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Laboratory of Lymphocyte Signaling and Development, Molecular Immunology Program, The Babraham Institute, Cambridge, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain phosphorylation and subsequent -associated protein of 70
kDa (ZAP-70) tyrosine kinase recruitment were not significantly
affected by CTLA-4 engagement. However, the association of
p56lck with ZAP-70 was inhibited following
CTLA-4 ligation, correlating with reduced actions of
p56lck in the ZAP-70 immunocomplex. Moreover,
CTLA-4 ligation caused the selective inhibition of CD3-mediated
phosphorylation of the positive regulatory ZAP-70 Y319 site. In
addition, we demonstrate protein tyrosine phosphatase activity
associated with the phosphorylated CTLA-4 cytoplasmic tail. The major
phosphatase activity was attributed to Src homology protein 2
domain-containing tyrosine phosphatase 1, a protein tyrosine
phosphatase that has been shown to be a negative regulator of multiple
signaling pathways in hemopoietic cells. Collectively, our findings
suggest that CTLA-4 can act early during the immune response to
regulate the threshold of T cell activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and
AP-1 activation (5), and inhibits cell cycle progression
(6), IL-2 secretion, and T cell proliferation
(7). In addition, CTLA-4 blockade in vivo increases T cell
responses to antigenic challenges (8), enhances T
cell-mediated tumor rejection (9), and exacerbates
autoimmune disease (10). The most compelling evidence for
a negative regulatory function for CTLA-4 has come from
CTLA-4-/- mice that develop fatal
lymphoproliferative disease, a phenotype that results from polyclonal
activation of peripheral T cells that infiltrate and cause multiorgan
destruction (11). Like CD28, CTLA-4 shares the same
ligands, namely B7-1 (CD80) and B7-2 (CD86). However, CTLA-4 binds with
an affinity 10- to 20-fold higher than that of CD28 (12).
Because CTLA-4 is not readily detectable on resting T cells and is
up-regulated after activation, there has been a prevailing idea that
CTLA-4 terminates on-going T cell responses, possibly by opposing
CD28-mediated costimulation by competing for the CD80/CD86 ligands
and/or by actively blocking CD28-induced signals (13).
However, other reports have emphasized a role for CTLA-4 during the
first 24 h of activation, suggesting that CTLA-4 may be important
for setting thresholds for T cell responses by down-regulating early
manifestations of T cell activation, including CD69 and CD25 expression
as well as IL-2 secretion (6). The molecular basis for these outcomes and the signaling mechanisms induced by CTLA-4 remain poorly understood. CTLA-4 has a 36-aa cytoplasmic tail that is 100% conserved in mammalian species. It contains two tyrosines at positions Y201 and Y218, which are present in the YVKM and YFIP motifs, respectively. In addition, it possesses one proline-rich motif that could recruit SH3 domain-containing signaling molecules, suggesting that the CTLA-4 cytoplasmic tail may play a role in signaling and be important for its function.
Phosphatidylinositol 3-kinase (14) and the protein
tyrosine phosphatase (PTPase) Src homology protein 2 domain-containing
tyrosine phosphatase 2 (SHP-2) (15) become recruited to
the phosphorylated pY201VKM motif in the CTLA-4 tail. In addition,
interaction of CTLA-4 with the TCR -chain (16) and with
the serine/threonine phosphatase PP2A (17) have been
described. However, despite the identification of associated
kinases/PTPases with CTLA-4, the mechanism of negative signaling by
CTLA-4 remains elusive. One model that explains the inhibitory function
of CTLA-4 proposes that recruitment of SHP-2 to CTLA-4 mediates
dephosphorylation of the CD3 -chains, thereby interfering with the
phosphorylation of crucial effector molecules essential for TCR
signaling (15, 16). However, this model has not been
validated in normal T cells and has been challenged by the observation
that SHP-2 acts as a positive regulator of the Ras/mitogen-activated
protein kinase pathway of the TCR signaling cascade (18).
Thus, the biochemical events that mediate CTLA-4 inhibitory function
remain controversial.
Little information is available about CTLA-4 signaling in primary human
T cells, and the present study was designed to address this question.
