- Email: [email protected]
Natural cytotoxicity receptors that trigger human NK-cell-mediated cytolysis
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42 Geginat, J. et al. (1999) Anchorage dependence of mitogen-induced G1 to S transition in primary T lymphocytes. J. Immunol. 162, 5085Ð5093 43 Sedwick, C.E. et al. (1999) TCR, LFA-1, and CD28 play unique and complementary roles in signaling T cell cytoskeletal reorganization. J. Immunol. 162, 1367Ð1375 44 Kennedy, J.S. et al. (1999) Signaling scaffolds in immune cells. Cell Calcium 26, 227Ð235 45 Gallego, M. et al. (1997) Defective actin reorganization and polymerization of WiskottÐAldrich T cells in response to
CD3-mediated stimulation. Blood 90, 3089Ð3097 46 Zhang, J. et al. (1999) Antigen receptor-induced activation and cytoskeletal rearrangement are impaired in WiskottÐAldrich syndrome protein-deficient lymphocytes. J. Exp. Med. 190, 1329Ð1342 47 Grakoui, A. et al. (1999) The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221Ð227 48 Caplan, S. and Baniyash, M. (1995) Multisubunit receptors in the immune system and their association with the cytoskeleton: in search of functional significance. Immunol. Res. 14, 98Ð118
Natural cytotoxicity receptors that trigger human NK-cell-mediated cytolysis Alessandro Moretta, Roberto Biassoni, Cristina Bottino, Maria C. Mingari and Lorenzo Moretta Natural killer (NK) cells can detect whether cells have undergone tumour transformation or viral atural killer (NK) cells Triggering receptors on human NK cells were originally described infection. The discovery of specific Surface molecules expressed by NK cells on the functional basis of inhibitory receptors for major that can trigger the cells have been known their ability to lyse certain for a long time, including CD2 (Ref. 13), tumour cells in the absence of prior stimuhistocompatibility complex class I CD16 (Ref. 14) and CD69 (Ref. 15). However, lation1,2. The molecular mechanism that premolecules clarified the basis of this none of these molecules appears to be invents NK cells from killing indiscriminately, discrimination. However, the volved directly in triggering natural cytotoxsparing normal cells, has recently been clariicity, although a possible role as a co-receptor fied. NK cells express a number of inhibitory receptors responsible for NK-cell has been proposed for CD2 (Ref. 16). Studies receptors that recognize major histocompatitriggering in the process of natural suggest that, in humans, the activating forms bility complex (MHC) class I molecules excytotoxicity remained elusive until of the HLA-C-specific receptor17,18 (p50) and pressed on normal cells. The lack of expresthe CD94ÐNKG2C heterodimer19 play a role sion of one or more MHC class I alleles or the recently. Here, Alessandro Moretta in the NK cytotoxicity against HLA class I1 expression of insufficient amounts of class I and colleagues describe the target cells. Both p50 and NKG2C receptors molecules leads to NK-mediated target-cell identification and characterization were found to be associated with a novel lysis, as originally proposed by Karre and 12 kDa signal-transducing molecule that was Coll in the Ômissing-self hypothesisÕ3Ð5. of several such receptors. In humans, different receptors have been termed KARAP (Ref. 20) and contains an identified that are specific for groups of immunoreceptor tyrosine-based activating HLA types A, B or C alleles. These receptors belong to the immotif (ITAM). KARAP has been cloned and is now referred to as munoglobulin superfamily (Ig-SF) and are characterized by two or DAP-12 (Ref. 21). three extracellular Ig-like domains5Ð9. A second type of receptor speHowever, a major characteristic of NK cells is the efficient lysis of cific for HLAs is formed by the association of CD94 molecules with target cells that are deficient or completely lacking in HLA class I members of the NKG2 family10,11. Remarkably, the various inmolecules10,22. Moreover, NK-cell-mediated lysis of HLA class I1 tarhibitory receptors of NK cells can be expressed by some activated get cells can be induced by masking the HLA class I molecules on cytolytic T cells12. Although detailed information is now available target cells with monoclonal antibodies5 (mAbs). These observations on the receptors and molecular mechanisms that lead, upon recogimply the existence of NK-cell receptors that are responsible for the nition of HLA class I molecules, to NK-cell inactivation, the cellinduction of NK-cell triggering in an HLA-independent fashion. surface receptors responsible for NK-cell triggering during natural Indeed, different receptorÐligand interactions are probably responcytotoxicity have remained largely unknown. sible for NK-cell activation upon interaction with target cells. For
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example, NK-cell clones can be heterogeneous in their ability to lyse different HLA class I2 target cells23. These data agree with the concept that NK-cell triggering in an HLA-independent context is the result of multiple interactions between NK receptors and their ligands on target cells. In the search for receptors responsible for NK-cell triggering in the process of nonMHC-restricted natural cytotoxicity, three novel NK-cell-specific triggering surface molecules have been identified that appear to play a critical role in the induction of NK-cell-mediated cytotoxicity24Ð28. These molecules represent the first members of a novel emerging group of receptors that we termed Ônatural cytoxicity receptorsÕ (NCRs).
Strategy for identifying NCRs in human NK cells A putative NCR should satisfy the following requirements: (1) its expression should be mostly restricted to NK cells; (2) its mAbmediated crosslinking in a redirected killing assay should trigger NK-cell cytotoxicity; (3) mAb-mediated masking of the NCR should inhibit the NK-cell-mediated cytotoxicity. The search for NCRs would be facilitated if different target cells were heterogeneous in their surface expression of relevant NCR ligands. In this context, we hypothesized the existence of at least three potential situations (Fig. 1): (1) target cells displaying a complete set of ligands for the various NCRs expressed by a given NK-cell clone; (2) target cells that express only a limited number of ligands for the NCRs; (3) target cells that only express a single ligand for an NCR.
suggested that NKp46 is the only triggering receptor expressed by human NK cells that is capable of recognizing a ligand on the surface of murine cells23Ð27. This means that the ligand(s) for NKp46 might be conserved between human and mouse. As discussed below, an NKp46 homologue has been cloned from the mouse28, which suggests that at least some NCRÐligand interactions that mediate NKcell triggering and target-cell lysis are conserved between species. In this context, and different from NKp46, the inhibitory (or activating) receptors that recognize the allelic determinants of MHC class I molecules seem to be highly species specific. This could be the result of adapting to the polymorphism of MHC class I molecules, which are known to display relatively rapid and independent evolution in different species9. Although NKp46 was highly expressed at the NK-cell surface (as measured by brightness of luminescence) in the majority of donors, some individuals had a proportion of NK cells (which varied up to 90%) expressing a ÔdullÕ NKp46 phenotype23. In general, these donors displayed a low cytolytic activity against a variety of NK-susceptible tumour targets. The NKp46dull phenotype detected in fresh NK cells was also maintained in cultured NK cells or NK-cell clones. NKp46dull clones displayed a low cytolytic activity compared with that of NKp46bright clones (isolated from either the same or different individuals). Taken together, these data support the notion that NKp46 plays a central role in the physiological triggering of NK cells and, as a consequence, in allowing the NK-cell-mediated clearance of HLA class I2 cells.
