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Originally published online as doi:10.1189/jlb.1103597 on May 3, 2004

Published online before print May 3, 2004
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(Journal of Leukocyte Biology. 2004;76:116-124.)
© 2004 by Society for Leukocyte Biology

Cross-linking of MHC class I molecules on human NK cells inhibits NK cell function, segregates MHC I from the NK cell synapse, and induces intracellular phosphotyrosines

Gonzalo Rubio*, Xavier Férez{dagger}, María Sánchez-Campillo{dagger}, Jesús Gálvez{ddagger}, Salvador Martí*, Rocío Verdú*, Trinidad Hernández-Caselles{dagger} and Pilar García-Peñarrubia{dagger},1

* Division of Immunology, Miguel Hernández University, San Juan de Alicante, Spain;
{dagger} Department of Biochemistry and Molecular Biology B and Immunology, School of Medicine, Murcia, Spain; and
{ddagger} Laboratory of Physical Chemistry, Faculty of Science, Murcia, Spain

1Correspondence: Department of Biochemistry and Molecular Biology B and Immunology, School of Medicine, 30100 Espinardo, Murcia, Spain. E-mail: pigarcia{at}um.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Engagement of major histocompatibility complex (MHC) class I molecules on immune cells, where they are usually highly expressed, induces signal transduction events of unclear significance. We show here that antibody-mediated cross-linking of MHC-I molecules on human natural killer (NK) cells inhibits their cytotoxic activity against tumor target cells. Inhibition by anti-MHC class I monoclonal antibody exhibits molecular specificity and is an isotype and Fc-independent process. Physical hindrance of specific molecular recognition, induction of apoptosis, or reciprocal NK cell killing, which could be induced by cross-linking of MHC I molecules, has also been ruled out as putative mechanisms of inhibition. Confocal microscopy analysis revealed that MHC class I molecules on the surface of NK cells colocalize constitutively with GM1, a marker of lipid rafts. Cross-linking of MHC class I resulted in the asymmetric redistribution of GM1-enriched raft domains, which are concentrated to the immunological synapse, and MHC I molecules, which segregate to the opposite pole. Also, the cross-linking of MHC I on NK cells induced intracellular tyrosine phosphorylations. These results suggest that MHC I molecules on NK cells could transmit inhibitory signals upon engagement with putative ligands expressed on the surface of those cells that need to be protected from natural cytotoxicity.

Key Words: rafts colocalization • cytotoxicity • E:T conjugation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Natural killer (NK) cells are cytotoxic effector lymphocytes able to recognize and to induce the lysis of a variety of target cells, including primarily virus-infected cells as well as tumor cells [1 , 2 ]. Multiple families of receptors regulate NK cell cytotoxicity. The interactions of these receptors with their ligands control different inhibiting/activating signal pathways, and it is the balance of these signals that determines the behavior of the NK cell. Thus, it is known that NK cells recognize major histocompatibility complex (MHC) class I molecules through surface receptors [lectin-like receptors and killer cell immunoglobulin (Ig)-like receptors (KIRs)], delivering signals that inhibit NK cell function [2 3 4 ]. Hence, NK cells lyse those target cells that have lost or express insufficient amounts of MHC I proteins. In turn, the activating receptors include a group of "natural cytotoxicity receptors", NKp46 and NKp30, which are constitutively expressed, and the inducible NKp44 among others [2 ]. Also, many different costimulatory or adhesion molecules have been shown to induce NK cell-mediated cytotoxicity [2 3 4 ].

MHC class I molecules are expressed on all nucleated cells, and the major function of these molecules is to present peptide fragments of antigens to T cells. The known ligands of the complex MHC I plus peptide are the T cell receptor, the coreceptor CD8, the lectin-like receptors, the KIRs, and other molecules codified in the leukocyte receptor complex of Ig-related genes [2 3 4 5 6 ]. However, there are many reports suggesting that aggregation of MHC I is able to induce positive and negative intracellular signals in T and B lymphocytes as well as in NK cells, resulting in tyrosine phosphorylation of multiple proteins [7 8 9 ], increases of intracellular Ca2+, interleukin (IL)-2 production and proliferation [10 , 11 ], T cell apoptosis [9 , 12 ], inhibition of T and B cell activation [13 , 14 ], and inhibition of NK cell lytic activity [15 ]. These data suggest that MHC I could not only be ligands of antigen-recognition and signaling receptors but also signaling molecules by themselves, likely through membrane colocalization with supramolecular activation clusters [16 ] or association with coreceptors more directly involved in signal transduction. The aim of this paper is to assess the effect on cell-to-cell conjugation and lysis mediated by NK cells after engagement with monoclonal antibodies (mAb) recognizing monomorphic determinants of MHC I. To this end, quantitative standard methods described previously to study effector-target (E:T) cell conjugation as well as 51Cr-release cytotoxicity assays and confocal microscopy analysis have been performed with a NK cell line and highly purified human NK cells and their targets in the absence or presence of mAb anti-MHC I. The results showed a significant inhibition of conjugation and lysis when MHC I molecules were cross-linked on the surface of NK cells. Cross-linking also induced the redistribution of MHC I molecules that segregated to the opposite pole of the immune synapse and the phosphorylation of intracellular substrates. This indicates that MHC I molecules could play a regulatory function on NK cells, providing inhibitory signals after engagement with a putative ligand(s) on target cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and reagents
mAb W6/32 (IgG2a), which react with monomorphic determinants of human leukocyte antigen (HLA) class I molecules, and mAb BBM.1 (IgG2b) against human ß2microglobulin were obtained from American Type Culture Collection (ATCC; Manassas, VA). mAb HP-1F7 (IgG1) anti-HLA class I and HP-3B1 (anti-CD94) were kindly provided by Miguel Lopez-Botet (Universitat Pompeu Faora, Barcelona, Spain). mAb RP2/21 (anti-CD45) and HP2/19 (anti-CD50) were a gift from Francisco Sanchez-Madrid (Hospital de la Princesa, Madrid, Spain). mAb p282 (anti-CD59) was from PharMingen (San Diego, CA), and mAb 71C03 (anti-CD71) was obtained from NeoMarkers (Fremont, CA). F(ab')2 fragments of W6/32 were prepared as described previously [17 ].

