Evidence That the Cellular Ligand for the
Human NK Cell Activation Receptor NKp30
Is Not a Heparan Sulfate Glycosaminoglycan
This information is current as
of June 18, 2017.
Hilary S. Warren, Allison L. Jones, Craig Freeman, Jayaram
Bettadapura and Christopher R. Parish
J Immunol 2005; 175:207-212; ;
doi: 10.4049/jimmunol.175.1.207
http://www.jimmunol.org/content/175/1/207
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References
The Journal of Immunology
Evidence That the Cellular Ligand for the Human NK Cell
Activation Receptor NKp30 Is Not a Heparan Sulfate
Glycosaminoglycan1
Hilary S. Warren,2 Allison L. Jones, Craig Freeman, Jayaram Bettadapura, and
Christopher R. Parish
N
atural killer cells are cells of the innate immune response
essential for the early response to virally infected cells
and tumor cells. NK cells interact with dendritic cells
(DC)3 to shape the adaptive immune response (1–3) and with CD4
T cells to facilitate immune responses to recall Ags (4, 5). NK cell
killing is dependent on activation receptors interacting with their
relevant ligands on target cells, with this activity controlled
through the class I HLA-binding inhibitory receptors namely, the
killer Ig-like receptors (CD158a, b1, b2, f, e1, k, and z), CD94/
NKG2A, and CD85j (ILT2) (6). NK cell killing is permitted only
with reduced levels of expression of class I HLA Ags on target
cells that can occur as a result of viral infection and malignant
transformation and is a feature of immature DC. Key receptors
involved in triggering NK cell lysis against tumor cells are the NK
cell-specific natural cytotoxicity receptors (NCR), NKp46 (NCR1,
CD335), NKp44 (NCR2, CD336), and NKp30 (NCR3, CD337)
(7). NKp46 and NKp30 are expressed on resting and activated NK
cells, and NKp44 is expressed only on activated NK cells. NKp30
is the only NCR implicated in the cross-talk between NK cells and
DC (8).
Division of Immunology and Genetics, John Curtin School of Medical Research,
Australian National University, Canberra, Australian Capital Territory, Australia
Received for publication February 23, 2005. Accepted for publication April 12, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This study was supported by grants from the National Health and Medical Research
Council of Australia through a Project grant and Fellowship grant (to H.S.W.), and a
Program grant (to C.R.P.). H.S.W. is a Visiting Fellow of the Australian National
University Medical School and a Visiting Research Fellow at the Canberra Hospital.
2
Address correspondence and reprint requests to Dr. Hilary S. Warren, Division of
Immunology and Genetics, John Curtin School of Medical Research, Australian National University, PO Box 334, Canberra, Australian Capital Territory 2601, Australia. E-mail address: [email protected]
3
Abbreviations used in this paper: DC, dendritic cell; NCR, natural cytotoxicity
receptor; GAG, glycosaminoglycan; HS, heparan sulfate; CHO, Chinese hamster
ovary.
Copyright © 2005 by The American Association of Immunologists, Inc.
Despite the importance of NCR, there is limited information on
their ligands. There is evidence that NKp46 and NKp44, but not
NKp30, bind viral hemagglutinins (9 –11). However, the nature of
the endogenous cellular ligands for the NCR remains unknown.
Cellular cytotoxicity assays identify cells that are killed by NK
cells through an NCR-dependent process (12, 13). Binding studies
with recombinant NCR fusion proteins (9, 14) also have identified
cells expressing ligands for these receptors. By these assays, ligands for the NKp46 and/or NKp30 are detected on human, simian, and hamster cells. Evidence that the sulfated glycosaminoglycan (GAG), heparan sulfate (HS), is involved in the recognition of
cellular targets by NKp46 and NKp30 was recently reported (15).
Such a widely distributed carbohydrate epitope expressed on a
variety of protein and/or lipid structures would nicely explain the
broad cellular distribution of the ligands for the NCR.