NKp46, the prototype NCR
NKp44: an NCR selectively expressed by activated NK cells
The experimental approaches based on the above strategy led to the identification of NKp46, which is the prototype NCR. Analysis of the tissue distribution using the reverse-transcriptase polymerase chain reaction (RT-PCR) and specific mAbs showed that NKp46 is expressed by all NK cells (both resting and activated) but is absent from all other cell types analysed24Ð27. The relevance of this finding is related to the fact that none of the markers available so far is truly NK-specific. In addition, NKp46 appears to be a major triggering receptor of NK cells, which, under crosslinking conditions, leads to Ca21 mobilization, cytotoxicity and cytokine release24. More importantly, it appears to have a central role as a receptor in the lysis of different targets, including normal and tumour-transformed cells of autologous, allogeneic or xenogeneic origin23. Indeed, NKp46 masking by specific mAbs inhibited lysis of the majority of human tumours belonging to different histotypes, including lung, liver and breast carcinomas, melanoma, and EpsteinÐ Barr virus (EBV)-transformed cell lines23Ð27. However, correct evaluation of the actual involvement of NKp46 in the triggering of cytotoxicity (as assessed by mAb-mediated-masking experiments) might require accurate selection of the target cells analysed (Fig. 1). In particular, the use of murine-tumour target cells (including Bw1502 and YAC cells) provided direct evidence that NKp46 is sufficient to mediate NK cytotoxicity (that is, it does not necessarily require coengagement of other triggering receptors). In addition, it
It is well known that culture with interleukin 2 (IL-2) increases the ability of NK cells to mediate non-MHC-restricted tumour-cell lysis2. In addition to the upregulation of cytolytic activity against NKsusceptible target cells, NK cells acquire the ability to lyse target cells that are resistant to fresh NK cells. This phenomenon may reflect, at least in part, the de novo expression of novel NCRs that allow activated NK cells to recognize additional ligands, resulting in moreefficient NK-cell triggering. In this context, we identified a novel 44 kDa surface molecule (NKp44) that is absent in freshly isolated peripheral-blood lymphocytes but is progressively expressed by all NK cells in vitro upon culture in IL-2; indeed, all NK-cell clones analysed so far express NKp44 (Ref. 25). Unlike other markers of lymphocyte activation, NKp44 is absent from activated T cells or T-cell clones; indeed, like NKp46, NKp44 was absent from all the other cell lineages analysed. Therefore, NKp44 appears to be the first marker specific for activated human NK cells. The only exceptions so far are two T-cell receptor gd1 clones derived from a melanoma patient that expressed very low levels of NKp44 at their surface25. Masking of NKp44 mediated by mAbs partially inhibited cytolytic activity against certain (HLA class I2 FcgR2) tumour target cells25. Remarkably, the degree of inhibition was greatly increased by the simultaneous masking of NKp46. Therefore, NKp44 appears to function as an NCR that is selectively expressed by activated NK
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NKp30: a third NCR
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Fig. 1. The identification of putative natural cytoxicity receptors (NCRs), which can trigger natural killer (NK) cells. This approach was based on the assumption that tumour target cells susceptible to NK-cell-mediated cytotoxicity might differ greatly in the surface expression of ligands for the NCRs. The representative NK-cell clone Y, used as a source of cytolytic effector cells, is shown to express several different NCRs, which recognize ligands that are expressed differently by a panel of allogeneic or xenogeneic tumour cells. Three representative target cells are illustrated. (a) A target that expresses the complete set of ligands recognized by the various NCRs of clone Y. The occurrence of multiple NCRÐligand interactions is likely to result in optimal NK-cell triggering and thus in the efficient lysis of that target. In this case, a monoclonal antibody (mAb) directed to a given NCR would be expected to inhibit the lysis of target cells only marginally, because efficient NK-cell triggering might still occur via the other NCRÐligand interactions. (b) A target that does not express one or more of the ligands recognized by the NCRs of clone Y, in which case the addition of a mAb directed to a given NCR should result in a sharp, but still incomplete, inhibition of the NK-cell-mediated target-cell lysis. Under these conditions, the number of NCRÐligand interactions should be strongly reduced. (c) A target that expresses only one relevant ligand for the NCRs of clone Y. In this case, mAb-mediated masking of the relevant NCR should virtually abolish cytotoxicity because, under these conditions, no relevant NCRÐligand interactions would remain available. The right-hand panels show the actual data obtained in chromium release cytotoxicity assays in which the ability of clone Y to kill the different types of target cells was evaluated (51Cr is released by target cells if damaged by clone Y) in either the absence (white) or the presence (blue) of the BAB281 (anti-NKp46) mAb. The A549 lung carcinoma (representative of target-type A) is lysed efficiently by clone Y and this lysis is only marginally inhibited by anti-NKp46 mAb. The melanoma M14 (representative of target-type B) is also lysed efficiently, but, in this case, the addition of anti-NKp46 mAb results in a substantial inhibition of lysis. The murine thymoma BW1502 (representative of target-type C) is lysed less efficiently by the human clone Y (compare the E:T ratios). Adding anti-NKp46 mAb completely inhibits lysis, thus strongly suggesting that the ligand for NKp46 may be the only one expressed by these cells and that, in this case, NKp46 is the only NCR responsible for the induction of cytotoxicity. Abbreviations: Ab, antibody; E:T, effector:target cell ratio.
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cells and that might cooperate with other NCRs in the process of non-MHC-restricted lysis. An example of the complementary role of NKp44 and NKp46 molecules in the lysis of an HLA class I2 FcgR2 tumour target cell (highly susceptible to lysis by a representative NK-cell clone) is shown in Fig. 2.
Although the NK-mediated lysis of various allogeneic tumour cell lines was sharply inhibited by mAb-mediated masking of NKp46 or NKp44, the lysis of other tumour cell lines was only marginally affected. This suggests that there are additional NCRs that play a prominent role in the induction of NK-cellmediated cytotoxicity against these target cells. Indeed, an additional NCR, termed NKp30, has recently been identifed and characterized using specific mAbs (Ref. 26). NKp30 was found to cooperate with NKp46 and NKp44 in the induction of cytotoxicity against a variety of target cells. Moreover, NKp30 is the major receptor responsible for killing some tumour target cells, for which NKp46 or NKp44 do not appear to play a significant role26. NKp30, like NKp46, is selectively expressed by all NK cells, both freshly isolated and cultured in IL-2. Remarkably, the surface expression of NKp30 parallels that of NKp46, as NK cells displaying an NKp46dull or an NKp46bright phenotype were also characterized by ÔdullÕ or ÔbrightÕ fluorescence with NKp30 (Ref. 26). The demonstration that NK cells express parallel densities of different triggering receptors might explain the existence of NK-cell subsets displaying different ÔnaturalÕ cytolytic activity. Thus, it is now possible to identify NK cells displaying an NCRbright phenotype (i.e. high surface density of NKp46, NKp44 and NKp30) and NK cells characterized by an NCRdull phenotype.
Molecular structure of NCRs The molecular cloning of NKp4627, NKp4429 and NKp3026 revealed novel members of the Ig-SF with no homology to each other and a low degree of homology to known human molecules (Fig. 3). When NKp46 was cloned27, it was found to be a type I transmembrane glycoprotein with two C2-type
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2B4 functions as a co-receptor in NK-cell triggering Studies of mAb-mediated masking of NCR and functional analysis of numerous NK-cell clones suggested that other receptors should cooperate with NKp46, NKp44 and NKp30 to induce optimal NK-cell triggering. A good candidate for this function was the 2B4 molecule, which activates NK-cell cytotoxicity
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Ig-like domains in its extracellular portion, a transmembrane region containing a positively charged amino acid (Arg), which might be involved in stabilizing the interaction(s) with transmembrane adapter molecules, and a cytoplasmic portion (30 amino acids long) that does not contain sequence motifs typically involved in the activation of the signal cascade(s), such as an ITAM. Biochemical analysis revealed that NKp46 molecules are coupled to the intracytoplasmic signal-transduction machinery with the ITAM-containing CD3z and/or FceRg adapter polypeptides24,29. These signaltransducing molecules become tyrosine phosphorylated after NKp46 crosslinking by specific mAbs (Refs 25, 27, 29). As with other members of the Ig-SF expressed on NK cells [including KIR, KAR (Ref. 6Ð8) and ILT (Refs 30, 31)], the NKp46-encoding gene maps to human chromosome 19 (Ref. 27). Recently, the murine28 and rat32 homologues of the human NKp46 receptor have been cloned. Remarkably, the murine gene for NKp46 maps to mouse chromosome 7, which is syntenic to human chromosome 19. NKp44 is characterized by a single extracellular V-type domain29. Its transmembrane region contains the charged amino acid Lys, which is likely to mediate association with the ITAM-bearing adapter molecules KARAP/ DAP-12 (Refs 25Ð29). The NKp44 gene has been mapped to human chromosome 6 (Ref. 29). NKp30 was found to be the product of 1C7, a gene previously mapped to human chromosome 6, in the HLA class III region41. NKp30 contains a single V-type domain and has a charged residue (Arg) in its transmembrane portion26. As revealed by biochemical analysis, and similar to NKp46, NKp30 associates with the CD3z chain-adapter molecule responsible for signal transduction26.
