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*
Immunology Program, Sloan-Kettering Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, NY 10021;
Department of Pathology, University of Chicago, Chicago, IL 60637; and
Skirball Institute of Biomolecular Medicine, New York University Medical Center, New York, NY 10016
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-12
during infection by viruses and other intracellular pathogens. They
represent an interface between innate and adaptive immunity by their
rapid production of cytokines, such as IFN-
and TNF-. NK cells
are also cytotoxic to certain tumor cells and virally infected cells
(1). The cytolytic activity is tightly regulated by
inhibitory receptors with ligand specificity for MHC class I Ags
(2, 3, 4). The MHC class I-dependent regulation of NK cell
cytotoxicity was first conceptualized by Ljunggren and Karre
(5) as the missing self hypothesis, which states that
target cell loss of self MHC class I will remove the inhibitory signals
for NK cytotoxicity. The dominating NK receptors with ligand
specificity for murine H-2 class I MHC Ags are the C-type lectin
receptors of the Ly49 family (3, 6), while the
corresponding human NK receptors belong to the Ig superfamily of
molecules and are named killer cell Ig-like receptors
(KIR)3
(2, 3, 4). Other NK receptors have ligand specificity for MHC
class Ib molecules: the Qa1b in the mouse and HLA-E in man (7, 8). These NK receptors are heterodimers composed of the C-type
lectin molecules NKG2A and CD94. Other NK receptors, such as Ig-like
transcripts have been identified, and some of these also have ligand
specificity for MHC class I Ags (3, 4, 9). NK receptors
for MHC class I Ags exist as pairs of inhibiting and activating
receptors with highly homologous extracellular ligand binding domains.
It has been shown that the inhibitory KIR and CD94/NKG2 receptors have
significantly higher ligand affinity than the corresponding activating
receptors (10, 11). Interactions between NK cells and
autologous target cells of hemopoietic origin are dominated by the
inhibitory signals preventing autocytotoxicity (2, 3, 4).
Inhibitory receptors contain in their cytoplasmic domain one or more
immunoreceptor tyrosine-based inhibitory motifs, which, upon tyrosine
phosphorylation, recruit and activate cytoplasmic tyrosine
phosphatases. NK receptors predominantly recruit the Src homology
(SH)2-containing protein tyrosine phosphatase (PTP)-1 (SHP-1)
(12, 13, 14, 15). Biochemical analyses of signal transduction
pathways mediated by inhibitory NK receptors have demonstrated a
central role for SHP-1 in dephosphorylation of activating receptors
such as 2B4 (16) and inactivation of downstream targets
mediating the cytolytic granule exocytosis pathways (12, 13, 14, 15, 17, 18, 19). Unidirectional killing by cytolytic effector lymphocytes is known to involve accumulation of actin, talin, and cytolytic granules at the cell-cell contact site as well as reorientation of microtubule-organizing center (MTOC) (20, 21, 22, 23, 24). In recent studies Ras-independent mitogen-activated protein kinase signals and phosphoinositide-3 kinase were shown to critically control lytic function and perforin/granzyme B polarization in NK-92 cells during tumor cell lysis (25, 26).
Direct analysis of signaling events in single NK cell-target cell conjugates regulated by MHC class I is limited to date. It has been shown that lipid rafts, enriched in signaling molecules, become polarized to the cell-cell contact area in NK cell conjugates with sensitive tumor cells, and this redistribution requires activation of Src and Syk kinases (27). It has also been shown that translocation of the MTOC is associated with activation of the tyrosine kinase, PYK-2 (28). In two other recent reports the inhibitory NK receptors and their corresponding MHC class I ligands were shown to colocalize in the contact area between NK cells and target cells (29, 30).
More information has been obtained from analysis of TCR-mediated
signaling events in single Th cell conjugates interacting with
peptide-MHC specific APCs (31, 32). It has been
demonstrated that a highly structured intercellular interface, termed
the supramolecular activation cluster (SMAC) (33, 34) or
immunological synapse (35, 36) is formed, in which the
adhesion molecule LFA-1 and the actin binding protein talin accumulate
in the peripheral SMAC (pSMAC), while the TCR and the signaling
molecule protein kinase C-
(PKC-) were shown to cluster in the
central SMAC (cSMAC) (33, 34). This structure is thought
to be critical for optimizing the interactions between signaling
molecules and their substrates. Although the distribution of only a
small number of molecules has been reported to date, it is highly
characteristic that LFA-1 and talin form a ring, which encloses TCR/MHC
complexes and PKC-. The SMAC forms only under conditions where T
cells receive a productive signal. When APCs lack peptide or when
antagonistic peptide is used, no molecular segregation has been
observed (34, 35, 36, 37, 38). Recently, in Jurkat T cell-APC
conjugates, PKC- was shown to translocate to the membrane lipid
rafts in the immune synapse (39). Furthermore, two recent
studies in T cell-B cell conjugates and T cell-dendritic cell
conjugates have shown an active role of the target cell in formation of
the T cell immune synapse (40, 41).
In the present study we have performed an analysis of the NK cell immune synapse (NKIS) in cytolytic and noncytolytic interactions with target cells. Two different in vitro systems have been investigated. In the first system polyclonal human NK cells were used as effector cells, and an autologous EBV-transformed B lymphoblastoid cell line (BLCL) was considered the nonsusceptible target and compared with the MHC class I-deficient B cell line 721.221, which is susceptible to NK cell-mediated cytotoxicity. In the second system NK cell-target cell conjugates were analyzed using KIR2DL3-positive NK clones that in the in vitro cytotoxicity assay were noncytolytic against 721.221 transfected with HLA-Cw*0304 and were cytolytic against untransfected 721.221 targets. In the first model system all the inhibitory interactions between NK cells and MHC class I ligands are assessed simultaneously in conjugates with autologous BLCL, while none of these interactions can occur with the 721.221 target. In the second system the target cells differ for only a single HLA class I allele, which is the ligand for the inhibitory KIR molecule, KIR2DL3, present on the NK clone. In both systems the inhibition of NK cytotoxicity is lifted in the presence of anti-HLA class I mAb (42, 43, 44).
