The leukocyte Ig-like receptor (LIR)-1 for the cytomegalovirus UL18 protein displays a broad specificity for different HLA class I alleles: analysis of LIR-1+ NK cell clones

Massimo Vitale1, Roberta Castriconi4, Silvia Parolini2, Daniela Pende1, Mei-Ling Hsu3, Lorenzo Moretta1,4, David Cosman3 and Alessandro Moretta4

1 Istituto Nazionale per la Ricerca sul Cancro e Centro Biotecnologie Avanzate, 16132 Genova, Italy
2 Dipartimento di Scienze Biomediche e Biotecnologie, Università di Brescia, Brescia, Italy
3 Immunex Corp., Seattle, WA 98101, USA
4 Dipartimento di Medicina Sperimentale, Università degli Studi di Genova, 16132 Genova, Italy

Correspondence to: L. Moretta, Laboratorio di Immunopatologia, Centro Biotecnologie Avanzate, L. go Rosanna Benzi 10, 16132 Genova, Italy


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Leukocyte Ig-like receptor (LIR)-1 is a member of the Ig superfamily which has been shown to bind the human cytomegalovirus MHC class I homologue UL-18 protein. In this study, we have analyzed the expression and function of LIR-1 in human NK cells. We show that LIR-1 is expressed by a subset of NK cells variable in size among different donors. When compared to the known HLA class I-specific NK receptors, the expression of LIR-1 was found to be partially overlapped with that of CD94–NKG2A or with that of killer inhibitory receptors (KIR) belonging to the Ig superfamily. The use of the soluble form of UL-18 molecule revealed, in double fluorescence analysis, a selective binding to LIR-1+ cells while no correlation was observed between expression of either KIR or CD94–NKG2A molecules and ability to bind UL18. We further determined whether LIR-1 could also function as receptor for HLA class I molecules. To this end, we assessed the capability of LIR-1+ NK cell clones of lysing HLA class I target cells transfected with different class I alleles, including HLA-A, -B, -C and -G alleles. Data revealed that LIR-1 functions as a broad HLA class I-specific inhibitory receptor recognizing different alleles coded for by different HLA loci.

Keywords: cytotoxicity, HLA class I, inhibitory receptors, NK cell


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
It is now well established that NK cells express MHC class I-specific inhibitory receptors (NKR). The interaction between NKR and MHC class I molecules leads to inhibition of NK cell function including cytolytic activity and cytokine production. In humans, NKR are represented by members of the Ig superfamily [killer inhibitory receptors (KIR)] (18) or by the covalently associated type II proteins CD94 and NKG2A (912). While KIR precisely recognize given groups of HLA class I allotypes (8), the CD94–NKG2A complex recognizes target cells transfected with different HLA class I molecules (9,12), characterized by a leader sequence that can allow expression of endogenous HLA-E molecules (13). Indeed it has been shown that CD94–NKG2A recognizes HLA-E rather than classical HLA class I alleles (1416). Both KIR and CD94–NKG2A are expressed on NK cells and on some activated T lymphocytes (17). Recently, another surface molecule homologous to KIR has been identified and cloned. This molecule, termed leukocyte Ig-like receptor (LIR)-1/Ig-like transcript (ILT)-2, displays a different tissue distribution as it is expressed on B cells and monocytes while its expression and function in NK and T lymphocytes has not been clearly established (18,19). Amino acid sequence analysis revealed, in the cytoplasmic tail of LIR-1, a typical immunoreceptor tyrosine-based inhibitory motif (ITIM) which upon tyrosine phosphorylation associates with the tyrosine phosphatase SHP-1 (19), a member of the inhibitory signal cascade (2023). This suggests that LIR-1 may function as an inhibitory receptor in different cell types. Importantly, LIR-1 was found to function as receptor for the cytomegalovirus (CMV) UL18 protein (19,2425). This implies that UL18 may negatively signal different (LIR-1+) cell types involved in the immune response. However, a discrepancy exists regarding the identification of the UL-18 receptor since another laboratory reported that the UL18-specific receptor is represented by CD94 (26).