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Fig. 2. The synergistic effect of natural cytoxicity receptors (NCRs) in the natural killer (NK)-cellmediated lysis of a representative HLA class I2 human tumour. The representative NK-cell clone Y expresses all known NCR molecules (a property common to all activated NK-cell populations and clones). The A549 lung carcinoma target cell (representative of targets ÔAÕ from Fig. 1) is highly susceptible to NK-cell-mediated lysis. The left-hand side of the figure shows the different experimental conditions used Ð addition to the cytolytic test of: (a) anti-NKp46 mAb alone; (b) anti-NKp44 mAb alone; (c) a mixture of the two mAbs. Clone Y expressed, in addition to NKp46 and NKp44, at least one additional NCR (NKp30). The right-hand side of the figure shows the actual data: the addition of either anti-NKp46 (blue) or anti-NKp44 (red) alone induces a low degree of inhibition of target-cell lysis; the combined use of the two mAbs (green) induces a strong, but not complete, inhibition. The residual cytolytic effect observed upon mAb-mediated masking of NKp46 and NKp44 is mostly due to the interaction of NKp30 with its ligand on target cells. Indeed, the combined use of mAbs against three different NCRs resulted in complete inhibition (d). Abbreviations: see Fig. 1 legend.
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Box 1. Characteristics of the natural cytoxicity receptors ¥ Natural cytoxicity receptors (NCRs) are selectively expressed by natural killer (NK) cells and thus represent the most reliable NK-cell markers. In particular, NKp46 and NKp30 are expressed by all resting and activated NK cells, whereas NKp44 is expressed by activated NK cells only. ¥ All NCRs belong to the immunoglobulin superfamily but have little homology with known human cell-surface molecules or with each other. ¥ NKp46 is conserved in other species, including the mouse and rat. ¥ Upon crosslinking, NCRs mediate NK-cell triggering, leading to target-cell lysis and cytokine production. ¥ The function of NCRs is normally downregulated by coaggregation with killer inhibitory receptors (KIRs). Thus, the induction of NK-cell activation via NCRs is possible only in the absence of a KIRÐHLA class I interaction. ¥ NCRs are associated with different signal-transducing molecules. ¥ Disruption of the NCRÐligand interactions by mAb-mediated masking of NCRs inhibits NK-cell-mediated cytotoxicity. ¥ The surface density of NCRs might differ between NK cells. The density correlates directly with their natural cytotoxicity. ¥ The nature of the NCR-specific ligands has still to be defined. However, experimental evidence suggests that their expression is different in different target cells. ¥ NKp46 recognizes ligands expressed on both normal and tumour cells, including xenogeneic cells.
in both mouse and human when either crosslinked with mAbs or engaged with its specific ligand (CD48)33Ð36. In contrast to NCRs, 2B4 is expressed not only by all NK cells but also by a subset of CD81 T cells, monocytes and basophils. Structurally, human 2B4 belongs to the CD2 subfamily of the Ig-SF and contains a membrane-distal IgV domain and a single membrane-proximal type-C2 Ig-like domain; the cytoplasmic tail of 2B4 contains tyrosine-based motifs34. In cell transfectants, these motifs allow 2B4 to interact with the Src homology 2 (SH2) domaincontaining molecule SH2D1A (Ref. 37). This interaction appears to prevent 2B4 from associating with the cytoplasmic tyrosine phosphatase SHP2. At present, the precise mechanisms by which 2B4 triggering leads to NK-cell activation remain largely unknown. As the 2B4ÐSH2D1A interaction requires tyrosine phosphorylation of 2B4 molecules, it will be important to define the in vivo role of different kinases. Recently, 2B4 has been shown to function as a co-receptor in human NK-cell activation induced by different NCRs or CD16 (Ref. 38). In this context, previous studies showed a clonal heterogeneity of NK cells in the response to anti-2B4 mAbs in redirected killing assays, which might suggest a functional heterogeneity of the 2B4 molecules themselves. However, the ability of NK clones to
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respond to anti-2B4 mAb was found to correlate strictly with their NCR phenotype38. Thus, NK-cell clones with a high surface density of NKp46 (NKp46bright) were efficiently triggered by anti-2B4 mAb. By contrast, NKp46dull clones were not, in spite of the fact that they had a similar surface density of 2B4. Modulation of NKp46 surface molecules in NKp46bright clones by mAbs had no effect on the expression of 2B4 but prevented cells from subsequently being triggered by anti-2B4 mAb. Moreover, anti-2B4 mAb could trigger NKp46dull NKcell clones in a redirected killing assay, provided that suboptimal doses of anti-NKp44 or anti-CD16 mAb were added38. These results indicate that NK-cell responses to the engagement of 2B4 depend upon the coengagement of other triggering receptors38.
Does NKG2D have a role in NK-cell-mediated cytotoxicity? A role in NK-cell triggering has also recently been suggested for NKG2D, a receptor for the stress-inducible ligand MICA (Ref. 39) (an MHC-related protein that is broadly expressed on epithelial tumours). NKG2D is a 42 kDa transmembrane molecule belonging to the C-type lectin superfamily, which maps to chromosome 12. Signalling via NKG2D appears to be mediated by a novel polypeptide, called DAP-10 and characterized by an SH2-domain-binding site in its cytoplasmic tail that recruits the p85 subunit of phosphoinositide 3-kinase (PI-3 kinase)40. At least three differences exist between NCRs and NKG2D: (1) NKG2D is expressed on most gd and virtually all CD81 ab T cells; (2) the ligand recognized by NKG2D is not expressed on normal cells and its expression on tumour cells is restricted to certain histological types; (3) triggering by NKG2D would appear to over-rule the inhibitory signals delivered by the KIR and HLA class I interactions, which makes sense as MICA is not expressed on normal cells24Ð26,40. The precise contribution of NKG2D to NK-cell function has yet to be determined. Indeed, its expression and triggering effects on NK cells have been analysed in detail only in a tumour NK-cell line. NKG2D might play a dominant role in the NK-cell-mediated lysis of rare target cells that appear to be killed by NCR-independent mechanisms.