F(ab')2 fragments of sheep anti-mouse IgG (SAM) were purchased from Zymed (South Francisco, CA) and Sigma Chemical Co. (St. Louis, MO). Secondary antibody goat anti-mouse IgG conjugated to Alexa 568 and cholera toxin subunit B (CTXB) conjugated to Alexa 488 were from Molecular Probes (Eugene, OR). IgG from normal mouse serum was used as a control. Anti-p-Tyr mAb (PY99) was from Santa Cruz Biotechnology (Santa Cruz, CA). Immunofluorescence staining methods have been described previously [17 , 18 ]. FACSort and a FACSVantage flow cytometer (Becton Dickinson, San Jose, CA) were used throughout the study.

E:T cells
Human-purified, fresh NK cells as well as the NKL cell line were used as effector cells. Highly enriched populations of NK cells were purified from heparinized blood obtained from healthy donors aged between 20 and 40 years, kindly supplied from the Blood Bank Center of Murcia (Spain), as described previously [18 ]. NKL is a human leukemia NK cell line kindly donated by Dr. Michael J. Robertson (Indiana University Medical Center, Indianapolis, IN) [19 ], which conjugates and kills NK-sensitive target cells [18 ]. For routine conjugation experiments, NKL cells were grown in RPMI 1640 (BioWhittaker, Walkersville, MD), supplemented with 10% fetal calf serum (FCS; Seromed Biochrom, Berlin, Germany), 2% heat-inactivated human serum, 2 mM L-glutamine (Seromed Biochrom), and 10 U/ml recombinant (r)IL-2, generously provided by Hoffmann La Roche (Nutley, NJ). For cytotoxicity assays, as well as for some conjugation experiments to K562 or U-937 cell lines, NKL cells were grown in 1000 U/ml rIL-2 and 10% heat-inactivated human serum or plasma instead of FCS. Under the latter conditions, NKL exhibits increased killer activity. Human cell lines K562 (erythroleukemia) and U-937 (myelomonocytic) were obtained from ATCC and were used as target cells. The Fc receptor for IgG (Fc{gamma}R)II-negative (CD32–) subclone of the K562 cell line, termed K562.S5 subclone, was obtained by repetitive sorting of K562 cells expressing the lowest level of CD32 and single-cell cloning. All target cells were grown in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, and penicillin-streptomycin (BioWhittaker), here referred to as complete medium. Prior to use, all cell lines were washed twice at room temperature; viability was assessed by trypan blue exclusion and was higher than 95%.

E:T conjugation and data analysis
Conjugation between E:T cells was measured by flow cytometry in normalized assays, as described previously [18 ]. Briefly, effector cells were labeled green with calcein acetoxymethylester (Ca-AM) purchased from Molecular Probes, and target cells were labeled red with hydroethidine (HE) from Molecular Probes. For labeling, E:T cells were washed twice in phosphate-buffered saline (PBS) and incubated (2x106 cells/ml) with Ca-AM, 400 nM for 30 min, or HE, 253 µM for 1 h, respectively, in PBS. The labeling reaction was stopped by washing twice with PBS containing 2% FCS. Effector cells were resuspended at 106 cells/ml in complete medium containing 20 mM HEPES (Gibco, Paisley, Scotland, UK). Where indicated, Ca-AM-labeled effector cells (2.5x106 cells in 0.5 ml PBS, 2% FCS) were incubated and protected from the light with mAb at a final concentration ranging from 0.25 to 25 µg/ml for 20 min at room temperature and washed twice. For cross-linking of the first mAb, cells were treated with F(ab')2 fragments of SAM IgG at a final concentration of 10 µg/ml for 20 min at 37°C and washed twice. Next, standard procedures to facilitate E:T conjugation were performed [20 , 21 ]. Equal volumes (100 µl) of effector (0.1x106 cells) and target cells (adjusted to concentrations ranging between 5x106 and 0.25x106/ml with complete medium, 20 mM HEPES) were added to wells of 96-well U-bottom microtiter plates, incubated at 24°C for 20 min, and centrifuged at 50 g for 5 min. The pellets were gently resuspended three to four times with a micropipette and kept on ice until they were acquired in the fluorescein-activated cell sorter. Setting of the flow cytometer to measure E:T conjugates has been described previously in detail [18 ]. For each sample, 8000 events were acquired. A light-scatter gate was designed to exclude cell debris, and the percentage of effector cells conjugated was determined by gating the green and dual-labeled events. The frequencies of conjugation of effector cells ({alpha}) at different values of R (0.2, 0.5, 1, 2, and 4) were obtained by analyzing the total number of effector cells bound to target cells. In these experiments, the total number of effector cells was kept constant (E=105 cells/tube). The results were expressed as the percentage of bound effector cells in relation to the total number of acquired effector cells. All data pairs ({alpha}i, Ri) were the mean values of two or three wells. Binding isotherms were obtained by plotting {alpha} versus 1/R (i.e., vs. the E:T ratio) [20 ]. For routine assays, the area under binding isotherm (AUI) was calculated. AUI can be considered as an abbreviated conjugation index that allows expression of the overall binding efficiency, which is a consequence of the total expression and affinity status of pairs of adhesion molecules expressed by NK and target cells, as previously detailed [21 ]. Thus, looking at the AUI values obtained, the conjugation of different E:T systems can readily be compared.