In this study, we show that LIR-1 is expressed on variable proportions of resting or activated NK cells. By the use of purified NK cell populations expressing (in different proportions) both LIR-1 and CD94–NKG2A we show that soluble UL-18 (19) binds to LIR-1+ cells, while no correlation exists between binding of soluble UL18 and CD94–NKG2A expression. Finally, by cytolytic assays using HLA class I target cells transfected with different HLA class I alleles, we show that LIR-1 functions as a broad specificity receptor recognizing not only classical (mainly HLA-A and -B) but also non-classical (HLA-G) HLA class I molecules (2728).


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
mAb, purified proteins and cytofluorimetric analysis
The following mAb were used in this study: M401 (IgG1 anti-LIR-1) (19), Z27 (IgG1, anti-p70: HLA-Bw4-specific NKR), Q66 (IgM, anti-p140: HLA-A3/A11-specific NKR), XA141 (IgM anti-p58.1) y249 (IgM anti-p58.2), Y9 (IgM, anti-CD94), XA185 (IgG1, anti-CD94), Z199 (IgG2b, anti-NKG2A), Z270 (IgG1, anti-NKG2A), C218 (IgG1, anti-CD56, Immunotech, Marseilles, France), A6/136 (IgM, anti-HLA class I), D1.12 (IgG2a, anti-HLA-DR), JT3A (IgG2a, anti-CD3) and AZ158 mAb (IgG2a specific for an epitope common to p70 and p140). Soluble UL18–Fc fusion protein was previously described (19). Specific binding was visualized with phycoerythrin (PE)-conjugated, Fc-specific mouse anti-human IgG (Southern Biotechnology Associates, Birmingham, AL).

Cytofluorimetric analysis of NK cell populations or NK cell clones was performed using the relevant mAb or soluble protein followed by the PE- or FITC-conjugated specific second reagent as previously described (29).

NK cell cloning and cytolytic assays
NK cell clones were obtained by limiting dilution as described (29). The cytolytic activity of cloned NK cells was assessed in a 4 h 51Cr-release assay in which effector cells were tested against P815 mouse cell line or the C1R human cell line transfected or not with various HLA class I genes (kindly provided by B. Biddison, National Institutes of Health Bethesda, MD; J. A. López De Castro, Foundaction Yimenez Diaz, Madrid; and P. Creswell, Yale University, New Haven, CT). Other target cells used in these studies were represented by the human HLA class I LCL 721.221 cell line either untransfected or transfected with various classical HLA class I alleles (kindly provided by Dr R. Biassoni, Genova) or the non-classical HLA-G1 transcript (27).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Expression of LIR-1 or HLA class I-specific NKR in human NK cells
In these experiments, we analyzed by indirect immunofluorescence and FACS analysis the LIR-1 surface expression on either fresh or cultured lymphocyte populations depleted of CD3+ and HLA-DR+ cells (i.e. enriched in NK cells) isolated from different donors. These experiments showed a high degree of variability in LIR-1 expression ranging from 5 to 85% of NK cells in different donors (not shown). Moreover, the percentage of LIR-1+ cells and their fluorescence intensity was not substantially modified following culture in IL-2 (for up to 15 days of culture). Since, similar to CD94–NKG2A or to KIR belonging to the Ig superfamily, LIR-1 is expressed by a subset of NK cells, the expression of these various molecules has been comparatively analyzed in polyclonal NK cell populations cultured in the presence of exogenous IL-2. Figure 1Go (upper panel, A), shows a representative experiment in which LIR-1 expression is analyzed by double fluorescence, in comparison with NKG2A. It can be seen that LIR-1+ cells may or may not co-express NKG2-A. In Fig. 1Go(upper panel, B) cells were stained with M401 (anti-LIR-1 mAb), and with a mixture of mAb recognizing all of the known KIR including p58, p70 and p140 (17). Again, LIR-1+ cells were either KIR+ or KIR. Therefore, no correlation appears to exist between the expression of LIR-1 and the various HLA class I-specific NKR. Figure 1Go(upper panel, C) shows the same NK cell population analyzed for the expression of KIR in comparison with that of NKG2A.