NCR ligands The discovery of novel surface molecules expressed by NK cells that are responsible for triggering the natural cytotoxicity has shed new light on our understanding of how NK cells function. NCRs appear to act as triggering receptors in the lysis of MHC class I2 allogeneic and xenogeneic tumour cells in vitro. However, what could the role of NCRs be under more-physiological conditions (i.e. against autologous cells)? The involvement of NCRs was analysed in the lysis of EBV-transformed autologous cell lines and autologous phytohaemagglutinin (PHA) blasts23,26. Remarkably, these cells, unlike tumour cells, are protected from NK-cell-mediated lysis because they express a normal level of (self) HLA class I molecules. To overcome this experimental difficulty, in these studies, the HLA class I molecules were masked by suitable anti-HLA class I
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Fig. 3. The molecular structure of natural cytoxicity receptors (NCRs) and their association with distinct signal-transducing molecules. NKp46 has an extracellular portion characterized by two immunoglobulin-like (Ig-like) domains of type C2. NKp30 and NKp44 have an extracellular region containing a single Ig-like domain of type V and NKp44 displays a membrane-proximal region with an extended open conformation typical of hinge-like sequences. NCR transmembrane portions contain positively charged amino acids that are thought to be crucial for their association with distinct signal-transducing molecules bearing immunoreceptor tyrosine-based activating motifs. Putative N- or O-linked glycosylation sites are indicated as horizontal or angled lines, respectively. mAb to prevent inhibition mediated by killer inhibitory receptors. Under these conditions, NK cells efficiently lysed autologous target cells. Moreover, mAb-mediated masking of NCRs resulted in a substantial (50Ð80%) inhibition of lysis in this experimental setting. These data imply that NCRs are involved in the recognition of ligands that are also expressed on autologous untransformed cells23,26. Although the ligands for NCRs appear to be expressed on both normal and tumour cells, it is possible that their expression could be modified by cellular stress, cell activation or tumour transformation. Moreover, as discussed above, some tumour cells appear to lack certain NCR ligands. This could be the result of the in vivo selection of antigen-loss mutants, possibly reflecting a mechanism of tumour escape from NK-mediated attack. If this is true, tumour cells could escape not only cytotoxic-Tcell-mediated control (selecting MHC class I loss variants) but also NK-cell-mediated control (upon loss of NCR-specific ligands). In order to identify the NCR ligands, different approaches are being attempted, mainly based on the use of soluble receptors. The identification of the NCR ligands and the definition of their cellular distribution, as well as of possible mechanisms leading to their expression, will help to identify tissues and cell types that might be susceptible to NK-cell lysis.
cytotoxicity. In order to prevent damage to normal cells, NCRs are usually under the control of HLA class I-specific inhibitory receptors that provide the ÔoffÕ signals. NCRs are strictly NK-cell specific and recognize ligands expressed on both normal and tumour cells. However, the existence of target cells such as certain T-cell-leukaemia lines that are lysed independently of NKp46, NKp44 or NKp30 implies a role for additional, still undefined, triggering receptors. Based on the strict correlation between NCR phenotype and the magnitude of the natural cytotoxicity, the expression of these stillunknown receptors might be confined to NK cells with the NCRbright phenotype. Alternatively, these receptors could be uniformly expressed by all human NK cells but require for their function a concomitant engagement of other NCRs; they would thus be co-receptors. Indeed, a co-receptor function has recently been demonstrated for the human 2B4 molecule: even though all NK cells express comparable surface amounts of 2B4, the ability to trigger cytotoxicity upon ligand binding by 2B4 varies with the NCRbright or NCRdull phenotype. In conclusion, the emerging family of NCRs and co-receptors is still growing, underscoring the complexity of NK-cell activation and regulation.
The authorsÕ work was supported by grants awarded by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), Istituto Superiore di Sanitˆ (I.S.S.),
Concluding remarks
Ministero della Sanitˆ and Ministero dellÕUniversitˆ e della Ricerca Scientifica
The recent identification and molecular cloning of NCRs shed new light on our understanding of NK-cell function (Box 1; Fig. 3). Indeed, they play a major role in mediating the ÔonÕ signals during natural
e Tecnologica (M.U.R.S.T.), and Consiglio Nazionale delle Ricerche, Progetto Finalizzato Biotecnologie. The financial support of Telethon-Italy (grant no. E.0892) is gratefully acknowledged.
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Alessandro Moretta and Lorenzo Moretta ([email protected]. unige.it) are at the Dipartimento di Medicina Sperimentale, University of Genova, Genova, Italy; Roberto Biassoni, Cristina Bottino and Maria Mingari are at the Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; Maria Mingari is also at the Dipartimento di Oncologia, Biologia e Genetica, University of Genova, Genova, Italy. Lorenzo Moretta is also at the Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy.
References 1 Herberman, R.B. et al. (1976) Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumours, I: distribution of reactivity and specificity. Int. J. Cancer 16, 216Ð229 2 Trinchieri, G. (1989) Biology of natural killer cells. Adv. Immunol. 47, 187Ð376 3 Ljunggren, H.G. and Karre, K. (1990) In search of the Ômissing selfÕ: MHC molecules and NK cell recognition. Immunol. Today 11, 237Ð244 4 Moretta, L. et al. (1994) Human NK cells: origin, clonality, specificity and receptors. Adv. Immunol. 55, 341Ð380 5 Moretta, A. et al. (1996) Receptors for HLA-class I-molecules in human natural killer cells. Annu. Rev. Immunol. 14, 619Ð648 6 Lanier, L.L. (1998) NK cell receptors. Annu. Rev. Immunol. 16, 359Ð393 7 Long, E.O. (1999) Regulation of immune responses through inhibitory receptors. Annu. Rev. Immunol. 17, 875Ð904 8 Colonna, M. (1997) Specificity and function of immunoglobulin superfamily NK cell inhibitory and stimulatory receptors. Immunol. Rev. 155, 127Ð133 9 Valiante, N.M. et al. (1997) Killer cell receptors: keeping pace with MHC class I evolution. Immunol. Rev. 155, 155Ð164 10 Moretta, A. et al. (1997) Major histocompatibility complex class I-specific receptors on human natural killer and T lymphocytes. Immunol. Rev. 155, 105Ð117 11 Lopez-Botet, M. et al. (1997) Structure and function of the CD94 C-type lectin receptor complex involved in recognition of HLA class I molecules. Immunol. Rev. 155, 165Ð174 12 Mingari, M.C. et al. (1998) Regulation of KIR expression in human T lymphocytes: a safety mechanism which may impair protective T cell responses. Immunol. Today 19, 153Ð157 13 Bolhuis, R.L.H. et al. (1986) Induction and blocking of cytolysis in CD21, CD32 NK and CD21, CD31 cytotoxic T lymphocytes via CD2 50 kD sheep erythrocyte receptor. J. Immunol. 136, 3939Ð3944 14 Lanier, L.L. et al. (1988) Functional and biochemical analysis of CD16 antigen on natural killer cells and granulocytes. J. Immunol. 141, 3478Ð3485 15 Moretta, A. et al. (1991) CD69-mediated pathway of lymphocyte activation: anti-CD69 mAbs trigger the cytolytic activity of different lymphoid effector cells with the exception of cytolytic T lymphocytes expressing TCR a/b. J. Exp. Med. 174, 1393Ð1398 16 Lanier, L.L. et al. (1997) Arousal and inhibition of human NK cells. Immunol. Rev. 155, 145Ð154 17 Moretta, A. et al. (1995) Existence of both inhibitory (p58) and activatory (p50) receptors for HLA-C molecules in human natural killer cells. J. Exp. Med. 182, 875Ð884 18 Biassoni, R. et al. (1996) The human leukocyte antigen (HLA)-Cspecific ÔactivatoryÕ or ÔinhibitoryÕ natural killer cell receptors display highly homologous extracellular domains but differ in their transmembrane and intracytoplasmic portions. J. Exp. Med. 183, 645Ð650 19 Cantoni, C. et al. (1998) The activating form of CD94 receptor complex: the CD94-associated Kp39 protein represents the product of the NKG2-C gene. Eur. J. Immunol. 28, 327Ð338
