International Immunology, Vol. 25, No. 12, pp. 671–681
doi:10.1093/intimm/dxt028
Advance Access publication 18 September 2013
© The Japanese Society for Immunology. 2013. All rights reserved.
For permissions, please e-mail: [email protected]
CD28 signaling in primary CD4+ T cells: identification
of both tyrosine phosphorylation-dependent and
phosphorylation-independent pathways
Shuhei Ogawa1,2, Masashi Watanabe3, Yuichi Sakurai4, Yu Inutake4, Shiho Watanabe1, Xuguang Tai3
and Ryo Abe1,2,4
Research Institute for Biomedical Sciences, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan
Center for Technologies against Cancer, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan
3
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
4
Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan
1
2
Correspondence to: R. Abe; E-mail: [email protected]
Received 24 September 2012, accepted 14 May 2013
Abstract
In addition to TCR signaling, the activation and proliferation of naive T cells require CD28-mediated
co-stimulation. Once engaged, CD28 is phosphorylated and can then activate signaling pathways
by recruiting molecules to its YMNM motif and two PxxP motifs. In this study, we analyzed the
relationship between tyrosine phosphorylation and the co-stimulatory function of CD28 in murine
primary CD4+ T cells. Tyrosine phosphorylation is decreased in CD28 where the N-terminal PxxP
motif is mutated (nPA). In cells expressing nPA, activation of Akt and functional co-stimulation
were decreased. In contrast, where the C-terminal PxxP motif is mutated, tyrosine phosphorylation
and activation of the ERK, Akt and NF-κB were intact, but proliferation and IL-2 production were
decreased. Using the Y189 to F mutant, we also demonstrated that in naive CD4+ T cells, tyrosine
at position 189 in the YMNM motif is critical for both tyrosine phosphorylation and the functional
co-stimulatory effects of CD28. This mutation did not affect unfractionated T-cell populations.
Overall, our data suggest that CD28 signaling uses tyrosine phosphorylation-dependent and
phosphorylation-independent pathways.
Keywords: CD28, co-stimulation, phosphorylation, T-cell activation
Introduction
Two signals are required for full activation and differentiation of
naive T cells. The first involves antigen-specific TCR signaling,
while the second is antigen-non-specific and requires signaling through co-stimulatory receptors. In naive T cells, TCR
recognition of MHC peptide expressed by antigen-presenting
cells (APCs) is insufficient to induce full activation and results
in an unresponsive state known as anergy (1, 2). The bestcharacterized co-stimulatory system is that mediated by the
interaction of CD28 on T cells with CD80 and CD86 on APCs.
In naive T cells, signaling through TCR and CD28 induces proliferation, IL-2 production and protects from apoptosis (3–5).
The CD28 molecule contains a short cytoplasmic region
composed of four tyrosine residues, as well as YMNM, and
two PxxP motifs as functional domains (6). Upon CD28
engagement, protein tyrosine kinases (PTKs) are recruited
to the CD28 cytoplasmic tail where they then phosphorylate
CD28 (7–10). Next, CD28 recruits several adaptor proteins,
including the p85 regulatory subunit of PI3K, Grb2 and Gads,
to its YMNM motif in a phosphorylation-dependent manner (9,
11–14). In turn, these interactions activate MAPK, Akt, NFAT
and NF-κB, leading to T-cell activation (6).
The C-terminal region of CD28 also contains two prolinerich segments that are required for phosphorylation by tyrosine kinases and essential for CD28’s co-stimulatory function
(15–19). This region, and especially the portion containing
the C-terminal PxxP motif, could bind PTKs through their
SH3 domains (7, 20, 21). The tyrosine kinase Lck is thought
to phosphorylate CD28 at Y189MNM and PY207AP (7, 9, 22).
Mutagenesis has shown that the C-terminal PxxP motif is
important in regulating CD28-dependent T-cell responses,
which include cytokine production, proliferation, regulatory
T-cell development, antibody production, allergy and autoimmune disease (17–19). The N-terminal PxxP motif binds the
SH3 domains of Itk and Tec (9, 20).
Despite much study, the detailed mechanisms of CD28
signaling remain unclear (7–9, 23–26). PI3K activity and PIP3
672 Tyrosine phosphorylation and co-stimulation of CD28
have received much attention as downstream messengers
(27–30). CD28-dependent PI3K activity helps recruit
molecules such as phosphoinosotide-dependent kinase 1
(PDK1), Akt, Itk and Vav, that contain a PH domain, which
then recruits PKCθ to the membrane (29, 31–33). Grb2 family
molecules are also involved. We previously demonstrated
that the association of Gads with CD28 is critical for CD28dependent NF-κB promoter activity, since it induces the
formation of CARMA1–Bcl-10–Malt1 complexes (13, 34,
35). In addition, the association of Grb2 but not Gads with
CD28 can activate NFAT (36). These and other studies have
focused on the YMNM motif, which may recruit PI3K and
Grb2/Gads. However, results from mutant CD28-expressing
cells have been controversial (11, 15–18, 28, 37–42).
