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*
Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark; and
Basel Institute for Immunology, Basel, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and linker for activation of T cells,
which led to reduced TCR-ZAP70 complex formation, as well as
extracellular signal-related kinase 1 and 2 activation. Furthermore,
ILT2 inhibited both superantigen and anti-TCR Ab-induced
rearrangement of the actin cytoskeleton. The inhibitory effect mediated
by ILT2 is probably concentrated at the APC-T cell interface because
both TCR and ILT2 were strongly polarized toward the APC upon
engagement by their specific ligands. Thus, ILT2 inhibits both
signaling and cellular events involved in the activation of T
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production
(6, 7, 8). The accumulation of actin in a tight collar at the
T cell-APC interface is thought to stabilize a continuous contact
between T cells and APCs and to allow the clustering of TCR, adhesion,
and costimulatory molecules into supramolecular activation clusters
(9, 10). In addition, the remodeling of the actin
cytoskeleton, as well as polarization of the microtubule-organizing
center toward the contact site, is thought to position the T cell
secretory apparatus into close proximity with the APC, thereby enabling
a polarized release of cytotoxic mediators and cytokines (11, 12). TCR signaling and polarization of the actin cytoskeleton
are linked via a chain of intracellular molecules that includes
Vav, the Rho family GTPases (RacI, Cdc42), SLP76, and Nck. These
molecules recruit the Wiskott-Aldrich syndrome protein-Arp2/3 complex,
which regulates the dynamics of the actin cytoskeleton (7, 8, 13). In subsets of CTLs, TCR-mediated activation is counterbalanced by inhibitory signals that are transduced by receptors specific for MHC class I molecules (14, 15, 16, 17, 18, 19, 20, 21, 22). Such receptors were originally identified on NK cells and are characterized by a significant diversity with respect to their structure and specificity (23). In humans, inhibitory receptors for MHC class I molecules include the killer cell Ig-like receptors (KIRs), the CD94/NKG2A heterodimer, and Ig-like transcript 2 (ILT2)/leukocyte Ig-like receptor 1 (LIR1). KIRs recognize specific polymorphisms on the classical MHC class I molecules HLA-A, -B, and -C. CD94/NKG2A recognizes the nonclassical class I molecule HLA-E, which is assembled with a peptide derived from the processed leader sequence of classical class I molecules (24, 25). ILT2/LIR-1 is expressed not only on subsets of NK and T cells, but also on myeloid cells. It recognizes a broad range of cellular MHC class I molecules (22), as well as the viral class I-like molecule UL18 (26). All of these receptors contain one or more immunoreceptor tyrosine-based inhibition motifs (ITIMs) in their cytoplasmic tails. ITIMs generally consist of a YxxL/V motif preceded by a hydrophobic (I, V, or L) residue at position Y-2 (27, 28). Upon tyrosine phosphorylation, ITIMs recruit phosphatases Src homology protein (SHP)-1 and/or -2, which can dephosphorylate molecules involved in immuno-tyrosine activation motif (ITAM)-induced signaling pathways (29, 30, 31). Although it has been shown that KIR can block cytokine production and cytotoxicity of T cells (16, 21), not much is known about the molecular mechanisms by which inhibitory receptors affect TCR signaling/T cell activation in primary T cells. Moreover, it is still unclear whether MHC class I inhibitory receptors can modulate cellular and morphological events linked to T cell activation and cytotoxicity, such as TCR-induced rearrangement of the cytoskeleton. To address these questions, we analyzed the inhibitory function of ILT2 in both primary and transfected T cells at a molecular and cellular level.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Jurkat and J. CaM 1.6 (deficient of
p56lck (32)) cells were grown in
RPMI-1640/10% FCS. ILT2 and CD4- cDNA was transfected in Jurkat or
J. CaM 1.6 cells by electroporation as previously described
(33), and stable transfectants were selected in
G418-containing medium. ILT2 and CD4- expression on transfected
cells was assessed by FACS analysis and immunoblot using mAb GHI/75 or
GKI1.5. 721.221 (or 721.221 transfected with HLA-B*2705) cells are MHC
class I-deficient EBV-transformed human B cell lines (22).
LOQ22.7 and OKT8–24
CD8+ILT2+ T cell clones
were isolated and maintained as previously described
(22).