Cytotoxicity
Cytotoxic activity was assessed in standard 4 h 51Cr release assays as described previously [22 ]. NKL cells were always used as effector cells. Where indicated, effector cells were preincubated with antibodies at the same doses as those indicated for conjugation assays, and target cells were incubated with mAb HP-1F7 at a final concentration of 5 µg/ml. Results have been reported as the percentage of specific cytotoxicity according to the following equation: % Lysis = (cpmexperimental–cpmspontaneous) x 100/(cpmmaximum–cpmspontaneous).

In this expression, cpmexperimental corresponds to wells containing E:T cells, cpmspontaneous, to wells with target cells only, and cpmmaximum, to wells with target cells lysed by HCl 0.1 M.

Apoptosis detection assay
After treatment with mAb W6/32 as described before, cells were incubated at 37°C for 4 h, harvested, washed twice with cold PBS, resuspended in binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 2.5 mM CaCl2) at a density of 1 x 106 cells/ml, and stained with Annexin-V fluorescein isothiocyanate (FITC) and propidium iodide (PI) in accordance with the manufacturer’s procedure (PharMingen). Cells were analyzed with the flow cytometer. A positive control for apoptosis was obtained by incubating NKL cells for 24 h in the absence of IL-2.

Immunofluorescence staining and confocal microscopy
Cells were treated with the corresponding primary antibody (W6/32, CD45, or CD71, 10 µg/ml, or isotype-matched antibody control) for 20 min at room temperature, washed three times, stained with Alexa Fluor 568 goat anti-mouse secondary antibody at 10 µg/ml for 20 min at room temperature, and washed in cold medium. Direct immunofluorescence staining was performed with R-phycoerythrin (R-PE)-conjugated anti-CD59 at 10 µg/ml, incubated for 20 min at room temperature. Cells were adhered to 3-aminopropyltriethoxysilane (Sigma Chemical Co.)-coated coverslips at 37°C and CO2 rich environment for 40 min and were fixed with paraformaldehyde 2%, 10 min. Finally, the cells were stained with CTXB subunit conjugated to Alexa Fluor 488 at 10 µg/ml for 10 min at room temperature. The coverslips were then washed once and mounted with fluorescent mounting medium (Dako, Carpinteria, CA). Cells were imaged by laser-scanning confocal fluorescence microscopy (TrueConfocal scanner TCSSP2, Leica, Knowlhill, UK). For dual-color analysis, cells were excited at 488 and 568 nm, and Alexa Fluor 488 (green) and Alexa Fluor 568 (red) fluorescence was detected simultaneously. All images represent Z-series pileups of several transverse slices acquired at 0.1–0.5 µm intervals from the top to the bottom of the cell. Confocal sections were obtained through a x100 (numerical aperture=1.4) objective. Colocalization of pairs of cell-surface molecules was determined by importing the digitized images to ImageJ, a public domain image-processing program inspired by National Institutes of Health Image, which runs on any platform with Java and along with all required plugins, can be freely downloaded from the Website http://rsb.info.nih.gov/ij. ImageJ reads and collects all intensity data pairs of the slices that form the three-dimensional arrays of the double-labeled cells. With this purpose, conical areas of colocalization were selected by using the threshold values (t1,t2; or a fraction of them=f1) provided by ImageJ as coordinates of the vertex of the area of colocalization (AOC). These values were also used to set the coordinates of the mask applied to the scatterplot as a fraction (=f2<f1) of (t1,t2). Then, files containing data pixels obtained from ImageJ were transferred to Mathematica v4.01 for computation and statistical analysis of percentages of colocalized intensity. These values were determined for each channel in the masked scatterplot by using the equation: Ii= Saoc (i) x 100/S(si≥ti). In this expression, Ii is the percentage of signal intensity in channel i above threshold ti colocalized; Saoc(i) is the sum of signal intensity of the colocalized pixels in channel i (i.e., of those pixels within the AOC); and S(si≥ti) is the sum of signal intensity of all pixels in channel i with values (si≥ti).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of MHC class I engagement and cross-linking by mAb on the parameters of conjugation of NKL cells
mAb directed against monomorphic determinants of HLA-ABC (W6/32, IgG2a isotype) were used to determine whether they have modulatory effects on the capacity of conjugation of the NKL cell line. To this end, NKL cells were preincubated for 20 min at room temperature with saturating concentrations (12.5 µg/ml) of W6/32 complete molecule or F(ab')2 fragments and were then conjugated with K562 cells as described in Materials and Methods.