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20 Olcese, L. et al. (1997) Human killer cell activatory are included in a multimeric complex expressed by natural killer cells. J. Immunol. 158, 5083Ð5086 21 Lanier, L.L. et al. (1998) Immunoreceptor DAP12 bearing a tyrosinebased activation motif is involved in activating NK cells. Nature 391, 703Ð707 22 Moretta, L. et al. (1992) Allorecognition by NK cells: nonself or no self? Immunol. Today 13, 300Ð306 23 Sivori, S. et al. (1999) NKp46 is the major triggering receptor involved in the natural cytotoxicity of fresh of cultured human NK cells: correlation between surface density of NKp46 and natural cytotoxicity against autologous, allogeneic or xenogeneic target cells. Eur. J. Immunol. 29, 1647Ð1655 24 Sivori, S. et al. (1997) p46, a novel natural killer cell-specific surface molecule which mediates cell activation. J. Exp. Med. 186, 1129Ð1136 25 Vitale, M. et al. (1998) NKp44, a novel triggering surface molecule specifically expressed by activated natural killer cells is involved in non-MHC restricted tumor cell lysis. J. Exp. Med. 187, 2065Ð2072 26 Pende, D. et al. (1999) Identification and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells. J. Exp. Med. 190, 1505Ð1516 27 Pessino, A. et al. (1998) Molecular cloning of NKp46: a novel member of the immunoglobulin superfamily involved in triggering of natural cytotoxicity. J. Exp. Med. 188, 953Ð960 28 Biassoni, R. et al. (1999) The murine homologue of the human NKp46, a triggering receptor involved in the induction of natural cytotoxicity. Eur. J. Immunol. 29, 1014Ð1020 29 Cantoni, C. et al. (1999) NKp44, a triggering receptor involved in tumor cell lysis by activated human natural killer cells, is a novel member of the immunoglobulin superfamily. J. Exp. Med. 189, 787Ð796 30 Cosman, D. et al. (1997) A novel immunoglobulin superfamily receptor for cellular and viral MHC class I molecules. Immunity 7, 273Ð282 31 Samaridis, J. and Colonna, M. (1997) Cloning of novel immunoglobulin superfamily receptors expressed on human myeloid and lymphoid cells: structural evidence for new stimulatory and inhibitory pathways. Eur. J. Immunol. 27, 660Ð665 32 Falco, M. et al. (1999) Identification of the rat homologue of the human NKp46 triggering receptor. Immunol. Lett. 68, 411Ð414 33 Moretta, A. et al. (1992) Novel surface molecules involved in human NK cell activation and triggering of the lytic machinery. Int. J. Cancer 7, 6Ð10 34 Mathew, P.A. et al. (1993) Cloning and characterization of the 2B4 gene encoding a molecule associated with non-MHC-restricted killing mediated by activated natural killer cells and T cells. J. Immunol. 151, 5328Ð5337 35 Valiante, N.M. and Trinchieri, G. (1993) Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes. J. Exp. Med. 178, 1397Ð406 36 Nakajima, H. et al. (1999) Activating interactions in human NK cell recognition: the role of 2B4ÐCD48. Eur. J. Immunol. 29, 1676Ð1683 37 Tangye, S.G. et al. (1999) Human 2B4, an activating NK cell receptor, recruits the protein tyrosine phosphatase SHP-2 and the adaptor signaling protein SAP. J. Immunol. 162, 6981Ð6985 38 Sivori, S. et al. (2000) 2B4 functions as a co-receptor in human natural killer cell activation. Eur. J. Immunol. 30, 787Ð793 39 Bauer, S. et al. (1999) Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285, 727Ð729 40 Wu, J. et al. (1999) An activating immunoreceptor complex formed by NKG2D and DAP-10. Science 285, 730Ð732 41 Nalabolu, S.R. et al. (1996) Genes in a 220-Kb region spanning the TNF cluster in human MHC. Genomics 31, 215Ð222
42 Geginat, J. et al. (1999) Anchorage dependence of mitogen-induced G1 to S transition in primary T lymphocytes. J. Immunol. 162, 5085Ð5093 43 Sedwick, C.E. et al. (1999) TCR, LFA-1, and CD28 play unique and complementary roles in signaling T cell cytoskeletal reorganization. J. Immunol. 162, 1367Ð1375 44 Kennedy, J.S. et al. (1999) Signaling scaffolds in immune cells. Cell Calcium 26, 227Ð235 45 Gallego, M. et al. (1997) Defective actin reorganization and polymerization of WiskottÐAldrich T cells in response to
CD3-mediated stimulation. Blood 90, 3089Ð3097 46 Zhang, J. et al. (1999) Antigen receptor-induced activation and cytoskeletal rearrangement are impaired in WiskottÐAldrich syndrome protein-deficient lymphocytes. J. Exp. Med. 190, 1329Ð1342 47 Grakoui, A. et al. (1999) The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221Ð227 48 Caplan, S. and Baniyash, M. (1995) Multisubunit receptors in the immune system and their association with the cytoskeleton: in search of functional significance. Immunol. Res. 14, 98Ð118
Natural cytotoxicity receptors that trigger human NK-cell-mediated cytolysis Alessandro Moretta, Roberto Biassoni, Cristina Bottino, Maria C. Mingari and Lorenzo Moretta Natural killer (NK) cells can detect whether cells have undergone tumour transformation or viral atural killer (NK) cells Triggering receptors on human NK cells were originally described infection. The discovery of specific Surface molecules expressed by NK cells on the functional basis of inhibitory receptors for major that can trigger the cells have been known their ability to lyse certain for a long time, including CD2 (Ref. 13), tumour cells in the absence of prior stimuhistocompatibility complex class I CD16 (Ref. 14) and CD69 (Ref. 15). However, lation1,2. The molecular mechanism that premolecules clarified the basis of this none of these molecules appears to be invents NK cells from killing indiscriminately, discrimination. However, the volved directly in triggering natural cytotoxsparing normal cells, has recently been clariicity, although a possible role as a co-receptor fied. NK cells express a number of inhibitory receptors responsible for NK-cell has been proposed for CD2 (Ref. 16). Studies receptors that recognize major histocompatitriggering in the process of natural suggest that, in humans, the activating forms bility complex (MHC) class I molecules excytotoxicity remained elusive until of the HLA-C-specific receptor17,18 (p50) and pressed on normal cells. The lack of expresthe CD94ÐNKG2C heterodimer19 play a role sion of one or more MHC class I alleles or the recently. Here, Alessandro Moretta in the NK cytotoxicity against HLA class I1 expression of insufficient amounts of class I and colleagues describe the target cells. Both p50 and NKG2C receptors molecules leads to NK-mediated target-cell identification and characterization were found to be associated with a novel lysis, as originally proposed by Karre and 12 kDa signal-transducing molecule that was Coll in the Ômissing-self hypothesisÕ3Ð5. of several such receptors. In humans, different receptors have been termed KARAP (Ref. 20) and contains an identified that are specific for groups of immunoreceptor tyrosine-based activating HLA types A, B or C alleles. These receptors belong to the immotif (ITAM). KARAP has been cloned and is now referred to as munoglobulin superfamily (Ig-SF) and are characterized by two or DAP-12 (Ref. 21). three extracellular Ig-like domains5Ð9. A second type of receptor speHowever, a major characteristic of NK cells is the efficient lysis of cific for HLAs is formed by the association of CD94 molecules with target cells that are deficient or completely lacking in HLA class I members of the NKG2 family10,11. Remarkably, the various inmolecules10,22. Moreover, NK-cell-mediated lysis of HLA class I1 tarhibitory receptors of NK cells can be expressed by some activated get cells can be induced by masking the HLA class I molecules on cytolytic T cells12. Although detailed information is now available target cells with monoclonal antibodies5 (mAbs). These observations on the receptors and molecular mechanisms that lead, upon recogimply the existence of NK-cell receptors that are responsible for the nition of HLA class I molecules, to NK-cell inactivation, the cellinduction of NK-cell triggering in an HLA-independent fashion. surface receptors responsible for NK-cell triggering during natural Indeed, different receptorÐligand interactions are probably responcytotoxicity have remained largely unknown. sible for NK-cell activation upon interaction with target cells. For
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example, NK-cell clones can be heterogeneous in their ability to lyse different HLA class I2 target cells23. These data agree with the concept that NK-cell triggering in an HLA-independent context is the result of multiple interactions between NK receptors and their ligands on target cells. In the search for receptors responsible for NK-cell triggering in the process of nonMHC-restricted natural cytotoxicity, three novel NK-cell-specific triggering surface molecules have been identified that appear to play a critical role in the induction of NK-cell-mediated cytotoxicity24Ð28. These molecules represent the first members of a novel emerging group of receptors that we termed Ônatural cytoxicity receptorsÕ (NCRs).
