained CD28.
Individual T cells are shown and the thick line represents the population
median (n ? 34). The thin line at 60% represents the cutoff used for
scoring in C. One experiment representative of three is shown. The
population distribution was compared using the nonparametric Mann-
Whitney U test (p ? 0.0001).
Mutation of tyrosine at position 188 disrupts the ability of
7644 CD28 LOCALIZATION TO THE IMMUNOLOGICAL SYNAPSE
by guest on June 13, 2013
stained for CD28, PKC?, and MHC class II and analyzed by three-
dimensional reconstruction (Fig. 7E). Class II localization serves
as a positive marker for the cSMAC region, because it labels the
class II molecules that are juxtaposed to the TCR in the cSMAC.
In T cells expressing WT CD28, both CD28 and PKC? colocalized
with class II in the cSMAC. In contrast, in T cells expressing
Y188F, a cSMAC is found, as indicated by central and focused
within the IS. CD28-deficient T cells were retrovirally transduced with WT
or Y188F CD28. A and B, T cell:APC conjugates with ProAd (fibroblast
expressing MHC class II but not CD80) or ProAd-B7 (fibroblast expressing
MHC class II and CD80) were analyzed for the ability to recruit CD28 and
PKC? to the IS. Representative images (A) and frequency of conjugates
that have recruited PKC? to the IS (B) are shown (n ? 50). ?, p ? 0.0001
by two-population proportion Z-Student statistical test. The T cell is ori-
ented toward the top left in each panel. C and D, Mutation at Y188 in CD28
results in a failure to concentrate PKC? in the cSMAC. C, The distribution
of PKC? within the IS is represented by the percentage of the IS that
contains PKC? (n ? 50). Individual T cells are shown and lines represent
the population median. The population distribution was compared using the
nonparametric Mann-Whitney U test (p ? 0.0001). D, Direct correlation
between the relative distribution of CD28 and PKC? within the IS is shown
(n ? 50). The E represent individual T cells and the gray line represents
the linear regression for WT CD28 T cells, while the F represent individual
T cells and the black line represents the linear regression for Y188F-ex-
pressing T cells. Note that the majority of WT T cells are clustered at the
bottom left (where both CD28 and PKC? are focused, while the majority of
Y188F T cells are clustered in the upper right (where neither CD28 not
PKC? are focused). Strikingly, the linear regression lines for these two
populations are essentially identical. E and F, Conjugates between WT or
Y188F-expressing T cells with ProAdB7 APC were stained for CD28
(green), PKC? (blue), and MHC class II (red). Three-dimensional recon-
structions of the interaction site between the T cell and the APC of indi-
vidual channels (top) and composites of two channels (bottom) are shown
CD28 localization determines the distribution of PKC?
(E). Additional examples of three-dimensional reconstructions of the T
cell-:APC interaction site of Y188F-expressing T cells stained for CD28
(top row) and PKC? (bottom row) are shown (F). Animated rotations of the
three-dimensional representations shown in the first three panels are in-
cluded in videos 4–6 in supplemental data. Note that the distribution of
CD28 and PKC? are more diffuse in Y188F-expressing T cells and that the
overall distribution of PKC? mirrors the distribution of CD28 at the IS.
clear translocation. CD28-deficient T cells were retrovirally transduced
with WT CD28, Y188F CD28, or empty vector (MIG). CD28?/?and
CD28-deficient (CD28KO) T cells were included as controls. Conjugates
with these T cells and Ag-pulsed ProAd-ICAM-B7 were stained for CD28
(green), NF-?B p65 (red), and the nucleus (blue) 45 min after conjugate
formation. A, Representative images are shown. The T cell is oriented
toward the top in each panel. B, Percentage of conjugates that translocated
visually detectable levels of NF-?B into the nucleus (n ? 40 conjugates).
CD28?/?vs CD28KO (p ? 0.0001), WT vs Y188F (p ? 0.121), and
Y188F vs MIG (p ? 0.0001) by two- population proportion Z-Student
statistical test. C, The intensity of NF-?B staining in the nucleus of indi-
vidual T cells is shown and the thick line represents the median of each
population sample. The thin line across all of the samples represents the
limit of detection for visual identification of NF-?B translocation into the
nucleus. Although Y188F CD28 did not impact on the frequency of T cells
that activated NF-?B, the magnitude of NF-?B nuclear translocation was
significantly reduced compared with T cells expressing WT CD28. One
experiment representative of three is shown. The population distribution
was compared using the nonparametric Mann-Whitney U test; statistical
significance between relevant samples is shown.
