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Departments of
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Molecular Biology,
Biomolecular Screening,
Protein Chemistry, and
Immunobiology, Immunex, Seattle, WA 98101
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production are blocked by inhibitors of PI 3-kinase, consistent with
the known binding of PI 3-kinase to DAP10, the membrane-bound
signal-transducing subunit of the NKG2D receptor. While all three ULBPs
activate the same signaling pathways, ULBP3 was found to bind weakly
and to induce the weakest signal. In summary, we have shown that NKG2D
is the ULBP counterstructure on primary NK cells and for the first time
have identified signaling pathways that are activated by NKG2D ligands.
These results increase our understanding of the mechanisms by which
NKG2D activates immune effector cells and may have implications for
immune surveillance against pathogens and tumors. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 domain, and do
not associate with 
2-microglobulin
(1). UL16 also binds to a member of another family of
nonclassical MHC class I proteins, MICB (2). MICB and the
closely related MICA protein share some similar properties with the
ULBPs, as discussed below.
NK cells are a key component of the innate immune system that recognize
and lyse virally infected and neoplastic cells. Recent studies indicate
that ULBPs are important activators of NK cells. Soluble recombinant
forms of ULBPs bind to human NK cells and stimulate NK cytotoxicity
against tumor targets (3). Soluble ULBPs also induce
production of the cytokines IFN-, GM-CSF, TNF-, and TNF-, and
the chemokines macrophage-inflammatory protein (MIP)-1, MIP-1,
and I-309. In all cases, costimulation with IL-12 has a superadditive
effect on the production of these factors (1, 3).
NK cells recognize cellular targets through the use of receptors that are specific for MHC class I molecules on the target cell. These NK cell receptors belong to the killer cell Ig-like receptor, Ly49, and NKG2 families and, depending on their structure, deliver either activating or inhibitory signals to the NK cell (4). Until recently, it was thought that signals generated by inhibitory NK cell receptors were dominant over those generated by any activating receptor, so that cells with down-regulated MHC class I levels would be killed, the missing-self hypothesis (5). However, expression of ULBPs or MHC class I-related chain molecules (MICs) on NK cell-resistant, MHC class I-expressing, target cells confers susceptibility to NK cell killing (1, 6). Thus, ULBPs and MICs can transduce an activating signal to NK cells that can override a negative signal generated by engagement of inhibitory receptors for MHC class I. Whether the signal delivered by ULBPs and MICs is sufficient to cause activation of NK cells in vivo is likely to depend on the relative numbers of NK cell-activating and inhibitory receptors expressed and on the avidity of receptor engagement.
MICA expression is up-regulated in certain epithelial tumors, in HCMV-infected cells, and in response to stress (2, 7). In contrast to the MICs, ULBP messages are expressed by a wide range of cells, tissues, and tumors, and ULBP proteins are expressed on various cell lines (1). Thus, several types of cells may potentially deliver ULBP-mediated signals to NK cells and be targets of ULBP-mediated killing.
Although the amino acid sequences of the ULBPs and MICs are only distantly related, both families of proteins deliver an activating signal to NK cells apparently by binding to NKG2D/DAP10 heterocomplexes. NKG2D is a homodimeric, C-type lectin that is expressed not only on NK cells, but also on T cells and activated macrophages (6, 8). In T cells, NKG2D has been shown to act as a costimulatory receptor, in a similar manner as CD28 (9). The cytoplasmic domain of NKG2D is short, and signaling appears to be mediated through its association with the DAP10 membrane adapter protein (10). DAP10 can bind the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) and the adapter protein Grb2 (10, 11). However, despite the likely importance of NKG2D in the activation of innate and adaptive immune responses to pathogens and tumors, nothing else is known about how NKG2D transmits its signal.
