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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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2 integrin LFA-1 (CD11a/CD18) mediates adhesion
of lymphocytes to cells expressing ICAM. The strength of this adhesion
is regulated by different signals delivered by cytokines and
chemokines, and by the TCR in the case of T cells. To determine the
receptor-ligand interactions required for adhesion of resting NK cells,
Drosophila cells expressing different combinations of
ligands of human NK cell receptors were generated. Expression of
ICAM-1 alone was sufficient for an adhesion of resting NK cells that
was sensitive to inhibitors of src family kinase and of
phosphatidylinositol 3-kinase. Binding of resting NK cells to
solid-phase ICAM-1 showed similar signaling requirements. A pulse of
either IL-2 or IL-15 to resting NK cells resulted in strongly enhanced,
actin-dependent adhesion to insect cells expressing ICAM-1 alone.
Coexpression of either LFA-3 (CD58) or CD48 with ICAM-1 resulted in
strong adhesion by resting NK cells, even in the absence of cytokines.
Therefore, receptors for LFA-3 and CD48 on resting NK cells strengthen
the adhesion mediated by LFA-1. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and cellular cytotoxicity (1). The rapid response
of resting NK cells to cytokines is well documented. Type I IFN,
produced during virus infections, stimulates the proliferation of NK
cells and augments their cytotoxic activity. IL-15, secreted by a
number of different cell types, activates NK cell proliferation,
cytotoxicity, and cytokine production (2). NK cells
secrete large amounts of IFN- in response to IL-12 and IL-18, which
are produced during infections by various intracellular pathogens
(3). NK cells are recruited to sites of infections by
chemokines, such as macrophage-inflammatory protein-1
, during
liver infection by mouse CMV (4). In addition to signals
received from soluble mediators, NK cells are also activated through
cell contact by receptors that recognize ligands on other cells.
However, little is known about activation of resting NK cells by such
receptors.
For practical reasons, most studies on NK-target cell interactions have
relied on NK cells expanded in vitro by continuous stimulation with
IL-2 or IL-15. The cytotoxicity of such activated NK cells is induced
by several activating receptors that bind ligands expressed at the
surface of other cells. For instance, human NK-sensitive tumor cells
express various combinations of ligands of the activation receptors
NKp30, NKp44, and NKp46 (5). Expression of MICA, MICB, and
UL16 binding protein molecules on stressed cells or tumor cells
leads to activation of NK cells through the receptor complex
NKG2D/DAP10 (6). Some receptors act as costimulators of
natural cytotoxicity. For instance, CD2, which binds to LFA-3 (CD58),
has long been known to costimulate NK cell cytotoxicity (7, 8). 2B4 (CD244), which binds to CD48, costimulates activation
signals delivered through the immunoreceptor tyrosine-based activation
motif
(ITAM)3-containing
signaling subunits associated with CD16 and NKp46 (9), and
through receptor KIR2DL4 (10). Conjugate formation between
NK cells and target cells is a prerequisite to target cell lysis and is
critically dependent on engagement of the 2
integrin LFA-1 (11), a heterodimer of CD11a and CD18 which
binds ICAM-1, ICAM-2, and ICAM-3. LFA-1-dependent adhesion of NK cells
to target cells is inhibited by killer cell Ig-like receptors upon
binding to MHC class I ligands on the target cells
(12).
To test how specific receptor-ligand interactions activate resting NK cells during cell contacts, we expressed ligands of human NK cell receptors in Drosophila cells. Unlike mammalian cells, insect cells do not express a multitude of ligands for adhesion and activation receptors of human NK cells, and are therefore well suited to study the contribution of individual receptors to NK cell activation. As a first step, we investigated the requirements for adhesion of resting NK cells to a target cell. Adhesion of T cells to APC depends on LFA-1 and on TCR signals that regulate the affinity and avidity of LFA-1 for its ICAM ligands (13, 14, 15, 16). T cell adhesion can be strengthened by signals from coreceptors CD2 and CD28 (17, 18). Adhesion of NK cells to target cells is also dependent on LFA-1 (11). Using transfected insect cells, we now show that expression of ICAM-1 is sufficient to initiate a signal-dependent adhesion of resting NK cells which can be greatly enhanced either by a pulse of IL-2 or IL-15, or by coexpression of LFA-3 or CD48 with ICAM-1, even in the absence of cytokines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Drosophila Schneider cell 2 (SC2; a gift from L. Teyton, Scripps Research Institute, La Jolla, CA) was maintained in Schneider’s medium with L-glutamine (Life Technologies, Rockville, MD) plus 10% FBS (Life Technologies). Human NK cells were isolated from peripheral blood using an NK cell isolation kit (Miltenyi Biotec, Auburn, CA). Activated NK cells were expanded in Iscove’s modified essential medium (Life Technologies), supplemented with 10% human serum (Life Technologies), 2 mM glutamine (Life Technologies), 100 U/ml rIL-2 (Hoffmann-LaRoche, Nutley, NJ), and 10% purified human IL-2 (Hemagen, Columbia, MD). Resting NK cells were resuspended in the same medium without IL-2, and were used within 1 or 2 days after isolation, only when <5% positive for the activation markers CD25 and CD69. rIL-15 was from PeproTech (Rocky Hill, NJ).
