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.

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.

Surface receptors involved in natural killer (NK) cell triggering during the process of tumor cell lysis have recently been identified. Of these receptors, NKp44 is selectively expressed by IL-2– activated NK cells and may contribute to the increased efficiency of activated NK cells to mediate tumor cell lysis. Here we describe the molecular cloning of NKp44. Analysis of the cloned cDNA indicated that NKp44 is a novel transmembrane glycoprotein belonging to the Immunoglobulin superfamily characterized by a single extracellular V-type domain. The charged amino acid lysine in the transmembrane region may be involved in the association of NKp44 with the signal transducing molecule killer activating receptor–associated polypeptide (KARAP)/DAP12. These molecules were found to be crucial for the surface expression of NKp44. In agreement with data of NKp44 surface expression, the NKp44 transcripts were strictly confined to activated NK cells and to a minor subset of TCR-γ/δ+ T lymphocytes. Unlike genes coding for other receptors involved in NK cell triggering or inhibition, the NKp44 gene is on human chromosome 6.

NKp46 is a novel triggering receptor expressed by all human NK cells that is involved in natural cytotoxicity. In this study we show that the surface density of NKp46 may vary in different NK cells and that a precise correlation exists between the NKp46 phenotype of NK clones and their natural cytotoxicity against HLA-class I-unprotected allogeneic or xenogeneic cells. Thus, NKp46bright clones efficiently lysed human and murine tumor cells while NKp46dull clones were poorly cytolytic against both types of target cells. We also show that the NKp46 phenotype of NK clones correlates with their ability to lyse HLA-class I-unprotected autologous cells. Finally, NKp46 was found to be deeply involved in the natural cytotoxicity mediated by freshly derived NK cells. This was indicated both by the inhibition of cytolysis after monoclonal antibody-mediated masking of NKp46 and by the correlation existing between the natural cytotoxicity of fresh NK cells derived from different donors and their NKp46 phenotype. In conclusion, these studies strongly support the concept that NKp46 plays a central role in the physiological triggering of NK cells and, as a consequence (in concert with killer inhibitory receptors), in the NK-mediated clearance of abnormal cells expressing inadequate amounts of HLA-class I molecules.

Background: Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating and dose-limiting complication of cancer treatment. Thus far, the impact of CIPN has not been studied in a systematic clinimetric manner. The objective of the study was to select outcome measures for CIPN evaluation and to establish their validity and reproducibility in a cross-sectional multicenter study. Patients and methods:After literature review and a consensus meeting among experts, face/content validity were obtained for the following selected scales: the National Cancer Institute-Common Toxicity Criteria (NCI-CTC), the Total Neuropathy Score clinical version (TNSc), the modified Inflammatory Neuropathy Cause and Treatment (INCAT) group sensory sumscore (mISS), the European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30, and CIPN20 quality-of-life measures. A total of 281 patients with stable CIPN were examined. Validity (correlation) and reliability studies were carried out.Results: Good inter-/intra-observer scores were obtained for the TNSc, mISS, and NCI-CTC sensory/motor subscales. Test-retest values were also good for the EORTC QLQ-C30 and CIPN20. Acceptable validity scores were obtained through the correlation among the measures. Conclusion:Good validity and reliability scores were demonstrated for the set of selected impairment and quality-of-life outcome measures in CIPN. Future studies are planned to investigate the responsiveness aspects of these measures.

In an attempt to identify the amino acid position(s) of the HLA-C-specific p58.1/p50.1 natural killer cell receptors that determine the binding affinity for their ligand, we used soluble fusion proteins formed by the ectodomain of either receptor and the Fc portion of human IgG1. We show that the soluble p50.1 (activating) receptor binds weakly to 221-Cw4 transfectants. In contrast, the soluble p58.1 (inhibitory) receptor binds with high affinity. A single amino acid mutation at position 70, obtained by site-directed mutagenesis, was found to affect the binding affinity of both the p50.1 and the p58.1 receptors. Thus, substitution in p50.1 of lysine 70 by threonine (typical of the inhibitory p58.1 molecule) resulted in a dramatic increase in binding affinity, comparable to that of the p58.1 molecule. On the other hand, substitution of threonine 70 by lysine in p58.1 almost abolished binding to 221-Cw4 cells. Our present data indicate that a single amino acid difference greatly influences the p58.1/p50.1 affinity for their HLA-C ligand and suggests a possible role of position 70 as a contact site in the natural killer cell receptor/major histocompatibility complex class I interaction.

NK cells display several killer inhibitory receptors (KIR) specific for different alleles of MHC class I molecules. A family of KIR are represented by type I transmembrane proteins belonging to the immunoglobulin superfamily (Ig-SF). Besides cDNA encoding for these KIR, additional cDNA have been identified which encode for Ig-SF receptors with still undefined specificity. Here we analyze one of these cDNA, termed cl.15.212, which encodes a type I transmembrane protein characterized by two extracellular Ig-like domains and a 115-amino acid cytoplasmic tail containing a single immuno-receptor tyrosine-based inhibitory motif (ITIM) which is typical of KIR. cl.15.212 cDNA displays approximately 50 % sequence homology with other Ig-SF members. Different from the other KIR, cl.15.212 mRNA is expressed by all NK cells and by a fraction of KIR + T cell clones. cl.15.212 cDNA codes for a membranebound receptor displaying an apparent molecular mass of 49 kDa, thus termed p49. To determine the specificity of the cl.15.212-encoded receptor, we generated soluble fusion proteins consisting of the ectodomain of p49 and the Fc portion of human IgG1. Soluble molecules bound efficiently to 221 cells transfected with HLA-G1, -A3, -B46 alleles and weakly to -B7 allele. On the other hand, they did not bind to 221 cells either untransfected or transfected with HLA-A2, -B51, -Cw3 or -Cw4. The binding specificity of soluble p49-Fc was confirmed by competition experiments using an anti-HLA class I-specific monoclonal antibody. Finally, different cDNA encoding for molecules homologous to cl.15.212 cDNA have been isolated, two of which lack the sequence encoding the transmembrane portion, thus suggesting they may encode soluble molecules.

The NKp46 molecule has been proposed to play the role of triggering receptor in the natural cytotoxicity mediated by human NK cells. In this study we have identified the gene encoding the murine NKp46 homologue that we termed MAR-1. The MAR-1 gene is localized on chromosome 7 that is synthenic to the human chromosome 19 where the NKp46 gene is located. MAR-1 encodes a type I transmembrane glycoprotein belonging to the immunoglobulin (Ig) superfamily that, like human NKp46, is characterized by two C2-type Ig-like domains, a transmembrane portion containing a positively charged residue and a cytoplasmic tail lacking the immunoreceptor tyrosine-based activation motif (ITAM). The MAR-1 protein is expressed on the surface of cell transfectants and displays a molecular mass of approximately 46 kDa similar to that of its human counterpart. Semiquantitative RT-PCR analysis showed that MAR-1, similar to the human NKp46, is selectively expressed by NK cells. The MAR-1 protein displays 58 % identity with the human NKp46 receptor. This high homology together with the presence of a charged amino acid (Arg) in the transmembrane portion suggest that MAR-1 may associate at the cell membrane into a multimeric complex with ITAM containing polypeptides.