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The Journal of Immunology, 2003, 170: 6107-6114.
Copyright © 2003 by The American Association of Immunologists

Spontaneous Clustering and Tyrosine Phosphorylation of NK Cell Inhibitory Receptor Induced by Ligand Binding

Mathias Faure1, Domingo F. Barber2, Stephenie M. Takahashi, Tian Jin and Eric O. Long3

Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20852


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inhibition of NK cell cytotoxicity by killer cell Ig-like receptors (KIR) depends on phosphorylation of cytoplasmic tyrosines in KIR, which recruit tyrosine phosphatase Src homology protein tyrosine phosphatase 1. It is not clear how KIR, whose function lies downstream of a tyrosine kinase, succeeds in blocking proximal NK cell activation signals upon binding HLA class I on target cells. Here we show that mixing NK cells with insect cells expressing HLA-C was sufficient to induce clustering of KIR, and phosphorylation of KIR and SHP-1. Transient phosphorylation of KIR was detected in the presence of pervanadate, an inhibitor of protein tyrosine phosphatases, at suboptimal concentration. Phosphorylation of KIR was specifically induced by ligand binding because it was detected only when HLA-C was loaded with a peptide that permits KIR binding. KIR phosphorylation was not dependent on ICAM-1-mediated adhesion and was not blocked by inhibition of actin polymerization, but required Zn2+. Fluorescence resonance energy transfer between HLA-C molecules revealed close molecular interactions induced by KIR binding. These results demonstrate tight clustering of KIR and rapid KIR phosphorylation induced simply by binding to HLA-C. The unique property of KIR to become phosphorylated in the absence of adhesion and of actin cytoskeleton rearrangement explains how KIR can efficiently block early activation signals during NK-target cell contacts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Inhibitory receptors specific for MHC class I regulate the cytotoxic activity of NK cells toward target cells (1, 2). Engagement of such receptors blocks proximal activation signals, upstream of Ca2+ flux (3, 4). Inhibition depends on the recruitment of tyrosine phosphatases Src homology protein tyrosine phosphatase 1 (SHP-1)4 or SHP-2 to phosphorylated immunoreceptor tyrosine-based inhibition motifs (ITIM) (3) in the cytoplasmic tail of inhibitory receptors (5, 6, 7). It is still unclear how the ITIM get phosphorylated and how the tyrosine phosphatases block early activation signals during NK-target cell contacts. Tyrosine phosphorylation of inhibitory killer cell Ig-like receptors (KIR) can be detected after KIR cross-linking with Abs or after treatment of cells with the tyrosine phosphatase inhibitor pervanadate, but not after KIR engagement by HLA class I on target cells (5, 8, 9, 10).

Activation of NK cell cytotoxicity can be induced by a variety of receptors that recognize different ligands on target cells (11, 12). In every case, activation involves tyrosine kinase-dependent signals, which are transmitted through immunoreceptor tyrosine-based activation motif (ITAM)-dependent and ITAM-independent pathways (11, 12, 13). A proposed mechanism for ITIM phosphorylation is that it is achieved by those tyrosine kinases that are involved in the activation pathway. Such a trans-phosphorylation mechanism would explain the requirement for coclustering of inhibitory and activation receptors to produce inhibition, as seen in Ab-induced cross-linking experiments (14). However, receptor-ligand interactions at the site of NK-target cell contacts are not random and result in specific segregation of molecules (15, 16, 17). At the NK-target cell interface inhibitory receptors cluster around areas that contain the {beta}2 integrin LFA-1 (15, 18). LFA-1 is essential for NK cell adhesion to target cells (19). The relative distribution of inhibitory KIR and activation receptors at the NK-target cell interface is not known yet.

A dependency of inhibitory receptors on proximal signals from other receptors for phosphorylation of the cytoplasmic ITIMs would not be compatible with an efficient mechanism to block those early signals. As ITIMs of NK cell inhibitory receptors are not constitutively phosphorylated, an alternative mechanism would be that ITIMs become phosphorylated upon MHC class I ligand binding, independently of other activation signals. The difficulty in determining requirements for ITIM phosphorylation lies in the multiple receptor-ligand interactions between mammalian NK cells and target cells. Here, we used transfected insect cells expressing ligands of human NK cell receptors (20) to dissect the contribution of individual receptor-ligand interactions to ITIM phosphorylation. Our results revealed that engagement of inhibitory receptor by its MHC class I ligand was sufficient to induce ITIM phosphorylation, even in the absence of ICAM-1-mediated adhesion. Furthermore, fluorescence resonance energy transfer (FRET) analysis of MHC class I molecules implied very tight clustering upon receptor engagement.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and Abs