As can be observed from Table 1 and Figure 1A , pretreatment of NKL cells with anti-MHC I W6/32 mAb (complete molecule) induced increases of percentages of conjugation, and F(ab')2 fragments slightly decreased the ability of conjugation to K562 target cells. These contradictory results suggest that the enhancer effect could be mediated by molecular bridging constructed between the Fc regions of mAb anti-MHC I and the Fc{gamma}RII (CD32) receptors, which are the only FcRs expressed on K562 target cells (Fig. 1B) . Additionally, we confirmed the Fc-mediated effect by obtaining a CD32– subclone of the K562 cell line (K562.S5; Fig. 1D ), which was used for similar conjugation experiments. As can be observed in Figure 1C , treatment of NKL cells with W6/32 complete molecule induced an inhibitory effect on conjugation to CD32– target cells. Next, we tested if the modulatory effect on E:T conjugation induced by mAb anti-MHC I was related to the density of expression of receptors recognized by the mAb. To this end, we performed similar experiments with mAb with the same isotype (IgG2a) but recognizing CD50 and CD94, whose NKL expression assessed by flow cytometry was lower than MHC I (data not shown). The results showed that anti-CD50 and anti-CD94 pretreatment of NKL cells did not modify the corresponding conjugation parameters (data not shown), suggesting that the increase of conjugation induced by anti-MHC I treatment, which is a Fc-mediated phenomenon, was related directly to the density of MHC I molecules expressed on the membrane of NK cells, and the inhibitory effect seems to be a specific effect.


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  		Table 1. Conjugation of NK Cells Pretreated with the Indicated mAb to Target Cells

 


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  		Figure 1. Binding isotherms for NKL cells conjugated to K562 parental target cells (A) or CD32– K562 subclone S5 (C). Effector cells were pretreated with (A) anti-MHC class I mAb W6/32 complete molecule (12.5 µg/ml), F(ab')2 fragments of W6/32 (12.5 µg/ml), F(ab')2 fragments of W6/32 (12.5 µg/ml) cross-linked with F(ab')2 fragments of SAM IgG (10 µg/ml), and F(ab')2 fragments of SAM IgG (10 µg/ml; Control). (B) Expression of the Fc{gamma}RII (CD32) molecule on parental K562 cells. Gray histograms represent isotype control. (C) Anti-MHC class I mAb W6/32 complete molecule (12.5 µg/ml), W6/32 (12.5 µg/ml) cross-linked with SAM IgG (10 µg/ml), and SAM IgG (10 µg/ml; Control). (D) Expression of the Fc{gamma}RII (CD32) molecule on the CD32– subclone 562.S5. NK effector cells, previously stained with Ca-AM, were incubated or not with the corresponding mAb as described, washed, and then seeded at 105 cells per well. K562 target cells stained with HE were added to each well at densities ranging from 5 x 105 to 0.25 x 105 cells per well to obtain E:T ratios of 0.2, 0.5, 1, 2, and 4, and the plate was processed for conjugation as described in Materials and Methods. Cells of every E:T ratio were harvested and analyzed by flow cytometry to obtain the corresponding frequency of conjugation of effector cells ({alpha}). R, E:T ratio. Each point represents the mean of duplicate wells with a resulting deviation lower than 15%. Isotherms have been fitted using nonlinear regression. One experiment out of three with similar results is depicted. The AUI for this and other experiments is shown in Table 1 .

 
Next, to further explore the effect of mAb anti-MHC I on NK cell conjugation, NKL cells were incubated with W6/32 complete molecule or F(ab')2 fragments for 20 min at room temperature, washed, and treated for 20 min at 37°C with F(ab')2 fragments of SAM IgG (10 µg/ml) to induce cross-linking of MHC I molecules. Then, the cells were washed and used in conjugation assays against K562 cells (Fig. 1A) and against the CD32– K562 subclone (Fig. 1C) .

The results showed that when MHC I molecules on NKL cells were cross-linked, the parameters of conjugation were lower than those corresponding to NKL effector cells pretreated with the primary antibody W6/32 or untreated cells (Fig. 1A and 1C , and Table 1 ).

Cytolytic activity of NK cells after treatment with mAb anti-MHC I and its F(ab')2 fragments
To determine if the modulatory effect on NK/target cell conjugation induced by mAb anti-MHC I correlated to a modulatory effect on the cytolytic activity, we set up cytotoxicity assays under the conditions assayed above for the NKL/K562 E:T system. As shown in Figure 2A , neither treatment of NKL cells with mAb anti-MHC I W6/32 nor with their F(ab')2 fragments induced significant variations on the cytotoxic activity of the NKL cell line. However, and in the same line of evidence as for conjugation assays, when MHC I proteins were cross-linked with SAM F(ab')2, the cytolytic activity of NKL cells was significantly inhibited with W6/32 (complete molecule) or its F(ab')2 fragments. The inhibitory effect was more significant when MHC I molecules were engaged with W6/32 F(ab')2 fragments.



