Cutting Edge: NKG2D-Dependent Cytotoxicity Is
Controlled by Ligand Distribution in the Target Cell
Emily Martinez,* Joseph A. Brzostowski,†Eric O. Long,* and Catharina C. Gross*,‡
Although the importance of membrane microdomains
in receptor-mediated activation of lymphocytes has
been established, much less is known about the role
of receptor ligand distribution on APC and target
cells. Detergent-resistant membranedomains, intowhich
GPI-linked proteins partition, are enriched in choles-
terol and glycosphingolipids. ULBP1 is a GPI-linked
ligand for natural cytotoxicity receptor NKG2D. To in-
vestigate how ULBP1 distribution on target cells affects
tracellular domain of ULBP1 to the transmembrane
domain of CD45. Introduction of this transmembrane
domain eliminated the association of ULBP1 with the
detergent-resistant membrane fraction and caused a
significant reduction of cytotoxicity and degranulation
by NK cells. Clustering and lateral diffusion of ULBP1
was not affected by changes in the membrane anchor.
These results show that the partitioning of receptor
ligands in discrete membrane domains of target cells is
an important determinant of NK cell activation.
Journal of Immunology, 2011, 186: 5538–5542.
including adhesion of NK cells to target cells, synapse for-
mation, polarization of cytolytic granules toward the target
cells, and granule exocytosis (2). Whereas binding of LFA-1
on human NK cells to ICAM on target cells induces adhesion
and polarization of lytic granules (3), degranulation is trig-
gered by low-affinity FcgR CD16 or by synergistic combi-
nations of coactivation receptors, such as 2B4 and NKG2D
NKG2D is a C-type lectin coactivation receptor expressed
as a disulfide-linked homodimer on NK cells, NKT cells,
and some T cells (6). In humans, NKG2D binds to stress-
atural killer cellsareasubsetofcytotoxic lymphocytes
that recognize and kill tumor cells and virus-infected
cells (1). Lysis of target cells is a multistep process
inducible members of the polymorphic MHC class I-related
chain A/B (MICA/B) family and the multigene family of
UL16-binding proteins (ULBPs; RAET1A-E). NKG2D ligands
are expressed in multiple types of tumors and play an impor-
tant role in immunosurveillance of cancer (7). However, by
shedding NKG2D ligands from their cell surface, tumor
cells may escape the antitumor response mediated by NKG2D
Within the lipid bilayer, proteins and lipids are segregated
laterally, leading to functional subcompartmentalization of
the plasma membrane (8). Lipid rafts are membrane micro-
domains enriched in glycosphingolipids, sphingomyelins,
and cholesterol. The role of membrane microdomains in pro-
moting receptor-mediated lymphocyte activation has been
well established (9, 10). Much less is known about how the
distribution of receptor ligands on target cells affects lym-
phocyte function, although studies have suggested that it may
be an important parameter for lymphocyte activation. For
example, the cytoplasmic tail of CD80 (B7-1), a CD28 ligand
expressed in APCs, is required for proper segregation of
CD28 at the immunological synapse and for full T cell ac-
tivation (11). Furthermore, although expression of MICA on
resistant target cells could overcome MHC class I-dependent
inhibitory signaling in NK cells, a truncated form of MICA
lacking a potential acylation site could not (12), suggesting
that NKG2D ligand distribution may play a role in over-
coming NK cell inhibition.
In this study, we tested ULBP1 as a ligand to investigate the
role of ligand distribution in NKG2D-dependent human NK
cell activation. To do so independently of HLA class I ligands
for inhibitory receptors, we expressed ULBP1 in a mouse
cell line. A chimera consisting of the extracellular portion of
ULBP1 and the transmembrane region of CD45 was gener-
ated. Its expression resulted in the localization of the normally
fractions to detergent-soluble fractions. This redistribution of
ULBP1 caused a reduction in cytotoxicity and degranulation
*Molecular and Cellular Immunology Section, Laboratory of Immunogenetics, National
Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville,
MD 20852;†Imaging Facility, Laboratory of Immunogenetics, National Institute of
Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852;
and‡Department of Neurology—Inflammatory Diseases of the Nervous System and
Neurooncology, University Hospital Mu ¨nster, 48149 Mu ¨nster, Germany
Received for publication July 7, 2010. Accepted for publication March 11, 2011.
