PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine

Abstract

PD-1 is an immunoreceptor that belongs to the immunoglobulin (Ig) superfamily and contains two tyrosine residues in the cytoplasmic region. Studies on PD-1-deficient mice have shown that PD-1 plays critical roles in establishment and/or maintenance of peripheral tolerance, but the mode of action is totally unknown. To study the molecular mechanism for negative regulation of lymphocytes through the PD-1 receptor, we generated chimeric molecules composed of the IgG Fc receptor type IIB (Fc gamma RIIB) extracellular region and the PD-1 cytoplasmic region and expressed them in a B lymphoma cell line, IIA1.6. Coligation of the cytoplasmic region of PD-1 with the B cell receptor (BCR) in IIA1.6 transformants inhibited BCR-mediated growth retardation, Ca(2+) mobilization, and tyrosine phosphorylation of effector molecules, including Ig beta, Syk, phospholipase C-gamma 2 (PLC gamma 2), and ERK1/2, whereas phosphorylation of Lyn and Dok was not affected. Mutagenesis studies indicated that these inhibitory effects do not require the N-terminal tyrosine in the immunoreceptor tyrosine-based inhibitory motif-like sequence, but do require the other tyrosine residue in the C-terminal tail. This tyrosine was phosphorylated and recruited src homology 2-domain-containing tyrosine phosphatase 2 (SHP-2) on coligation of PD-1 with BCR. These results show that PD-1 can inhibit BCR signaling by recruiting SHP-2 to its phosphotyrosine and dephosphorylating key signal transducers of BCR signaling.

Full text

PD-1 immunoreceptor inhibits B cell receptor-
mediated signaling by recruiting src homology
2-domain-containing tyrosine phosphatase 2
to phosphotyrosine
Taku Okazaki*, Akito Maeda
†
, Hiroyuki Nishimura*, Tomohiro Kurosaki
‡
, and Tasuku Honjo*
§
*Department of Medical Chemistry and †Department of Molecular Immunology and Allergy, Graduate School of Medicine, Kyoto University,
Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan; and ‡Department of Molecular Genetics, Institute for Liver Research, Kansai Medical
University, Moriguchi 570-8506, Japan
Contributed by Tasuku Honjo, September 17, 2001
PD-1 is an immunoreceptor that belongs to the immunoglobulin
(Ig) superfamily and contains two tyrosine residues in the cyto-
plasmic region. Studies on PD-1-deficient mice have shown that
PD-1 plays critical roles in establishment and兾or maintenance of
peripheral tolerance, but the mode of action is totally unknown. To
study the molecular mechanism for negative regulation of lym-
phocytes through the PD-1 receptor, we generated chimeric mol-
ecules composed of the IgG Fc receptor type IIB (Fc
␥
RIIB) extracel-
lular region and the PD-1 cytoplasmic region and expressed them
in a B lymphoma cell line, IIA1.6. Coligation of the cytoplasmic
region of PD-1 with the B cell receptor (BCR) in IIA1.6 transformants
inhibited BCR-mediated growth retardation, Ca
2ⴙ
mobilization,
and tyrosine phosphorylation of effector molecules, including Ig
␤
,
Syk, phospholipase C-
␥
2 (PLC
␥
2), and ERK1兾2, whereas phosphor-
ylation of Lyn and Dok was not affected. Mutagenesis studies
indicated that these inhibitory effects do not require the N-
terminal tyrosine in the immunoreceptor tyrosine-based inhibitory
motif-like sequence, but do require the other tyrosine residue in
the C-terminal tail. This tyrosine was phosphorylated and recruited
src homology 2-domain-containing tyrosine phosphatase 2 (SHP-2)
on coligation of PD-1 with BCR. These results show that PD-1 can
inhibit BCR signaling by recruiting SHP-2 to its phosphotyrosine
and dephosphorylating key signal transducers of BCR signaling.
The immunoreceptor PD-1 belongs to the Ig superfamily (1).
