Engagement of CD28 Outside of the Immunological Synapse
Results in Up-Regulation of IL-2 mRNA Stability but Not
Mariano Sanchez-Lockhart and Jim Miller2
During T cell activation by APC, CD28 is colocalized with TCR in the central supramolecular activation cluster (cSMAC) region
of the immunological synapse. CD28 signaling through PI3K results in the recruitment of protein kinase C (PKC)? to the cSMAC,
activation of NF-?B, and induction of IL-2 transcription. These results suggest that localized engagement of CD28 within the
cSMAC may be required for CD28 activation and/or signal integration with TCR signals. To test this model we have examined
the mechanism of CD28-mediated induction of IL-2 secretion when CD28 is engaged outside of the immunological synapse. CD4
T cells were stimulated with Ag presented by B7-negative APC and CD28 costimulation was provided in trans by anti-CD28-coated
beads or by class II-negative, B7-positive cells. We show that induction of IL-2 secretion under these conditions did not require
expression of PKC? and did not induce NF-?B activation or IL-2 transcription. In contrast, CD28 costimulation in trans did induce
IL-2 mRNA stability, accounting for the up-regulation of IL-2 secretion. These data indicate that the ability of CD28 to up-regulate
IL-2 transcription requires colocalization of TCR and CD28 at the plasma membrane, possibly within the cSMAC of the immu-
nological synapse. In contrast, the ability of CD28 to promote IL-2 mRNA stability can be transduced from a distal site from the
TCR, suggesting that signal integration occurs downstream from the plasma membrane. These data support the potential role of
trans costimulation in tumor and allograft rejection, but limit the potential functional impact that trans costimulation may have
on T cell activation. The Journal of Immunology, 2006, 176: 4778–4784.
CD28 leads to T cell anergy rather than activation and only co-
stimulation through CD28, and not other costimulatory molecules,
can protect against anergy induction (5, 6). CD28 costimulation
leads to a dramatic up-regulation in IL-2 expression mediated by
both enhanced transcription and induction of mRNA stabilization
(7, 8). CD28 costimulation also plays an important role in T cell
survival, inducing expression of the antiapoptotic protein Bcl-xL
plays a key role on the generation of Th2 responses (11). In combi-
nation, these effects resulted in a dramatic loss in T cell expansion
and effective immune responses in CD28-deficient mice.
Despite the well-recognized functional importance of CD28, the
biochemical signaling pathways induced downstream of CD28 are
still not completely understood (1, 4). This result may be due, in
part, to the ability of CD28 to amplify TCR-initiated signaling
events (1). CD28 has been shown to lower the threshold of TCR
engagement (12) and T cell responses are diminished, but not ab-
sent, in CD28-deficient T cells. Both protein profiling of signaling
t has been shown that T cell costimulation through CD28 can
have a dramatic impact on T cell activation, differentiation,
and tolerance (1–4). T cell stimulation in the absence of
intermediates (13) and genetic profiling of changes in gene expres-
sion (14, 15) have suggested that CD28 costimulation functions
primarily to modify those signaling pathways that can be induced
by the TCR itself and it has been difficult to identify a unique
contribution of CD28.
One potential site where CD28 could impact on TCR signaling
is within the central supramolecular activation cluster (cSMAC)3
of the immunological synapse (16–19). Although T cells express
a number of protein kinase C (PKC) isoforms, PKC? is selectively
activated and recruited to the immunological synapse, where it is
colocalized with TCR and CD28 in the cSMAC (20, 21). PKC?
plays an essential role in transducing TCR-mediated activation of
NF-?B (22–24). Expression of CD28 is required for the targeting
of PKC? to the cSMAC. In the absence of CD28, PKC? is re-
cruited to the immunological synapse, but it is diffusely distributed
across the synapse and is not focused into the cSMAC (25, 26).