The human NK cell line YTS-2DL1 (21) and the B cell lines 721.221, 721.221-Cw3, and 721.221-Cw15 (22) were maintained in Iscove’s medium supplemented with 10% FCS and L-glutamine. Drosophila Schneider cell 2 (SC2, a gift from L. Teyton, Scripps Research Institute, La Jolla, CA) were maintained in Schneider’s medium with L-glutamine (Life Sciences, Gaithersburg, Maryland) and 10% FCS (Life Sciences). The human embryonic kidney cell line 293T/17 (American Type Culture Collection, Manassas, VA) was maintained in DMEM supplemented with 10% FCS and L-glutamine. The following Abs were used: anti-KIR2DL1, mAb EB6 (Beckman Coulter, Miami, FL), and a rabbit polyclonal Ab specific for the cytoplasmic tails of KIR2DL1/KIR2DL2 (23); anti-SHP-1 (rabbit IgG; Upstate Biotechnology, Lake Placid, NY); and biotin-conjugated antiphosphotyrosine mAb PY-20 (Transduction Laboratories, Lexington, KY). For FACS analysis and cell sorting the following directly conjugated mouse mAbs were used: R-PE-conjugated (R-PE)-CD80 (MAB104; Beckman Coulter), R-PE-CD54 (HA58), CyChrome-CD54 (HA58), FITC-conjugated anti-HLA-A,B,C (G46-2.6; all from BD PharMingen, San Diego, CA). FITC-CD48 (J4-57) was obtained from Beckman Coulter. Isotype-matched PE-, FITC-, or CyChrome-conjugated mAbs were purchased from BD PharMingen.

Inhibitors

Pervanadate was prepared fresh by mixing a 100-mM solution of NaVO3 in water with H2O2 to a final concentration of 2% H2O2 and 80 mM NaVO3. This mixture was diluted to a 4-mM pervanadate concentration in cell medium just before use. Pervanadate was added to cells on ice 10 min before incubation at 37°C. Other inhibitors used were cytochalasin D (10 µM), PP1 (10 µM), herbimycin A (2 µM), wortmannin (1 µM), colchicine (10 µM), and 1,10-phenanthroline (2 mM), all from Biomol Research Laboratories (Plymouth Meeting, PA). YTS-2DL1 cells were preincubated for 45 min at 37°C with inhibitors and assayed in the continuing presence of inhibitors.

Expression of human proteins in Drosophila SC2 cells

cDNA clones for B7.1 (CD80), human {beta}2-microglobulin, HLA-Cw*0304, and HLA-Cw*0401 were obtained by PCR amplification from total RNA isolated from the human cell line 721.221 or HLA-C-transfected 721.221 cells using the RNeasy kit (Qiagen, Valencia, CA). The following primers pairs were used: B7.1, 5'-ATTGGATCCGAAGTGCCCTGGTCTTACTT-3' and 5'-AAGTCGACTAGTTCATGATCCCCACGTCCAT-3'; {beta}2-microglobulin, 5'-ATTGGATCCGCATTCCTGAAGCTGACAGCA-3' and 5'-AAGTCGACTAGTTGGAGCAACCTGCTCAGATAC-3'; and HLA-C, 5'-ATTGGATCCCGGACTCAGAATCTCCCCAGA-3' and 5'-AAGTCGACTAGTTCAGGCTTTACAAGCGATGAG-3'. PCR products were gel purified and extracted from agarose using the Wizard PCR prep system (Promega, Madison, WI), cloned into pGEM-T-Easy (Promega), from which they were subcloned using BamHI/SalI sites into the insect expression vector pRmHa3 (24) (a gift from L. Teyton) under control of the metallothionein promoter. All cDNAs were verified by sequencing. The pRmHa3 vectors encoding ICAM-1 (CD54) and CD48 have been described previously (20). Transfection of SC2 cells and selection of cells expressing the cDNAs of interest were performed as previously described (20). HLA-C*0304 on SC2 was loaded with 10 µM of the peptide GAVDPLLAL (a gift from P. Sun, National Institute of Allergy and Infectious Diseases, Bethesda, MD) during the last 20 h of a 40-h induction period in 1 mM CuSO4. HLA-C*0401 on SC2 cells was loaded with 2 µM of the peptide QYDDAVYKL (peptide 1) or QYDDAVYEL (peptide K8E) (25). Expression of transfected human cDNAs was monitored before every experiment by flow cytometry.

Cell mixing, immunoprecipitation, and Western blotting

For cell mixing, 1.6 x 106 YTS-2DL1 cells were mixed with 3 x 106 (unless indicated otherwise) target cells in 100 µl of medium and were allowed to sediment and interact with each other for 10 min at 4°C. Cells were then rapidly warmed at 37°C in the presence of 1 mM pervanadate (final volume, 200 µl), incubated for the indicated time, and centrifuged for 1 min at 4°C. The pellet was lysed in ice-cold lysis buffer (150 mM NaCl, 50 mM Tris (pH 7.4), 2 mM EGTA, 0.5% Triton X-100, 1 mM NaVO3, 1 mM PMSF, and 1 mM NaF). KIR2DL1 or SHP-1 was immunoprecipitated with 2 µg of mAb EB6 or rabbit polyclonal anti-SHP-1 Abs, respectively, and protein G agarose (Life Technologies) or protein A-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ). Samples were analyzed by SDS-PAGE and Western blotting as previously described (7), using biotinylated PY-20 Ab and peroxidase-coupled streptavidin (Amersham Pharmacia Biotech, Arlington Heights, IL). To control for loading, blots were reprobed with the rabbit polyclonal antiserum against the cytoplasmic tail of KIR2DL1. In some experiments results are expressed as arbitrary units of OD, as determined with NIH Image 1.62 software.

