The Journal of Immunology
Tethering of Intercellular Adhesion Molecule on Target Cells
Is Required for LFA-1–Dependent NK Cell Adhesion and
Catharina C. Gross,* Joseph A. Brzostowski,†Dongfang Liu,* and Eric O. Long*
aLb2integrin (LFA-1) has an important role in the formation of T cell and NK cell cytotoxic immunological synapses and in
target cell killing. Binding of LFA-1 to ICAM on target cells promotes not only adhesion but also polarization of cytolytic granules
in NK cells. In this study, we tested whether LFA-1–dependent NK cell responses are regulated by the distribution and mobility of
ICAM at the surface of target cells. We show that depolymerization of F-actin in NK-sensitive target cells abrogated LFA-1–
dependent conjugate formation and granule polarization in primary NK cells. Degranulation, which is not controlled by
LFA-1, was not impaired. Fluorescence recovery after photobleaching experiments and particle tracking by total internal re-
flection fluorescence microscopy revealed that ICAM-1 and ICAM-2 were distributed in largely immobile clusters. ICAM clusters
were maintained and became highly mobile after actin depolymerization. Moreover, reducing ICAM-2 mobility on an NK-
resistant target cell through expression of ezrin, an adaptor molecule that tethers proteins to the actin cytoskeleton, enhanced
LFA-1–dependent adhesion and granule polarization. Finally, although NK cells kept moving over freely diffusible ICAM-1 on
a lipid bilayer, they bound and spread over solid-phase ICAM-1. We conclude that tethering, rather than clustering of ICAM,
promotes proper signaling by LFA-1 in NK cells. Our findings suggest that the lateral diffusion of integrin ligands on cells may be
an important determinant of susceptibility to lysis by cytotoxic lymphocytes.
phocytes to target cells, which is essential for the cytotoxic activity
of T cells and NK cells (2–4). LFA-1 plays a central role in the
organization of immunological synapses formed by T cells and
NK cells (5–10). LFA-1 on resting T cells is kept in an inactive,
mation through “inside-out” signals delivered by other receptors
(11). In contrast, resting NK cells bind directly to ICAM-1 in a sig-
naling dependent way (12, 13). Furthermore, LFA-1 binding to
NKG2D, induces granule polarization (16).
Whereas polarization of cytolytic granules is induced by LFA-1
in NK cells, degranulation is triggered by the low-affinity FcgR
CD16 or by synergistic combinations of coactivation receptors, in-
dependently of LFA-1 (10, 13, 14, 17). This uncoupling of signals
for granule polarization and degranulation observed in NK cells is
very different from cytotoxic T cells, in which these two functions
The Journal of Immunology, 2010, 185: 2918–2926.
he aLb2integrin LFA-1 (a heterodimer of CD11a/CD18)
binds to ICAMs and mediates arrest of rolling leukocytes
in blood vessels (1) and tight adhesion of cytotoxic lym-
are controlled centrally by the TCR and are costimulated by other
receptors including CD28 and LFA-1 (9, 18, 19). The ability of
LFA-1 in NK cells to signal autonomously make NK cells an excel-
lent tool to study LFA-1–dependent functions.
Lateral segregation of proteins within cell membranes leads to
functional subcompartementalization of the lipid bilayer (20, 21).
How receptor distribution at the plasma membrane and receptor
binding to the cytoskeleton determines signaling output has been
function is controlled by the distribution of ligands on target cells. A
few studies have suggested that ligand distribution on target cells
may be important for proper recognition by T cells and NK cells. A
lighting an unexpected contribution of the intracellular portion of
a ligand to synapse organization and to signaling in another cell
(29). A mutation in the acylation site of MICA, an NKG2D receptor
ligand on target cells, resulted in diminished NKG2D-dependent
killing by NK cells, suggesting that sorting of NKG2D ligands into
naling by NKG2D in NK cells (30). An earlier study suggested that
sion of the ezrin/radixi/moesin protein ezrin rendered the cells more
clustering, or mobility of ICAM at the surface of target cells that is
critical for LFA-1–dependent responses is not known.
In this study, we tested how the distribution of ICAM on
functions, namely adhesion to target cells and polarization of
cytolytic granules. We have used several approaches to manipulate
the attachment of ICAM-1 and ICAM-2 to the cytoskeleton or to
artificial surfaces. Our results show that it is the immobilization of
ICAM, rather than polarization to one end of the cell or clustering,
that is required for functional interaction with LFA-1.
*Molecular and Cellular Immunology Section and†Imaging Facility, Laboratory of
Immunogenetics, National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Rockville, MD 20852
Received for publication March 8, 2010. Accepted for publication June 28, 2010.
This work was supported by the Intramural Research Program of the National In-
stitute of Allergy and Infectious Diseases, National Institutes of Health.
Address correspondence and reprint requests to Dr. Eric O. Long, Laboratory of
Immunogenetics, National Institute of Allergy and Infectious Diseases, National
Institutes of Health, 12441 Parklawn Drive, Rockville, MD 20852. E-mail address:
The online version of this article contains supplemental material.
Abbreviations used in this paper: CDF, cumulative distribution function; FRAP,
fluorescence recovery after photobleaching; Jasp, jasplakinolide; LatA, latrunculin
A; TIRF, total internal reflection fluorescence.
Materials and Methods
Human NK cells were isolated from peripheral blood cells by negative
selection using an NK cell isolation kit (Miltenyi Biotec, Auburn, CA).
Resting NK cells (95–99% CD32CD56+) were resuspended in IMDM
(Invitrogen, Carlsbad, CA) supplemented with 10% human serum (Valley
Biomedical, Winchester, VA) and used 1–2 d after isolation. Polyclonal
IL-2–activated NK cells were expanded in the presence of feeder cells
(0.5 3 106PBLs/ml, gamma-irradiated with 4.5K) with IMDM supple-
mented with 10% human serum, 100 IU/ml rIL-2 (Hoffman-La Roche,
Basel, Switzerland), and 10% purified human IL-2 (Hemagen Diagnostics,
Columbia, MD). IL-2–activated cells were used 2–3 wk after isolation.
The B cell lymphoma line 721.221 was cultivated in IMDM supplemented
with 10% heat-inactivated FBS (Hyclone, Logan, UT). The mouse thymoma
cell line BW5147 (a gift from T. Kamala, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, MD) was culti-
vated in IMDM supplemented with 10% heat-inactivated FBS.
