The Rockefeller University Press $30.00
J. Cell Biol. Vol. 192 No. 4 675–690
T.P. Abeyweera and E. Merino contributed equally to this paper.
Correspondence to M. Huse: firstname.lastname@example.org
Abbreviations used in this paper: DMNB, dimethoxy-nitrobenzyl; Fmoc,
9-fluorenylmethoxycarbonyl; HLA, human leukocyte antigen; ICAM, intercellular
adhesion molecule; ITIM, immunotyrosine-based inhibitory motif; KIR, killer Ig
receptor; MHC, major histocompatibility complex; MSCV, murine stem cell virus;
NK, natural killer; SA, streptavidin; TIRF, total internal reflection fluorescence.
Natural killer (NK) lymphocytes play a crucial role in antiviral
and anticancer responses by killing infected or tumorigenic tar-
get cells and also by secreting inflammatory cytokines. They are
activated by a diverse set of transmembrane receptors that rec-
ognize cell surface proteins characteristic of infected or trans-
formed tissue (Lanier, 2005). Ligand binding triggers the
elevation of intracellular calcium (Ca2+), the up-regulation of
integrin-mediated adhesion, and cytoskeletal reorganization
leading to the formation of a radially symmetric cell–cell con-
tact called the cytolytic synapse (Burshtyn et al., 2000; Orange
et al., 2003; Barber et al., 2004; Bryceson et al., 2005; Stinchcombe
and Griffiths, 2007). Soluble cytotoxic agents, such as perforin
and granzyme, are then secreted by the NK cell into the synapse
to kill the target (Stinchcombe and Griffiths, 2007).
Activating NK receptors are opposed by a group of in-
hibitory receptors that contain a cytoplasmic-signaling motif
known as an immunotyrosine-based inhibitory motif (ITIM).
Although ITIM receptors regulate multiple cell types, they are
particularly important for the control of lymphocyte activity
and the prevention of autoimmunity (Long, 2008). In NK cells,
they block the cytolysis of normal healthy tissue by recognizing
class I major histocompatibility complex (MHC), which is ex-
pressed on the surface of most cell types and serves as a marker
for “self” (Lanier, 2005; Long, 2008). MHC binding induces
ITIM phosphorylation and the subsequent recruitment of the
tyrosine phosphatases SHP-1 and SHP-2 (Burshtyn et al., 1996;
Olcese et al., 1996; Bruhns et al., 1999), which dephosphorylate
signaling molecules required for activating responses (Binstadt
et al., 1998; Stebbins et al., 2003).
Studies suggest that ITIM signaling in NK cells disrupts
activating pathways at a level close to the activating receptor
itself (Kaufman et al., 1995; Valiante et al., 1996; Guerra et al.,
2002; Krzewski et al., 2006; Masilamani et al., 2006). Precisely
how inhibitory signals interface with their activating counterparts
within a cell–cell contact, however, is not understood. In T cells
and B cells, activated antigen receptors signal from plasma
immune response. To prevent targeting healthy tissue,
NK cells also express numerous inhibitory receptors that
signal through immunotyrosine-based inhibitory motifs
(ITIMs). Precisely how signals from competing activating
and inhibitory receptors are integrated and resolved is not
understood. To investigate how ITIM receptor signaling
impinges on activating pathways, we developed a photo-
chemical approach for stimulating the inhibitory receptor
atural killer (NK) lymphocytes use a variety
of activating receptors to recognize and kill
infected or tumorigenic cells during an innate
KIR2DL2 during ongoing NK cell–activating responses
in high-resolution imaging experiments. Photostimulation
of KIR2DL2 induces the rapid formation of inhibitory re-
ceptor microclusters in the plasma membrane and the
simultaneous suppression of microclusters containing
activating receptors. This is followed by the collapse of
the peripheral actin cytoskeleton and retraction of the
NK cell from the source of inhibitory stimulation. These
results suggest a cell biological basis for ITIM receptor
signaling and establish an experimental framework for
Inhibitory signaling blocks activating receptor
clustering and induces cytoskeletal retraction in
natural killer cells
Thushara P. Abeyweera, Ernesto Merino, and Morgan Huse
Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065
© 2011 Abeyweera et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub-
lication date (see http://www.rupress.org/terms). After six months it is available under a
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as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
T H E J O U R N A L O F C E L L B I O L O G Y
JCB • VOLUME 192 • NUMBER 4 • 2011 676
and inhibitory pathways. To circumvent this problem, we devel-
oped a photochemical approach that enabled us to stimulate
inhibitory signaling after the onset of activating signals in high-
resolution imaging experiments. We prepared a semisynthetic
peptide–MHC complex that is nonstimulatory to the inhibitory
NK receptor KIR2DL2 until it is irradiated with UV light. Photo-
stimulation of KIR2DL2-expressing NK cells on surfaces
containing this reagent triggered the formation of inhibitory re-
ceptor microclusters and suppressed the formation of new acti-
vating receptor microclusters. This was followed by rapid
reorganization of the actin cytoskeleton and retraction of the
cells from the stimulatory surface. These results establish a cell
biological basis for ITIM receptor signaling and provide insight
into the mechanisms of signal integration in NK cells.
KIR2DL2 signaling blocks the initiation of
KIR2DL2 recognizes a subset of human class I MHC mole-
cules, including human leukocyte antigen (HLA)-Cw1, -Cw3,
-Cw7, and -Cw8, and transduces inhibitory signals via two
cytoplasmic ITIMs (Lanier, 2005). To analyze the effects of
KIR2DL2 signaling on NK cell activation, we stably transduced
the receptor into the human NK cell line NKL, which does not
express any endogenous inhibitory KIR proteins (Robertson
et al., 1996). To stimulate KIR2DL2 signaling, we used a mu-
tant form of HLA-Cw3 that binds to KIR2DL2 but does not
bind to ILT2, another inhibitory receptor for MHC that is ex-
pressed by NKL cells (Fig. 1 A). This HLA-Cw3 mutant, which
membrane microclusters that traffic centripetally toward the
center of the synapse between the lymphocyte and the antigen-
presenting cell (Harwood and Batista, 2010; Yokosuka and
Saito, 2010). Given the similarities between NK cells and other
lymphocytes, it is likely that activating NK receptors also form sig-
naling microclusters, which would presumably need to be neu-
tralized by inhibitory receptors to block activating responses.
In vivo, NK cells must eliminate rare target cells that are
surrounded by healthy tissue expressing high levels of class I
MHC. In this context, it would presumably be important to re-
strict the scope of inhibitory signals to avoid blocking activating
responses against bona fide targets. In vitro experiments have
shown that NK cells can form cytolytic synapses at one cell–cell
interface while receiving inhibitory stimulation from other sites
(Eriksson et al., 1999; Vyas et al., 2001), suggesting that they
can indeed limit the extent of inhibitory signals. Furthermore,
inhibitory killer Ig receptors (KIRs) have been observed to clus-
ter and undergo tyrosine phosphorylation exclusively at inter-
faces containing their cognate MHC ligands (Davis et al., 1999;
Faure et al., 2003; Vyas et al., 2004; Treanor et al., 2006). How
signals emanating from these receptors are also spatially con-
strained, however, is not known. Also unclear is how long NK
cells remain sensitive to inhibitory stimulation from before they
become committed to a killing response. Precise quantification
of this window of responsiveness, if it exists, would provide in-
sight into the mechanisms that govern the integration of activat-
ing and inhibitory signals.
The analysis of signal integration in NK cells has been
complicated by the speed and breadth of inhibitory responses,
which make it difficult to observe interactions between activating
Figure 1. KIR2DL2 signaling blocks activating re-
sponses. (A) Control NKL cells (left) and NKL cells
expressing KIR2DL2 (right) were stimulated with
-NK and either wild-type (WT) HLA-Cw3 or HLA-
Cw3 containing the ILT2-binding mutation (IBM) as
indicated. Both HLA-Cw3 proteins contained the
importin- peptide with p8 Ala. IFN- secretion was
quantified by ELISA. Two independent experiments
are shown for each cell type. Although maximal
IFN- secretion varied from day to day, the relative
differences in cytokine production between different
stimulus conditions were consistent. All other figures
presented in this paper used HLA-Cw3 containing
the ILT2-binding mutation. (B–D) NKL cells express-
ing wild-type KIR2DL2 (B–D) or KIR2DL2(mut) (D)
were added to plastic wells containing immobilized
-NK and the indicated HLA-Cw3 proteins. (B) IFN-
secretion from NKL cells expressing KIR2DL2, mea-
sured by ELISA. Two independent experiments are
shown. (C) Representative degranulation responses
measured by surface expression of CD107a.