A detailed immunofluorescence analysis of the spatial recruitment of
early signal transduction molecules, cytoskeletal elements, and
secretory organelles in conjugates formed between NK cells and target
cells was assessed. Selection of Abs for the characterization of NKIS
was based on information gained from biochemical analysis of signal
transduction in NK cells (12, 13, 14, 15, 16, 17, 18, 19, 27, 45, 46, 47) and previous
studies of the immune synapses formed in Ag-specific Th cell
interactions with APC (33, 36, 37, 38). We anticipated that
the cytolytic NKIS would have features in common with the Ag-specific T
cell immune synapse. Therefore, Abs detecting Src kinases, Syk kinases,
adaptor molecules, PKC-, and Itk were included in this analysis and
expected to be present in the cytolytic cSMAC. In contrast, activation
and recruitment of the cytoplasmic tyrosine phosphatase SHP-1 were
expected to dominate the cSMAC in the noncytolytic NKIS.
Redistribution of LFA-1 and talin into the pSMAC of the Ag-specific T
cell immune synapse has been observed (33, 34, 35, 36, 37, 38); therefore,
Abs to these molecules were also included in the present study. Our
analysis establishes that two distinctly different reorganizations
occur in cytolytic and noncytolytic NKIS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Polyclonal NK cells (i.e., NK cell lines) were generated and maintained as previously described (48). Briefly, FACS-sorted NK cells (CD56+, CD3-) were cocultured with irradiated allogeneic PBMC and EBV-transformed allogeneic BLCL (JY) and activated with 300 IU/ml IL-2 (provided by the National Cancer Institute/Biological Response Modifiers Program, Frederick, MD). NK clones were generated after FACS sorting for CD56+CD3- subpopulation of lymphocytes from freshly isolated PBMC of a donor homozygous for HLA-Cw*0304. The postsort purity of NK cells was confirmed. NK cells were then plated in limiting dilution at 0.3 cells/well in 40-µl tissue culture plates (Robbins Scientific, Sunnyvale, CA). NK cell clones were cultured in IMDM (Life Technologies, Gaithersburg, MD) with 10% FCS containing heat-inactivated human AB serum (Pel-Freez Biologicals, Rogers, AR) and 300 IU/ml IL-2. The cells were cultured at 37°C in 7.5% CO2. At the start of the culture and weekly thereafter until expanded into 48-well culture plates, the clones were cocultured with 1 x 106/ml irradiated allogeneic PBMC and 1 x 105/ml irradiated EBV-BLCL (JY). Coculturing with PBMC and BLCL-JY was discontinued once cultures were expanded into 24-well plates. All clones were then allowed to grow in 24-well plates until an adequate number of cells was reached, usually within 5–7 days. Clones were then characterized for receptor phenotype (DX9, GL183, EB6, and CD94) and used for 51Cr release cytotoxicity and conjugation assays. NK clones selected for further studies were CD3-CD56+, and GL183+ and mediated NK cytolytic function against 721.221 (class I-negative EBV-BLCL), but protected autologous BLCL and .221-Cw*0304 cells (self-allele transfectant). The HLA class I-negative cell line, 721.221, was used as an NK-sensitive target, while autologous BLCL and .221-Cw*0304 cells were used as an NK-protected target as previously described (42). The .221-Cw*0304 cell line was a gift from Dr. P. Parham (Stanford University, Stanford, CA). For conjugation assays, the target cells were preincubated with CellTracker Blue CMAC (Molecular Probes, Eugene, OR) for 30 min at 37°C and washed in serum-free medium before use. This allowed easy identification of target cell in an NK-target conjugate. In three cell conjugates only autologous BLCLs were prelabeled blue, and 721.221 cells were unlabeled. The 721.221 cells were identified by their large size and lack of LFA-1 staining, whereas NK cells stained for CD11a.
Antibodies
Primary.
NK cells were phenotyped with anti-KIR3DL1 (DX9),
anti-KIR2DL3/2DS2/2DS3 (GL183), anti-KIR2DL1/2DS1 (EB6), and
anti-human CD94 mAbs, which were purchased from Immunotech
(Marseilles, France). Anti-CD56 and anti-CD3 were purchased from BD
Biosciences (San Jose, CA). Mouse monoclonal anti-human talin and
-tubulin (identifies tubules and MTOC) were purchased from Chemicon
International (Temecula, CA) and Amersham Pharmacia Biotech
(Piscataway, NJ), respectively. Goat polyclonal anti-human Itk,
talin, PKC-, ZAP-70, mouse monoclonal anti-human phospholipase
C-1 (PLC-1), rabbit
polyclonal anti-human SHP-1, Fyn, Lck, and SYK were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA).
Goat anti-B cell linker protein (anti-BLNK) affinity-purified antiserum raised against the C-terminal 19 aa of human BLNK and a mouse mAb (IgG2a) detecting BLNK were both used in the experiments (Santa Cruz Biotechnology). The mouse anti-BLNK Ab identifies the epitope corresponding to aa 4–205 mapping at the N terminus of BLNK of human origin. A sheep anti-human SH2 domain-containing leukocyte protein of 76 kDa (SLP-76) was a gift from Dr. G. Koretzky (University of Pennsylvania, Philadelphia, PA). Mouse anti-human LAMP-1 (H4A3; identifies lysosomes) was obtained from the Developmental Studies Hybridoma Bank, Department of Biological Sciences, University of Iowa (Ames, IA). Mouse anti-CD11a was obtained from the Monoclonal Antibody Core Facility, Sloan-Kettering Institute (New York, NY). Anti-HLA class I mAb, DX17 (IgG1) was a gift from Dr. L. Lanier (University of California, San Francisco, CA) and Dr. J. Phillips (DNAX Research Institute, Palo Alto, CA). Isotype control IgG1 Ab was purchased from BD Biosciences (Mountain View, CA). Cells were labeled with a maximum of four primary Ab combinations of mouse, rabbit, goat, and sheep.