This work was supported by the Intramural Research Program of the National Institute
of Allergy and Infectious Diseases, National Institutes of Health.
Address correspondence and reprint requests to Dr. Eric O. Long or Dr. Catharina C.
Gross, Laboratory of Immunogenetics, NIAID-NIH, 12441 Parklawn Drive, Rockville,
MD 20852 (E.O.L.) or Department of Neurology—Inflammatory Diseases of the Ner-
vous System and Neurooncology, University Hospital Mu ¨nster, ICB, Mendelstrasse 7,
48149 Mu ¨nster, Germany (C.C.G.). E-mail addresses: firstname.lastname@example.org (E.O.L.) or
The online version of this article contains supplemental material.
Abbreviations used in this article: DRM, detergent-resistant membrane; MICA, MHC
class I-related chain A; PI-PLC, phosphatidylinositol-specific phospholipase C; TIRF,
total internal reflection fluorescence; ULBP, UL16-binding protein.
by NK cells, implying a role for receptor ligand distribution in
the activation of NK cell responses.
Materials and Methods
Resting human NK cells were isolated from peripheral blood cells by negative
selection using an NK cell isolation kit (Stem Cell Technologies). Freshly
isolated resting NK cells (95–99% CD32CD56+) were resuspended in IMDM
(Invitrogen) supplemented with 10% human serum (Valley Biomedical) and
used 1–2 d after isolation. Polyclonal IL-2–activated NK cells were cultured as
described previously (3). P815 cells were cultured in IMDM supplemented
with 10% heat-inactivated FBS (Thermo Scientific). For phospholipase treat-
ment, P815-ULBP1 and P815-ULBP1-CD45TM cells were treated with 2 IU/
ml phosphatidylinositol-specific phospholipase C (PI-PLC) (Sigma) for 1 h
at 37˚C and 5% CO2, and surface protein levels were measured by flow cyto-
metry using an R-PE–conjugated ULBP1 mAb (R&D Systems). The ULBP1
mAb was conjugated with a Phycolink R-PE kit (Prozyme).
Transfection of P815 cells
P815 cells were transfected with human ULB1 or ULBP1-CD45TM (Sup-
plemental Fig. 1) using the Bio-Rad Gene Pulser (10 mg of each DNA, 260 V,
960 mF). Transfected cells were selected in IMDM supplemented with 10%
heat-inactivated FBS and 800 mg/ml Geneticin (Invitrogen), and subcloned.
Different clones were tested for ULBP1 expression and in functional assays,
and representative clones from each cell line were selected for further use.
DRM preparation was performed as described previously (13), except that
OptiPrep (Axis-Shield) was used instead of sucrose. Fractions 4–11 were
separated on 12% SDS NuPAGE gels (Invitrogen) and transferred to poly-
vinylidene difluoride membrane (Invitrogen). The membrane was blocked
with Odyssey blocking buffer (LI-COR Biosciences) for 1 h at room tem-
perature and incubated with biotinylated goat anti-human ULBP1 Ab (R&D
System) overnight at room temperature. After washing, the membrane was
stained with IRDye 680-labeled streptavidin (LI-COR Biosciences) for 1 h at
room temperature, and bands were detected using the Odyssey infrared im-
aging system (LI-COR Biosciences). In the case of latrunculin A treatment,
cells were incubated with either 0.3% DMSO carrier or 3 mM latrunculin A
(Calbiochem) for 40 min at 37˚C and 5% CO2.
Total internal reflection fluorescence microscopy
Cell surface ULBP1 or ULBP1-CD45TM on P815 cells was fluorescently
labeled with PE-conjugated ULBP1 mAb (R&D). For latrunculin A treat-
ment, labeled cells were incubated for 40 min at 37˚C and 5% CO2with
either 0.3% DMSO carrier or 3 mM latrunculin A before analysis by total
internal reflection fluorescence (TIRF) microscopy. TIRF imaging and anal-
ysis was performed as described previously (14).
