Natural killer cells can discriminate between normal cells and cells that do not express adequate amounts of major histocompatibility complex (MHC) class I molecules. The discovery, both in mouse and in human, of MHC-specific inhibitory receptors clarified the molecular basis of this important NK cell function. However, the triggering receptors responsible for positive NK cell stimulation remained elusive until recently. Some of these receptors have now been identified in humans, thus shedding some light on the molecular mechanisms involved in NK cell activation during the process of natural cytotoxicity. Three novel, NK-specific, triggering surface molecules (NKp46, NKp30, and NKp44) have been identified. They represent the first members of a novel emerging group of receptors collectively termed natural cytotoxicity receptors (NCR). Monoclonal antibodies (mAbs) to NCR block to differing extents the NK-mediated lysis of various tumors. Moreover, lysis of certain tumors can be virtually abrogated by the simultaneous masking of the three NCRs. There is a coordinated surface expression of the three NCRs, their surface density varying in different individuals and also in the NK cells isolated from a given individual. A direct correlation exists between the surface density of NCR and the ability of NK cells to kill various tumors. NKp46 is the only NCR involved in human NK-mediated killing of murine target cells. Accordingly, a homologue of NKp46 has been detected in mouse. Molecular cloning of NCR revealed novel members of the Ig superfamily displaying a low degree of similarity to each other and to known human molecules. NCRs are coupled to different signal transducing adaptor proteins, including CD3ζ, Fc∍RIγ, and KARAP/DAP12. Another triggering NK receptor is NKG2D. It appears to play either a complementary or a synergistic role with NCRs. Thus, the triggering of NK cells in the process of tumor cell lysis may often depend on the concerted action of NCR and NKG2D. In some instances, however, it may uniquely depend upon the activity of NCR or NKG2D only. Strict NKG2D-dependency can be appreciated using clones that, in spite of their NCR^dullphenotype, efficiently lyse certain epithelial tumors or leukemic cell lines. Other triggering surface molecules including 2B4 and the novel NKp80 appear to function as coreceptors rather than as true receptors. Indeed, they can induce natural cytotoxicity only when co-engaged with a triggering receptor. While an altered expression or function of NCR or NKG2D is being explored as a possible cause of immunological disorders, 2B4 dysfunction has already been associated with a severe form of immunodeficiency. Indeed, in patients with the X-linked lymphoproliferative disease, the inability to control Epstein-Barr virus infections may be consequent to a major dysfunction of 2B4 that exerts inhibitory instead of activating functions.

▪ Abstract Natural killer cells are likely to play an important role in the host defenses because they kill virally infected or tumor cells but spare normal self-cells. The molecular mechanism that explains why NK cells do not kill indiscriminately has recently been elucidated. It is due to several specialized receptors that recognize major histocompatibility complex (MHC) class I molecules expressed on normal cells. The lack of expression of one or more class I alleles leads to NK-mediated target cell lysis. During NK cell development, the class I–specific receptors have adapted to self–class I molecules on which they recognize epitopes shared by groups of class I alleles. As such, they may fail to recognize either self-molecules that bound unusual peptides or allogeneic class I molecules unrelated to self-alleles. Different types of receptors specific for groups of HLA-C or HLA-B alleles have been identified. While in most instances, they function as inhibiting receptors, an activating form of the HLA-C-specific receptors has been identified in some donors. Molecular cloning of HLA-C- and HLA-B-specific receptors has revealed new members of the immunoglobulin superfamily with two or three Ig-like domains, respectively, in their extracellular portion. While the inhibiting form is characterized by a long cytoplasmic tail associated with a nonpolar transmembrane portion, the activating one has a short tail associated with a Lys-containing transmembrane portion. Thus, these human NK receptors are different from the murine Ly49 that is a type II transmembrane protein characterized by a C type lectin domain. A subset of cytolytic T lymphocytes expresses NK-type class I–specific receptors. These receptors exert an inhibiting activity on T cell receptor–mediated functions and offer a valuable model to analyze the regulatory mechanisms involved in receptor-mediated cell activation and inactivation.

The surface density of the triggering receptors responsible for the natural killer (NK)-mediated cytotoxicity is crucial for the ability of NK cells to kill susceptible target cells. In this study, we show that transforming growth factor β1 (TGFβ1) down-regulates the surface expression of NKp30 and in part of NKG2D but not that of other triggering receptors such as NKp46. The TGFβ1-mediated inhibition of NKp30 surface expression reflects gene regulation at the transcriptional level. NKp30 has been shown to represent the major receptor involved in the NK-mediated killing of dendritic cells. Accordingly, the TGFβ1-dependent down-regulation of NKp30 expression profoundly inhibited the NK-mediated killing of dendritic cells. On the contrary, killing of different NK-susceptible tumor cell lines was variably affected, reflecting the differential usage of NKp30 and/or NKG2D in the lysis of such tumors. Our present data suggest a possible mechanism by which TGFβ1-producing dendritic cells may acquire resistance to the NK-mediated attack.

Two major receptors involved in human natural cytotoxicity, NKp46 and NKp44, have recently been identified. However, experimental evidence suggested the existence of additional such receptor(s). In this study, by the generation of monoclonal antibodies (mAbs), we identified NKp30, a novel 30-kD triggering receptor selectively expressed by all resting and activated human natural killer (NK) cells. Although mAb-mediated cross-linking of NKp30 induces strong NK cell activation, mAb-mediated masking inhibits the NK cytotoxicity against normal or tumor target cells. NKp30 cooperates with NKp46 and/or NKp44 in the induction of NK-mediated cytotoxicity against the majority of target cells, whereas it represents the major triggering receptor in the killing of certain tumors. This novel receptor is associated with CD3ζ chains that become tyrosine phosphorylated upon sodium pervanadate treatment of NK cells. Molecular cloning of NKp30 cDNA revealed a member of the immunoglobulin superfamily, characterized by a single V-type domain and a charged residue in the transmembrane portion. Moreover, we show that NKp30 is encoded by the previously identified 1C7 gene, for which the function and the cellular distribution of the putative product were not identified in previous studies.

Summary: NKp46 has been shown to represent a novel, natural killer (NK) cell–specific surface molecule, involved in human NK cell activation. In this study, we further analyzed the role of NKp46 in natural cytotoxicity against different tumor target cells. We provide direct evidence that NKp46 represents a major activating receptor involved in the recognition and lysis of both human and murine tumor cells. Although NKp46 may cooperate with other activating receptors (including the recently identified NKp44 molecule) in the induction of NK-mediated lysis of human tumor cells, it may represent the only human NK receptor involved in recognition of murine target cells. Molecular cloning of the cDNA encoding the NKp46 molecule revealed a novel member of the immunoglobulin (Ig) superfamily, characterized by two C2-type Ig-like domains in the extracellular portion. The transmembrane region contains the positively charged amino acid Arg, which is possibly involved in stabilizing the association with CD3ζ chain. The cytoplasmic portion, spanning 30 amino acids, does not contain immunoreceptor tyrosine-based activating motifs. Analysis of a panel of human/hamster somatic cell hybrids revealed segregation of the NKp46 gene on human chromosome 19. Assessment of the NKp46 mRNA expression in different tissues and cell types unambiguously confirmed the strict NK cell specificity of the NKp46 molecule. Remarkably, in line with the ability of NKp46 to recognize ligand(s) on murine target cells, the cDNA encoding NKp46 was found to be homologous to a cDNA expressed in murine spleen. In conclusion, this study reports the first characterization of the molecular structure of a NK-specific receptor involved in the mechanism of NK cell activation during natural cytotoxicity.