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Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several of the NK cell-activating receptors signal by association with
immunoreceptor tyrosine-based activation motif
(ITAM)2-containing
partner chains (4). For instance, CD16 associates with the
-chain, NKp46 associates with the FcR 
-chain, while NKp44 and
KIR2DS pair with DAP12. The noncovalent association of the
ITAM-containing chains is controlled by a basic amino acid in the
transmembrane region of the receptor that is compensated by an acidic
amino acid at a similar position in the adaptor protein. Thus, the
presence of a charged residue in the transmembrane region of an
immunoreceptor indicates a potential for an activating function. Other
NK cell-activating receptors, such as 2B4 (CD244) and CD2, carry
signaling motifs in their own cytoplasmic tail (4).
To override potentially harmful activation signals delivered by ITAM-containing molecules, NK cell activation is suppressed by inhibitory signals transduced by immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptors (5). In human NK cells, killer cell Ig-like receptors (KIR) and the lectin-like CD94/NKG2A receptor are mainly responsible for such inhibition. Some members of the KIR family carry two consecutive ITIM in their cytoplasmic tail and bind to classical HLA class I molecules. The ITIM is defined by the consensus sequence I/V/LxYxxL/V (6). Following tyrosine phosphorylation, the tandem ITIM recruits and activates tyrosine phosphatases that dephosphorylate components of the activation pathway. Src homology 2-containing phosphatase (SHP)-1 is thought to be the major phosphatase recruited in vivo by KIR, but the related phosphatase SHP-2 also binds phosphorylated ITIM in vitro and in vivo (7, 8, 9, 10). Although the precise molecular target(s) of SHP-1 still remains to be determined, ITAM-induced positive signals as well as signals from the activating receptor 2B4 are sensitive to ITIM-mediated inhibition (3). In contrast, the signaling pathway induced by the NKG2D/DAP10 receptor complex is not as sensitive to ITIM-mediated inhibition as that induced by ITAM-containing receptors (11, 12, 13, 14).
Among KIR family members, KIR2DL4, which was assigned the new CD designation CD158d (15), displays unusual characteristics. First, KIR2DL4 carries a single ITIM (the tyrosine of the second ITIM is substituted by a cysteine residue). Second, in addition to the presence of an inhibitory motif, KIR2DL4 contains a charged residue in its transmembrane region, suggesting the possibility of an activating function for this receptor (16). These two properties may explain earlier observations on the function of KIR2DL4. Indeed, both inhibitory (17) and activating (18) functions have been described for KIR2DL4. Third, the mRNA of KIR2DL4 is present in every NK cell, in contrast to all other KIR that are expressed in overlapping subsets of NK cells (19, 20). Fourth, the two Ig-like extracellular domains of KIR2DL4 and of KIR2DL5 have the unusual D0-D2 configuration, in contrast to the usual D1-D2 and D0-D1-D2 organization of Ig domains in other KIR2D and KIR3D receptors, respectively. Finally, KIR2DL4 binds to cells expressing HLA-G (20, 21), a nonclassical HLA class I molecule expressed by trophoblast cells that invade the maternal decidua (22), by activated monocytes (23, 24), by thymic epithelial cells (25), and by certain tumor cells (26).
In the present study, mutated KIR2DL4 receptors lacking either the charged residue in the transmembrane region or lacking the cytoplasmic ITIM were tested for their ability to activate or to inhibit NK cells. We report that KIR2DL4 lacking the ITIM is still activating NK cell cytotoxicity and that KIR2DL4 lacking the charged residue in the transmembrane region can deliver inhibitory signals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human NK populations were purified as described (27). Briefly, NK cells were isolated from human PBL using the magnetic activated cell sorter NK cell isolation kit (Miltenyi Biotec, Auburn, CA). Human NK cells were maintained in Iscove’s medium supplemented with 10% human serum (BioWhittaker, Walkersville, MD), L-glutamine, 10% purified IL-2 (Hemagen Dignostics, Columbia, MD), and 100 U/ml rIL-2. The human NK cell line YTS (gift from G. Cohen, Massachusetts General Hospital, Charlestown, MA) and the mastocytoma cell line P815 (American Type Culture Collection, Manassas, VA) were maintained in Iscove’s medium supplemented with 10% FCS and L-glutamine. The following Abs were used: anti-CD16 (3G8, mouse IgG1), MOPC-21 (control mouse IgG1), all from BD PharMingen (San Diego, CA); anti-hemagglutinin (HA) mouse ascites fluid (16B12, IgG1; Covance, Richmond, CA); control mouse ascites fluid (NS-1, IgG1; Sigma-Aldrich, St. Louis, MO); anti-2B4 (C1.7.1, mouse IgG1), anti-KIR2DL1 (CD158a) (EB6, mouse IgG1), anti-KIR2DL3 (CD158b2) (GL183, mouse IgG1), all from Beckman Coulter (Miami, FL); anti-Src homology 2-containing inositol 5'-phosphatase (SHIP) (rabbit polyclonal IgG), anti-SHP-1 (rabbit IgG), anti-insulin receptor substrate-1 (IRS-1; rabbit IgG), biotin-conjugated anti-phosphotyrosine (4G10, mouse IgG2b), all from Upstate Biotechnology (Lake Placid, NY); and anti-SHP-2 (79, mouse IgG1; Transduction Laboratories, Lexington, KY).