Transfection of BW5147 cells with hEzrin-EGFP
BW5147 cells were transfected with hEzrin-EGFP (pHJ421) human ezrin
coding sequence subcloned into pEGFP-N1 eukaryotic expressionvector [a
gift from J.-J. Hao and S. Shaw, National Cancer Institute, National Insti-
tutes of Health (32)], using the BTX machine (ECM830, settings: 230 V, 10
ms, 1 pulse; Harvard Apparatus, Holliston, MA). Cells were recovered for
16 h at 37˚C and 5% CO2, and hEzrin-EGFP–positive cells were selected
by FACS. Stable transfectants were generated from the FACS-sorted cells
by cultivating them in IMDM/10% FBS/1 mg/ml Geneticin (Life Technol-
ogies, Grand Island, NY) and subcloning.
Pretreatment of target cells with inhibitors
latrunculin A (LatA) or jasplakinolide (Jasp) (both Calbiochem, San Diego,
CA) treatment, 10 3 106721.221 or K562 target cells were resuspended in
1 ml IMDM supplemented with 10% FBS containing 2% DMSO as a neg-
ative control to match the final concentration of carrier in treated cells. For
the experiments, cells were treated 0.5, 3, or 20 mM LatA or 0.5, 1, or 2
mM Jasp. Staining F-actin with phalloidin revealed that 3 mM LatA was
required to disrupt F-actin completely and that the effect of Jasp was
evident at a concentration as low as 0.5 mM (data not shown). Cells were
incubated for 40 min at 37˚C. Inhibitors were washed away prior mixing
with NK cells for the different assays.
Conjugate formation between NK cells and target cells was determined as
previously described (12) with minor modifications. Briefly, NK cells
were labeled with 1 mg/ml Cell Tracker Green CMFDA (Invitrogen) for
30 min at 37˚C and 5% CO2, washed, and incubated for another 30 min at
37˚C and 5% CO2. If BW5147 cells were used as targets, NK cells were
labeled with 20 ml/ml anti-CD56 allophycocyanin (BD Biosciences, San
Jose, CA) for 15 min at 37˚C and 5% CO2. Target cells were labeled with
PKH26 Red (721.221, K562) or PKH67 Green (BW5147 cells) (both
Sigma-Aldrich) for 5 min at room temperature, washed extensively, and
recovered for 30 min at 37˚C and 5% CO2. NK cells and target cells were
mixed at a 1:2 E:T cell ratio with 1 3 105NK cells and 2 3 105target-
cells at 4˚C. Cells were spun down at 20 3 g for 3 min, and conjugate
formation was stopped by vortexing and fixation of cells using 0.5%
paraformaldehyde solution (Electron Microscopy Sciences, Hatfield,
PA) after 0-, 5-, 10-, 20-, 30-, or 60-min incubation at 37˚C. Conjugate
formation was determined by flow cytometry (FACSCalibur; BD Bio-
sciences) and is represented as the fraction of NK cells that shifted into
two-color conjugates. For blocking of LFA-1 on NK cells, 1 3 106Cell
Tracker Green-labeled NK cells were pretreated with 20 mg/ml IgG2a
(HOPC-1; Sigma-Aldrich) or anti-human CD18 (Calbiochem) for 15
min at 4˚C.
The degranulation assay was performed as described previously (14).
Briefly, 2 3 105NK cells were added to 4 3 105721.221 or K562 cells in
a total volume of 200 ml IMDM supplemented with 10% heat-inactivated
FBS. Cells were mixed and incubated for 1 h at 37˚C and 5% CO2. After-
ward, the cells were spun down and stained with PE-conjugated anti-CD56
(BD Biosciences) and FITC-conjugated anti-CD107a (BD Biosciences) Ab
for 45 min at 4˚C. Degranulation of CD56-positive NK cells was analyzed
by flow cytometry.
Perforin polarization assay
The polarization assay was performed as described previously (15). Briefly,
NK cells and target cells (721.221, K562, or BW5147) were mixed at a 1:1
E:T ratio, with 1 3 106of each cell type per sample. Cells were incubated
for 20 min at 37˚C and 5% CO2. Cells were resuspended and put onto
a poly-D-lysine–coated 2-well Culture Slide (BD Biosciences). Cells were
allowed to settle down for 1 h at room temperature and fixed using 4%
paraformaldehyde (Electron Microscopy Sciences). Cells were permeabi-
lized using 0.5% Triton X-100 and stained using an anti-human perforin
Ab (Pierce, Rockford, IL) and an Alexa 488-conjugated secondary goat
anti-mouse IgG Ab (Molecular Probes, Eugene, OR). Cells were imaged
by confocal microscopy (LSM510; Zeiss, Oberkochen, Germany) with
a 340, 1.3 NA Plan Neofluar (Zeiss) oil immersion objective lens. Fifty
to 200 NK cells in contact with target cells were analyzed for polarization
of perforin containing granules.
Fluorescence recovery after photobleaching analysis
ICAM-1 on 721.221 cells was stained using a PE-conjugated anti–ICAM-
was performed using a confocal microscope (LSM510; Zeiss). A region of
the cell surface was bleached using the 488-nm laser. The recovery of the
assay, cells were maintained at 37˚C. The fluorescence intensities of the
bleached region and an unbleached region of the same cell over time were
analyzed using the LSM510 software (Zeiss). The medians of the relative
intensities of different cells were plotted over time.
Total internal reflection fluorescence microscopy
Endogenous cell surface ICAM-1 or ICAM-2 on 721.221 or BW5147 cells
was fluorescently labeled with PE-conjugated anti-human ICAM-1 (BD
Biosciences), anti-human ICAM-2 (Beckman Coulter, Fullerton, CA), or
anti-mouse ICAM-2 (BD Pharmingen) Ab. Total internal reflection fluores-
cence (TIRF) imaging was performed using an Olympus IX81 TIRF micro-
scope equipped with a 561-nm diode-pumped laser (Cobolt, Stockholm,
Sweden), 3100 1.45 NA Olympus TIRF microscopy lens, and a Cascade
II 1024B EM-CCD camera (Photometrics, Tucson, AZ). Cells were main-
tained at 37˚C using a LiveCell environmental chamber (Pathology Devi-
ces, Westminster, MD). Images were captured at 32 frames/s using
MetaMorph software (Molecular Devices, Sunnyvale, CA) for 250 frames.
The movement of labeled ICAM molecules was automatically tracked
using code developed for MatLab software [http://physics.georgetown.
edu/matlab (33)]. The code was modified to refine particle positioning with
a two-dimensional Gaussian fit (34) and to determine intensities of in-
dividual particles. Mean square displacements were determined from
positional coordinates to calculate short-range diffusion coefficients of
individual particles for five time intervals. The resulting diffusion coef-
ficients were plotted as a cumulative distribution function (CDF). A CDF
plots the same information as a histogram for a given data set; however,
where the histogram plots the frequency distribution (y-axis) as a func-
tion of the binned data set (x-axis), the data are not binned for a CDF
plot. The CDF plot is generated as follows: the data set is sorted from the
smallest to greatest value. Next, the number of data points is summed,
and the value of the position of a data point on the sorted list is divided
by the sum to yield the probability value (y-axis). Finally, based on its
position on sorted list, the probability is plotted against the value of
the data point. The CDF displays the distribution of the data set from
the smallest (from left on the x-axis) to greatest value (at the right of the
x-axis) and provides the probability (y-axis) of whether a particular value
will occur at or less than a specified point on the x-axis. Moreover, a CDF
plot allows for the rapid identification of the median value of the data set
by interpolation at 50% on the y-axis, it is not prone to binning artifacts,
and the separation of the data set by log scale is more apparent for visual
inspection. In addition, graphical comparisons between data sets are
generally more easily interpretable relative to overlaying histograms.
