By
From the * Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Rockville, Maryland 20852; Laboratory of Allergy and Immunology,
Department of Pathology, Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts
02215; and the § Fred Hutchinson Cancer Research Center, Seattle, Washington 98104
Inhibition of natural killer (NK) cells by the killer cell inhibitory receptor (KIR) involves recruitment of the tyrosine phosphatase SHP-1 by KIR and is prevented by expression of a dominant negative SHP-1 mutant. Another inhibitory receptor, the low affinity Fc receptor for immunoglobulin G (IgG) (FcRIIb1), has been shown to bind SHP-1 when cocross-linked with
the antigen receptor on B cells (BCR). However, coligation of Fc
RIIb1 with BCR and with Fc
RI on mast cells leads to recruitment of the inositol 5
phosphatase SHIP and to inhibition
of mast cells from SHP-1-deficient mice. In this study, we evaluated the ability of these two
inhibitory receptors to block target cell lysis by NK cells, and the contribution of SHP-1 and
SHIP to inhibition. Recombinant vaccinia viruses encoding chimeric receptors and dominant
negative mutants of SHP-1 and SHIP were used for expression in mouse and human NK cells. When the KIR cytoplasmic tail was replaced by that of Fc
RIIb1, recognition of HLA class I
on target cells by the extracellular domain resulted in inhibition. A dominant negative mutant
of SHP-1 reverted the inhibition mediated by the KIR cytoplasmic tail but not that mediated
by Fc
RIIb1. In contrast, a dominant negative mutant of SHIP reverted only the inhibition
mediated by the Fc
RIIb1 tail, providing functional evidence that SHIP plays a role in the
Fc
RIIb1-mediated negative signal. These data demonstrate that inhibition of NK cells by
KIR involves primarily the tyrosine phosphatase SHP-1, whereas inhibition mediated by
Fc
RIIb1 requires the inositol phosphatase SHIP.
Activation of different types of cells in the immune system shares common signal transduction pathways,
such as activation of tyrosine kinases, turnover of phosphoinositides, and calcium mobilization. The description of
several inhibitory receptors that can interrupt the activation
process in different cell types has generated interest in the
mechanism of inhibition and raised questions about features
that may be shared by the different receptors. It has been
proposed that both the inhibitory Fc receptor Fc Fc The KIR expressed on human natural killer (NK) cells
deliver a negative signal upon recognition of MHC class I
molecules on target cells (13, 14). Tyrosine phosphorylation of the ITIMs in the cytoplasmic tail of KIR is critical
for recruitment of SHP-1 and for inhibition (2, 15). Expression of a dominant negative mutant of SHP-1 in NK
cells prevented the inhibition mediated by KIR, suggesting
an important role for SHP-1 in the negative signal (2). The
possibility that SHIP may also be involved in the inhibition
of NK cells mediated by KIR has not been investigated. To
test rigorously whether KIR and Fc Cells and Antibodies.
The human NK cell line NK92 (a gift
from H.-G. Klingemann, The Terry Fox Laboratory, Vancouver,
Canada) (16) was maintained in Myelocult H5100 medium (Stem
Cell Technologies, Inc., Vancouver, Canada) and supplemented
with 100 U/ml rIL-2. The B lymphoblastoid cell line 721.221 and its HLA class I transfectants .221-Cw3 and .221-Cw4 were provided by J. Gumperz and P. Parham (Stanford University, Stanford, CA). Mouse NK populations were generated from C57BL/6 mice as described (17). The anti-p58-KIR mAb GL183 (18) was provided by A. Moretta and C. Bottino (University of Genova,
Italy) and anti-p58-KIR mAb EB6 was obtained from Immunotech,
Inc. (Westbrook, ME). The FITC-conjugated F(ab cDNAs and Construction of Chimeras.
The chimeric KIR-6/
RIIb1 cDNA was constructed by ligation of fragments encoding
the extracellular and transmembrane regions of KIR-6 (a p58-KIR
reactive with mAb GL183) with the cytoplasmic region of human Fc Dominant Negative Mutants of SHP-1 and SHIP.