Antibodies
PY20, anti-SHP-1, and anti-ZAP70 mAbs were obtained from
BD Transduction Laboratories (Lexington, KY). Anti-CD3 mAb was kindly
donated by A. Lanzavecchia (Institute for Research in Biomedicine,
Bellinzona, Switzerland). F(ab')2 of goat
anti-mouse (GAM) IgG H+L or mouse anti-human IgG Fc-specific
were from Jackson ImmunoResearch (West Grove, PA). HRP-conjugated goat
anti-mouse and PE-conjugated anti-CD69, FITC-conjugated CD25,
or anti-TCR mAb were obtained from Immunotech (Marseille,
France). Anti-phospho-extracellular signal-related kinase (ERK) or ERK
Abs were obtained from New England Biolabs (Beverly, MA). Anti-LAT Ab
was obtained from Upstate Biotechnology (Lake Placid, NY). Src kinase
inhibitors PP1 and PP2 were obtained from Calbiochem (La Jolla, CA).
Alexa-conjugated phalloidin was obtained from Molecular Probes
(Eugene, OR).
Immunoprecipitation and immunoblotting
Cells treated with mAbs or pervanadate (PV) (200 µM sodium orthovanadate and 200 µM H2O2 at 37°C for 10 min) as indicated were lysed in 1% Triton X-100 or 1% Brij97 lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl with added inhibitors, 0.75 µM aprotinin, 10 µM leupeptin, 3 µM pepstatin A, 1 mM PMSF, 0.4 mM EDTA). PV-treated cells were precleared with protein G beads (Amersham Pharmacia, Uppsala, Sweden). Thereafter, lysates were subjected to immunoprecipitation with the indicated mAbs as previously described (34). For whole-cell lysate analysis cells were lysed in Laemmli sample buffer. Immunoprecipitates and whole-cell lysates were separated by standard SDS-PAGE, transferred to polyvinylidene difluoride membranes (Amersham Pharmacia) and immunoblotted with the indicated mAbs. Bound Abs were visualized using ECL (Amersham Pharmacia).
Cell stimulations
Two million cells/ml were incubated at 37°C with the indicated mAbs and F(ab')2 GAM Ab (Jackson ImmunoResearch) as cross-linker. After stimulation, cell aliquots were lysed and subjected to anti-phosphotyrosine and anti-ERK blotting using PY20 (BD Transduction Laboratories) and either anti-phospho-ERK or ERK Abs (New England Biolabs). Alternatively, after different time periods of stimulation, cell aliquots were fixed, permeabilized, stained with Alexa-conjugated phalloidin (Molecular Probes), and analyzed by FACS. In some experiments, cells were pretreated with 10 µg/ml of Src kinase inhibitors PP1 or PP2 for 30 min at 37°C. For stimulation with toxic shock syndrome toxin-1 (TSST-1), 106 OKT8-24 cells were incubated with 721.221 cells or an HLA-B27 transfectant of 721.221 pulsed with TSST-1 at 37°C for 10 min. Both target cells expressed equivalent levels of MHC class II molecules. Thereafter, cells were transferred to polylysine-coated slides, fixed, permeabilized, and stained with anti-CD3 mAbs, ILT2 mAbs, or phalloidin. In other experiments, GAM Dynabeads M-450 (Dynal, Oslo, Norway) were loaded with mAbs as indicated and incubated with cells in a ratio of two beads/cell at 37°C for 15 min. Thereafter, bead-cell conjugates were transferred to polylysine-coated slides and analyzed as described above in confocal microscopy.