Unstim., unstimulated. As with IFN- secretion, maxi-
mal degranulation responses were quite variable.
However, the relative differences between stimulus
conditions were consistent. (D) Dose–response curves
showing induced degranulation from NKL cells ex-
pressing either wild-type KIR2DL2 or KIR2DL2(mut)
(both Tyr 302 and Tyr 332 mutated to Phe) as a
function of the concentration of HLA-Cw3(Ala) used
during protein immobilization. In A and B, error
bars represent SEM between replicates, with n = 3.
All data are representative of at least two indepen-
dent experiments. P-values were calculated using
Student’s t test.
677Inhibitory signaling triggers NK cell retraction • Abeyweera et al.
Hence, we also prepared HLA-Cw3 containing importin- pep-
tides with Ser or Tyr in the p8 position.
To evaluate the potency of these HLA-Cw3 reagents, we
determined whether they could inhibit responses triggered by
the activating receptor NKG2D. Although previous studies had
will be called HLA-Cw3 hereafter, was purified and complexed
with a nonamer peptide derived from importin- (GAVDPLLAL).
Previous experiments had demonstrated that the side chain in
the p8 position of this peptide must be small (either Ala or Ser)
to accommodate KIR2DL2 binding (Boyington et al., 2000).
Figure 2. KIR2DL2 signaling inhibits cell spreading and the initiation of Ca2+ flux. (A and B) NKL cells expressing KIR2DL2 were stained with PKH26
and imaged using TIRF microscopy on lipid bilayers containing the indicated proteins. (A) Representative time-lapse montages (90-s intervals) under
both activating (top) and inhibitory (bottom) conditions. (B) Bar graph representing the distribution of cell behavior on surfaces containing the indicated
ligands. Only cells visible in the imaging field for ≥5 min were analyzed. Cells were described as spread if they formed a stationary footprint at least 10 µm
in diameter (yellow arrow in A), collapsed if they engaged in minimal dynamic interactions with the membrane (magenta arrow in A), or motile if they
exhibited directional migration (cyan arrow in A). Occasionally, cells would display two phenotypes during the imaging period. (C) NKL cells express-
ing KIR2DL2 (KIR-WT) or KIR2DL2(mut) (KIR-Mut) were loaded with Fura-2AM and imaged on lipid bilayers containing ULBP3, ICAM, and the indicated
HLA-Cw3 proteins. (right) Representative time-lapse montages (4-min intervals) showing a pseudocolored Fura-2AM ratio (warmer colors indicate higher
intracellular Ca2+ concentrations). (left) Background-corrected mean Fura-2AM ratios for all imaging fields are plotted versus time for each condition. Error
bars show SEM. All data are representative of at least two independent experiments. Bars, 10 µm.
JCB • VOLUME 192 • NUMBER 4 • 2011 678
(HLA-Cw3(cage)) had little to no effect on IFN- secretion and
degranulation induced by activating ligands (Fig. 3, B and C).
However, substantial inhibition was observed if the peptide–
MHC was exposed to UV light before NK cell stimulation
(Fig. 3, B and C), indicating that HLA-Cw3(cage) is a photo-
inducible ligand for KIR2DL2.
KIR2DL2 photostimulation induces
receptor microcluster formation and
Having validated that HLA-Cw3(cage) could induce UV-
dependent inhibitory signaling, we used this reagent in imaging
experiments to stimulate KIR2DL2 during ongoing activating
responses (Fig. S2 A). NKL cells expressing GFP-labeled
KIR2DL2 were imaged by TIRF microscopy on bilayers con-
taining ULBP3, ICAM, and HLA-Cw3(cage). These bilayers
induced symmetric cell spreading characteristic of activation,
reflecting the fact that HLA-Cw3(cage) does not inhibit activat-
ing responses before UV exposure (Fig. 4 A and Video 3). Sub-
sequent UV irradiation of the surface induced the rapid (<3 s)
formation of KIR2DL2 microclusters, particularly in an annular
zone at the periphery of the contact (Figs. 4, A and B; and S2 B;
and Video 3). This was followed, in most experiments, by the
retraction of the cell from the bilayer. Retraction occurred pri-
marily in the peripheral region enriched in KIR2DL2 microclus-
ters, and the process tended to eliminate these clusters. In
most experiments, we observed low levels of UV-independent
KIR2DL2 clustering on membranes containing HLA-Cw3(cage)
(Fig. 4 A), which was most likely a result of background levels
indicated that NKG2D signaling alone was insufficient to trig-
ger NK cell activation (Bryceson et al., 2006, 2009), we found
that the responsiveness of NKL cells to NKG2D was enhanced
in the presence of interleukin-2 (IL-2). Under these conditions,
incubation of NKL cells expressing KIR2DL2 in plastic wells
coated with an antibody against NKG2D (-NK) induced both
degranulation and the secretion of IFN- (Fig. 1, B and C). Co-
immobilization of HLA-Cw3(Ala) or HLA-Cw3(Ser) in the
stimulatory wells inhibited these responses (Fig. 1, B and C).
HLA-Cw3(Tyr) had no effect, confirming that a small residue in
the p8 position is necessary for KIR2DL2 stimulation. NKL
cells expressing a mutant of KIR2DL2 in which the crucial Tyr
residues of both cytoplasmic ITIMs were replaced with Phe
(called KIR2DL2(mut) hereafter) were markedly less sensitive
to HLA-Cw3 (Fig. 1 D), indicating that the disruption of acti-
vating responses required ITIM signaling from KIR2DL2.
To examine the cell biological basis for this inhibition, we
stained KIR2DL2-expressing NKL cells with the vital mem-
brane dye PKH26 and imaged them using total internal reflec-
tion fluorescence (TIRF) microscopy as they interacted with
supported lipid bilayers containing activating and inhibitory li-
gands. Consistent with previous work (Culley et al., 2009; Liu
et al., 2009), NKL cells formed stable, synapselike contacts on
bilayers containing ULBP3 (an NKG2D ligand), the intercellu-
lar adhesion molecule (ICAM), and the nonfunctional HLA-
Cw3(Tyr) (Fig. 2, A and B; and Video 1). Substitution of
HLA-Cw3(Tyr) with the functional HLA-Cw3(Ser) dramati-
cally altered this spreading behavior; most cells did not spread
at all, instead forming small, dynamic contacts (Fig. 2, A and B;
and Video 2). A subset of cells exhibited a highly mobile crawl-
ing phenotype (Fig. 2 A, cyan arrow), which was consistent
with the notion that inhibitory receptors block the ability of ac-
tivating signals to arrest cell motility (Culley et al., 2009).
Activating bilayers also induced robust Ca2+ flux in
KIR2DL2-expressing NKL cells. This response was blocked in
the presence of HLA-Cw3(Ser) (Fig. 2 C). NKL cells express-
ing KIR2DL2(mut) were insensitive to HLA-Cw3(Ser), indi-
cating that inhibition required KIR2DL2 signaling (Fig. 2 C).
Collectively, these data showed that KIR2DL2-mediated inhibi-
tion of cytokine secretion and degranulation was associated
with a block in the initiation of cell spreading and Ca2+ flux.
A photocaged ligand for KIR2DL2
The lack of observable activating responses in the presence of in-
hibitory ligands (Figs. 1 and 2) complicated our attempts to analyze
the cellular mechanisms of inhibitory signaling. To circumvent
this issue, we developed a photochemical approach that allowed
us to stimulate KIR2DL2 after activating signals had begun.