A recombinant
vaccinia virus encoding a mutant form of SHP-1 with a point mutation in the catalytic site (cysteine 453 to serine) has been described (2). A cDNA encoding SHIP was used to make a deletion
mutant containing the SH2 domain alone (referred to as SHIP-
SH2) by PCR amplification from the start site to leucine 166 followed by a stop codon and a SalI site. The PCR fragment was
cloned into a modified version of the vaccinia vector pSC66, which contains at its NH2 terminus an ATG initiation site in a suitable context, a Flag epitope (21), and a NotI site in-frame with the coding sequence.
Vaccinia Virus Infections.
Purified viruses encoding KIR-6,
KIR-6/RIIb1, KIR-6tr, KIR-42, KIR-42/RIIb1, SHP-1C453S,
and SHIP-SH2 were used to infect mouse NK populations or the
human cell line NK92 as described (14).
Cytotoxicity Assays.
The ability of mouse NK and human
NK92 cells to lyse targets was measured in a 4-h 51Cr release assay
as described (14).
To test whether the cytoplasmic tail of Fc
Expression of KIR-6 on mouse NK cells inhibits the antibody-dependent cell-mediated cytotoxicity of HLA-Cw3
positive .221 targets (15). NK1.1+, TCR
Expression of a catalytically inactive mutant of SHP-1 in
human NK cells reverts the KIR-mediated inhibition of target cell lysis (2, 6). KIR-6, KIR-42, and their chimeric counterparts KIR-6/RIIb1 and KIR-42/RIIb1 were expressed
in the human NK cell line NK92 either individually or together with the dominant negative mutant of SHP-1. The
surface staining of Vac-6 and Vac-6/RIIb1 infected cells by
GL183 and that of Vac-42 and Vac-42/RIIb1 infected cells
by EB6 was comparable as seen by the mean fluorescence
intensities (Fig. 3). Coinfection of NK92 cells with the receptors along with dominant negative SHP-1 did not alter
their surface expression (Fig. 3). NK92 cells infected with
Vac-6 or with Vac-6/RIIb1 killed .221 and .221-Cw4 targets but were inhibited from lysing .221-Cw3 targets. Reciprocally, cells infected with Vac-42 and Vac-42/RIIb1 lysed .221 and .221-Cw3 but not .221-Cw4 targets (Fig.
3). Therefore, as shown in mouse NK cells, the cytoplasmic tail of Fc
A recent report showed that mast cells from SHP-1-deficient me/me mice are inhibited by Fc
These data provide functional evidence that SHIP is required for the negative signal delivered by Fc Stimulation of B cells by receptor cross-linking involves
rapid release of Ca2+ from intracellular stores and a sustained influx of extracellular calcium (25). A significant feature of Fc The evolution of distinct mechanisms to block lymphocyte activation may relate to the consequences of partial activation of various types of lymphocytes. Cocross-linking
of surface Ig with FcRIIb1 and the killer cell inhibitory receptor (KIR) mediate inhibition by recruitment of the SH2-containing protein tyrosine phosphatase SHP-1 (1). However, it has also been
shown that Fc
RIIb1 can inhibit mast cells that do not express SHP-1 (7). The aim of this study was to compare the
inhibition mediated by Fc
RIIb1 and KIR in the same cell
type and to evaluate the contribution of SHP-1 and SH2-containing inositol polyphosphatase 5
phosphatase (SHIP)
to these negative signals.
RIIb1 inhibits activation responses when cocross-linked with the B cell, T cell, and mast cell antigen receptors (8). The cytoplasmic tail of Fc
RIIb1 contains an
immunoreceptor tyrosine-based inhibitory motif (ITIM),
which is necessary for the inhibitory function of the receptor (9, 10). In vitro, the phosphorylated Fc
RIIb1 ITIM
associates with SHP-1 and to a 145-kD protein designated as SHIP (7). SHIP is involved in inhibition of growth factor and cytokine responses (11, 12). Coligation of Fc
RIIb1
with the IgE receptor on bone marrow-derived mast cells
from me/me mice, which lack SHP-1, still resulted in inhibition of degranulation (7), demonstrating that SHP-1 was
not necessary for inhibition and suggesting that SHIP might
be involved in the negative signal mediated by Fc
RIIb1.