Imaging of NK cells on ICAM-1 bound on lipid bilayers or
formed between a coverslip and the microaqueduct slide of a Bioptechs par-
allel plate flow chamber-FCS2 (Bioptechs, Butler, PA) as described previ-
ously (10, 11). A human ICAM-1-Fc fusion protein was coated on
coverslips in 100 mM sodium bicarbonate (pH 9.2) at 10 ng/ml, 100 ng/
ml, 1 mg/ml, and 10 mg/ml at 4˚C overnight. Five-percent FBS-containing
culture medium was used to block nonspecific binding. The ICAM-1-Fc
fusion protein cloned in the CD5lneg1 vector (35) was produced by
The Journal of Immunology2919
transfection into 293T cells, as described previously (36). To avoid stimula-
tion of NK cells via CD16 by the human IgG1 Fc portion of the ICAM-1-Fc
Fc domain were mutated as Leu235to Gly235and Gly236to Leu236. Whereas
unmutated ICAM-1-Fc fusion protein that was attached to plates triggered
degranulation in NK cells, the ICAM-1-Fc protein carrying the mutated
CD16 binding site did not. The movement of NK cells was measured
frames by Dynamic Image Analysis System (37).
Disruption of actin filaments in target cells prevents conjugate
formation with NK cells
adhesion and granule polarization. The B cell lymphoma cell line
721.221 (38), which does not express HLA-A, HLA-B, and
HLA-C, is sensitive to lysis by NK cells. To test whether conjugate
formation of NK cells is dependent on an intact cytoskeleton in
with different concentrations of LatA (0.5, 3, and 20 mM) prior to
Conjugate formation of freshly isolated, primary NK cells was
inhibited by LatA treatment of 721.221 cells (Fig. 1A, left panel).
Moreover, conjugate formation of IL-2–activated NK cells with
721.221 cells was also prevented by pretreatment of target cells
with LatA (Supplemental Fig. 1). These results suggest that either
an intact actin cytoskeleton or actin cytoskeleton remodeling is
required for proper conjugate formation.
To distinguish between these two possibilities, F-actin filaments
in target cells were stabilized by treatment with Jasp (Fig. 1A, right
panel). Jasp stabilizes existing F-actin but prevents actin cytoskel-
eton remodeling. Because Jasp had no effect on conjugate forma-
tion with NK cells (Fig. 1A, right panel), cytoskeleton dynamics
in target cells do not seem to be important for conjugate formation
of NK cells. In addition, disruption of microtubules in target cells
with Nocodazole had no effect on conjugate formation with rest-
ing NK cells (data not shown). We conclude that an intact cyto-
skeleton, but not cytoskeleton dynamics, in target cells is impor-
tant for NK cell conjugate formation.
To test whether disruption of F-actin in target cells was prevent-
ing conjugate formation specifically, or whether it had more global
inhibitory effects, the ability of NK cells to degranulate in response
to target cells was evaluated. Whereas conjugate formation of
NK cells with ICAM-expressing target cells is strictly dependent
target cells prevents conjugate formation with
NK cells. A, 721.221 cells pretreated with DMSO
carrier alone (d), LatA (left panel), or Jasp (right
panel) at indicated concentrations for 40 min at
37˚C were tested for conjugate formation with
resting NK cells. Error bars indicate the SD (n =
3 individual NK cell donors in three independent
experiments). B, 721.221 target cells treated as in
A were mixed with resting NK cells, and de-
granulation was measured after 1 h at 37˚C by
staining for CD107a at the surface of NK cells.
Each symbol indicates one of the six (left panel)
or four (right panel) individual NK cell donors.
Six (left panel) or four (right panel) independent
experiments were performed.
Disruption of actin filaments in
with DMSO carrier (A, B, filled histograms) or with 3 mM (A) or 20 mM (B)
to poly-D-lysine–coated culture slides and incubated for 1 h at room
temperature before fixation, permeabilization, and staining for perforin.
Granule polarization is expressed as the fraction of cells in contact with
perforin clustered at the NK-target cell interface. Error bars indicate the
SD (n = 3 individual donors in three independent experiments).
Disruption of actin filaments in 721.221 target cells inhibits
2920 ICAM MOBILITY CONTROLS LFA-1–DEPENDENT NK CELL FUNCTION
on LFA-1 (Supplemental Fig. 2) (39), degranulation is LFA-1 in-
dependent (13, 14). 721.221 cells treated with lower doses of LatA
induced about twice as much degranulation by NK cells as un-
treated 721.221 cells (Fig. 1B, left panel). Our laboratory has
observed that, under certain conditions, engagement of LFA-1
by ICAM-1 reduces the amount and delays the onset of degranu-
lation induced by activation receptors (10, 14). The enhanced de-
granulation seen in this study after latrunculin treatment of
target cells may be due to a reversal of LFA-1–mediated inhi-
bition of degranulation. In agreement with this hypothesis, block-
ing of LFA-1 with an Ab resulted in a 2-fold increase of degran-
ulation by NK cells (Supplemental Fig. 3). At the highest dose
(20 mM), however, LatA blocked degranulation by NK cells (Fig.
1B, left panel). Stabilization of F-actin in 721.221 cells by Jasp
(Fig. 1B, right panel), or disruption of microtubules with Nocoda-
zole (data not shown), had no effect on degranulation by NK cells.
We conclude that LFA-1–dependent conjugate formation, but not
LFA-1–independent degranulation, requires an intact cytoskeleton
on target cells.
Disruption of F-actin in target cells inhibits polarization of
cytolytic granules in NK cells
The sensitivity of conjugate formation but not degranulation of
NK cells to the disruption of F-actin in target cells suggested that
a functional LFA-1 interaction with ICAM is dependent on intact
F-actin in target cells. To test this possibility, we evaluated the im-
dependent function in NK cells—granule polarization toward
target cells (14, 15). Pretreatment of 721.221 cells with LatA
resulted in diminished granule polarization in NK cells (Fig. 2).