Several studies concluded that the YMNM motif is important
for CD28 activation of signaling pathways (26, 34, 43–45),
co-localization of PKCθ with CD28 (28, 30, 46, 47), the
induction of Bcl-xL expression (15, 38, 42), T-cell migration
(40), early IL-2 production and the induction of acute GVH
disease (GVHD) (37). However, other studies concluded that
the YMNM motif plays only a limited role in regulating CD28
co-stimulation (15, 16, 38, 48, 49). Recently, a study of Y189F
mutant knock-in mice concluded that T-cell proliferation and
cytokine production were normal even though phosphorylation
of Akt was diminished (48).
We have now generated transgenic mice expressing a
series of mutant CD28 molecules in order to define the structural requirements for the specific domains needed for tyrosine phosphorylation of CD28 and for co-stimulatory signaling
in primary T cells. Our findings show that both the N-terminal
PxxP motif and Y189 in the YMNM motifs are critical for inducing phosphorylation. Such phosphorylation is required for
the proliferation of naive CD4+ T cells, but not unfractionated
T cells. Surprisingly, the C-terminal PxxP mutant showed
intact tyrosine phosphorylation, and activation of ERK,
Akt and NF-κB pathways, but impaired CD28-dependent
T-cell proliferation and IL-2 production. These data suggest that CD28-mediated co-stimulatory signaling occurs
via both phosphorylation-dependent and phosphorylation-­
independent mechanisms.
Methods
Construction of transgenes and generation of
transgenic mice
Six CD28 transgenic strains were used: wild-type (WT), YF
(the YF strain has Y189 in YMNM mutated to F), nPA (nPA
has the N-terminal PxxP site mutated to AxxA), cPA (cPA has
the C-terminal PxxP site mutated to AxxA), ncPA (ncPA has
both the PxxP motifs mutated to AxxA) and TM (TM lacks
the cytoplasmic portion). Murine CD28 cDNA was kindly
donated by K. Lee (University of Miami, Miami, FL, USA). It
was sub-cloned into pBluescript (Stratagene, La Jolla, CA,
USA). Mutant CD28 constructs were generated by oligonucleotide-directed site-specific mutagenesis and verified by
sequencing. WT, YF and TM constructs were sub-cloned into
a human CD2 expression cassette and introduced into a fertilized mouse embryo [(BDF1 × B6.CD28KO)F1]. Additional
lines of transgenic mice expressing nPA, cPA and ncPA
CD28 were generously provided by Dr Alfred Singer. Each
transgene-expressing mouse line was backcrossed with
B6.CD28KO mice at least 10 times. All animals were housed
in a dedicated pathogen-free facility at the Tokyo University
of Science. All experiments followed the guidelines of the
University’s Animal Care and Use Committee.
Immunoprecipitation and blotting for phosphorylated
tyrosine
For immunoprecipitation, 3 × 107 pre-activated CD4+ T
cells from mutant mice were stimulated with anti-CD28
mAb (5 µg ml−1) followed by anti-hamster IgG (Jackson
ImmunoResearch, #127-005-099) (15 µg ml−1) for 5 min at
37°C. Cells were then washed thoroughly with PBS and lysed
with RIPA buffer (50 mM Tris–HCl pH 7.4, 1% Nonidet P-40,
0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride, 1 mM Na3VO4 and 1 mM NaF)
for 1 h at 4°C. Lysates were centrifuged at 14 000 × g for
10 min at 4°C and then incubated with anti-CD28 mAb-conjugated protein A-Sepharose beads (GE Healthcare, 17-528004) at 4°C for 1 h. Beads were washed with RIPA buffer and
pelleted by centrifugation. Proteins were released by incubation with 5× sample buffer, resolved on 12.5% polyacrylamide
gels and transferred to polyvinylidene difluoride membranes.
Membranes were incubated with 5% BSA in Tris-buffered
saline–Tween buffer for 1 h. Biotinylated anti-phosphotyrosine
antibodies (4G10) were added for 1 h; the membranes were
then washed and incubated with HRP-conjugated streptavidin
(Sigma, A3151) for 1 h. The signal was detected by enhanced
chemiluminescence. Blots were stripped with stripping buffer
(Pierce, #21059) and then reprobed with anti-CD28 or antip85 mAbs and HRP-conjugated donkey anti-rabbit IgG.