However, no functional evidence has been obtained for a
role of SHIP in the inhibition mediated by Fc
RIIb1.
RIIb1 can deliver inhibitory signals in the same cell type through similar or distinct signaling pathways, and whether SHIP may be involved in the KIR-mediated inhibition of NK cells, we have
constructed chimeric KIR molecules with the cytoplasmic
tail of Fc
RIIb1 and assessed their ability to inhibit NK
cells. We report here that SHIP, but not SHP-1, is required for the negative signal transmitted by Fc
RIIb1 and
that the reverse is true for KIR.
)2 goat anti-
mouse IgG was from Jackson ImmunoResearch (West Grove, PA).
RIIb1. The cytoplasmic tail of Fc
RIIb1 was amplified
from a cDNA molecule containing the transmembrane and cytoplasmic regions of human Fc
RIIb1 (a gift from J. Altrichter, National Institute of Allergy and Infectious Diseases, Bethesda, MD)
using the forward primer 5
-TTGATCTACCTTAGGAAAAA
GCGGATTTCAG-3
containing a Bsu36I restriction site and
reverse primer 5
-TGCACCGAATTCAGACTAAATACGG-3
containing a stop codon followed by an EcoRI restriction site. The fragment encoding KIR-6 used for ligation was amplified
from the plasmid pSPORT-p58-cl6 (19), using forward primer
5
-AGCTCCCGGAGCTCCTATGACATG-3
containing a SacI
site and the reverse primer 5
-GCAGCAGGATCCCCTAAGGAGAAAGAAGAGGAG-3
containing a Bsu36I site and a
BamHI site for cloning into pBluescript. The Bsu36I-EcoRI
fragment encoding the cytoplasmic tail of Fc
RIIB1 was ligated
to the Bsu36I-EcoRI digested pBluescript-cl6, followed by excision of the SacI-EcoRV chimeric insert and subcloning into the
SacI-StuI sites of pSPORT-cl6. The amino acids at the KIR-6/ Fc
RIIb1 boundary are FFLL/RKKR. Chimeric KIR-42/RIIb1
was made by replacing the extracellular segment of KIR-6 between the restriction sites SalI-BstEII with that of KIR-42 (a
p58-KIR reactive with mAb EB6). The BstEII site encodes the
amino acids glycine and asparagine that are five residues upstream
from the start of the transmembrane region in both KIR-6 and
KIR-42. A truncated version of KIR-6 was generated by amplifying a fragment of pSPORT-cl6 (from the initiation codon to
asparagine 258 of KIR-6) using the forward primer 5
-GTCAAACTCGAGTGACCCAC-3
containing the XhoI site and
the reverse primer 5
-CTGTTCTCCCGGGTCATCCTTGGTTGTCCATTACAACAGC-3
containing StyI site followed
by a stop codon and a SmaI site. The XhoI-SmaI fragment was
then ligated back into the XhoI-StuI sites of pSPORT-cl6 such
that the cytoplasmic tail is 17 amino acids long (the last three residues, asparagine, glutamine, and glycine were added before the
stop codon during PCR amplification) and lacks the two ITIM
sequences. The truncated KIR-6 (KIR-6tr) and the chimeric
constructs KIR-6/RIIb1 and KIR-42/RIIb1 were subcloned
into the plasmid pSC66 (gift of B. Moss, National Institute of Allergy and Infectious Diseases, Bethesda, MD) and used to generate recombinant vaccinia viruses as described (20).
RIIb1 may
inhibit NK cells, and to address the role of the tyrosine
phosphatase SHP-1 and inositol phosphatase SHIP in the
negative signal delivered by these receptors, chimeric molecules with the cytoplasmic tail of Fc
RIIb1 and the extracellular domains of KIR-6 or KIR-42 were constructed
(Fig. 1). As a control, a truncated version of KIR-6 was
made (Fig. 1), which lacks both ITIM sequences that are
essential for KIR-mediated inhibition (4, 15). KIR-6 prevents the lysis of HLA-Cw3 positive target cells, whereas
KIR-42 prevents lysis of targets that express HLA-Cw4
(14). Recombinant vaccinia viruses were made that express
the chimeric KIR-6/RIIb1, KIR-42/RIIb1, and KIR-6tr
molecules.