We show that HS expressed on 293-EBNA cells and other human cells, and on Chinese hamster ovary (CHO)-K1 cells, is not a
ligand for NKp30. First, complete removal of HS by treatment
with mammalian heparanase did not prevent binding of rNKp30/
human IgG1 Fc chimera complexes or liposome-rNKp30 complexes to 293-EBNA cells. Second, NK cell killing of 293-EBNA
cells and other human cell lines was unaffected by heparanase
treatment, and killing of untreated and treated cells was inhibited
by an anti-NKp30 mAb. Third, the GAG-deficient hamster pgsA745 cells, which lack cell surface HS, are killed by NK cells to the
same extent as the GAG-expressing parent CHO-K1 cells, and
killing of both cell lines is inhibited to the same extent by an
anti-NKp30 mAb. These studies do not support the notion that HS
is a ligand for NKp30.
Materials and Methods
Cell lines and culture conditions
NK-92, an activated human NK cell line (16), and the J-774 monocytic
macrophage cell line were obtained from L. Sullivan (University of Melbourne, Parkville, Victoria, Australia). NK-92 was grown in H-5100 Myelocult medium (StemCell Technologies) supplemented with 1% conditioned medium from the J-774 cell line. The 293-EBNA cell line (derived
0022-1767/05/$02.00
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NKp30 (NCR3, CD337) is a natural cytotoxicity receptor, expressed on subsets of human peripheral blood NK cells, involved in
NK cell killing of tumor cells and immature dendritic cells. The cellular ligand for NKp30 has remained elusive, although evidence
that membrane-associated heparan sulfate (HS) proteoglycans are involved in the recognition of cellular targets by NKp30 was
recently reported. The data presented in this report show conclusively that HS glycosaminoglycans (GAG) are not ligands for
NKp30. We show that removing HS completely from the cell surface of human 293-EBNA cells with mammalian heparanase does
not affect binding of rNKp30/human IgG1 Fc chimera complexes or binding of multimeric liposome-rNKp30 complexes. Removing
HS from 293-EBNA cells, culture-generated DC, MM-170 malignant melanoma cells, or HeLa cells does not affect the NKp30dependent killing of these cells by NK cells. We show further that the GAG-deficient hamster pgsA-745 cells that lack HS and the
GAG-expressing parent CHO-K1 cells are both killed by NK cells, with killing of both cell lines inhibited to the same extent by
anti-NKp30 mAb. From these results we conclude that HS GAG are not ligands for NKp30, leaving open the question as to the
nature of the cellular ligand for this important NK cell activation receptor. The Journal of Immunology, 2005, 175: 207–212.
208
Abs and enzymes
The anti-HS mAbs F58-10E4 (cat. no. 370255-1) and HepSS-1 (cat. no.
270426-1) were obtained from Seikaguku Kogyo. The rNKp30/human
IgG1 Fc chimera (cat. no. 1849-NK-025) and the anti-NKp30 mAb (clone
210845, IgG2a) were purchased from R&D Systems. Human IgG was
purchased from the Commonwealth Serum Laboratories. PE- and FITCconjugated mAbs were anti-NKp30 PE (cat. no. IM3709) and anti-CD94
PE (cat. no. IM2276) from Beckman Coulter; anti-HLA-DR PE (cat. no.
347367), Simultest (anti-CD3 FITC/anti-CD16 PE/anti-CD56 PE, cat. no.
340042) and anti-CD14 FITC (cat. no. 347493) from BD Biosciences; and
anti-CD1c PE (anti-BDCA-1, cat. no. 130-090-508) from Miltenyi Biotec.
mAb to class I HLA (DX17, IgG1) was a gift from Prof. L. Lanier (University of California, San Francisco, CA). mAb to NKG2D (clone 149804,
IgG1) was a gift from Dr. J. P. Houchins (R&D Systems, Minneapolis,
MN). The anti-CD56 (WV3, IgG1) was made in our laboratory. FITCconjugated sheep anti-mouse Ig F(ab⬘)2 (cat. no. AQ326) was purchased
from Chemicon International. FITC-conjugated anti-human IgG (cat. no.
62-8411) and biotinylated anti-human IgG (cat. no. 62-8440) were purchased from Zymed Laboratories. Streptavidin-PE was purchased from BD
Pharmingen (cat. no. 554061).