As expected, because of inhibition of tight conjugate formation,
fewer NK cells were found in conjugates with 721.221 cells in the
presence of LatA. However, the reduction of polarization was not
a consequence of reduced conjugate formation as polarization was
scored only in NK cells that had formed tight contact with target
cells. Therefore, in addition to the LFA-1–dependent conjugate
formation, another LFA-1–dependent process, polarization of
cytolytic granules, is dependent on an intact cytoskeleton in target
Disruption of F-actin changes the lateral mobility of
ICAM-1 and ICAM-2
How could disruption of F-actin in target cells result in defective
ICAM interactions with LFA-1 on NK cells? As ICAM is linked
to the actin cytoskeleton by direct binding to a-actinin (a-actinin-
1 and a-actinin-4) (40–42) and to the ezrin/radixi/moesin protein
ICAM-1 mobility. ICAM-1 mobility was determined by FRAP. Cells were
stained with a PE-labeled anti–ICAM-1 Ab (original magnification 380).
Fluorescence recovery in the bleached area of 721.221 cells either untreated
(A, upperpanel; scale bar, 5 mm; B, d) or LatA treated (A, lower panel; B, s)
is displayed. Fluorescence intensity of a nonbleached area of the 721.221 cells
either untreated (B, n) or LatA treated (B, N) served as a control. Error bars
indicate the SD (untreated cells, n = 7; LatA-treated cells, n = 8).
Disruption of actin filaments in target cells increases
ICAM-1 on K562 cells and decreases conjugate formation with NK cells.
The mobility of ICAM-1 was determined by FRAP. Cells were stained
with a PE-labeled anti–ICAM-1 Ab (original magnification 380). Fluo-
rescence recovery in the bleached area of K562 cells either untreated (A,
upper panel; scale bar, 5 mm; B, d) or LatA treated (A, lower panel; B, s)
is displayed. Fluorescence intensity of a nonbleached area of the K562
cells either untreated (B, n) or LatA-treated (B, N) served as a control.
Error bars indicate the SD (untreated cells: n =9; LatA–treated cells, n = 6).
C, K562 cells pretreated with DMSO carrier alone (d) and LatA at indicated
concentrations for 40 min at 37˚C were tested for conjugate formation with
resting NK cells. Error bars indicate the SD (n = 3 individual NK cell donors
in three independent experiments). D, K562 target cells treated as in C were
mixed with resting NK cells, and degranulation was measured after 1 h at 37˚C
by staining for CD107a at the surface of NK cells. Each symbol indicates one
of the four individual NK cell donors (n = 4 independent experiments).
Disruption of actin filaments increases the mobility of
The Journal of Immunology 2921
FRAP (Fig. 3, Supplemental Videos 1, 2). ICAM-1 molecules on
and a region of the cell surface was bleached using a 488-nm laser
(Fig. 3A, white arrow). The recovery of the bleached region of
untreated (Fig. 3A, upper panel; 3B, d, Supplemental Video 1)
2) 721.221 cells was captured during 5 min. Photobleaching as
a result of image acquisition was monitored in untreated and
LatA-treated 721.221 cells to normalize recovery curves (Fig. 3B,
n and N, respectively). On untreated 721.221 cells, no recovery of
the bleached region was observed (Fig. 3A, upper panel, 3B, d,
Supplemental Video 1), suggesting that ICAM-1 is immobilized on
721.221 cells. However, after disruption of F-actin with 20 mM
LatA, recovery of the bleached region (Fig. 3A, lower panel, 3B,
of ICAM-1 from the cytoskeleton. Moreover, ICAM-1, which was
polarized to one side of untreated 721.221 cells (Fig. 3A, upper
panel, Supplemental Video 1), was evenly distributed after disrup-
tion of F-actin with LatA (Fig. 3A, lower panel, Supplemental
rather than or in addition to ICAM-1 tethering, was important for
LFA-1–dependent adhesion of NK cells, we examined the cell
line K562, which does not exhibit polarity of ICAM-1 at the cell
surface (Fig. 4A). Treatment of K562 cells with LatA-released
ICAM-1 form the cytoskeleton and increased its mobility (Fig.
4B). In accordance with the 721.221 data, the release of ICAM-1
from the cytoskeleton of K562 target cells prevented conjugate
formation in freshly isolated primary NK cells (Fig. 4C) and polar-
ization of cytolytic granules (data not shown), indicating that
ICAM-1 tethering rather than polarization is important for
LFA-1–dependent signaling in NK cells. The LFA-1–independent
target cells with different concentrations of LatA (Fig. 4D) ex-
cluding more global inhibitory effects of the drug.
We next used TIRF microscopy to visualize the distribution and
mobility of ICAM molecules in the plasma membrane of
target cells and to understand how the actin cytoskeleton affects
ICAM mobility by treating target cells with either LatA or Jasp
to disrupt or stabilize the actin cytoskeleton, respectively. TIRF
inates interference from bulk fluorescence that may be present
within cells to allow for the detection of fluorophores proximal
to and within the plasma membrane of cells adhered to glass
panel, D) was determined by TIRF microscopy (original magnification 3100). The movement of ICAM particles labeled with ICAM-1 and ICAM-2 PE-
panel) particles are shown in CDF plots. Plots represent one of two representative experiments. The total number of ICAM-1 particles analyzed over
determined by Kolmogorov-Smirnov test. Similarly, ∼32,700 ICAM-2 particles in latrunculin-treated cells and ∼23,600 particles in Jasp-treated cells were
analyzed. C, The average intensity of ICAM-1 and ICAM-2 particles was measured in a 5 3 5 pixel grid centered over the peak of the Gaussian distribution
are indicated in parentheses.
2922 ICAM MOBILITY CONTROLS LFA-1–DEPENDENT NK CELL FUNCTION
coverslips (45). Endogenous, cell surface ICAM-1 or ICAM-2 on
721.221 cells were fluorescently labeled with PE-conjugated anti-
ICAM Abs. Although individual ICAM proteins were labeled with
a single PE-fluorophore (see Materials and Methods), photo-
bleaching characteristics (the presence of multiple-step bleaching
events over long track lengths; data not shown) of fluorescent PE-
labeled particles suggest that ICAM proteins were mostly ob-
served as clusters and not single molecules (data not shown).