This disruption in PKC? localization in the absence of CD28 cor-
relates with a loss in PKC?-dependent induction of NF-?B and
IL-2 transcription. Interestingly, all of these functions of CD28
(recruitment of PKC? to the cSMAC, activation of NF-?B, and
up-regulation of IL-2 transcription) are lost by a single amino acid
mutation of the PI3K interaction site in the cytosolic tail of CD28
(26). These results suggest that CD28-mediated activation of PI3K
leads to a localized concentration of phosphatidylinositol-3,4,5-
trisphosphate (PIP3) at the synapse that results in the recruitment
and activation of PKC? and subsequent induction of IL-2 tran-
scription. Recent data on the recruitment of a GFP-PH domain
fusion protein to the synapse (27–29) and the possible role for
phosphoinositide-dependent protein kinase PDK1 in PKC? activa-
tion (23) support this model. Taken together, these results suggest
David H. Smith Center for Vaccine Biology and Immunology, Aab Institute of Bio-
medical Sciences and the Department of Microbiology and Immunology, University
of Rochester, Rochester, NY 14642
Received for publication December 1, 2005. Accepted for publication January
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 Grants RO1-AI48237 and RO1-AI063418 from the
National Institutes of Health (to J.M.), M.S.-L. was supported by National Institutes
of Health Training Grant T32-AI007169.
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: cSMAC, central supramolecular activation cluster;
PKC?, protein kinase C?; WT, wild type; ARE, AU-rich element; UTR, untranslated
region; TTP, tristetraprolin.
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc.0022-1767/06/$02.00
that signal integration between TCR and CD28 may occur within
and through the spatial organization of proteins in the immuno-
In addition, CD28 has been shown to transduce costimulatory
signals in trans, i.e., from a separate site on the cell surface from
TCR engagement. This was first shown by the ability of MHC-
disparate APC to provide T cell costimulation and protect against
clonal anergy (30). Later, trans costimulation was shown to be
mediated through CD28 (31–33). In vivo, trans costimulation may
contribute to T cell activation to Ags expressed by nonhemopoietic
cells, such as in tumor or allograft rejection (34). These functional
studies suggested that CD28 did not have to be localized to the
immunological synapse to provide costimulatory signals to T cells.
In this study we show that when CD28 is provided in trans, the
induction of IL-2 expression is not mediated through PKC? acti-
vation and induction of IL-2 transcription. Rather, CD28 costimu-
lation in trans effectively enhances T cell activation through the
induction of IL-2 mRNA stability.
Materials and Methods
Cell lines and T cell stimulation
6132 Pro cell transfectants expressing class II (I-Ad) in combination with
ICAM-1 (ProAd-ICAM) or with ICAM-1 and B7-1 (ProAd-ICAM-B7)
(35) and the class II-negative fibroblast L cell line (DAP-3), which con-
stitutively expresses B7 (36), were maintained in DMEM (Invitrogen Life
Technologies) supplemented with 10% FCS, 2 mM glutamine, 0.1 mM
nonessential amino acids, 40 ?g/ml gentamicin, and 50 ?M 2-ME. To
maintain selection of transfected genes, G418 (200 ?g/ml) and/or MXH (6
?g/ml mycophenolic acid, 250 ?g/ml xanthine, 15 ?g/ml hypoxanthine)
were added as appropriate. CD4-positive lymph node T cells were purified
as previously described (35) from wild-type (WT), CD28-deficient, PKC?-
deficient (24), and IL-2-luciferase reporter transgenic (26, 37, 38) DO11.10
TCR transgenic mice. Freshly isolated DO11.10 T cells were assayed di-
rectly or after in vitro activation with Ag presented by irradiated BALB/c
splenocytes under neutral conditions. Previously activated T cells are es-
sential 100% CD4 and KJ1.26-positive and contain a mixture of IL-4 and
IFN-?-secreting cells (typically 10–20% IL-4 and 40–60% IFN-?-secret-
ing). Previously activated T cells were rested for 7–14 days before use in
experiments. For retroviral transduction, freshly isolated DO11.10 T cells
were stimulated with Ag as shown and 1 day later were transduced with a
retrovirus encoding an NF-?B p65-GFP fusion protein (39). For trans co-
stimulation with anti-CD28-coated beads, surfactant-free surface latex
beads were first coated with goat anti-Syrian hamster Ab at room temper-
ature overnight, and after blocking with BSA, anti-CD28 mAb (37.51) was
captured at room temperature for 4 h. T cell to bead ratio was 1:3 in all
IL-2 ELISA and luciferase assay
T cells were stimulated with Ag in the absence of CD28 costimulation
(ProAd-ICAM), in the presence of CD28 in cis (ProAd-ICAM-B7), or by
providing CD28 in trans (ProAd-ICAM with anti-CD28-coated beads or
B7-positive L cells). Supernatants were collected at 24 or 48 h and IL-2
was measured by a capture ELISA. For luciferase activity, T cells were
stimulated under the same conditions, but after 24 h of stimulation, T cells
were lysed and luciferase activity was determined according the manufac-
turer’s instructions (Promega).