Intracellular staining to detect phosphorylated ERK and Akt
CD4+ T cells were stimulated by plate-bound anti-TCR mAb
(5 µg ml−1) for 12 h and then cultured with human recombinant
IL-2 (25 units ml−1) for 3 days. Cells were harvested, washed
and re-cultured with IL-2 for an additional 3 days. At day 6,
CD4+ T cells were harvested and treated with anti-CD3ε mAb
and/or anti-CD28 mAb for 20 min on ice. Cells were centrifuged and cross-linked with anti-hamster IgG (15 µg ml−1) for
various times at 37°C. Reactions were stopped by the addition of cold PBS containing 1 mM EDTA and 1 mM Na3VO4.
After centrifugation, cells were fixed with 4% paraformaldehyde at 37°C for 10 min, permeabilized with 90% methanol
on ice for 30 min and then stained with anti-phospho-ERK or
anti-phospho-Akt (Ser473) mAbs (Cell Signaling, #4377 and
#4058, respectively). Staining was visualized by incubating
cells with Alexa 488-conjugated anti-rabbit IgG (Invitrogen,
A-11008) and was measured by flow cytometry.
Preparation of CD44low naive CD4+ T cells
Enriched T cells were prepared by incubating spleen and
lymph node cells on plates coated with anti-mouse MHC
class II mAb for 30–60 min at 4°C and then collecting the nonadherent cells. CD4+ T cells were purified by positive panning
on plates coated with anti-CD4 mAb. Naive CD4+ T cells were
enriched for CD44low by negative selection with anti-CD44conjugated magnetic beads (Qiagen, 310104). The purity of
CD44low CD4+ T cells from all mutant Tgs was found to be
Tyrosine phosphorylation and co-stimulation of CD28 673
>93% (Supplementary Figure 1, available at International
Immunology Online).
EMSA
CD44low naive CD4+ T cells were stimulated with antiCD3ε mAb (2 µg ml−1) in the presence or absence of antiCD28 mAb (5 µg ml−1) for 24 h. After stimulation, nuclear
extracts were prepared (Sigma, NXTRAXT). The extracts
(1 µg) were incubated with a 32P-labeled NF-κB probe
(5′-AGATGAGGGGACTTTCCCAGGC-3′) for 30 min at room
temperature and then subjected to electrophoresis in a 5%
polyacrylamide gel followed by autoradiography.
In vitro proliferation assay and measurement of IL-2
CD44low naive CD4+ T cells were stimulated with plate-bound
anti-CD3ε mAb or PMA plus soluble anti-CD28 mAb. Cultures
were pulsed with tritiated thymidine for the final 8–12 h of
culture. Supernatants were collected before pulsing with
tritiated thymidine, and IL-2 concentrations were analyzed
by ELISA according to the manufacturer’s instructions
(PharMingen, San Diego, CA, USA)
Statistical methods
Analyses used an unpaired two-tailed Student’s t-test.
Values of P < 0.05 were considered to indicate statistical
significance.
Results
The N-terminal PxxP motif and Y189 are critical for tyrosine
phosphorylation of CD28
In response to CD28 ligation, phosphorylation of tyrosine
residues of CD28 has been considered to play a key role in
delivering specific intracellular signals by recruiting various
molecules to its SH2 domain. However, the regulation and
effects of tyrosine phosphorylation of CD28 remain controversial (7–10, 22, 24). To investigate how CD28’s different
domains contribute to phosphorylation, and co-stimulation
in primary CD4+ T cells, we generated transgenic mice with
mutant CD28 (Fig. 1). Cell surface expression of transgenic
CD28 in the different mouse lines is shown in Fig. 1B. These
mutant mice were healthy and their phenotype was normal.
CD4+ T cells from WT and mutant mice were stimulated with
anti-CD28 mAb and anti-hamster IgG secondary antibody,
and then tyrosine phosphorylation of CD28 and its interaction with p85 were examined by western blotting. Tyrosine
phosphorylation of CD28 was induced by CD28 stimulation
in T cells from WT transgenic mice (Fig. 2). Phosphorylation
was not augmented by co-cross-linking with TCR (data not
shown). As shown in Fig. 2, compared with WT CD28, the
N-terminal PxxP mutant (nPA) showed weak phosphorylation, whereas the C-terminal PxxP mutant (cPA) showed
strong phosphorylation even though it expressed slightly less
CD28. If both the N- and C-terminal PxxP motifs were mutated
(ncPA), phosphorylation was weak.
Fig. 1. Generation of CD28 transgenic mice. (A) Amino acid sequences of WT or mutant CD28 and their associated molecules. These CD28
transgenes were expressed under the control of the human CD2 promoter and crossed onto a CD28−/− background. (B) Expression of CD28 on
CD4+ T cells from transgenic mice (solid line). Cells were stained with allophycocyanin-conjugated anti-CD4 mAb and biotinylated anti-CD28
mAb followed by PE-conjugated streptavidin. The filled histogram indicates cells from a CD28−/− mouse. The dotted line indicates cells from a
transgenic mouse with WT CD28. Numbers represent mean fluorescence intensity.