Secondary. Affinity-purified second Abs and species-absorbed conjugates (FITC, Cy3, Cy5 (near-infrared emission), aminomethyl coumarinacetic acid (UV)) for multiple labeling were purchased from Chemicon International. CellTracker Blue CMAC and Orange CMTMR were purchased from Molecular Probes.
Western blot analysis
The presence of BLNK was assessed in NK, T, and B cells. NK cells, Jurkat T cells, and EBV-transformed B cells were washed and lysed with the lysis buffer (1% Triton X-100, 0.1% SDS, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, and 1 µg/ml leupeptin). Lysates were run on 10% SDS-PAGE. Proteins were transferred to nitrocellulose membrane (Bio-Rad, Richmond, CA), and the blots were blocked with 5% milk. Membranes probed with a 1/200 dilution of mouse anti-human BLNK mAb (Santa Cruz Biotechnology) were then washed and incubated with a 1/10,000 dilution of goat anti-mouse HRP secondary Ab (Bio-Rad). Following multiple washes the membranes were developed with ECL (NEN Life Sciences, Boston, MA). Cell lysates stained only with goat anti-mouse HRP secondary Ab were used as controls.
Flow cytometry
Analysis of NK cell conjugates using polyclonal NK cell lines and NK clones was performed on FACScan (BD Biosciences) as previously described (49). Target cells were labeled with CellTracker Orange CMTMR, and NK cells with FITC-labeled anti-CD56. Polyclonal NK cells were used to form conjugates with .221 and autologous BLCL, and NK clones were used to form conjugates with .221 and .221-Cw*0304. Aliquots of effector-target cell suspensions were removed after incubation at 37°C for 1, 3, 5, 10, 15, and 30 min and were placed on ice for immediate FACS analysis. Effectors and targets mixed in the presence of EDTA, which prevents conjugation, served as a control sample. The percentage of conjugated effector cells was determined after dividing the number of dual-labeled particles by the total number of effector lymphocytes and multiplying the result by 100 (49).
Cytotoxicity assay
Cell-killing assays were performed using polyclonal NK cells and NK clones as effectors and 51Cr-labeled autologous BLCL, 721.221, and .221-Cw*0304 as targets. Assays were performed in triplicate for 4 h at an E:T ratio of 7:1. The percent specific lysis was calculated as previously described (48). Autologous BLCL and .221-Cw*0304 targets were tested in the presence (10 µg/ml) or the absence of anti-HLA class I mAb, DX17 and its isotype control Ab IgG1. DX17 is known not to induce ADCC (42, 43, 44). For cold target cell inhibition, either unlabeled autologous BLCL or 721.221 were added at increasing E:T cell ratios to 51Cr-labeled 721.221.
Conjugation assay
NK cells and targets were prepared for conjugation after washing in serum-free medium and adjusting the concentration to 1 x 106/ml. Effector and target cells were mixed at an E:T cell ratio of 3:2 and spun at 500 rpm for 5 min at room temperature. For triple-cell conjugates equal numbers of autologous BLCL and .221 were mixed with NK cells. Before mixing, autologous BLCL cells were prelabeled with blue cell tracker dye while keeping .221 cells unlabeled, allowing easy identification of the two targets in the triple-cell conjugate. After incubating at 37°C for 5 min, cells were gently resuspended and transferred to poly-L-lysine-coated slides (Labscientific, Livingston, NJ), briefly cytocentrifuged, and fixed in 3% paraformaldehyde. After permeabilizing both with Triton X-100 (0.2% in PBS) for 1 min and 0.01% saponin/0.25% gelatin/0.02% NaN3 (in PBS) for 20 min, cells were incubated with primary Abs. For visualizing cell surface molecules such as LFA-1, fixed cells were labeled before membrane permeabilization. After multiple washes with serum-free medium, cells were incubated with secondary Abs. Cells mounted with ProLong antifade kit (Molecular Probes) were then analyzed with fluorescent microscopy.
Fluorescent microscopy
In all experiments an Intelligent Imaging Innovations imaging system (Intelligent Imaging Innovations, Denver, CO) with a Zeiss Axioplan 2 microscope and AttoArc mercury light source (Zeiss, New York, NY), which includes motorized 0.1-µm linear encoders on x- and y-axes, harmonic drive z-focusing, Nomarski optics, motorized filter turret, a Sensicam SVGA high performance camera to record fluorescence, and differential interference contrast images, was used. Based on the Nomarski imaging, cells that were clearly conjugated were examined for their fluorescence and selected for scoring analysis using SlideBook analysis software (Intelligent Imaging Innovations). Images were obtained in both two-dimensional (2D) (x-y-axis) and in three-dimensional (3D) (x-z-axis) (34). Sixty to 70 serial optical sections of 0.1-µm thickness were acquired for each label. The digital recorded data were then deconvolved using Nearest Neighbor Deconvolution, giving the fluorescent image representative of the label in the entire cell and not just in a single plane. The contact areas (synapses) were acquired using both the mask function and the three-view function of the SlideBook software, which displays all three (xy, xz, yz) orthogonal planes.
Analysis of immune synapses
Based on the Nomarski images, cells that were clearly conjugated were selected for fluorescent analysis. Fifty to 70 conjugates were randomly selected for each label and target combination from two to four independent experiments. It is specifically stated in Results when <50 conjugates were analyzed. Two- and three-dimensional images of all such conjugates were acquired and analyzed. The scoring of the synaptic regions was made following acquisition, deconvolution, and rendering of 60–70 z-stack images. Such projections in the z-axis of the contact areas yielded three distinct patterns: 1) formation of ordered SMAC structures with peripheral and central clusters of molecules, 2) absence of the ordered SMAC structures resulting in homogenous distribution of the molecules, and 3) uninterpretable SMACs. SMAC was considered uninterpretable when the x-y-axis of the deconvolved fluorescent image was not congruous with the plane of the three-view function, thus allowing only partial visualization of the projections in the z-axis. Uninterpretable SMACs were observed in 14% (range, 8–20%) of the randomly selected conjugates.