We have previously shown that ULBPs bind to recombinantly expressed NKG2D/DAP10 heterodimers and that an anti-NKG2D antiserum partially blocks ULBP binding to NKL cells (1). In this report we provide proof that NKG2D is indeed the ULBP receptor expressed on primary human NK cells. We also identify signal transduction pathways that are activated by ULBPs in NK cells. These results provide a basis for comparison with CD28 signaling pathways and give insight into the mechanisms by which NKG2D ligands can provide a powerful stimulatory signal to activate NK cell effector functions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Soluble ULBP-leucine zipper (LZ), MIC-LZ, UL16-LZ, ULBP-Fc, MIC-Fc, human NKG2D-Fc, and p7.5-Fc fusion proteins were prepared as previously described (1). F(ab')2 forms of the M15 anti-LZ mAb were prepared by digestion with Ficin (Sigma, Milwaukee, WI) and then purified using a protein A-Sepharose column (Amersham Pharmacia Biotech, Piscataway, NJ). The 4G10 anti-phosphotyrosine mAb, the p85 anti-PI 3-kinase mAb used for immunoblotting, and rabbit Abs used to immunoprecipitate Janus kinase (JAK)2 and PI 3-kinase were purchased from Upstate Biotechnology (Lake Placid, NY). Rabbit Abs used to immunoblot phosphorylated JAK2 (Tyr1007/1008) were obtained from BioSource International (Camarillo, CA). Rabbit anti-phospho-STAT5 (Tyr694), phospho-mitogen-activated protein (MAP) kinase kinase 1/2 (phospho-MEK1/2; Ser217/221), phospho-extracellular signal-regulated kinase (phospho-ERK; Thr202/Tyr204), and phospho-Akt (Ser473) were purchased from Cell Signaling Technology (Beverly, MA). Rabbit anti-ERK1 (Sc-94) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), whereas anti-STAT5, anti-Akt, and anti-p70 S6 kinase were obtained from Cell Signaling Technology.
mAbs M585 and M580 to human NKG2D were generated by injecting BALB/c
mice at 0, 2, and 6 wk with 10 µg of NKG2D-Fc protein in Titermax
adjuvant (Immunex, Seattle, WA) as previously described
(12). Positive supernatants were subsequently confirmed by
flow cytometry and immunoprecipitation using CV-1 cells cotransfected
with cDNAs encoding full-length NKG2D and DAP10. Monoclonal cultures
were expanded, and mAbs were purified using protein A-agarose (Bio-Rad,
Hercules, CA). The purified anti-NKG2D mAbs bind essentially all
CD56+CD3- NK cells and
CD8+ T cells, but few CD4+
T cells, and show no detectable binding to CD19+
B cells. Therefore, the binding patterns of these Abs fit the known
expression patterns of human NKG2D. For cell stimulation, recombinant
human (rhu)IL-4, IL-10, IL-12, and IFN- were from Genzyme
(Cambridge, MA), whereas rhuIL-15 was obtained from Immunex.
Purification of NK cells
Primary human NK cells were obtained as previously described (13). Briefly, peripheral blood was layered over isolymph and centrifuged. PBMC from the resulting interphase were depleted of monocytes by monocyte adherence to tissue culture dishes. NK cells were expanded by coculture with irradiated RPMI 8866 cells. On day 8 or 9 of culture NK cells were depleted of contaminating T cells by magnetic negative selection using a MACS column (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s protocol.
Flow cytometry
To determine whether soluble NKG2D-Fc blocks ULBP binding to
primary NK cells, 1 µg/ml ULBP-LZ protein or MIC-LZ protein was mixed
with either 50 µg/ml NKG2D-Fc or p7.5-Fc control protein for 30 min
at room temperature. Cells (3 x 105) were
then resuspended in the NKG2D-Fc/ULBP-LZ mixture and incubated for 45
min on ice. Specific binding of LZ proteins was detected with a
combination of biotin-conjugated M15 anti-LZ Ab (Immunex), followed
by PE-conjugated streptavidin (Jackson ImmunoResearch Laboratories,
West Grove, PA). To determine whether NKG2D-specific mAbs block ULBP
binding, NK cells were treated for 45 min on ice with 4 µg/ml M585 or
M580 anti-human NKG2D mAb (Immunex) or M90 anti-CD40 ligand
(anti-CD40L) control mAb (Immunex), followed by 10 µg/ml ULBP-Fc
or MIC-Fc protein. Binding of Fc proteins was detected with
PE-conjugated, goat anti-murine Fc-specific Ab (Jackson
ImmunoResearch Laboratories). To determine the effect of cytokine
stimulation on NKG2D expression, NK cells were cultured for 20 h
with IL-4 at 10 ng/ml, IL-10 at 10 ng/ml, IL-12 at 1 ng/ml, IFN- at
1000 U/ml, or IL-15 at 50 ng/ml. Cells were washed and stained with
M585 anti-NKG2D mAb, followed by detection with PE-conjugated, goat
anti-mouse IgG (Sigma). After staining, cells were analyzed for
ULBP-LZ, MIC-LZ, ULBP-Fc, and MIC-Fc binding or for NKG2D expression
using a FACScan (BD Biosciences, Mountain View, CA).