PD-1 contains two tyrosine residues in its cytoplasmic re-
gion, the N-terminal of which is embedded in a sequence defined
as the immunoreceptor tyrosine-based inhibitor y motif (ITIM),
I兾L兾VXYXXL兾V (1–4). The expression of PD-1 can be de-
tected at certain developmental stages of thymocytes (i.e.,
double-negative to double-positive stages) and on the activated
peripheral T and B lymphocytes (5, 6).
PD-1-deficient mice exhibit splenomegaly, selective augmen-
tation of IgG3 Ab response to a T-independent type II antigen,
and enhanced proliferative responses of B cells and myeloid cells
by anti-IgM and granulocyte colony-stimulating factor stimula-
tion, respectively (ref. 7 and unpublished data). Thus, PD-1
appears to inhibit immune responses in vivo, and abrogation of
this inhibition would result in development of autoimmune
diseases. Indeed, PD-1-deficient C57BL兾6 mice spontaneously
develop typical lupus-like glomerulonephritis and destructive
arthritis (8). In addition, we have recently reported that PD-1-
deficient BALB兾c mice die of autoantibody-mediated dilated
cardiomyopathy (9). Because PD-L1, a ligand of PD-1, is highly
expressed on heart (10), and the antigen recognized by the
autoantibody is strictly restricted to heart, we assume that the
direct interaction between the heart tissue and B cells by means
of PD-1兾PD-L1 is responsible for the prevention of this deadly
disease. So the negative effect of PD-1 on B cell receptor (BCR)
signaling is strongly suggested, although no direct evidence of the
inhibitory effect has been obtained.
Because the sequence surrounding the N-terminal tyrosine
fulfills the requirement of ITIM, identified in the cytoplasmic
region of immunoinhibitory receptors such as IgG Fc receptor
type IIB (Fc
␥
RIIB), killer Ig-like receptor (KIR), CD22, and the
paired Ig-like receptor B, it is quite natural to speculate that
PD-1 mediates its negative signal by means of this tyrosine
residue. On stimulation, tyrosine residues of ITIMs are usually
phosphorylated to recruit Src homology 2 (SH2)-containing
phosphatases that play critical roles in negative regulation of
cellular activities. Among ITIM-bearing immunoinhibitory
receptor-deficient mice, only Fc
␥
RIIB-deficient mice spontane-
ously develop autoimmune diseases (11–14). In C57BL兾6 back-
ground, Fc
␥
RIIB-deficient mice develop glomerulonephritis,
which is similar to but different from that of PD-1-deficient mice.
In the BALB兾c background, Fc
␥
RIIB-deficient mice are re-
ported to be healthy, whereas PD-1-deficient mice develop
autoimmune dilated cardiomyopathy (9, 14), suggesting that
PD-1 and Fc
␥
RIIB regulate the immune system in a similar but
distinct manner.
According to the multistep model for BCR signaling (15),
BCR stimulation first activates Lyn, which, in turn, phosphory-
lates the tyrosine residue in the immunoreceptor tyrosine-based
activation motif of the Ig
␣
and Ig
␤
, resulting in recruitment of
another protein tyrosine kinase, Syk. Activated protein tyrosine
kinases also phosphorylate a tyrosine residue in ITIM, which
recruits SH2-containing phosphatases to dephosphorylate and
deactivate signal transducers. Thus regulation of protein tyrosine
phosphorylation is important in both the activation and inhibi-
tion of B cells. Among ITIM-bearing immunoinhibitory recep-
tors, Fc
␥
RIIB is reported to recruit SH2-domain-containing
phosphatidylinositol 5-phosphatase (SHIP) on coligation with
BCR, engagement of which leads to deactivation of CD19 and
inhibition of signaling downstream of CD19 (16, 17). On the
other hand, paired Ig-like receptor B is reported to recruit src
homology 2-domain-containing tyrosine phosphatase 1 (SHP-1)
and SHP-2, engagement of which leads to dephosphorylation of
a wider variety of molecules, including Ig
␣
兾
␤
, Syk, Btk, and
phospholipase C-
␥
2 (PLC
␥
2) (18). Most of ITIM-bearing im-
munoinhibitory receptors such as Fc
␥
RIIB, paired Ig-like re-
ceptor B, and CD72 are expressed constitutively on B cells,
whereas PD-1 is expressed only on activated B cells, suggesting
that PD-1 may have some unique and important roles in the
Abbreviations: ITIM, immunoreceptor tyrosine-based inhibitory motif; Fc
␥
RIIB, IgG Fc
receptor type IIB; BCR, B cell receptor; PLC
␥
2, phospholipase C-
␥
2; SH2, src homology 2; SHP,
SH2-domain-containing protein tyrosine phosphatase; KIR, killer immunoglobulin-like
receptor; SHIP, SH2-domain-containing phosphatidylinositol 5-phosphatase.