The lateral movement of labeled ICAM-1 (Fig. 5A, upper panel,
5B, left panel, Supplemental Videos 3–5) and ICAM-2 (Fig. 5A,
lower panel, 5B, right panel) particles recorded by TIRF micros-
copy was automatically tracked using an algorithm developed for
MatLab software (33), which was further modified to refine par-
ticle positioning with a two-dimensional Gaussian fit (34). Short-
range mean square displacements were determined from posi-
tional coordinates of particles tracked for five frames [.160 ms
(34)] and were linearly dependent on time under all conditions
measured, consistent with a simple diffusion model. Mean square
displacement versus time plots clearly showed that diffusion
increases, as indicated by the slope of the line, with LatA concen-
tration (Supplemental Fig. 4). Short-range diffusion coefficients
were then determined for thousands of particles in multiple cells
and graphed as a cumulative distribution function (CDF) to rep-
resent the frequency of diffusion coefficients for the entire pop-
ulation of tracked particles (Fig. 5B) (34). Consistent with our
FRAP data above, disrupting the actin cytoskeleton with LatA
incurred a dose-dependent shift toward the mobile population
for both ICAM-1 and ICAM-2 particles (Fig. 5B, Supplemental
Videos 3, 4). The median diffusion coefficient for ICAM-1 and
ICAM-2 in untreated control cells was calculated to be 0.013 and
0.030 mm2/s, respectively, and after the maximum dose of LatA
treatment, mobility increased ∼10- and 5-fold, respectively (0.132
mm2/s for ICAM-1 and 0.146 mm2/s for ICAM-2) (Fig. 5B). In
accordance with these results, binding of NK cells to 721.221
target cells was inhibited by disruption of F-actin filaments in
mobility of mouse ICAM-2. A, Lysates of BW5147 cells and BW5147
cells transfected with human ezrin-EGFP where probed with anti-ezrin,
anti-GFP, and anti–a-tubulin Ab. B and C, The mobility of ICAM-2 on
BW5147 cells (C, black line) and BW5147 cells expressing human ezrin
(C, gray line) was determined by TIRF microscopy as described in Fig. 5
using a PE-labeled anti-mouse ICAM-2 Ab. Scale bar, 5 mm; original
magnification 3100. Plots represent the cumulative frequency of diffusion
coefficients of ICAM-2 particles of one of two representative experiments.
Approximately 3100 and 2700 particles in untransfected and ezrin-transfected
cells, respectively, were analyzed. D, The intensity of ICAM-2 clusters on
BW5147 cells (black line) and BW5147 cells expressing human ezrin (gray
line) was analyzed as described in Fig. 5. Median intensities are indicated in
Expression of human ezrin in BW5417 cells reduces the
interaction with LFA-1. A, Conjugate formation of IL-2–activated NK cells
2–activated NK cells mixed with either BW5147 (n) or BW5147-hEzrin (N)
cells was measured as described in Fig. 2. Error bars indicate the SD (n = 3
individual NK cell donors in three independent experiments).
containing ICAM-1 bound to either lipid bilayers or glass surfaces, respec-
stack is the cell from the last frame. A, Engagement of LFA-1 by diffusible
ICAM-1 on lipid bilayer promoted active movement of NK cells. B, Engage-
ment of LFA-1 by solid-phase ICAM-1 resulted in arrest and stable binding
(left panel) or spreading and slow movement of NK cells (right panel).
Movement of human resting NK cells over mobile and im-
The Journal of Immunology 2923
721.221 cells (Fig. 1A, left panel). Although LatA reduced the
intensity of the brightest ICAM-1 clusters, it did not appreciably
alter the median intensity of either ICAM-1 or ICAM-2 particles
(Fig. 5C), suggesting that ICAM cluster size on target cells does
not affect NK cell response.
Both our FRAP and TIRF microscopy results indicate that
ICAM-1 and ICAM-2 are tethered to the actin cytoskeleton, which,
in turn, slows their lateral mobility in the plasma membrane; if so,
then one would predict that ICAM mobility would not increase,
and may be reduced even further, after treatment with the actin-
stabilizing drug Jasp. Indeed, the mobility of ICAM-1 (Fig. 5D,
left panel, Supplemental Video 5) and ICAM-2 (Fig. 5D, right
panel) was slightly reduced with Jasp and stabilization of F-actin
in target cells had little effect on LFA-1–dependent signaling
(Fig. 1A, right panel). In addition, disruption of the microtubule
network with Nocodazole did not affect the mobility of ICAM-1
(data not shown).
Expression of ezrin in BW5147 cells restores ICAM-2 tethering
and functional interaction with LFA-1
Our results so far indicate that a higher mobility of ICAM in the
plasma membrane hinders its interaction with LFA-1 on NK cells.
However, the contribution of other changes induced by LatA in
target cells that could impact on LFA-1–ICAM interactions cannot
be ruled out. We therefore carried out gain-of-function experiments
change in the distribution of ICAM-2 on the mouse BW5147
expression of human ezrin has been reported (31). We generated
BW5147 cells that stably express human ezrin tagged with GFP
(Fig. 6B, 6C, Supplemental Videos 6, 7). As shown in Fig. 6B and
Supplemental Video 6, ICAM-2 was evenly distributed and mobile
band) in BW5147 cells decreased the mobility of ICAM-2 (Fig. 6C,
Supplemental Videos 6, 7) from 0.053 to 0.027 mm2/s. Moreover,
ICAM-2 became polarized (Fig. 6B) and colocalized with the dis-
tribution of ezrin-GFP (data not shown). However, clustering of
ICAM-2 molecules did not change (Fig. 6D).
To test whether the reduced mobility of ICAM-2 as a result of
ezrin expression had an impact on functional interaction with
LFA-1 on NK cells, conjugate assays were performed. Conjugate
formation between human primary IL-2–activated NK cells and
BW5147 cells was increased (Fig. 7A) when ICAM-2 mobility
was reduced through ezrin expression (Fig. 6C). Moreover, expres-
sion of human ezrin in BW5147 target cells increased polarization
of cytolytic granules in IL-2–activated NK cells (Fig. 7B). We
conclude that tethering of ICAM-2 to the cytoskeleton of BW5147
cells by expression of human ezrin restores functional interaction
Rapid movement of NK cells over ICAM-1 inserted into lipid
of NK cells to mobile ICAM-1, a histidine-tagged form of
iological concentration of 250 molecules/mm2. Under these condi-
tions, ICAM-1 exhibits high lateral mobility (10, 46). Primary
resting NK cells on ICAM-1–coated lipid bilayers displayed rapid
movement and failed to stop (Fig. 8A, Supplemental Video 8). A
large fraction of NK cells displayed directed movement (Fig.