We found that CTLA-4 engagement inhibited TCR-stimulated events, as
evidenced by reduced protein tyrosine phosphorylation and inhibition of
ERK-1/2 activation. Analysis of proximal TCR signaling revealed that
both TCR -chain phosphorylation and -associated protein of 70 kDa
(ZAP-70) recruitment remained intact upon CTLA-4 ligation, whereas
there was a marked inhibition of the association of
p56lck tyrosine kinase with ZAP-70. Furthermore,
CTLA-4 engagement resulted in marked inhibition of CD3-induced
phosphorylation of tyrosine 319 in the interdomain B of ZAP-70. Because
this phosphorylation site constitutes a binding site for the SH2 domain
of p56lck and because
p56lck/ZAP-70 association is thought to be
critical in promoting TCR coupling to intracellular signals
(19), our findings provide a novel molecular explanation
for attenuated TCR signaling and subsequent inhibition of IL-2
production resulting from CTLA-4 engagement. The inhibitory effects of
CTLA-4 were evident in resting CD4+ T cells,
suggesting that CTLA-4 can act early during the immune response to
regulate the threshold of T cell activation. In addition,
phosphorylated versions of the cytoplasmic domain of CTLA-4 were used
as affinity reagents to screen lysates for PTPase activities. We
demonstrate in in vitro studies that SHP-1, but not SHP-2, accounts for
the major PTPase activity associated with CTLA-4 peptides containing
both the phosphorylated Y218 and the doubly phosphorylated Y201 and
Y218 motifs. Because SHP-1 has been implicated in negatively regulating
the signaling thresholds that influence TCR signaling and activation
(20, 21, 22), the potential formation of a CTLA-4/SHP-1
signaling complex may provide an explanation for CTLA-4-mediated T cell
suppression in human T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following Abs were used in this study: CD3 mAb (UCHT-1;
Serotec, Oxford, U.K.); CD28 mAb (clone 9.3; Dr. M. Glennie,
Tenovus Laboratory, Southampton, U.K.); CTLA-4 mAb (clone BI3;
Immunotech, Marseilles, France); isotype control trinitrophenol (TNP),
PE-labeled-TNP, and CTLA-4 mAbs (BD PharMingen, San Diego, CA); rabbit
antiserum to the SH2 domains of SHP-1 (Dr. J. Matthews, Cardiff
University, Cardiff, U.K.); TCR- Ab (Prof. C. Terhorst, Beth Israel
Hospital, Boston, MA); TCR- mAb (Zymed Laboratories, South San
Francisco, CA); rabbit p56lck Ab (from BD
PharMingen and Dr. L. Samelson, National Institutes of Health,
Bethesda, MD); ERK-2, SHP-1, and ZAP-70 mAbs (Transduction
Laboratories, Lexington, KY); rabbit ZAP-70 and SHP-2 Ab (Santa Cruz
Biotechnology, Santa Cruz, CA); p95vav and
phosphotyrosine 4G10 mAbs (Upstate Biotechnology, Lake Placid, NY);
phosphospecific rabbit ERK-1/2 and (Y319) ZAP-70 Abs (Cell Signaling
Technology, Beverly, MA); HRP-conjugated goat anti-mouse
(DAKO, Glostrup, Denmark); and the following mouse anti-human mAbs:
CD14, CD16, CD33, and glycophorin (Serotec); CD8 and CD11b (European
Collection of Cell Cultures, Porton Down, U.K.), and CD19
(provided by Prof. P. Beverley, Jenner Institute, Compton, U.K.).
Preparation of CD4+ T lymphocytes
CD4+ T cells were obtained from buffy coats originating from healthy donors (National Blood Transfusion Center, Brentwood, U.K.). PBMC were isolated by density gradient centrifugation using Lymphoprep (Nycomed, Oslo, Norway) and washed three times with PBS. Adherent cells were removed by incubation of cells in plastic culture flasks for 60 min at 37°C. Cells were incubated for 20 min at 4°C in RPMI 1640 with saturating concentrations of CD8, CD11, CD14, CD16, CD19, CD33, and glycophorin mAbs. Cells were washed and incubated with sheep anti-mouse IgG-coupled magnetic Dynabeads (Dynal Biotech, Oslo, Norway) for 45 min. CD4+ T cells were isolated by removing the supernatant containing nonmagnetized cells. Cells were rested for 1 h at 37°C in RPMI/2% FCS before stimulation.
Preparation of mAb-coated microspheres
Latex microspheres (1 µm in diameter; Sigma, Poole, U.K.) were coated with CD3, CD28, CTLA-4, or mouse isotype control IgG (TNP) mAb in place of anti-CTLA-4. For beads used in IL-2 cytokine assays, 106 beads/ml PBS were coated with CD3 mAb at 1 µg/ml, CD28 mAb at 2.5 µg/ml, and CTLA-4 or TNP mAb at 10 µg/ml in the presence of 1 mg/ml BSA. Washed beads were resuspended in 100 µl complete RPMI 1640 and added to 100 µl of the CD4+ T cell suspension. For stimulating cells before biochemical analysis, beads (109) were coated with CD3 mAb or CTLA-4/TNP mAb (all at 20 µg/ml) for 1 h, washed twice with PBS, and then resuspended in 2 ml serum-free RPMI 1640. FACS analysis of beads was used to demonstrate that the CTLA-4 and TNP mAbs bound at comparable levels and did not reduce the binding of the CD3 mAb (data not shown).
IL-2 cytokine assay
CD4+ T cells (2 x 105 cells/well in complete RPMI 1640) were cocultured with mAb-coated microbeads (bead:cell ratio, 5:1) in round-bottom 96-well plates in a total volume of 200 µl complete RPMI 1640. Supernatants from duplicate cultures were collected after 30 h and assayed for IL-2 using an ELISA kit (BioSource, Nivelles, Belgium).