Mammalian heparanase was prepared from human platelets as previously described (20). Cells, at a concentration of 5 ⫻ 106/ml, were incubated with heparanase, at a final concentration of 2 ␮g/ml, in PBS/0.1%
BSA for 90 min at 37°C. The cells were washed three times in PBS/0.1%
BSA before subsequent assays. In some experiments bacterial heparinase-1
(cat. no. H-2519; Sigma-Aldrich) was used at a concentration of 3 U/ml.
were incubated with an anti-NKp30 mAb (10 ␮g/ml) or a mouse isotype
control (10 ␮g/ml) before incubating with the cells.
Liposome-rNKp30 constructs
The extracellular domain of rNKp30 (1C7c) was prepared with a 6-His tag
in a baculovirus expression system. Sequences corresponding to the extracellular domain of NKp30 were amplified from cDNAs prepared from
mRNAs of NK cells. The PCR primers used for amplification were based
on the cDNA sequence for NKp30 (21) (EMBL/GenBank/DDBJ accession
number AJ223153). PCR primers also amplified a signal sequence at the 5⬘
end and sequences for a 6-His tag at the 3⬘ end of the cDNA. The PCR
products were cloned into the pFASTbac vector and expressed in a baculovirus system using the Bac-to-Bac system (cat. no. 10359-016; Invitrogen Life Technologies). The recombinant protein was purified from insect
culture supernatants by Ni-NTA affinity chromatography and Dynabeads
TALON (Dynal Biotech) according to the manufacturer’s instructions. Purity of the recombinant proteins was assessed by SDS-PAGE. Correct folding of the recombinant protein was shown by the ability of the protein to
block binding of PE-conjugated anti-NKp30 mAb to NK cells. The 6-His
rNKp30 protein was bound via the 6-His tag to nitrilotriacetic acid groups
embedded in the lipid matrix of fluorescent liposomes (22) by incubation
at room temperature for 1 h with mixing every 15 min (23). Control liposomes were prepared without 6-His rNKp30. The fluorescent liposomerNKp30 complexes and control liposomes were added to cells prepared in
RPMI 1640 containing 5 mM HEPES, 10% heat-inactivated FCS, and 5%
BSA. The cells and liposomes were incubated at room temperature for 1 h
with mixing every 15 min. Cells were washed in PBS containing 5% heatinactivated FCS, and analyzed by flow cytometry. To ensure binding was
specific, the rNKp-30 liposome complexes were incubated with an antiNKp30 mAb (10 ␮g/ml) or a murine isotype control (10 ␮g/ml) before
incubating with the cells.
Cytotoxicity assays
Target cells were labeled with 51Cr by incubation with Na251CrO3 (25
␮Ci/0.5 ⫻ 106 cells/0.1 ml volume) for 90 min, with and without heparanase, as previously detailed. After washing, 5000 51Cr-labeled target cells
were combined with NK-92 cells or polyclonal NK cells (at E:T cell ratios
between 1:1 and 4:1) in a final volume of 0.1 ml of HBSS containing 10%
heat-inactivated FCS. For assays containing polyclonal NK cells and human target cells, anti-class I HLA mAb was preincubated with the target
cells for 15 min at room temperature before combining with NK cells, to
prevent class I HLA binding the inhibitory class I HLA receptors. Also,
before combining target cells with NK cells, anti-NKG2D mAb was preincubated with NK cells for 15 min at room temperature to prevent
NKG2D ligands on target cells activating NK cell killing. Cultures were set
up in triplicate using 96-round-well trays. After 4 h, an extra 0.1 ml of
medium was added and the cells pelleted at 250 ⫻ g for 2 min. Supernatant
(100 ␮l) was harvested and counted in a Packard gamma counter. Percentage of specific lysis was calculated after subtracting the amount of 51Cr
released spontaneously from target cells alone, compared with 51Cr released from target cells in the presence of 1% Triton X-100. The spontaneous release from target cells was ⬃10% in different experiments.