674 Tyrosine phosphorylation and co-stimulation of CD28
Fig. 2. Tyrosine phosphorylation of mutant CD28. CD4+ T cells from mutant CD28 transgenic mice were stimulated with anti-TCR mAb (5 µg
ml−1) and IL-2 (25 units ml−1). At 3 days after stimulation, the cells were washed and re-cultured with IL-2 (25 units ml−1) for an additional 3 days.
At day 6, cells were untreated or treated with anti-CD28 mAb (10 µg ml−1) on ice, centrifuged and cross-linked with anti-hamster IgG antibody
(15 µg ml−1) for 5 min at 37°C. Immunoprecipitates of CD28 were analyzed by immunoblotting with anti-phosphotyrosine and reprobed with
anti-CD28 and anti-p85. Numbers show band intensities of CD28 phosphotyrosine and p85 relative to intensities seen in CD28-WT mice as
calculated with ImageGauge (FUJIFILM co.). The data represent six independent experiments.
Fig. 3. Activation of MAPK and Akt after CD28 co-stimulation. Pre-activated CD4+ T cells from mutant CD28 mice were incubated in medium alone
(dotted line), anti-CD3ε mAb (gray filled) and anti-CD3ε plus anti-CD28 mAbs (solid line) on ice for 20 min and then cross-linked with anti-hamster
IgG for 2 or 5 min. After fixation, permeablized cells were stained with anti-phospho-ERK (A) or anti-phospho-Akt (B) followed by Alexa 488-conjugated anti-rabbit IgG. Numbers represent mean fluorescence intensity of pERK (A) and pAkt (B). Data represent three independent experiments.
It has been well documented that CD28 ligation induces the
association of the p85 regulatory subunit of PI3K through its
SH2 domain in a tyrosine phosphorylation-dependent manner. Accordingly, the association of p85 with mutant CD28
was examined. The nPA mutant exhibited a weak association
with p85, whereas association remained strong in the cPA
mutant. These results are consistent with the idea that the
N-terminal PxxP motif is important for CD28 tyrosine phosphorylation and that the C-terminal PxxP motif has a minimal
effect. Similar results were seen with stimulation by natural
ligands B7-1 and/or B7-2 (data not shown).
We next studied Y189 in the YMNM motif by using transgenic
mice with Y mutated to F (YF strain). As shown in Fig. 2, neither tyrosine phosphorylation of CD28 nor association with
p85 was observed, although expression of CD28 on T cells
was the highest among all mice studied. This suggested that
Y189 could be the only phosphorylation site in CD28 or that
its phosphorylation is critical for phosphorylation elsewhere.
In CD28 mutant CD4+ T cells, activation of ERK, Akt and
NF-κB signaling pathways correlates with CD28 tyrosine
phosphorylation
It is generally accepted that after CD28 ligation, tyrosine
phosphorylation leads to the recruitment of signaling molecules that activate specific signaling pathways leading to
gene transcription and full T-cell activation. Furthermore, it is
well documented that MAPK, Akt and NF-κB pathways are
involved in CD28-mediated co-stimulation upon engagement
of TCR and CD28. We next examined, therefore, the relationship between tyrosine phosphorylation of CD28 and activation of cytoplasmic signaling molecules (Figs 3 and 4).
Tyrosine phosphorylation and co-stimulation of CD28 675
not shown). The co-stimulatory effect on NF-κB activation was
almost absent in CD4+ T cells from ncPA and YF mutant mice.
These results suggest that NF-κB activation also correlates
with the phosphorylation of Y189 in YMNM (Figs 2 and 4).
Fig. 4. NF-κB activation in naive CD4+ T cells. Naive CD4+ T cells
from CD28 transgenic mice were stimulated with either plate-bound
anti-CD3ε (2 µg ml−1) alone or plate-bound anti-CD3ε (2 µg ml−1) plus
soluble anti-CD28 (5 µg ml−1) mAbs for 24 h. Nuclear extracts were
subjected to EMSA with a probe specific for the NF-κB binding site
of the IL-2 promoter region. The band specifically disappeared in
the presence of excess cold probe. Band intensities were calculated
with ImageGauge (FUJIFILM co.) and are shown as a ratio for the two
treatments (anti-CD3ε + anti-CD28):(anti-CD3ε alone). Results represent four independent experiments.