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Fig. 1. Analysis of different NK subsets for the expression of LIR-1 and for the ability to bind the UL18 protein. (Upper panel) A representative polyclonal NK cell population was analyzed in double fluorescence by FACS analysis. (A and B) LIR-1 expression (M401 mAb) was analyzed in comparison to that of NKG2A (Z199 mAb) and KIR respectively. (C) The expression of NKG2A (Z199 mAb) was compared to that of KIR. (D–F) The cells were stained with soluble UL18–Fc fusion protein and then analyzed for the expression of LIR-1 (M401), NKG2A (Z199) or KIR respectively. In all instances, staining of KIR was obtained by the simultaneous use of XA141 (anti-p58.1), Y249 (anti-p58.2) and AZ158 (anti-p70 and anti-p140) mAb. (Lower panel) Comparative analysis of the ability of NK cell clones or peripheral blood NK cells to bind UL18 fusion protein or anti-LIR-1 (M401) mAb. Two representative NK clones are shown, one of which (A62-56) is LIR-1+ and one (TB-14) is LIR-1. In the two right panels the distribution of LIR-1+ cells and of cells able to bind the UL18 fusion protein is analyzed on peripheral blood lymphocytes (B.C. A39).

 
Soluble UL18 binds to LIR-1 but not to CD94–NKG2A or KIR
Conflicting data have been reported regarding the identification of the receptor for the human CMV UL18 protein. Thus, although LIR-1 has been shown to bind UL18 (19), CD94 also has been reported to recognize UL18 protein (26). CD94 is known to form different heterodimers with different members of the NKG2 family; however, only the CD94–NKG2A complex (expressed by a subset of NK cells) functions as a HLA class I-specific inhibitory receptor (912). Therefore, we analyzed the ability of soluble UL18 to bind to NK cells expressing LIR-1, NKG2A, CD94 or KIR respectively. As shown in Fig. 1Go(upper panel, D) when NK cells were stained with soluble UL18 and anti-LIR-1 mAb a diagonal distribution of stained cells was observed while no single-positive populations could be detected. This suggests that both soluble UL18 protein and M401 mAb bind to the same surface molecule. In addition, as previously reported by Cosman et al. (19), the binding of anti-LIR-1 mAb was partially inhibited by the binding of soluble UL18 resulting in a reduced fluorescence intensity of cells stained by the M401 mAb. In contrast, binding of UL18 did not correlate with the expression of either NKG2A or KIR. Thus, cells binding UL18 did not necessarily express either NKG2A or KIR and, vice versa, only a fraction of NKG2A+ or KIR+ cells bound UL18 (Fig. 1Go, upper panel, E and F). Notably, pictures obtained in experiments of double fluorescence using UL18 versus KIR or NKG2A (Fig. 1Go, upper panel, E–F) were remarkably similar to those obtained in Fig. 1Go(upper panels, A and B) in which the red fluorescence was represented by the anti-LIR-1 mAb. That the ability to bind soluble UL18 is a characteristic confined to LIR-1+ NK cells was also demonstrated by the analysis of a large panel of NK clones. The expression of LIR-1 and the ability to bind UL18–Ig was evaluated on LIR-1+ and LIR-1 NK clones; as shown in Fig. 1Go(lower panel left and central square), LIR-1+, but not LIR-1 clones, bound soluble UL18. Finally, similar proportions of LIR-1+ and sUL18+ cells were consistently detected in peripheral blood lymphocytes (Fig. 1Go, lower panel right squares shows a representative experiment). Altogether, these data, while confirming the identity of the UL18 receptor with the LIR-1 molecule, also indicate that no correlation exists between binding of soluble UL18 and expression of other known inhibitory NK receptors. Although not shown, binding of UL18 did not correlate with expression of CD94 (which is expressed by most NK cells).