§To whom reprint requests should be addressed. E-mail: honjo@mfour.med.kyoto-u.ac.jp.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
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regulation of B cell activation, which leads to the prevention of
autoimmune diseases.
We report here that PD-1 signaling inhibited growth retar-
dation, protein tyrosine phosphor ylation, and Ca
2⫹
mobilization
of antigen-stimulated B lymphoma cell line IIA1.6. Unexpect-
edly, coligation of PD-1 and BCR recruited SHP-2 preferentially
to the C-terminal phosphotyrosine in its cytoplasmic tail, result-
ing in dephosphorylation of effector molecules and down-
regulation of downstream molecules.
Materials and Methods
Cells, Expression Constructs, and Antibodies. Murine B cell line
IIA1.6 was maintained in RPMI medium 1640 supplemented
with 10% FCS, 50 mM 2-mercaptoethanol, 2 mM L-glutamine,
and antibiotics. Fc
␥
RIIB and FcKIR constructs were as de-
scribed (16). The mouse PD-1 cDNA fragment downstream of
the EcoRV site was fused with the mouse Fc
␥
RIIB cDNA
fragment upstream of the ApaI site by blunting ligation to obtain
FcPD chimera. One copy of a flag tag was added at the C
terminus of all constructs by PCR methods as shown in Fig. 1A.
Introduction of mutations at tyrosine residues and truncation of
the cytoplasmic region were carried out by PCR methods. The
resulting constructs were confirmed by DNA sequencing and
subcloned into the pMX-IRES-EGFP vector (EGFP, enhanced
green fluorescence protein) (19). BOSC23 cells were transfected
by these vectors to obtain retroviruses, which were subsequently
used to infect IIA1.6 cells. EGFP-positive infectants were sorted
by FACS Vantage (Becton Dickinson) to obtain an almost 100%
pure population. Intact Abs and F(ab⬘)
2
fragments of rabbit
anti-IgG (H ⫹L) (Zymed), anti-IgG2a, hamster anti-CD3
(PharMingen), anti-SHP-2, anti-SHIP, anti-Lyn, anti-Syk, anti-
PLC
␥
, anti-Dok, anti-RasGAP, anti-ERK1, anti-ERK2 (Santa
Cruz Biotechnology), anti-f lag (Sigma), anti-phospho-ERK
(New England Biolabs), anti-phosphotyrosine (Transduction
Laboratories, Lexington, KY), and streptavidin (Vector Labo-
ratories) were purchased. Anti-mouse PD-1 mAb (J43) (5),
anti-SHP-1, and anti-Ig
␤
were as described elsewhere (18).
Calcium Measurements. Cells (2 ⫻10
6
) were loaded with 4
␮
M
fura-2兾AM (Molecular Probes) in Krebs Hepes-buffered saline
without calcium at 37°C for 45 min. After the cells were washed
twice, they were resuspended with Krebs Hepes-buffered saline
supplemented with 1 mM CaCl
2
. Cytosolic calcium concentra-
tions of 5 ⫻10
5
cells were measured with a CAF 110 spectro-
photometer (Jasco, Tokyo) as described (20). PD-1-expressing
cells were stimulated with 2
␮
g兾ml biotinylated anti-PD-1 mAb
and 8
␮
g兾ml biotinylated anti-IgG2a mAb, followed by the
addition of 6
␮
g兾ml streptavidin to coligate PD-1 and BCR. For
BCR-only ligation, anti-PD-1 mAbs were replaced with 2
␮
g兾ml
biotinylated hamster anti-CD3 mAb. Intact Abs or F(ab⬘)
2
fragments of rabbit anti-mouse IgG (10
␮
g兾ml) were used,
respectively, for coligation of Fc chimera and BCR or ligation of
BCR only.