We initially considered if mutations of the N-terminal or
C-terminal PxxP motifs would affect Akt and ERK phosphorylation. CD4+ T cells expressing WT CD28 showed increased
phosphorylation of ERK and Akt when stimulated with antiCD3ε and anti-CD28 mAbs, and cPA transgenic mice
showed comparable results (Fig. 3A and B). These results
demonstrated that the C-terminal PxxP motif is not required
for the phosphorylation of ERK or Akt. In contrast, the nPA
mutant showed slightly less phospho-ERK and significantly
less phospho-Akt. Activation of ERK and Akt correlated with
tyrosine phosphorylation of CD28.
Since both tyrosine phosphorylation and the association
with p85 were abrogated in CD28 YF mutant CD4+ T cells,
we next investigated the role of the YMNM motif on phosphorylation of ERK and Akt. We found this phosphorylation to be
absent in T cells from either YF or TM mutant mice. Taken
together, these results suggest that the induction of ERK and
Akt phosphorylation following CD28 ligation correlates with
phosphorylation within Y189MNM. These results are consistent with the idea that the N-terminal PxxP motif, but not the
C-terminal PxxP motif, plays a critical role in the activation of
ERK and Akt after CD28 ligation and that binding of YMMN to
Grb2/Gads and to p85 forms bridges to activate ERK and Akt,
respectively.
The translocation of NFAT, NF-κB and AP-1 to the nucleus
is a key event in CD28-mediated co-stimulation. Moreover,
NF-κB activation is essential for CD28-dependent IL-2
production (35). We next examined the contribution of each
cytoplasmic motif to NF-κB activation (Fig. 4). Purified naive
CD4+ T cells from each type of mutant mouse were stimulated
with plate-bound anti-CD3ε mAb in the presence or absence
of the soluble form of anti-CD28 mAb. Activation of NF-κB
was examined by EMSA using a probe specific for the NF-κB
binding site in the IL-2 promoter. Strong NF-κB activation
was observed when WT CD4+ T cells were stimulated with
anti-CD3ε plus anti-CD28 mAbs. The co-stimulatory effect on
NF-κB activation was comparable to that in naive CD4+ T cells
from WT and from the cPA mutant. On the other hand, the
nPA mutant showed a modest reduction in NF-κB activation.
When the CD28RE/AP-1 site, another NF-κB binding site
within the IL-2 promoter (34, 50), was used, the cPA mutant
did not show a significant defect in NF-κB activation (data
Both phosphorylation-dependent and -independent
pathways are required for CD28-dependent proliferation
and IL-2 production by naive CD4+ T cells
The experiments above demonstrated a correlation between
tyrosine phosphorylation and activation of ERK, Akt and NF-κB.
We next considered if the amount of tyrosine phosphorylation
influences CD28-dependent T-cell proliferation and IL-2
production. As can be seen in Fig. 5, mutations in the Nor C-terminal PxxP motifs impaired proliferation and IL-2
production by naive CD4+ T cells stimulated with anti-CD3ε plus
anti-CD28 (Fig. 5A) or with PMA plus anti-CD28 (Fig. 5B). CD4+
T cells with mutations in both PxxP motifs showed a significantly
decreased co-stimulatory effect on proliferation and on IL-2
production. The YF mutation also substantially impaired the
co-stimulatory function of CD28, as assessed by proliferation
and by IL-2 production (Fig. 5A, B and D). As described
above (Fig. 2), in naive CD4+ T cells, not only the nPA and
YF mutants (which had diminished tyrosine phosphorylation)
but also the cPA mutant (which had normal phosphorylation)
had weaker CD28-dependent T-cell responses. These results
indicate that in naive T cells, the induction of co-stimulation
by CD28 depends on phosphorylation-dependent and
phosphorylation-independent pathways.