Cell stimulation and preparation of cell lysates
Primary human NK cells were treated overnight with RPMI 1640 containing 50 ng/ml rhuIL-15, 1% heat-inactivated FBS, and 2 mM L-glutamine. The cells were serum-starved for an additional 4 h in RPMI 1640 supplemented with 2 mM L-glutamine, then resuspended in RPMI 1640 to 3 x 107/ml. Where indicated, cells were pretreated for 30 min with 1 µM of the PI 3-kinase inhibitor wortmannin (Upstate Biotechnology), 50 µM of the PI 3-kinase inhibitor Ly294002 (Upstate Biotechnology), 5 µM of the MEK1 inhibitor PD98059 (New England Biolabs, Beverly, MA), or DMSO vehicle alone. For the NKG2D-Fc blocking experiments, cells were pretreated for 30 min with various concentrations of NKG2D-Fc or with p7.5-Fc control protein.
Cells were stimulated with 1 µg/ml ULBP-LZ fusion protein in the presence of 1 µg/ml M15 F(ab')2 (anti-LZ) cross-linking Ab (Immunex). UL16-LZ and CD40L-LZ were used as negative control LZ proteins. Reactions were stopped by adding cold PBS containing 1 mM Na3VO4 and then centrifuging the cells for 3 min in the cold. The cells were solubilized in 20 mM Tris-HCl (pH 7.4), 1% Triton X-100, 10% glycerol, 137 mM NaCl, 2 mM EDTA, 1 mM Na3VO4, 1 mM Na3MoO4, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and 1 mM PMSF. After 10 min on ice, detergent-insoluble material was removed by centrifugation. Protein concentrations were determined using the bicinchoninic acid assay (Pierce, Rockford, IL).
Immunoprecipitations
Cell lysates (0.4–0.75 mg of protein) were precleared by mixing with 15 µl of protein A-Sepharose for 1 h at 4°C. JAK2 or PI 3-kinase was immunoprecipitated by incubating the precleared lysates for 3 h at 4°C with anti-JAK2 or anti-p85 Ab, respectively. Immune complexes were collected by mixing the lysate with 15 µl of protein A-Sepharose for 1 h at 4°C. The beads were washed four times with lysis buffer before eluting bound proteins with SDS-PAGE sample buffer.
Immunoblotting
Proteins (20 µg of total cell lysate or immunoprecipitates) were separated on 1-mm-thick, 4–20% Tris-glycine gels and then transferred to nitrocellulose. Equal protein loading in each of the lanes was confirmed by staining the filters with Ponceau S (Sigma). The filters were blocked with 5% nonfat dry milk powder in TBS (10 mM Tris-HCl (pH 8) and 150 mM NaCl). Primary Abs were diluted 1/1000 in TBS containing 3 mg/ml BSA (ICN Biomedicals, Aurora, OH) and 0.02% sodium azide and incubated with the filters overnight at 4°C. After washing with TBS/0.1% Tween 20 (TBST), the filters were incubated 1 h with HRP-conjugated goat anti-rabbit IgG (1/2,500 in TBST) or HRP-conjugated sheep anti-mouse IgG (1/10,000). Filters were washed extensively with TBST, and immunoreactive bands were visualized by ECL (Amersham Pharmacia Biotech).
PI 3-kinase enzyme assays
Lysates (300 µg) from NK or 293 cells were immunoprecipitated as described above, and kinase assays were performed as described previously (14).