Proliferation Assay. IIA1.6 transformants (1 ⫻10
4
) were incu-
bated in 96-well U-bottomed culture dishes for 48 h with the
indicated amount of intact Abs or F(ab⬘)
2
fragments of anti-
mouse IgG Abs, pulsed with 0.5
␮
Ci of [methyl-
3
H]thymidine per
well (Amersham Pharmacia) for the last 4 h, and then harvested
with Ready Filter (Beckman Instruments, Fullerton, CA) for
Skatron (Skatron Instruments, Lier, Norway) followed by scin-
tillation counting. The percentage of
3
H incorporation was
Fig. 1. Strategies to dissect inhibitory mechanisms of PD-1. (A) Fc chimera and PD-1 constructs. The cytoplasmic regions of PD-1, its mutants, and KIR were fused
with the extracellular region of Fc
␥
RIIB. A flag tag was added to each Fc chimera. (B) Cell surface expression of Fc chimeras and PD-1. Sorted Fc chimera and PD-1
transformants were stained with anti-Fc
␥
RIIB and anti-PD-1 mAb, respectively. GFP, green fluorescence protein. (C) Schematic representation of stimulation
strategy. Incubation with intact anti-mouse IgG Ab results in coligation of Fc chimera and BCR (Right), whereas a F(ab⬘)2fragment of anti-mouse IgG Ab results
in BCR-only ligation (Left).
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calculated by dividing incorporation counts per minute (cpm) in
the presence of Abs by that in the absence of Abs.
Immunoprecipitation,
in Vitro
Kination, and Western Blot Analysis.
IIA1.6 transformants (1.5 ⫻10
7
cells per ml) were incubated for
2 min at 37°C with 25
␮
g兾ml intact Abs or F(ab⬘)
2
fragments of
rabbit anti-IgG Abs. Cells were solubilized in a lysis buffer
containing 1% Nonidet P-40, 20 mM Tris䡠HCl (pH 7.4), 100 mM
NaCl, 5 mM EDTA, 50 mM NaF, 1 mM sodium vanadate, and
protease inhibitor mixture (Roche Molecular Biochemicals).
Precleared cell lysates were incubated with agarose-conjugated
anti-flag mAb at 4°C for 1 h. Immunoprecipitates were separated
by SDS兾PAGE, transferred to poly(vinylidene dif luoride) mem-
brane, and detected by appropriate Abs with an enhanced
chemiluminescence system (Amersham Pharmacia). An in vito
kination assay was performed as described (18).
Flow Cytometric Analysis. Cells were stained with phycoerythrin-
conjugated anti-Fc
␥
RIIB mAb (2.4G2) (PharMingen) and an-
alyzed by FACSCalibur (Becton Dickinson).
Results
PD-1 Engagement Inhibits Antigen-Stimulated Growth Retardation of
B Cells. To test the molecular mechanism of PD-1 signaling, we
constructed a series of chimeric molecules that consisted of the
extracellular region of Fc
␥
RIIB and the cytoplasmic region of
Fc
␥
RIIB, KIR, and PD-1 or its mutants, as illustrated in Fig. 1A.
The chimera of the PD-1 mutant with N-terminal, C-terminal, or
both tyrosines replaced by phenylalanine was designated
FcPDF1, FcPDF2, or FcPDF1F2, respectively. The chimera of a
PD-1 mutant with deletion of the total cytoplasmic region is
called FcPDTM. These constructs were introduced into
Fc
␥
RIIB-negative mutant cells of the mouse A20 B cell lym-
phoma IIA1.6 by retroviral infection, followed by f luorescence-
activated cell sorting. Expression levels of these chimeric recep-
tors and cistronic enhanced green f luorescence protein were
almost the same among these transformants (Fig. 1B). These
transformants were stimulated with intact anti-IgG Ab, resulting
in coligation of BCR and chimeric receptors. As a control,
F(ab⬘)
2
fragments of anti-IgG Ab, which cannot crosslink BCR
and chimeric receptors, were used (BCR only) (Fig. 1C).