Infection with recombinant vaccinia viruses and cytotoxicity assays
KIR2DL4 carrying an HA epitope tag at the N terminus (HA-2DL4) and an arginine-tyrosine to glycine-threonine (RY-GT) mutant of HA-2DL4 have been described (18). The unique tyrosine of the cytoplasmic tail of HA-KIR2DL4 was mutated to a phenylalanine residue using the oligonucleotide 5'-GGAGGTGACATTCGCACAGTTGG-3' and its complement with the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The mutation was confirmed by sequencing, and the mutant HA-KIR2DL4 Y-F was cloned into the plasmid pSC65 and used to generate recombinant vaccinia viruses (Vac), as previously described (28). Recombinant vaccinia viruses encoding KIR2DL1 and KIR2DL3 Y312F have been described (28, 29). Purified viruses were used to infect human NK cells at 20 PFU/cell, and cell surface expression of recombinant proteins was monitored by FACS analysis. Infected or uninfected human NK cells were preincubated for 15 min at room temperature with Abs, and tested for their ability to kill 51Cr-prelabeled P815 cells (2500 cells/well) in a 3-h 51Cr release assay, as described (27). All points were conducted in triplicate, and every experiment was repeated at least three times.
GST fusion proteins
A series of GST fusion proteins with the cytoplasmic tails of different KIR were produced. Templates corresponding to the cloned cDNA of KIR2DS4 (CD158i), KIR2DL1 (CD158a), KIR2DL3 (CD158b2), KIR2DL4, KIR3DL1 (CD158e1), and KIR3DL2 (CD158k) were used for PCR amplification. The primers used to amplify the cytoplasmic tails of these individual KIRs are the following: common antisense primer, 5'-GCATCTCGAGGCCTTCAGATTCCAGCTGCTGG-3'; sense primers to amplify, 2DS4, 5'-GCATGGATCCCATCGCTGGTGCTCCGACAA-3'; 2DL1, 5'-GCATGGATCCCATCGCTGGTGCTCCAACAAA-3'; 2DL3, 5'-GCATGGATCCCATCGCTGGTGCTGCAACAAA-3'; 2DL4, 5'-GCATGGATCCCATCGCTGGTGCTCCAAAAAA-3'; 3DL1, 5'-GCATGGATCCCATCTCTGGTGCTCCAACAAA-3'; 3DL2, 5'-GCATGGATCCTATCGCTGGTGCTCCAACAAA-3'. PCR amplification was performed as follows: 28 cycles at 94°C for 25 s, 60°C for 45 s, and 72°C for 45 s. PCR products were digested with XhoI and BamHI and cloned in the pGEX-4T-1 vector (Pharmacia Biotech, Piscataway, NJ). GST fusion proteins were purified as described (30). Nonphosphorylated GST fusion proteins were produced in BL21(DE3) bacteria (Stratagene, La Jolla, CA), whereas tyrosine-phosphorylated GST fusion proteins were prepared from transformed TKB1 bacteria (Stratagene). The TKB1 strain of Escherichia coli can be induced with indole-acrylic acid to express a protein tyrosine kinase, which phosphorylates on tyrosine fusion proteins expressed in the same cell. Phosphorylation of the GST fusion proteins was confirmed by Western blot with 4G10 mAb (Upstate Biotechnology). No tyrosine phosphorylation of GST fusion proteins isolated from BL21(DE3) bacteria was detected.