In vitro binding studies using beads coated with peptides
Peptides corresponding to the four cytoplasmic tyrosine motifs
in ILT2 bound to vinyl-activated Sepharose 4B beads at a density of
1–2 µmol/ml Sepharose gel were obtained from Schafer-N
(Copenhagen, Denmark). The peptides were denoted pY1 (EENLpYAAVKHTQ),
pY2 (DPQAVTpYAEVKHSR), pY3 (APQDVTpYAQLHSLT), pY4 (VPSIpYATLAIH), and
con (ESSNpYMAPYDNY), where pY denotes a phosphorylated tyrosine residue
and con a control peptide sequence from the platelet-derived growth
factor receptor. All peptides were coupled to the beads via an
N-terminal linker of four amino acids (EACA) and a cysteine residue
(bead-Cys-linker-peptide). The coupling procedure via the
amino-terminal cysteine resulted in immobilization of monomeric
peptides with freely exposed carboxyl termini. To prepare T cell
cytosol, 4 x 107 Jurkat cells were washed
three times in PBS and lysed in 1% Triton X-100 with added inhibitors
(0.75 µM aprotinin, 10 µM leupeptin, 3 µM pepstatin A, 1 mM PMSF,
0.4 mM EDTA). Beads were incubated with T cell cytosol for 2 h at
37°C, washed six times in PBS at 4°C, and bound material was eluted
by boiling the beads in low salt buffer with 2% 2-ME. Following
SDS-PAGE, the eluted material was transferred to polyvinylidene
difluoride membranes, and immunoblotting was performed using
anti-SHP-1 mAb (BD Transduction Laboratories) followed by
peroxidase-conjugated rabbit anti-mouse Abs (Immunotech). Bound Abs
were visualized using ECL (Amersham Pharmacia).
Confocal microscopy
Cells were washed in PBS and fixed for 10 min with 1% paraformaldehyde/PBS. The cells were permeabilized for 10 min at room temperature with washing buffer (HEPES-buffered PBS, containing 0.1% saponin) and stained with primary Abs at room temperature for 10 min. Cells were washed three times in washing buffer and stained with FITC-conjugated secondary Abs at room temperature for 10 min. In experiments involving stimulation of cells with Ab-coated beads or TSST-1-pulsed APCs, cell-bead/APC conjugates were attached to polylysine (1 mg/ml)-coated coverslips, fixed, permeabilized, and stained with Alexa-conjugated phalloidin or the indicated mAbs. Confocal microscopy was performed on an MRC-1000 (Bio-Rad, Richmond, CA) connected to an Axiovert 100 M microscope (Zeiss, Oberkochen, Germany).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ILT2 was previously shown to inhibit superantigen-induced T
cell-mediated cytotoxicity by a subset of CD8+ T
cells (22). To investigate the molecular mechanisms
responsible for this inhibitory function, we first examined the
phosphorylation status of ILT2 in T cells after cross-linking ILT2 with
the TCR (with specific mAbs and a cross-linker) or following treatment
of cells with the phosphatase inhibitor PV. Jurkat T cells transfected
with ILT2 cDNA (Jurkat-ILT2) were either treated with PV or subjected
to ILT2-TCR cross-linking. Thereafter, ILT2 was immunoprecipitated from
cell lysates, and its phosphorylation status was analyzed by Western
blot. Both PV treatment and ILT2-TCR cross-linking induced substantial
tyrosine phosphorylation of ILT2. Some ILT2 phosphorylation was also
observed following ligation of the TCR alone (data not shown). In
contrast, ILT2 was not phosphorylated following cross-linking of ILT2
alone (Fig. 1
A). To analyze
which tyrosine kinase was involved in the phosphorylation of ILT2,
Jurkat-ILT2 cells were pretreated with an inhibitor of Src-tyrosine
kinase p56lck (PP2) before stimulation with PV.
This treatment significantly reduced the phosphorylation of ILT2 (Fig. 1B). The same result was obtained with Src-tyrosine kinase
p56lck inhibitor PP1 (data not shown). In
another approach, ILT2 was transfected into J. CaM 1.6, a mutant of
Jurkat that lacks expression of the tyrosine kinase
p56lck. No phosphorylation of ILT2 was observed
upon PV treatment or after TCR-ILT2 cross-linking in the J. CaM
1.6-ILT2 transfectants (Fig. 1, C, and D). Thus,
p56lck is required for phosphorylation of ILT2.