As stated in the previous section, the side chain in the p8
position of the importin- peptide must be small to allow
KIR2DL2 binding to HLA-Cw3 (Boyington et al., 2000). Ac-
cordingly, we synthesized a derivative of the peptide containing
a photocaged Ser residue (Veldhuyzen et al., 2003) in the p8
position (Figs. 3 A and S1), reasoning that the presence of a
bulky caging group would sterically block KIR2DL2 binding
to HLA-Cw3 and that UV irradiation would relieve this
blockade. Indeed, HLA-Cw3 containing the photocaged peptide
Figure 3. Preparation of photocaged HLA-Cw3. (A) Photocaged Ser
was synthesized and incorporated into the importin- peptide, which
was then refolded with purified HLA-Cw3 and 2m. UV irradiation of
HLA-Cw3(cage) yields stimulatory HLA-Cw3(Ser). (B and C) IFN- secre-
tion (B) and degranulation (C) of KIR2DL2-expressing NKL cells stimulated
on plastic surfaces coated with the indicated activating and inhibitory
molecules. HLA-Cw3(cage) was either UV irradiated or left untreated
before immobilization on the stimulatory surfaces. Asterisks in B denote
P < 0.001 (Student’s t test). Unstim, unstimulated. Error bars show SEM
between replicates, with n = 3. Data are representative of at least three
679Inhibitory signaling triggers NK cell retraction • Abeyweera et al.
is closely related to KIR2DL2 and shares the same ligand speci-
ficity (Lanier, 2005). These cells spread and fluxed Ca2+ on
bilayers containing ULBP3 and ICAM as the sole activating
ligands (unpublished data), which was consistent with previous
studies showing that culturing NK cells in IL-2 enhances
NKG2D expression and responsiveness (Bryceson et al., 2006;
Decot et al., 2010). Importantly, UV irradiation on bilayers con-
taining HLA-Cw3(cage), but not HLA-Cw3(Tyr), induced cel-
lular retraction (Fig. 5). These responses were qualitatively
similar to those observed with NKL cells but tended to be
weaker quantitatively. This could be caused by differences in
the inhibitory potency of KIR2DL3 relative to KIR2DL2. It is
also possible that primary NK cells express a composition of
cytoplasmic signaling regulators that makes them less respon-
sive to inhibitory stimulation than NKL cells. Nevertheless,
these results indicate that the retraction response to inhibitory
KIR stimulation is not unique to the NKL cell line.
ITIM signaling is required for retraction but
not microcluster formation
To determine whether KIR2DL2 microcluster formation and
cellular retraction required KIR2DL2 signaling, we photostimu-
lated NKL cells expressing KIR2DL2(mut)-GFP. KIR2DL2(mut)
formed microclusters in response to photostimulation that were
enriched in the periphery of the contact, similar to wild-type
of decaging that were insufficient to promote retraction. Impor-
tantly, photostimulation on bilayers containing HLA-Cw3(Tyr)
instead of HLA-Cw3(cage) induced neither KIR2DL2 cluster-
ing nor cellular retraction, indicating that both responses re-
quired HLA-Cw3(cage) (Fig. 4, C and D).
To better characterize the speed and prevalence of retrac-
tion after KIR2DL2 stimulation, we performed lower resolution
TIRF experiments in which multiple cells were imaged simulta-
neously. PKH26-labeled NKL cells expressing KIR2DL2 were
allowed to spread on bilayers containing ULBP3, ICAM, and
either HLA-Cw3(Tyr) or HLA-Cw3(cage) for a defined period
of time and then UV irradiated as a group. KIR2DL2-mediated
retraction tended to be quite rapid. Of the cells that collapsed
in response to photostimulation (65% of total cells), close to
half did so within 5 min of UV exposure (Fig. 4 E). We also
asked how the duration of activating signals before photostimu-
lation affected subsequent retraction responses to investigate
whether there was a finite period of sensitivity to inhibitory sig-
nals. Our data, which included cells that landed up to 15 min
before UV irradiation, revealed no evidence for a loss in respon-
siveness over time (Fig. 4 E). Hence, NK cells retract within
minutes of KIR2DL2 stimulation, and they remain sensitive to
inhibitory signals at least 15 min after initial activation.
Photostimulation experiments were also performed using
cultured primary human NK cells expressing KIR2DL3, which
Figure 4. Photostimulation of KIR2DL2 induces receptor microclusters and cellular retraction. (A–D) NKL cells expressing KIR2DL2-GFP were imaged using
TIRF microscopy and UV irradiated on bilayers containing ULBP3, ICAM, and either HLA-Cw3(Tyr) or HLA-Cw3(cage). (A and C) Representative time-lapse
montages (25-s intervals) showing NKL cells responding to photostimulation on surfaces containing the indicated proteins. UV irradiation is denoted in
magenta. (B) Graph showing the change in KIR2DL2 microclusters and cell contact area after photostimulation on bilayers containing HLA-Cw3(cage). Data
were derived from seven cells. (D) Graph showing mean cell contact area before and after photostimulation on bilayers containing the indicated HLA-Cw3
proteins. Each curve was derived from at least nine cells. (E) PKH26-stained NKL cells expressing KIR2DL2 were imaged on bilayers containing ULBP3,
ICAM, and the indicated HLA-Cw3 proteins for up to 15 min before UV irradiation. Cells on the HLA-Cw3(cage) bilayer were grouped based on when they
formed stable contacts with the bilayer (early, first 5 min; late, between 5 and 15 min) and how quickly they collapsed after UV (within 5 or 17.5 min).
A timeline for the experiment is shown on the right in a gray box. Cells were defined as collapsed once their footprint on the bilayer shrank to <50% of its
value before UV irradiation. Error bars represent SEM. Purple lines in graphs denote UV irradiation. All data are representative of at least two independent
experiments. norm., normalized. Bars, 5 µm.
JCB • VOLUME 192 • NUMBER 4 • 2011 680
Culley et al., 2009), where it is thought to be important for cell
spreading and target cell adhesion.
To investigate the effects of ITIM receptor signaling on
actin structure, we transduced NKL cells with the Lifeact pep-
tide (Riedl et al., 2008), which binds specifically to filamentous
actin. When cells expressing KIR2DL2-GFP and Lifeact-RFP
contacted bilayers containing ULBP3, ICAM, and HLA-
Cw3(cage), the Lifeact-RFP probe accumulated in a ring at
the periphery of the synapse, which is characteristic of an acti-
vated lymphocyte. Subsequent UV irradiation dramatically
altered this configuration (Fig. 7 A and Video 5). As the con-
tact retracted, the actin ring “filled in” so that the intensity of
the Lifeact-RFP probe became uniform over the entire inter-
face. UV-induced dissolution of the actin ring was far less
frequent on bilayers containing HLA-Cw3(Tyr) instead of
HLA-Cw3(cage) (Fig. 7 B). This remodeling response was also
impaired by NSC87877 (Fig. 7 C). Hence, the reorganization of
actin induced by KIR2DL2 requires both ITIM signaling and
“Outside-in” signals from integrins play an important role
in leukocyte adhesion and cell spreading by promoting the
polymerization and stabilization of actin (Abram and Lowell,
2009). To determine whether integrin signaling to the cytoskel-
eton could counteract the effects of KIR2DL2, we performed
photostimulation experiments in the presence of manganese
(Mn2+), a divalent cation that up-regulates integrin-mediated ad-
hesion and enhances outside-in signaling. Indeed, UV-induced
retraction was diminished by Mn2+ treatment (Fig. S3), suggest-
ing that integrin and KIR2DL2 signals intersect at the level of
actin. Interestingly, however, Mn2+ did not block the dissolution
of the peripheral actin ring (Fig. 7 D). Hence, there are certain
aspects of KIR2DL2-mediated actin remodeling that are not re-
versed by integrin signaling.
KIR2DL2 (Figs. 6, A and B; and S2 C; and Video 4). However,
no significant retraction was observed, and peripheral micro-
clusters tended to persist for the duration of the experiment. Hence,
ITIM signaling is required for retraction but not for KIR2DL2
In NK cells, ITIM-induced dephosphorylation of activat-
ing signaling proteins is mediated by SHP-1 and SHP-2 (Burshtyn
et al., 1996; Olcese et al., 1996). To assess the role of these
phosphatases downstream of KIR2DL2, photostimulation ex-
periments were performed in the presence of NSC87877, a
SHP-1/2 inhibitor. NSC87877 substantially impaired retraction re-
sponses but did not affect KIR2DL2 microcluster formation
(Figs. 6 C and S2 D), suggesting that recruitment and activation
of SHP-1/2 is required for mediating ITIM-induced changes
in synaptic structure.