Pull-down experiments, depletion, and immunoprecipitation
A total of 90 x 106 IL-2-activated human NK cells was radiolabeled with [35S]methionine (0.1 mCi/5 x 106 cells/1 ml; Amersham, Arlington Heights, IL) for 7 h. The cells were then lysed in lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.4, 2 mM EGTA, 1% Triton X-100, 1 mM sodium orthovanadate), and, following preclearing with purified GST, the total cellular protein lysate was aliquoted and incubated with each described GST fusion protein (5 nmol/10 x 106 cells) in a final volume of 1 ml. After a 2-h incubation at 4°C, 50 µl glutathione-Sepharose (Pharmacia Biotech, Piscataway, NJ) was added for 45 min. Bound proteins were washed three times with lysis buffer, eluted with reduced glutathione (Pharmacia Biotech), resolved on SDS-polyacrylamide gels, fixed in a 10% acetic acid/50% methanol solution, and revealed by autoradiography. To deplete the total lysate of SHP-1, total cellular proteins were subjected to three consecutive immunoprecipitations with 20 µg anti-SHP-1 (or anti-IRS-1 as control). After each round, protein A-Sepharose (Pharmacia Biotech) was used to remove Ab-coated SHP-1. Efficient depletion of SHP-1 was confirmed by immunoprecipitation of the phosphatase from SHP-1- or IRS-1-depleted total proteins. Binding of SHP-1, SHP-2, and SHIP to GST-KIR fusion proteins was measured by Western blot from total lysate of unlabeled YTS cells. Western blot was performed as described (29) with specific primary Ab and goat anti-rabbit IgG peroxidase (Amersham) or goat anti-mouse IgG peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) as the secondary Ab and Super Signal as the substrate (Pierce, Rockford, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using an anti-KIR2DL4-specific mAb and P815 as target cells in
a redirected Ab-dependent cellular cytotoxicity (rADCC) assay, we have
recently reported that KIR2DL4 is an activating receptor in NK cells
(18). Wild-type HA-tagged KIR2DL4 (to distinguish mutated
from endogenous receptors) and a mutation of the arginine-tyrosine
motif to glycine-threonine (RY-GT) in the transmembrane region of
HA-KIR2DL4 expressed by recombinant vaccinia viruses (Fig. 1
) showed the requirement of the
arginine-tyrosine motif to induce activating signals of KIR2DL4
(18). The charged arginine residue is thought to mediate
interaction of KIR2DL4 with a partner chain that would be responsible
for the activating signal. Using the same technical approach, we show
in this study that NK cells expressing HA-2DL4 wild type (Fig. 2A) kill P815 targets at the
same level whether triggered with an anti-HA mAb or through
engagement of the activating receptor 2B4 (CD244) (Fig. 2Bb). Anti-HA mAb did not enhance lysis of P815 by
uninfected NK cells, compared with control IgG, whereas a strong lysis
of P815 was obtained in response to anti-CD16 mAb (Fig. 2Ba). Coengagement of HA-2DL4 wild type with 2B4 led to an
additive effect and induced a level of lysis of P815 equivalent to that
obtained by triggering the activating receptor CD16 (Fig. 2Bb).
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) was expressed
on NK cells using vaccinia viruses. Infection of NK cells with
Vac-HA-2DL4 Y-F resulted in uniform expression of HA-2DL4 Y-F at the
cell surface, equivalent to that of HA-2DL4 wild type, as detected by
FACS analysis (Fig. 2A). In a rADCC assay, NK cells infected
with Vac-HA-2DL4 Y-F lysed P815 cells in the presence of anti-HA
mAb to the same extent as NK cells infected with Vac-HA-2DL4 wild type
(Fig. 2Bc). Lysis obtained through ligation of CD16 alone
was not affected by coligation of HA-2DL4 Y-F (Fig. 2Bc). As
with HA-2DL4 wild type, coligation of 2B4 and HA-2DL4 Y-F resulted in
an additive effect on P815 lysis (data not shown). Specific lysis
induced by HA-2DL4 Y-F was observed over a wide range of anti-HA
mAb concentration (Fig. 2Bd). (All other experiments with
HA-2DL4 in this study were conducted at a 1/200 dilution of anti-HA
mAb.) These results show that KIR2DL4 delivers activating signals that
do not require the unique ITIM present in the cytoplasmic
tail. KIR2DL4 is sensitive to ITIM-mediated inhibition
HA-2DL4 wild type delivers activating signals to NK cells, despite
the presence of an ITIM in its cytoplamic tail. We therefore asked
whether KIR2DL4 was resistant to inhibition by ITIM-containing
receptors. NK cells were coinfected with Vac-HA-2DL4 Y-F and Vac-2DL1.