LIR-1 functions as an inhibitory receptor for different HLA class I alleles
The LIR-1 molecule is characterized by four ITIM in its cytoplasmic tail (19). Moreover, previous studies indicated that LIR-1 associates with SHP-1 phosphatase (19). These data strongly suggested that LIR-1 could represent an additional inhibitory receptor, capable of down-regulating NK cell function upon cross-linking. Indeed, anti-LIR1 mAb inhibited the cytolytic activity of LIR-1+ NK cell clones when analyzed in a redirected killing assay against the Fc{gamma}R+ P815 murine target cells (not shown). Thus, the cross-linking of LIR-1 by specific mAb results in down-regulation of cytolytic activity similar to that obtained under the same experimental conditions with mAb directed to KIR. In addition, experiments using a soluble form of LIR-1 molecule suggested that LIR-1 could also function as a receptor for different HLA class I molecules since anti-HLA class I mAb strongly inhibited the binding of soluble LIR-1 to HLA class I+ cell lines (19). Therefore, we analyzed a series of LIR-1+ NK cell clones to assess whether LIR-1 could function as a HLA class I-specific inhibitory receptor. Target cells were represented by HLA class I cell lines transfected with different HLA class I alleles. LIR-1+ clones were selected on the basis of the lack of expression of known KIR belonging to the Ig superfamily. Notably all the LIR-1+KIR NK clones analyzed expressed the inhibitory CD94–NKG2A complex.

The representative clone A62-6 (LIR-1+, CD94–NKG2A+) has been tested against C1R target cells (which only express low levels of HLA-Cw4 molecules) either untransfected or transfected with HLA-B27 (Bw4) or -B44 (Bw4), or -A1. As shown in Fig. 2(A)Go, untransfected C1R cells were efficiently lysed by clone A62-6, whereas the various C1R transfectants analyzed were highly (B27 and B44) or partially (A1) resistant to lysis. In all instances, addition of the anti-HLA class I mAb A6-136 (IgM) (30) to the cytolytic test, resulted in increments of cytotoxicity against cell transfectants. Similarly, addition of M401 mAb induced lysis of all cell transfectants, although the magnitude of this effect was slightly below that obtained with anti-HLA class I mAb. Addition of the anti-CD94 (Y9) mAb also induced increments of cytotoxicity against cell transfectants; however, this was marginal in the case of cells transfected with Bw4 alleles while it was more evident in the case of cells transfected with HLA-A1. The low levels of reconstitution of cytotoxicity observed against Bw4 transfectants in the presence of anti-CD94 mAb are likely to be consequent to the fact that C1R cells, used in these studies, constitutively express HLA-Cw4 molecules. Indeed these data are in agreement with our previous results indicating that the Bw4 alleles are poorly recognized by CD94–NKG2A receptor (9,31). This has been recently clarified by studies showing that HLA-E expression is not induced in cells which express alleles belonging to the HLA-Bw4 supertype (14). The simultaneous addition of M401 and Y9 mAb resulted in increments of cytotoxicity comparable to that obtained in the presence of anti-class I mAb. Clone A62-6 is representative of 10 distinct LIR-1+, CD94–NKG2A+, KIR clones isolated from different donors.



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Fig. 2. LIR-1 recognizes classical and non classical HLA class I molecules. (A) The representative clone A62/6 (phenotype: p58.1, p58.2, p70, p140, CD94–NKG2A+, LIR-1+) was assessed for cytolytic activity against the C1R target cell line either untransfected or transfected with A1, B27 or B44 HLA class I alleles. (B) The cytolytic activity of clone A62/6 was assessed against the class I 221 target cell line untransfected or transfected with A2, A3, B7 or G1 different HLA class I alleles. (C) The cytolytic activity of clone A62/602 (p58.1, p58.2, NKG2A, LIR-1+) was tested against 221 target cells either untransfected or transfected with Cw3 or Cw4 HLA class I alleles or with HLA-G1 molecules. The cytolytic assays were performed either in the absence ({square}) or in the presence of different mAb: , M401 mAb (anti-LIR-1) at the final concentration of 2,5 µg/ml; , y9 mAb (anti-CD94) (at the final concentration of 1 µg/ml; , a mixture of y9 and M401 mAb; {blacksquare}, A6/136 (anti-class I) (at the final concentration of 1 µg/ml). The cytolytic assays were performed in triplicates; results are expressed as a percent specific 51Cr release ± SD at an E:T ratio of 3:1.