Because it was previously shown that phosphorylated ILT2 recruits SHP-1
in B cells and NK cells (22), we verified whether this
also occurs in T cells. SHP-1 clearly associated with ILT2 following PV
treatment (Fig. 1E). This association was dependent upon
p56lck activity, because no association was
observed in J. CaM 1.6-ILT2 transfectants (Fig. 1E). We
finally examined which of the four cytoplasmic tyrosine-based motifs of
ILT2 bind to SHP-1. Phosphorylated and unphosphorylated peptides
spanning the four tyrosine motifs were conjugated with Sepharose beads
and incubated at 37°C with lysate from Jurkat-ILT2 cells for 2
h. The association of SHP-1 to the peptides was then analyzed by
Western blot. SHP-1 bound to the peptides in the following order: pY2
>> pY3 > pY1, whereas little or no binding was observed with
pY4, control peptide, or nonphosphorylated forms of the peptides (Fig. 1F, and data not shown). Taken together, our results show
that in T cells ILT2 is tyrosine phosphorylated following cross-linking
to the TCR. Tyrosine-phosphorylated ILT2 recruited SHP-1, which
preferentially bound to the VxYxxV motif in ILT2 in vitro. Tyrosine
phosphorylation of ILT2 and the association with SHP-1 required the
presence and activity of p56lck.
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, LAT, and ERK1/2
Because ILT2 recruited the phosphatase SHP-1 upon phosphorylation,
we next examined whether this would affect the phosphorylation of
proteins involved in TCR-induced signaling pathways. TCR was
cross-linked either alone, with ILT2, or with another cell surface
protein, MHC class I, in Jurkat-ILT2 cells. Cell lysates from
stimulated cells were analyzed by Western blot using an
anti-phosphotyrosine mAb. The results showed that the
phosphorylation of two proteins of molecular mass 20 and 36–44 kDa was
clearly decreased following TCR-ILT2 coligation as compared with TCR
cross-linking alone or with TCR-MHC-I coligation (Fig. 2, A and B). To
assess whether the proteins of molecular mass 20 and 36 kDa
corresponded to TCR and LAT, tyrosine-phosphorylated proteins were
precipitated from stimulated Jurkat-ILT2 cells and analyzed by Western
blot analysis using an anti-TCR mAb or a LAT Ab. Alternatively,
TCR and LAT were precipitated, and the phosphorylation state of
these proteins was analyzed by Western blot. In control experiments,
TCR was cross-linked with MHC-I, which was highly expressed on Jurkat
cells. The results showed that the phosphorylation of both TCR (Fig. 2, C–E) and LAT (Fig. 2, F–I) was reduced upon
TCR-ILT2 ligation. In addition, the reduction in TCR phosphorylation
was associated with reduced recruitment of ZAP70 (Fig. 2E).
In control experiments, TCR-MHC-I cross-linking did not reduce
phosphorylation of TCR or LAT (Fig. 2, D, G,
and I). ILT2-induced reduction of LAT and TCR
phosphorylation was also observed in a
CD8+ILT2+ T cell clone
(LOQ22.7) (Fig. 2I, and data not shown). Because
phosphorylated LAT links to activation of the mitogen-activated protein
kinases ERK1 and 2, we also examined whether TCR-ILT2 coligation
affected the activation of ERK1 and 2. Activation of both ERK1 and ERK2
in LOQ22.7 cells decreased following ILT2-TCR cross-linking as compared
with cross-linking of TCR alone or TCR-MHC-I cross-linking (Fig. 3). This was also observed in Jurkat-ILT2
cells (data not shown). Taken together, these experiments show that
TCR-ILT2 coligation reduces TCR-mediated phosphorylation of TCR,
LAT, and ERK1 and 2, as well as the TCR-ZAP70 complex formation.
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One of the consequences of TCR engagement is modification of the
the actin cytoskeleton, which is absolutely required for T cell
activation and cytotoxicity (7, 35, 36, 37). Because TCR-ILT2
coligation decreased T cell activation, we speculated that it might
also affect polymerization of actin following engagement of TCR. To
examine this possibility, cells were incubated with beads coated with
either anti-CD3 mAb, anti-ILT2 mAb, anti-CD3 and -ILT2
mAbs, or anti-CD3 and -MHC-I mAbs and analyzed by confocal
microscopy using phalloidin, which binds to polymerized actin
(F-actin). Upon triggering with anti-CD3 or anti-CD3/MHC-I-coated
beads, actin strongly accumulated at the bead-cell contact region. In
contrast, when cells were incubated with anti-CD3/ILT2 beads, we
observed a significant reduction in the accumulation and polarization
of actin (Fig. 4A). Anti-ILT2
beads did not induce polarization of actin. This was observed with
Jurkat-ILT2 and OKT8-24 cells (Fig. 4, A and B).