Adhesion assays

Conjugate formation between YTS-2DL1 cells and SC2 cells was determined as previously described (26) with the following modifications. SC2 cells were resuspended in HBSS medium (BioSource, Camarillo, CA) and 5% FCS, and 1 x 105 effector cells and 2 x 105 target cells were mixed in a 200-µl final volume. The percentage of effector cell events bound to SC2 cells was determined of the total numbers of effector cell events.

Immunostaining and confocal microscopy

YTS-2DL1 cells (1 x 106) were resuspended with 1 x 106 721.221, 721.221-Cw15, SC2, or SC2-Cw4 (loaded with peptide 1 or peptide K8E) in HBSS/3% FCS and centrifuged at 4°C for 3 min at 300 rpm. Samples were placed at 37°C for 20 min. Cells were allowed to settle on polylysine (Roche, Indianapolis, IN)-coated coverglasses for 1 h before fixation in PBS/4% paraformaldehyde. Unspecific sites were saturated, and cells were permeabilized 30 min with PBS/10% normal donkey serum/0.5% Triton X-100, stained with either 50 µg/ml mAb EB6 or 5 µg/ml of the rabbit Ab specific for the cytoplasmic tail of KIR2DL1, and revealed with goat anti-mouse Alexa-568 or goat anti-rabbit Alexa-488 secondary Abs (Molecular Probes, Eugene, OR) in PBS/3% normal donkey serum/0.5% Triton X-100. After three washes with PBS, the cells were mounted on slides using the Prolong antifade kit (Molecular Probes). Images were collected on a Leica TCS-NT/SP confocal microscope (Leica Microsystems, Exton, PA) using a x100 oil immersion objective (NA 1.4, zoom 2). Differential interference contrast (DIC) images were collected simultaneously with the fluorescence images. For three-dimensional reconstructions, 30 z sections were collected at 0.4-µm intervals. Images were processed using the Leica TCS-NT/SP software (version 1.6.587), Imaris 3.0.6 (Bitplane, Zurich Switzerland), and Adobe Photoshop 6 (Adobe Systems, Mountain View, CA).

Expression of CFP- and YFP-tagged HLA-Cw4

HLA-Cw4 was amplified by PCR from the pGEM-T-Easy-HLA-Cw4 containing vector with the following primers: 5'-CCGGTCGACCGGACTCAGAATCTCCCCAG-3' and 5'-CCCGGATCCGGCTTTACAAGCGATGAGAG-3'. Amplified product was ligated into vectors pCFP-N3 and pYFP-N3 containing the HLA-A2 cDNA in-frame (a gift from M. Edidin, The Johns Hopkins University, Baltimore, MD), generated as previously described (27). HLA-A2 was removed and replaced by HLA-Cw4 using the SalI and BamHI restriction sites. The constructs were then excised out of the N3 vectors with SalI and XbaI and cloned into a modified pBABE vector (21). Each construct was confirmed by sequencing and used for transient transfection into 293T cells with the Lipofectamine 2000 reagent kit (Invitrogen, Carlsbad, CA).

FRET measurement

Transfected 293T cells were mixed with YTS-2DL1 cells for 30 min at 37°C, and conjugates were allowed to settle on polylysine (Roche)-coated chambers for 30 min at room temperature before fixation in PBS and 1% paraformaldehyde. Cells were imaged on a confocal laser scanning microscope 510 META (Zeiss, Thornwood, NY). The cells were excited with a 454-nm laser line using a Plan-Neofluar objective x40 NA 1.3 Oil Dic. To clearly separate multifluorescence signals, each of the fluorescence images was collected using Lambda Stack acquisition. The spectral distributions of fluorescence images were simultaneously recorded in a CHS-1 from 458–640 nm. The spectra of the cells expressing HLA-Cw4-CFP or HLA-Cw4-YFP only were obtained and were used as references for linear unmixing. Digital images of the cells coexpressing HLA-Cw4-CFP and HLA-Cw4-YFP were collected as a time series with 10 frames. FRET signals were monitored as intensity increase in the CFP signals after photobleaching of the YFP. Cells expressing HLA-Cw4-YFP and HLA-Cw4-CFP were excited with a 514-nm laser line 20 times with 100% power to photobleach YFP. Photobleaching started after the second image of the time series, and eight more images were collected after the photobleaching. Digital images of the time series were processed with linear unmixing function (LSM 510 META program), using the reference spectra of CFP and YFP, and the spectrally separated CFP and YFP images were shown in the two channels. The intensities of CFP and YFP in each frame of the time series, before and after photobleaching, were analyzed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tyrosine phosphorylation of KIR2DL1 induced by HLA-C