 
Next, the same clone was assessed for cytolytic activity against the class I 221 cells transfected with different class I alleles including HLA-A2, -A3, -B7 (Bw6); -Cw3, -Cw4 (9,32) and -G1 (27).

As shown in Fig. 2(B)Go, clone A62-6 efficiently killed the untransfected 221 target cells, whereas it was unable to kill all the 221 cell transfectants analyzed. Reconstitution experiments in the presence of M401 mAb indicated that LIR-1 was involved in the recognition of HLA-G1, -B7, -A3 and -A2. However, the magnitude of reconstitution was consistently lower than that obtained with anti-HLA class I mAb. It is of note, however, that the cytotoxicity mediated by clone A62-6 against the different cell transfectants was partially reconstituted also by anti-CD94 mAb. Interestingly, only the combined use of mAb directed to both receptors (anti-LIR-1 + anti-CD94) could reconstitute cytotoxicity in a manner comparable in magnitude to that observed with anti-HLA class I mAb. This would indicate that the two receptors, when co-expressed by the same NK cell, cooperate in the generation of the protective effects.

In the case of 221 target cells transfected with HLA-C, most of the inhibitory effect appeared to be mediated by the CD94–NKG2A receptors since reconstititution could only be achieved by the use of anti-CD94 but not by anti-LIR-1 mAb (not shown). In order to better define whether LIR-1 was able or not to recognize HLA-C molecules, we further isolated LIR-1+ NK clones which did not express the inhibitory CD94–NKG2A complex. These clones expressed a CD94 molecular complex unreactive with the NKG2A-specific Z270 mAb (9) and were not inhibited by the cross-linking mediated by anti-CD94 mAb (in redirected killing assays). Since these clones expressed, in all instances, one or another inhibitory receptor belonging to the Ig superfamily, we further selected those lacking the expression of p58.1 and p58.2 receptors specific for HLA-C molecules. As shown in Fig. 2(C)Go, the representative LIR-1+ clone A62-602, although lacking the expression of both CD94–NKG2A and p58 receptors, was partially inhibited by both HLA-Cw3 and -Cw4 molecules. Anti-LIR-1 mAb reconstituted this partial inhibition of cytotoxicity, thus suggesting that LIR-1 may also recognize HLA-C molecules, although with low efficiency.

The low degree of inhibition of lysis of HLA-C+ 221 cell transfectants does not reflect an unefficient transduction of inhibitory signals in these clones since other 221 cell transfectants (including the 221-G1 transfectant shown in Fig. 2CGo) were efficiently protected from lysis.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the present study we show that LIR-1 molecule expressed by NK cell clones is functioning as a HLA class I-specific inhibitory receptor. Remarkably, different from the other known KIR belonging to the Ig superfamily, LIR-1 displays a broad specificity for different HLA class I molecules including HLA-G, a non-classical HLA class I molecule.

In a first set of experiments, the expression of LIR-1 was evaluated in freshly derived or cultured polyclonal NK cell populations. A remarkable variability was observed among different individuals in the percent of NK cells stained by the LIR-1-specific M401 mAb. Comparative analysis of the expression of LIR-1 and of the other known HLA class I-specific inhibitory receptors showed no preferential co-expression of LIR-1 with one or another receptor. On the other hand, while all LIR-1+ NK cells were stained by soluble UL18 protein, no such correlation could be demonstrated between the expression of the CD94–NKG2A and the ability to bind UL18 (19). This finding is in contrast with a previous study reporting that NK cells recognize UL18 via the CD94–NKG2A complex (26). This discrepancy may, at least in part, be explained by differences between the membrane bound form of UL18 molecule and the UL18 fusion protein. Thus our present results do not exclude that the CD94–NKG2A receptor might recognize UL18 when expressed at the surface of cell transfectants. Alternatively the CD94–NKG2A receptor may recognize UL18+ cell transfectants not via a direct interaction with the UL18 protein but rather via the recognition of HLA-E molecules. At present, however, it is not known whether the expression of endogenous HLA-E molecules might be induced by the leader peptide of UL18 (14). Recently, Leong et al. (33) reported that no correlation exists between functional CD94 surface expression and ability of NK cells to kill HCMV infected target cells. Moreover, in this study, UL18, when transfected in target cells, was shown to enhance rather than inhibit NK-mediated target cell lysis. On the other hand, since the NK cells used in this study were not analyzed for surface expression of LIR-1/ILT2, we cannot exclude the existence of activating forms of this receptor (belonging to the LIR/ILT molecules family?).