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  		Figure 2. NK cell-mediated lysis of target cells decreases upon MHC class I cross-linking on NK cell membrane. Target cells K562 (A), CD32– subclone K562.S5 (B), and U-937 (C) were labeled with 51Cr, and then mAb, pretreated or not, NKL cells (A and C) or freshly purified NK cells (B) were added at different E:T ratios, and cytotoxicity was determined in a standard 4-h 51Cr-release assay. (C) MHC I expression on U-937 target cells was masked with HP1F7 mAb. Data are shown as the mean ± SD (error bars) of three assays. (A and C) Significantly different than control values (without mAb): *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t-test). (B) Two representative experiments are shown.

 
Next, to further explore whether primary NK cells react the same as the NKL cell line to cross-linking with W6/32 mAb, we performed cytotoxicity experiments by using fresh, purified human NK cells and the CD32– K562 subclone as target cells. Figure 2B shows that MHC class I cross-linking on the surface of primary NK cells inhibits their cytolytic activity. Additionally, cross-linking of a different, abundant molecule expressed on NKL cells (i.e., CD71) had no significant effect on NKL-mediated lysis of the CD32– K562 subclone (data not shown).

Inhibition of NK cell cytotoxicity induced by engagement of MHC I is not caused by steric hindrance
Next, we tried to rule out if the inhibitory effect on NKL cell activity induced by ligation of MHC I molecules could be caused by a decreased availability of NK cell-activating receptors, which could have been shielded by anti-MHC I treatment. To this end, we designed cytotoxicity assays masking MHC I on appropriate target cells trying to know whether NK cell-activating receptors were hindered to interact with their MHC I ligands. These experiments were performed with a U-937 cell line, a target cell with low NK susceptibility (as this cell line expresses MHC class I). As expected, U-937 cells were more resistant to lysis mediated by NKL cells (Fig. 2C) . In the same line of evidence as with K562, cross-linking of MHC I molecules on NKL cells with mAb W6/32 significantly decreased percentages of lysis against U-937. Furthermore, addition of anti-HLA class I mAb HP-1F7, which is able to interfere with recognition of MHC class I molecules by NK inhibitory receptors [23 ], was not only able to increase percentages of killing to U-937, according to the missing self-hypothesis, but was also able to revert the inhibitory effect induced by MHC I cross-linkage (Fig. 2C) . These results strongly suggest that MHC I cross-linking does not interfere with NK cell recognition of the putative(s), activating ligands on the membrane of target cells.

MHC I-mediated inhibition of NK cell cytolytic activity is neither caused by induction of apoptosis on effector cells nor reciprocal killing
As it has been described that aggregation of MHC I on T cell lines induces apoptosis [9 , 12 ], we also tested whether inhibition of conjugation and cytotoxicity induced by cross-linking of MHC I molecules on NK cells could be caused by induction of apoptosis of the effector population. To this end, we assayed and analyzed for apoptosis by Annexin V staining and flow cytometry under MHC class I cross-linking or not. Figure 3 reveals that the percentages of apoptotic population are not significantly different among untreated, MHC I-coated, and MHC I-cross-linked NKL cells. In fact, NKL cells were resistant to apoptotic treatments assayed in a 4-h experiment (wortmanin, 100 nM; camptotecin, 12 µM; anti-CD95, IL-2, and serum deprivation). For this reason, the positive control of apoptosis in Figure 3 is referred to 24 h incubation in the absence of IL-2. Thus, these experiments allow us to rule out an apoptotic-mediated inhibition of the NK cell function. Finally, we also excluded the possibility that masking MHC I molecules on NK cells could induce a reciprocal NK cell killing by performing cytotoxic assays using 51Cr-labeled NKL cells as target cells. Under these conditions, the percentages of lysis were insignificant (data not shown).



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  		Figure 3. Detection of apoptosis by flow cytometry after 4 h incubation at 37°C of NKL cells previously treated with anti-MHC class I mAb W6/32 F(ab')2 fragments, W6/32 F(ab')2 fragments cross-linked with SAM F(ab')2 fragments, SAM, untreated control (None), and 24 h incubation in the absence of IL-2 (as positive control). MHC class I mAb, treated or not, NKL were stained with Annexin-FITC and PI, as described in Materials and Methods, and were analyzed by flow cytometry. Eight thousand events were acquired from each sample. Data are shown as the mean ± SD (error bars) of percentages of apoptotic NKL cells from three assays.