First, PD-1 signaling effects on cell growth were determined
by [
3
H]thymidine incorporation at various concentrations of
anti-IgG Ab. Cell growth of mock transformants was retarded by
BCR stimulation, whereas coligation of FcPD with BCR inhib-
ited this growth retardation in a manner similar to that of
coligation of Fc
␥
RIIB or FcKIR with BCR, indicating that
PD-1-BCR coligation counteracts BCR signaling (Fig. 2).
Requirement of the C-Terminal Tyrosine Residue of PD-1 for Its
Inhibition of BCR-Stimulated Ca
2ⴙ
Mobilization in B Cells. To see that
PD-1 inhibits more proximal events of antigen stimulation, we
next examined the effect of PD-1 engagement on Ca
2⫹
mobili-
zation, with the use of fura-2 as an indicator. IIA1.6 cells,
expressing various chimeric molecules illustrated in Fig. 1A,
were stimulated by coligation of BCR and chimeric receptors.
Like Fc
␥
RIIB and FcKIR (16), FcPD inhibited BCR-mediated
Ca
2⫹
mobilization when coligated with BCR (Fig. 3A). FcPDF1
strongly, but not completely, blocked BCR-mediated Ca
2⫹
mo-
bilization, whereas FcPDF2 showed almost no inhibitory effect,
and FcPDF1F2 or FcPDTM did not block BCR-mediated Ca
2⫹
mobilization at all, suggesting that C-terminal but not N-
terminal tyrosine residue is indispensable for the inhibitory
effect of PD-1 on BCR-stimulated Ca
2⫹
mobilization (Fig. 3B).
FcPD inhibited Ca
2⫹
mobilization in the presence of EGTA as
FcKIR did. In contrast, the inhibitory effect of Fc
␥
RIIB is
partially attenuated in the presence of EGTA, as reported (16).
These results indicate that PD-1 inhibits Ca
2⫹
release from the
intracellular pool (Fig. 3C).
To demonstrate that these inhibitory effects are not caused by
the artifact of chimeric molecules, the effect of full-length PD-1
itself was examined (Fig. 3D). The transformants of the full-
length PD-1 and its tyrosine mutant (PDF1F2) were stimulated
by biotinylated anti-IgG2a mAb and anti-PD-1 mAb (J43),
followed by crosslinking of BCR and PD-1 with streptavidin.
PD-1 transformants showed reduced mobilization of Ca
2⫹
by
coligation of BCR and PD-1 as compared with BCR ligation. In
mock and PDF1F2 transformants, the coligation did not inhibit
Ca
2⫹
mobilization as compared with stimulation by anti-IgG2a
mAb alone. These results showed that the assays with the
chimeric molecules of Fc
␥
RIIB and PD-1 can replace those with
full-length PD-1 in a facilitated way.
Association of SHP-2 with Phosphorylated PD-1. FcPD transformants
of IIA1.6 were stimulated for the indicated times, and phos-
phorylation of tyrosine residues was examined by immunopre-
cipitation with anti-f lag mAb and Western blotting with anti-
phosphotyrosine mAb. PD-1 was tyrosine phosphorylated only
when coligated to BCR, and this tyrosine phosphorylation could
be detected as early as8safterstimulation and lasted as long as
8 min (Fig. 4B). Mutation of either of the tyrosine residues
reduced tyrosine phosphor ylation in FcPD, suggesting that both
tyrosine residues are phosphorylated (Fig. 4C). Strong phos-
phorylation of the tyrosine residues of PD-1 suggests its asso-
ciation with SH2-containing signaling molecules on coligation
with BCR. A possible association of SHP-1, SHP-2, or SHIP with
PD-1 was tested, because these phosphatases are reported to be
recruited by ITIM-bearing immunoreceptors and critical for
their inhibitory function. SHP-2 but neither SHP-1 nor SHIP was
coimmunoprecipitated with FcPD when BCR and FcPD were
coligated (Fig. 4A). Control experiments confirmed association
of Fc
␥
RIIB and KIR with SHIP and SHP-1, respectively.