Analysis of polarization event
Localization of MTOC, talin, and lysosomes in the NK-target cell conjugates was evaluated to determine the presence or absence of polarization of these organelles toward the contact site. NK cell was divided into three zones: 1) proximal one-third, as an area in the cell closest to the cell-cell contact with the target; 2) distal one-third, as an area most distant from the cell-cell contact; and 3) middle one-third, as an area in between the two zones. MTOC and lysosomes were considered polarized to the cell-cell contact when they were located in the proximal one-third of the NK cell. Any other observations, i.e., localization in the middle or distal one-third of the cell from the contact area, were considered nonpolarized events. Talin was considered polarized when it was seen to cluster at the cell-cell contact, as determined by the fluorescent intensity of the molecule being highest in the cell-cell contact area compared with the rest of the cell. Clustering of talin in any other site except the contact zone and equal distribution of talin around the cell were both considered nonpolarized events. Scoring analysis of the polarization events was performed after conducting three independent experiments with each target using either polyclonal NK cells or NK clones in conjugates with .221, .221-Cw*0304, and autologous BLCL target cells. Fifty conjugates were scored in each of the three experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two experimental models were applied to analyze differences in the
immune synapses during the cytolytic and noncytolytic interactions of
human NK cells. In the first model polyclonal NK cells (i.e., NK cell
lines) generated from healthy donors were used as effectors. The
targets in this assay included the human major histocompatibility Ag
(HLA) class I-negative cell line 721.221 (.221) as a susceptible target
and in vitro established autologous EBV-transformed BLCL as a
nonsusceptible target. As shown in Fig. 1
A, NK cells do not mediate
cytotoxicity against the autologous BLCL, while the HLA class
I-deficient target .221 is susceptible to NK cell-mediated lysis. When
BLCL target is tested in the presence of the anti-HLA class I mAb
DX17, which does not bind to FcR (42, 43, 44), the protection
against NK cytotoxicity is removed. However, this inhibition is not
lifted in the presence of the isotype control Ab IgG1 (Fig. 1A, left panel) or anti-CD56 (data not
shown). Therefore, the protection of autologous target is MHC class
I-regulated and mediated by the inhibitory signals. Lack of
cytotoxicity against autologous BLCL target cells is not due to lack of
NK cell-target cell conjugate formation. Conjugates are formed both
with the autologous BLCL as well as with 721.221. However, the number
of conjugates formed with autologous BLCL at different time points is
smaller than the number of conjugates formed with the susceptible
target .221 (Fig. 1A, right panel). The
differences in the number of conjugates formed with the two cell lines
are not due to differences in expression of intercellular adhesion
molecules ICAM-1 and -2 (data not shown). However, the
origins of autologous BLCL and 721.221 target cells are different, and
this could potentially account for the differences observed in the
number of conjugates formed. The receptor-ligand interactions
responsible for the lack of cytotoxicity against autologous BLCL are
probably multiple, as are the signals that induce cytotoxicity against
.221. Therefore, this model is used to identify the general differences
in NKIS between cytolytic and noncytolytic conjugates.
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B, left panel) or
anti-CD56 (data not shown). Protection of .221-Cw*0304 was also
lifted with mAb GL 183 (data not shown), consistent with previous
studies (42). Therefore, the ligand-receptor pair of
HLA-Cw*0304 and KIR2DL3 that controls the inhibition of NK cytotoxicity
is directly tested in this model system. The number of conjugates
formed with the nonsusceptible target, .221-Cw*0304, was fewer than the
number obtained with the susceptible target, .221 (Fig. 1B,
right panel), which is similar to the findings made in the
first model (Fig. 1A, right panel). A higher
number of conjugates was formed at each time point using NK clones
compared with polyclonal NK cells (Fig. 1, right
panels). Cytoskeletal remodeling and polarization events in the cytolytic and noncytolytic conjugates
Conjugates with the susceptible and nonsusceptible target cells
using polyclonal NK cell lines and NK clones were analyzed for
localization of MTOC and lysosomes (cytolytic granules) (22, 23) as well as the clustering of actin and talin. The data in
Fig. 2 are representative of 150
conjugates analyzed for each label and target combination. Conjugates
formed using the polyclonal NK cell line that is cytotoxic for .221 and
noncytotoxic for autologous BLCL were initially analyzed. Polarization
toward .221 cells was observed for the MTOC in 80% (n
= 78–82), lysosomes in 78% (n = 76–80), and
talin in 75% (n = 70–78) of the conjugates (Fig. 2, second row). The results for redistribution of actin and
talin were similar (data not shown). In contrast, <25%
(n = 18–30) of NK cell conjugates showed polarization
of any of these elements toward autologous BLCL targets (Fig. 2, first row). In the second dataset conjugates formed using
GL183+ NK clones that are cytotoxic to .221 and
noncytotoxic to .221-Cw*0304 were analyzed. Here, polarization toward
.221 cell was observed for the MTOC in 84% (n =
82–86), lysosomes in 80% (n = 78–82), and talin in
78% (n = 75–80) of the conjugates (Fig. 2, third row). In contrast, <20% (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) of NK
cell conjugates showed polarization of any of these elements toward
.221-Cw*0304 target cells (Fig. 2, fourth row). Therefore,
conjugates with autologous BLCL and .221-Cw*0304 expressing a single
self HLA-Cw allele gave similar results in cortical cytoskeletal
remodeling and polarization events in both polyclonal NK cells and NK
clones. These results demonstrate that inhibitory signaling does not
require large scale remodeling of the cortical cytoskeleton, nor does
it involve changes in cytoplasmic structures normally associated with
activation of the cytolytic machinery.