Thus, TCR-ILT2 coligation specifically inhibited TCR-mediated actin
polymerization. To quantify the changes in actin polymerization,
Jurkat-ILT2 or OKT8-24 cells were stimulated with soluble anti-TCR
and/or anti-ILT2 mAbs and cross-linker for different time periods,
stained with phalloidin, and analyzed by FACS. Engagement of TCR alone
or together with MHC-I led to a 50% increase in the amount of
F-actin after 5 min, which declined with time (Fig. 4C).
Upon TCR-ILT2 coligation, only small increases in F-actin were
observed, in agreement with the results obtained with Ab-coated beads
(Fig. 4, C and D). Cross-linking of ILT2 alone
did not increase F-actin (data not shown). To determine whether ILT2
could also inhibit actin polymerization induced by engagement of the
TCR with superantigen, OKT8-24 cells were incubated with 721.221 or
721.221 cells transfected with the ligand for ILT2, HLA-B27. Both types
of cell had been pulsed with the superantigen TSST-1. Binding of TCR to
TSST-1-pulsed 721.221 induced strong actin accumulation at the contact
site as compared with OKT8-24 cells incubated with nonpulsed 721.221
cells (Fig. 5A). FACS analysis
showed a 30–40% increase in F-actin in superantigen-stimulated
OKT8-24 cells. However, upon TSST-1 presentation by HLA-B27-positive
cells, actin polymerization was strongly reduced (Fig. 5B).
Taken together, these results demonstrate that ILT2 inhibits
TCR-induced actin polymerization.
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and LAT, are involved in actin
polymerization
ILT2-mediated reduction of TCR-induced actin polymerization could
be due to reduced tyrosine phosphorylation of TCR, LAT, or other
substrates. In support of a role for tyrosine phosphorylation,
TCR-induced actin polymerization was strongly reduced in cells with
impaired tyrosine phosphorylation of TCR (and other proteins). These
included cells treated with an inhibitor (PP2) of
p56lck, which is responsible for TCR
phosphorylation (38), and cells deficient in
p56lck (Fig. 6A). In direct support of a
role for TCR in actin polymerization, Ab-mediated cross-linking of a
fusion protein containing the extracellular and transmembrane region of
murine CD4 and the cytoplasmic tail of TCR was sufficient to induce
actin polymerization in Jurkat (Fig. 6B). Another target of
ILT2-mediated dephosphorylation that could be involved in actin
polymerization is LAT. Although the role of LAT in TCR-induced actin
polymerization is not known, LAT has been reported to interact with
proteins involved in cytoskeletal changes (5, 39). To
examine whether LAT is required for actin polymerization, we used the
LAT-deficient Jurkat cell line (ANJ3) (5). Stimulation of
this cell line with an anti-TCR mAb, either in soluble form or
coated on beads, did not induce actin polymerization. In contrast,
transfection of ANJ3 cells with LAT cDNA completely restored
TCR-induced actin polymerization (Fig. 6, C and
D). Thus, actin polymerization following TCR stimulation
most probably involves tyrosine phosphorylation and at least two of the
targets for ILT2, LAT and TCR.
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We finally analyzed the localization of both ILT2 and TCR on T
cells upon recognition of their respective ligands, HLA-B27 and TSST-1,
on APCs. OKT8-24 cells were incubated with 721.221 cells or an HLA-B27
transfectant of 721.221 pulsed with TSST-1. Thereafter, cells were
analyzed by confocal microscopy. As expected, TCR was clearly recruited
to the contact region between the T cell and the TSST-1-pulsed APC.