T cells were centrifuged with peptide-pulsed APC with and without anti-
CD28-coated beads or B7-positive L cells for 20 s at relative centrifugal
force of 2000. The cell pellet was incubated for 5 min at 37°C, resuspended
in DMEM, and plated on poly-L-lysine-coated coverslips for 10 min at
37°C. Cells were fixed in 3% (w/v) paraformaldehyde, and stained with
rabbit anti-PKC? (C-18; Santa Cruz Biotechnology) and anti-rabbit-FITC
or anti-rabbit-Cy3 (Jackson ImmunoResearch Laboratories). For NF-?B
nuclear localization, the incubation time on poly-L-lysine-coated coverslips
was increased to 25 min and cells were fixed in 3% (w/v) paraformalde-
hyde and permeabilized in 0.3% Triton X-100. Localization of NF-?B was
detected with rabbit anti-p65 (SC-109; Santa Cruz Biotechnology) or in
retrovirally infected cells by detection of the p65-GFP fusion protein. After
staining, nuclei were labeled with Hoestch dye. Samples were analyzed on
a Zeiss Axiovert microscope controlled by SlideBook software (Intelligent
Activated T cells (1 ? 106) were stimulated with Ag presented by ProAd-
ICAM alone, ProAd-ICAM, and anti-CD28-coated beads (trans costimu-
lation) or ProAd-ICAM-B7 (cis costimulation) for 4 h and further IL-2
transcription was inhibited by the addition of 1.0 ?g/ml cyclosporin A
(Calbiochem). Total RNA was isolated using TRIzol (Invitrogen Life
Technologies) and reverse transcribed into cDNA. The level of IL-2
mRNA was determined by real-time PCR following normalization to a T
cell-specific gene, CD3?, using the ??CTmethod for relative quantitation
with IL-2 mRNA levels in the absence of Ag as the calibrator. TaqMan
probe and primers for IL-2 and CD3? were obtained from Applied
CD28 costimulation in trans is not mediated through PKC?
CD28 has the unique ability among potential costimulatory mol-
ecules to function when it is engaged on the T cell at a site that is
distal to the TCR (in trans). CD28 costimulation in trans can lead
to the up-regulation of IL-2 secretion (Fig. 1), but it is not clear
how signals from CD28 in trans are integrated with TCR-derived
signals. We have recently found that there are two independent
pathways for CD28 costimulation (26). One pathway is mediated
through PI3K and leads to the cSMAC localization of PKC?, ac-
tivation of NF-?B, and the transcriptional up-regulation of IL-2.
The second pathway is not dependent on CD28-mediated activa-
tion of PI3K and results in the up-regulation of IL-2 secretion
through the induction of IL-2 mRNA stability. To determine
whether CD28 in trans can induce one or both of these pathways,
we initially examined the localization of PKC? within the immu-
nological synapse (Fig. 2). WT DO11.10 T cells were stimulated
with Ag presented by ProAd-ICAM. LFA-1 costimulation in the
absence of CD28 costimulation can induce PKC? recruitment to
the immunological synapse, but not to the cSMAC (26). To de-
termine whether CD28 must be engaged within the immunological
synapse to impart this effect, PKC? localization was assayed after
providing CD28 costimulation in trans. When anti-CD28-coated
beads were added to allow the formation of T cell:APC:bead con-
jugates, the level of IL-2 secretion was increased (Fig. 1), but
PKC? remained diffusely localized to the synapse and was not
focused into the cSMAC (Fig. 2C). Instead, recruitment of PKC?