The actin motor protein myosin II has been impli-
cated in retraction responses in several systems (Small and
Resch, 2005). However, KIR2DL2-induced collapse of the
contact region was unaffected by the myosin inhibitor blebbi-
statin (Fig. S2 E), suggesting that it operates via a myosin-
KIR2DL2 signaling induces remodeling of
the actin cytoskeleton
The observation that KIR2DL2 photostimulation triggered re-
traction suggested that ITIM signaling might be altering the
underlying actin cytoskeleton. Cytolytic synapse formation is
accompanied by a burst of actin polymerization (Orange et al.,
2003), and agents that disrupt filamentous actin block killing
and other activating responses (Watzl and Long, 2003; Barber
et al., 2004; Endt et al., 2007). Previous studies have demon-
strated that actin accumulates in an annular zone at the periph-
ery of the synapse (Bunnell et al., 2001; Arana et al., 2008;
Figure 5. Photostimulation of KIR2DL3 triggers retraction in primary human NK cells. KIR2DL3+ NK cells were stained with PKH26 and photostimulated
on bilayers containing ULBP3, ICAM, and either HLA-Cw3(cage) or HLA-Cw3(Tyr). (left) A time-lapse montage (25-s intervals) showing a representative
photostimulation experiment on a bilayer containing HLA-Cw3(cage). UV irradiation is indicated in magenta. (right) A graph showing the mean cell contact
area before and after photostimulation on bilayers containing the indicated HLA-Cw3 proteins. The purple line denotes UV irradiation. Each curve was
derived from ≥15 cells. Error bars show SEM. Data are representative of two independent experiments. norm., normalized. Bars, 5 µm.
681Inhibitory signaling triggers NK cell retraction • Abeyweera et al.
lipid bilayers containing ULBP3 and ICAM, microclusters of
DAP10 formed that could be imaged by TIRF microscopy
(Fig. 8 A and Video 6). These microclusters were not observed
on bilayers containing ICAM alone (Fig. S4), indicating that
they required NKG2D stimulation. Microclusters adopted
one of two behaviors (Fig. 8 A). One group of clusters formed
in the periphery and migrated centripetally toward the center of
the contact. The second group was relatively immobile and
tended to be distributed closer to the central region. The mobile
clusters either vanished as they approached the center or merged
with other clusters and became immobile (Video 6). Interest-
ingly, neither the immobile nor the mobile clusters fused into
a single central accumulation over time. This distinguishes
NKG2D-DAP10 microclusters from antigen receptor micro-
clusters in T cells and B cells, which coalesce into a central supra-
molecular activation cluster as the synapse matures (Harwood
and Batista, 2010; Yokosuka and Saito, 2010).
To determine which pool of DAP10 microclusters was ac-
tively engaged in signaling, we imaged DAP10-GFP–expressing
NKL cells that were fixed on bilayers containing ULBP3 and
ICAM and then stained with antibodies against phosphotyro-
sine. Although microclusters near the center of the contact tended
to contain more DAP10, phosphotyrosine staining was enriched
in the peripheral microclusters (Fig. 8, B–D). These results,
which are similar to what has been observed for antigen recep-
tor clusters in T cells (Campi et al., 2005; Yokosuka et al., 2005;
Varma et al., 2006), suggested that signaling is mediated pre-
dominantly by peripheral clusters containing NKG2D-DAP10.
KIR2DL2 signaling inhibits the formation of
activating receptor microclusters
Next, we imaged DAP10-GFP and KIR2DL2-mCherry in the
same cells to assess the interplay between the two receptors. As
described in previous sections, photostimulation on bilayers
containing ULBP3, ICAM, and HLA-Cw3(cage) induced the
rapid formation of KIR2DL2 microclusters, particularly in the
periphery of the contact. This ring of KIR2DL2 showed little
overlap with the relatively stable immobile pool of DAP10 clus-
ters located in the central region (Fig. 9 A). Indeed, many of
these immobile DAP10 clusters persisted for the duration of the
retraction response, even as the annular zone of KIR2DL2 accu-
mulation collapsed inward. In contrast, KIR2DL2 stimulation
dramatically affected the mobile pool of DAP10 microclusters
in the periphery. Although these clusters were readily apparent
before photostimulation (Fig. 9 A, arrows), they tended to be ab-
sent after UV irradiation, particularly in peripheral regions rich
in KIR2DL2 (Fig. 9 A, brackets). Particle tracking revealed that
the formation of peripheral DAP10 clusters was strongly inhib-
ited by UV irradiation (Fig. 9, B and C; and Video 7). Peripheral
clusters were not suppressed by UV irradiation on surfaces con-
taining HLA-Cw3(Tyr) instead of HLA-Cw3(cage) (Fig. 9,
B and C), indicating that the response required KIR2DL2 stimu-
lation. Hence, the formation of KIR2DL2 microclusters at the
periphery of the synapse is associated with the suppression of
new DAP10 microclusters in the same domain.
Because we had also observed that KIR2DL2 photostimula-
tion alters actin structure (Fig. 7), we explored the possibility that
The activating receptor NKG2D forms
The aforementioned imaging experiments showed that KIR2DL2
stimulation could reverse cell-spreading responses induced by
the activating receptor NKG2D. To visualize NKG2D dynamics
directly, we fluorescently labeled DAP10, a small signaling
adaptor that constitutively associates with NKG2D (Lanier,
2005). When NKL cells expressing DAP10-GFP were added to
Figure 6. ITIM signaling and SHP-1/2 activity are required for cellular
retraction. (A and B) NKL cells expressing KIR2DL2(mut)-GFP were imaged
using TIRF microscopy and UV irradiated on bilayers containing ULBP3,
ICAM, and HLA-Cw3(cage). (A) Time-lapse montage (25-s intervals)
showing a representative response UV irradiation, which is indicated in
magenta. (B) Graph showing the cell contact area and the change in
KIR2DL2 microcluster number after photostimulation. Data were derived
from 12 cells. (C) NKL cells expressing wild-type KIR2DL2 were photostimu-
lated on bilayers containing ULBP3, ICAM, and the indicated HLA-Cw3
proteins in the presence or absence of NSC87877. Mean cell contact area
is graphed both before and after UV irradiation. Each curve was derived
from at least seven cells. Throughout the figure, purple lines indicate UV
irradiation. Error bars show SEM. All data are representative of at least
two independent experiments. norm., normalized. Bars, 5 µm.
JCB • VOLUME 192 • NUMBER 4 • 2011 682
actin is necessary for NKG2D-DAP10 microcluster assembly and
trafficking. These results are similar to what has been observed for
antigen receptor microclusters in T cells (Varma et al., 2006). Col-
lectively, our data indicated that KIR2DL2 signaling inhibits acti-
vating receptor microcluster formation, possibly by influencing
the underlying actin cytoskeleton.
actin is required for DAP10 microcluster dynamics. NKL cells
expressing DAP10-GFP were imaged on bilayers containing
ULBP3 and ICAM both before and after the addition of the actin-
depolymerizing agent latrunculin. Treatment with latrunculin
abolished the formation and centripetal migration of peripheral
DAP10 microclusters (Fig. 9, C and D), indicating that filamentous
Figure 7. KIR2DL2 photostimulation induces actin remodeling. (A–D) NKL cells expressing KIR2DL2 and Lifeact-RFP were imaged using TIRF microscopy
and UV irradiated on bilayers containing ULBP3, ICAM, and either HLA-Cw3(cage) (A, C, and D) or HLA-Cw3(Tyr) (B). Photostimulation was performed
using cells left untreated (A and B) or treated with NSC87877 (C) or Mn2+ (D). For each panel, a time-lapse montage (75-s intervals) is shown (top) along
with an associated kymograph. UV irradiation is indicated by magenta text in the time lapse and by a magenta line in the kymograph. Kymographs were
generated using the yellow line in the first image of each time lapse. Shown on the bottom in each panel, normalized mean fluorescence intensity of Lifeact-
RFP in the center of the contact is graphed as a function of time together with cell area. The contact center is indicated by a cyan ellipse in each time lapse.
Data are representative of at least two independent experiments. F/F, normalized fluorescence intensity. Bars, 5 µm.
Inhibitory signaling triggers NK cell retraction • Abeyweera et al.
on both surfaces after UV irradiation were essentially super-
imposable (Fig. 10 C).