The cell surface level of HA-2DL4 Y-F was much lower after coinfection
with Vac-2DL1 (Fig. 3A) than
after infection with Vac-HA-2DL4 Y-F alone (Fig. 2A).
Despite low cell surface levels, activation through HA-2DL4 Y-F was
still strong in the P815-redirected lysis assay, reaching a level
comparable with that with anti-CD16 (Fig. 3Ba).
Cocross-linking of KIR2DL1, which was expressed at a high level,
blocked activation through HA-2DL4 Y-F as efficiently as that through
CD16 (Fig. 3Ba). To test whether activation through HA-2DL4
Y-F was sensitive to coengagement of a receptor carrying a single ITIM
(as in KIR2DL4), NK cells were coinfected with Vac-HA-2DL4 Y-F and a
vaccinia virus encoding Vac-2DL3 Y312F (an inhibitory KIR lacking the
tyrosine in the second ITIM (Fig. 1)). Coligation of KIR2DL3 Y312F
completely inhibited the P815 cell lysis induced by HA-2DL4 Y-F and
that induced by CD16 (Fig. 3Bb). These results show that
activation through KIR2DL4 is sensitive to inhibition induced by a
conventional KIR2DL and by a KIR containing only the
membrane-proximal ITIM.
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The sensitivity of KIR2DL4 activation signals to inhibition by a
receptor carrying only the membrane-proximal ITIM suggested that the
membrane-proximal ITIM of KIR2DL4 itself may not be functional. We
asked whether the ITIM of KIR2DL4 could provide inhibitory function by
using the activation-deficient HA-2DL4 RY-GT mutant expressed with a
recombinant vaccinia virus (Fig. 4A). As we reported,
triggering HA-2DL4 RY-GT with an anti-HA mAb did not induce lysis
of P815 cells (18) (Fig. 4Ba). Coligation of
HA-2DL4 RY-GT with CD16 did not inhibit the CD16-induced
lysis of P815 cells (Fig. 4Ba). In this experiment, CD16 was
cross-linked with a saturating concentration of the anti-CD16 mAb
3G8 (1 µg/ml). KIR2DL1, containing two ITIMs, blocked the CD16
activation signal completely (Fig. 4, A and Bb),
as did KIR2DL3 Y312F (Fig. 4, A and Bc). As
KIR2DL4 and KIR2DL3 Y312F have the same conserved ITIM sequence (Fig. 1), the lack of inhibition by HA-2DL4 RY-GT is not simply due to the
lack of a second ITIM. The inhibitory effect of HA-2DL4 RY-GT was then
tested under conditions of limiting signals through CD16. In
control experiments with uninfected cells, anti-HA ascites or control
ascites did not affect the anti-CD16 dose-response profile (Fig. 4Ca). In contrast, 5–10 times more anti-CD16 Ab was
required when HA-2DL4 RY-GT was cocross-linked, to match the level of
P815 lysis obtained in the presence of control ascites (Fig. 4Cb). The partial inhibition triggered by KIR2DL4 RY-GT may
be due to lower vaccinia virus-mediated receptor expression and/or to a
weaker affinity of the HA-specific Ab than that of EB6 for KIR2DL1 or
GL183 for KIR2DL3. Alternatively, the cytoplasmic tail of KIR2DL4 might
be intrinsically less inhibitory than that of other KIR2DL receptors.
Altogether, these results indicate that the cytoplasmic tail of KIR2DL4
has inhibitory potential.