Fig. 1.
Schematic representation of the chimeric receptors. Hybrid
molecules bearing the cytoplasmic tail of human FcRIIb1 and the extracellular domains of KIR-cl6 or KIR-cl42 were generated as described.
Open, hatched, and shaded boxes represent sequences from KIR-6, KIR-42, and Fc
RIIb1, respectively. Closed boxes represent transmembrane
regions and open bars in the cytoplasmic tails indicate the position of ITIMs.
[View Larger Version of this Image (18K GIF file)]
NK cells, prepared from the spleens of C57Bl/6 mice were infected with Vac-6, Vac-6/RIIb1, and Vac-6tr and tested for their
ability to lyse .221 and .221-Cw3 target cells. The infected
cells expressed comparable levels of the three receptors
(Fig. 2 A). NK cells expressing KIR-6/RIIb1 lysed .221 cells but had reduced cytotoxic activity against .221-Cw3
cells. Uninfected cells and those infected with Vac-6tr lysed
both .221 and .221-Cw3 to the same extent (Fig. 2 B). Thus,
the cytoplasmic tail of Fc
RIIb1 can deliver a signal that
inhibits the cytotoxic function of NK cells.
Fig. 2.
The cytoplasmic tail of human FcRIIb1 can deliver an inhibitory signal in mouse NK cells. (A) Surface expression of KIR-6, KIR-6/RIIb1, and KIR-6tr on cells infected with 5, 5, and 10 PFU/cell of the
indicated recombinant vaccinia viruses, respectively. (B) Specific lysis of
the B cell line .221 (open circles), and its HLA-Cw3 transfectant (closed circles) by uninfected mouse NK cells or those infected with Vac-6, Vac-6/
RIIb1 or Vac-6tr. ADCC was induced by precoating the targets with 0.1 µg/ml of anti-HLA-DR mAb L243 for 30 min on ice. Effectors and targets were plated at the indicated ratios.
[View Larger Version of this Image (19K GIF file)]
RIIb1 was able to deliver an inhibitory signal in human NK cells. Expression of dominant negative
SHP-1 in NK92 reverted the KIR-mediated inhibition of
target cell lysis but not that mediated by the cytoplasmic tail
of Fc
RIIb1 (Fig. 3). Thus, SHP-1 appears to play an important role in KIR-mediated inhibition of NK cells, as reported earlier (2), but not in Fc
RIIb1-mediated inhibition
of NK cells.
Fig. 3.
SHP-1 is not required for inhibition of NK cells by
FcRIIb1. The KIR-negative human NK92 cell line was infected with
recombinant vaccinia viruses expressing KIR-6, KIR-42, or their chimeric derivatives bearing the cytoplasmic tail of human Fc
RIIb1 (KIR-6/RIIb1 and KIR-42/RIIb1), alone or with dominant negative SHP-1
(dnSHP-1), as indicated. The mean fluorescence intensity of staining with mAbs GL183 (for KIR-6 and KIR-6/RIIb1) and EB6 (for KIR-42 and KIR-42/RIIb1) is indicated next to each bar in the .221 panel. Lysis of
.221, .221-Cw3, and .221-Cw4 targets was determined in a 4-h 51Cr release assay at an E/T of 4. Similar results were observed at an E/T of 1 and in two independent experiments.
[View Larger Version of this Image (28K GIF file)]
RIIb1 (7). Coclustering Fc
RIIb1 with the BCR or Fc
RI led to recruitment of
SHIP, suggesting that SHIP is involved in inhibition mediated
by Fc
RIIb1 (7). We tested functionally whether SHIP has
a role in Fc
RIIb1-mediated inhibition and whether it
may also contribute to KIR-mediated inhibition of NK
cells. KIR or chimeric KIR were expressed in NK92 cells together with a dominant negative mutant of SHIP containing the SH2 domain alone. Expression of SHIP-SH2 in
NK92 caused some reduction in the ability of NK92 to lyse
target cells, as well as a small reduction in the expression of
KIR during coinfections (Fig. 4). The partial inhibition
mediated by KIR-6 when NK92 were tested with .221-Cw4
cells has not been consistently observed. Coexpression of
SHIP-SH2 prevented the inhibition mediated by KIR-6/
RIIb1 and KIR-42/RIIb1 but not that mediated by KIR-6
or by KIR-42 (Fig. 4).