Antibodies
For FACS analysis and cell sorting, the following directly conjugated mouse mAbs were used: R-PE-conjugated (R-PE)-CD2 (clone RPA-2.10), R-PE-CD11a (clone Hl11), R-PE-CD18 (clone 6.7), R-PE-CD25 (clone M-A251), R-PE-CD54 (clone HA58), CyChrome-CD54 (clone HA58), R-PE-CD58 (clone 1C3), and FITC-conjugated CD58 (clone 1C3) (BD PharMingen, San Diego, CA). R-PE-CD3 (clone UCHT1), FITC-CD48 (clone J4-57), R-PE-CD56 (clone N901), and R-PE-CD69 (clone FN50), and R-PE-2B4 (clone C1.7) were from Beckman Coulter (Miami, FL). FITC-CD58 (clone TS2/9.1.4.3) was from Ancell (Bayport, MN). Isotype-matched PE-, FITC-, or CyChrome-conjugated mAbs were from BD PharMingen.
Expression of human proteins in Drosophila SC2 cells
cDNA clones for LFA-3 (CD58), CD48, and ICAM-1 (CD54) were obtained by RT-PCR amplification from total RNA isolated from the human cell line 721.221 using the RNeasy kit (Qiagen, Valencia, CA). The following primer pairs were used: LFA-3, 5'-ATTGGATCCCGAGCATGGTTGCTGGGAGC-3' and 5'-AAGTCGACTAGTTCAATTGGAGTTGGTTCTGTC-3'; CD48, 5'-ATTGGATCCTCTAGCCAGGCTCTCAACTGTCT-3' and 5'-AAGTCGACTAGTGTGAGGAGCATGATCACCAACAG-3'; and ICAM-1, 5'-ATTGGATCCGAGCTCCTCTGCTACTCAGAGTTG-3' and 5'-AAGTCGACTAGTATCTGACTGAGGACAATGCCCTGT-3'. PCR products were gel-purified using the Wizard PCR system (Promega, Madison, WI), cloned into pGEM-T-Easy (Promega), from which they were subcloned using BamHI/SalI sites into the insect expression vector pRmHa3 (19) (a gift from L. Teyton) under control of the metallothionein promoter. All cDNAs were verified by sequencing. Three million SC2 cells (1 x 106/ml) in a well of a six-well plate were transfected with a total of 30 µg of recombinant DNA and 1 µg of the selection vector pNeofly (20) (a gift from L. Teyton) using a calcium phosphate transfection kit (Invitrogen, Carlsbad, CA). Stable transfectants were selected and maintained in 0.5 mg/ml G418 (Life Technologies). Expression of the transfected cDNAs was induced by incubation for 24 h with 1 mM CuSO4. SC2 cells expressing the transfected cDNAs were selected by fluorescence-activated cell sorting (FAST Systems, Gaithersburg, MD). Expression of transfected human cDNAs was monitored before every experiment by flow cytometry.
ICAM-1 binding and adhesion assays
Soluble ICAM-1 was produced as a fusion protein with the Fc
portion of human IgG1, using the ICAM-1-receptor globulin
construct (a gift of W. Kolanus, University of Munich, Munich, Germany)
after transfection into COS/7 cells (21). Flat-bottom
96-well plates were coated with goat anti-human IgG
(Fc-specific) Ab at 1 µg/well for 90 min at 25°C, blocked
overnight with 1% BSA in PBS, incubated with purified ICAM-1-receptor
globulin for 30 min at 24°C, and washed with PBS. NK cells were
labeled with 12 µg/ml bisbenzimide H33342 fluorochrome
trihydrochloride (Calbiochem, La Jolla, CA) for 30 min at 37°C,
collected by centrifugation, resuspended in HBSS, and plated at
1.5 x 105/well. Where indicated, cells were
incubated with different inhibitors 45 min before, and during the
binding assay. Cells were then allowed to adhere for 30 min at 37°C.