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The cytoplasmic tails of several KIRs were produced as GST fusion
proteins to test for their association with cytoplasmic proteins. The
cytoplasmic tails of KIR2DL1, KIR2DL3, KIR3DL1 (which contain two
ITIMs), of KIR3DL2 (which has a nonstandard second ITIM and 11 unique
C-terminal amino acids), and of KIR2DS4 (which carries a short
ITIM-less cytoplasmic tail) were compared with the single
ITIM-containing cytoplasmic tail of KIR2DL4 (Fig. 5). As KIR2DL4 carries a unique
C-terminal sequence of 37 aa not found in other KIRs, we were
interested in the possibility of specific protein associations with the
tail of KIR2DL4. Either tyrosine-phosphorylated or nonphosphorylated
GST fusion proteins were produced in E. coli.
Phosphorylation of each GST-KIR fusion protein was confirmed by Western
blot using a phosphotyrosine-specific mAb (data not shown).
IL-2-activated human NK cells were labeled with
[35S]methionine, and proteins from total
lysates were pulled down with GST-KIR fusion proteins attached to
glutathione-Sepharose. Associated proteins were analyzed by SDS-PAGE
and autoradiography (Fig. 6A).
No specific protein association was detected with the unphosphorylated
cytoplasmic tails of any of the KIRs (data not shown). In contrast,
tyrosine-phosphorylated tails of each KIR, except for KIR2DS4, bound
protein(s) with an apparent molecular mass of 62–64 kDa. This
protein(s) did not bind to GST alone (Fig. 6A). Therefore,
one predominant protein species associated with all the phosphorylated
ITIM-containing tails, and no unique association was detected with the
phosphorylated GST-2DL4 fusion protein.
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B, arrow 1). Another protein of 
64 kDa remained after
SHP-1 depletion (Fig. 6B, arrow 2). This protein corresponds
in size to the phosphatase SHP-2. Western blotting was performed to
confirm association of SHP-1 and to test whether SHP-2 was also bound
to the phosphorylated cytoplasmic tails of KIR2DL1 and KIR2DL4. SHP-1
and SHP-2 were specifically pulled down by glutathione-Sepharose coated
with tyrosine-phosphorylated GST-2DL1 and GST-2DL4 (Fig. 6C). Western blotting analysis with Abs specific for the
inositol phosphatase SHIP did not reveal association of this protein
after pull down with phosphorylated GST-2DL1 or GST-2DL4
glutathione-Sepharose (Fig. 6C). SHIP binds a
tyrosine-phosphorylated sequence motif distinct from, but similar to
the SHP-1-binding motif (31). Our results show that, when
phosphorylated, the ITIM of KIR2DL4 can bind the phosphatase SHP-1, as
do inhibitory KIR with two ITIMs, confirming the inhibitory potential
of KIR2DL4. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion in both resting and activated peripheral
NK cells (18).
These different results underscore the importance of determining the
outcome of signaling by KIR2DL4. We report in the present study that
KIR2DL4 has the potential for both activating and inhibitory functions.
Target cell lysis by activated NK cells induced by KIR2DL4 in a
redirected lysis assay does not require an intact cytoplasmic ITIM, but
an intact transmembrane region that includes an arginine residue (Ref.
18 , and this study). Target cell lysis induced by KIR2DL4
with a mutated ITIM in a rADCC assay was as efficient as that induced
by wild-type KIR2DL4. This result suggests that the ITIM may serve
other functions, such as inhibition of other signals. Indeed, as shown
in this study, the single ITIM of KIR2DL4 can bind tyrosine
phosphatases and deliver negative signals. The nature of the signal
delivered by KIR2DL4 may depend on the type of cell expressing KIR2DL4.
For instance, it is interesting that KIR2DL4 engagement on resting NK
cells results in IFN- production, but not cytotoxicity, whereas
IL-2-activated NK cells respond to KIR2DL4 by increasing both IFN-
secretion and cytotoxicity (18). Whether natural mutations
that disrupt the arginine in the transmembrane region of KIR2DL4 exist
is not known. This type of mutation has been described for the
activating receptor KIR2DS2 (36). As a result, the mutant
KIR2DS2, lacking the lysine residue in the transmembrane region, failed
to associate with DAP12 and to deliver activation signals.