Staining with mAb
Cells were incubated with mAb in PBS containing 0.1% BSA, for 30 min
on ice. Cells were washed three times after incubation with mAb before
adding the FITC-conjugated second reagent. Cells were fixed in 1% paraformaldehyde and analyzed on a FACScan (BD Biosciences), and the data
were processed using WinMDI 2.8 software (具facs.scripps.edu/典). All reagents were titrated for optimum concentrations.
rNKp30/human IgG1 Fc/anti-human IgG complexes
Complexes were prepared in PBS/0.1% BSA by combining rNKp30/human IgG1 Fc chimera (final concentration 2.5 ␮g/ml) or human IgG (final
concentration 5 ␮g/ml) with FITC-conjugated anti-human IgG (final concentration 20 ␮g/ml). After 45 min on ice, the solution was diluted 4-fold
and 25 ␮l was added to 104 cells pelleted in V wells. This dilution of the
preformed complex was predetermined as the optimum concentration for
binding. Cells were incubated for 45 min on ice, washed in PBS/0.1%
BSA, and fixed in 1% paraformaldehyde in PBS/0.05% BSA before analysis by flow cytometry. In some experiments the complexes were preformed using biotinylated anti-human IgG instead of FITC-conjugated antihuman IgG. In this case, after incubation with cells and washing, the bound
complexes were detected by incubating the cells with streptavidin-PE for
30 min on ice. To ensure binding was specific, the preformed complexes
Results
293-EBNA cells express a ligand for NKp30, which is not HS
In initial experiments we showed that 293-EBNA cells bind the
extracellular domain of NKp30 (Fig. 1). For these experiments two
different rNKp30 preparations were used, a commercially prepared
rNKp30/human IgG1 Fc chimera and a 6-His rNKp30 prepared in
our laboratory. Preliminary studies showed that binding of
rNKp30/human IgG1 Fc chimera to cells, detected with FITCconjugated anti-human IgG, is weak (2-fold above background).
However when soluble complexes of the rNKp30/human IgG1 Fc
chimera are prepared with FITC-conjugated anti-human IgG before incubation with the cells, the efficacy of binding is substantially increased, presumably due to a more multivalent structure.
This binding is specific because preincubation of the soluble complexes with anti-NKp30 mAb before incubation with the cells inhibits binding by 95% (Fig. 1). In the case of the 6-His rNKp30,
the protein was attached via nitrilotriacetic acid groups to fluorescent liposomes to achieve a multimeric complex (22, 23). These
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from American Type and Culture Collection (ATCC) CRL-1573, transformed primary human embryonal kidney fibroblast) was obtained from
Dr. B. Loveland (Austin Research Institute, Heidelberg, Victoria, Australia). Other cell lines were HeLa cells obtained from S. Ford (John Curtin
School of Medical Research, Australian National University, Canberra,
Australian Capital Territory, Australia), and MM-170 malignant melanoma
cells (17) obtained from Dr. R. Whitehead (Ludwig Institute for Cancer
Research, Melbourne, Victoria, Australia). Cells lines were cultured in
RPMI 1640 containing 10% heat-inactivated FCS. The CHO-K1 cell line
(ATCC CCL-61) and the GAG-deficient pgsA-745 cell line (ATCC CRL2242) derived from CHO-K1 cells were obtained from Dr. E. Lee (John
Curtin School of Medical Research, Australian National University). The
CHO-K1 and pgsA-745 cells were grown in a 50:50 mixture of HAMSF12 and DMEM with 10% heat-inactivated FCS. Adherent cells were released from plastic culture flasks after washing in PBS and incubation at
37°C for 2–5 min in PBS containing 0.9 mM EDTA.
Polyclonal NK cells were generated from purified peripheral blood NK
cells by culture with gamma-irradiated MM-170 malignant melanoma cells
and rIL-2 (18). The cultured cells were entirely NK cells as they lacked cell
surface CD3 and expressed CD16 and/or CD56 and/or CD94. DC were
generated from plastic adherent PBMC by culture for 7 days with rGMCSF and rIL-4 (19). The cultured cells lacked CD14, and expressed CD1c
and high levels of HLA-DR, consistent with an immature DC phenotype.