Unbound cells were washed off with 300 µl of HBSS three times. The
proportion of bound cells was calculated by resuspending the cells in
100 µl of 2% paraformaldehyde in PBS and using a fluorescence plate
reader (Victor2 1420 multilabel counter; Wallac,
Gaithersburg, MD). The signal of 1.5 x 105
cells/well at 490 nm represents 100% of binding. Each determination
was conducted in triplicate. Conjugate formation between NK cells and
SC2 cells was determined as described (12), with the
following modifications: SC2 cells were resuspended in HBSS medium
(BioSource, Camarillo, CA) plus 5% FBS, and 4 x
105 cells were mixed with 1 x
105 effector cells in a final volume of 200 µl.
Insect cells grow at temperatures below 30°C and undergo a heat shock
reaction above 32°C. However, the short incubation times (up to
1 h) at 37°C of the assays used here were not sufficient to
cause detectable increase of propidium iodide uptake in insect
cells.
Actin polymerization
NK cells (1 x 106) and SC2 cells (1 x 106) were mixed in a 400-µl final volume in 12 x 75 mm round-bottom polystyrene tubes (BD Falcon, Franklin Lakes, NJ), and centrifuged at 4°C for 3 min at 300 rpm (25 x g). Samples were placed at 37°C for various times. The reaction was stopped by brief vortexing. Cells were fixed by adding 400 µl of 8% paraformaldehyde in Dulbecco’s PBS for 20 min at room temperature, and permeabilized by adding 800 µl of 4% paraformaldehyde in PBS plus 0.25% Triton X-100, for 20 min at room temperature. After three washes with Dulbecco’s PBS/0.5%BSA, the cells were resuspended in 100 µl of washing buffer and kept at room temperature for 20 min. Each sample was incubated for 30–60 min at 4°C in the dark with 5 µl of Oregon green-conjugated phalloidin (Molecular Probes, Eugene, OR) and 10 µl of PE-conjugated anti-CD18. After three washes, samples were analyzed by FACS. NK cells were gated by side scatter and by PE fluorescence.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Resting NK cells, freshly isolated from peripheral blood, were
compared with IL-2-activated NK cells for their ability to bind to
ICAM-1 using two different assays. First, fluorescently labeled NK
cells were added to plates coated with goat anti-human IgG Fc to
which soluble ICAM-1, produced as a fusion protein with the Fc portion
of human IgG1, was bound. The proportion of cells that remained bound
after washing was determined with a fluorescence plate-reader. About
20% of resting NK cells and 80% of activated NK cells remained
attached (Fig. 1
A). Second,
adhesion of NK cells (labeled with a green dye) to insect SC2 cells
(labeled with a red dye) expressing human ICAM-1 was determined by a
conjugation assay using two-color flow cytometry. Conjugate formation
with resting NK cells peaked at 20 min, with 
20% of NK cells in
conjugates with SC2-ICAM-1 cells (Fig. 1B). In contrast,
within 5 min, 60% of activated NK cells had already formed
conjugates with SC2-ICAM-1 cells (Fig. 1B). No conjugate
formation was detected with untransfected SC2 cells and either
activated or resting NK cells.
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A).
In contrast, binding of resting NK cells was severely inhibited by PP1
and Wortmannin, but not by cytochalasin D and colchicine (Fig. 2B). Adhesion of resting NK cells to SC2-ICAM-1 cells was
also inhibited by PP1 and Wortmannin (Fig. 2C). Adhesion of
activated NK cells was not sensitive to these inhibitors (data not
shown). These results suggest that binding of resting NK cells to
ICAM-1 requires src family kinase and PI3K signals. These
signals may be transmitted directly by LFA-1 upon binding to ICAM-1.
However, it is formally possible that a constitutive, basal activity of
src family kinase and PI3K in resting NK cells is required
for LFA-1 binding to ICAM-1. We conclude that the binding of resting NK
cells to ICAM-1 is signaling-dependent, and differs from that of
IL-2-activated NK cells.