Target cell lysis by the NK cell line YTS transduced with KIR2DL1 (YTS-2DL1) is inhibited by expression of group 2 HLA-C allotypes, which include HLA-Cw4 and HLA-Cw15 (21, 26). Tyrosine phosphorylation of KIR2DL1 is required for its inhibitory function (7), but is not detectable after mixing NK cells with target cells that express a KIR ligand (data not shown). A possible reason for the lack of detectable phosphorylated KIR is that it is rapidly dephosphorylated by tyrosine phosphatases in NK cells. The tyrosine phosphatase inhibitor pervanadate was therefore used at different concentrations to find a suboptimal dose that still permitted regulated, yet detectable, phosphorylation of KIR. Partial phosphorylation of KIR2DL1 was induced at 1 mM pervanadate, whereas maximal phosphorylation was reached at 6 mM pervanadate (data not shown). A concentration of 1 mM pervanadate was used in all additional experiments. A similar approach had been used to detect regulated phosphorylation of the signal regulatory protein-{alpha} after mixing with CD47+ cells (28). The NK-sensitive human target cell 721.221 (which lacks HLA-A, -B, and -C genes) and transfected 721.221 cells expressing either HLA-Cw3 (221-Cw3) or HLA-Cw15 (221-Cw15; Fig. 1a) were used in mixing experiments with YTS-2DL1 cells. HLA-Cw3 is a group 1 allotype and is not recognized by KIR2DL1. Increased KIR2DL1 phosphorylation was obtained after incubation with 221-Cw15 cells, compared with the phosphorylation observed after incubation with target cells that lacked the ligand (Fig. 1b). The increased phosphorylation is striking, considering that KIR2DL1 recovery was lower in the sample incubated with 221-Cw15 cells (Fig. 1b). Therefore, tyrosine phosphorylation of KIR2DL1 is still regulated by ligand binding in the presence of 1 mM pervanadate. These results suggest that the ITIMs of KIR are subject to rapid dephosphorylation in vivo. We conclude that ligand binding by KIR2DL1 induces tyrosine phosphorylation of its ITIM.



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  		FIGURE 1. KIR2DL1 phosphorylation induced by ligand binding. a, HLA class I on 721.221 cells and HLA-Cw3- or HLA-Cw15-transfected 721.221 cells was determined by FACS analysis. {square}, Isotype controls; {blacksquare}, anti-HLA class I. b, YTS-2DL1 cells (1.6 x 106) were mixed with 3 x 106 of the indicated 721.221 cells for 3.5 min at 37°C in the presence of 1 mM pervanadate. Cell lysates were immunoprecipitated with anti-KIR2DL1 mAb and analyzed by sequential Western blotting with antiphosphotyrosine-specific mAb (pTYR; upper panel) and with rabbit anti-KIR2DL1 Ab (2DL1; lower panel). Note that KIR2DL1 recovery was lower in the sample incubated with 221-Cw15 cells. c, Coexpression of ICAM-1, B7.1, and HLA-Cw4 on transfected SC2 cells detected by FACS analysis ({blacksquare}). Open profiles are isotype controls. d, YTS-2DL1 cells (1.6 x 106) were mixed with SC2-ICAM-1/B7.1/Cw4 cells (loaded with peptide 1) and incubated for 2, 5, or 15 min at 37°C at two different ratios in the presence of 1 mM pervanadate. Cells were lysed, and tyrosine-phosphorylated KIR2DL1 was detected as described in b.

 
To determine the requirements for KIR2DL1 phosphorylation and to reduce the complexity of the multiple receptor-ligand interactions between human NK cells and target cells, we used Drosophila SC2 cells expressing specific ligands of NK cell receptors (20). Human ICAM-1 (CD54) and B7.1 (CD80) were chosen as ligands of activation receptors because activation of YT cells depends on coengagement of LFA-1 and CD28 (29, 30, 31). HLA-C and {beta}2-microglobulin were cotransfected along with ICAM-1 and B7.1. As HLA class I molecules do not acquire endogenous peptides in insect cells, peptides must be loaded exogenously (32). HLA-Cw4 expression at the surface of SC2 cells was stabilized by addition of a specific peptide (peptide 1), compatible with recognition by KIR2DL1 (25). SC2 cells coexpressing ICAM-1, B7.1, and HLA-Cw4 were selected by cell sorting (Fig. 1c). Despite a low level of surface HLA-Cw4, mixing of SC2-ICAM-1/B7.1/Cw4 cells with YTS-2DL1 cells resulted in tyrosine phosphorylation of KIR2DL1 (Fig. 1d). KIR2DL1 phosphorylation was not detectable at a low target/NK cell ratio. KIR2DL1 phosphorylation was transient (Fig. 1d), indicating that tyrosine phosphatases were still active at 1 mM pervanadate. The inducible phosphorylation of KIR2DL1 by insect cells expressing ligands of human NK cell receptors opened the way to a more detailed analysis of this process.

HLA-C on insect cells induces phosphorylation of KIR2DL1

Transfected SC2 cells expressing individual ligands ICAM-1, B7.1, CD48, HLA-Cw3, or HLA-Cw4 were selected (Fig. 2a) and incubated with YTS-2DL1 cells. Two different peptides were used to stabilize cell surface HLA-Cw4, one (no. 1) that is compatible with recognition by KIR2DL1, and another (K8E) that is not (25). Only HLA-Cw4 loaded with peptide 1 induced tyrosine phosphorylation of KIR2DL1 (Fig. 2b). No other SC2 cell transfectant induced KIR phosphorylation, including 221-Cw4 cells loaded with peptide K8E. Phosphorylation of KIR2DL1 was transient, reaching a maximum at 3.5 min, and was no longer detectable at 10 min (Fig. 2c). Therefore, binding of KIR2DL1 to peptide-loaded HLA-Cw4 on insect cells is sufficient to induce tyrosine phosphorylation of KIR2DL1. In a separate study we have recently shown that binding of KIR2DL1 to HLA-C on insect cells resulted in a functional inhibitory signal that blocked NK cell activation induced by ligands of activation receptors (D. F. Barber, M. Faure, and E. O. Long, unpublished observations). Consistent with this, we show here that binding of KIR2DL1 to HLA-Cw4 on insect cells is also sufficient to induce tyrosine phosphorylation of SHP-1 (Fig. 3), suggesting that the phosphorylated ITIM of KIR2DL1 recruited and activated SHP-1.