The procedures for obtaining peripheral blood were approved by the Human Ethics Committees of the Australian National University and the Australian Capital Territory Department of Health and Community Care.
THE LIGAND FOR NKp30 IS NOT HS
The Journal of Immunology
liposome-rNKp30 complexes also bound specifically to 293EBNA cells in that preincubation of the liposome-rNKp30 with
anti-NKp30 mAb before incubation with cells abolished binding
FIGURE 2. HS is not a NKp30 ligand
on 293-EBNA cells. A, 293-EBNA cells
were treated with mammalian heparanase
and then tested for their reactivity with the
anti-HS mAbs F58-10E4 and HepSS-1,
mAb binding being detected with FITCconjugated anti-mouse Ig (filled histograms). The line histogram shows staining
with the FITC-conjugated secondary reagent alone. B, Binding of NKp30 complexes to 293-EBNA cells before and after
treatment with mammalian heparanase
(filled histograms). Data for the rNKp30/
human IgG1 Fc/anti-human IgG complexes and liposome-rNKp30 complexes
are shown. The line histograms show cells
incubated with a control human Ig-antihuman IgG complex or incubated with liposomes without added recombinant protein. C, Killing of 293-EBNA cells by
NK-92 cells or polyclonal NK cells without a mAb (Medium), with a control antiCD56 mAb or with an anti-NKp30 mAb,
as indicated. The E:T cell ratio was 4:1.
The lower limit in the assays was 1.3 (untreated cells) and 1.6% (heparanase-treated
cells) determined at 2 SD above the spontaneous release of 51Cr from target cells in
the absence of NK cells. FL1 and FL1-H,
Fluorescence intensity channel 1.
(Fig. 1). These experiments show that NKp30 ligands can be detected on 293-EBNA cells using multimeric complexes of NKp30.
To evaluate HS as a ligand for NKp30, 293-EBNA cells were
treated with the mammalian HS degrading endoglycosidase,
heparanase (20). The data in Fig. 2A show that treatment with
heparanase totally removes cell surface HS detected with two antiHS-specific mAbs, F58-10E, and HepSS-1. F58-10E reacts with an
epitope present in many types of HS, which include N-sulfated
glucosamine residues (24), whereas HepSS-1 recognizes a HSspecific epitope containing O-sulfated and N-acetylated glucosamine residues (25). Treatment of 293-EBNA cells with bacterial heparinase-1 only partially removed HS (data not shown), so
this enzyme was not used in further studies. Having established the
efficacy of mammalian heparanase treatment of 293-EBNA cells,
the treated and untreated cells were compared for their ability to
bind rNKp30. Data in Fig. 2B show that binding of the liposomerNKp30 complexes and the rNKp30/human IgG1 Fc/anti-human
IgG complexes to heparanase-treated 293-EBNA cells is equivalent to their binding to untreated cells. In all cases binding was
inhibited by ⬎95% by preincubation of the complexes with antiNKp30 mAb (data not shown). These data show that NKp30 does
not bind to HS on 293-EBNA cells.
To further substantiate that HS is not a ligand for NKp30, we
tested the ability of NK cells to kill untreated and mammalian
heparanase-treated 293-EBNA cells (Fig. 2C). These experiments
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FIGURE 1. Multimeric complexes of NKp30 detect NKp30 ligands on
293-EBNA cells. The filled histograms show binding of preformed complexes of rNKp30/human IgG1 Fc chimera and FITC-conjugated antihuman IgG (rNKp30/human IgG1 Fc/anti-human IgG), or fluorescent liposome-rNKp30 complexes, to 293-EBNA cells. Binding in the presence
of an anti-NKp30 mAb (dotted line histogram) and in the presence of
control IgG2a mAb (filled histogram) coincident with the histograms
shown were obtained. The line histogram shows cells incubated with a
control human IgG/anti-human IgG complex (left) or cells incubated with
liposomes without added recombinant protein (right). FL1, Fluorescence
intensity channel 1.