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). The surface level of LFA-1 is very
high on resting NK cells and is even higher on activated NK cells.
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The stronger binding to ICAM-1 and the greater adhesion to
SC2-ICAM-1 cells by IL-2-activated NK cells (Fig. 1
) could be due to a
stable alteration in the adhesive properties of IL-2-activated NK
cells, to a specific signal transmitted by IL-2 and IL-15, or to a
combination of both. We tested whether a 45-min pulse with 100 U/ml of
IL-2 or IL-15 was sufficient to alter adhesion by resting NK cells.
IL-2 and IL-15 each enhanced the binding of resting NK cells to ICAM-1
on plates (Fig. 4A) and to
SC2-ICAM-1 cells (Fig. 4B). Adhesion to SC2-ICAM-1 cells
occurred faster and reached higher levels in the presence of these
cytokines. Binding of IL-2-pulsed resting NK cells to solid-phase
ICAM-1 and adhesion to SC2-ICAM-1 cells were strongly inhibited by PP1,
Wortmannin, and cytochalasin D (Fig. 4, C and D).
Colchicine had only a minor effect: it slowed down the initial phase of
adhesion to SC2-ICAM-1 cells (Fig. 4D). Similar results were
obtained with these inhibitors during IL-15-induced adhesion to
SC2-ICAM-1 cells (data not shown). Thus, not only src family
kinase and PI3K activity but also actin polymerization appears to be an
important requirement for the cytokine-induced binding to ICAM-1. These
results show that IL-2 and IL-15 enhance adhesion of resting NK cells
through an actin-dependent pathway that does not overcome the
requirement for src family kinase and PI3K activity.
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To test whether other receptors on resting NK cells could
contribute to adhesion, we generated Drosophila SC2 cell
transfectants that expressed human LFA-3 (CD58) and CD48, either alone
or in combination with ICAM-1 (Fig. 5).
Cell sorting was used to obtain SC2 cells with matched levels of
expression of the same ligand on different transfectants. CD2 and 2B4
(CD244), the receptors for LFA-3 and CD48, respectively, are expressed
on NK cells. Whereas expression of 2B4 is uniformly high, and
comparable between resting and activated NK cells, CD2 expression is
higher on activated NK cells (Fig. 3). A minority fraction of resting
NK cells, which varies among individuals, is negative for CD2 surface
expression (Fig. 3). Very little, if any, conjugate formation occurred
between resting NK cells and SC2 cells expressing only LFA-3 or CD48
(Fig. 6A). However,
coexpression of either LFA-3 or CD48 with ICAM-1 resulted in a much
higher level of adhesion than that obtained with SC2-ICAM-1 cells (Fig. 6A). Conjugates increased gradually for 40 min, reaching
60% of resting NK cells after incubation with SC2-ICAM-1/LFA-3 and
SC2-ICAM-1/CD48 cells. In contrast, as shown in Fig. 1, activated NK
cells formed conjugates very rapidly with SC2-ICAM-1 cells.
Coexpression of LFA-3 or CD48 with ICAM-1 resulted in minimal
enhancement of adhesion by activated NK cells (Fig. 6B). As
observed with resting cells, activated NK cells formed very few, if
any, conjugates with insect cells expressing only LFA-3 or CD48 (Fig. 6B). We conclude that, even in the absence of exogenous
signals such as cytokines, resting NK cells are characterized by their
ability to adhere to target cells expressing ICAM-1, and to adhere
strongly to target cells that coexpress LFA-3 or CD48 with ICAM-1.
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), as adhesion to SC2-ICAM-1 cells was,
in the absence or presence of IL-2 (Figs. 2 and 4). In contrast,
adhesion to SC2-ICAM-1/LFA-3 and to SC2-ICAM-1/CD48 cells was not as
sensitive to Wortmannin and was only partially sensitive to
cytochalasin D (Fig. 7). The effect of Wortmannin was mainly to reduce
the number of conjugates at all time points whereas cytochalasin D
caused a delay in conjugate formation. Partial sensitivity to the
microtubule inhibitor colchicine was also observed (Fig. 7). Therefore,
the signaling requirements for adhesion of resting NK cells to target
cells expressing ICAM-1 together with either LFA-3 or CD48 are
different from those for adhesion to ICAM-1 alone and for adhesion to
ICAM-1 in the presence of IL-2. These results suggest that CD2 and 2B4,
upon engagement by ligand on target cells, provide signals that enhance
LFA-1-mediated adhesion.