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  		FIGURE 2. HLA-Cw4 on insect cells induces tyrosine phosphorylation of KIR2DL1. a, Expression of the indicated molecules on individual SC2 cell transfectants detected by FACS analysis. Cell surface HLA-C was stabilized by addition of the indicated peptides. {square}, Isotype controls; {blacksquare}, specific Abs, as indicated above the panels. The dashed line in the first panel represents anti-HLA class I staining of SC2-Cw4 cells that were not loaded with peptide. The profile is almost identical with the negative control. b, YTS-2DL1 cells were mixed with the indicated SC2 cell transfectants and incubated for 3.5 min at 37°C in the presence of 1 mM pervanadate. Tyrosine-phosphorylated KIR2DL1 was detected as described in Fig. 1. c, YTS-2DL1 cells were mixed with SC2-Cw4 cells (loaded with peptide 1) for the indicated times in the presence of 1 mM pervanadate. The increase in tyrosine phosphorylation of KIR2DL1 was normalized for equivalent KIR2DL1 loading and expressed as arbitrary units.

 


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  		FIGURE 3. HLA-Cw4 on insect cells induces tyrosine phosphorylation of SHP-1. YTS-2DL1 cells were mixed with the indicated SC2 cell transfectants for 3.5 min at 37°C in the presence of 1 mM pervanadate. SHP-1 was immunoprecipitated from cell lysates and tested for tyrosine phosphorylation by sequential Western blotting with antiphosphotyrosine-specific mAb (pTYR; upper panel) and rabbit anti-SHP-1 Ab (SHP-1; lower panel).

 
The stability of the interaction between YTS-2DL1 and SC2 cells expressing HLA-Cw4 was tested using a two-color conjugation assay by flow cytometry. The expression of peptide-loaded HLA-Cw4 alone did not result in detectable conjugate formation (Fig. 4). In contrast, ~25% of YTS-2DL1 cells formed conjugates with SC2 cells expressing ICAM-1 (Fig. 4). The ICAM-1-mediated adhesion was not inhibited by coengagement of KIR2DL1 with peptide-loaded HLA-Cw4 (Fig. 4). This result is consistent with our earlier finding that KIR engagement inhibits the adhesion of NK cells with human target cells, but not beyond a basal level (also ~25% of YTS-2DL1 cells) of LFA-1-dependent adhesion (26). We conclude that tyrosine phosphorylation of KIR2DL1 and SHP-1 is independent of strong adhesion, such as that mediated by ICAM-1.



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  		FIGURE 4. SC2 cells do not adhere to YTS-2DL1 cells in the absence of ICAM-1. Untransfected SC2 cells ({square}), SC2-ICAM-1 cells ({diamondsuit}), SC2-Cw3 cells loaded with peptide GAV ({triangleup}), SC2-Cw4 cells loaded with peptide 1 ({circ}), SC2-Cw4 cells loaded with peptide K8E ({triangledown}), and SC2-ICAM-1/Cw4 cells loaded with peptide 1 (•) were tested for conjugate formation with YTS-2DL1 cells using a two-color flow cytometry assay.

 
The contribution of other receptor-ligand interactions to phosphorylation of KIR2DL1 was tested with another set of SC2 cell transfectants. SC2 cells expressing HLA-Cw4 either alone, together with ICAM-1, or together with ICAM-1 and B7.1 were selected that had similar levels of HLA-Cw4 expression (Fig. 5a). Coexpression of ICAM-1 or ICAM-1 and B7.1 with HLA-Cw4 did not induce greater phosphorylation of KIR2DL1 and SHP-1 than that induced by HLA-Cw4 alone (Fig. 5b and data not shown). These results show that coengagement of LFA-1 and CD28 does not enhance the phosphorylation of KIR2DL1 induced by binding to HLA-Cw4. However, the presence of ICAM-1 may provide for a more sustained phosphorylation of KIR2DL1 (compare Fig. 1d and Fig. 2c).



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  		FIGURE 5. Coexpression of ICAM-1 and B7.1 with HLA-Cw4 does not enhance tyrosine phosphorylation of KIR2DL1. a, HLA-Cw4 expression on the indicated SC2 cell transfectants loaded with peptide 1. {square}, Isotype controls; {blacksquare}, anti-HLA class I. b, YTS-2DL1 cells were mixed with the indicated SC2 cell transfectants (loaded with peptide 1) at the indicated ratios and incubated for 3.5 min at 37°C in the presence of 1 mM pervanadate. The increase in tyrosine phosphorylation of KIR2DL1 was normalized for equivalent KIR2DL1 loading and expressed as arbitrary units.