That KIR2DL2 photostimulation did not inhibit ongoing
Ca2+ responses was surprising, given that KIR2DL2 did block
the initiation of Ca2+ flux when triggered concurrently with ac-
tivating receptors (Fig. 2 C). We sought to confirm this ob-
servation using a flow cytometry–based approach. NKL cells
expressing KIR2DL2 were preincubated with biotinylated mouse
antibodies against two activating receptors, NKG2D and 2B4,
either in the presence or absence of an unbiotinylated mouse
antibody against KIR2DL2. Streptavidin (SA) was then used to
trigger activating signals and an anti–mouse secondary antibody
(-Mouse) to induce inhibition (Fig. S5). Consistent with previous
KIR2DL2 signaling does not block ongoing
We also examined the ability of KIR2DL2 photostimulation to
inhibit ongoing Ca2+ responses. NKL cells expressing KIR2DL2
displayed robust Ca2+ flux upon contact with bilayers containing
ULBP3, ICAM, and either HLA-Cw3(Tyr) or HLA-Cw3(cage)
(Fig. 10 A). Intracellular Ca2+ concentrations typically peaked
in the first few minutes of the response before entering a phase
of sustained Ca2+ elevation (Fig. 10 B). UV irradiation, which
we delivered during the sustained phase in some cells and the
early phase in others, did not significantly affect the intensity
of Ca2+ responses on HLA-Cw3(cage) surfaces relative to
HLA-Cw3(Tyr) controls (Fig. 10, A and B). Mean Ca2+ levels
Figure 8. NKG2D stimulation induces the forma-
tion of activating receptor microclusters. (A) NKL cells
expressing DAP10-mCherry were imaged using TIRF
microscopy on bilayers containing ULBP3 and ICAM.
(left) A kymograph showing centripetally mobile and
stationary DAP10 clusters, indicated by the cyan
arrow and arrowhead, respectively. The line used to gen-
erate the kymograph is shown on the right. (B–D) NKL
cells expressing DAP10-GFP were fixed and stained
with antibodies against phosphotyrosine (pY) on bilay-
ers containing ULBP3 and ICAM. (B) Representative
images showing DAP10 fluorescence (left), phospho-
tyrosine fluorescence (right), and the overlay (center).
(C) Linescans depicting DAP10 and phosphotyrosine
fluorescence (yellow and blue, respectively) in specific
microclusters within the contact region. The lines used
for each linescan are shown in the central image in B.
(D, left) Schematic showing how images were divided
into central and peripheral zones for quantification.
(right) Before and after graph showing the ratio of nor-
malized phosphotyrosine fluorescence intensity (FpY) to
normalized DAP10-GFP fluorescence intensity (FDAP10)
for peripheral and central regions. Ratios calculated
from the same cell are connected by lines. All data
are representative of at least two independent experi-
ments. arb., arbitrary. Bars, 5 µm.
JCB • VOLUME 192 • NUMBER 4 • 2011 684
Figure 9. KIR2DL2 photostimulation blocks the formation of activating receptor microclusters. (A) NKL cells expressing DAP10-GFP and KIR2DL2-mCherry
were imaged in TIRF mode and photostimulated on bilayers containing ULBP3, ICAM, and HLA-Cw3(cage). (top) A representative time-lapse montage
(80-s intervals), with UV irradiation indicated by magenta text. (bottom) Two single-cell kymographs showing KIR2DL2-mCherry and DAP10-GFP clusters
685Inhibitory signaling triggers NK cell retraction • Abeyweera et al.
Varma et al., 2006). Thus, sustained signaling is dependent on
the continuous formation of new peripheral microclusters. Using
DAP10 as a probe, we observed that the NKG2D receptor com-
plex forms two kinds of clusters, a mobile variety that is gener-
ated in the periphery and migrates centripetally and an immobile
variety that accumulates closer to the center. Interestingly,
NKG2D-DAP10 does not coalesce into a central supramolecu-
lar activation cluster over time, possibly because NKG2D sig-
naling is mediated by Tyr-Ile-Met-Asn motifs in DAP10 rather
than the immunotyrosine-based activation motifs contained in
antigen receptors. Nevertheless, peripheral DAP10 microclus-
ters contain higher levels of phosphotyrosine, suggesting that
they mediate most of the NKG2D-dependent signaling. Strik-
ingly, it is in this peripheral zone that we observed UV-induced
accumulation of KIR2DL2 microclusters, suppression of DAP10
microclusters, and actin remodeling.
It is well established that filamentous actin at the synapse
is required for receptor-proximal activating signals in NK cells
(Orange et al., 2003; Watzl and Long, 2003; Barber et al., 2004;
Endt et al., 2007). Consistent with these studies, we observed
that disrupting synaptic actin with latrunculin blocked DAP10
microcluster formation and movement. Interestingly, when
cells were subjected to KIR2DL2 photostimulation, suppres-
sion of activating microclusters occurred within seconds, before
the dissolution of the peripheral actin ring, indicating that
cytoskeletal retraction per se is not responsible for micro-
cluster suppression. It is possible, however, that the dramatic actin
reorganization induced by KIR2DL2 signaling is preceded by a
period of actin destabilization that is more difficult to detect and
that this initial actin destabilization is sufficient to suppress ac-
The pathway linking inhibitory receptors to actin remod-
eling remains unclear but is likely to involve Vav-1, a guanine
nucleotide exchange factor that is phosphorylated and activated
during synapse formation (Bustelo, 2000; Riteau et al., 2003).
Vav-1 stimulates the Rho family GTPase Rac1, which is thought
to promote cytolytic function and target cell adhesion by trig-
gering actin polymerization and the up-regulation of integrins
(Billadeau et al., 1998; Galandrini et al., 1999; Riteau et al.,
2003; Nolz et al., 2008). It is known that SHP-1 directly de-
phosphorylates Vav-1 downstream of ITIM receptors (Stebbins
et al., 2003), which could conceivably lead to the actin remodel-
ing we have observed.
Integrins play a critical role in NK cell function by pro-
moting synapse formation and the polarization of cytolytic
granules (Barber et al., 2004; Bryceson et al., 2005). Our obser-
vations that KIR2DL2 signaling induced retraction and that
studies (Binstadt et al., 1996; Bléry et al., 1997; Bruhns et al.,
1999; Bryceson et al., 2006), clustering of NKG2D and 2B4
with SA induced rapid Ca2+ flux (Fig. 10 D). This response was
inhibited by simultaneous co–cross-linking of KIR2DL2, but
not KIR2DL2(mut), with -Mouse, indicating that KIR2DL2
signaling impairs the initiation of Ca2+ responses (Fig. 10 D,
top). To test whether KIR2DL2 could inhibit ongoing Ca2+ re-
sponses, -Mouse was added either 75 or 150 s after initial
NKG2D and 2B4 cross-linking. Only weak inhibition was
observed relative to control cells in both of these experiments
(Fig. 10 D, bottom). These data confirmed that whereas
KIR2DL2 can inhibit the induction of Ca2+ flux, it is substan-
tially less effective at curtailing ongoing Ca2+ responses.
The cell biological basis of ITIM receptor signaling in NK cells
has remained largely unexplored because of difficulties in visu-
alizing inhibitory and activating signals simultaneously. Using a
photoinducible ligand for KIR2DL2, we were able to separate
the stimulation of activating and inhibitory pathways in time
and establish a spatial and temporal window of sufficient size to
actually observe interactions between them. Our work provides
insight into the mechanisms of ITIM receptor signaling and
signal integration in NK cells.
Photostimulation of KIR2DL2 during ongoing activating
responses is admittedly an imperfect model for the simultane-
ous triggering of activating and inhibitory receptors that presum-
ably occurs in many NK cell–target cell synapses. Nevertheless,
we feel that our results reflect a biologically relevant mecha-
nism for ITIM receptor signaling for the following reasons.
First, the retraction response we observe requires ITIM signal-
ing and SHP-1/2 activity, the same molecular determinants nec-
essary for KIR-mediated inhibition in other systems. Second,
our observation that NK cells remain responsive to inhibitory
stimulation well after the onset of activating signals is consis-
tent with previous experiments showing that NK cells modify
the morphology of a growing synapse upon encountering ITIM
receptor ligands (Culley et al., 2009). In vivo, the ability to re-
spond to fresh inhibitory stimulation in this manner is likely
important for keeping growing cytolytic synapses with bona
fide targets from spilling over onto adjacent bystander cells.