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, A and B). The
maximal number of conjugates was reached within 10 min, instead of 40
min in the absence of cytokine. Further evidence for a cooperation
between cytokine signals and coreceptor engagement was obtained by
quantitating polymerized F-actin (Fig. 8C). Phalloidin
binding to fixed, permeabilized cells was determined by flow cytometry.
Some variability in the assay may have been contributed by phalloidin
binding to SC2 cells. Therefore, the only interpretable changes in
F-actin were those observed between samples that formed a similar
number of conjugates. In the absence of IL-2, no significant change in
F-actin content was observed after incubation of resting NK cells with
any of the SC2 cells. In contrast, incubation of IL-2-pulsed resting NK
cells with SC2-ICAM-1/LFA-3 and SC2-ICAM-1/CD48 cells resulted in a
doubling of F-actin content (Fig. 8C). This increase was
significantly greater than that observed with SC2 and SC2-ICAM-1 cells,
or with the double-transfected SC2 cells incubated with resting NK
cells that had not been pulsed with IL-2. Therefore, combined signals
from activation receptors CD2 or 2B4 and the IL-2R led to enhanced
actin polymerization in resting NK cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 integrins
such as LFA-1 is a regulated process that controls leukocyte migration
and function (22). Expression of ICAM-1 is up-regulated on
endothelial cells during inflammatory responses. Specific signals in T
cells and neutrophils, delivered by various receptors for cytokines,
chemokines, and for ligands on other cells, regulate the affinity and
avidity of 2 integrins for ICAM. In this
study, we investigated the regulation of adhesion of resting NK cells
to ICAM-1. We found that resting NK cells have ICAM-binding properties
different from those of activated NK cells, and from those reported for
T cells. Resting NK cells bound to ICAM-1 and formed conjugates with
insect cells expressing ICAM-1 alone, in the absence of exogenous
signals. This binding to ICAM-1 is an active process that was sensitive
to inhibitors of src family kinases and of PI3K. Adhesion of resting NK cells to ICAM-1 was enhanced by two different types of receptors. First, IL-2- or IL-15-pulsed resting NK cells adhered much more strongly than resting NK cells. This enhanced adhesion was sensitive to inhibitors of src family kinases, PI3K, and actin polymerization. Therefore, in response to a signal from IL-2 or IL-15, resting NK cells up-regulate their ICAM-1 binding properties through an actin-dependent mechanism that does not bypass the requirement for src family kinase and PI3K signals. In contrast, activated NK cells, which were expanded in IL-2 for several days, bound very strongly to ICAM-1 by a mechanism that was not sensitive to inhibitors of src family kinases and of PI3K. Therefore, the different adhesive properties of IL-2-activated NK cells must be due to stable alterations during expansion in IL-2. Second, resting NK cells adhered strongly to SC2 cells that expressed either LFA-3 or CD48 with ICAM-1. This enhanced adhesion could be due simply to the additive contribution of a second receptor-ligand interaction without specific signals, or it may require signals delivered by CD2 and 2B4 that enhance adhesion.
Our results support a signaling role of CD2 and 2B4 in adhesion to ICAM-1 by resting NK cells. Adhesion of resting NK cells to SC2-ICAM-1/LFA-3 and to SC2-ICAM-1/CD48 cells was only partially sensitive to inhibition of PI3K and of actin polymerization. The sensitivity of the CD2 and 2B4-dependent signals to PP1 indicated that these receptors deliver a src family kinase-dependent signal that reduces the dependence of adhesion to ICAM-1 on PI3K and actin polymerization. By comparison, the marked contribution of IL-2 and IL-15 to ICAM-1 binding, which is not due to an additive receptor-ligand interaction, did not overcome inhibition of src family kinases and PI3K, but had the additional requirement for actin polymerization. Signals through CD2 and 2B4 were also evident when measuring actin polymerization in resting NK cells incubated with SC2 cells expressing ICAM-1 with LFA-3 or CD48. Only with the combination of 1) coengagement of LFA-1 with CD2 or 2B4 and 2) an IL-2 or IL-15 signal was actin polymerization significantly increased.