8A, Supplemental Video 8), whereas some moved randomly. The
moving NK cells have a distinguished morphology consisting of
a uropod and a dominant pseudopod at the leading edge of the cell
(Fig. 8A, Supplemental Video 8). NK cells did not contact lipid
bilayers in the absence of ICAM-1 (data not shown). At concentra-
tions of 200 and 500 ICAM-1 molecules/mm2, NK cells showed
a similar phenotype than at 250 molecules/mm2. The inability of
accumulation of IgG1 Fc into tight clusters (10). To monitor inter-
action with immobile ICAM-1, an ICAM-1-Fc fusion protein, in
which the CD16 binding site had been mutated, was attached to
coverslips. NK cells did not contact the coverslips in the absence
of ICAM-1 or coverslips that had been coated with ICAM-1 at
a concentration of 10 and 100 ng/ml (data not shown). However,
ICAM-1 coating at concentrations of 1 and 10 mg/ml resulted in
NK cells that were either attached and spread on the coverslips
(Fig. 8B, left panel, Supplemental Video 9) or attached and spread
NK cells that moved a little (Fig. 8B, right panel, Supplemental
Video 10). In contrast to the persistent movement over ICAM-1–
coated lipid bilayers, movementof NK cells over ICAM-1–coated
coverslips was nondirectional and slower (Fig. 8B, Supplemental
Videos 9, 10). A recent study reported that the NK cell line NKL
moved over plate-bound ICAM-1-Fc (47). The difference may be
primary NK cells when placed over lipid bilayers carrying ligands
of NK cell receptors (D. Liu, unpublished observations). Taken
ment, whereas immobile ICAM-1 promotes stable binding of pri-
mary NK cells.
Lateral mobility and clustering of transmembrane receptors and
their segregation within membrane domains are often essential for
proper signaling (26–28). Less is known about the importance of
receptor ligand distribution at the surface of opposing cells (48).
We addressed the question of how the distribution or mobility of
ligands affect receptor signaling in the context of b2integrin
LFA-1 in NK cells and its functional interaction with ligands
ICAM-1 and ICAM-2 on target cells. Earlier work established that
the binding of LFA-1 on NK cells to ICAM-1 on target cells was
sufficient for the formation of tight conjugates and polarization of
cytolytic granules toward the NK-target cell interface (4, 12, 14,
15, 39). Because LFA-1 signaling for adhesion and granule polar-
ization in human NK cells is uncoupled from signaling by other
activation receptors (13, 14), it was possible to focus on NK cell
functions controlled uniquely by LFA-1.
Two techniques, FRAP and TIRF microscopy, were used to
visualize and quantify the distribution and movement of ICAM-1
by LatA. The two-dimensional mobility of ICAM-1 and ICAM-2
at the surface of target cells was greatly enhanced after depolymer-
ization of F-actin and correlated with reduced LFA-1–dependent
NK cell responses. ICAM mobility, rather than clustering or
polarization, was the key parameter for adhesion and granule
polarization induced by LFA-1. LatA had minimal effect on the
intensity of ICAM-1 and ICAM-2 clusters. Polarization of ICAM
to one end of the cell, such as the one induced by ezrin, is not
necessary for functional LFA-1–dependent responses because the
target cell K562, which is highly sensitive to lysis by NK cells,
does not display polarized ICAM-1 and stimulates strong LFA-1–
dependent responses, which were lost upon release of ICAM-1
from cytoskeletal constraints.
2924 ICAM MOBILITY CONTROLS LFA-1–DEPENDENT NK CELL FUNCTION
To address the possibility that the reduction in NK cell adhesion
to a change other than ICAM distribution, gain-of-function experi-
ments were carried out with the NK-sensitive mouse thymoma cell
line BW5147, which expresses ICAM-2. Transfection of human
ezrin into BW5147 causes a redistribution of ICAM-2 into a uro-
pod-like cell extension and an increase in sensitivity to killing by
NK cells (31). As reported in this paper, the lateral mobility of
ICAM-2, but not the intensity of ICAM-2 clusters, was reduced
by transfection of ezrin, which correlated with increased NK cell
adhesion and granule polarization. Although ICAM clustering and
activation, our data show that tethering and reduced mobility of
ICAM are required for proper signaling by LFA-1.
ICAM-1 in the complete absence of other receptor–ligand interac-
tions, purified rICAM-1 was inserted into artificial planar lipid
bilayers, on which it is freely diffusible, or attached to glass. Pri-
mary, resting NK cells readily bound and formed tight contact with
ICAM-1 on the solid support but moved continuously on lipid
bilayers carrying diffusible ICAM-1. Therefore, ICAM-1 tethering
is required for primary NK cells to form stable contacts, such as
those occurring during NK-target cell interactions.
Adhesion of NK cells to target cells is accompanied by the for-
49, 50). At inhibitory NK cell immunological synapses, LFA-1 on
NK cells and ICAM-1 on target cells are excluded from a central
region where inhibitory receptors accumulate (8, 51). These results
suggest that ICAM-1 acquires mobility during synapse formation.
Engagement of ICAM-1 initiates signaling, which leads to recruit-
ment of lipid rafts and MHC molecules (48). Whether or not
ICAM-1 signaling is required for its peripheral distribution at
NK cell cytotoxic immunological synapses remains to be tested.
Cellular signaling in response to mechanical forces exerted
through cell surface receptors and the cytoskeleton, referred to as
“mechanotransduction,” has been gaining recognition (52–54).
Cells sense force at points of attachment, such as b1integrin
affinity changes in integrins (56, 57). As force sensing by LFA-1
would depend on tethering of its ligands, it is possible that a loss of
mechanotransduction as a result of release of ICAM from the cy-
toskeleton in target cells is at the base of adhesion and granule
polarization defects in NK cells.
We have shown that low mobility of ICAM on target cells is
important for conjugate formation and adhesion of NK cells,
suggesting that changes in the cytoskeleton could render target
cells less sensitive to killing by NK cells. Changes in ICAM-1 mo-
bility, such as those occurring during cell senescence (58), could
have a direct impact on the sensitivity of cells to killing by NK
cells. The importance of the cytoskeleton in cells that activate
lymphocytes was illustrated recently by the effect of Schistosoma
mansoni T2 RNase on dendritic cells, in which cytoskeletal
changes were accompanied by deficient interaction with CD4+
T cells (59).
We thank J.-J. Hao and S. Shaw for the hEzrin-EGFP construct, T. Kamala
for the BW5147 cells, M. Peterson for mutagenesis of the Fc fusion vector,
for the MatLab code and technical discussions.
The authors have no financial conflicts of interest.
1. Springer, T. A. 1994. Traffic signals for lymphocyte recirculation and leukocyte
emigration: the multistep paradigm. Cell 76: 301–314.
2. Davignon, D., E. Martz, T. Reynolds, K. Ku ¨rzinger, and T. A. Springer. 1981.
Lymphocyte function-associated antigen 1 (LFA-1): a surface antigen distinct
from Lyt-2,3 that participates in T lymphocyte-mediated killing. Proc. Natl.
Acad. Sci. USA 78: 4535–4539.
3. Helander, T., T. Timonen, P. Kallioma ¨ki, and J. Schro ¨der. 1991. Recognition of
chromosome 6-associated target structures by human lymphokine-activated
killer cells. J. Immunol. 147: 2063–2067.