Association between SHP-2 and FcPD was abrogated when
C-terminal tyrosine was mutated to phenylalanine (FcPDF2),
suggesting that this association may depend on the C-terminal
phosphotyrosine (Fig. 4C). Coligation of PD-1 and BCR induced
Fig. 2. FcPD coligation with BCR inhibited BCR-mediated growth retardation
in IIA1.6 cells. Mock, Fc
␥
RIIB, FcKIR, and FcPD transformants were stimulated
with indicated concentrations of anti-mouse IgG Abs, and cell growth was
determined by [3H]thymidine incorporation. Relative growth was calculated
by dividing thymidine incorporation of stimulated cells by that of unstimu-
lated cells. Each percentage is the mean of triplicate wells.
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Fig. 3. Inhibition of BCR-mediated Ca2⫹mobilization by coligation with FcPD and PD-1. Cytosolic calcium concentrations of Fc-chimera transformants were
measured with a KAF 110 spectrophotometer. Each transformant was stimulated by coligation of Fc chimera and BCR or ligation of BCR only. (A) FcPD inhibited
BCR-mediated Ca2⫹mobilization as efficiently as Fc
␥
RIIB and FcKIR. (B) FcPDF1 inhibited BCR-mediated Ca2⫹mobilization, whereas FcPDF2, FcPDF1F2, and
FcPDTM could not. (C) FcPD inhibited BCR-mediated Ca2⫹mobilization in the presence of EGTA. (D) Inhibitory effect of full-length PD-1.
Fig. 4. Recruitment and phosphorylation of SHP-2 to tyrosine-phosphorylated FcPD. (A) IIA1.6 cells expressing FcPD, Fc
␥
RIIB, and FcKIR were stimulated, and
cell lysates were immunoprecipitated (IP) by anti-flag mAb and analyzed for interaction with SHP-2 by Western blotting. (B) FcPD-expressing IIA1.6 cells were
stimulated with intact Abs or F(ab⬘)2fragments of anti-mouse IgG for the indicated intervals. Tyrosine phosphorylation of FcPD, association between FcPD and
SHP-2, and tyrosine phosphorylation of SHP-2 were determined as described in Materials and Methods.(C) Tyrosine phosphorylation of tyrosine mutants of FcPD
and association between tyrosine mutants of FcPD and SHP-2 were determined as above. Closed and open arrowheads indicate full-length and truncated FcPD
chimeras, respectively.
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not only recruitment of SHP-2 but also tyrosine phosphorylation
of SHP-2. Both the association with PD-1 and SHP-2 phosphor-
ylation took place in parallel with phosphorylation of PD-1,
which was detectable as early as 8 s after BCR stimulation and
lasted longer than 8 min (Fig. 4B).
PD-1 Signaling Reduces BCR-Mediated Tyrosine Phosphorylation of
Various Molecules. BCR stimulation leads to activation of several
protein tyrosine kinases, including Lyn, Syk, and Btk. Because
the inhibitory effect of PD-1 is likely to be mediated by the
phosphatase activity of SHP-2, effects of FcPD coligation on
BCR-mediated tyrosine phosphor ylation of various molecules
were determined. Tyrosine phosphor ylation levels of p150, p95,
p85, p75, and p70 were reduced by coligation of FcPD with BCR,
whereas phosphorylation of p65, which is most likely FcPD itself,
was augmented (Fig. 5A). Lyn, Ig
␤
, Syk, PLC
␥
2, phosphatidyl-
inositol 3-kinase (PI3K), and vav are well-known positive signal
transducers that reside downstream of BCR signaling. We
examined tyrosine phosphor ylation levels of these molecules and
kination activity of Lyn. FcPD coligation with BCR markedly
reduced tyrosine phosphor ylation levels of Ig
␤
, Syk, PLC
␥
2,
PI3K, and vav. Because this reduction could not be observed by
FcPDF1F2 coligation, the involvement of the phosphatase ac-
tivity of SHP-2 is strongly suggested (Fig. 5 C–E and data not
shown). The kinase activity of Lyn against enolase was slightly
up-regulated by BCR engagement, and this augmentation was
not affected by coligation of FcPD. The phosphorylation level of
Lyn was not affected by coligation of FcPD with BCR (Fig. 5B).