Activation of CD4+ T cells for biochemical analysis, immunoprecipitation, and immunoblotting
Resting CD4+ T cells
(108 per stimulation) were activated with TNP,
CD3 plus TNP, or CD3 plus CTLA-4 mAb-coated beads (bead/cell ratio,
100/1) in 3 ml serum-free RPMI 1640 for 5 min at 37°C. Cells were
centrifuged and lysed in ice-cold lysis buffer containing 1% Nonidet
P-40, 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM
Na3VO4, 1 mM
4-[2-aminoethylbenzenesulfonyl]fluoride (AEBSF), 1 mM EGTA, 1
mM EDTA, and protease inhibitor cocktail (Roche, Lewes, U.K.). After 15
min of incubation on ice, lysates were prepared by centrifugation and
precleared by incubation with protein G-Sepharose (Amersham Pharmacia
Biotech, Little Chalfont, U.K.) for 45 min at 4°C. Lysates from
3 x 106 cell equivalents were boiled in 3x
SDS sample buffer and subjected to immunoblotting with phospho-ERK and
phosphotyrosine mAbs following transfer to polyvinylidene difluoride
membranes (Millipore, Bedford, U.K.). For immunoprecipitations, cleared
cell lysates were incubated for 1–24 h at 4°C with TCR-,
p56lck, ZAP-70, or phosphospecific (Y319) ZAP-70
Abs bound to protein G-Sepharose. The immune complexes were washed
three times in Nonidet P-40 lysis buffer and either subjected to
SDS-PAGE for immunoblotting or prepared for an immunocomplex in vitro
kinase assay. Bands were visualized by ECL (Amersham Pharmacia Biotech)
or by the addition of the alkaline phosphatase-specific substrate
5-bromo-4-chloro-3 indolyl phosphate/nitro blue tetrazolium
(Roche).
Immunocomplex in vitro kinase assays
TCR-, ZAP-70, and p56lck
immunoprecipitates were prepared as described above and washed in
buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM
Na3VO4, and 1 mM AEBSF). To
preclear lysates of p56lck, unstimulated samples
were lysed and subjected to three consecutive rounds of
p56lck immunoprecipitation using
p56lck Ab. Depleted samples were then used for
ZAP-70 immunoprecipitation, washed, and subjected to in vitro kinase
assays. Precipitates were resuspended in 40 µl kinase assay buffer
(50 mM PIPES (pH 7.4), 10 mM MgCl2, 10 mM
MnCl2, 1 mM
Na3VO4, 1 mM AEBSF, 2 mM
DTT, 4.5 µM cold ATP, and 5 µCi
[
-32P]ATP), and kinase reactions were
performed for 10 min at room temperature. The reaction was stopped by
adding 50 µl of 3x SDS reducing sample buffer. Samples were boiled,
and proteins were resolved on 7–15% SDS-PAGE, followed by
autoradiography and immunoblotting.
CTLA-4 cytoplasmic tail peptide precipitation and in vitro PTPase assay
Peptides representing the complete CTLA-4 cytoplasmic tail were synthesized and HPLC-purified by the Microchemical Facility (Babraham Institute, Cambridge, U.K.). The amino acid sequences of the peptides used were as follows: KMLKKRSPLTTGYVKMPPTEPECEKQFQPYFIPIN (unphosphorylated peptide), KMLKKRSPLTTGpYVKMPPTEPECEKQFQPpYFIPIN (pY201 and pY218 peptide), KMLKKRSPLTTGpYVKMPPTEPECEKQFQPYFIPIN (pY201 peptide), and KMLKKRSPLTTGYVKMPPTEPECEKQFQPpYFIPIN (pY218 peptide). Peptides were covalently bound to cyanogen bromide-activated Sepharose beads (Amersham Pharmacia Biotech) according to the manufacturer’s protocol. Peptides were subsequently used to precipitate PTPase activities from whole cell lysates originating from DT40 and SHP-2-deficient DT 40 cells (from Prof. T. Kurosaki, Kansai Medical University, Japan), from primary, human CD4+ T cells, and from motheaten (me/me) mice (from Dr. J. Matthews).
PTPase activity was measured using the Malachite green PTPase assay kit (Upstate Biotechnology). Postnuclear lysates from 4–6 x 107 cell equivalents were prepared as described above, but without the PTPase inhibitor Na3VO4, and were incubated with 30 µl Sepharose slurry for 1 h at 4°C. Samples were incubated for 1 h at 4°C with 30 µl CTLA-4 tail peptide Sepharose slurry. To deplete lysates of SHP-1, samples originating from human CD4+ T cells were subjected to four consecutive rounds of SHP-1 immunoprecipitation using SHP-1 Ab coupled to protein G-Sepharose beads. SHP-1-depleted lysates were then exposed to CTLA-4 tail homologs. Precipitates were washed three times with lysis buffer, followed by three washes in PTPase assay buffer containing 25 mM HEPES (pH 7.2), 50 mM NaCl, 5 mM DTT, 2.5 mM EDTA, and 1 mg/ml BSA. Samples were resuspended in PTPase assay buffer and incubated for 10 min at 37°C with 250 µM phosphopeptide (TSTEPQpYQPGENL) as the substrate. After centrifugation, the supernatants were transferred to microtiter wells and incubated with 100 µl Malachite Green solution for 15 min, and PTPase activity was quantitated by measuring the absorbance at 630 nm using a microtiter plate reader. All assays were performed in duplicate in parallel with assay blanks containing CTLA-4 synthetic tail peptide beads, PTPase assay buffer, and phosphopeptide substrates. PTPase activity was calculated from standard curves after subtracting background values. The remaining peptide pellets were resuspended in 3x SDS sample buffer, boiled, and subjected to SDS-PAGE and Western blot analysis as described above.