A role of CD2 in adhesion of resting NK cells had been suggested by Ab
blocking experiments (23). Adhesion of resting NK cells to
a T lymphoma was blocked partially by anti-CD18, and less so by
anti-CD2; but complete inhibition was obtained by blocking both
receptors. In addition, the adhesion necessary for Ab-dependent
cell-mediated cytotoxicity by a human NK clone was contributed
by a combination of CD2 and CD18, whereas CD16 was not necessary for
conjugate formation (24). 2B4 has been described as a
costimulatory receptor that enhances signals from ITAM-containing
receptors (9). In this study, we show that it can provide
a signal that enhances adhesion through 2
integrin, a process that is most likely ITAM independent.
The signal-dependent adhesion of resting NK cells to insect cells
expressing ICAM-1 alone suggests that 2
integrin itself can signal upon binding ICAM-1 to up-regulate its own
adhesion. However, the possibility of an evolutionarily conserved
structure on insect cells that is recognized by a receptor on human NK
cells cannot be excluded. If such a receptor were to exist, it is
unable to provide the signals that resting NK cells receive through the
IL-2/IL-15 receptor, CD2, or 2B4. The transfected insect cells
described here provide a useful system to dissect the contribution of
individual ligand-receptor interactions to the activation of NK cells.
The very large number of activation and inhibitory receptors on NK
cells, and the fairly ubiquitous expression of their ligands on various
cell types, complicates the analysis of the role of specific receptors
in NK cell responses. Additional ligands of NK cell receptors can be
expressed in SC2 insect cells to study their contribution to different
types of NK cell responses.
The physiological importance of NK cell regulation by cell contact is
illustrated by the activation of resting NK cells by human dendritic
cells. Within 1 wk of coculture, resting NK cells underwent several
cell divisions and began producing IFN- (25). The
unique ability of resting NK cells to initiate adhesion upon contact
with ICAM-1-expressing cells, in the absence of exogenous signals, may
be relevant to their important role as innate effector cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Eric O. Long, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook II, 12441 Parklawn Drive, Rockville, MD 20852-1727. E-mail address: elong{at}nih.gov
3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; PI3K, phosphatidylinositol 3-kinase.
Received for publication July 23, 2002. Accepted for publication November 1, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L 2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1, a cytoplasmic regulatory molecule. Cell 86:233.[Medline]
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D. F. Barber, M. Faure, and E. O. Long LFA-1 Contributes an Early Signal for NK Cell Cytotoxicity J. Immunol., September 15, 2004; 173(6): 3653 - 3659. [Abstract] [Full Text] [PDF]
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A. Gismondi, L. Cifaldi, C. Mazza, S. Giliani, S. Parolini, S. Morrone, J. Jacobelli, E. Bandiera, L. Notarangelo, and A. Santoni Impaired natural and CD16-mediated NK cell cytotoxicity in patients with WAS and XLT: ability of IL-2 to correct NK cell functional defect Blood, July 15, 2004; 104(2): 436 - 443. [Abstract] [Full Text] [PDF]
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A. Fuchs, M. Cella, E. Giurisato, A. S. Shaw, and M. Colonna Cutting Edge: CD96 (Tactile) Promotes NK Cell-Target Cell Adhesion by Interacting with the Poliovirus Receptor (CD155) J. Immunol., April 1, 2004; 172(7): 3994 - 3998. [Abstract] [Full Text] [PDF]
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B. Riteau, D. F. Barber, and E. O. Long Vav1 Phosphorylation Is Induced by {beta}2 Integrin Engagement on Natural Killer Cells Upstream of Actin Cytoskeleton and Lipid Raft Reorganization J. Exp. Med., August 4, 2003; 198(3): 469 - 474. [Abstract] [Full Text] [PDF]
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M. Faure, D. F. Barber, S. M. Takahashi, T. Jin, and E. O. Long Spontaneous Clustering and Tyrosine Phosphorylation of NK Cell Inhibitory Receptor Induced by Ligand Binding J. Immunol., June 15, 2003; 170(12): 6107 - 6114. [Abstract] [Full Text] [PDF]
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, •) and
activated (
, 
) NK cells labeled with a green dye were incubated
with SC2 cells (
), PP1 (
), or Wortmannin (
) for 45 min and tested for
binding to plate-bound ICAM-1 (A) or to SC2-ICAM-1 cells
(B) as described in Fig. 1