PD-1 Inhibits BCR-Mediated Activation of Mitogen-Activated Protein
Kinase in a Manner Different from That of Fc
␥
RIIB or KIR. PD-1 effects
on another signaling pathway leading to cell growth were also
examined. BCR-mediated activation of ERK1 and ERK2 was
inhibited by coligation of BCR with FcPD, as reported for
Fc
␥
RIIB (21) and KIR (Fig. 5F). Fc
␥
RIIB was shown to
suppress BCR-mediated activation of mitogen-activated protein
kinase by augmentation of Dok tyrosine phosphorylation in the
SHIP兾Dok兾RasGAP pathway (21). On the other hand, SHP-1,
which is recruited to KIR, was reported to suppress phosphor-
ylation of Dok (22). Unlike Fc
␥
RIIB and FcKIR, FcPD did not
affect the phosphorylation status of Dok, suggesting that PD-1
inhibits activation of mitogen-activated protein kinase by a
pathway different from that of Fc
␥
RIIB or KIR (Fig. 5G). As
reported, activation of shc p52, association between Dok and
RasGAP, and association between shc and SHIP were observed
only by coligation of BCR with Fc
␥
RIIB but not w ith FcPD (data
not shown). SHP-2 was not coimmunoprecipitated with Dok or
shc by coligation with FcPD (data not shown).
Discussion
Because PD-1-deficient mice spontaneously develop autoim-
mune diseases and the sequence surrounding the N-terminal
tyrosine residue of PD-1 fulfills the requirement of ITIM, PD-1
has been thought to negatively regulate immune responses (8, 9).
Indeed, splenic B cells from PD-1-deficient mice exhibit aug-
mented proliferative responses on BCR stimulation, suggesting
that PD-1 may inhibit BCR signaling (7). But recent isolations of
PD-1 ligands have made the story complicated (10, 23–26). We
have recently reported that the ligands of PD-1 transduce
negative signals on T cells (10, 23). But there are other reports
that the ligands of PD-1 are costimulatory (24–26). Here we
report that PD-1 actually can inhibit BCR signaling and that
PD-1 inhibits antigen-stimulated B cell activation according to
Fig. 5. Coligation of PD-1 with BCR inhibited BCR-mediated tyrosine phosphorylation of various molecules. (A) Mock and FcPD transformants were stimulated,
and cell lysates were immunoprecipitated (IP) with anti-phosphotyrosine Ab (anti-pY) and examined for phosphotyrosine contents by Western blotting. (B–G)
Mock, FcPD, FcPDF1F2, Fc
␥
RIIB, or FcKIR transformants were stimulated under indicated conditions. Cell lysates were immunoprecipitated with Abs against Lyn
(B), Ig
␤
(C), Syk (D), PLC
␥
2(E), and Dok (G); resolved by SDS兾PAGE; transferred to membrane; and probed (immunoblotted, IB) with the Abs indicated. The closed
arrowhead indicates the tyrosine-phosphorylated Ig
␤
in C. The kinase activity of Lyn on enolase was also measured (B). Cell lysates were probed with anti-pERK1兾2
(F). pERK1兾2 represents the p44兾42 ERK1 and ERK2, which are phosphorylated at Thr-202 and Tyr-204 and thus are activated. Closed and open arrowheads
indicate ERK1 and ERK2, respectively, in F.