 
Clustering of KIR2DL1 in the absence of ICAM-1-mediated adhesion

Incubation of YTS-2DL1 cells with human target cells expressing an HLA-C ligand results in extensive clustering of KIR2DL1 at the cell interface (15). Can KIR2DL1 form such clusters in the absence of ICAM-1-mediated adhesion? Confocal microscopy analysis was conducted to test this. As expected, KIR2DL1 was distributed uniformly at the surface of YTS-2DL1 cells in contact with target cells that were not expressing HLA-C, but clustered at the interface between YTS-2DL1 cells and human 221-Cw15 cells (Fig. 6a). Likewise, KIR2DL1 clustered when YTS-2DL1 cells were incubated with SC2 cells expressing HLA-Cw4 alone, loaded with peptide 1 (Fig. 6a). In contrast, clustering did not take place after incubation with the same SC2 cells loaded this time with peptide K8E (Fig. 6a). Therefore, KIR2DL1 clustering is induced directly by recognition of HLA-Cw4 and occurs in the absence of adhesion mediated by ICAM-1.



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  		FIGURE 6. HLA-Cw4 on insect cells induces clustering of KIR2DL1. KIR2DL1 localization is shown either in a single confocal plane section (a) or as a three-dimensional view (z stack) of the entire cell conjugate (b). DIC images of the cell conjugates are shown on the left, with YTS-2DL1 cells marked by a white asterisk. YTS-2DL1 cells mixed with the indicated cells and incubated for 20 min at 37°C were allowed to adhere on poly-D-lysine-coated coverslips, fixed, permeabilized, and stained with anti-KIR2DL1 mAb EB6 and goat anti-mouse Alexa-568 (red) or with a rabbit polyclonal Ab specific for the cytoplasmic tail of KIR2DL1 and anti-rabbit Alexa-488 (green)-conjugated secondary Abs.

 
High resolution analysis of the contact zone between NK cells and human target cells has revealed segregation of KIR from other molecules within the inhibitory NK cell immune synapse (15, 18). We collected z section images to reconstruct a three-dimensional view of the KIR2DL1 distribution at the interface of YTS-2DL1 cells and insect cells expressing an HLA-C ligand. A fairly uniform distribution of KIR2DL1 was observed throughout the contact zone with SC2-Cw4 cells (Fig. 6b). A similar KIR2DL1 distribution was revealed using either a mAb specific for the extracellular domain of KIR2DL1 or a rabbit Ab specific for the cytoplasmic tail (Fig. 6b). In contrast, and as expected (15, 18), a three-dimensional view, reconstructed from z section images, showed that KIR2DL1 was concentrated in specific areas of the interface between YTS-2DL1 cells and 221-Cw15 cells, with gaps clearly visible between clusters (Fig. 6b). Therefore, concentration of KIR in specific areas of the contact zone is induced either by HLA-C in the B cell line (but not by HLA-C in insect cells) or, more likely, by the interaction of additional receptor-ligand combinations between human NK and target cells.

Phosphorylation of KIR2DL1 requires an Src kinase and Zn2+

Zn2+ is required for the inhibitory function of KIR2DL1 and for clustering of KIR2DL1 at the NK immune synapse (15, 33, 34). The precise role of Zn2+ in KIR function is still unknown. Zn2+ and the related metal Co2+ mediate dimerization of soluble, recombinant KIR2DL1 molecules (35). The Zn2+ chelator 1,10-phenanthroline was used to test whether Zn2+ was also required for the phosphorylation of KIR2DL1. KIR phosphorylation was completely blocked by Zn2+ chelation (Fig. 7). The specificity of the inhibition by 1,10-phenanthroline was confirmed by addition of divalent metals. Excess Zn2+, but not Ca2+ or Mg2+, restored KIR2DL1 phosphorylation (data not shown). As expected, KIR phosphorylation was also sensitive to PP1, an inhibitor of Src family kinases (Fig. 7). Inhibition was also observed with another inhibitor of Src family kinases, herbimycin A (data not shown). In contrast, ligand-induced phosphorylation of KIR was not blocked by inhibitors of actin polymerization (cytochalasin D), PI3K (wortmannin), and tubulin polymerization (colchicine; Fig. 7). At the concentrations used, PP1 and cytochalasin D completely blocked lysis of sensitive target cells by NK cells and YTS cells (data not shown). Therefore, Src kinase activity and Zn2+ are required for tyrosine phosphorylation of KIR2DL1 induced by ligand binding.



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  		FIGURE 7. Inhibition of KIR2DL1 phosphorylation. YTS-2DL1 cells were preincubated for 45 min with DMSO, PP1, cytochalasin D (Cyto. D), wortmannin (Wort.), colchicine (Colch.), or 1,10-phenanthroline (1, 10-Phen.); mixed with SC2-Cw4 cells (loaded with the indicated peptide); and incubated for 3.5 min at 37°C in the presence of 1 mM pervanadate. Tyrosine-phosphorylated KIR2DL1 was detected as described in Fig. 1.

