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: email@example.com
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
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
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.
689 Inhibitory signaling triggers NK cell retraction • Abeyweera et al.
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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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