viously activated DO11.10 T cells were unstimulated (?Ag) or stimulated
with Ag (?) presented by ProAd-ICAM (?CD28), ProAd-ICAM and anti-
CD28-coated beads (trans), or ProAd-ICAM-B7 (cis). T cells were cul-
tured for 48 h, and supernatants were assayed for IL-2 secretion by capture
ELISA. Data are the mean and SD from a total of eight independent ex-
periments. Both CD28 costimulation in trans and in cis enhanced IL-2
secretion (p ? 0.002 and p ? 0.001, respectively), and CD28 in cis resulted
in significantly higher levels of IL-2 secretion than in trans (p ? 0.001).
CD28 costimulation in trans promotes IL-2 secretion. Pre-
4779The Journal of Immunology
to the site of TCR engagement at the APC was actually diminished
(Fig. 2B). In no case was PKC? detected at the site of CD28 en-
gagement, whether the T cells were in contact with beads alone or
with both APC and beads. To confirm that trans costimulation
mediated by anti-CD28-coated beads mimicked CD28 engagement
by natural ligand, B7-positive, class II-negative L cells were used
to provide CD28 costimulation in trans. CD4?T cells costimu-
lated in trans by L cells displayed the same phenotype, as was
observed when CD28 costimulation was provided by anti-CD28-
coated beads (Fig. 2). Thus, unlike when CD28 costimulation was
provided at the same site as TCR engagement, CD28 costimulation
in trans did not direct the localization of PKC? to the cSMAC.
These results suggested that the ability of CD28 to costimulate
IL-2 expression in trans was not mediated through activation of
PKC?. To test this possibility directly, we compared the ability of
CD28 in trans to costimulate CD4 T cells from WT and PKC?-
deficient DO11.10 mice (Fig. 3A). PKC? was required for optimal
IL-2 secretion when CD28 was provided in cis, as predicted from
previous studies (24). However, T cells from WT and PKC?-de-
ficient mice induced similar levels of IL-2 secretion when CD28
was provided in trans. Similar results were obtained when CD28
costimulation in trans was provided by anti-CD28-coated beads or
A, Previously activated T cells from WT (f), PKC?-deficient (u), or
CD28-deficient (?) DO11.10 mice were unstimulated (no Ag) or stimu-
lated with Ag presented by ProAd-ICAM (Ag), by ProAd-ICAM in the
presence of either anti-CD28-coated beads (beads) or L cells, or by ProAd-
ICAM-B7 (cis). T cells were cultured for 48 h, and supernatants were
assayed for IL-2 by capture ELISA. B, Freshly isolated T cells were ana-
lyzed as in A, except that trans costimulation was only provided by anti-
CD28-coated beads. Data are the mean and SD of three independent ex-
periments (except for L cells, which were only included in two of the three
experiments). The absence of PKC? does not significantly reduce the level
of IL-2 secretion induced by CD28 costimulation in trans. In contrast,
expression of PKC? is required for the enhanced IL-2 secretion seen when
CD28 costimulation is provided in cis (p ? 0.03 in A and 0.01 in B).
CD28 costimulation in trans is largely PKC? independent.
L cells, n ? 87; and cis, n ? 156. CD28 costimulation in trans significantly
inhibits PKC? recruitment to the immunological synapse (p ? 0.001). In
contrast, costimulation in cis enhances both recruitment of PKC? to the
synapse and localization to the cSMAC (p ? 0.001).