The CD28-mediated tyrosine phosphorylation-dependent
pathway is critical for proliferation of naive CD4+ but not
unseparated T cells
In the above, we concluded that in naive CD4+ T cells, Y189
is necessary for tyrosine phosphorylation of CD28, for the
activation of several signaling pathways, for co-stimulationinduced proliferation and for IL-2 production. However, several other groups have reported that Y189 was not necessary
for these co-stimulation-induced activities (15–18, 38, 39). In
those studies, the authors used unfractionated T cells or retrovirally transduced CD4+ T cells, which contain memory and
effector T cells. As these different types of cells have different
requirements for activation, we tested whether cell type (unfractionated or naive to be more specific) influences the requirement for CD28 tyrosine phosphorylation (Fig. 6). Consistent
with other reports, when unfractionated T cells from WT, YF
or nPA mice were stimulated with PMA plus anti-CD28 mAb,
their proliferative responses were comparable (Fig. 6A). In
contrast, when CD44low naive CD4+ T cells were stimulated,
cells from YF and nPA mice showed less proliferation (Fig. 6B,
right). In cPA and ncPA mice, a weak co-stimulatory effect was
observed in CD44low naive CD4+ T cells and also in unfractionated T cells. Activated CD4+ T cells, stimulated with anti-TCR
mAb and expanded with recombinant IL-2 from Y189F mutant
CD28 Tg mice, showed co-stimulatory function comparable to
that seen in WT CD28. However, T cells from the cPA mutant,
even when activated, showed a weak proliferative response
(Supplementary Figure 2, available at International Immunology
Online). These results suggest that for naive T cells, tyrosine
676 Tyrosine phosphorylation and co-stimulation of CD28
Fig. 5. Two PxxP motifs and a YMNM motif are important for proliferation and IL-2 production of naive CD4+ T cells. CD44low naive CD4+ T cells
were stimulated with plate-bound anti-CD3ε mAb (1 µg ml−1) (A) or PMA (10 ng ml−1) (B) in the absence (open) or presence (filled) of soluble
anti-CD28 mAb (0.8 µg ml−1: gray; 5 µg ml−1: black). Cell proliferation was measured by [3H]thymidine incorporation at 30 h. (C) Proliferative
response of naive CD4+ T cells from mutant CD28 transgenic mice after 30-h stimulation with PMA (10 ng ml−1) plus ionomycin (400 ng ml−1). (D)
Naive CD4+ T cells were stimulated as in (A), and at 64 h after stimulation, IL-2 production was determined by ELISA. Data are averages from
three individual mice (n = 3) and represent two independent experiments. An unpaired two-tailed Student’s t-test was used to compare differences with respect to WT transgenic mice. *P < 0.05 and **P < 0.01. Error bars indicate the SEM.
Tyrosine phosphorylation and co-stimulation of CD28 677
Fig. 6. Proliferative responses of unfractionated T cells and naive CD4+ T cells. Unfractionated splenic T cells (A) or CD44low naive CD4+ T cells
(B) from CD28 transgenic mice were stimulated with PMA (10 ng ml−1) in the absence (open) or presence (filled) of soluble anti-CD28 mAb
(0.25 µg ml−1). Unfractionated T cells (C) or naive CD4+ T cells (D) were stimulated with PMA (10 ng ml−1) plus ionomycin (400 ng ml−1). Cell
proliferation was measured by [3H]thymidine incorporation at 30 h. Data represent five experiments. Error bars indicate the standard deviation.
phosphorylation of CD28 is essential, whereas for unfractionated T cells, presumably including memory/effector T cells, the
phosphorylation-dependent pathway was dispensable and the
C-terminal PxxP-mediated phosphorylation-independent pathway was sufficient.
Discussion
Phosphorylation, especially tyrosine phosphorylation, is
considered to be key in signal transduction after receptor
engagement. Identification of the tyrosine residue(s) and
kinase(s) involved, as well as the underlying mechanism, is
important for a full understanding of intracellular signaling
after receptor ligation.
There are four conserved tyrosine residues in the cytoplasmic domain of CD28 as well as two PxxP motifs that can
serve as docking sites for tyrosine kinases. To the best of our
knowledge, the relationship between CD28 phosphorylation
and CD28-mediated co-stimulation has not been studied in
primary T cells. Therefore, we used primary T cells isolated
from transgenic mice expressing mutant CD28 to identify the
contributions of the YMMN motif, the N-terminal PxxP motif
and the C-terminal PxxP motif in the regulation of tyrosine
phosphorylation and the co-stimulatory function of CD28.
With mutation in the C-terminal PxxP motif, tyrosine phosphorylation was fully preserved (Fig. 2), but IL-2 production
and proliferation of T cells were severely diminished (Fig. 5).
These results indicate that CD28-mediated co-stimulation
can occur without CD28 tyrosine phosphorylation. On the
other hand, mutations at Y189 and in the N-terminal PxxP motif,
which prevent or weaken CD28 tyrosine phosphorylation
(Fig. 2), also diminished IL-2 production and proliferation of
naive T cells (Fig. 5). This indicates that CD28 ligation triggers
tyrosine phosphorylation-dependent and phosphorylation-­
independent pathways.
The CD28 tyrosine phosphorylation-deficient mutant YF
(and nPA) exhibited defects in CD28-mediated co-stimulation
of proliferation and IL-2 production in naive T cells but not in
unfractionated T cells, whereas the cPA mutant showed these
defects in both naive (Fig. 5) and unfractionated T-cell populations (Fig. 6). Thus, the two pathways appear to perform
different functions.