Chemokine and cytokine assays
Primary human NK cells were cultured in the presence of rhuIL-15
(50 ng/ml) for 20 h and washed. The cells were then stimulated
with ULBP-LZ fusion proteins, IL-12 (1 ng/ml), or the combination of
ULBP-LZ and IL-12 in the presence of 0.2 µM wortmannin (Upstate
Biotechnology), 50 µM Ly294002 (Upstate Biotechnology), 1 µM
PD98059 (New England Biolabs), or DMSO vehicle alone. GM-CSF, MIP-1,
and IFN- levels in tissue culture supernatants were determined by
ELISA. GM-CSF levels were determined using the M8 and P5 paired Abs
(Immunex), whereas MIP-1 and IFN- levels were determined using
paired Abs from R&D Systems (Minneapolis, MN). Assays were performed in
triplicate wells, and results are presented as the mean from a
representative experiment.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have shown earlier that ULBPs bind to recombinantly expressed
NKG2D/DAP10 heterocomplexes and that an antiserum raised against
recombinant NKG2D-Fc fusion protein partially blocks binding of ULBPs
to NKL cells (1). In addition, another group has shown
that anti-NKG2D mAbs block MIC binding to NKL cells
(6). To determine whether NKG2D/DAP10 is the ULBP receptor
on primary human NK cells, we tested the effects of soluble NKG2D-Fc
fusion proteins and mAbs against NKG2D on the binding of soluble forms
of the ULBPs to these cells. We found that NKG2D-Fc (Fig. 1
A) and anti-NKG2D mAb
M585 (Fig. 1B) completely block binding of ULBP1, ULBP2,
ULBP3, and the MICs to primary NK cells. In contrast, a control Fc
fusion protein (Fig. 1A) and an isotype-matched control mAb
(Fig. 1B) did not block ULBP or MIC binding. These results
demonstrate that NKG2D is the major binding component of the ULBP
counterstructure on primary NK cells. However, there appear to be
differences in the NKG2D epitopes used for binding individual NKG2D
ligands. For example, we found that anti-NKG2D mAb M580 partially
blocks binding of ULBP2 (Fig. 1C) and MICA (M. Kubin,
unpublished observation) to NK cells. In contrast, M580 consistently
causes a slight increase in binding of ULBP1, ULBP3 (Fig. 1C), and MICB (M. Kubin, unpublished observation) to NK
cells, perhaps by causing a conformational change in NKG2D that favors
binding of these ligands.
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D). NKG2D
expression and ULBP binding are also up-regulated in response to IL-10,
IFN-, and IL-12, but weakly or not at all in response to IL-4
treatment (Fig. 1D). ULBPs stimulate marked protein tyrosine phosphorylation
Protein tyrosine phosphorylation is an important mechanism used by
many receptors to transmit signals into cells. To determine whether
ULBPs induce activation of protein tyrosine kinases, we performed
anti-phosphotyrosine immunoblots on lysates from ULBP-LZ-treated NK
cells. Both ULBP1 and ULBP2 induced marked protein tyrosine
phosphorylation within 1 min (Fig. 2A). The most prominent
tyrosine-phosphorylated proteins present in response to ULBP treatment
had apparent molecular masses of approximately 40, 45, 100, and 120 kDa
(Fig. 2A). UL16-LZ control protein, which shows no
detectable binding to NK cells, had little or no effect on protein
tyrosine phosphorylation (Fig. 2A). We have shown that ULBPs
synergize strongly with IL-12 in inducing IFN- production from NK
cells (3). In this study we found that IL-12 has no
obvious effect on the pattern of ULBP-induced protein tyrosine
phosphorylation (Fig. 2A). Together, these data suggest that
protein tyrosine kinase activation may be a signaling mechanism used by
ULBPs to transmit their effects on NK cells.
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After determining that ULBPs cause marked protein tyrosine
phosphorylation, we set out to identify ULBP-activated tyrosine
kinase(s). We focused on the JAKs and their downstream effectors, the
STAT transcription factors, because JAKs and STATs are activated by
tyrosine phosphorylation, and major phosphotyrosine proteins identified
in Fig. 2A have similar sizes, 
110–130 kDa, as JAK and
STAT proteins. We assessed JAK activation by stimulating NK cells
with ULBP2-LZ, followed by immunoprecipitation of the JAKs from cell
lysates and assessment of their activation states by immunoblotting
with Abs specific for the tyrosine-phosphorylated, active forms of
these kinases. We found that ULBP2-LZ induces phosphorylation of JAK2
(Fig. 2B) but has no detectable effect on
phosphorylation of JAK1, JAK3, and Tyk2 (data not shown).
To determine whether ULBPs activate the main downstream effectors of
the JAKs, the STATs, NK cells were stimulated with ULBPs for 5 min, and
then cell lysates were immunoblotted with phosphospecific STAT1, STAT3,
STAT5, or STAT6 Abs. Alternatively, STAT4 was immunoprecipitated and
then immunoblotted with 4G10 anti-phosphotyrosine mAb. We found
that STAT5 is strongly phosphorylated in response to ULBP1 and ULBP2
(Fig. 2C) and weakly phosphorylated in response to ULBP3
(data not shown). In contrast to STAT5, we found that ULBP1 and ULBP2
weakly phosphorylate STAT1 but have no apparent effect on
phosphorylation of STAT3, STAT4, and STAT6 (C. L. Sutherland,
unpublished observations). NKG2D-Fc completely blocks ULBP-induced
STAT5 phosphorylation (Fig. 2D), consistent with the idea
that STAT5 is activated in response to ULBP engagement of NKG2D.