of PKC? to the cSMAC of the immunological synapse. T cells were stim-
ulated with Ag presented by ProAd-ICAM in the absence of CD28 co-
stimulation (none), with ProAd-ICAM in the presence of CD28 costimu-
lation in trans with either anti-CD28-coated beads (beads) or B7-positive
L cells (L cells), or with ProAd-ICAM-B7 to provide CD28 costimulation
in cis (cis). T cell conjugates were stained for PKC? (green) and analyzed
by immunofluorescent microscopy. L cells were prelabeled with 7-amino-
4-chloromethylcoumarin (CMAC; blue) to distinguish them from the
ProAd-ICAM cells. A, Differential interference contrast images (DIC) and
immunofluorescent images (PKC?) are shown for representative conju-
gates. The position of T cells (T), ProAd-ICAM cells (APC), L cells (L),
and anti-CD28-coated beads (B) are overlaid on the differential interfer-
ence contrast image. Note that there are two T cells in the example with L
cells providing CD28 costimulation in trans, only one of which is in contact
with L cells. Both T cells display a diffuse pattern of PKC? localization across
the immunological synapse, unlike the focused localization of PKC? in the
cSMAC seen when CD28 costimulation is provided in cis. B and C, Individual
(B) and for localization of PKC? to the cSMAC (C). Data are shown as the
percentage and SD of conjugates displaying PKC? recruitment or cSMAC
localization, respectively. The total number of conjugates scored over two to
three separate experiments were as follows: none, n ? 223; beads, n ? 132;
CD28 costimulation in trans does not promote localization
4780CD28 COSTIMULATION IN trans
with cells expressing the natural ligand for CD28 (B7-positive L
cells). CD28-deficient T cells were included as a negative control,
as these cells are unable to respond to either cis or trans CD28
costimulation. Similar results were obtained when we tested pre-
viously activated and freshly isolated lymph node T cells (Fig. 3B).
Interestingly, the greater potency of cis costimulation compared
with trans costimulation was lost in the PKC?-deficient T cells,
suggesting that this increased potency of cis costimulation may be
dependent on CD28-mediated recruitment of PKC? to the cSMAC.
Together, these results indicate that CD28 costimulation in trans is
mediated through a PKC?-independent pathway.
CD28 costimulation in trans does not induce NF-?B nuclear
translocation or IL-2 transcription
As discussed, CD28 costimulation through PI3K and the recruit-
ment of PKC? to the cSMAC has been implicated in the ability of
CD28 to up-regulate IL-2 transcription. Because CD28 costimu-
lation in trans is independent of PKC?, it suggests that the ability
of CD28 in trans to up-regulate IL-2 secretion is not mediated
through the up-regulation of IL-2 transcription. To test the possi-
bility directly, we first assessed nuclear localization of NF-?B
(Fig. 4). CD28 costimulation in cis resulted in the induction of
nuclear localization of NF-?B. In contrast, CD28 in trans had little
effect on NF-?B activation. Similar results were obtained when
nuclear localization of endogenous NF-?B was monitored by im-
munofluorescent staining (Fig. 4, A and B) or when an NF-?B
p65-GFP fusion protein was retrovirally transduced to follow
NF-?B activation (Fig. 4C). To assay, IL-2 transcription directly
we used DO11.10 T cells that contain a transgenic IL-2-luciferase
reporter construct (38). We have previously established that this
transgene is a good indicator of endogenous IL-2 transcriptional
activity (37). As expected from previous studies, cis costimulation
resulted in transcriptional activation of the IL-2 enhancer (26). In
contrast, only a modest increase in IL-2 transcriptional activation
was detected with trans costimulation (Fig. 5). Collectively, these
data indicate that CD28 engagement outside of the immunological
synapse does not result in PKC?-dependent activation of NF-?B
and subsequent up-regulation of IL-2 transcription.
CD28 costimulation in trans can increase the stability of IL-2
The ability of CD28 costimulation in trans to up-regulate IL-2
secretion, but not IL-2 transcription, indicates that trans costimu-
lation must be mediated through posttranscriptional mechanisms.