Coligation of a nonactivating mutant of KIR2DL4 (the HA-tagged RY-GT mutant) with CD16 resulted in a dose-response shift in target cell lysis induced by CD16, in a rADCC assay. Greater concentrations of anti-CD16 mAb were required when HA-2DL4 RY-GT was cocross-linked to achieve the same level of target cell lysis than that induced by engaging CD16 alone. However, this inhibition was not as strong as that obtained by cocross-linking wild-type KIR2DL1. The presence of a single ITIM in KIR2DL4, as opposed to the two ITIMs found in inhibitory KIR2DL receptors, does not explain the weaker inhibition because a mutated KIR2DL3 carrying only the first ITIM was fully inhibitory in the same assay. It is possible that cross-linking HA-2DL4 RY-GT through the N-terminal HA epitope tag does not generate the full inhibitory signal. Alternatively, the context of the unique KIR2DL4 cytoplasmic tail may dampen the inhibitory function of the ITIM. As the inhibitory function of the KIR2DL4 cytoplasmic tail was tested in the context of a mutated receptor that was unable to activate, it is not known whether this inhibitory signal can be delivered by a wild-type KIR2DL4.
The normal function of the ITIM in KIR2DL4 is not known. Although the
level of killing induced by wild-type KIR2DL4 and the KIR2DL4 Y-F
mutant is similar, it is possible that the ITIM serves to down-modulate
activation signals transmitted by KIR2DL4 itself. Such modulation may
contribute to the unique ability of KIR2DL4 to induce IFN- secretion
without inducing cytotoxic activity in resting NK cells
(18). Alternatively, recruitment of SHP-1 and SHP-2 by the
KIR2DL4 ITIM may serve to terminate the activation signal, in a manner
similar to the negative autoregulation of the erythropoietin receptor
by SHP-1 recruitment (37). It is also possible that the
ITIM of KIR2DL4 does not recruit SHP in intact cells because of
structural constraints imposed by the putative partner chain or by the
cytoplasmic tail itself. The activation signal transmitted by wild-type
KIR2DL4 leading to IFN- secretion by activated NK cells is sensitive
to inhibition by ITIM-containing receptors, such as CD94/NKG2A
(18). In this study, coligation of the activating KIR2DL4
Y-F with either a wild-type KIR2DL1 or a mutated KIR2DL3 carrying only
the first ITIM resulted in complete inhibition of the KIR2DL4 signal,
indicating that KIR2DL4 is potentially sensitive to the ITIM sequence
present in its own cytoplasmic tail.
KIR2DL4 contains a charged amino acid (arginine) in its transmembrane
domain, reminiscent of the lysine residue found in the transmembrane
domain of numerous NK cell-activating receptors, such as NKp30, NKp46,
KIR2DS, and NKG2C, that pair with ITAM-containing signaling partner
chains (4). It is possible that the arginine mediates a
noncovalent interaction with an as yet unidentified partner chain that
transmits the positive signal. Consistent with this hypothesis,
disruption of the arginine-tyrosine motif in the transmembrane domain
abolishes the ability of KIR2DL4 to induce target cell lysis by
IL-2-activated NK cells (Fig. 4Ba) (18). Once
this putative signaling subunit is identified, it will be possible to
test its effect on and sensitivity to the ITIM of KIR2DL4. KIR2DL4
might have a costimulatory function in activation of natural
cytotoxicity, as described for other receptors such as 2B4, CD2, and
NKp80 (2). The mouse target cell line P815 used in our
rADCC experiments expresses ligands for the human-activating receptor
NKp46, and LFA-1 (38). The enhanced lysis of P815 induced
by cross-linking KIR2DL4 is similar to that obtained by cross-linking
2B4 and may result from costimulation of signals from NKp46 and LFA-1.
Interestingly, coengagement of KIR2DL4 and 2B4 resulted in additive
levels of target cell lysis, suggesting that those receptors induce
different signaling pathways. Distinct signaling by these two receptors
was observed in resting NK cells: cross-linking KIR2DL4 induced IFN-
secretion, but not cytotoxicity, whereas the opposite response was
obtained after cross-linking 2B4 (18).