Flow cytometry
Cells were incubated for 30 min at 4°C with PE-conjugated CTLA-4 or isotype-specific TNP mAb in PBS/1% BSA/0.1% sodium azide or fixed with 1% paraformaldehyde and permeabilized with 0.3% saponin before incubation with mAb for intracellular CTLA-4 staining. Cells were analyzed on a FACScan flow cytometer (BD Biosciences, Mountain View, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Freshly purified human CD4+ T cells were
stimulated with CD3/CD28 and CTLA-4 mAbs coimmobilized on microbeads,
and culture supernatants were assayed for IL-2 cytokine production.
Stimulation using CD3 mAb alone caused little IL-2 secretion, but this
was greatly increased upon CD3 and CD28 costimulation (Fig. 1
). Concomitant ligation of CTLA-4 with
CD3/CD28 consistently resulted in a striking reduction (50–70%
inhibition) of IL-2 secretion. This effect was specific to CTLA-4,
because the irrelevant TNP control mAb had no effect when
co-cross-linked with CD3/CD28 (data not shown). These results confirm
an earlier report (23) and establish that in our
experimental system engagement of human CTLA-4 exerts an inhibitory
effect on CD3/CD28-mediated T cell activation events resulting in
diminished IL-2 production.
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It has been suggested that CTLA-4 is expressed at detectable
levels at the T cell surface only after activation (24).
Previous studies have shown that CTLA-4 is located primarily in
endosomal vesicles, from where it cycles continuously to and from the
cell surface (25). To measure cell surface and cytoplasmic
CTLA-4 expression, FACS analysis was conducted on intact and
permeabilized resting, purified human CD4+ T
cells. Table I lists the 23 blood donors
who were analyzed for CTLA-4 expression, and Fig. 2 shows a representative FACS profile of
CTLA-4 receptor staining. In most instances (17 of 23 donors) flow
cytometric analysis of CD4+ T cells revealed low,
but detectable, levels of surface CTLA-4 (7 ± 1% positive cells;
Table I and Fig. 2A). As expected, a larger proportion of
CTLA-4 could be detected intracellularly after treatment of cells with
saponin (34 ± 5% positive cells; Table I and Fig. 2B). However, we noticed considerable variation in CTLA-4
expression levels among individual CD4+ T cell
preparations originating from various blood donors (Table I),
presumably due to the different immune histories of the
donors. The observation that CTLA-4 was detectable on resting
CD4+ T cells raises the possibility that it
may also be operational and functional, resulting in inhibition of the
very earliest stages of T cell activation.
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The aim of our investigation was to elucidate the molecular mechanism of action by which CTLA-4 engagement inhibits TCR-mediated activation signals in primary cells.
It is unknown whether CTLA-4 interferes with TCR-mediated protein
tyrosine phosphorylation in primary resting human
CD4+ T cells. Therefore, we evaluated the effects
of CTLA-4 ligation on TCR signaling by analyzing changes in tyrosine
phosphorylation in whole cell lysates. Fig. 3A shows that TCR stimulation
caused increased protein tyrosine phosphorylation of proteins migrating
in the range of 
34–40 and 75–150 kDa and that most
phosphorylation events were much reduced upon CD3/CTLA-4 coligation.
Stripping and reprobing the membrane with p95vav
mAb demonstrated comparable loading between lanes. Minor qualitative
variations in tyrosine-phosphorylated substrates were noted between
different donors, but CTLA-4-mediated inhibition of phosphorylation was
consistently noted in five separate experiments. Intriguingly, as Fig. 3A illustrates, basal tyrosine phosphorylation was also
reduced by CTLA-4 ligation in T cells prepared from several different
donors. The striking generalized reduction in protein tyrosine
phosphorylation suggests that CTLA-4 interferes with TCR signal
transduction at a very early stage in the initiation of the signaling
cascade.
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B shows that coengagement of CTLA-4 with
the TCR in primary resting CD4+ T cells also
results in significant inhibition (80%) of CD3-stimulated ERK-1/2
activation. Subsequent reprobing of the membrane with an ERK-2 Ab
revealed comparable loading of protein between lanes. CTLA-4-mediated
inhibition of ERK-1/2 activity was also demonstrated at later time
points, excluding the possibility of a modified kinetic response
induced by CTLA-4 (data not shown). Thus, our results suggest that
coengagement of TCR and CTLA-4 perturbs proximal TCR signal
transduction independently of CD28 engagement and upstream or at the
level of ERK-1/2 activation.