 
Quantitative analysis of confocal images reveals a high degree of colocalization of MHC I molecules with membrane rafts
Once we ruled out steric hindrance, apoptosis, or reciprocal killing as putative mechanisms of MHC I-induced inhibition of NK cell function, our next goal was to test whether MHC class I on NK cells could be associated with the glycolipid-enriched membrane microdomains (GEMs), also known as lipid rafts [24 ]. To this end, NKL cells were treated with mAb W6/32 (10 µg/ml) for 20 min at room temperature and were then stained with Alexa Fluor 568 goat anti-mouse for 20 min at room temperature. Next, cells were adhered to coverslips and fixed with paraformaldehyde, and then, membrane rafts were revealed by incubating with CTXB-Alexa Fluor 488, which binds to GM1, a glycolipid concentrated within membrane rafts [24 ]. As shown in Figure 4A 4f , large patches of MHC I molecules are uniformly distributed on the surface of NKL cells, and it presents high percentages of colocalization with lipid rafts as revealed by GM1 stained with CTXB-Alexa Fluor 488 (Fig. 4B) . The patterns of surface distribution of CD45 and CD71 (the cell-surface transferrin receptor), which have been described to locate outside the rafts [25 , 26 ], were determined as negative controls. As positive controls, we used anti-CD59 {specific for a surface receptor for complement, also known as protectin, which is a glycosylphosphatidylinositol (GPI)-anchored lipid rafts molecule; [27 ]}. As displayed in Figure 4A 4a and 4B , CD45 does not colocalize with GM1, and CD59 is highly colocalized with lipid rafts (Fig. 4A 4c and 4B) . Next, we determined the partitioning of cross-linked MHC I, CD45, and CD59 with lipid rafts. With this purpose, MHC I, CD45, and CD59 were cross-linked on NK cells before fixation using the corresponding, primary antibody for 20 min at room temperature, then, Alexa Fluor 568 goat anti-mouse secondary antibody for 20 min at 37°C, and finally, fixed with paraformaldehyde and stained with CTXB-Alexa 488. Figure 4A 4g and 4B shows that cross-linked MHC I are significantly excluded from GEMs, reaching percentages of colocalized intensity similar to negative controls CD45 (Fig. 4A 4a and 4b) and CD71 (Fig. 4A 4e) . Once established that cross-linked MHC I is excluded from lipid rafts, we wanted to determine the location of cross-linked MHC I in the membrane of NKL conjugated to K562 target cells. With this purpose, mAb-treated NKL cells (before and after cross-linking conditions) were conjugated with K562 at 0.5 E:T ratio for 20 min at room temperature. Then, they were carefully adhered to the coverslip, fixed with paraformaldehyde, and finally, stained with CTXB-Alexa Fluor 488 (Fig. 5 ). From each individual experiment, more than 20 conjugates were analyzed. Two- and three-dimensional images of all conjugates were acquired and analyzed. The scoring of the synaptic regions was made from 40 to 50 z-stack images using ImageJ. The percentages of conjugates including or excluding MHC I molecules at the synaptic area are displayed in Figure 5 , which shows that MHC class I molecules are constitutively included in the immunological synapse in 100% of conjugates analyzed. However, cross-linked MHC I molecules not only segregate from lipid rafts (85% of conjugates) but also locate to the opposite pole of the synapse (Fig. 5) .



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  		Figure 4. MHC class I molecules constitutively colocalize with GM1 glycolipid, a marker of membrane rafts, and cross-linked MHC class I is excluded from membrane rafts. (A) Two-color staining of GM1 (green) and mAb (red) against CD45 (rows a and b) and CD71 (row e) used as negative controls of colocalization, CD59 (rows c and d used as positive control), and MHC class I (rows f and g) of NKL cells under the following experimental procedures: (a) NKL cells were stained red by indirect immunofluorescence using anti-CD45 (RP2/21) primary antibody and Alexa Fluor 568 goat anti-mouse secondary antibody. (b) CD45 was cross-linked on NKL cells using anti-CD45 primary antibody as above and then Alexa Fluor 568-conjugated secondary antibody for 20 min at 37°C. (c) NKL cells were stained red with R-PE-conjugated anti-CD59 (p282). (d) CD59 was cross-linked on NK cells using R-PE-conjugated anti-CD59 primary antibody as above and then goat anti-mouse secondary antibody for 20 min at 37°C. (e) NKL cells were stained red by indirect immunofluorescence with the primary mAb anti-CD71 (71C03) and secondary antibody Alexa Fluor 568 goat anti-mouse. (f) NKL cells were stained red by indirect immunofluorescence with the primary mAb anti-MHC I (W6/32) and the secondary antibody Alexa Fluor 568 goat anti-mouse. (g) MHC I proteins were cross-linked on NKL cells using mAb W6/32 as above and Alexa Fluor 568-conjugated goat anti-mouse secondary antibody for 20 min at 37°C. In all cases, after the red staining, the cells were adhered to coverslips, fixed with paraformaldehyde, and then stained green with CTXB-Alexa Fluor 488. (B) Percentages of colocalized fluorescence intensity between GM1 molecules (a marker of lipid rafts) and MHC class I as compared with CD45, CD59, and CD71. NKL cells were double-labeled green and red as described in A, acquired by confocal microscopy, and analyzed by ImageJ, as described in Materials and Methods. Bars represent mean ± SD of at least three independent experiments. For each condition, more than 10 cells were acquired, and for each cell, more than 15 slices were analyzed.