Cytotoxicity assays were performed as described previously (15). Degranu-
lation assays were performed as described previously (4) with minor changes.
In brief, 2 3 105NK cells were added to 4 3 105P815 target cells in a total
volume of 200 ml IMDM medium supplemented with 10% heat-inactivated
FBS, 6 mg/ml monensin (Calbiochem), 20 ml/ml FITC-conjugated
CD107a mAb (Becton Dickinson), and 20 ml/ml PE-conjugated CD56
mAb (Becton Dickinson). Cells were mixed and incubated for 1 h at 37˚C
and 5% CO2. Afterward, cells were spun down and expression of CD107a on
CD56+cells was determined by flow cytometry. In case of latrunculin A
pretreatment of the target cells, cells were incubated with either 0.3% DMSO
carrier (Sigma) or 3 mM latrunculin A (Calbiochem) for 40 min at 37˚C at
5% CO2and washed extensively before use in the assay.
Results and Discussion
Linking the extracellular portion of ULBP1 to the transmembrane
region of CD45 changes its localization within the membrane
To change the distribution of the NKG2D ligand ULBP1
within the plasma membrane, we generated a chimera con-
sisting of the extracellular portion of ULBP1 and the trans-
Fig. 1). In brief, the extracellular portion of ULBP1 including
the GPI anchor site (Supplemental Fig. 1B) was fused to the
transmembrane region of CD45 that includes only two ex-
tracellular amino acids and four amino acids in the cytosolic
portion for anchoring purposes (Supplemental Fig. 1B). The
GPI-linked ULBP1 and the recombinant ULBP1-CD45TM
were transfected into the mouse mastocytoma cell line P815,
and clones with similar expression levels were selected (Fig.
1A). To verify that ULBP1-CD45TM had lost the GPI an-
chor, P815 cells expressing ULBP1 and ULBP1-CD45TM
were treated with PI-PLC, which cleaves GPI anchors.
ULBP1 was sensitive to PI-PLC, but ULBP1-CD45TM
was not, indicating a loss of the GPI anchor in the chimera
(Fig. 1A). The incomplete cleavage of ULBP1 (Fig. 1A),
which could be caused by limited accessibility to phospholi-
pase, is consistent with the low amount of ULBP1 shedding
observed in other target cells (16).
To test whether linkage to the transmembrane domain of
CD45 changed the localization of ULBP1, we prepared DRM
fractions from P815-ULBP1 and P815-ULBP1-CD45TM
cells. Whereas ULBP1 was almost exclusively localized in
the DRM fraction, the ULBP1-CD45TM protein was asso-
ciated with the soluble fraction (Fig. 1B). Association of
ULBP1 with the DRM fraction was even stronger than that of
the DRM marker flotillin 1 (Fig. 1B). Therefore, linking the
extracellular portion of ULBP1 to the transmembrane region
of CD45 changed its localization from the DRM fraction to
the detergent-soluble membrane fraction.
Targeting of ULBP1 to the detergent-soluble membrane fraction
reduces the sensitivity of target cells to lysis by NK cells
It has been shown that redistribution of ICAM-2 on tumor
cells via ezrin renders these cells more sensitive to lysis by NK
cells (17). To test whether distribution of ULBP1 in either the
membrane domain of CD45 alters its distribution in P815 cells. A, P815 cells
transfected with GPI-linked ULBP1 (left panel) or ULBP1-CD45TM (right
panel) were either untreated (solid line) or PI-PLC–treated (dashed line), and
analyzed with a PE-conjugated ULBP1 Ab or an IgG2A isotype control
(shaded). Data are representative of nine individual experiments. B, Fractions
4–11 of DRM preparations from P815 cells expressing either GPI-linked
ULBP1 (left panel) or ULBP1-CD45TM (right panel) were analyzed by im-
munoblotting with Abs to ULBP1 (upper lane), raft-associated Flotillin-1
(Flot-1, middle lane), and detergent-soluble membrane transferring receptor
(TfR, bottom lane). Data are representative of two individual experiments.