IL-2 can be regulated at several posttranscriptional events, includ-
ing mRNA elongation, mRNA stability, translation, and secretion
(7, 8, 40–42), but the best-described effect of CD28 costimulation
is on mRNA stability. To determine the half-life of IL-2 mRNA, T
cells were stimulated for 4 h, transcription was then blocked by
addition of cyclosporin A, and IL-2 mRNA levels were determined
by real-time PCR at different time points (Fig. 6). In the absence
of CD28 costimulation IL-2 mRNA rapidly decays with a half-life
of ?30 min. When costimulation is provided by CD28 in cis the
half-life increases to ?90 min. Importantly, the increase in the
half-life of IL-2 mRNA is similar when CD28 costimulation is
provided in cis or in trans. Thus, engagement of CD28 outside of
the immunological synapse is not sufficient to induce up-regulation
of IL-2 transcription, but is able to transduce signals necessary for
induction of IL-2 mRNA stability.
?B. A, T cell conjugates with ProAd-ICAM (none), with ProAd-ICAM and
anti-CD28-coated beads (trans), or ProAd-ICAM-B7 (cis) were stained for
p65 NF-?B (red). Nuclei were stained with Hoechst dye (blue). Examples
of individual conjugates are shown. The T cell is the smaller cell orientated
toward the left in each panel. Nuclear localization of NF-?B is detected by
overlay of red p65 staining on the blue Hoechst stained nuclei. The APC
on the right have constitutively active NF-?B. B, The percentage and SD
of conjugates (n ? 40–60 conjugates) displaying nuclear localization of
NF-?B is shown. CD28 costimulation in cis, but not in trans, results in a
significant increase in activation of NF-?B (p ? 0.001). C, Conjugates
were analyzed as described in A, except that T cells expressing a p65-GFP
fusion protein were used to localize NF-?B. The T cell is the smaller cell
orientated toward the lower left in each panel. The localization of the p65-
GFP chimera is easier to discern because there is no staining in the APC.
CD28 in trans does not induce nuclear localization of NF-
viously activated T cells from DO11.10 mice carrying the luciferase gene
under the control of the IL-2 enhancer were unstimulated (?Ag) or stim-
ulated with Ag (?) presented by ProAd-ICAM alone (?CD28), by ProAd-
ICAM in the presence of anti-CD28-coated beads (trans), or by ProAd-
ICAM-B7 (cis). T cells were cultured for 24 h, then lysed, and luciferase
activity was measured in the supernatants. Luciferase activity is shown as
the mean and SD of the fold induction above unstimulated T cells from
three independent experiments. CD28 costimulation in trans results in a
marginal increase in IL-2-luciferase expression (p ? 0.04 in a one-tailed t
test), whereas CD28 in cis results in a dramatic increase in IL-2-luciferase
expression compared with both no CD28 (p ? 0.007) and CD28 in trans
(p ? 0.005).
CD28 in trans does not up-regulate IL-2 transcription. Pre-
4781The Journal of Immunology
We have recently proposed that there are two distinct pathways for
CD28-mediated costimulation (26). The first is mediated through
recruitment of PI3K to the cSMAC of the immunological synapse
and results in the activation of PKC?, nuclear translocation of NF-
?B, and up-regulation of IL-2 transcription. Costimulation through
this pathway may reflect integration of plasma membrane-proxi-
mal signals mediated through TCR and CD28 within the context of
the immunological synapse. The second pathway of CD28 co-
stimulation is not affected by mutation of the PI3K interaction site
in CD28 and drives IL-2 production through stabilization of IL-2
mRNA. In this study, we show that when CD28 is engaged outside
of the immunological synapse, only the second pathway of CD28
costimulation is induced. Thus, CD28-mediated up-regulation of
IL-2 transcription requires membrane colocalization with TCR,
possibly within the cSMAC of the immunological synapse. In con-
trast, CD28 signals that mediate IL-2 mRNA stability do not need
to be colocalized with TCR, supporting the model that costimula-
tion through this pathway may reflect signal integration down-
stream of the plasma membrane.
In addition to providing costimulation, there is evidence that
CD28 can activate T cells in the absence of TCR engagement.
Early studies showed that anti-CD28 cross-linking could induce a
calcium response and anti-CD28 in combination with phorbol es-
ters could induce IL-2 secretion and T cell proliferation (43, 44).