Tyrosine phosphorylation of CD28 is thought to contribute to CD28-mediated co-stimulation by activating various
678 Tyrosine phosphorylation and co-stimulation of CD28
signaling pathways. Much attention has focused on the
phosphorylation of Y189 in the YMNM motif in the cytoplasmic
domain. The binding of CD28 with its ligand B7, as well as
antibody cross-linking, recruits the p85 subunit of PI3K and
Grb2/Gads to Y189MNM (44, 45, 51) and subsequently activate Akt, NF-κB and Bcl-xL, which play critical roles in CD28mediated co-stimulation.
The mechanisms underlying CD28 tyrosine phosphorylation have not been well understood, and the kinase has not
been firmly established. Lck-deficient Jurkat cells exhibit little tyrosine phosphorylation of CD28 (25), and CD28 crosslinking in T cells leads to the association of CD28 with Lck
as well as to Lck activation (52, 53). Also, Lck could bind to
the C-terminal PxxP motif of the CD28. However, we found
that in cPA mutants, where this motif is mutated, tyrosine
phosphorylation equaled or was higher than that in WT cells.
This makes it unlikely that Lck is essential in primary CD4+T
cells. Consistent with this, we found that in this mutant, Y189
phosphorylation-dependent associations with the p85 subunit of PI3K as well as the activation of Akt were intact (Figs
2–4). This conclusion is also supported by experiments using
C-terminal PxxP mutant knock-in mice (48). Alternatively, a
recent report demonstrated that the SH2 domain of Lck binds
to a phosphorylated tyrosine residue in CD28, and this interaction led to the recruitment of PKCθ to CD28 (54). Further
experiments are needed to confirm the importance of the Lck
SH2–CD28 pY interaction in the phosphorylation of CD28 and
CD28-mediated co-stimulation.
In terms of tyrosine phosphorylation, the N-terminal PxxP
motif seems critical for the regulation of tyrosine kinase activity, because mutation of this motif caused weak phosphorylation of CD28 and association with p85 (Fig. 2). Previous
reports showed that Itk and Tec could bind to this motif (55,
56) and that Itk could phosphorylate all four tyrosine residues
of the CD28 cytoplasmic region (8), although one study demonstrated that Itk had little effect (9). We are currently examining whether Itk binds to the N-terminal PxxP motif.
Our results clearly demonstrate that C-terminal PxxP motifs
do not participate in tyrosine phosphorylation of CD28, but
evidence has been increasing that this motif is indispensable for a variety of CD28-dependent T-cell responses. Tai
et al. (17) reported that Treg-cell generation in the thymus and
induction of autoimmune disease in CTLA-4-deficient mice
(18) require the motif. Furthermore, mice with a knock-in
CD28 gene, whose C-terminal PxxP motif was mutated, had
impaired ability to form antibodies (19) and exhibited induced
experimental allergic encephalomyelitis (EAE) (48). T cells
from these mice showed a profound decrease in IL-2 secretion (19), which is consistent with our observation that mutation of the PxxP motif diminished both CD28-dependent T-cell
proliferation and IL-2 production (Fig. 5).
It has been shown that the C-terminal PxxP motif is required
for association of CD28 with Lck and subsequent activation of
Lck (10). Dodson et al. (48) demonstrated that C-terminal PxxP
motif is directly responsible for tyrosine phosphorylation of
PDK1, PKCθ and glycogen synthase kinase 3β (GSK3β) and
for threonine phosphorylation of Akt. Furthermore, Yokosuka
et al. (46) demonstrated that the co-localization of CD28 with
PKCθ decreased following deletion of the C-terminal region
of CD28. These observations suggest that the C-terminal
PxxP motif may help to recruit signaling molecules to CD28
and help them interact by forming an optimal microenvironment that might include immunological synapses and a signalsome. Further studies are needed on the mechanisms
underlying these phosphorylation-independent pathways.
In this study, we confirmed the importance of the tyrosine phosphorylation-dependent pathway. We showed that
loss of Y189 phosphorylation (by mutating Y to F), as well
as alteration of the N-terminal PxxP motif (by mutating P to
A), caused significant defects in the activation of Akt, ERK
and NF-κB. In naive CD4+ T cells, these changes reduced
CD28-dependent proliferation and IL-2 production (summarized in the Supplementary Table, available at International
Immunology Online). These results are consistent with other
reports showing that the YMNM motif is important for in vitro
T-cell activation and survival (30, 38, 44) and in vivo for the
induction of GVHD (37) and for thymic T-cell development
(57). These results suggest that both phosphorylationdependent and phosphorylation-independent pathways are
utilized, but within different immunological settings. Thus, the
factors that govern preferential usage of these two pathways
require consideration.