Fusion of the ULBP1 extracellular domain with the trans-
The Journal of Immunology 5539
DRM fraction or the detergent-soluble membrane fraction had
any functional consequence for sensitivity to NKG2D-depen-
dent cytotoxicity, P815-ULBP1 and P815-ULBP1-CD45TM
cells were used as targets in a 2-h lysis assay (Fig. 2). Expres-
sion of ULBP1 on P815 cells rendered them more sensitive to
lysis by primary, resting NK cells (Fig. 2A), and IL-2 acti-
vated NK cells (Fig. 2B). Expression of ULBP1-CD45TM on
P815 cells resulted in a lower sensitivity to lysis by NK cells,
as compared with ULBP1 on P815 cells (Fig. 2). These re-
sults indicate that distribution of ULBP1 within the mem-
brane may be important for proper NK cell function.
We next tested which step in NK cell cytotoxicity was
sensitive to changes in the distribution of ULBP1. Expression
of ULBP1 in the detergent-soluble membrane fraction of P815
cells resulted in reduced degranulation of resting NK cells (Fig.
2C) and of IL-2–activated NK cells (Fig. 2D). Expression of
ULBP1 and ULBP1-CD45TM on P815 cells had only a
minor enhancing effect on the polarization of lytic granules
toward the NK–P815 cell contact (not shown). We conclude
that a change of the localization of ULBP1 from the DRM
domains to the detergent-soluble membrane fraction reduces
cytotoxicity of NK cells at the level of degranulation.
Clustering and lateral diffusion of ULBP1 and ULBP1-CD45TM in
the plasma membrane are similar
We next tested how the segregation of ULBP1 into DRM and
detergent-soluble domains affected the extent of ULBP1 clus-
tering and lateral mobility within the plasma membrane. To
investigate these parameters, we labeled ULBP1 and ULBP1-
CD45TM with a PE-conjugated Ab to ULBP1, and their
distribution and mobility were visualized by TIRF micro-
scopy. TIRF microscopy is a spatially limited, high-contrast
technique that eliminates interference from bulk fluores-
fluorophores proximal to and within the plasma membrane
of cells on glass coverslips (18). Both ULBP1 and ULBP1-
CD45TM were distributed into small clusters at the surface
of P815 cells (Fig. 3A). Although individual ULBP1 and
ULBP1-CD45TM proteins were labeled with a single PE-
fluorophore, photobleaching characteristics (the presence of
multistep bleaching events over long track length, data not
shown) of fluorescent PE-labeled particles suggested that
ULBP1 and ULBP1-CD45TM were observed primarily as
clusters and not single molecules. Cluster analysis by fluo-
reduces the cytotoxicity and degranulation of NK cells. Susceptibility of P815
(open circles), P815-ULBP1 (black circles), and P815-ULBP1-CD45TM
(gray circles) cells by freshly isolated resting (A) or IL-2–activated NK cells (B)
was measured in a 2-h assay. Bars indicate SD of independent experiments
with three NK cell donors. Degranulation of freshly isolated resting NK cells
(C) or IL-2–activated NK cells (D) in response to P815, P815-ULBP1, and
P815-ULBP1-CD45TM cells was measured in a 1-h degranulation assay with
CD107a mAb. Each symbol represents an individual donor among three (C)
or five (D), which were tested in independent experiments. Paired t test was
performed. *p # 0.01.