Mutation at Y188 diminishes the efficiency of NF-?B nu-
7645 The Journal of Immunology
by guest on June 13, 2013
distribution of class II, but CD28 and PKC? are broadly distributed
across the IS. Y188F CD28 and PKC? were not excluded from the
cSMAC, but not preferentially concentrated in the cSMAC as in
the majority of the WT CD28 T cell conjugates. In many conju-
gates, Y188F was not evenly distributed across the IS and formed
multiple regions of higher concentration. Interestingly, in these
cases, PKC? distribution mirrored CD28 localization; PKC? and
CD28 colocalized in these multiple foci (Fig. 7F and videos 4–6
in supplemental data). These results further support the idea that
CD28 determines the localization of PKC?.
Mislocalization of Y188F CD28 correlates with reduced
NF-?B nuclear translocation
To evaluate signals downstream from CD28 and PKC?, we an-
alyzed NF-?B nuclear translocation. WT or Y188F CD28 ex-
pressing T cell conjugates with Ag-pulsed ProAd-ICAM-B7
were analyzed at 45 min for nuclear translocation of NF-?B
(Fig. 8, A and B). No significant defect in the frequency of T
cells that had activated NF-?B was noted in T cells expressing
Y188F compared with WT CD28 (Fig. 8B). However, a differ-
ence in the magnitude of NF-?B translocation was apparent
(Fig. 8A). To better quantify NF-?B nuclear translocation, we
measured the intensity of NF-?B staining inside the nucleus of
individual T cells (Fig. 8C). Y188F CD28 was sufficient to
drive NF-?B nuclear translocation in the majority of T cells
compared with CD28-deficient T cells, but the magnitude of
NF-?B activation in T cells expressing Y188F was significantly
decreased compared with WT T cells. Although it remains pos-
sible that this represents a decrease in CD28 signaling in
Y188F, the correlation with Y188F and PKC? mislocalization
suggests that the disruption of cSMAC localization of CD28
may diminish CD28-mediated costimulation through PKC? and
The mechanisms that mediate CD28 localization to the cSMAC
and the functional consequences of CD28 localization to the
cSMAC are not understood. In this report, we show that CD28
recruitment and persistence at the IS requires TCR signals, CD80
engagement, and sequences within the CD28 cytosolic tail. The
requirement for both TCR and CD28 engagement indicates that
there may be more cross-talk between TCR and CD28 signaling
than would be predicted by the simple two signal model, where
CD28 only provides costimulatory signals for TCR. Our data in-
dicate that TCR signals are required to allow for CD28 ligand
interaction, suggesting that TCR may also provide costimulatory
functions to enhance CD28 signaling. We also show that a single
point mutation at tyrosine 188 in the cytosolic tail disrupts the
ability of CD28 to preferentially accumulate at the cSMAC. This
provides the first indication of a cSMAC localization signal within
CD28. Interestingly, PKC? distribution at the IS mirrors CD28
localization, even when CD28 adopts a more diffuse distribution
compared with TCR. This indicates that CD28 itself determines
the location of PKC? and there are no additional factors within the
cSMAC that are required for PKC? recruitment. Finally, mutation
of Y188 diminishes CD28-dependent activation of NF-?B, sug-
gesting that the colocalization of CD28 and PKC? specifically to
the cSMAC region is important for efficient signal transduction.
Taken together, these results provide new insight into the regula-
tion of CD28 localization to the cSMAC and the functional con-
sequences of specific protein organization within the IS.