209
210
Other human cells lines express a ligand for NKp30, which also
is not HS
We next showed that HS is not a ligand for NKp30 on culturegenerated DC, MM-170 malignant melanoma cells, and HeLa cells
(Fig. 3). All cells express HS, although at different levels, and
FIGURE 3. HS is not a NKp30 ligand
on DC, MM-170, or HeLa cells. A, Cells
were tested for their reactivity with the antiHS mAb HepSS-1 before and after treatment with mammalian heparanase. mAb
binding was detected with FITC-conjugated anti-mouse Ig (FL1). Staining of untreated cells (filled histogram) and of mammalian heparanase-treated cells (dotted
line histogram), or staining with the FITCconjugated secondary reagent alone (line
histogram) is shown. B, Killing of DC,
MM-170, or HeLa cells by polyclonal NK
cells without a mAb (Medium), with a control anti-CD56 mAb or with an anti-NKp30
mAb, as indicated. The E:T cell ratio was
1:1. The lower limit in the assays for untreated and treated cells, determined at 2 SD
above the spontaneous release of 51Cr from
target cells in the absence of NK cells, was
1.3 and 1.8% for DC, 1.1 and 2.8% for MM170, and 4.5 and 2.4% for HeLa cells.
treatment with mammalian heparanase removed HS from the cell
surface (Fig. 3A). The data in Fig. 3B show that killing of DC,
MM-170, and HeLa cells by polyclonal NK cells is unaffected by
heparanase treatment, and that killing of both untreated and
heparanase-treated cells is partially inhibited by NKp30 mAb. In
these experiments we verified that HS was not regenerated on the
heparanase-treated cells during the 4 h cytotoxicity assay (data not
shown). Therefore a number of different human cell lines in addition to 293-EBNA are killed by NK cells through NKp30, and the
NKp30 ligands on these cells are also not HS.
An NKp30 ligand is present on CHO-K1 cells and the GAGdeficient pgsA-745 cells
To definitively establish that HS is not a ligand for NKp30 we
tested CHO-K1 cells and the mutant GAG-deficient pgsA-745
cells derived from the CHO-K1 cell line, for their ability to bind
multimeric NKp30 complexes and to be killed by NK cells. We
first established that CHO-K1 cells express cell surface HS detected with the F58-10E mAb (Fig. 4A) and HepSS-1 mAb (data
not shown), and that the GAG-deficient mutant lacks cell surface
HS (Fig. 4A). We next showed that both CHO-K1 and pgsA-745
cells bind NKp30 complexes (Fig. 4B). In this case we were unable
to measure binding of the liposome-rNKp30 complexes as these
cells exhibited very high background binding of liposomes lacking
attached recombinant protein. However we were able to detect
weak and specific binding of rNKp30/human IgG1 Fc/anti-human
IgG complexes, provided that biotinylated anti-human IgG was
used to make the preformed complexes. In this case bound complex was detected with streptavidin-PE. This detection system is
more sensitive compared with complexes prepared with FITC-conjugated anti-human IgG. Both CHO-K1 and pgsA-745 cells bound
the NKp30 complexes. We next compared the ability of CHO-K1
with pgsA-745 cells to be killed by NK92 cells and polyclonal NK
cells. The extent of NK cell killing of CHO-K1 and pgsA-745 cells
was similar and importantly, killing of both cell lines was inhibited
to a similar extent by anti-NKp30 mAb and was not inhibited by
the control anti-CD56 mAb.
Discussion
The conclusion from this study that HS is not a ligand for NKp30
is based on data using cells that were either replete or deficient in
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used the NK-92 cell line (16) and polyclonal NK cells established
from human peripheral blood (18). The NK-92 cell line and the
polyclonal NK cells are both effective at killing 293-EBNA cells.
The NK-92 cells express 6-fold higher levels of NKp30 compared
with the polyclonal NK cells as assessed by anti-NKp30 mAb
staining (data not shown). NK-92 is a convenient cell line in cytotoxicity experiments with human target cells as it lacks inhibitory receptors for MHC class I (16) and has only weak expression
of the NKG2D activation receptor (H. S. Warren, unpublished observation). By contrast the polyclonal NK cells express a range of
class I HLA inhibitory receptors (KIR, CD94/NKG2A, ILT2) and
a fully functional NKG2D activation receptor (data not shown).