Exclusion of ULBP1 from the DRM fraction of target cells
A, Pictures of P815-ULBP1 (left panel) and P815-ULBP1-CD45TM (right
panel) cells labeled with PE-conjugated Ab to ULBP1. Scale bars, 5 mm. B,
The intensity of ULBP1 (blue) and ULBP1-CD45 (green) particles was
measured in a 5 3 5-pixel grid centered over the peak of the Gaussian dis-
tribution calculated for the particle in the first frame in which it appeared in
the tracking algorithm. Average particle intensities are shown in cumulative
probability plots. Median intensities are indicated in parentheses. C, The
movement of ULBP1 and ULBP1-CD45TM particles labeled with PE-
conjugated Ab to ULBP1 was tracked by capturing TIRF images at 35
frames/s for 100 frames. Diffusion coefficients of ULBP1 (blue, n = 3532)
and ULBP1-CD45TM (green, n = 2295) particles are shown in cumulative
probability plots. Data are from one of seven representative experiments. D,
Movement of DMSO-treated P815-ULBP1 (blue, n = 2826) and P815-
ULBP1-CD45TM (green, n = 2921), and latrunculin A–treated P815-
ULBP1 (red, n = 4114) and P815-ULBP1-CD45TM (aqua, n = 3240) cells
was tracked by TIRF as described in B. Data are from one of eight in-
dependent experiments. E, Degranulation of IL-2–activated NK cells stimu-
lated with P815-ULBP1 and P815-ULBP1-CD45TM cells pretreated with
DMSO carrier or 3 mM latrunculin A, as described in Fig. 2B. Each symbol
represents an individual donor among six, which were tested in independent
experiments. Paired t test was performed. **p # 0.0025, ***p # 0.0004.
Lateral diffusion of ULBP1 and ULBP1-CD45TM is similar.
5540CUTTING EDGE: CYTOTOXICITY CONTROLLED BY LIGAND DISTRIBUTION
rescence intensity measurements revealed no significant dif-
ference in ULBP1 and ULBP1-CD45TM clusters, with a
median intensity of 1283 and 1204, respectively (Fig. 3B).
Therefore, the distribution of ULBP1 in different membrane
domains did not have a detectable impact on the number of
ULBP1 molecules per cluster. Furthermore, the cluster in-
tensity for both molecules was homogenous (Fig. 3B). We
conclude that the difference in sensitivity to NKG2D-depen-
dent cytotoxicity is not due to a change in the number of
ULBP1 molecules per cluster.
The lateral movement of labeled ULBP1 and ULBP1-
CD45TM particles recorded by TIRF microscopy was tracked
automatically using an algorithm developed for MatLab
software (19), which was further modified to refine particle
positioning with a two-dimensional Gaussian fit (20). Short-
range mean square displacements were determined from po-
sitional coordinates of particles tracked for five frames (over
160 ms) (20) and were linearly dependent on time under
all conditions measured, consistent with a simple diffusion
model for this range of movement. Short-range diffusion
coefficients were then determined for thousands of particles
in multiple cells and graphed either in cumulative probabi-
lity plots (also known as cumulative distribution function) to
represent the frequency of diffusion coefficients for the entire
population of tracked particles (Fig. 3C), or median scattered
plots (Supplemental Fig. 2A). Each one of several thousand
particles is represented as a separate point in the cumulative
probability plot. This type of graph can visually resolve small
differences between samples even when extensive overlap
occurs. ULBP1 clusters displayed a high lateral mobility at the
surface of P815 cells, with a median diffusion coefficient of
0.122 mm2/s. Lateral mobility of ULBP1-CD45TM, with
a median diffusion coefficient of 0.075 mm2/s, was reduced
compared with ULBP1. Single-particle tracking experiments
have shown that diffusion rate at the plasma membrane is
reduced when proteins associate with lipid rafts (21) or
with protein complexes (22). The distribution of ULBP1-
CD45TM in the detergent-soluble membrane fraction may
have resulted in intermolecular interactions that reduced
ULBP1 mobility even further than the association of ULBP1
with DRM domains.
To test whether the lateral mobility of ULBP1 and ULBP1-
CD45TM was controlled by the actin cytoskeleton, we tracked
mobility on P815 cells treated with either DMSO carrier alone
or 3 mM latrunculin A (Fig. 3D, Supplemental Fig. 2B).
Whereas the mobility of ULBP1 did not change after treat-
ment with latrunculin A, the mobility of ULBP1-CD45TM
increased from a median diffusion coefficient of 0.065 mm2/s
to a diffusion coefficient of 0.093 mm2/s, which was close to
the mobility of ULBP1 (0.118 mm2/s) after treatment (Fig.