More recently, it has been shown that CD28 cross-linking, in the
absence of TCR engagement, can be sufficient to activate Vav and
SLP-76 (45). However, these effects of anti-CD28 cross-linking
can be restricted to certain cells and mAb epitopes. Recent data has
provided some insight into this issue (46, 47). There are at least
two major mAb epitopes expressed on CD28. Conventional Ab
epitopes are localized near the B7 binding site and are thought to
mimic natural ligand binding. The 37.51 mAb used in the present
studies is a conventional Ab. Superagonists Ab epitopes have been
mapped to a site that is proximal to the membrane and are func-
tionally mitogenic, inducing IL-2 secretion and T cell prolifera-
tion. Interestingly, T cell activation by superagonist Abs can be
mediated through PKC? and NF-?B and up-regulation of IL-2
transcription (48). This suggests that CD28 is capable of indepen-
dently activating T cells, but it is not clear whether these same
signaling pathways mediate CD28 costimulation of TCR-derived
signals. Furthermore, the relationship between CD28 engagement
by superagonist Abs and by natural ligand binding is not under-
stood. In the experiments reported, we show that trans costimula-
tion using a conventional mAb or natural ligand is not mediated
through PKC? and NF-?B, but rather enhances IL-2 secretion
through mRNA stability.
CD28 has been shown to function as a signal amplifier for TCR-
transduced signals (1). Thus, the current model would suggest that
colocalization of TCR and CD28 during cis costimulation would
facilitate CD28 amplification of proximal TCR signals. The re-
quirement for CD28-mediated activation of PI3K is consistent
with this model because the product of PI3K, PIP3, would enhance
the recruitment of PH domain-containing proteins, such as VAV,
Itk, Akt, and PDK1, to the site of activation. In this case, colocal-
ization of TCR and CD28 would create a signaling subdomain
within the immunological synapse. However, Hu ¨nig and col-
leagues (47–49) have suggested an additional role for TCR/CD28
colocalization. They have shown that expression of the superago-
nist epitope on CD28 is enhanced following T cell activation, and
activated T cells are more responsive to activation by superagonist
anti-CD28 mAb. Thus, TCR signaling may induce a conforma-
tional change in CD28, which reveals the mitogenic Ab epitope
and potentiates CD28 signaling. In this case, localization of CD28
to the site of TCR engagement may promote CD28 activation. This
activity could be mediated by CD28 association with lipid raft
domains and/or by access of CD28 to Lck. Lck binding to the
polyproline rich region has been proposed to be the first step in
CD28 signaling. Lck is then thought to phosphorylate Y170 on the
CD28 cytosolic tail creating the binding site for the Src homology
2 domain of PI3K (50, 51). Thus, TCR/CD28 colocalization within
the cSMAC may have a dual function, first in the activation of
CD28 and subsequently in the signal integration of TCR- and
In addition to enhancing proximal TCR-derived signals, CD28
signaling can also induce mRNA stability. The regulation of
mRNA stability is largely controlled by AU-rich elements (ARE)
within the 3? untranslated region (UTR). ARE-mediated mRNA
degradation plays an important role in regulation of many genes
(52, 53), including cytokines (8). The current model for regulated
mRNA stability is that AU-binding proteins that induce mRNA
instability, such as tristetraprolin (TTP), bind to the 3? UTR in
unstimulated cells. TTP recruits the multicomponent exosome, al-
lowing for deadenylation and 3? exonuclease digestion of the
mRNA (54, 55). In the absence of ARE-mediated mRNA degra-
dation, either by genetic disruption of TTP expression (56) or the
deletion of the ARE from TNF (57), overexpression of TNF results
in the induction of autoimmune inflammatory diseases. The sta-
bility of ARE-containing mRNAs can also be enhanced during cell
activation events, although the mechanisms that mediate this sta-
bilization are not well understood. One model that has been pro-
posed is that cell signaling induces the recruitment of different
AU-binding proteins, such as HuR, that may compete with TTP for
binding to the 3? UTR and, thus, interfere with TTP-dependent
recruitment of the exosome. T cell activation leads to an increase
in expression of TTP and HuR, and TTP can bind to the AU-rich
region in the IL-2 3? UTR and drive IL-2 mRNA degradation (58,
59). However, HuR does not recognize the specific AU-rich region
in the IL-2 mRNA and another AU-binding protein, NF90, that can
compete with TTP binding, has been implicated in signal-depen-
dent IL-2 mRNA stabilization (60). Access of HuR and NF90 to
target mRNA may be regulated by shuttling these nuclear proteins
to the cytosol, and this process could be mediated by signal-de-
pendent association of HuR with nuclear shuttle proteins (60, 61).