Strategy for identifying NCRs in human NK cells A putative NCR should satisfy the following requirements: (1) its expression should be mostly restricted to NK cells; (2) its mAbmediated crosslinking in a redirected killing assay should trigger NK-cell cytotoxicity; (3) mAb-mediated masking of the NCR should inhibit the NK-cell-mediated cytotoxicity. The search for NCRs would be facilitated if different target cells were heterogeneous in their surface expression of relevant NCR ligands. In this context, we hypothesized the existence of at least three potential situations (Fig. 1): (1) target cells displaying a complete set of ligands for the various NCRs expressed by a given NK-cell clone; (2) target cells that express only a limited number of ligands for the NCRs; (3) target cells that only express a single ligand for an NCR.
suggested that NKp46 is the only triggering receptor expressed by human NK cells that is capable of recognizing a ligand on the surface of murine cells23Ð27. This means that the ligand(s) for NKp46 might be conserved between human and mouse. As discussed below, an NKp46 homologue has been cloned from the mouse28, which suggests that at least some NCRÐligand interactions that mediate NKcell triggering and target-cell lysis are conserved between species. In this context, and different from NKp46, the inhibitory (or activating) receptors that recognize the allelic determinants of MHC class I molecules seem to be highly species specific. This could be the result of adapting to the polymorphism of MHC class I molecules, which are known to display relatively rapid and independent evolution in different species9. Although NKp46 was highly expressed at the NK-cell surface (as measured by brightness of luminescence) in the majority of donors, some individuals had a proportion of NK cells (which varied up to 90%) expressing a ÔdullÕ NKp46 phenotype23. In general, these donors displayed a low cytolytic activity against a variety of NK-susceptible tumour targets. The NKp46dull phenotype detected in fresh NK cells was also maintained in cultured NK cells or NK-cell clones. NKp46dull clones displayed a low cytolytic activity compared with that of NKp46bright clones (isolated from either the same or different individuals). Taken together, these data support the notion that NKp46 plays a central role in the physiological triggering of NK cells and, as a consequence, in allowing the NK-cell-mediated clearance of HLA class I2 cells.
NKp46, the prototype NCR
NKp44: an NCR selectively expressed by activated NK cells
The experimental approaches based on the above strategy led to the identification of NKp46, which is the prototype NCR. Analysis of the tissue distribution using the reverse-transcriptase polymerase chain reaction (RT-PCR) and specific mAbs showed that NKp46 is expressed by all NK cells (both resting and activated) but is absent from all other cell types analysed24Ð27. The relevance of this finding is related to the fact that none of the markers available so far is truly NK-specific. In addition, NKp46 appears to be a major triggering receptor of NK cells, which, under crosslinking conditions, leads to Ca21 mobilization, cytotoxicity and cytokine release24. More importantly, it appears to have a central role as a receptor in the lysis of different targets, including normal and tumour-transformed cells of autologous, allogeneic or xenogeneic origin23. Indeed, NKp46 masking by specific mAbs inhibited lysis of the majority of human tumours belonging to different histotypes, including lung, liver and breast carcinomas, melanoma, and EpsteinÐ Barr virus (EBV)-transformed cell lines23Ð27. However, correct evaluation of the actual involvement of NKp46 in the triggering of cytotoxicity (as assessed by mAb-mediated-masking experiments) might require accurate selection of the target cells analysed (Fig. 1). In particular, the use of murine-tumour target cells (including Bw1502 and YAC cells) provided direct evidence that NKp46 is sufficient to mediate NK cytotoxicity (that is, it does not necessarily require coengagement of other triggering receptors). In addition, it
It is well known that culture with interleukin 2 (IL-2) increases the ability of NK cells to mediate non-MHC-restricted tumour-cell lysis2. In addition to the upregulation of cytolytic activity against NKsusceptible target cells, NK cells acquire the ability to lyse target cells that are resistant to fresh NK cells. This phenomenon may reflect, at least in part, the de novo expression of novel NCRs that allow activated NK cells to recognize additional ligands, resulting in moreefficient NK-cell triggering. In this context, we identified a novel 44 kDa surface molecule (NKp44) that is absent in freshly isolated peripheral-blood lymphocytes but is progressively expressed by all NK cells in vitro upon culture in IL-2; indeed, all NK-cell clones analysed so far express NKp44 (Ref. 25). Unlike other markers of lymphocyte activation, NKp44 is absent from activated T cells or T-cell clones; indeed, like NKp46, NKp44 was absent from all the other cell lineages analysed. Therefore, NKp44 appears to be the first marker specific for activated human NK cells. The only exceptions so far are two T-cell receptor gd1 clones derived from a melanoma patient that expressed very low levels of NKp44 at their surface25. Masking of NKp44 mediated by mAbs partially inhibited cytolytic activity against certain (HLA class I2 FcgR2) tumour target cells25. Remarkably, the degree of inhibition was greatly increased by the simultaneous masking of NKp46. Therefore, NKp44 appears to function as an NCR that is selectively expressed by activated NK
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Fig. 1. The identification of putative natural cytoxicity receptors (NCRs), which can trigger natural killer (NK) cells. This approach was based on the assumption that tumour target cells susceptible to NK-cell-mediated cytotoxicity might differ greatly in the surface expression of ligands for the NCRs. The representative NK-cell clone Y, used as a source of cytolytic effector cells, is shown to express several different NCRs, which recognize ligands that are expressed differently by a panel of allogeneic or xenogeneic tumour cells. Three representative target cells are illustrated. (a) A target that expresses the complete set of ligands recognized by the various NCRs of clone Y. The occurrence of multiple NCRÐligand interactions is likely to result in optimal NK-cell triggering and thus in the efficient lysis of that target. In this case, a monoclonal antibody (mAb) directed to a given NCR would be expected to inhibit the lysis of target cells only marginally, because efficient NK-cell triggering might still occur via the other NCRÐligand interactions. (b) A target that does not express one or more of the ligands recognized by the NCRs of clone Y, in which case the addition of a mAb directed to a given NCR should result in a sharp, but still incomplete, inhibition of the NK-cell-mediated target-cell lysis. Under these conditions, the number of NCRÐligand interactions should be strongly reduced. (c) A target that expresses only one relevant ligand for the NCRs of clone Y. In this case, mAb-mediated masking of the relevant NCR should virtually abolish cytotoxicity because, under these conditions, no relevant NCRÐligand interactions would remain available. The right-hand panels show the actual data obtained in chromium release cytotoxicity assays in which the ability of clone Y to kill the different types of target cells was evaluated (51Cr is released by target cells if damaged by clone Y) in either the absence (white) or the presence (blue) of the BAB281 (anti-NKp46) mAb. The A549 lung carcinoma (representative of target-type A) is lysed efficiently by clone Y and this lysis is only marginally inhibited by anti-NKp46 mAb. The melanoma M14 (representative of target-type B) is also lysed efficiently, but, in this case, the addition of anti-NKp46 mAb results in a substantial inhibition of lysis. The murine thymoma BW1502 (representative of target-type C) is lysed less efficiently by the human clone Y (compare the E:T ratios). Adding anti-NKp46 mAb completely inhibits lysis, thus strongly suggesting that the ligand for NKp46 may be the only one expressed by these cells and that, in this case, NKp46 is the only NCR responsible for the induction of cytotoxicity. Abbreviations: Ab, antibody; E:T, effector:target cell ratio.