For polyclonal NK cells, blocking class I HLA on 293-EBNA cells
with an anti-class I HLA mAb and blocking NKG2D on NK cells
with an anti-NKG2D mAb was necessary to demonstrate NKp30dependent killing of 293-EBNA cells. The results in Fig. 2C show
that NK-92 cells and polyclonal NK cells kill 293-EBNA cells and
that killing is unaffected following treatment of 293-EBNA cells
with mammalian heparanase. NK-92 killing of both untreated and
mammalian heparanase-treated 293-EBNA cells was almost totally inhibited by anti-NKp30 mAb. In the case of polyclonal NK
cells, killing was partially inhibited in the presence of anti-NKp30
mAb, with the extent of inhibition being similar for both the untreated and heparanase-treated cells. As a control in this experiment we showed that killing was not inhibited by a mAb to CD56,
a receptor highly expressed by NK-92 cells. It is also of interest
that both the NKp30-dependent and the residual NKp30-independent killing of 293-EBNA cells was not affected by removal of HS.
Therefore ligands on 293-EBNA cells for other NK cell activation
receptors on polyclonal NK cells are also not HS. In these experiments we established, by mAb staining, that HS was not regenerated on the cell surface during the 4 h incubation used for the
cytotoxicity assay (data not shown). Therefore 293-EBNA cells
are killed by NK cells through NKp30, and the NKp30 ligand is
not HS.
THE LIGAND FOR NKp30 IS NOT HS
The Journal of Immunology
cell surface HS. In one series of experiments, human cell lines
were treated with mammalian heparanase, which totally removes
HS from the cell surface. In a second series of experiments
CHO-K1 cells were compared with mutant pgsA-745 cells that are
deficient in their ability to synthesize GAG including HS.
NKp30 complexes bound strongly to 293-EBNA cells irrespective of whether cell surface HS was present or absent. Similarly,
NKp30 complexes bound equally well to GAG-expressing
CHO-K1 cells and GAG-deficient pgsA-745 cells. In all cases
binding was specific in that it was inhibited by an anti-NKp30
mAb. The NKp30 complexes were generated in two ways. The
first was a complex prepared by preincubating rNKp30/human
IgG1 Fc chimera with FITC-conjugated anti-human IgG, using
commercially available reagents. The second was a fluorescent liposome-rNKp30 complex prepared using fluorescent liposomes
(23) and a 6-His-tagged rNKp30 prepared in our laboratory. Both
complexes gave equivalent and specific binding to 293-EBNA
cells. In the case of the CHO-K1 and the pgsA-745 cells, only
weak binding was detected and only with the rNKp30/human IgG1
Fc/anti-human IgG complexes.
NK cell killing of 293-EBNA cells, culture-generated DC, MM170 cells, and HeLa cells was not dependent on the presence of cell
surface HS. Similarly, killing of GAG-expressing CHO-K1 cells
was equivalent to killing of the GAG-deficient pgsA-745 cells. In
all cases killing was inhibited by an anti-NKp30 mAb, the extent
of inhibition presumably related to the presence or absence of
other activating NK cell receptor/ligand interactions involved in
cytotoxicity. Killing of 293-EBNA cells by the NK-92 cells was
almost entirely NKp30-dependent, whereas killing of the various
human cell lines by polyclonal NK cells was only partly NKp30dependent. The fact that the HS-replete and HS-depleted human
cell lines are killed to the same extent by polyclonal NK cells, and
that the NKp30-dependent killing was similar shows that HS is not
involved in other activating NK cell receptor/ligand interactions
involved in NK cell cytotoxicity. Interestingly, killing of CHO-K1
and pgsA-745 cells is mostly NKp30-dependent, and this was the
case using either NK-92 cells or polyclonal NK cells. These data
show that a limited number of human NK cell activation receptors
are involved in NK cell killing of these hamster cell lines. These
studies show conclusively for a range of cell lines that the ligand
for NKp30 is not HS.