One factor may be differences in the type of T cell. We
demonstrated that in CD28-dependent T-cell proliferation, the
requirement for the YMNM motif differs in naive CD4+ T cells
and in unfractionated T cells. In naive CD4+ T cells, either
signaling pathway can be used. In unfractionated cells, which
include effector/memory cells, the phosphorylation-independent pathway is sufficient to promote proliferation, making
the phosphorylation-dependent pathway redundant. These
results suggest that pre-activation of T cells could alter the
microenvironment surrounding CD28, which compensates for
the tyrosine phosphorylation-dependent pathway. Consistent
with this hypothesis, we previously demonstrated that in the
YF mice, IL-2 production was reduced after 24-h stimulation
but gradually increased to that of WT transgenic mice (37).
Further experiments are needed to confirm the contribution of
the two pathways, particularly in mutant CD28 knock-in mice
whose CD28 expression is regulated by endogenous genetic
elements.
Alternatively, TCR-mediated signaling could be involved.
Sanchez-Lockhart et al. demonstrated that both TCR- and
CD28-mediated signals are required for sustained translocation of CD28 in cSMAC. Yokosuka et al. showed that a TCR
signal is required for the formation of micro-clusters, where
CD28 interacts with PKCθ. Microenvironments created by
TCR signaling could provide conditions in which molecules
binding to the C-terminal region of CD28 trigger phosphorylation-independent CD28 signaling (15, 16). If, for example,
the intracellular environment in naive T cells is not suitable
to trigger this, the phosphorylation-dependent pathway could
compensate. It should be noted that CD28 ligation, but not
TCR ligation, can induce tyrosine phosphorylation, and thus
the phosphorylation-dependent pathway would not be influenced by the strength of signal 1.
The same line of reasoning can explain the different
requirements for these two pathways in in vivo immune
responses. In germinal center formation and in EAE, the
phosphorylation-dependent pathway has been shown to
be dispensable (48). On the other hand, in an animal model
Tyrosine phosphorylation and co-stimulation of CD28 679
Fig. 7. A model for CD28-mediated co-stimulation. CD28 tyrosine phosphorylation-dependent pathway: CD28 engagement leads to the association of PTKs with the N-terminal PxxP motif (PRRP). PTKs phosphorylate tyrosine residues at position 189 within the YMNM motif. Tyrosine
phosphorylation recruits PI3K and Grb2/Gads to the YMNM motif. This subsequently activates Akt, ERK and NF-κB. CD28 tyrosine phosphorylation-independent pathway: TCR engagement recruits Lck to an immunological synapse where Lck interacts with CD28 through association
of its SH3 domain with the C-terminal PxxP motif of CD28. The activated Lck further activates other kinases. (A) In naive T cells, both pathways
are required for efficient proliferation. (B) In pre-activated T cells including effector/memory cells, the phosphorylation-dependent pathway is
not needed. CD28’s cytoplasmic tail may have become more accessible to Lck, creating a different form of signalsome with a lower threshold.
Alternatively, more signaling molecules, such as PKCθ, may be involved.
of GVH responses, either pathway can serve (37). In these
immune responses, different inflammatory environments
and T-cell populations may cause different patterns of CD28
signaling.
Finally, we propose a model for CD28-mediated costimulation (Fig. 7). In naive T cells, engagement of CD28
with B7 induces phosphorylation of the tyrosine residue at
position 189. For this to occur, the N-terminal PxxP motif is
critical. Then, PI3K and Grb2/Gads interact with CD28 in a
phosphorylation-dependent manner. The association of PI3K
and/or Grb2/Gads with CD28 activates Akt, ERK and NF-κB.
TCR engagement recruits Lck to an immunological synapse,
where Lck interacts with CD28 through association of its SH3
domain with the C-terminal PxxP motif of CD28 or its SH2
domain with the phosphorylated tyrosine (7, 58). Activated
Lck then activates other kinases, such as PDK1, GSK3β and
PKCθ (48). In other types of T cells, including effector/memory cells, which are already activated, the phosphorylationdependent pathway is not needed. In these cells, CD28’s
cytoplasmic tail may have become more accessible to Lck.
This could create a different form of signalsome, with a lower
threshold or with more signaling molecules. These two models, in unactivated T cells and activated T cells, clearly merit
further investigation.
Supplementary data
Supplementary data are available at International Immunology
Online.
Funding
Japan Society for the Promotion of Science (C; 21590541).
Acknowledgements
We thank Drs Richard Hodes, Karen Hathcock, Alfred Singer,
Hidehiro Kishimoto and Yohsuke Harada for helpful discussions.
We also thank Mr N. Ito, Ms T. Adachi, Mr Y. Koh, Ms S. Kagaya,
Mr K. China, Mr W. Izumi, Ms Y. Miura and Ms A. Katayama for
their technical assistance. The authors gratefully acknowledge the
members of Science Service, Inc. for their care of the experimental
animals.
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