3D). A higher dose of latrunculin A (10 mM) did not increase
the mobility of ULBP1-CD45TM any further and had no
effect on the mobility of ULBP1 (data not shown).
Previous work from our group has shown that immobili-
zation of ICAM on target cells, rather than its clustering,
promotes proper LFA-1–dependent conjugate formation
and granule polarization in primary NK cells (14). To test
whether changes in lateral diffusion of ULBP1 were respon-
sible for the difference in sensitivity to lysis by NK cells (Fig.
2), we took advantage of the similar lateral diffusion of
ULBP1 and ULBP1-CD45TM after treatment with latrun-
culin A. If lateral diffusion of ULBP1 was the main deter-
minant of the functional difference (i.e., greater ULBP1
mobility leading to increased NKG2D-dependent degranu-
lation), treatment of the target cells with latrunculin A should
equalize the response to P815-ULBP1 and P815-ULBP1-
CD45TM cells. As seen earlier in the absence of DMSO (Fig.
2), in the presence of DMSO, ULBP1 induced significantly
greater degranulation than ULBP1-CD45TM (Fig. 3E).
Treatment of P815 cells with latrunculin A did not equalize
the response induced by ULBP1 and ULBP1-CD45TM:
degranulation induced by ULBP1 did not change, but that in-
duced by ULBP1-CD45TM was reduced even further (Fig.
3E). Therefore, NKG2D-dependent cytotoxicity is controlled
by the distribution of NKG2D ligands into separate mem-
brane domains, independently of the number of ligand mol-
ecules per cluster, and of the lateral mobility of clusters in the
The molecular basis for the change in NKG2D-dependent
responses when ULBP1 is moved to a different membrane
environment is still unknown. This change could be relevant,
as the related NKG2D ligand ULBP2 is expressed as both
a GPI-linked form and a transmembrane form (23). However,
expression of the transmembrane form of ULBP2 on the NK-
sensitive CHO cell line had a similar small enhancing effect
on sensitivity to lysis by NK cells, as expression of both forms
(23). Potential cis interactions of ULBP1 with cell surface
proteins are different in the DRM domains than in the rest
of the plasma membrane. The slower mobility of ULBP1-
CD45TM, as compared with ULBP1, and the recovery to
a similar lateral mobility after inhibition of F-actin suggest
that ULBP1-CD45TM interacts with molecules tethered to
the cytoskeleton. Receptor ligands on target cells may often
exist within the context of larger protein complexes, and the
role of these complexes in ligand recognition should be given
greater consideration. The overall extent of basal ULBP1
clustering, before contact of target cells with NK cells, was
virtually identical for ULBP1 and ULBP1-CD45TM. Nev-
ertheless, despite a similar number of molecules per cluster,
the molecular and biophysical properties of ULBP1 and
ULBP1-CD45TM clusters may be different because of the
unique properties of membrane subdomains. Such differences
may change the interaction of receptor NKG2D with its li-
gands at immunological synapses, the organization of which
plays an important role in lymphocyte responses (24). Dis-
tribution of ULBP1 either within or outside of DRM do-
mains may also affect trogocytosis, a process by which
cell surface proteins are transferred between target cells and
lymphocytes (25–27). Whether intercellular transfer of a li-
gand for an activation receptor from a target cell to an ef-
fector cell leads to amplification of the response or to
desensitization of the receptor is unclear. It would be in-
teresting to investigate whether the distribution of ULBP1 in
different membrane domains has an impact on its transfer to
Our data suggest that ligand distribution into distinct mem-
brane domains in general may play an underappreciated role
in the activation of NK cells. Given the potential of tumor
cells or virus-infected cells to alter ligand distribution at the
plasma membrane and to escape immune responses, it will be
important to investigate how the distribution of other ligands
impacts the activation of lymphocytes.
The Journal of Immunology 5541
We thank M. Vales-Gomez and H. Reyburn for advice and comments and
M. Peterson and M. March for discussion.
The authors have no financial conflicts of interest.
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