TCR signals are essential to initiate the formation of the IS
structure. We show here that, even after a mature IS has been
formed, sustained TCR signaling is required to maintain CD28
localization at the IS. Addition of anti-class II mAb results in a
rapid cessation of proximal TCR signaling followed by the dis-
persion of CD28 from the IS. Both of these events occur well
before T cell:APC conjugates are disrupted, indicating that avail-
ability of B7 ligand is insufficient to retain CD28 at the IS. This
apparent TCR dependence for CD28-B7 interactions is reminis-
cent of the inside-out signaling associated with integrin activation
(41). In this regard, TCR signaling may have a direct effect on
CD28 ligand binding, possibly through modulation of the cytosolic
tail. Y188 can be phosphorylated upon T cell activation (42) and
the conservative structural mutation (Y188F) supports a role for
phosphorylation. Thus, it is possible that TCR-mediated phosphor-
ylation of Y188 is required to efficiently recruit and/or sustain
CD28 within the IS. Thus far, phosphorylated Y188-interacting
proteins have not been clearly identified, although it has been sug-
gested that phosphorylation of Y188 may create a SH2 docking
site for Lck (43). Alternatively, CD28 localization at the IS may be
dependent on TCR-induced changes in membrane domains. Al-
though the biophysical properties of lipid rafts in living cell mem-
branes and their functional importance in T cell activation remains
controversial (44–46), Y188 has been implicated in the ability of
CD28 to partition into detergent-resistant membrane fractions (47).
Finally, Y188F may control CD28 interactions with specific
adapter proteins that facilitate or promote cSMAC localization,
such as Filamin A, that can be associated with CD28 and impact
on CD28-mediated lipid raft accumulation at the IS (48).
CD28 ligand binding is also required to recruit and sustain
CD28 in the IS. CD28 is a disulfide-linked dimer and the conven-
tional model would suggest that ligand binding transduces a con-
formational change through the dimer to alter the orientation of the
cytosolic tails to allow for protein interactions that could mediate
IS localization and/or signaling. However, ligand binding is not
sufficient to induce CD28 localization to the IS and additional sig-
nals mediated through the TCR are required. This cross-talk be-
tween TCR and CD28 could be mediated through the inside-out
model discussed above. Alternatively, CD28 ligand binding may
function to retain CD28 in the IS. In this case, disruption of CD28
localization following the addition of anti-CD80 mAb might reflect
the low affinity of CD28-CD80 interactions and the ability of this
the released CD28 would simply diffuse away from the IS. This pos-
sibility will favor a more dynamic model for CD28 localization at the
IS, where the pool of CD28 at the cSMAC is constantly turning over
and the steady-state localization of CD28 at the IS depends on con-
stant recruitment from the periphery.
It has recently been suggested that sustained TCR signaling is
mediated through the continued formation of new TCR microclus-
ters at the periphery of the IS (29–32). These microclusters me-
diate TCR signaling as they transit through the pSMAC en route to
the cSMAC region. Addition of anti-class II mAb is thought to
disrupt the formation of new microclusters and so inhibit sustained
TCR signaling (32). TCR and CD28 have been shown to cocluster
in the immature IS, before the formation of an organized cSMAC
region (49). It is possible that CD28 is also corecruited with the
TCR to the peripheral microclusters that continuously form after
the mature IS has been established. In this case, anti-class II-me-
diated inhibition of new microcluster formation and sustained TCR
signaling would result in a loss in the influx of CD28 to the
cSMAC. In this microcluster model, the rapid dispersion of CD28
from the IS following the addition of anti-class II mAb would
imply that CD28 retention in the cSMAC is transient. For example,
CD28 may be rapidly internalized following TCR activation, li-
gand binding, and/or recruitment to the cSMAC region. This raises
the possibility that CD28 follows a reciprocal pathway compared
7646 CD28 LOCALIZATION TO THE IMMUNOLOGICAL SYNAPSE
by guest on June 13, 2013
with CTLA-4. In resting activated T cells, CTLA-4 resides pri-
marily within intracellular compartments due to rapid internaliza-
tion. Upon T cell activation, tyrosine phosphorylation disrupts an
internalization motif and redistributes CTLA-4 to the cell surface
(50, 51). It is possible that TCR activation induces CD28 internal-
ization. As long as sufficient antigenic peptide-MHC complexes
remain on the cell surface, new TCR microclusters form, recruiting
new CD28 molecules, and providing TCR signaling in the context
of costimulation. Once the concentration of Ag drops below the
threshold to drive new microcluster formation, new TCR/CD28
signaling would stop and the high concentration of CTLA-4 at the
cell surface could inhibit any residual ongoing T cell activation.