In other studies (our unpublished observations) we have established that treatment of 293-EBNA cells with neuraminidase does
not affect binding of NKp30 complexes or their ability to be killed
by NK cells. However treatment of 293-EBNA cells with trypsin
abolishes binding of NKp30 complexes. Therefore we conclude
that the ligand for NKp30 is a protein, and that ligand binding is
not dependent on sialic acid or HS moieties.
Our conclusion that HS is not a ligand for NKp30 contrasts with
that of Bloushtain et al. (15). In studies with CHO-K1 cells and the
mutant pgsA-745 cells, this group showed that binding of an
rNKp30/human IgG1 Fc chimera was substantially less on the mutant cells. However the NKp30 specificity of this binding was not
established. In our studies, binding of an rNKp30/human IgG1 Fc
chimera was seen only when using a preformed complex with
anti-human IgG and when the sensitivity of detection was increased using biotinylated anti-human IgG and streptavidin-PE. In
other studies (data not shown) using 293-EBNA cells we observed
that binding of the rNKp30/human IgG1 Fc chimera to the cells
was in part nonspecific because binding was only partially inhibited by preincubating the rNKp30/human IgG1 Fc chimera with an
anti-NKp30 mAb. Interestingly, nonspecific binding was eliminated by preforming rNKp30/human IgG1 Fc complexes with antihuman IgG before incubation with the cells. These experiments
illustrate that specificity must be controlled in binding studies using
recombinant protein/human IgG1 Fc chimeras. In fact, nonspecific
binding of PECAM-1/Fc chimeras was noted in an early study by Sun
et al. (26), and the potential for nonspecific binding of Fc chimeras to
FcRs was noted in a recent study by Stark et al. (14).
The identity of the cellular ligands for NKp30 and other NK cell
activation receptors has remained elusive. The identity is not revealed probably because of the low affinity of the receptor ligand
interactions and difficulties with nonspecific binding of soluble recombinant protein/human IgG1 Fc chimeras. Enhanced avidity of
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FIGURE 4. An NKp30 ligand is present on both GAG-expressing
CHO-K1 cells and GAG-deficient pgsA-745 cells. A, Staining of CHO-K1
and pgsA-745 cells with the anti-HS mAb F58-10E4 detected with FITCconjugated anti-mouse Ig (filled histogram) and staining with the FITCconjugated secondary reagent alone (line histogram) are shown. B, Binding
of rNKp30/human IgG1 Fc/anti-human IgG complexes to CHO-K1 and
pgsA-745 cells. In these experiments preformed complexes of rNKp30/
human IgG1 Fc-biotinylated anti-human IgG were incubated with the cells,
and binding was detected with streptavidin-PE (FL2). Binding of the complexes after preincubation with control mouse IgG2a (filled histogram) and
after their preincubation with anti-NKp30 mAb (dotted line histogram) are
shown. The line histogram shows cells alone. C, Killing of CHO-K1 and
mutant pgsA-745 cells by NK-92 cells and polyclonal NK cells, without a
mAb (Medium), with a control anti-CD56 mAb, or with an anti-NKp30
mAb. The E:T cell ratio was 1:1 for NK-92 cells and 4:1 for polyclonal NK
cells. The lower limit in the assays, which was 2 SD above the spontaneous
release of 51Cr from target cells in the absence of NK cells, was 0.6 (CHOK1) and 1.3% (pgsA-745). FL1-H and FL2, Fluorescence intensity channels 1 and 2.
211
212
binding can be achieved using multimeric arrays of recombinant
proteins, as shown with studies on the interaction of CD2 (27, 28)
and CD4 (23) with their low affinity ligands. The use of complexes
of recombinant proteins as in this study, or the use of trimeric
isoleucine zipper-fusion proteins described recently (14), should
facilitate the isolation of ligands for NK cell activation receptors.
Acknowledgments
We thank Dr. Joseph Altin for providing fluorescent liposomes, and we
acknowledge the assistance of Margaret Hilton in the preparation of recombinant NKp30, and the assistance of Kimberly Hewitt in the binding
studies.
Disclosures
THE LIGAND FOR NKp30 IS NOT HS
13.
14.
15.
16.
17.
The authors have no financial conflict of interest.
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