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cells and that might cooperate with other NCRs in the process of non-MHC-restricted lysis. An example of the complementary role of NKp44 and NKp46 molecules in the lysis of an HLA class I2 FcgR2 tumour target cell (highly susceptible to lysis by a representative NK-cell clone) is shown in Fig. 2.
Although the NK-mediated lysis of various allogeneic tumour cell lines was sharply inhibited by mAb-mediated masking of NKp46 or NKp44, the lysis of other tumour cell lines was only marginally affected. This suggests that there are additional NCRs that play a prominent role in the induction of NK-cellmediated cytotoxicity against these target cells. Indeed, an additional NCR, termed NKp30, has recently been identifed and characterized using specific mAbs (Ref. 26). NKp30 was found to cooperate with NKp46 and NKp44 in the induction of cytotoxicity against a variety of target cells. Moreover, NKp30 is the major receptor responsible for killing some tumour target cells, for which NKp46 or NKp44 do not appear to play a significant role26. NKp30, like NKp46, is selectively expressed by all NK cells, both freshly isolated and cultured in IL-2. Remarkably, the surface expression of NKp30 parallels that of NKp46, as NK cells displaying an NKp46dull or an NKp46bright phenotype were also characterized by ÔdullÕ or ÔbrightÕ fluorescence with NKp30 (Ref. 26). The demonstration that NK cells express parallel densities of different triggering receptors might explain the existence of NK-cell subsets displaying different ÔnaturalÕ cytolytic activity. Thus, it is now possible to identify NK cells displaying an NCRbright phenotype (i.e. high surface density of NKp46, NKp44 and NKp30) and NK cells characterized by an NCRdull phenotype.
Molecular structure of NCRs The molecular cloning of NKp4627, NKp4429 and NKp3026 revealed novel members of the Ig-SF with no homology to each other and a low degree of homology to known human molecules (Fig. 3). When NKp46 was cloned27, it was found to be a type I transmembrane glycoprotein with two C2-type
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2B4 functions as a co-receptor in NK-cell triggering Studies of mAb-mediated masking of NCR and functional analysis of numerous NK-cell clones suggested that other receptors should cooperate with NKp46, NKp44 and NKp30 to induce optimal NK-cell triggering. A good candidate for this function was the 2B4 molecule, which activates NK-cell cytotoxicity
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Ig-like domains in its extracellular portion, a transmembrane region containing a positively charged amino acid (Arg), which might be involved in stabilizing the interaction(s) with transmembrane adapter molecules, and a cytoplasmic portion (30 amino acids long) that does not contain sequence motifs typically involved in the activation of the signal cascade(s), such as an ITAM. Biochemical analysis revealed that NKp46 molecules are coupled to the intracytoplasmic signal-transduction machinery with the ITAM-containing CD3z and/or FceRg adapter polypeptides24,29. These signaltransducing molecules become tyrosine phosphorylated after NKp46 crosslinking by specific mAbs (Refs 25, 27, 29). As with other members of the Ig-SF expressed on NK cells [including KIR, KAR (Ref. 6Ð8) and ILT (Refs 30, 31)], the NKp46-encoding gene maps to human chromosome 19 (Ref. 27). Recently, the murine28 and rat32 homologues of the human NKp46 receptor have been cloned. Remarkably, the murine gene for NKp46 maps to mouse chromosome 7, which is syntenic to human chromosome 19. NKp44 is characterized by a single extracellular V-type domain29. Its transmembrane region contains the charged amino acid Lys, which is likely to mediate association with the ITAM-bearing adapter molecules KARAP/ DAP-12 (Refs 25Ð29). The NKp44 gene has been mapped to human chromosome 6 (Ref. 29). NKp30 was found to be the product of 1C7, a gene previously mapped to human chromosome 6, in the HLA class III region41. NKp30 contains a single V-type domain and has a charged residue (Arg) in its transmembrane portion26. As revealed by biochemical analysis, and similar to NKp46, NKp30 associates with the CD3z chain-adapter molecule responsible for signal transduction26.
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Fig. 2. The synergistic effect of natural cytoxicity receptors (NCRs) in the natural killer (NK)-cellmediated lysis of a representative HLA class I2 human tumour. The representative NK-cell clone Y expresses all known NCR molecules (a property common to all activated NK-cell populations and clones). The A549 lung carcinoma target cell (representative of targets ÔAÕ from Fig. 1) is highly susceptible to NK-cell-mediated lysis. The left-hand side of the figure shows the different experimental conditions used Ð addition to the cytolytic test of: (a) anti-NKp46 mAb alone; (b) anti-NKp44 mAb alone; (c) a mixture of the two mAbs. Clone Y expressed, in addition to NKp46 and NKp44, at least one additional NCR (NKp30). The right-hand side of the figure shows the actual data: the addition of either anti-NKp46 (blue) or anti-NKp44 (red) alone induces a low degree of inhibition of target-cell lysis; the combined use of the two mAbs (green) induces a strong, but not complete, inhibition. The residual cytolytic effect observed upon mAb-mediated masking of NKp46 and NKp44 is mostly due to the interaction of NKp30 with its ligand on target cells. Indeed, the combined use of mAbs against three different NCRs resulted in complete inhibition (d). Abbreviations: see Fig. 1 legend.
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Box 1. Characteristics of the natural cytoxicity receptors ¥ Natural cytoxicity receptors (NCRs) are selectively expressed by natural killer (NK) cells and thus represent the most reliable NK-cell markers. In particular, NKp46 and NKp30 are expressed by all resting and activated NK cells, whereas NKp44 is expressed by activated NK cells only. ¥ All NCRs belong to the immunoglobulin superfamily but have little homology with known human cell-surface molecules or with each other. ¥ NKp46 is conserved in other species, including the mouse and rat. ¥ Upon crosslinking, NCRs mediate NK-cell triggering, leading to target-cell lysis and cytokine production. ¥ The function of NCRs is normally downregulated by coaggregation with killer inhibitory receptors (KIRs). Thus, the induction of NK-cell activation via NCRs is possible only in the absence of a KIRÐHLA class I interaction. ¥ NCRs are associated with different signal-transducing molecules. ¥ Disruption of the NCRÐligand interactions by mAb-mediated masking of NCRs inhibits NK-cell-mediated cytotoxicity. ¥ The surface density of NCRs might differ between NK cells. The density correlates directly with their natural cytotoxicity. ¥ The nature of the NCR-specific ligands has still to be defined. However, experimental evidence suggests that their expression is different in different target cells. ¥ NKp46 recognizes ligands expressed on both normal and tumour cells, including xenogeneic cells.