We have shown that ULBPs synergize strongly with IL-12 in inducing
IFN- production from NK cells (3). To analyze the
mechanism underlying the synergistic effect of ULBPs and IL-12 on NK
cells, we tested the effect of ULBP and IL-12 cotreatment on STAT
activation. We were particularly interested in determining whether
ULBPs synergized with IL-12 for STAT4 activation, because STAT4 is
required for IL-12-induced IFN- production (15, 16).
While IL-12 caused a small increase in ULBP-induced STAT5
phosphorylation (Fig. 2C), it had no detectable effect on
the ability of ULBPs to induce phosphorylation of STAT1, STAT3, STAT4,
or STAT6 (C. L. Sutherland, unpublished observations).
ULBPs induce activation of the ERK MAP kinase pathway
After finding that ULBPs induce activation of JAK2 and STAT5 in NK
cells, we tested whether ULBPs activate the ERK, c-Jun N-terminal
kinase (JNK), and p38 MEKs. We were interested in testing whether ULBPs
activate MEKs, because these serine/threonine kinases play key roles in
transducing extracellular signals from the cytosol to the nucleus. In
particular, ERK2 has recently been shown to play a critical role in
driving NK cell lysis of tumor targets (17). After
stimulating cells with ULBP-LZ proteins, MEK activity was assessed by
immunoblotting cell lysates with Abs specific for the phosphorylated,
activated forms of ERK, JNK, and p38. We found that ULBP1-LZ, ULBP2-LZ,
and the MICs induce marked phosphorylation of p42 ERK2 and modest
phosphorylation of p44 ERK1 within 1 min (Fig. 3, upper panels). In contrast,
ULBP3-LZ only weakly phosphorylates the ERKs, and a UL16 control LZ
protein has no effect (Fig. 3, upper panels). Consistent
with the finding that ULBPs activate ERK1 and ERK2, ULBPs induce
phosphorylation of kinases upstream of ERK1 and ERK2, MEK1/2 (Fig. 3, lower panel).
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) and Ly294002 (data
not shown) completely blocked ULBP-induced phosphorylation of ERK1/2
and MEK1/2 (Fig. 3). Examples of PI 3-kinase-dependent activation of
ERK have been cited previously in the literature (18, 19).
In contrast to the ERKs, we did not detect ULBP-induced activation of
JNK or p38 in NK cells (data not shown). ULBPs induce activation of PI 3-kinase and the antiapoptotic kinase, Akt
The receptor for MICA and MICB is a heterocomplex composed of the
C-type lectin, NKG2D, and the DAP10 adapter protein (6).
Studies in pervanadate-treated NKL cells (10) and in
CD4-DAP10 chimera-transfected Jurkat cells (11) have shown
that DAP10 can recruit the p85 subunit of PI 3-kinase. However, it is
not known whether NKG2D engagement by ULBPs or MICs induces PI 3-kinase
activation. To determine whether ULBP triggering induces
phosphorylation and activation of PI 3-kinase, NK cells were treated
with ULBP2-LZ, and then the p85 subunit of PI 3-kinase was
immunoprecipitated from cell lysates. We found that both the p85 and
p110 subunits of PI 3-kinase are phosphorylated within 2 min of ULBP
addition, whereas CD40L-LZ control protein has no effect (Fig. 4A). Furthermore, ULBP
triggering induces activation of PI 3-kinase, as assessed by in vitro
lipid kinase assay (Fig. 4B).