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To address whether the signaling molecules do or do not passively
move along with the MTOC in the conjugates, the IL-2-activated NK cells
were dual-labeled with Abs against a variety of signal transduction
molecules and -tubulin. Dual-labeling with closely related molecules
such as the two Syk kinases, SYK and ZAP-70, and two adaptor molecules,
SLP-76 and BLNK, was also evaluated to study the possible redundancy of
such molecules in NK signaling. Digital immunofluorescence microscopy
was used to acquire fluorescent images keeping MTOC and the labeled
molecule in the same focal plane. The data in Fig. 3 are representative of 20 NK cells
analyzed for each Ab combination. The distribution of signaling
molecules in relation to MTOC was different for each molecule. The
distribution of SHP-1 and SLP-76 was distinct from the location of MTOC
(in green, Fig. 3, rows 3 and 4). Lck, ZAP-70,
BLNK, Itk, and PKC- have some portion of their molecules that
colocalize with MTOC (in yellow, Fig. 3, panels 1,
2, 5, 6, and 7, overlay).
These results indicate that at least a subset of some of the signal
transduction molecules colocalizes with MTOC. Therefore, they may be
translocated with MTOC to the contact site with susceptible targets
similar to recent findings observed for the tyrosine kinase PYK 2
(28). It is also possible that the molecules colocalized
with MTOC exist as preformed complexes in IL-2-activated NK cells.
Furthermore, NK cells have closely related signaling molecules that
otherwise are predominantly expressed in either B cells (e.g., BLNK and
SYK) or T cells (e.g., SLP-76 and ZAP-70). The presence of BLNK was
confirmed by Western blot analysis, in which an appropriate size band
(68 kDa) was detected in human NK cells and EBV-transformed B cells,
but not in Jurkat T cells (Fig. 4).
Cellular localization of the signaling molecules was also determined.
In activated NK cells the distributions of Lck, ZAP-70, and SYK were
similar, having both cytoplasmic and perimembraneous localizations (in
red, Fig. 3, rows 1, 2, and
8). SHP-1 and SLP-76 have predominantly perimembraneous
localization (in red, Fig. 3, rows 3 and 4),
whereas BLNK, Itk, and PKC- have predominantly cytoplasmic
localization (in red, Fig. 3, rows 5–7). Although, SYK and
ZAP-70 have mostly overlapping distribution (in yellow, Fig. 3, row 8, overlay), there are areas where the two molecules are
not colocalized.
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NKIS visualized as projections in the z-axis were
analyzed using the 3D digital immunofluorescence microscopy. The word
synapse is used to describe structures visualized in 3D
(x-z-axis) and not in 2D
(x-y-axis) fluorescent images (34).
In the figure the fluorescent distributions of the signaling molecules
are shown to be discrete and clumped, as the panels represent only the
most intense signals within the NK cell obtained following
deconvolution of 60–70 images. In one set of experiments analysis of
the immune synapses was made in conjugates between polyclonal NK cells
and either .221 or autologous BLCL (Fig. 5, a–c), and in the other set
of experiments conjugates between NK clones and either .221 or
.221-Cw*0304 (self allele) were analyzed (Fig. 5, d–f). The
results of synaptic molecular redistribution obtained in both
experimental models were similar. Data in the figure represent 75–80%
of 50–70 conjugates analyzed for each Ab and target combination
obtained in at least two independent experiments (see Materials
and Methods). Recruitment to the immune synapse of the signaling
molecules, PKC-, PLC-1, Itk, and SLP-76,
which are expected to be involved in the granule exocytosis activation
pathways, was evaluated. BLNK was selected to determine the involvement
of other adaptor molecules in NK signaling (50, 51). LFA-1
and components of the cortical cytoskeleton, actin and talin, are known
to mediate essential functions during T cell interactions with target
cells and APCs and to accumulate at the contact sites
(35). Here we demonstrate that talin is clustered at the
contact site with the susceptible target (i.e., .221) as seen in 2D
fluorescent images (Fig. 5, panels 2 and 4,
b and e) and is also redistributed in the
cytolytic synapses (3D images) of both NK cell lines (Fig. 5, panels 2 and 4c) and NK clones (Fig. 5, panels 2 and 4f). LFA-1 did not cluster at the
cell-cell contact of the cytolytic conjugates when analyzed as 2D
images (Fig. 5, panels 6, 8, and 10,
b and e); however, there was a distinct
redistribution of LFA-1 in the cytolytic synapses (3D images), similar
to that observed for talin (Fig. 5, panels 6, 8,
and 10, c and f). Both LFA-1 and talin
are localized in the periphery of the synapse (pSMAC), while PKC-
(Fig. 5, panel 2, c and f),
PLC-1 (Fig. 5, panel 4,
c and f), Itk (Fig. 5, panel 6,
c and f), SLP-76 (Fig. 5, panel 8,
c and f) and BLNK (Fig. 5, panel 10,
c and f) are localized in the center (cSMAC). In
contrast, redistribution in the noncytolytic immune synapse of these
molecules was not observed with either autologous BLCL (Fig. 5, panels 1, 3, 5, 7, and
9c) or .221-Cw*0304 (Fig. 5, panels 1,
3, 5, 7, and 9f). This
demonstrates that cytolytic effector function is associated with
dramatic changes in the plasma membrane at the contact sites between NK
cells and susceptible targets and can be visualized in 3D images of the
projections in the x-z-axis.
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The early recruitment of PTKs, such as the Src kinases Lck and
Fyn, and the Syk kinases SYK and ZAP-70, was visualized in cytolytic
and noncytolytic NK cell conjugates formed between polyclonal NK cells
and either .221 or autologous BLCL and analyzed after 10 min of NK
cell-target cell mixing. The data in Fig. 6 represent 75–80% of 50–70 conjugates
analyzed collectively from two to four independent experiments for each
Ab and target combination. Analysis of the immune synapses in cytolytic
interactions with .221 showed all four PTKs as well as BLNK to be
clustered in the center of the synapse (cSMAC) surrounded by LFA-1,
which is localized in the pSMAC (Fig. 6, panels 1–3C). It
was also demonstrated by independent visualization of signaling
molecules obtained after removing either red or blue colors that there
was some compartmentalization within the cytolytic cSMAC.