Pioneering imaging studies in T cells have indicated that
activating signals are initiated by antigen receptor microclusters
at the periphery of the synapse and that, in most cases, signaling
is down-regulated as these clusters approach the center (Campi
et al., 2005; Mossman et al., 2005; Yokosuka et al., 2005;
both before and after UV irradiation, which is indicated by the magenta line. Mobile clusters of DAP10 are denoted by arrows. Brackets indicate areas of
the kymographs showing peripheral regions rich in KIR2DL2 microclusters but devoid of mobile DAP10 microclusters. Lines used for kymographs are shown
in the first image of the time lapse. (B and D) Tracks of DAP10 microclusters in representative single cells. (B) Cells were imaged and UV irradiated on bilay-
ers containing ULBP3, ICAM, and either HLA-Cw3(Tyr) (left) or HLA-Cw3(cage) (right). Paths traveled before UV irradiation are shown in red, and paths
after UV irradiation are shown in blue. (D) Cells were treated with latrunculin (Lat) on bilayers containing ULBP3 and ICAM. Paths traveled before latrunculin
addition are shown in red, and paths after latrunculin addition are shown in blue. (C) Bar graphs showing the relative amounts of mobile versus immobile
DAP10 microclusters. (left and center) Cells were photostimulated on bilayers containing either HLA-Cw3(Tyr) (left) or HLA-Cw3(cage) (center). (right) Cells
were treated with latrunculin on bilayers containing ULBP3 and ICAM. The total number of analyzed microclusters is indicated above each bar, and the
number of analyzed cells for each experiment is shown between bars. The mean starting ratio of mobile to immobile microclusters differed from experiment
to experiment. Hence, two independent experiments, each derived from cells imaged the same day, are shown for each condition. Bars, 5 µm.
JCB • VOLUME 192 • NUMBER 4 • 2011 686
previously suggested (Barber et al., 2004). This result also im-
plies that there is at least some down-regulation of integrin
affinity taking place in response to ITIM signaling. That Mn2+
does not block the dissolution of the actin ring, however, indi-
cates that outside-in signaling alone is insufficient to counteract
the effects of KIR2DL2.
Actin remodeling and concomitant retraction are well
suited as mechanisms for NK cell inhibition for two reasons.
First, by targeting the integrity of the synapse, which is required
by most, if not all, activating receptors, actin remodeling pro-
vides an elegant way to block effector responses that is indepen-
dent of the specific activating pathways involved. Second, because
retraction breaks cell–cell contact and hence the receptor–
ligand interactions that drive the response, it is self-limiting
Mn2+ blocked this response are consistent with previous work
suggesting a direct link between ITIM-dependent signaling and
the regulation of integrins (Burshtyn et al., 2000; Bryceson
et al., 2009). The formation and maintenance of integrin-
mediated cell–cell contacts require Vav and the Rho family
GTPases, and also depend on a strong physical linkage between
ligand-bound integrins and the underlying actin cytoskeleton
(Swat and Fujikawa, 2005; Abram and Lowell, 2009). KIR2DL2-
induced actin remodeling would presumably weaken this adhe-
sive network either by breaking contacts between integrins and
the cytoskeleton or by somehow inducing affinity down-regulation
of integrins. That Mn2+ treatment preserves the contact area
in photostimulation experiments is consistent with the notion
that outside-in signaling is affected by ITIM receptors, as
Figure 10. Photostimulation of KIR2DL2 does
not block ongoing Ca2+ responses. Fluo-4AM–
loaded NKL cells expressing KIR2DL2 were
imaged and UV irradiated on bilayers con-
taining the indicated proteins. (A) Time-lapse
montages (4-min intervals) showing Fluo-4AM
responses before and after UV irradiation. Fluo-
4AM fluorescence is proportional to intracellu-
lar Ca2+ concentration. (B) Ca2+ responses of
two individual cells, which are indicated by
asterisks in A. (C) Mean calcium responses for
the entire population of cells. Each curve was
derived from ≥30 cells. Error bars show SEM.
In B and C, shaded purple bars denote UV ir-
radiation. (D) Antibody cross-linking of KIR2DL2
does not inhibit ongoing Ca2+ responses. Fluo-
4AM–loaded NKL cells expressing either wild-
type KIR2DL2 or KIR2DL2(mut) were incubated
with the indicated antibodies and subjected to
flow cytometry. (top) SA (to cross-link -NK and
-2B4) and -Mouse (to cross-link -KIR with
-NK and -2B4) were added simultaneously as
indicated. (bottom) -Mouse was added after
SA as indicated. All data are representative of
at least two independent experiments. Norm.,
normalized. Bars, 10 µm.
687Inhibitory signaling triggers NK cell retraction • Abeyweera et al.
the Fmoc-Ser(DMNB)-OH was further purified over a cartridge (Sep-Pak
C18; Waters) using a reversed-phase solvent system: 0.1% aqueous trifluoro-
acetic acid (solvent A) versus 90% acetonitrile plus 0.1% trifluoroacetic
acid (solvent B). Purified Fmoc-Ser(DMNB)-OH was validated by electro-
spray mass spectrometry and nuclear magnetic resonance, and it was then
incorporated into a modified importin- peptide at the p8 position by solid-
phase peptide synthesis. After trifluoroacetic acid–mediated cleavage from
the resin, the caged peptide was purified by reversed-phase HPLC using a
C18 column in the absence of UV detection. Other peptides were synthe-
sized by Fmoc chemistry either by our laboratory or by the Microchemistry
and Proteomics Core Facility at the Memorial Sloan-Kettering Cancer Cen-
ter. To assess UV decaging, the caged importin- peptide was irradiated
for 20 min with a 365-nm light using a handheld UV lamp (UVGL-25;
Thermo Fisher Scientific). The irradiated peptide was then compared with
the unirradiated peptide as well as the importin- peptide with Ser in the
p8 position by analytical reversed-phase HPLC (Fig. S1).
HLA-Cw3, 2m, and ULBP3 were isolated from inclusion bodies under
denaturing conditions. HLA-Cw3 was refolded together with 2m and
importin- peptide by rapid dilution into buffer containing 100-mM Tris,
pH 8.0, 400-mM arginine, 5-mM reduced glutathione, 0.5-mM oxidized
glutathione, and protease inhibitors. ULBP3 was refolded by rapid dilution into
buffer containing 100-mM Tris, pH 8.0, 500-mM arginine, 5-mM reduced
glutathione, 2.5-mM oxidized glutathione, and protease inhibitors. After
refolding, HLA-Cw3 and ULBP3 were biotinylated using the BirA enzyme
and purified by size exclusion chromatography. The extracellular domain
of ICAM fused to a C-terminal histidine tag and a BirA recognition sequence
was expressed in SF9 cells by baculoviral transduction and purified by
Ni2+ affinity and anion-exchange chromatography followed by BirA-mediated
biotinylation and size exclusion chromatography. In general, purified pro-
teins were stored at 20°C in the presence of 50% glycerol. It was found,
however, that prolonged storage of HLA-Cw3(cage) under these conditions
increased its inhibitory activity in the absence of UV light, presumably the
result of slow cleavage of the DMNB group. Hence, subsequent prepara-
tions were snap frozen in liquid nitrogen and stored at 80°C.
cDNAs encoding full-length KIR2DL2 (gift from L. Lanier, University of Cali-
fornia, San Francisco, San Francisco, CA) and DAP10 were subcloned
into an murine stem cell virus (MSCV) retroviral plasmid (Quann et al.,
2009) upstream of either GFP, mCherry, or a Myc epitope tag. The pres-
ence of these tags on the C termini of either KIR2DL2 or DAP10 did not
affect their inhibitory or activating functions, respectively. Lifeact fused to RFP
(gift from R. Wedlich-Soldner, International Max Planck Research School
for Molecular and Cellular Life Sciences, Munich, Germany) was sub-
cloned as a single fragment into pMSCV. HLA-Cw3 (gift from P. Parham,
Stanford University, Stanford, CA) and ULBP3, both containing C-terminal
BirA recognition sequences, were expressed in Escherichia coli using
pET28 and pET30 expression plasmids, respectively. Mutagenesis of the
ITIM Tyr residues of KIR2DL2 and the ILT2 binding site of HLA-Cw3 was
performed by PCR using the Quikchange protocol (Agilent Technologies).
The ILT2 binding site mutation replaces amino acid residues 194VSDHE198
of wild-type HLA-Cw3 with 194RSPGF198. The baculoviral expression con-
struct for ICAM containing a C-terminal BirA recognition sequence has
been previously described (Lillemeier et al., 2010), as has the human 2m
expression vector (Garboczi et al., 1992).