The specificity of LIR-1 is reminiscent of that described for CD94–NKG2A since both receptors are characterized by the ability to recognize target cell transfected with different HLA class I molecules. However, LIR-1, unlike CD94–NKG2A, also recognizes cells transfected with HLA-Bw4 alleles. These alleles are known to lack leader peptide allowing surface expression of HLA-E molecules. This implies that LIR-1 molecules directly recognize HLA-Bw4 molecules. Similar results have recently been obtained by Colonna et al. (34) who analyzed the specificity of the LIR-1/ILT-2 receptor by using the NK-L cell line (instead of NK clones as in the present study) as effector cells. Differently from the above study, we find that LIR-1 may also recognize HLA-C. This apparently conflicting result may be explained by the fact that the inhibitory effect of HLA-C alleles is marginal and only detectable with NKG2A NK clones. It is of note that the NK-L cell line used by Colonna et al. was LIR-1+ NKG2A+. The absence of a direct binding of soluble LIR-1/ILT-2 molecule to HLA-Cw3+ cell transfectants (34) would confirm that the affinity of this receptor for HLA-C molecules is rather low.

Interestingly, LIR-1 also recognizes HLA-G molecules. In this context, previous studies indicated that cells transfected with HLA-G are recognized by NK cell clones and that the inhibitory receptor involved is, in most instances, represented by the CD94–NKG2A complex (12,3536). However, the leader peptide of HLA-G can induce expression of HLA-E (14), thus suggesting that, also in this case, the molecule recognized on cell transfectants by the CD94–NKG2A receptor may be HLA-E. In addition, a fraction of NK clones displaying HLA-G specificity did not express NKG2A (12). These findings suggested the existence of a different inhibitory receptor specific for HLA-G. Our present study strongly supports the concept that LIR-1 represents a novel inhibitory receptor involved in HLA-G recognition. It is also of note that the magnitude of the inhibition induced by cross-linking of LIR-1, mediated either by HLA class I ligands (expressed on cell transfectants) or by anti-LIR-1 mAb (in redirected killing assays), is generally lower than that of other inhibitory receptors. In addition, the degree of inhibition varied among different LIR-1+ NK clones and was clearly related to the density of LIR-1 expression at the cell surface (not shown). Along this line, it is important to note that LIR-1 is often co-expressed at the NK cell surface with other inhibitory receptors including CD94–NKG2A. In these clones, the function of the two receptors appears to be complementary since reconstitution experiments required the simultaneous presence of anti-LIR-1 and anti-CD94 mAb in order to achieve maximal effects. In light of the recent demonstration that the CD94–NKG2A complex recognizes HLA-E rather than classical HLA class I molecules, it is not surprising that reconstitution of lysis requires masking of both receptors. In conclusion, the heterogeneity of LIR-1-mediated inhibitory effects may depend upon the co-expression and cooperation of one or another inhibitory receptor belonging either to the Ig superfamily or to the CD94 family.


    Acknowledgments
 
This work was supported by grants awarded by the Associazione Italiana per la Ricerca sul Cancro (AIRC), Istituto Superiore di Sanità (ISS), Consiglio Nazionale delle Ricerche (CNR) and Progetto Finalizzato ACRO


    Abbreviations
 
CMVcytomegalovirus
ILTIg-like transcript
ITIMimmunoreceptor tyrosine-based inhibitory motif
KIRkiller inhibitory receptor
LIRleukocyte Ig-like receptor
NKRNK receptor
PEphycoerythrin

    Notes
 
Transmitting editor: G. Doria

Received 8 July 1998, accepted 21 September 1998.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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