4. Krensky, A. M., F. Sanchez-Madrid, E. Robbins, J. A. Nagy, T. A. Springer, and
S. J. Burakoff. 1983. The functional significance, distribution, and structure of
LFA-1, LFA-2, and LFA-3: cell surface antigens associated with CTL-target
interactions. J. Immunol. 131: 611–616.
5. Somersalo, K., N. Anikeeva, T. N. Sims, V. K. Thomas, R. K. Strong, T. Spies,
T. Lebedeva, Y. Sykulev, and M. L. Dustin. 2004. Cytotoxic T lymphocytes form
an antigen-independent ring junction. J. Clin. Invest. 113: 49–57.
6. Suzuki, J., S. Yamasaki, J. Wu, G. A. Koretzky, and T. Saito. 2007. The actin
cloud induced by LFA-1–mediated outside-in signals lowers the threshold for
T-cell activation. Blood 109: 168–175.
7. Sims, T. N., and M. L. Dustin. 2002. The immunological synapse: integrins take
the stage. Immunol. Rev. 186: 100–117.
8. Almeida, C. R., and D. M. Davis. 2006. Segregation of HLA-C from ICAM-1 at
NK cell immune synapses is controlled by its cell surface density. J. Immunol.
9. Anikeeva, N., K. Somersalo, T. N. Sims, V. K. Thomas, M. L. Dustin, and
Y. Sykulev. 2005. Distinct role of lymphocyte function-associated antigen-1 in
mediating effective cytolytic activity by cytotoxic T lymphocytes. Proc. Natl.
Acad. Sci. USA 102: 6437–6442.
10. Liu, D., Y. T. Bryceson, T. Meckel, G. Vasiliver-Shamis, M. L. Dustin, and
E. O. Long. 2009. Integrin-dependent organization and bidirectional vesicular
traffic at cytotoxic immune synapses. Immunity 31: 99–109.
11. Dustin, M. L., and T. A. Springer. 1989. T-cell receptor cross-linking transiently
stimulates adhesiveness through LFA-1. Nature 341: 619–624.
12. Barber, D. F., and E. O. Long. 2003. Coexpression of CD58 or CD48 with in-
tercellular adhesion molecule 1 on target cells enhances adhesion of resting
NK cells. J. Immunol. 170: 294–299.
13. Bryceson, Y. T., H. G. Ljunggren, and E. O. Long. 2009. Minimal requirement
for induction of natural cytotoxicity and intersection of activation signals by
inhibitory receptors. Blood 114: 2657–2666.
14. Bryceson, Y. T., M. E. March, D. F. Barber, H. G. Ljunggren, and E. O. Long.
2005. Cytolytic granule polarization and degranulation controlled by different
receptors in resting NK cells. J. Exp. Med. 202: 1001–1012.
15. Barber, D. F., M. Faure, and E. O. Long. 2004. LFA-1 contributes an early signal
for NK cell cytotoxicity. J. Immunol. 173: 3653–3659.
16. Mace, E. M., S. J. Monkley, D. R. Critchley, and F. Takei. 2009. A dual role for
talin in NK cell cytotoxicity: activation of LFA-1–mediated cell adhesion and
polarization of NK cells. J. Immunol. 182: 948–956.
17. Bryceson, Y. T., M. E. March, H. G. Ljunggren, and E. O. Long. 2006. Synergy
among receptors on resting NK cells for the activation of natural cytotoxicity and
cytokine secretion. Blood 107: 159–166.
18. Mor, A., M. L. Dustin, and M. R. Philips. 2007. Small GTPases and LFA-1;
reciprocally modulate adhesion and signaling. Immunol. Rev. 218: 114–125.
19. Yokosuka, T., and T. Saito. 2009. Dynamic regulation of T-cell costimulation
through TCR-CD28 microclusters. Immunol. Rev. 229: 27–40.
20. Lingwood, D., and K. Simons. 2010. Lipid rafts as a membrane-organizing
principle. Science 327: 46–50.
21. Lillemeier, B. F., M. A. Mo ¨rtelmaier, M. B. Forstner, J. B. Huppa, J. T. Groves,
and M. M. Davis. 2010. TCR and Lat are expressed on separate protein islands
on T cell membranes and concatenate during activation. [Published erratum
appears in 2010 Nat. Immunol. 11: 543.] Nat. Immunol. 11: 90–96.
22. Watzl, C., and E. O. Long. 2003. Natural killer cell inhibitory receptors block
actin cytoskeleton-dependent recruitment of 2B4 (CD244) to lipid rafts. J. Exp.
Med. 197: 77–85.
23. Marwali, M. R., M. A. MacLeod, D. N. Muzia, and F. Takei. 2004. Lipid rafts
mediate association of LFA-1 and CD3 and formation of the immunological
synapse of CTL. J. Immunol. 173: 2960–2967.
24. Sheets, E. D., D. Holowka, and B. Baird. 1999. Critical role for cholesterol in
Lyn-mediated tyrosine phosphorylation of FcεRI and their association with
detergent-resistant membranes. J. Cell Biol. 145: 877–887.
25. Tolar, P., H. W. Sohn, and S. K. Pierce. 2008. Viewing the antigen-induced
initiation of B-cell activation in living cells. Immunol. Rev. 221: 64–76.
26. Dykstra, M., A. Cherukuri, H. W. Sohn, S. J. Tzeng, and S. K. Pierce. 2003.
Location is everything: lipid rafts and immune cell signaling. Annu. Rev.
Immunol. 21: 457–481.
27. Harder, T., C. Rentero, T. Zech, and K. Gaus. 2007. Plasma membrane segre-
gation during T cell activation: probing the order of domains. Curr. Opin.
Immunol. 19: 470–475.
28. Sengupta, P., B. Baird, and D. Holowka. 2007. Lipid rafts, fluid/fluid phase
separation, and their relevance to plasma membrane structure and function.
Semin. Cell Dev. Biol. 18: 583–590.
29. Tseng, S. Y., M. L. Liu, and M. L. Dustin. 2005. CD80 cytoplasmic domain
controls localization of CD28, CTLA-4, and protein kinase Cu in the immuno-
logical synapse. J. Immunol. 175: 7829–7836.
30. Eleme, K., S. B. Taner, B. Onfelt, L. M. Collinson, F. E. McCann, N. J. Chalupny,
D. Cosman, C. Hopkins, A. I. Magee, and D. M. Davis. 2004. Cell surface
The Journal of Immunology2925
organization of stress-inducible proteins ULBP and MICA that stimulate human Download full-text
NK cells and T cells via NKG2D. J. Exp. Med. 199: 1005–1010.