The cytoplasmic tail of KIR2DL4 resembles that of inhibitory KIRs, except for a unique C-terminal extension and for the presence of a cysteine in place of the tyrosine in the second ITIM. The first ITIM is within a stretch of amino acids that is conserved among inhibitory KIRs in primate species (39, 40, 41). Inhibitory KIRs with a mutation in the second ITIM (tyrosine to either phenylalanine or serine) retain inhibitory function in NK cells (29, 42 , and this study) and in a transfected rat basophil cell line (10). Using chimeric receptors expressed in RBL cells, Bruhns et al. (10) reported that a cytoplasmic tail of KIR lacking the tyrosine of the second ITIM recruits tyrosine phosphatase SHP-2 preferentially over SHP-1. In this study, cytoplasmic tails of several KIRs were produced as GST fusion proteins, either phosphorylated or not on tyrosine. When used in pull-down experiments with lysates of NK cells, the phosphorylated tail of KIR2DL4 bound SHP-1 and SHP-2, as did the tail of KIR2DL1. Therefore, both SHP-1 and SHP-2 may be recruited in vivo by the single intact membrane-proximal ITIM of KIR2DL4. The C-terminal extension of the KIR2DL4 cytoplasmic tail is conserved among higher primates and may serve some unique function (39, 40, 41), even though we did not detect other cellular proteins besides SHP-1 and SHP-2 that bound to the cytoplasmic tail of KIR2DL4.
In conclusion, the activating and inhibitory properties of KIR2DL4 endow this receptor with the potential to regulate different types of NK cell responses.
Note added in proof.
Using a different approach, Yusa et al. (43) also concluded that the cytoplasmic tail of KIR2DL4 has inhibitory function.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; HA, hemagglutinin; IRS-1, insulin receptor substrate-1; ITIM, immunoreceptor tyrosine-based inhibition motif; KIR, killer cell Ig-like receptor; rADCC, redirected Ab-dependent cellular cytotoxicity; SHIP, Src homology 2-containing inositol 5'-phosphatase; SHP, Src homology 2-containing phosphatase; Vac, vaccinia virus.
Received for publication February 1, 2002. Accepted for publication April 17, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production but not cytotoxicity by the killer cell Ig-like receptor KIR2DL4 (CD158d) in resting NK cells. J. Immunol. 167:1877.. J. Immunol. 156:4224.[Abstract]
-subunit of the interleukin-3 receptor. J. Biol. Chem. 272:16189.RIIB. J. Biol. Chem. 275:37357.This article has been cited by other articles:
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  A. Iannello, O. Debbeche, S. Samarani, and A. Ahmad Antiviral NK cell responses in HIV infection: I. NK cell receptor genes as determinants of HIV resistance and progression to AIDS J. Leukoc. Biol., July 1, 2008; 84(1): 1 - 26. [Abstract] [Full Text] [PDF]
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 S. M. Shahjahan Miah, T. L. Hughes, and K. S. Campbell KIR2DL4 Differentially Signals Downstream Functions in Human NK Cells through Distinct Structural Modules J. Immunol., March 1, 2008; 180(5): 2922 - 2932. [Abstract] [Full Text] [PDF]
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E. Estefania, R. Flores, N. Gomez-Lozano, H. Aguilar, M. Lopez-Botet, and C. Vilches Human KIR2DL5 Is an Inhibitory Receptor Expressed on the Surface of NK and T Lymphocyte Subsets J. Immunol., April 1, 2007; 178(7): 4402 - 4410. [Abstract] [Full Text] [PDF]
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K. Wendt, E. Wilk, S. Buyny, J. Buer, R. E. Schmidt, and R. Jacobs Gene and protein characteristics reflect functional diversity of CD56dim and CD56bright NK cells J. Leukoc. Biol., December 1, 2006; 80(6): 1529 - 1541. [Abstract] [Full Text] [PDF]
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B. C. Viertlboeck, F. A. Habermann, R. Schmitt, M. A. M. Groenen, L. Du Pasquier, and T. W. Gobel The Chicken Leukocyte Receptor Complex: A Highly Diverse Multigene Family Encoding at Least Six Structurally Distinct Receptor Types J. Immunol., July 1, 2005; 175(1): 385 - 393. [Abstract] [Full Text] [PDF]