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C). ULBP-induced
activation of Akt is dependent on PI 3-kinase, because the PI 3-kinase
inhibitors wortmannin and Ly294002 block Akt phosphorylation (Fig. 4C). In addition to blocking ULBP binding to NK cells (Fig. 1A), we found that NKG2D-Fc blocks ULBP-induced
phosphorylation of Akt (Fig. 4D). PI 3-kinase is required for ULBP-induced cytokine and chemokine production
We have shown that ULBPs induce NK cells to produce a variety of
cytokines and chemokines. IL-12 cotreatment greatly enhances the
production of these factors and has a synergistic effect on IFN-
production (1, 3). To determine whether the PI 3-kinase
and ERK pathways mediate ULBP-induced cytokine and chemokine
production, we tested the effects of inhibitors of these pathways on
GM-CSF, MIP-1, and IFN- synthesis. We found that the PI 3-kinase
inhibitor, wortmannin (0.2 µM), completely blocked ULBP2-induced
GM-CSF production and caused a 60–70% reduction of MIP-1 and
IFN- production (Fig. 5). Wortmannin
also caused a large reduction in IFN- synthesis induced by ULBP2 and
IL-12 cotreatment (Fig. 5). While wortmannin had a dramatic effect on
ULBP2-induced cytokine and chemokine induction, it did not affect NK
cell proliferation in response to IL-15, suggesting that there was no
nonspecific cytotoxic effect at this dose (data not shown). The PI
3-kinase inhibitor, Ly294002, had similar effects as wortmannin on
ULBP-induced cytokine production (data not shown). Compared with PI
3-kinase inhibitors, the MEK1/2 inhibitor PD98059 (1 µM) weakly
inhibited production of GM-CSF, MIP-1, and IFN- (Fig. 5).
Although the data shown here are for ULBP2, wortmannin and PD98059 had
similar effects on ULBP1- and ULBP3-induced GM-CSF, MIP-1, and
IFN- production (data not shown). Taken together, these results
suggest that PI 3-kinase is a major mediator of ULBP-induced cytokine
and chemokine production in NK cells, whereas the ERK pathway plays at
most a minor role.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our findings that both soluble NKG2D-Fc and anti-NKG2D mAb M585
completely block binding of ULBP1, ULBP2, and ULBP3 to NK cells (Fig. 1, A and B) demonstrate that NKG2D is the ULBP
counterstructure on primary human NK cells. Further evidence that NKG2D
is the functional ULBP receptor on primary NK cells is that NKG2D-Fc
blocks ULBP-induced signaling in these cells (Figs. 2D and
4D). Although the ULBPs and MICs all bind to NKG2D on NK
cells, there appear to be some differences in the NKG2D epitopes used
for binding. For example, while anti-NKG2D mAb M585 blocks binding
of all five proteins to NK cells (Fig. 1B), mAb M580
partially blocks binding of ULBP2 (Fig. 1C) and MICA (M.
Kubin, unpublished observations) but has no effect on the binding of
ULBP1, ULBP3, and MICB. IL-15, IL-12, and IFN- all up-regulate both
NKG2D expression and ULBP binding to NK cells derived from coculture on
irradiated RPMI 8866 cells (Fig. 1D). NKG2D is expressed on
essentially all CD56+CD3-
NK cells from freshly isolated PBMC (our unpublished observations).
Experiments are in progress to determine whether coculture of NK cells
with irradiated RPMI 8866 cells influences the level of ULBP receptors
expressed relative to freshly isolated NK cells.
Previous work has established the association of the
membrane-bound signal transducer DAP10 with NKG2D (10).
Studies in CD4-DAP10 chimera transfectants and pervanadate-treated
NKG2D-DAP10 transfectants have shown that DAP10 is capable of
recruiting the p85 subunit of PI 3-kinase (10, 11). In
this study we found, using soluble versions of the natural ligands for
NKG2D, that ULBPs induce phosphorylation and activation of PI 3-kinase
as well as activation of the PI 3-kinase target, Akt, in NK cells (Fig. 4). Furthermore, the PI 3-kinase inhibitors wortmannin and LY294002
block ULBP-induced activation of Akt (Fig. 4C). Although
several types of receptors are capable of activating Akt, these data
are consistent with the hypothesis that, like MICA, ULBPs may bind to
and signal through a DAP10-containing receptor complex. Future
experiments will reveal whether NKG2D can partner with other adapters
besides DAP10, and if the ULBPs bind to receptors other than NKG2D in
different cell types. Another membrane adapter protein expressed in NK
cells, DAP12, does not associate with NKG2D and does not contain a PI
3-kinase binding site (20). We are currently testing
whether, in addition to NK cells, ULBPs bind to and activate other
cells that express NKG2D, such as CD8+ T
cells, 
T cells, and activated macrophages (6, 8).
We found that ULBPs and MICs induce a marked increase in intracellular
calcium and that inhibition of PI 3-kinase with either Ly294002 or
wortmannin attenuates ULBP-induced calcium mobilization (our
unpublished observations). Thus, PI 3-kinase activity is required to
couple NKG2D/DAP10 to calcium mobilization. In addition to calcium
flux, ULBPs induce rapid tyrosine phosphorylation of several proteins
(Fig. 2A). Although qualitatively similar, ULBP1 causes a
more intense tyrosine phosphorylation pattern than ULBP2 (Fig. 2A). Furthermore, for all signaling events tested, ULBP1 and
ULBP2 clearly induce stronger signaling responses than ULBP3.