 


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  		Figure 5. MHC class I proteins are located in the NKL/K562 immune-cell synapse, and cross-linked MHC class I is excluded. Shown is two-color staining of GM1 (green) and MHC class I proteins (red) in NKL/K562 conjugates and their corresponding synapses under the conditions described in Materials and Methods. Synapses are visualized as projections in x–zaxes of the conjugates. Data represent 20–30 immune synapses analyzed for each condition from two independent experiments. Percentages show the relative frequency of synapses including or excluding MHC I molecules.

 
MHC I cross-linking on NK cells induces intracellular tyrosine phosphorylation
Finally, we tried to test if cross-linking of MHC class I molecules on NK cells could transduce intracellular signals. With this purpose, we treated NKL cells with the mAb anti-MHC I W6/32 under the conditions described above. Then, the cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min, incubated overnight with the anti-p-Tyr antibody (10 µg/ml) at 4°C, and finally, stained with Alexa Fluor 568-conjugated goat anti-mouse and CTXB-Alexa Fluor 488. Figure 6 shows that when MHC class I molecules on NKL cells were cross-linked, intracellular tyrosine phosphorylation was induced. However, engagement of MHC class I proteins in absence of cross-linkage was not able to induce any detectable tyrosine phosphorylation.



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  		Figure 6. Cross-linking of MHC class I molecules on NKL cells induce intracellular phophotyrosines. Confocal laser-scanning microscopy detection of NKL double-labeled green with CTXB-Alexa Fluor 488 and red with primary antibody anti-p-Tyr and Alexa Fluor 568 goat anti-mouse secondary antibody. After treatment with F(ab')2 fragments of mAb W6/32 (10 µg/ml), cross-linked or not cross-linked, cells were permeabilized with Triton X-100 0.5% and treated with mAb anti-p-Tyr followed by secondary antibody and finally, with CTXB-Alexa Fluor 488, as described in Materials and Methods.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
MHC class I molecules are widely expressed on all nucleated cells. These molecules function as restricting elements and adhesion receptors for T cell activation and target-cell recognition or as inhibitory signaling receptors when interacting with MHC class I-specific inhibitory receptors on the surface of a NK cell [2 3 4 5 ]. Engagement of MHC I molecules on effector cells has been described to result in positive and negative effects on T and B cells, likely depending on the density of MHC I expression, mAb fine specificity, the degree of class I MHC molecules cross-linking, or the state of activation [28 ]. Thus, anti-MHC I mAb have been described to induce proliferation of peripheral T cells [10 , 11 ], activation of nuclear factor-{kappa}B [29 ], increases of intracellular Ca2+, and phosphorylation of p56lck in T cells [11 ], phospholipase C{gamma}1 [8 ], p56lck and {zeta}-associated protein-70 [9 ], p53/56lyn and p72syk in B cells [30 ], and Src, paxilin, and focal adhesion kinase in an actin-dependent manner in human endothelial cells [31 ]. Conversely, inhibition of CD3-mediated activation and more recently, inhibition of CD94-mediated lysis on a cytolytic T cell clone have also been reported [13 , 32 ]. Similarly, several authors have described that engagement of MHC I on NK cells results in an inhibitory or stimulating effect on their killing capacity [15 , 33 ] or even induction of apoptosis of the fraction of CD56+ cells in peripheral blood mononuclear cells after 8 days of culture in the presence of mAb anti-MHC I by a Fas-independent pathway [12 ]. Thus, a functional association between MHC class I molecules expressed on effector NK cells and cytolytic activity has been suggested [15 , 34 ]. Contrary to this hypothesis, Jones et al. [33 ] described that inhibition of NK cell cytotoxicity by rabbit antibodies to human ß2microglobulin is dependent on a membrane-associated rabbit Fc moiety, as the use of mAb anti-MHC I alone not only failed to reduce polymorphonuclear cell lytic activity but also increased the lytic effect. Similarly, pretreatment of highly purified NK cells with rabbit anti-ß2microglobulin did enhance target cell lysis.

In our hands, W6/32 as well as BBM.1 (anti-ß2microglobulin, data not shown) mAb promote a dose-response induction of NK/K562 E:T conjugation, which is attributable to direct interaction of the Fc region of mAb anti-MHC I with FcRs on target cells, as demonstrated by the results obtained with F(ab')2 fragments and CD32– target cells. However, when MHC class I proteins on the membrane of NK cells were cross-linked by F(ab')2 fragments of W6/32 mAb and F(ab')2 fragments of a secondary antibody, a significant inhibition of conjugation and lysis against K562 and U-937 target cells was produced in agreement with the results of Petersson et al. [15 ].