in both mouse and human when either crosslinked with mAbs or engaged with its specific ligand (CD48)33Ð36. In contrast to NCRs, 2B4 is expressed not only by all NK cells but also by a subset of CD81 T cells, monocytes and basophils. Structurally, human 2B4 belongs to the CD2 subfamily of the Ig-SF and contains a membrane-distal IgV domain and a single membrane-proximal type-C2 Ig-like domain; the cytoplasmic tail of 2B4 contains tyrosine-based motifs34. In cell transfectants, these motifs allow 2B4 to interact with the Src homology 2 (SH2) domaincontaining molecule SH2D1A (Ref. 37). This interaction appears to prevent 2B4 from associating with the cytoplasmic tyrosine phosphatase SHP2. At present, the precise mechanisms by which 2B4 triggering leads to NK-cell activation remain largely unknown. As the 2B4ÐSH2D1A interaction requires tyrosine phosphorylation of 2B4 molecules, it will be important to define the in vivo role of different kinases. Recently, 2B4 has been shown to function as a co-receptor in human NK-cell activation induced by different NCRs or CD16 (Ref. 38). In this context, previous studies showed a clonal heterogeneity of NK cells in the response to anti-2B4 mAbs in redirected killing assays, which might suggest a functional heterogeneity of the 2B4 molecules themselves. However, the ability of NK clones to
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respond to anti-2B4 mAb was found to correlate strictly with their NCR phenotype38. Thus, NK-cell clones with a high surface density of NKp46 (NKp46bright) were efficiently triggered by anti-2B4 mAb. By contrast, NKp46dull clones were not, in spite of the fact that they had a similar surface density of 2B4. Modulation of NKp46 surface molecules in NKp46bright clones by mAbs had no effect on the expression of 2B4 but prevented cells from subsequently being triggered by anti-2B4 mAb. Moreover, anti-2B4 mAb could trigger NKp46dull NKcell clones in a redirected killing assay, provided that suboptimal doses of anti-NKp44 or anti-CD16 mAb were added38. These results indicate that NK-cell responses to the engagement of 2B4 depend upon the coengagement of other triggering receptors38.
Does NKG2D have a role in NK-cell-mediated cytotoxicity? A role in NK-cell triggering has also recently been suggested for NKG2D, a receptor for the stress-inducible ligand MICA (Ref. 39) (an MHC-related protein that is broadly expressed on epithelial tumours). NKG2D is a 42 kDa transmembrane molecule belonging to the C-type lectin superfamily, which maps to chromosome 12. Signalling via NKG2D appears to be mediated by a novel polypeptide, called DAP-10 and characterized by an SH2-domain-binding site in its cytoplasmic tail that recruits the p85 subunit of phosphoinositide 3-kinase (PI-3 kinase)40. At least three differences exist between NCRs and NKG2D: (1) NKG2D is expressed on most gd and virtually all CD81 ab T cells; (2) the ligand recognized by NKG2D is not expressed on normal cells and its expression on tumour cells is restricted to certain histological types; (3) triggering by NKG2D would appear to over-rule the inhibitory signals delivered by the KIR and HLA class I interactions, which makes sense as MICA is not expressed on normal cells24Ð26,40. The precise contribution of NKG2D to NK-cell function has yet to be determined. Indeed, its expression and triggering effects on NK cells have been analysed in detail only in a tumour NK-cell line. NKG2D might play a dominant role in the NK-cell-mediated lysis of rare target cells that appear to be killed by NCR-independent mechanisms.
NCR ligands The discovery of novel surface molecules expressed by NK cells that are responsible for triggering the natural cytotoxicity has shed new light on our understanding of how NK cells function. NCRs appear to act as triggering receptors in the lysis of MHC class I2 allogeneic and xenogeneic tumour cells in vitro. However, what could the role of NCRs be under more-physiological conditions (i.e. against autologous cells)? The involvement of NCRs was analysed in the lysis of EBV-transformed autologous cell lines and autologous phytohaemagglutinin (PHA) blasts23,26. Remarkably, these cells, unlike tumour cells, are protected from NK-cell-mediated lysis because they express a normal level of (self) HLA class I molecules. To overcome this experimental difficulty, in these studies, the HLA class I molecules were masked by suitable anti-HLA class I
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Immunology Today
Fig. 3. The molecular structure of natural cytoxicity receptors (NCRs) and their association with distinct signal-transducing molecules. NKp46 has an extracellular portion characterized by two immunoglobulin-like (Ig-like) domains of type C2. NKp30 and NKp44 have an extracellular region containing a single Ig-like domain of type V and NKp44 displays a membrane-proximal region with an extended open conformation typical of hinge-like sequences. NCR transmembrane portions contain positively charged amino acids that are thought to be crucial for their association with distinct signal-transducing molecules bearing immunoreceptor tyrosine-based activating motifs. Putative N- or O-linked glycosylation sites are indicated as horizontal or angled lines, respectively. mAb to prevent inhibition mediated by killer inhibitory receptors. Under these conditions, NK cells efficiently lysed autologous target cells. Moreover, mAb-mediated masking of NCRs resulted in a substantial (50Ð80%) inhibition of lysis in this experimental setting. These data imply that NCRs are involved in the recognition of ligands that are also expressed on autologous untransformed cells23,26. Although the ligands for NCRs appear to be expressed on both normal and tumour cells, it is possible that their expression could be modified by cellular stress, cell activation or tumour transformation. Moreover, as discussed above, some tumour cells appear to lack certain NCR ligands. This could be the result of the in vivo selection of antigen-loss mutants, possibly reflecting a mechanism of tumour escape from NK-mediated attack. If this is true, tumour cells could escape not only cytotoxic-Tcell-mediated control (selecting MHC class I loss variants) but also NK-cell-mediated control (upon loss of NCR-specific ligands). In order to identify the NCR ligands, different approaches are being attempted, mainly based on the use of soluble receptors. The identification of the NCR ligands and the definition of their cellular distribution, as well as of possible mechanisms leading to their expression, will help to identify tissues and cell types that might be susceptible to NK-cell lysis.
cytotoxicity. In order to prevent damage to normal cells, NCRs are usually under the control of HLA class I-specific inhibitory receptors that provide the ÔoffÕ signals. NCRs are strictly NK-cell specific and recognize ligands expressed on both normal and tumour cells. However, the existence of target cells such as certain T-cell-leukaemia lines that are lysed independently of NKp46, NKp44 or NKp30 implies a role for additional, still undefined, triggering receptors. Based on the strict correlation between NCR phenotype and the magnitude of the natural cytotoxicity, the expression of these stillunknown receptors might be confined to NK cells with the NCRbright phenotype. Alternatively, these receptors could be uniformly expressed by all human NK cells but require for their function a concomitant engagement of other NCRs; they would thus be co-receptors. Indeed, a co-receptor function has recently been demonstrated for the human 2B4 molecule: even though all NK cells express comparable surface amounts of 2B4, the ability to trigger cytotoxicity upon ligand binding by 2B4 varies with the NCRbright or NCRdull phenotype. In conclusion, the emerging family of NCRs and co-receptors is still growing, underscoring the complexity of NK-cell activation and regulation.
The authorsÕ work was supported by grants awarded by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), Istituto Superiore di Sanitˆ (I.S.S.),
Concluding remarks
Ministero della Sanitˆ and Ministero dellÕUniversitˆ e della Ricerca Scientifica
The recent identification and molecular cloning of NCRs shed new light on our understanding of NK-cell function (Box 1; Fig. 3). Indeed, they play a major role in mediating the ÔonÕ signals during natural
e Tecnologica (M.U.R.S.T.), and Consiglio Nazionale delle Ricerche, Progetto Finalizzato Biotecnologie. The financial support of Telethon-Italy (grant no. E.0892) is gratefully acknowledged.
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Alessandro Moretta and Lorenzo Moretta ([email protected]. unige.it) are at the Dipartimento di Medicina Sperimentale, University of Genova, Genova, Italy; Roberto Biassoni, Cristina Bottino and Maria Mingari are at the Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; Maria Mingari is also at the Dipartimento di Oncologia, Biologia e Genetica, University of Genova, Genova, Italy. Lorenzo Moretta is also at the Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy.
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