Cell lines and retroviral transduction
NKL cells were maintained in complete RPMI 1640 (RPMI 1640 with 10%
FCS) supplemented with 200 IU/ml IL-2. Retrovirus was generated using
amphotropic Phoenix cells, which were grown in DME containing 10%
FCS. Phoenix cells were transfected with MSCV vectors and supplementary
plasmids encoding retroviral gag and pol using either calcium phosphate
or transfection reagent (FuGENE; Roche). Viral supernatants were col-
lected after 48 h at 37°C and concentrated using centrifugal filter devices
(Amicon Ultra; Millipore) with a 105-kD molecular mass cutoff. The virus
was then mixed with 106 NKL cells in 2 ml complete RPMI 1640 and cen-
trifuged at 1,400 g for 2 h in the presence of 8 µg/ml polybrene at 30°C.
After 48 h, transduction efficiency was assessed by flow cytometry (LSR II;
BD) using either the transduced fluorescent protein label or an antibody
against KIR2DL2/3 (clone DX27; BD) for detection. NKL cells expressing
the transduced protein (typically representing 2–10% of the total popula-
tion) were isolated by FACS 1–2 wk after transduction and maintained as
stable cell lines.
and more easily constrained in space and time. This would pre-
sumably facilitate efficient scanning of potential target cells in
vivo. In this context, retraction from inhibitory cells would not
only prevent inappropriate killing responses but also play an im-
portant role in directing those responses to the correct targets.
Using photostimulation as well as antibody-mediated re-
ceptor cross-linking, we found that KIR2DL2 only weakly in-
hibits ongoing Ca2+ responses despite the fact that it blocks the
initiation of Ca2+ flux when triggered concurrently with activat-
ing receptors. This result is intriguing, particularly because
KIR2DL2-induced retraction was not diminished, in our hands,
by prolonged exposure to activating signals. Collectively, our
data suggest that strong adhesion to the target cell is required
for the initiation, but not the maintenance, of Ca2+ signals. Pre-
cisely why KIR2DL2 stimulation does not block ongoing Ca2+
responses is unclear. It is possible that ITIM receptor signaling
disrupts early events, such as the activation of phospholipase C,
that are required for the initiation of Ca2+ flux but has less of an
effect on store-operated calcium channels or other downstream
components that have been implicated in the sustained phase of
the response (Lewis, 2001). Further studies will be required to
explore this issue. From a functional perspective, however, lim-
iting the scope of KIR2DL2 action could facilitate target cell
killing in vivo. Elevated cytoplasmic Ca2+ is required for the se-
cretion of lytic granules (Ostergaard et al., 1987; Takayama and
Sitkovsky, 1987; Esser et al., 1998). The insensitivity of on-
going Ca2+ responses to ITIM receptor signals would presumably
allow NK cells to mount a cytolytic response at one cell–cell
interface while receiving inhibitory signals at a distal contact site.
This model is consistent not only with our data but also with re-
cent experiments indicating that ITIM receptors are more effec-
tive at blocking lytic granule polarization toward the synapse
than at disrupting degranulation (Das and Long, 2010).
The extent to which the cellular consequences of KIR2DL2
signaling will apply to related signaling pathways in other cell
types remains to be seen. It is worth noting, however, that the
effectors of phosphorylated ITIMs, SHP-1 and particularly
SHP-2, are broadly expressed, as is the tyrosine kinase Ableson,
which is also required for inhibitory KIR function in NK cells
(Peterson and Long, 2008). Conceptually, localized retraction is
an elegant mechanism for the inhibition of signals delivered by
membrane-bound ligands, and it is conceivable that this mecha-
nism would be useful in processes such as cell migration and
neuronal path finding. In that regard, it is intriguing that the
ITIM receptor PirB was recently shown to promote axonal col-
lapse in sensory neurons (Atwal et al., 2008). Future mechanis-
tic studies will be required to determine whether or not cursory
similarities like these reflect a shared mechanism for the inhibi-
tory regulation of cell–cell interactions.
Materials and methods
9-fluorenylmethoxycarbonyl (Fmoc)–protected dimethoxy-nitrobenzyl (DMNB)
Ser was synthesized essentially as previously described (Veldhuyzen et al.,
2003). In brief, Fmoc-Ser allyl ester was reacted with 4,5-dimethoxy-2-
nitrobenzyl trichloroacetimidate using catalytic triflic acid under anhydrous
conditions to generate the DMNB-protected Ser derivative. After allyl de-
protection with palladium and purification by silica gel chromatography,
JCB • VOLUME 192 • NUMBER 4 • 2011 688
blebbistatin (Sigma-Aldrich) and 25-µM NSC87877 (Tocris), respectively,
were added to the imaging medium. 1–2-µM latrunculin and 1-mM MnCl2
(both obtained from Sigma-Aldrich) were used to disrupt actin and up-regulate
integrins, respectively. Mn2+ treatment was performed without depleting
Ca2+ and Mg2+. Live-imaging experiments used an inverted fluorescence
video microscope (IX-81; Olympus) attached to an EM charge-coupled de-
vice camera (ImagEM; Hamamatsu). 488- and 561-nm lasers (CVI Melles
Griot) were used for TIRF imaging of GFP and mCherry/RFP, respectively,
and a Xe lamp (DG-4; Sutter Instrument Co.) was used for epifluorescence
imaging. TIRF experiments used 150 or 60× objective lenses, 1.45 NA,
and Ca2+ imaging experiments used a 20× epifluorescence objective,
0.75 NA (all objectives were obtained from Olympus). Time-lapse record-
ings were made using Slidebook software (Intelligent Imaging Innovations).
All live imaging was performed at 37°C.
For high-resolution (150×) imaging of receptor dynamics, images
were acquired every 3 or 5 s for a total of 4 min. Photostimulation was
implemented using a digital diaphragm apparatus (Mosaic; Photonic Instru-
ments) attached to a mercury lamp (HBO; Olympus). Light from the HBO
lamp was filtered through a 350/50-nm bandpass to reduce photo-
damage. Cells were typically photostimulated during either the 25th or the
35th interval in the time lapse using a 2-s exposure.
For lower resolution TIRF imaging of cell spreading and Ca2+ imag-
ing, cells were stained with PKH26 (Sigma-Aldrich; using the manufacturer’s
protocol) or loaded with calcium dyes (5 µg/ml Fura-2AM or Fluo-4AM),
respectively. Fluo-4 loading and imaging were performed in the presence of
2.5-mM probenecid. Images were acquired every 30 or 60 s for 20–30 min
after cells had been added to the bilayer. Photostimulation, when neces-
sary, was performed using a handheld UV lamp positioned just above the
chamber slide. Cells were typically photostimulated after 5–15 min using
a 2-min exposure.
Data analysis was performed using Slidebook, Matlab (MathWorks), Prism
(GraphPad Software, Inc.), and Excel (Microsoft). To quantify cell contact
area, intensity thresholding was used to define the cell boundaries for every
frame or every other frame in the time lapse. Only cells that formed stable
symmetric contacts before UV irradiation were used for the analysis. The
quantification of actin remodeling shown in Fig. 7 was performed by cal-
culating the mean intensity of Lifeact-RFP within a central elliptical region
over the length of the time lapse. For graphical representation of area and
intensity data, values were normalized using images taken before UV irra-
diation. Microclusters of DAP10 and KIR2DL2 were traced and counted,
respectively, using Matlab scripts from D. Blair (Georgetown University,
Washington, DC) and E. Dufresne (Yale, New Haven, CT) based on code
from E. Weeks (Emory University, Atlanta, GA), J. Crocker (University of
Pennsylvania, Philadelphia, PA), and D. Grier (New York University, New
York, NY). Only particles that appeared in at least four consecutive frames
were used for tracing analysis. Mean microcluster velocity was derived
from particle traces using Matlab. Clusters with a mean velocity
<0.03 µm/s were classified as immobile, and the rest were classified as
mobile. Analysis of single-cell Ca2+ flux in Fluo-4–loaded cells was per-
formed by normalizing the fluorescence intensity of each cell using the last
image before the initial rise in Ca2+. The ensemble mean graph shown in
Fig. 2 C was computed using all cells in the imaging field. The curves shown
in Fig. 10 C were calculated using only cells that bound to the surface and
fluxed Ca2+ before UV irradiation.