 
FRET between HLA-C molecules engaged by KIR2DL1

The molecular interactions that lead to the formation of KIR clusters at the NK-target cell interface are not known. We performed FRET experiments to test whether KIR clustering involved homotypic interactions between ligand-engaged KIR molecules. Fusion proteins were constructed that carried either CFP or YFP at the C terminus of HLA-Cw4 and were coexpressed by transfection into 293T cells. An HLA-A molecule tagged the same way has shown normal cellular distribution (27). FRET was determined by simultaneous recording of spectral fluorescence images (from 458–640 nm on a LSM510 META Zeiss instrument), and by comparing the CFP signal before and after YFP photobleaching. Controls with cells expressing HLA-Cw4-CFP or HLA-Cw4-YFP alone and with cells coexpressing both, but in the absence of NK cells, did not show any difference in the CFP signal upon YFP photobleaching (data not shown). In contrast, a strong FRET signal was detected in cells coexpressing HLA-Cw4-CFP and HLA-Cw4-YFP that were in contact with YTS-2DL1 cells (Fig. 8). The increase in CFP signal upon YFP photobleaching was >20% at each one of the five cell contacts tested (Fig. 8 and data not shown). Note that dimerization would result in some HLA-Cw4 dimers carrying the same fluorophore (i.e., CFP with CFP, or YFP with YFP), which cannot produce FRET, as well as heterodimers with CFP and YFP, which can. These data demonstrate that a significant proportion of HLA-C molecules engaged by KIR are within <100 Å from each other.



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  		FIGURE 8. Fluorescence resonance energy transfer between HLA-C molecules. 293T cells cotransfected with HLA-Cw4-CFP and HLA-Cw4-YFP were mixed with YTS-2DL1 cells for 20 min at 37°C. Conjugates were fixed, and FRET analysis was performed. a, DIC image of a transfected 293T cell (marked with a white asterisk) in contact with several YTS-2DL1 cells (marked with dark asterisks). b, YTS-2DL1 cells visualized with a red fluorescent dye. c and d, CFP (donor; c) and YFP (acceptor; d) signals obtained by simultaneous recording of spectral distribution of fluorescence. e and f, CFP (e) and YFP (f) signals obtained after photobleaching of YFP. Images are pseudocolored for fluorescence intensity. The color scale is shown on the right.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several important conclusions can be drawn from our studies. First, clustering and tyrosine phosphorylation of KIR occur as a result of interaction with HLA-C on the target cell, independently of ICAM-1-mediated adhesion and of any other vertebrate molecule on the target cell. Second, the divalent metal Zn2+, which is required for the inhibitory function of KIR (33, 34), for clustering of KIR at the interface with a target cell expressing an HLA-C ligand (15), and for enhanced affinity/avidity binding to HLA-C (35, 36), is also required for tyrosine phosphorylation of KIR. Third, the clustering of KIR at the interface with HLA-C-expressing cells involves close homotypic molecular interactions, as demonstrated by FRET between HLA-C molecules. Tyrosine phosphorylation of an inhibitory KIR is therefore most likely autonomous, provided a ligand is present on the target cell.

Tyrosine phosphorylation of KIR2DL1 induced by HLA-Cw4 on insect cells occurred even in the absence of ICAM-1-mediated adhesion and was not increased further by coexpression of ICAM-1 with HLA-Cw4. Stable conjugates between NK cells and insect cells were not detected unless ICAM-1 was expressed on the insect cells. The use of insect cells to dissect functional receptor-ligand interactions between NK cells and target cells has been validated by our finding that KIR engagement by HLA-C on insect cells inhibits cytotoxicity of NK cells induced by ligands of activation receptors (D. F. Barber, M. Faure, and E. O. Long, unpublished observations). The ICAM-1-independent clustering and phosphorylation of KIR2DL1 can explain the rapid and signal-independent clustering of KIR2DL1 induced by HLA-C on human cells (15, 37). KIR phosphorylation induced by HLA-C on insect cells was sensitive to inhibitors of Src family kinase activity. Earlier work has shown that KIR can be phosphorylated by Src kinases, such as Lyn (5) and Lck (9). It is interesting to note that the sequence context of the KIR ITIMs is closely related to the consensus sequence for preferred substrates of Lck (38). We have not detected a direct association of KIR with a Src kinase either before or after mixing with HLA-C-expressing cells. As Lck and SHP-1 are both localized at the NK-target cell interface (39), Lck may be readily available for phosphorylation of the ITIMs upon receptor clustering. A simple redistribution of KIR may be sufficient to interfere with constitutive dephosphorylation and change the equilibrium in favor of phosphorylation.

Tyrosine phosphorylation of ITIM-containing KIR following incubation with HLA-C-expressing target cells is not detectable in the absence of tyrosine phosphatase inhibitor, but has been reported after KIR cross-linking with Abs (5, 8, 9, 10). Experiments using Abs are difficult to interpret because of possible engagement of Fc receptors and because they induce nonphysiological receptor cross-linking. Here we used a limiting concentration of pervanadate, which binds to the catalytic site of tyrosine phosphatases, to detect ligand-induced tyrosine phosphorylation of KIR2DL1. Several points indicate that pervanadate did not cause aberrant phosphorylation, but facilitated detection of physiological phosphorylation, as reported with the ITIM-containing receptor signal regulatory protein {alpha} (28). First, KIR2DL1 phosphorylation was clearly enhanced by recognition of HLA-Cw4 loaded with a peptide that permits binding of KIR2DL1. Second, phosphorylation was rapid and transient. The loss of phosphorylated KIR2DL1 within minutes after binding to HLA-C is more consistent with a dephosphorylation by tyrosine phosphatases than with a ligand-induced degradation of KIR2DL1. Finally, tyrosine phosphorylation of SHP-1 was completely dependent on KIR2DL1 binding to HLA-Cw4. Pervanadate alone did not induce phosphorylation of SHP-1. Pervanadate is not replacing the function of an NK cell activation receptor normally engaged by human target cells, because detection of KIR phosphorylation induced by HLA-C on human target cells is also dependent on pervanadate.