Similarly, we found that soluble ULBP1 and ULBP2 bind to NK cells from
several different donors to a greater extent than does soluble ULBP3
(3). In addition, our preliminary BiaCore analysis
indicates that ULBP1 binds NKG2D most strongly and with the slowest
off-rate, followed closely by ULBP2. ULBP3 shows significantly weaker
binding and has a faster off-rate than ULBP1 and ULBP2. These
observations are consistent with a model in which the strength of the
ULBP-mediated signal correlates with the stability of the
ligand-receptor complex. A recent study using soluble versions of NKG2D
and the murine NKG2D ligands, RAE-1 and H60, found that RAE-1 and H60
each bind to NKG2D tightly, but that H60 binds to NKG2D with
approximately 25-fold higher affinity (21). However, it
should be noted that the NKG2D ligands are normally membrane-bound
proteins, and it is possible that other cell surface proteins on both
effector and target cells may contribute as accessories to the overall
strength of the NKG2D/NKG2D ligand interaction.
We found that ULBPs cause a strong phosphorylation of STAT5 at tyrosine
residues that correlate with its activation (Fig. 4C). STAT5
exists as two closely related proteins, STAT5a and STAT5b
(22). Studies in STAT5-deficient mice indicate that STAT5a
is required for lactogenesis (23), whereas STAT5b is
essential for NK cell-mediated proliferation and cytotoxicity
(24). Both STAT5 proteins are expressed in primary NK
cells, and studies are underway to determine which form(s) is activated
by ULBPs. STAT5 activation is typically mediated by JAK2; however,
STAT5 can be directly activated by some receptor tyrosine kinases
(25, 26), and Src-dependent mechanisms for STAT5
activation also exist (27). We found that ULBPs induce
phosphorylation of JAK2 (Fig. 2B) but do not appear to
activate c-Src (C. L. Sutherland, unpublished observations).
Experiments are in progress to determine whether JAK2-specific
inhibitors block ULBP-induced activation of STAT5 and ULBP-induced NK
cytotoxicity against tumor targets.
The serine/threonine kinase, Akt, promotes cell survival in response to
various growth and survival factors (28). We found that
ULBPs and MICs stimulate phosphorylation of Akt in a pathway involving
PI 3-kinase (Fig. 4C). In contrast, we did not detect
ULBP-induced activation of NF-
B, an antiapoptotic transcription
factor that is a target of Akt (29, 30). Whether ULBPs
provide a survival signal to NK cells requires further study. However,
this concept is consistent with our unpublished findings that ULBPs
up-regulate messages for several antiapoptotic factors, including a
caspase inhibitor, AP12/cIAP2, and a Bcl-2 homolog, Bcl2A1.
ULBPs activate the ERK MAP kinase pathway (Fig. 3) but have no apparent
effect on JNK or p38. Our results suggest that transcription factors
targeted by ERK, such as Elk-1, c-Fos, and Ets-1, may be activated by
ULBPs and mediate functional responses in NK cells. ERK2 plays a
pivotal role in NK cytotoxicity, presumably by controlling mobilization
of perforin and granzyme B toward target cells (17).
Whether ERK2 mediates ULBP-induced NK cytotoxicity remains to be
tested. Our finding that MEK1/2 and ERK1/2 phosphorylations are blocked
in PI 3-kinase-inhibited NK cells (Fig. 3) is consistent with a recent
report that PI 3-kinase is the key upstream regulator of ERK in the NK
cytolytic process (31). Our data showing that the MEK1/2
inhibitor, PD98059, weakly inhibits production of GM-CSF, MIP-1, and
IFN- (Fig. 5) suggests that the ERK pathway may play a small role in
mediating ULBP-induced cytokine production.
We have previously shown that ULBPs synergize strongly with IL-12 for
production of IFN- by NK cells (3). While IL-12
enhances ULBP-induced phosphorylation of STAT5 (Fig. 2C), it
is unclear whether the STAT5 pathway contributes to IFN- production
in this system. In addition to modulating IL-12-initiated signals, the
ULBPs may function with IL-12 by activating signaling pathways that are
distinct from but complimentary to those activated by IL-12. For
example, in this study we found that ULBPs induce phosphorylation of
Akt (Fig. 4C), whereas IL-12, either alone or in combination
with ULBPs, has no effect on Akt phosphorylation (C. L.