TCR -chain tyrosine phosphorylation and ZAP-70 recruitment are
not inhibited by CTLA-4 engagement
To investigate whether CTLA-4 counteracted the most proximal step
in the TCR signaling cascade, we measured TCR -chain tyrosine
phosphorylation following CTLA-4 coengagement. Phosphorylation of
TCR- by p56lck causes binding of ZAP-70
tyrosine kinase via its SH2 domains, leading to its phosphorylation and
activation (27). Stimulation of the TCR complex resulted,
as expected, in generation of the TCR- p21 and p23 phosphoisomers,
but coengagement of CTLA-4 did not significantly change their
phosphorylation status (Fig. 4A, upper panel).
Likewise, CTLA-4 did not interfere with ZAP-70 recruitment to the TCR
complex, as assessed by immunoblotting TCR -chain immunoprecipitates
for ZAP-70 (Fig. 4B). In addition, ZAP-70 immunoprecipitates
were analyzed for coassociated TCR-. Fig. 4C shows that
CTLA-4-TCR coligation did not interfere with the ability of ZAP-70 to
interact with the TCR- p23-kDa phosphoisoform. Thus, TCR -chain
phosphorylation and ZAP-70 recruitment to the TCR -chain remained
unaltered by CTLA-4 engagement.
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To investigate whether CTLA-4 cross-linking interfered with the
recruitment and/or activation of kinases within TCR signaling
complexes, in vitro kinase assays were conducted on ZAP-70
immunoprecipitates originating from TCR/CTLA-4-stimulated cells. Fig. 5A shows that CTLA-4
engagement caused a striking reduction in the phosphorylation of
several proteins migrating at 70, 56, 28, and 25 kDa in the
immunocomplex. Immunoblot analysis revealed, as expected, that the
70-kDa protein was ZAP-70 (Fig. 5A, middle
panel). We speculated that the ZAP-70-associated 56-kDa protein
was p56lck based on previous work
(28); however, we were unable to identify this protein
unambiguously as p56lck by Western blotting due
to the presence of the IgG heavy chain. Instead we immunoprecipitated
ZAP-70 from p56lck-depleted lysates and
performed in vitro kinase assays in the ZAP-70 immunocomplex and in
p56lck immunoprecipitates as a control. The
results confirmed that the 56-kDa protein in the ZAP-70 immunocomplex
was indeed p56lck, as this protein was
undetectable in the immunoprecipitates prepared from
p56lck-depleted lysates (Fig. 5A,
lower panel). Our results show that CTLA-4 perturbs
phosphorylation of ZAP-70 and/or recruitment of several proteins
interacting with ZAP-70, including p56lck as
well as pp28 and pp25.
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B, upper panel). Both ZAP-70 and
p56lck were at comparable levels in the lysates
used for immunoprecipitation (Fig. 5B, lower
panels). To probe the regulation of ZAP-70 more selectively, we
examined the phosphorylation status of the particular pool of ZAP-70
found in TCR- immunocomplexes by in vitro kinase assay. Fig. 5C shows that TCR-induced phosphorylation of a 70-kDa
protein (upper panel), established by immunoblotting to be
ZAP-70 (middle panel), was reduced by 48 ± 10%
(n = 3) after co-cross-linking with CTLA-4 mAb.
Immunoblotting confirmed the findings shown in Fig. 4A that
CTLA-4 engagement reduced neither TCR- phosphorylation nor ZAP-70
recruitment per se (lower panels). Thus, reduced
interaction of ZAP-70 with p56lck correlates
with inhibited ZAP-70 phosphorylation. CTLA-4 ligation selectively inhibits phosphorylation of ZAP-70 Y319 but does not affect p56lck kinase activity
One mechanism that could explain the reduced
ZAP-70-p56lck association is that CTLA-4 might
inhibit the kinase activity of p56lck.
Alternatively, CTLA-4 could directly interfere with the phosphorylation
state of tyrosine residues present in ZAP-70. In particular,
phosphorylated Y319, which was shown to be a docking site for the SH2
domain of p56lck (19), could be a
target for CTLA-4. To distinguish these possibilities, in vitro kinase
assays were performed on p56lck
immunoprecipitates originating from CD3/CTLA-4-stimulated lysates. As
is shown in Fig. 6A,
p56lck kinase activity is not reduced following
CTLA-4 engagement, indicating that the activation of
p56lck, an upstream regulator of the TCR/ZAP-70
signaling complex, is not affected by CTLA-4. We next addressed
whether CTLA-4 interferes with phosphorylation of the ZAP-70 Y319 site.