mRNA stability. Previously activated T cells were stimulated with Ag pre-
sented by ProAd-ICAM (no CD28), by ProAd-ICAM with anti-CD28-
coated beads (trans), or by ProAd-ICAM-B7 (cis) for 4 h. IL-2 transcrip-
tion was blocked by the addition of cyclosporin A. Cyclosporin A rapidly
inhibits NFAT-dependent IL-2 transcription without affecting IL-2 mRNA
stability (37, 62, 74). Levels of IL-2 mRNA were measured every hour by
real-time PCR and shown as a percentage of IL-2 mRNA before the ad-
dition of cyclosporin A. The fold induction of IL-2 mRNA after the initial
4 h of activation was 3200 (no CD28), 5600 (trans), and 9800 (cis). This
experiment is one representative experiment of two completed.
Costimulation by CD28 in trans is mediated through IL-2
4782 CD28 COSTIMULATION IN trans
MAPK activation has been implicated in the induction of mRNA
stability. JNK can induce IL-2 and IL-3 mRNA stability (62–64);
p38 has been implicated in the stabilization of IL-2, IL-6, IL-8, and
TNF-? mRNA (65–67); and ERK can stabilize COX-2 mRNA in
smooth muscle cells. Although there is some evidence of signal-
dependent phosphorylation of AU-binding proteins, the exact role
for these phosphorylation events in mediating mRNA stability is
not clear (65, 68). Importantly, the proximal signals that are in-
duced uniquely by CD28 to up-regulate mRNA stability have not
Trans costimulation has been proposed to play a role in immune
responses to nonhemopoietic cells, such as in tumor and allograft
rejection and autoimmunity. Using genetically deficient cells that
eliminate the possibility of cis costimulation, it was directly dem-
onstrated that trans costimulation could result in rejection of car-
diac allografts (34). Nevertheless, any function of trans costimu-
lation in vivo would be limited by two key factors. First, it requires
separate interactions of a T cell with an Ag-bearing target cell and
a non-Ag-bearing, B7-positive cell. Second, we show that trans
costimulation does not induce a full component of CD28-mediated
signals. Trans costimulation does not result in enhanced transcrip-
tional activation and may be limited to the induction of mRNA
stability. This finding could still play a key immunoregulatory role,
not only to enhance IL-2 expression, but also to promote secretion
of other proinflammatory cytokines, in particular TNF-? and
IFN-?. Furthermore, the CD28 signaling pathway induced by trans
costimulation may be sufficient for other CD28-mediated immune
functions. Although it is not yet clear how many different signaling
pathways are induced by CD28 (69, 70), as we discussed the PI3K
pathway may be the major pathway associated with cis costimu-
lation. In vivo reconstitution of CD28-deficient mice with CD28
mutants that cannot activate the PI3K pathway (Y170F) restores
many, but not all, CD28-dependent functions. There are notable
defects in up-regulation of Bcl-xL, radiation resistance, and graft-
versus-host disease; however, T cell activation, IL-2 production,
and proliferation are largely intact in mice expressing the Y170F
mutation (71–73). In addition these mice generate WT levels of
Th2 and T cell-dependent B cell responses, and normal numbers of
CD25?regulatory T cells (69, 70). Whether all of these functions
can be induced by trans costimulation or are mediated through the
regulation of mRNA stability will require additional investigation.
We thank Dan Littman (New York University, New York, NY) for pro-
viding the PKC?-deficient mice, Jeff Hanke (Pfizer, Groton, CT) for IL-
2-LUC mice, and Ranjan Sen (Brandeis University, Waltham, MA) for
providing the p65-GFP construct.
The authors have no financial conflict of interest.
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