Further studies including fluorescence resonance energy transfer will
be needed to address issues regarding colocalization and coassociation
of molecules in the immune synapses. Recruitment to the synapse of the
PTKs or adaptor proteins is not identified in NK cell conjugates with
autologous cells or with .221-Cw*0304 (data not shown).
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Biochemical analysis of signal transduction events mediated by
inhibitory NK receptors for MHC class I Ags have clearly demonstrated
that SHP-1 is recruited to these receptors and activated by a tyrosine
phosphorylation-dependent mechanism (12, 13, 14, 15, 52).
Therefore, localization of SHP-1 in cytolytic and noncytolytic
conjugates was determined. Experiments were performed using both model
systems. In one experimental system .221 and autologous BLCL were used
as targets for polyclonal NK cells, and in the other .221 and
.221-Cw*0304 were used as targets for GL183+NK
clones. Fifty conjugates were collectively analyzed from two to four
independent experiments for each Ab and target combination. The results
obtained from the analysis of cytolytic conjugates with either .221
targets and NK cell lines or .221 targets and NK clones are
representative of 85–90% of the conjugates (Fig. 7A, top row). It is
demonstrated that SHP-1 and ZAP-70 are recruited to the cell-cell
contact site with .221 (Fig. 7A, b and
e, top row) and are clustered in the cytolytic
cSMAC (Fig. 7A, c and f, top
row). This is further demonstrated in a detailed four-color
analysis of NK cell conjugates, where SHP-1 is recruited to the contact
area with .221 target cells in the cytolytic conjugate (Fig. 7B, a–e). Here, SHP-1 is seen to occupy a
central position along with ZAP-70 and SLP-76 at the cell-cell contact
site (Fig. 7B, a–e).
|
A,
b and e, bottom row, and Fig. 7Bg) and is clustered in the cSMAC, while LFA-1
is redistributed in the pSMAC of the noncytolytic NKIS (Fig. 7A, c and f, bottom row,
and Fig. 7Bg, inset). Although only
32–40% of the noncytolytic conjugates demonstrated cSMAC with SHP-1,
the other conjugates did not demonstrate any redistribution of LFA-1 in
the synapse when analyzed at 10 min of conjugate formation. In contrast
to cytolytic conjugates formed with HLA class I-deficient target cells,
SHP-1 was not associated with ZAP-70 or SLP-76 in the noncytolytic
conjugates formed with either autologous BLCL or .221-Cw*0304, as
demonstrated by absence of any other molecules in the cSMAC after
removing the red color of SHP-1 (Fig. 7A, c and
f, bottom row). This is also illustrated in the
four-color immunofluorescence image of a noncytolytic conjugate, where
SLP-76 and ZAP-70 are located in the distal one-third of the NK cell
(Fig. 7B, f–h). This analysis demonstrates that
noncytolytic NK cell interactions with autologous target cells involve
the generation of an inhibitory immune synapse, which is dominated by
SHP-1, while downstream signaling molecules are excluded from the
contact region. Our results also implicate a role for SHP-1 in
cytolytic NK interactions. Unidirectional interactions in triple-cell conjugates
The ability of NK cells to identify a susceptible target in the
presence of a nonsusceptible target and deliver a unidirectional
cytolytic hit was examined in both the cold target inhibition assay and
the analyses of the three-cell conjugates. The specificity for .221
cytotoxicity is demonstrated by cold target cell inhibition, where a
100-fold excess of autologous BLCL is needed before any reduction in
cytotoxicity occurs, suggesting that NK cells, even in the presence of
an overwhelming number of autologous target cells, identify and lyse
susceptible target cells. Furthermore, NK cells also lyse susceptible
target cells (i.e.,721.221) when preincubated with autologous target
cells (i.e., BLCL) for 1, 2, and 3 h (data not shown). In
contrast, only a 10-fold increase in .221 cold target cells is
sufficient for inhibition of 51Cr-labeled 721.221
cells (Fig. 8A), probably due
to competition between 51Cr-labeled and unlabeled
721.221 target cells.
|
B, lower cell in differential
interference contrast overlay and three-color image). Here,
SLP-76 clusters in the cSMAC of the NK cell-contacting membrane with
.221, while this was not observed in the contacting membrane with
autologous BLCL (Fig. 8B). Triple-cell conjugates formed
between a single NK cell and two susceptible target cells demonstrate
different patterns dependent upon the indicator used for assessment of
recognition and the induction of cytotoxicity. SLP-76 and PKC- were
polarized toward only one of the two targets; in contrast, talin was
polarized toward both target cells (data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and PLC1, in the immune
synapse, and for synaptic redistribution of LFA-1 and the
actin-associated molecule, talin. Two different in vitro models yielded
similar results. It is demonstrated that two distinctly different NKIS
can be defined: the inhibitory NKIS and the cytolytic NKIS. The inhibitory NKIS is formed when polyclonal IL-2-activated NK cells form conjugates with autologous BLCL or when KIR2DL3-positive NK clones interact with .221 cells expressing the cognate ligand HLA-Cw*0304. When the target cell lacks self MHC, a cytolytic NKIS is formed at the contact site with the target cell. Here, the NK-target cell contact area assembles into topologically and spatially distinct regions characterized by redistribution of LFA-1 and talin in the pSMAC, and a multimolecular signaling complex including SHP-1 is assembled in the cSMAC. In contrast, the inhibitory NKIS is characterized by redistribution of LFA-1 in the pSMAC and clustering of only SHP-1 in the cSMAC. The cytolytic NKIS has several features in common with the TCR-specific immune synapse observed for Th lymphocytes interacting with their Ag-specific APCs (34, 35, 36, 37, 38).