Lysates from stimulated samples were subjected to immunoprecipitations
using an Ab specific for phosphotyrosine 319 in ZAP-70 and the amount
of pY319-ZAP-70 was then detected by ZAP-70 immunoblotting (Fig. 6B). Whereas CD3 stimulation induced phosphorylation of Y319
in ZAP-70, CTLA-4 engagement resulted in a striking inhibition of that
phosphorylation site. The same results were obtained in independent
experiments in which the phospho-ZAP-70 Ab was used to immunoblot
pY319-ZAP-70 immunoprecipitates (Fig. 6C). Reprobing of
whole cell lysates with a ZAP-70 Ab confirmed similar protein loading
between samples (Fig. 6, B and C, lower
panels). Our data show for the first time that CTLA-4 causes the
inhibition of CD3-mediated phosphorylation of the Y319 site present in
ZAP-70, which correlates with reduced
ZAP-70-p56lck association. Changes in total
ZAP-70 tyrosine phosphorylation, as measured using a phosphotyrosine
Ab, were not detectable in these experiments (data not shown). This is
consistent with previous results showing that pY319 represents only one
of multiple ZAP-70 phosphorylation sites and suggesting a highly
selective action of CTLA-4 on the pY319 site.
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Signaling pathways responsible for CTLA-4-mediated inhibition are
largely unknown. Examination of tyrosine phosphorylation levels in T
cells from CTLA-4-/- mice revealed that a
number of proteins implicated in TCR signaling, such as CD3-,
ZAP-70, Shc, Lck, and Fyn, are hyperphosphorylated and hyperactive
(15). These observations led to the suggestion that
tyrosine phosphatases may be involved in the inhibitory signal
transduced by CTLA-4. One candidate PTPase is SHP-2, which has been
shown to be recruited to CTLA-4 through the phosphorylated Y201VKM
motif in the cytoplasmic tail (15). However, the
involvement of SHP-2 in mediating CTLA-4-mediated inhibition and SHP-2
binding to CTLA-4 has been challenged in other studies (29, 30). Our observation that phosphorylation events were inhibited
after CTLA-4 cross-linking prompted us to investigate whether PTPases
could be recruited to CTLA-4. The complete CTLA-4 cytoplasmic tail was
synthesized in nonphosphorylated, doubly phosphorylated, or
mono-tyrosine-phosphorylated versions. These were used as affinity
reagents to precipitate PTPases from cellular lysates that were
subsequently analyzed for associated PTPase activity. Using Jurkat cell
lysates it was possible to detect PTPase activity associated with the
phosphorylated CTLA-4 tail containing the pY218 motif and the doubly
phosphorylated pY210 and pY218 motifs (data not shown). Comparable
results were obtained when the chicken DT40 cell line was used (Fig. 7A). Blotting the precipitates
for the presence of SHP-2, we found that SHP-2 associated predominantly
with the tyrosine-phosphorylated versions of the CTLA-4 tail (Fig. 7A). To further analyze the contribution of SHP-2 activity
associated with CTLA-4, we made use of a SHP-2-deficient DT40 cell
line. As expected, no SHP-2 was precipitated from the deficient cells
(Fig. 7A). Interestingly, however, there was no difference
in CTLA-4-associated PTPase activity between CTLA-4 tail precipitates
derived from wild-type and SHP-2-deficient DT40 cells (Fig. 7A). Similar results were obtained when the phosphorylated
TCR- peptide immunoreceptor tyrosine-based activation motif was used
as a substrate (data not shown). These findings suggest that SHP-2 does
not contribute to the PTPase activity associated with the cytoplasmic
tail of CTLA-4.
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B, left panel). Similar
amounts of proteins were subjected to the analysis, as confirmed by
blotting for growth factor receptor binding protein 2 (Grb-2; Fig. 7B). SHP-1 binding was dependent on phosphorylation of the
Y218 or Y201 and Y218 motifs within the CTLA-4 cytoplasmic tail.
Interestingly, however, little SHP-1 coassociated with the pY201VKM
motif. SHP-1 immunodepletion resulted in a reduction by 80% of SHP-1
binding to the peptides (Fig. 7B). This reduced SHP-1
binding correlated with a striking decrease in PTPase activity
associated with the pY218 (75% reduction) and pY201 and pY218 (50%
reduction) motifs compared with the nondepleted samples. These results
strongly support the view that SHP-1 represents the major PTPase
activity associated with the phosphorylated cytoplasmic tail of CTLA-4
(Fig. 7B).
To substantiate these findings, similar experiments were conducted on T
cell lysates originating from motheaten (me/me) mice, which
completely lack the expression of SHP-1, and from wild-type mice. We
used the nonphosphorylated and the doubly phosphorylated version of the
CTLA-4 peptide tail as the affinity reagents for these experiments.
Blotting the lysates for Grb-2 revealed that protein concentrations
were similar in wild-type and me/me samples (Fig. 7C). Consistent with the immunodepletion experiments, the
level of PTPase activity associated with the phosphorylated CTLA-4
cytoplasmic tail was reduced by 50–75% (n = 2)
compared with that in wild-type controls (Fig. 7C). Taken
together, these findings suggest that SHP-1 coassociates with CTLA-4
and constitutes an important PTPase activity in the CTLA-4 complex.