Cells not pulsed with TSST-1 did not induce TCR polarization (Fig. 7A). ILT2 was also polarized
toward the APC expressing an MHC-I ligand, whereas no polarization was
observed using class I-negative APCs (Fig. 7B). ILT2
polarization did not require TSST-1 stimulation (data not shown). This
demonstrated that upon recognition of their ligands both TCR and ILT2
are polarized toward the APC, indicating that the inhibitory effect
mediated by ILT2 is probably concentrated at the contact region between
T cell and APC.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we analyzed the interaction between the TCR and
ILT2, an inhibitory receptor that has a broad specificity for MHC class
I molecules. We found that, upon TCR-ILT2 cross-linking, ILT2 is
phosphorylated on tyrosines. In addition, using PV, we showed that
tyrosine phosphorylation and p56lck were
required for SHP-1 recruitment to ILT2. In vitro binding experiments
showed that SHP-1 binds preferentially to phosphopeptides spanning the
cytoplasmic ILT2 VxpYxxL/V motif. This indicates that the Src homology
2 domains of SHP-1 choose ITIMs, which are preceded by a V in the Y-2
position, in agreement with previous studies on KIRs and paired Ig-like
receptor B ITIMs (40, 41, 42, 43). TCR-ILT2 cross-linking resulted
in reduced phosphorylation of the ITAMs of TCR, reduced recruitment
of ZAP70, and also decreased phosphorylation of LAT and ERK1/2. Reduced
ZAP70 recruitment to TCR could explain the reduction of LAT
phosphorylation because LAT can be a substrate for ZAP70
(44). Reduced LAT phosphorylation as well as reduced
TCR-ZAP70 complex formation may in turn explain the deactivation of
ERK1 and 2 because both ZAP70 and LAT have proved important for
activation of ERKs (5, 45). However, both ERK1/2 and a
protein of 36 kDa (most probably LAT) have been shown to constitute
direct substrates for tyrosine phosphatases (46, 47, 48, 49). In
addition, ERK1- and ERK2-mediated phosphorylation of
p56lck influence the activity of this kinase
(50), which is known to phosphorylate TCR. Therefore,
our results do not exclude the possibility that ERK1 and 2 (and LAT)
are directly dephosphorylated by SHP-1, which in turn negatively
affects activation of p56lck and phosphorylation
of TCR ITAMs. Thus, although we favor the first model, future
experiments are required to identify precisely the mechanism
responsible for the observed reduction in tyrosine phosphorylation of
TCR, LAT, and ERK1/2.
In contrast to TCR-ILT2 coligation, engagement of ILT2 alone induced
neither ILT2 phosphorylation nor association with SHP-1. Upon
stimulation, TCR associates with Src kinase
p56lck (9, 38, 51), and this kinase
was also required for ILT2 phosphorylation. Thus, activation
(phosphorylation) of ILT2 required interaction with the TCR. In
addition, previous studies showed that in NK cells, phosphorylation of
KIR3DL1 and KIR2DL1 required p56lck and the
CD16- complex (29), whereas in B cells, phosphorylation
of paired Ig-like receptor B required the Src kinase Lyn and the
Fc
RI-Fc complex (52). Thus, the activity of
inhibitory receptors apparently requires ITAM-containing receptors and
their associated tyrosine kinases. One model that could explain this is
that the ITAM of the activating receptor (TCR/FcR/B cell receptor)
recruits the tyrosine kinase, which phosphorylates the ITIM. However,
following ITIM phosphorylation and recruitment of SHP-1, the ITAM
and/or associated proteins are dephosphorylated, thus preventing that
TCR/FcR/B cell receptor activation proceeds further. This model further
predicts: 1) the initial activation of TCR, involving recruitment of
kinases, cannot be inhibited by inhibitory receptors because it is in
fact required for their activation; 2) therefore, the phosphorylation
and activity of ITIMs are regulated by their own substrates, the ITAMs;
and 3) dephosphorylation of ITAM (by ITIM), resulting in loss of
associated proteins (including the kinases that activate the ITIM), may
down-regulate not only the ITAM but also the ITIM itself.
Modulation of TCR induced reorganization of the actin cytoskeleton by ILT2
This is the first study to demonstrate that an inhibitory receptor
can negatively affect cytoskeletal changes triggered by the TCR.
TCR-ILT2 cross-linking markedly reduced actin polymerization as
compared with T cells stimulated by cross-linking of TCR alone.
Furthermore, coengagement of ILT2 and TCR by HLA-B27 and TSST-1,
respectively, reduced TCR-mediated actin polymerization as compared
with T cells stimulated with TSST-pulsed class I-negative APCs. The
inhibition of actin polymerization may reduce the polarization of TCR
(and adhesion/costimulatory molecules) toward the target cell and/or
the generation of supramolecular activation clusters
(7, 8, 9). This in turn would affect the coordinated
recruitment of signaling proteins and thus the activation of the T
cell. The mechanism by which ILT2 inhibits TCR-triggered actin
polymerization is most likely based on dephosphorylation and subsequent
lack of recruitment/activation of proteins involved in this event.