Visualization of the signaling molecules included in the cytolytic
cSMAC provides further insight into the spatial organization of enzymes
and adaptor molecules needed for initiation of cytolytic effector
function. Translocation of signaling molecules from the cytosol to the
NK cell plasma membrane occurs within a limited region of contact with
the target cell. The complex consists of Src kinases, Syk kinases, the
Tec kinase Itk, adaptor molecules, PKC-, and
PLC-1. These findings are consistent with
expectations derived from biochemical analysis of receptor-mediated
signaling pathways in lymphocytes (12, 13, 14, 15, 16, 17, 18, 19, 27, 45, 46, 47, 50, 51, 52, 53, 54, 55). Although the molecular basis for NKIS formation and its
function remains to be defined, recent studies have suggested that the
compartmentalization of the plasma membrane imposed by
glycolipid-enriched microdomains (GEMs) is essential for TCR-mediated
signaling (56). GEMs are enriched in molecules involved in
signal transduction, such as the PTKs Lck and Fyn and the transmembrane
adaptor linker of activation in T cells (57). In contrast,
the PTPs CD45, SHP-1, and SHP-2 are excluded from the GEMs
(58), and such restricted distribution of PTPs may play a
critical role in the initiation of signaling.
Our results on clustering of the GEM-resident PTKs in the cSMAC of the
cytolytic conjugates are consistent with a model in which aggregated
GEMs get reorganized during cell-mediated cytotoxicity
(27). We also demonstrate that SLP-76 in the cytolytic
interaction is brought to the contact area in such a way that it could
provide the scaffold where Itk (54), PKC-
(55), and PLC-1
(17, 18, 19) are brought in contact with their substrates
(Fig. 7, A–E). Therefore, clustering of this activation
signaling complex to the cytolytic NKIS could occur by recruitment to
the GEM-resident anchoring molecule, linker of activation in T cells
(51), which is tyrosine phosphorylated in cytolytically
active NK cells (19). Given that the majority of SHP-1 in
T cell membranes is present outside the lipid rafts (58),
it was surprising to find SHP-1 in the cytolytic NKIS as a component of
the multimolecular signaling complex in the cSMAC. However, it has been
shown in Jurkat T cell lines that the predominantly cytosolic SHP-1
becomes enriched in membranes and GEMs in association with activated
Lck (58). Therefore, it is possible that the activated Lck
recruited to cytolytic NKIS will also recruit SHP-1 to
GEMs. Our analysis cannot determine whether SHP-1 is present in the
complex as an active enzyme. In this case SHP-1 could provide a
regulatory function by guiding tyrosine phosphorylation-dependent
pathways toward granule exocytosis and deactivation of signals that
could mediate apoptosis or cell proliferation via regulation of
mitogen-activated protein kinases (25, 26, 59). Direct
support for involvement of enzymatically active SHP-1 in cytolytic NK
cell effector function with MHC class I negative targets has recently
been provided by studies in mice transgenic for dominant-negative
SHP-1. Such mice display decreased NK cytotoxicity against MHC class
I-negative targets (60).
Visualization of NK cell interactions with target cells expressing self
HLA class I ligands for inhibitory NK receptors clearly demonstrates
that the cytolytic effector mechanisms are not being activated. MTOC,
lysosomes, and talin are not polarized to the contact site with the
target (Fig. 2), and the cascade of signaling events is interrupted
early in such interactions (this study and Ref. 16). This
deactivation of NK cell effector functions is mediated by the
interactions of inhibitory NK receptors for MHC class I, as evidenced
by the occurrence of cytotoxicity in the presence of anti-HLA class
I mAb (Fig. 1, A and B, left panels).
It has recently been shown that inhibitory KIR or CD94/NKG2 receptors
interacting with cognate MHC class I ligand interrupt tyrosine
phosphorylation of the activating NK receptor 2B4 (16).
The inhibitory NKIS has a well-defined pSMAC containing LFA-1, and a
cSMAC containing only SHP-1. Recruitment and activation of SHP-1 are
tyrosine phosphorylation dependent (12, 13, 14, 15, 52). The
initial NK cell activation will induce Src kinase-mediated tyrosine
phosphorylation (16), and activation of SHP-1 will then
occur when the inhibitory NK receptor is recruited into the synapse as
a consequence of cognate MHC-ligand interactions. The
perimembraneous components of Lck and SHP-1 would favor rapid
initiation of the inhibitory signal transduction pathway. This could
then facilitate interruption of the cytolytic signaling cascade, which
also depends on Src kinase activation.
Noncytolytic NK cell conjugates were analyzed at 10 min following effector cell-target cell mixing. The majority of these conjugates display a synaptic region with uniform distribution of LFA-1 and absence of SHP-1. It is possible that these conjugates represent cell-cell interactions where the self recognition process has been completed. This conclusion is supported by recent studies in T cells, where the dephosphorylation of proteins by SHP-1 and SHP-2 was completed within 5 min of incubation of the T cell membrane with ATP and SHP-1 (58). This hypothesis is further supported by three recent studies, one demonstrating down-regulation of integrin function upon inhibitory KIR interactions with cognate HLA-C molecules (61) and the others demonstrating redistribution of inhibitory Ly49 and KIR molecules on NK cells during interactions with their ligands on target cells (29, 30). Therefore, our analyses of noncytolytic NKIS support the results obtained in the other study where the inhibitory signals initiated by KIRs block aggregation and polarization of GEMs in an SHP-1-dependent manner (27).