31. Helander, T. S., O. Carpe ´n, O. Turunen, P. E. Kovanen, A. Vaheri, and T. Timonen.
32. Hao, J. J., Y. Liu, M. Kruhlak, K. E. Debell, B. L. Rellahan, and S. Shaw. 2009.
from lymphocyte membrane. J. Cell Biol. 184: 451–462.
33. Douglass, A. D., and R. D. Vale. 2005. Single-molecule microscopy reveals
plasma membrane microdomains created by protein-protein networks that ex-
clude or trap signaling molecules in T cells. Cell 121: 937–950.
34. Tolar, P., J. Hanna, P. D. Krueger, and S. K. Pierce. 2009. The constant region of
the membrane immunoglobulin mediates B cell-receptor clustering and signaling
in response to membrane antigens. Immunity 30: 44–55.
35. Aruffo, A., I. Stamenkovic, M. Melnick, C. B. Underhill, and B. Seed. 1990.
CD44 is the principal cell surface receptor for hyaluronate. Cell 61: 1303–1313.
36. Winter, C. C., and E. O. Long. 2000. Binding of soluble KIR-Fc fusion proteins
to HLA class I. Methods Mol. Biol. 121: 239–250.
37. Wessels, D. J., S. Kuhl, and D. R. Soll. 2009. Light microscopy to image and
quantify cell movement. Methods Mol. Biol. 571: 455–471.
38. Shimizu, Y., D. E. Geraghty, B. H. Koller, H. T. Orr, and R. DeMars. 1988.
Transfer and expression of three cloned human non-HLA-A,B,C class I major
histocompatibility complex genes in mutant lymphoblastoid cells. Proc. Natl.
Acad. Sci. USA 85: 227–231.
is disrupted by the killer cell inhibitory receptor. Curr. Biol. 10: 777–780.
40. Carpe ´n, O., P. Pallai, D. E. Staunton, and T. A. Springer. 1992. Association of
intercellular adhesion molecule-1 (ICAM-1) with actin-containing cytoskeleton
and a-actinin. J. Cell Biol. 118: 1223–1234.
41. Heiska, L., C. Kantor, T. Parr, D. R. Critchley, P. Vilja, C. G. Gahmberg, and
O. Carpe ´n. 1996. Binding of the cytoplasmic domain of intercellular adhesion
molecule-2 (ICAM-2) to a-actinin. J. Biol. Chem. 271: 26214–26219.
42. Celli, L., J. J. Ryckewaert, E. Delachanal, and A. Duperray. 2006. Evidence of
a functional role for interaction between ICAM-1 and nonmuscle a-actinins in
leukocyte diapedesis. J. Immunol. 177: 4113–4121.
43. Heiska, L., K. Alfthan, M. Gro ¨nholm, P. Vilja, A. Vaheri, and O. Carpe ´n. 1998.
Association of ezrin with intercellular adhesion molecule-1 and -2 (ICAM-1 and
ICAM-2): regulation by phosphatidylinositol 4, 5-bisphosphate. J. Biol. Chem.
44. Barreiro, O., M. Yanez-Mo, J. M. Serrador, M. C. Montoya, M. Vicente-
Manzanares, R. Tejedor, H. Furthmayr, and F. Sanchez-Madrid. 2002. Dynamic
interaction of VCAM-1 and ICAM-1 with moesin and ezrin in a novel endothelial
docking structure for adherent leukocytes. J. Cell Biol. 157: 1233–1245.
of PIP2 releasesERM proteins
45. Axelrod, D. 2001. Total internal reflection fluorescence microscopy in cell bi-
ology. Traffic 2: 764–774.
46. Grakoui, A., S. K. Bromley, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen,
and M. L. Dustin. 1999. The immunological synapse: a molecular machine
controlling T cell activation. Science 285: 221–227.
M. Mehrabi, M. P. Deonarain, D. S. Ushakov, et al. 2009. Natural killer cell signal
integration balances synapse symmetry and migration. PLoS Biol. 7: e1000159.
48. Lebedeva, T., M. L. Dustin, and Y. Sykulev. 2005. ICAM-1 co-stimulates
target cells to facilitate antigen presentation. Curr. Opin. Immunol. 17: 251–258.
49. Vyas, Y. M., K. M. Mehta, M. Morgan, H. Maniar, L. Butros, S. Jung,
J. K. Burkhardt, and B. Dupont. 2001. Spatial organization of signal transduction
molecules in the NK cell immune synapses during MHC class I-regulated non-
cytolytic and cytolytic interactions. J. Immunol. 167: 4358–4367.
50. Nedvetzki, S., S. Sowinski, R. A. Eagle, J. Harris, F. Ve ´ly, D. Pende,
J. Trowsdale, E. Vivier, S. Gordon, and D. M. Davis. 2007. Reciprocal regulation
of human natural killer cells and macrophages associated with distinct immune
synapses. Blood 109: 3776–3785.
51. Schleinitz, N., M. E. March, and E. O. Long. 2008. Recruitment of activation
receptors at inhibitory NK cell immune synapses. PLoS ONE 3: e3278.
52. Giannone, G., and M. P. Sheetz. 2006. Substrate rigidity and force define form
53. Schwartz, M. A. 2009. Cell biology: the force is with us. Science 323: 588–589.
54. Kim, S. T., K. Takeuchi, Z. Y. Sun, M. Touma, C. E. Castro, A. Fahmy,
M. J. Lang, G. Wagner, and E. L. Reinherz. 2009. The ab T cell receptor is an
anisotropic mechanosensor. J. Biol. Chem. 284: 31028–31037.
55. Friedland, J. C., M. H. Lee, and D. Boettiger. 2009. Mechanically activated
integrin switch controls a5b1function. Science 323: 642–644.
56. Astrof, N. S., A. Salas, M. Shimaoka, J. Chen, and T. A. Springer. 2006. Im-
portance of force linkage in mechanochemistry of adhesion receptors. Bio-
chemistry 45: 15020–15028.
57. Alon, R., and M. L. Dustin. 2007. Force as a facilitator of integrin conforma-
tional changes during leukocyte arrest on blood vessels and antigen-
presenting cells. Immunity 26: 17–27.
58. Zhou, X., F. Perez, K. Han, and D. A. Jurivich. 2006. Clonal senescence alters
endothelial ICAM-1 function. Mech. Ageing Dev. 127: 779–785.
59. Steinfelder, S., J. F. Andersen, J. L. Cannons, C. G. Feng, M. Joshi, D. Dwyer,
P. Caspar, P. L. Schwartzberg, A. Sher, and D. Jankovic. 2009. The major
component in schistosome eggs responsible for conditioning dendritic cells for
Th2 polarization is a T2 ribonuclease (v-1). J. Exp. Med. 206: 1681–1690.
2926 ICAM MOBILITY CONTROLS LFA-1–DEPENDENT NK CELL FUNCTION