However, SHP-1 is not solely responsible for the observed PTPase
activity, because 25–50% PTPase activity was still detectable even in
SHP-1-deficient cells.
In summary, this study shows for the first time that coengagement of CTLA-4 on resting human CD4+ T cells inhibits TCR induced p56lck-ZAP-70 complex formation, which correlates with reduced ZAP-70 Y319 phosphorylation, causing inhibition of downstream signaling events and of IL-2 secretion. The major active PTPase recruited to the phosphorylated CTLA-4 cytoplasmic tail appears to be SHP-1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). We noticed that CTLA-4 is constitutively
expressed, both intracellularly and on the surface of resting
CD4+ T cells (Fig. 2). However, there was
variation of CTLA-4 surface expression levels among the different blood
donors. There is still some controversy regarding the level and
kinetics of CTLA-4 expression. CTLA-4 is thought to be undetectable in
naive T cells, but is rapidly up-regulated upon T cell activation.
CTLA-4 mRNA can be readily detected within 1 h of TCR engagement
and peaks at 24–36 h (32). It is likely in the present
work that the donors had encountered Ag in vivo that may have mobilized
existing intracellular stores of CTLA-4 to the membrane. Thus, the
variation observed among the donors may reflect heterogeneity in the
immune histories of the different donors. Our observations suggest that
the low CTLA-4 expression level could be functionally significant in
inhibiting early T cell activation events. Interestingly, recent
studies have shown that CTLA-4 is detectable and constitutively
expressed on murine
CD25+CD45RBlowCD4+
T regulatory cells and that CTLA-4 is likely to be involved in their
immune-suppressive function (33). It remains to be
established whether expression of CTLA-4 is restricted predominantly to
human T regulatory cells.
Analysis of CTLA-4-mediated signaling events in resting human
CD4+ T cells showed that CD3-evoked protein
tyrosine phosphorylation events were decreased upon CTLA-4 ligation.
This was paralleled with reduced ERK activation (Fig. 3). These data
suggest that CTLA-4 interferes with proximal TCR signaling. However,
CD3-induced tyrosine phosphorylation of the TCR -chains and
subsequent ZAP-70 recruitment were not affected by CTLA-4 (Fig. 4),
indicating that CTLA-4 acts downstream of these molecules. Our results
are consistent with a previous study using preactivated, murine T
cells, which also found that these phosphorylation events and ZAP-70
recruitment remain unaltered upon CTLA-4 engagement (4).
In contrast, another report demonstrated reduced TCR -chain and
linker for activation of T cells phosphorylation following CTLA-4
cross-linking (16). However, these latter results were
obtained using preactivated murine T cells and different stimulation
conditions, and it is possible that T blasts may display different
signaling characteristics due to their altered activation state.
Because ZAP-70 recruitment to the TCR -chain was not affected by
CTLA-4 and because ZAP-70 has been implicated as a critical
intermediary between TCR stimulation and Ras activation
(34), we investigated whether recruitment and/or
activation of kinases in the ZAP-70 immunocomplex were altered by
CTLA-4. Phosphorylation of multiple ZAP-70 tyrosine residues, mediated
by autophosphorylation, Src kinases, or other cellular kinases, is
thought to result in the association of downstream signaling components
by SH2 domain-phosphotyrosine interactions (35).
Importantly, phosphorylation of Y319 was shown to be a binding site for
the SH2 domain of p56lck, and this interaction
is crucial for ZAP-70 activation and subsequent amplification of
downstream signaling (19, 34). In this study we
demonstrate for the first time that CTLA-4 interferes with the
regulation of ZAP-70. This was evidenced by reduced phosphorylation of
ZAP-70 (Fig. 5A) and diminished coassociation or reduced
phosphorylation of ZAP-70 with unknown proteins of 25 and 28 kDa (Fig. 5A). Moreover, the TCR--associated ZAP-70 pool was found
to be hypophosphorylated, as assessed by in vitro kinase assays of
TCR- immunoprecipitates (Fig. 5C). Current experiments
are in progress to identify pp25/pp28. One possible candidate could be
the p23 isoform of TCR-; however, immunoblotting for TCR- was
inconsistent with this idea (data not shown). More importantly, CTLA-4
ligation resulted in perturbed TCR-induced complex formation of
p56lck with ZAP-70 following ZAP-70 blotting of
p56lck immunoprecipitates (Fig. 5B).