Our studies also demonstrate that the NK cells express signaling
molecules characteristic for both T cell and B cell lineages. The two
Syk kinases, ZAP-70 and SYK, which have close functional similarity
(62), were recruited into the cSMAC of cytolytic NKIS
(Fig. 6). The two adaptor molecules, SLP-76 and BLNK, were also
recruited into the cSMAC of cytolytic NKIS. It has recently been
suggested that these adaptor molecules have overlapping, yet unique,
additional functions (50, 51, 63). It has previously been
reported that BLNK is not present in human lymphokine-activated killer
cells (64). However, our studies with two different
anti-BLNK Abs, including Western blot analysis of purified IL-2
activated human NK cells and NK clones did indicate the presence of
this adaptor molecule in NK cells (Fig. 4). The immunofluorescence
staining in NK cells of BLNK and SLP-76 demonstrate different patterns
(Fig. 3), further supporting that the Abs do not cross-react between
these two molecules. In another recent report SLP-76 and BLNK were both
expressed in murine macrophages and were linked to signaling via Fc
receptors (65). Our observation that a fraction of Lck,
ZAP-70, BLNK, Itk, and PKC- colocalized with MTOC in IL-2-activated
NK cells (Fig. 3, rows 1, 2, 5,
6, and 7) suggest that these signaling molecules
might be translocated along with MTOC to the contact area with target
cells during initiation of the cytolytic signaling cascade. The recent
finding of PYK-2 association and cotranslocation with MTOC in cytolytic
NK effector function supports this model for temporal and spatial
regulation of cytotoxicity (28).
The ability of NK cells to distinguish self from nonself based on the
presence or absence of autologous MHC class I Ags on target cells was
clearly illustrated in triple-cell conjugates (Fig. 8). A single NK
cell interacting simultaneously with an autologous and an MHC class
I-deficient target cell displays a unidirectional cytolytic immune
synapse with only the class I-negative target cell (Fig. 8B,
compare two right side images). The ability of an NK cell to
precisely direct its cytolytic machinery toward a susceptible target
when surrounded by overwhelming numbers of self targets is also
illustrated in the cold target inhibition assay (Fig. 8A).
These findings are in agreement with previous studies in cytolytic T
cells (21) and with recent studies applying video
microscopy of murine NK cell interactions with target cells
(66). Collectively, these results support the hypothesis
that the inhibitory signals within a single NK cell are spatially and
temporally restricted and are limited to interactions with
nonsusceptible, resistant target cells. This localized inhibition does
not lead to a general inactivation of the cytolytic effector function
of the cell (Fig. 8). However, a more detailed analysis of triple-cell
conjugates between a single effector cell and two susceptible target
cells, two effector cells and a single susceptible target cell, and
other permutations is needed to dissect the directional events required
for induction of cytotoxicity and simultaneous protection of self
targets. We observed polarization of talin toward two
susceptible targets (2D analysis, x-y-axis)
interacting simultaneously with a single effector cell, but the
reorganized cytolytic NKIS with PKC- in the cSMAC was only observed
for one of the two susceptible target cells. We have not attempted to
quantitate polarization of LFA-1 and/or ICAM-1 in cytolytic or
noncytolytic conjugates. Previous studies of triple-cell conjugates
between a single T cell and two APCs have demonstrated accumulation of
ICAM-1 at the contact sites of both APCs (67).
Our studies with IL-2-activated NK cells and NK clones have been limited to a single 10 min point. Others previously reported a temporal distribution of LFA-1 in the NKIS with central accumulation of LFA-1 (29). However, this study was performed with the CD28-positive NK-like cell line, YT, where induction of the cytolytic effector function depends on signals mediated by both CD28 and LFA-1/ICAM-1 (68, 69). Sequential analysis of the NKIS with simultaneous determination of localization of NK receptors, their ligands, and signaling molecules will provide further insight into the molecular mechanisms responsible for NK cell recognition of self vs nonself.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Bo Dupont, K-406, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. E-mail: b-dupont{at}ski.mskcc.org
3 Abbreviations used in this paper: KIR, killer cell Ig-like receptor; BLCL, B lymphoblastoid cell line; BLNK, B cell linker protein; SMAC, supramolecular activation cluster; cSMAC, central SMAC; 2D, two-dimensional; 3D, three-dimensional; GEM, glycolipid-enriched microdomain; MTOC, microtubule-organizing center; NKIS, NK cell immune synapse; PLC-1, phospholipase C-1; PKC-, protein kinase C-; pSMAC, peripheral SMAC; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; SH, Src homology; SHP-1, SH2-containing PTP-1; SLP-76, SH2 domain-containing leukocyte protein of 76 kDa.
Received for publication January 4, 2001. Accepted for publication August 7, 2001.
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E. Espinosa, J. Tabiasco, D. Hudrisier, and J.-J. Fournie Synaptic Transfer by Human {gamma}{delta} T Cells Stimulated with Soluble or Cellular Antigens J. Immunol., June 15, 2002; 168(12): 6336 - 6343. [Abstract] [Full Text] [PDF]
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S.-i. Yusa, T. L. Catina, and K. S. Campbell SHP-1- and Phosphotyrosine-Independent Inhibitory Signaling by a Killer Cell Ig-Like Receptor Cytoplasmic Domain in Human NK Cells J. Immunol., May 15, 2002; 168(10): 5047 - 5057. [Abstract] [Full Text] [PDF]
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D. HUDRISIER and P. BONGRAND Intercellular transfer of antigen-presenting cell determinants onto T cells: molecular mechanisms and biological significance FASEB J, April 1, 2002; 16(6): 477 - 486. [Abstract] [Full Text] [PDF]
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Y. M. Vyas, H. Maniar, and B. Dupont Cutting Edge: Differential Segregation of the Src Homology 2-Containing Protein Tyrosine Phosphatase-1 Within the Early NK Cell Immune Synapse Distinguishes Noncytolytic from Cytolytic Interactions J. Immunol., April 1, 2002; 168(7): 3150 - 3154. [Abstract] [Full Text] [PDF]
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M. S. Fassett, D. M. Davis, M. M. Valter, G. B. Cohen, and J. L. Strominger Signaling at the inhibitory natural killer cell immune synapse regulates lipid raft polarization but not class I MHC clustering PNAS, November 20, 2001; (2001) 211563598. [Abstract] [Full Text] [PDF]
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M. S. Fassett, D. M. Davis, M. M. Valter, G. B. Cohen, and J. L. Strominger Signaling at the inhibitory natural killer cell immune synapse regulates lipid raft polarization but not class I MHC clustering PNAS, December 4, 2001; 98(25): 14547 - 14552. [Abstract] [Full Text] [PDF]
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30% of noncytolytic
conjugates.