The requirement for the cytosolic tail of CD28 implies that ei-
ther ligand binding and/or TCR signals modify some molecular
aspects of the tail that will facilitate CD28 recruitment at the IS. As
discussed above, this could be a positive function of the cytosolic
tail, mediated through phosphorylation of Y188, lipid raft associ-
ation, and/or binding of an adaptor or cytoskeletal component. It
has been shown that in resting T cells, CD28 mobility in the
plasma membrane is reduced compared with other surface mole-
cules, suggesting that CD28 may be anchored to some cytoskeletal
elements in the absence of TCR signals or ligand binding (33).
Interestingly, deletion of the cytosolic tail increases mobility of
CD28 in the plasma membrane. Thus, it is possible that this pu-
tative interaction in resting T cells may regulate CD28 recruitment
to the cSMAC. Additional experiments to address the specific role
of TCR signaling, ligand binding, and cytosolic tail sequences,
including Y188, in the regulation of CD28 mobility in the plasma
membrane will be required to address this issue.
One function of CD28 is to recruit PKC? to the cSMAC (21,
34). We have shown that this is mediated through PI3K and mu-
tation of the PI3K interaction site on the cytosolic tail of CD28
results in a failure to recruit PKC?, to translocate NF-?B to the
nucleus, and to up-regulate IL-2 transcription (21). This is consis-
tent with the proposed role of PDK1 in PKC? recruitment to the
synapse (52). However, CD28 costimulation does not appear to
generate a localized concentration of phosphatidylinositol-(3,4,5)-
triphosphate (the product of PI3K) within the cSMAC (C. Baker
and J. Miller, unpublished data), suggesting that additional features
of the cSMAC or CD28 signaling may be required for PKC? re-
cruitment. Consistent with this idea, we found that mislocalization
of CD28 within the synapse by mutation of Y188 resulted in a
corresponding mislocalization of PKC?. A similar association of
PKC? with CD28 outside of the cSMAC regions was observed
using transfected Chinese hamster ovary cells as APC (53). These
results suggest that there are no unique features to the cSMAC that
allow for PKC? recruitment; rather PKC? recruitment appears to
be an intrinsic function of CD28. No direct association between
CD28 and PKC? has been reported. The SH2 and SH2 domain
docking sites on CD28 may recruit adaptor proteins that mediate
PKC? recruitment. For example, Filamin A has been shown to
localized with CD28 and PKC? and Filamin A knock-down in-
hibits PKC? recruitment to the IS (48, 54).
Activation of PKC? results in the subsequent activation of NF-
?B. Interestingly, the ability of CD28 to drive nuclear localization
of NF-?B is reduced by mutation of Y188. It is possible that in
addition to its effect on CD28 localization, Y188 may directly
modulate some aspect of CD28 signaling that impacts on NF-?B
activation. Alternatively, the correlation between CD28 and PKC?
mislocalization and diminished NF-?B activation suggests that the
ability of Y188 to direct CD28 localization itself may account for
the diminished signaling. Mutation of Y188 could effect CD28/
TCR association in microclusters, which would have a secondary
effect on CD28 cSMAC localization. Alternatively, CD28 signal-
ing through PKC? may be mediated at the cSMAC and reduced
cSMAC localization of Y188F may account for reduced CD28
Although the functional impact of CD28 on T cell activation can
be clearly seen in CD28-deficient T cells, analysis of the specific
elements in CD28 that mediate these functions have been difficult
to define (15–20). This may be a result of both functional redun-
dancy and in the ability of multiple elements within CD28 to syn-
ergize in the activation of specific signaling pathways and/or func-
tional readouts of T cell activation. The interplay of TCR and
CD28 in the localization of proteins within the immunological syn-
apse, in the induction of specific signaling events, and in the func-
tional activation/differentiation of T cells is more intricate than the
original two-signal model of costimulation. Future studies will re-
quire both a molecular breakdown of signaling pathways along
with sophisticated real-time imaging studies to define the specific
roles of TCR and CD28 in T cell activation.
We thank Ryo Abe and Jim Allison for providing DNA constructs, Tim
Bushnell in the URMC Flow Cytometry Core for help with the calcium
assays, and Nathan Laniewski for help with cell sorting.
The authors have no financial conflict of interest.
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7648 CD28 LOCALIZATION TO THE IMMUNOLOGICAL SYNAPSE
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