Sutherland, unpublished observations). Furthermore, PI 3-kinase
inhibitors block both ULBP-induced phosphorylation of Akt (Fig. 4C) and IFN- production induced by costimulation with
ULBPs and IL-12 (Fig. 5). A recent paper shows that Akt provides the
CD28 costimulatory signal that augments TCR-initiated production of
IFN- (32). Although additional studies remain to be
performed, our data are consistent with the hypothesis that, like the
CD28 pathway, Akt provides the ULBP costimulatory signal for
IL-12-initiated production of IFN-. The concept that NKG2D can
function as a costimulatory receptor is extended by the recent finding
that NKG2D can serve, in the absence of CD28, as an alternative
costimulator of TCR-dependent T cell activation (9).
Moreover, the parallel between CD28 and DAP10 signaling has been drawn
previously, as both contain a YxxM motif for binding the p85 subunit of
PI 3-kinase (33). Thus, our data further extend the
analogy between NKG2D/DAP10 and CD28 signaling.
Studies are in progress to further define the function of the
ULBPs in immune surveillance. At present, several findings suggest that
ULBPs, like MICs, play an important role in the activation of innate
immunity. For example, ULBPs induce NK cells to produce multiple
cytokines and chemokines, including GM-CSF, IFN-, TNF-, MIP-1,
and I-309 (1, 3). IFN- and TNF- are important
anti-viral cytokines (34), whereas GM-CSF, MIP-1,
and I-309 probably function in vivo to recruit and activate NK cells,
macrophages, and other components of both innate and adaptive immunity.
Besides inducing cytokine and chemokine production, ULBPs stimulate
potent NK cytotoxicity against tumor targets (1, 3).
Further investigation is required to understand how both ULBP expression and activation are regulated in normal cells and tumors. ULBP transcripts are expressed in a wide range of normal and tumor tissues (1). At the protein level, ULBPs are expressed in a variety of cell lines (1), whereas MIC expression is up-regulated on some epithelial tumors (2, 7). Murine ligands for NKG2D, the RAE-1 and H-60 proteins, are induced on some tumors (35, 36), and their ectopic expression on tumor cell lines mediates potent rejection of MHC class I-bearing tumors in vivo (37, 38). Given these recent findings, it is tempting to speculate that, similar to RAE-1 and H-60 in the mouse, ULBPs and MICs may stimulate tumor immunity in humans.
MIC expression is known to be up-regulated on HCMV-infected cells (9). How ULBP expression and ULBP and MIC functions are affected by pathological conditions requires further study. We have shown previously that the HCMV glycoprotein, UL16, blocks ULBP and MIC binding to NK cells, and ULBP-induced NK cell activation (1, 3). Both NK and T cells play important roles in controlling CMV infection (39, 40). Thus, another area of interest is to examine how UL16 masking of NKG2D ligands may protect a virus-infected cell against immune recognition.
In summary, we found that ULBPs bind to the NKG2D/DAP10-activating
receptor on primary human NK cells and induce calcium mobilization and
activation of the JAK2, STAT5, ERK, and PI 3-kinase/Akt signal
transduction pathways. By phosphorylating different substrates and
regulating different transcription factors, JAK2, ERK, and Akt may
mediate the effects of ULBPs on NK cell activation (Fig. 6). This study is the first, to our
knowledge, that characterizes the signaling pathways induced by
engagement of NKG2D by its ligands. NKG2D is emerging as an
important activating receptor that bridges the gap between innate and
adaptive immunity and that can act as a costimulatory molecule in a
similar manner as CD28. Understanding the mechanisms by which
ULBP/NKG2D interactions activate immune effector cells may have
implications for immune surveillance against pathogens and tumors.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: ULBP, UL16-binding protein; CD40L, CD40 ligand; ERK, extracellular signal-regulated kinase; HCMV, human CMV; JAK, Janus kinase; JNK, c-Jun N-terminal kinase; LZ, leucine zipper; MAP, mitogen-activated protein; MEK, MAP kinase kinase; MIC, MHC class I-related chain molecules; MIP, macrophage-inflammatory protein; PI 3-kinase, phosphatidylinositol 3-kinase; rhu, recombinant human.
Received for publication April 10, 2001. Accepted for publication November 12, 2001.
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