It is this reduction in p56lck-ZAP-70
association rather than any reduction in ZAP-70 activity that most
likely explains the reduced phosphorylation of ZAP-70 shown in Fig. 5A. When phosphospecific ZAP-70 Y319 Abs were used to
immunoprecipitate and to blot for phosphorylated ZAP-70, we were able
to demonstrate that CTLA-4 caused a striking inhibition of ZAP-70 Y319
phosphorylation. Because phosphorylation at this site has been shown to
be important for the induction of the NFAT transcription factor complex
(34), a reduction in Y319 phosphorylation could readily
explain the inhibition of IL-2 secretion (Fig. 1) that we observed upon
CTLA-4 engagement. The reduced phosphorylation could be explained by
inhibition of a kinase or increased actions of a phosphatase. We were
unable to detect any reduction in p56lck kinase
activity following CTLA-4 engagement (Fig. 6A). Because
p56lck is most likely the kinase that
phosphorylates Y319, this points to increased phosphatase action as the
possible explanation for the reduction in Y319 phosphorylation. A
CTLA-4-associated PTPase could dephosphorylate ZAP-70 Y319,
leading to diminished interaction with the
p56lck SH2 domain, resulting in inhibited TCR
signaling.
To identify potential PTPases that associate with CTLA-4 in an active
form, we took an approach using the CTLA-4 cytoplasmic tail in
nonphosphorylated and phosphorylated forms as affinity reagents to
precipitate PTPase activities. One candidate PTPase that has been shown
to interact with and negatively regulate ZAP-70 is SHP-1 (36, 37). By immunodepletion of SHP-1 from human
CD4+ T cell lysates and by using SHP-1-deficient
me/me cell lysates, we show that SHP-1 accounts for the
majority of the PTPase activity associated with the CTLA-4 cytoplasmic
tail phosphorylated at Y218 or at Y201 and Y218 (Fig. 7, B
and C). The preferential binding motif for SHP-1 appears to
be the pY218FIP motif, and we detected neither significant PTPase
activity nor SHP-1 binding to the pY201VKM motif (Fig. 7B
and data not shown). The CTLA-4 cytosolic domains do not possess
immunoreceptor tyrosine-based inhibition motifs, which could mediate
direct binding to SHP-1, indicating an indirect association. The
absence of direct SHP-1 binding to CTLA-4 does not preclude a
functional role, because the interaction may be mediated by one or more
adaptor molecules. We were unable to detect interaction between
endogenous CTLA-4 and SHP-1 in pervanadate treated primary T cell
blasts (data not shown), which may be due to the extremely small
amounts of CTLA-4 expressed in these cells.
From our results it is evident that SHP-1 does not constitute the sole
CTLA-4-associated PTPase, because appreciable activities (25–50%)
were detected in the me/me mouse cell lysates. Using the
SHP-2-deficient DT40 cell line we could exclude the contribution of
SHP-2 to the PTPase activity associated with CTLA-4, although SHP-2,
presumably catalytically inactive, binds to the phosphorylated
cytoplasmic tail (Fig. 7A). It is possible that the N-SH2
domain of SHP-2 interacts with its PTPase domain, and SHP-2 adopts a
closed conformation, thereby rendering the enzyme catalytically
inactive when bound to the CTLA-4 cytoplasmic tail.
Although it was impossible to directly prove the involvement of SHP-1
using primary human cells, we speculate that SHP-1 may be critical for
the observed CTLA-4-induced inhibition of TCR signaling. There is
evidence that CTLA-4 interacts with the TCR -chain
(16), and SHP-1 is normally excluded from lipid rafts for
initiation of TCR signaling to occur (38, 39). It is
possible that CTLA-4 targets SHP-1 to the TCR complex, thereby
interfering with the phosphorylation of signal effectors, e.g., ZAP-70,
and with its association state with p56lck. A
study using lymph node T cells from motheaten viable mice has shown
that proliferation and IL-2 production could still be inhibited by
CTLA-4 engagement (40), suggesting that SHP-1 may not be
essential for mediating the inhibitory effects of CTLA-4 in vivo.
Clearly, the availability of conditional knockout
SHP-1-/- mice would help to elucidate the
possible role of SHP-1 in vivo in mediating the negative effects of
CTLA-4 in the T cell lineage. The phenotype of the T cells from such
mice might provide a more complete understanding of SHP-1 function,
because the development of hemopoietic cell lineages are not expected
to be as severely affected as in motheaten viable mice. Furthermore,
our findings do not exclude other potential mechanisms for
CTLA-4-mediated inhibition, such as the possibility that CTLA-4
competes with CD28 for binding to CD80 and CD86 ligands.
In summary, our results using primary resting CD4+ T cells point to a highly selective molecular mechanism of action of CTLA-4 in down-regulating TCR signal transduction coupling.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Denis R. Alexander, Laboratory of Lymphocyte Signaling and Development, Molecular Immunology Program, The Babraham Institute, Cambridge CB2 4AT, U.K. E-mail address: denis.alexander{at}bbscrc.ac.uk
3 Abbreviations used in this paper: ERK, extracellular signal-regulated kinase; Grb-2, growth factor receptor binding protein 2; PTPase, protein tyrosine phosphatase; SHP, Src homology protein 2 domain-containing tyrosine phosphatase; ZAP-70, -associated protein of 70 kDa; AEBSF, 4-[2-aminoethylbenzenesulfonyl]fluoride.
Received for publication November 21, 2001. Accepted for publication February 25, 2002.
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