Staining and imaging of fixed cells
NKL cells expressing DAP10-GFP were added to bilayers containing
ULBP3 and ICAM and incubated at 37°C for 15 min before fixation with
2% paraformaldehyde. After permeabilization with Triton X-100 and
blocking with bovine serum albumin, the cells were stained with 1 µg/ml
antiphosphotyrosine antibody (clone 4G10; Millipore) followed by 1 µg/ml
Alexa Fluor 555–conjugated F(ab)2 goat anti–mouse IgG (Invitrogen).
After staining, the cells were imaged by TIRF microscopy at 150× magni-
fication. Single-cell images were divided into a central region containing
40% of the contact area and a peripheral region containing the remain-
ing 60%. Phosphotyrosine fluorescence per unit DAP10-GFP fluorescence
(FpY/FDAP10) was determined for both regions, and the ratio of periphery/
center was calculated. The mean value of this ratio for a dataset of
20 cells was 5.0 ± 1.4.
Online supplemental material
Fig. S1 shows the chemical validation of the photocaged importin- pep-
tide. Fig. S2 shows a schematic diagram of a typical KIR2DL2 photostimu-
lation experiment and some additional analyses of KIR2DL2 clustering
Primary human NK cells were isolated from peripheral blood from a
healthy donor by negative selection (Miltenyi Biotec). NK cells were resus-
pended in stem cell growth media (CellGro; CellGenix) supplemented with
10% human serum and 200 IU/ml IL-2, and they were split periodically to
maintain a density of 106 cells/ml. After ≥8 d at culture, KIR2DL3+ NK cells
were isolated by cell sorting using antibodies against CD3 (clone S4.1; In-
vitrogen), CD56 (clone B159; BD), and KIR2DL2/3. This procedure typi-
cally yielded >95% CD3CD56+KIR2DL3+ cells.
Stimulatory plastic surfaces for IFN- secretion and degranulation assays were
prepared using 96-well plates (Maxisorp; Thermo Fisher Scientific). First, a
layer of SA (Prozyme) was immobilized on the surfaces followed by incuba-
tion with biotinylated anti-NKG2D antibody (-NK, clone 1D11; Abcam) and
HLA-Cw3 protein. In general, -NK was used at 1 µg/ml, and HLA-Cw3 was
used at 1 µg/ml. In cases in which one or more of these constituents was left
out, a nonstimulatory biotinylated mouse MHC molecule (either I-Ek or H2-Db)
was added to bring the total protein concentration to 2 µg/ml. After protein
immobilization, 2–3 × 105 NKL cells were added to each well, and the plate
was incubated at 37°C for 30 min (for degranulation) or 16 h (for IFN- secre-
tion). For degranulation experiments, we found that a 30-min incubation time
in the absence of monensin or brefeldin yielded optimal responses. All func-
tional assays were performed in the presence of 200 IU/ml IL-2. IFN- secre-
tion experiments were performed in triplicate and quantified by ELISA using
a mouse monoclonal antibody (clone K3.53; R&D Systems) for capture and
a biotinylated affinity-purified goat IgG (R&D Systems) for detection. For
degranulation experiments, 5 µg/ml phycoerythrin-Cy5–conjugated anti-
CD107a antibody (clone H4A3; BD) was included at the start of the 37°C in-
cubation. After washing, CD107a staining was quantified by flow cytometry.
For the analysis shown in Fig. 1 D, the level of activation-induced degranula-
tion was determined by subtracting the percentage of CD107a+ cells in un-
stimulated samples. UV irradiation of HLA-Cw3(cage) was performed using a
handheld UV lamp for 20 min before protein immobilization. All flow cyto-
metric data were analyzed using FlowJo software (Tree Star, Inc.).
Flow cytometric analysis of Ca2+ signaling
Stimulation by antibody cross-linking was performed using -NK, biotinylated
anti-2B4 (-2B4, clone C1.7; eBioscience), and anti-KIR2DL2 (-KIR, clone
GL183; Beckman Coulter). -KIR was cross-linked with -NK and -2B4 using
F(ab)2 goat anti–mouse IgG (-Mouse; Jackson Immunoresearch Laborato-
ries, Inc.). For Ca2+ flux assays, 2–3 × 105 NKL cells were loaded with 5 µg/ml
Fluo-4AM (Invitrogen) and incubated in 50 µl of complete RPMI 1640 on
ice in the presence or absence of 10 µg/ml -NK, 10 µg/ml -2B4, and
20 µg/ml -KIR. After ≥15 min, cells were diluted in 500 µl of warm complete
RPMI 1640 and incubated at 37°C for 2 min followed immediately by flow
cytometric analysis. -NK and -2B4 were cross-linked by the addition of
20 µg/ml SA 1 min after the start of the flow cytometry experiment. -KIR
was cross-linked to -NK and -2B4 by the addition of 20 µg/ml -Mouse.
Data were collected for a total of 10 min. Fluo-4AM loading and flow cytom-
etry were performed in the presence of 2.5-mM probenecid (Invitrogen).
Supported lipid bilayers
A 10:1 mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine and biotinyl
cap phosphoethanolamine (both obtained from Avanti Polar Lipids, Inc.)
was resuspended in PBS and emulsified into small unilamellar vesicles
using a lipid extruder (Avanti Polar Lipids, Inc.). 8-well glass-chamber slides
(Thermo Fisher Scientific) were cleaned by sonication in 2% Hellmanex
(Helma Analytics) at 50°C followed by extensive washing in deionized
water. Small unilamellar vesicle suspensions were added to the cleaned
glass surfaces and allowed to form supported bilayers followed by a
30-min incubation with 20 µg/ml SA in PBS. After further washing in PBS,
a mixture of biotinylated NK receptor ligands was applied for 45 min. For
experiments examining KIR2DL2-mediated inhibition, ULBP3 was used at
0.5 µg/ml, ICAM was used at 1 µg/ml, and HLA-Cw3 was used at 2 µg/ml.
For experiments characterizing DAP10 clustering in response to activat-
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added at 2 µg/ml. For phosphotyrosine-staining experiments, ULBP3 was
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one or more of these constituents was left out, a nonstimulatory biotinylated
mouse MHC molecule (either I-Ek or H2-Db) was added to keep the total
protein concentration constant. After protein loading, bilayers were stored
at room temperature for ≤4 h before use.
Before imaging, cells were transferred into RPMI 1640 supplemented with
5% FCS and lacking phenol red. To inhibit myosin II and SHP-1/2, 50-µM
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and cellular retraction in photostimulated cells. Fig. S3 shows that Mn2+
impairs KIR2DL2-induced retraction. Fig. S4 shows that the formation of
DAP10 microclusters requires stimulation of NKG2D. Fig. S5 schematizes
the strategy used to cross-link activating and inhibitory receptors for flow
cytometry–based Ca2+ experiments. Videos 1 and 2 show the spreading
behavior of KIR2DL2-expressing NKL cells on activating and inhibitory bi-
layers, respectively. Videos 3 and 4 show NKL cells expressing KIR2DL2-GFP
and KIR2DL2(mut)-GFP, respectively, responding to KIR2DL2 photostimula-
tion. Video 5 illustrates the actin remodeling that takes place in response
to KIR2DL2 photostimulation. Video 6 shows the formation of DAP10 micro-
clusters in response to NKG2D stimulation. Video 7 demonstrates how
photostimulation of KIR2DL2 suppresses the formation of new DAP10 micro-
clusters in the periphery of the contact. Online supplemental material is avail-
able at http://www.jcb.org/cgi/content/full/jcb.201009135/DC1.
We thank D. Tan and members of his laboratory for help with synthetic proto-
cols, reagents, and equipment; G. Altan-Bonnet for assistance with Matlab;
L. Lanier, P. Parham, and R. Wedlich-Soldner for constructs; B. Dupont, A. Hall,
F. Giancotti, and members of their laboratories for advice; B. Driscoll for techni-
cal assistance; S.S. Yi and the Memorial Sloan-Kettering Cancer Center Micro-
chemistry Core Facility for peptide synthesis; H. Hang, K. Pham, J. Sun,
T. Muir, and A. Hall for critical reading of the manuscript; and members of the
M. Huse and M.O. Li laboratories for advice and encouragement.
This study was supported by a T32 postdoctoral training grant from the
National Institutes of Health (T.P. Abeyweera), the Spanish Ministry of Science
and Innovation (E. Merino), the Searle Scholars Program (M. Huse), and the
Cancer Research Institute (M. Huse).
Submitted: 28 September 2010
Accepted: 25 January 2011
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