The difficulty of detecting KIR2DL1 phosphorylation is not a general feature of ligand-induced receptor engagement on NK cells. For instance, the ITIM-containing receptor ILT2 becomes phosphorylated upon binding to HLA class I on target cells (40). Unlike KIR2DL1, ILT2 carries a non-ITIM cytoplasmic tyrosine that up-regulates phosphorylation of the ITIMs (40). Furthermore, tyrosine phosphorylation of activation receptor 2B4 is easily detected after mixing with target cells that express its ligand CD48 (41). 2B4 phosphorylation is dependent on cholesterol-rich membrane domains called lipid rafts, and phosphorylated 2B4 is found exclusively in those domains (42). KIR binding to HLA-C inhibits ligand-induced phosphorylation of 2B4 by preventing the recruitment of 2B4 to lipid rafts. KIR was not detected in lipid rafts during inhibition (42). Altogether, these data suggest that engagement of KIR by HLA-C on target cells results in rapid KIR clustering and phosphorylation of the ITIMs, which are then rapidly dephosphorylated by tyrosine phosphatases. Transient phosphorylation of the ITIMs may be sufficient to activate tyrosine phosphatases SHP-1 and SHP-2. Recent studies have shown that these two phosphatases remain in an active conformation when their SH2 domains form intramolecular bonds to two C-terminal phosphorylated tyrosines (43, 44). Therefore, it is not obligatory for SHP-1 or SHP-2 to remain attached to phosphorylated ITIMs to dephosphorylate their intended substrates.

Zn2+ was required for tyrosine phosphorylation of KIR2DL1. (Co2+, a metal with chemical properties similar to those of Zn2+, could substitute for Zn2+, but is much less physiological.) One interpretation of this result is that Zn2+-induced dimerization of KIR2DL1, as detected in solution (35), is required for receptor phosphorylation. However, it is also possible that in addition to an extracellular role in receptor clustering, Zn2+ is required intracellularly for tyrosine phosphorylation of the ITIM. For instance, it has been reported that Zn2+ can activate Src kinases (45) and mediate their association with receptors, such as CD4 and the epidermal growth factor receptor (46, 47). Further work is required to define the precise role of Zn2+ in the function of KIR.

FRET analysis was used to demonstrate that clustering of KIR2DL1 induced by binding to HLA-Cw4 involves some form of receptor dimerization or multimerization, rather than merely random concentration at the cell interface. Random clustering of HLA-C molecules in the NK-target cell interface among all the other cell surface proteins would not bring a high proportion of HLA-C molecules within <100 Å. The cytoplasmic tail of HLA-Cw4 was fused to YFP and CFP molecules to detect homotypic interactions among HLA-Cw4 molecules. We decided against fusing KIR with YFP and CFP for FRET analysis because C-terminal tagging of the long cytoplasmic tail may put the fluorophores beyond the FRET range, and because insertions near the transmembrane region may result in nonfunctional receptor, as was shown with green fluorescence protein-tagged erythropoietin receptor (48). As the stoichiometry of KIR2DL1:HLA-Cw4 complexes is clearly 1:1 (49, 50), the strong FRET signal detected between HLA-Cw4 molecules upon KIR2DL1 engagement implies close homotypic interactions between KIR2DL1 as well.

The demonstration of a ligand-induced phosphorylation of KIR2DL1, independent of actin polymerization and of ICAM-1-mediated adhesion, solves the apparent paradox of inhibition of tyrosine kinase-dependent activation signals by a receptor whose own function is downstream of tyrosine phosphorylation. Phosphorylation of KIR2DL1 is not only independent of adhesion, but is also likely to precede signaling through activation receptors. Indeed, clustering of KIR is independent of the cytoplasmic ITIMs and of actin polymerization (15, 37). In contrast, activation of T and NK cells through target cell contact is dependent on cytoskeleton reorganization (51, 52, 53, 54). The rapid clustering and tyrosine phosphorylation of KIR induced by ligand binding, independently of actin polymerization and of ICAM-1-mediated adhesion, are unprecedented among immune receptors. These unique properties provide a mechanism for having inhibitory receptors in place and ready before the activation signals that are targeted for inhibition.


   Acknowledgments
 
We thank G. Cohen, M. Edidin, P. Parham, P. Sun, and L. Teyton for gifts of reagents, and O. Schwartz for help with the Leica confocal microscope.


   Footnotes
 
1 Current address: Unité 548, Institut National de la Santé et de la Recherche Médicale, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble, France. Back

2 Current address: Centro Nacional de Biotecnologia, 28049 Madrid, Spain. Back

3 Address correspondence and reprint requests to Dr. Eric O. Long, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Bethesda, MD 20852. E-mail address: elong{at}nih.gov Back

4 Abbreviations used in this paper: SHP, Src homology protein tyrosine phosphatase; DIC, differential interference contrast; FRET, fluorescence resonance energy transfer; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibition motif; KIR, killer cell immunoglobulin-like receptor. Back

Received for publication November 8, 2002. Accepted for publication April 4, 2003.


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