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the following scheme: (i) coligation of PD-1 with BCR results in
phosphorylation of both tyrosines in PD-1 (Fig. 4); (ii) SHP-2 is
recruited to the C-terminal phosphotyrosine of PD-1 and phos-
phorylated (Fig. 4); (iii) phosphorylated SHP-2 dephosphory-
lates proximal signal transducers of BCR such as Syk and Ig
␣
兾
␤
,
which leads to deactivation of downstream molecules, including
PI3K, PLC
␥
2, and ERK (Fig. 5); (iv) deactivation of signal
transducers results in inhibition of acute-phase reactions such as
Ca
2⫹
mobilization (Fig. 3) as well as long-term effects such as
growth retardation (Fig. 2). The inhibition of Ca
2⫹
mobilization
should be explained by the reduced phosphorylation of PLC
␥
2
(Fig. 5E), which converts phosphatidylinositol 4,5-bisphosphate
into diacylglycerol and inositol 3,4,5-trisphosphate; each of these
products is responsible for protein kinase C activation and
intracellular [Ca
2⫹
] increase, respectively. The inhibition of
growth retardation should be explained by the reduced phos-
phorylation of mitogen-activated protein kinase (Fig. 5F), which
is involved in the proliferation and differentiation of immune
cells (27). The costimulatory effect of the PD-1 ligands may
come from another receptor that may share the ligands with
PD-1.
Although PD-1 engagement leads to dephosphorylation of
various molecules, including Ig
␤
, Syk, PLC
␥
2, and ERK1兾2, this
does not necessarily mean that PD-1-activated SHP-2 dephos-
phorylates these molecules directly. SHP-2 is likely to dephos-
phorylate a few molecules that reside more proximal to the BCR
signaling as shown in paired Ig-like receptor B-activated SHP-1
(18). The most probable candidates are Syk and Ig
␣
兾
␤
, because
Syk has been shown to activate all of the molecules mentioned
above and their upstream molecules (28), and Ig
␣
兾
␤
have the
immunoreceptor tyrosine-based activation motif in their cyto-
plasmic regions, to which Syk is recruited.
Between two tyrosine residues in the cytoplasmic region of
PD-1, the N-terminal tyrosine residue in the ITIM-like sequence
was almost dispensable, and the C-terminal one was necessary
for the inhibitory effects of PD-1 on antigen-stimulated Ca
2⫹
mobilization and growth retardation (Fig. 3 and dat a not shown).
It is not known why the N-terminal tyrosine of PD-1 does not
associate with either SHP-1 or SHP-2. A possible explanation is
the presence of alanine at ⫺1, because an ITIM mutant of KIR
that has alanine at ⫺1 is less efficient in the activation of SHP-1
than is the wild type (29). However, the conservation of the PD-1
ITIM in human and mouse (1, 2) and its significant phosphor-
ylation on stimulation (Fig. 4) imply that this ITIM may have
important roles other than recruitment of SHP-1 and SHP-2.
The amino acid sequence around the C-terminal tyrosine is also
well conserved between mouse and human PD-1 (TEYATIVF),
and a similar sequence (TEYASI) is found in signal regulatory
protein
␤
, which is reported to associate with SHP-1 and SHP-2
(30). These observations suggest that the sequence TEYAS(T)I
may be involved in binding of SHP-2 with phosphotyrosine.
PD-1 executes its inhibitory effect only when coligated to BCR
(Figs. 3 and 5), suggesting that PD-1 has to get some positive
signals to execute its inhibitory function. Immunoprecipitated
Lyn but not Syk tyrosine-phosphor ylated PD-1 (data not shown),
suggesting that on coligation of BCR and PD-1, PD-1 is tyrosine
phosphorylated by Lyn, which resides near BCR. Thus Lyn plays
critical roles in both the activation and regulation of BCR
signaling. Because PD-1 is phosphorylated only by coligation
with BCR and expressed only on activated cells (5), PD-1
probably functions in the down-modulation of excessive and
prolonged activation and inhibition and兾or suppression of inap-
propriate activation such as autoreactivities by elevating the
threshold for restimulation.
We thank Drs. S. Miwa and Y. Kawanabe for their technical assistance
on calcium measurements and Mses. Y. Tada, T. Toyoshima, and Y. Doi
for their technical support. We are grateful to Dr. J. L. Strominger and
K. Ikuta for their critical reading of the manuscript. This work was
supported by grants from the Ministr y of Education, Science, Sports, and
Culture of Japan. T.O. is a research fellow of the Japan Society for the
Promotion of Science.
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Okazaki et al. PNAS
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IMMUNOLOGY