ZAP70 was less recruited to TCR , and in support of a role for
TCR in the actin cytoskeleton reorganization we showed that TCR
ITAMs were indeed sufficient for induction of actin polymerization,
which is in agreement with a previous study (36).
Moreover, in experiments using J. CaM 1.6 cells or normal cells
pretreated with the tyrosine kinase inhibitor PP2, a failure to
tyrosine phosphorylate TCR and other adapter proteins was paralleled
by a lack of TCR-triggered actin polymerization (Fig. 6
A).
In addition, decreased LAT phosphorylation may lead to less recruitment
of SLP76, Vav, and Nck, all of which are involved in actin cytoskeleton
reorganization (7, 8, 39). In support of this, a
LAT-deficient cell line exhibited a defect in TCR-induced actin
polymerization (Fig. 6, C and D). Taken together,
these results may explain the inhibitory effect of ILT2 on actin
polymerization because we show that tyrosine phosphorylation, TCR,
and LAT not only have important roles in the regulation of the actin
cytoskeleton, but are all influenced by ILT2 ligation.
A physiological role for ILT2
Because increasing the amount of superantigen reduced the inhibitory effect of ILT2 on killing of target cells (22), this suggests that one function of ILT2 is to increase the activation threshold of T cells. Raising the activation threshold could prevent self-reactivity by low-affinity self-MHC-peptide complexes, resulting in only high-affinity MHC-peptide complexes being able to elicit a full immune response. Inhibition of self-reactivity by inhibitory receptors would in particular be expected to apply for CD8 effector/ memory T cells, which have acquired a lower activation threshold as compared with naive T cells. In support of this, the majority of T cells expressing ILT2 are indeed CD8 effector/memory cells (J. D. and M. C., unpublished observation), and similar expression patterns were observed for other inhibitory receptors (53). Another function for ILT2 and other inhibitory receptors may be to terminate an immune response. Following TCR stimulation, the TCR is internalized and targeted for lysosomal degradation (54). The decrease in surface-expressed TCRs during the course of T cell activation may change the balance in favor of inhibitory receptors and thus lead to down-regulation of the response. Finally, ILT2 may protect effector/memory T cells from activation-induced cell death, thereby preserving a pool of Ag-specific T cells.
Concluding remarks
In conclusion, we have described the molecular mechanisms behind
activation of ILT2 and showed that a cross-talk exists between ILT2 and
TCR, which may involve the regulation of ILT2-ITIM
activation/phosphorylation by TCR-ITAMs, as well as the TCR-ITAM
being a direct substrate for the ILT2-ITIM-SHP-1 complex. ILT2 ligation
inhibited both signaling and cellular events important for T cell
activation. Proximal signaling events including TCR phosphorylation
and recruitment of ZAP70 were inhibited, as were downstream events such
as LAT phosphorylation and mitogen-activated protein kinase activation.
Furthermore, TCR-induced reorganization of the actin cytoskeleton was
strongly reduced. Because cytoskeletal changes are involved in both T
cell- and NK cell-mediated killing, this may well represent a common
target for ILTs and KIRs in these cells.
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Acknowledgments |
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transfectants. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Jes Dietrich, Institute of Medical Microbiology and Immunology, University of Copenhagen, The Panum Institute, Building 18.3, Blegdamsvej 3C, DK-2200 Copenhagen, Denmark.
3 Abbreviations used in this paper: LAT, linker for activation of T cells; PV, pervanadate; GAM, goat anti-mouse; ERK, extracellular signal-related kinase; TSST-1, toxic shock syndrome toxin-1; MFI, mean fluorescence intensity; KIR, killer cell Ig-like receptor; ITIM, immunoreceptor tyrosine-based inhibition motif; ITAM, immuno-tyrosine activation motif; ILT2, Ig-like transcript 2; LIR1, leukocyte Ig-like receptor 1; SHP, Src homology protein.
Received for publication August 21, 2000. Accepted for publication December 5, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  S. Endo, Y. Sakamoto, E. Kobayashi, A. Nakamura, and T. Takai Regulation of cytotoxic T lymphocyte triggering by PIR-B on dendritic cells PNAS, September 23, 2008; 105(38): 14515 - 14520. [Abstract] [Full Text] [PDF]
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