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 L. Abi-Rached and P. Parham Natural selection drives recurrent formation of activating killer cell immunoglobulin-like receptor and Ly49 from inhibitory homologues J. Exp. Med., April 18, 2005; 201(8): 1319 - 1332. [Abstract] [Full Text] [PDF]
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A. Kikuchi-Maki, T. L. Catina, and K. S. Campbell Cutting Edge: KIR2DL4 Transduces Signals into Human NK Cells through Association with the Fc Receptor {gamma} Protein J. Immunol., April 1, 2005; 174(7): 3859 - 3863. [Abstract] [Full Text] [PDF]
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 N. Nikolaidis, I. Makalowska, D. Chalkia, W. Makalowski, J. Klein, and M. Nei Origin and evolution of the chicken leukocyte receptor complex PNAS, March 15, 2005; 102(11): 4057 - 4062. [Abstract] [Full Text] [PDF]
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 M. Draghi, N. Yawata, M. Gleimer, M. Yawata, N. M. Valiante, and P. Parham Single-cell analysis of the human NK cell response to missing self and its inhibition by HLA class I Blood, March 1, 2005; 105(5): 2028 - 2035. [Abstract] [Full Text] [PDF]
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M. I. Bonaparte and E. Barker Killing of human immunodeficiency virus-infected primary T-cell blasts by autologous natural killer cells is dependent on the ability of the virus to alter the expression of major histocompatibility complex class I molecules Blood, October 1, 2004; 104(7): 2087 - 2094. [Abstract] [Full Text] [PDF]
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S.-i. Yusa, T. L. Catina, and K. S. Campbell KIR2DL5 Can Inhibit Human NK Cell Activation Via Recruitment of Src Homology Region 2-Containing Protein Tyrosine Phosphatase-2 (SHP-2) J. Immunol., June 15, 2004; 172(12): 7385 - 7392. [Abstract] [Full Text] [PDF]
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K.-M. Lee, M. E. McNerney, S. E. Stepp, P. A. Mathew, J. D. Schatzle, M. Bennett, and V. Kumar 2B4 Acts As a Non-Major Histocompatibility Complex Binding Inhibitory Receptor on Mouse Natural Killer Cells J. Exp. Med., May 3, 2004; 199(9): 1245 - 1254. [Abstract] [Full Text] [PDF]
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N. Toyama-Sorimachi, Y. Tsujimura, M. Maruya, A. Onoda, T. Kubota, S. Koyasu, K. Inaba, and H. Karasuyama Ly49Q, a member of the Ly49 family that is selectively expressed on myeloid lineage cells and involved in regulation of cytoskeletal architecture PNAS, January 27, 2004; 101(4): 1016 - 1021. [Abstract] [Full Text] [PDF]
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K. S. Campbell, S.-i. Yusa, A. Kikuchi-Maki, and T. L. Catina NKp44 Triggers NK Cell Activation through DAP12 Association That Is Not Influenced by a Putative Cytoplasmic Inhibitory Sequence J. Immunol., January 15, 2004; 172(2): 899 - 906. [Abstract] [Full Text] [PDF]
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A. Kikuchi-Maki, S.-i. Yusa, T. L. Catina, and K. S. Campbell KIR2DL4 Is an IL-2-Regulated NK Cell Receptor That Exhibits Limited Expression in Humans but Triggers Strong IFN-{gamma} Production J. Immunol., October 1, 2003; 171(7): 3415 - 3425. [Abstract] [Full Text] [PDF]
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J. P. Goodridge, C. S. Witt, F. T. Christiansen, and H. S. Warren KIR2DL4 (CD158d) Genotype Influences Expression and Function in NK Cells J. Immunol., August 15, 2003; 171(4): 1768 - 1774. [Abstract] [Full Text] [PDF]
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),
1 µg/ml anti-CD16 (
), 1 µg/ml anti-2B4 (
), a 1/200
dilution of an anti-HA ascites (
), or with the same
concentration of anti-CD16 + anti-HA (•) or anti-2B4 +
anti-HA (
). Bd, Uninfected (open symbols) or
Vac-HA-2DL4 Y-F-infected (filled symbols) human NK cells were incubated
with a serial dilution of anti-HA ascites (squares) or control
ascites (circles), at a 3:1 E:T ratio. NK cells were then tested for
their ability to kill 51Cr-labeled FcR+ P815
target cells in a 3-h rADCC assay.
), 1 µg/ml anti-KIR2DL3 (
), a 1/200
dilution of an anti-HA ascites (
), anti-CD16 + anti-KIR2DL3 (
), anti-HA +
anti-KIR (
, KIR2DL1 or KIR2DL3 in a or
b, respectively). Lysis of P815 was detected as
indicated in Fig. 2
), at
a 1:1 E:T ratio. Lysis of P815 was detected as indicated in Fig. 2