The analysis of the mechanisms of inhibition of NK cell function by MHC I cross-linking revealed that steric hindrance of NK cell-activating receptors involved in recognition of target-cell ligands was not the case, as masking MHC I molecules on target cells reverts percentages of lysis, which demonstrated that activating receptors were still available for recognizing their specific ligands. We also ruled out induction of NK cell apoptosis under conditions of MHC I cross-linking. This is in agreement with previous studies describing that mAb W6/32 was lacking an apoptotic effect on T cells [35 ] and others, describing that spontaneous apoptosis in neutrophils is associated with reduced levels of MHC I expression, and it is prevented by ligation of surface HLA I with mAb W6/32, indicating that engagement of MHC I could transduce signals that inhibit apoptosis [36 ]. It is likely that recognition of different epitopes on MHC I molecules results in triggering different signals, perhaps by inducing lateral association with different surface molecules or by determining translocation of supramolecular complexes in or out of the rafts that have been shown to function as platforms coordinating the induction of signaling pathways [16 ]. In this context, we have shown here that MHC class I molecules present a high degree of colocalization with lipid rafts, which decrease dramatically following cross-linking of MHC I. The data showing that MHC I proteins constitutively locate within lipid rafts strongly suggest an important role of these molecules in NK cell function. This fact could enable them to respond faster to the interaction with a putative ligand than other signaling molecules that need to be recruited into lipid rafts by their ligands. Along this line, we have also shown here that intracellular tyrosine phosphorylation is triggered by MHC I cross-linking on NK cells. This strongly suggests that the inhibition of NK cell function induced by aggregation of MHC class I molecules is mediated by an intracellular inhibitory signal triggered by MHC I.

Our results agree and extend those of Vereb et al. [37 ] and Damjanovich et al. [38 ], which suggested that MHC proteins together with IL-2R{alpha} could be partially confined to small receptor islands and larger lipid rafts on T lymphoma cells. In these microdomains, they hypothesized that MHC proteins could provide a stabilizing effect through their direct association with cytoskeletal proteins [39 ] or even regulate signaling through IL2-R{alpha} by impeding its association with IL-2Rß and IL-2R{gamma}, which are located in soluble membrane fractions [40 ]. Additionally, Gur et al. [41 ] reported that the cytoplasmic domain of MHC class I molecules is not required for T cell signaling through these receptors, and the transmembrane region is always required for this effect [42 ]. This suggests that lateral association with a coreceptor connected to intracellular transduction pathways could be responsible for this phenomenon [31 ]. Among molecules reported to be associated with MHC I are the IL-2R{alpha} and the GPI-anchored raft protein CD48 in T cell lymphomas [37 ] and the adhesion molecule intercellular adhesion molecule-1 (CD54) in colon carcinoma cells [38 ]. Furthermore, we have shown here by confocal studies of conjugates formed between cross-linked MHC I NKL and K562 target cells that cross-linked MHC I proteins are significantly excluded from the immunological synapse, although this does not preclude the polarization of lipid rafts to the contact area, which is a process that requires activation of src and syk kinases [43 ].

At present, we have no information about the topology and temporal progression of the signaling events taking place at the NK cell synapse under the condition assayed in this paper. In this context, Vyas et al. [44 ] described that talin, Lck, and Src homology-2 (SH2) domain-containing inositol phosphatase-1 are recruited to the NK cell synapse within 1 min in cytolytic and noncytolytic conjugates. However, SH2-containing tyrosine phosphatase-1 clusters in the periphery of the cytolytic synapse, whereas it clusters in the center of the noncytolytic one. Along this line, we can hypothesize that segregation of cross-linked MHC I from lipid rafts could deprive the NK cell synapse of activating signals if some critical transduction molecules were directly or indirectly associated with MHC I proteins. In this regard, it has been described recently that cross-linking MHC I on a CD8+ T cell clone induced a selective inhibition of microtubule organizing center reorientation toward anti-CD94-coated beads upon coengagement of CD94 and MHC class I molecules [32 ].

Finally, one may ask what could be the significance of the inhibitory function induced through MHC class I molecules expressed on NK cells. We propose a model in which MHC I proteins expressed on the membrane of NK cells could modulate natural killing against self-hematopoietic cells, especially against those acting as professional antigen presenting cells (APCs) such as dendritic cells (DC), which could be attacked during a pathogen-induced, immune response. In this regard, APCs express subsets of leukocyte Ig-like receptors (LIR), which interact with classical and nonclassical MHC molecules [6 , 23 ]. Thus, one may hypothesize that the phenomena of MHC I location to the opposite pole of the synapse, here seen in NK cells after specific MHC I aggregation, could also occur in such APCs upon cognate interaction with appropriate MHC I ligands. This may turn a normal cell into a potential target for NK effector cells, because of the local reduction of MHC I proteins in the rear guard. Under these circumstances, MHC I ligands (i.e., LIR) expressed by the eventual target cell could act as a safeguard mechanism inhibiting NK cells as described here. Furthermore, it has been described recently that although immature DC have a preferential susceptibility to NK-mediated lysis, mature DC and bacterial-infected DC are more resistant to NK cell lysis as a consequence of the up-regulation of HLA class I expression on their surface [45 ] and hypothetically, by the increased expression of LIR. In conclusion, data presented here support a dual role of MHC I molecules in natural cytotoxicity, that is, protecting target cells from NK cell-mediated lysis and at the same time, inhibiting NK cells directly. These results suggest a novel function of the multifarious class I molecules that could have important implications in the study of regulatory signals that control and regulate NK cell activity.


   ACKNOWLEDGEMENTS
 
A grant from the Fundación Séneca (CARM) project number PB/27/FS/02 supported P. G-P. G. R. and X. F. contributed equally to this work. The authors thank Dr. M. Robertson for the NKL cell line and Drs. F. Sanchez-Madrid and M. Lopez-Botet for the gifts of antibodies.

Received November 26, 2003; revised March 15, 2004; accepted March 30, 2004.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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