Fig. 4.
SHIP is required for inhibition of NK cells by FcRIIb1 but
not by KIR. The experiment was as described in Fig. 3 except that a
dominant negative SHIP (dnSHIP) was used. The data shown were obtained at an E/T of 6. Similar results were obtained at an E/T of 1 and in
two independent experiments.
[View Larger Version of this Image (27K GIF file)]
RIIb1. In
addition, they demonstrate that two distinct signaling pathways can turn off the cytotoxic activity of NK cells upon
interaction with target cells. The inhibitory receptor normally used by human NK cells to detect self-MHC class I
on target cells requires interaction with the tyrosine phosphatase SHP-1 to deliver the inhibitory signal (2, 6). In
contrast, Fc
RIIb1, whose function is to inhibit B cell and
mast cell activation in the presence of soluble IgG, requires
the inositol phosphatase SHIP to inhibit NK cells. KIR
signaling interferes with early activation events, including
tyrosine phosphorylation of FcR-associated
chain, ZAP-70, and PLC-
2 (6), and calcium flux (22, 23). KIR recognition of HLA class I on target cells was also found to prevent association of the adaptor protein pp36 with PLC-
1
(23). The lack of a role for SHIP in KIR-mediated inhibition is consistent with the identification of SHP-1 as the
only major protein purified from lysates of T, B, and NK
cells by phosphopeptides corresponding to the KIR ITIMs
immobilized on beads (24). In contrast, a phosphopeptide corresponding to the ITIM of Fc
RIIb1 precipitated both
SHP-1 and SHIP from such cell lysates (24). However, the
functional evidence provided here indicates that association
of Fc
RIIb1 with SHP-1 is not necessary for inhibition of
NK cells.
RIIb1-mediated inhibition is a selective block
in calcium influx through the plasma membrane while not
affecting release from intracellular stores (7, 26, 27). The
enzymatic activities of SHIP include removal of the 5
phosphate moiety from substrates like phosphatidylinositol 3,4,5-trisphosphate (PIP3) and inositol 1,3,4,5-tetrakisphosphate (IP4) (11, 12). Therefore, downstream events dependent on these phosphoinositol metabolites are likely to be
inhibited by SHIP (28). Maximal tyrosine phosphorylation
of SHIP and association with Shc was observed in B cells
upon cocross-linking of the BCR with Fc
RIIb1 (29). Thus,
SHIP association with the Fc
RIIb1 ITIM could sequester
Shc and uncouple the early tyrosine phosphorylation events
from late events like p21ras activation (30).
RIIb1 results in accelerated entry into
apoptosis by B cells as compared with that caused by cross-linking surface Ig alone (31). The function of Fc
RIIb1 is to
inhibit Ig-producing and proliferating B cells after soluble
Ig has been produced. Therefore, apoptosis may be useful
to eliminate these cells. The function of KIR is to protect
normal cells from attack by NK cells. Therefore, it is essential for KIR to provide a complete and reliable shutdown of the NK cell. The most suited target in this inhibitory pathway would be tyrosine-phosphorylated substrates that are
proximal in the activation pathway. Recruitment of SHP-1
by KIR followed by rapid dephosphorylation of proximal
substrates may fulfill this requirement. Thus, the dichotomy in the use of SHP-1 by KIR and SHIP by Fc
RIIb1,
as demonstrated by our data, appears well suited to the
function of the cells in which these receptors operate.
Address correspondence to E.O. Long, LIG-NIAID-NIH Twinbrook II, 12441 Parklawn Drive, Rockville, MD 20852-1727. E-mail: elong{at}nih.gov
Received for publication 2 May 1997.
The authors wish to thank M. Weston for technical assistance, J. Altrichter for helpful discussions and a plasmid, H.-G. Klingemann for the cell line NK92, B. Moss for the plasmid pSC66, A. Moretta and C. Bottino for the mAb GL183, and J. Gumperz and P. Parham for the 721.221 HLA transfectants.
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