After culture in interleukin (IL)-2, natural killer (NK) cells acquire an increased capability of mediating non–major histocompatibility complex (MHC)–restricted tumor cell lysis. This may reflect, at least in part, the de novo expression by NK cells of triggering receptors involved in cytolysis. In this study we identified a novel 44-kD surface molecule (NKp44) that is absent in freshly isolated peripheral blood lymphocytes but is progressively expressed by all NK cells in vitro after culture in IL-2. Different from other markers of cell activation such as CD69 or VLA.2, NKp44 is absent in activated T lymphocytes or T cell clones. Since NKp44 was not detected in any of the other cell lineages analyzed, it appears as the first marker specific for activated human NK cells. Monoclonal antibody (mAb)–mediated cross-linking of NKp44 in cloned NK cells resulted in strong activation of target cell lysis in a redirected killing assay. This data indicated that NKp44 can mediate triggering of NK cell cytotoxicity. mAb-mediated masking of NKp44 resulted in partial inhibition of cytolytic activity against certain (FcγR-negative) NK-susceptible target cells. This inhibition was greatly increased by the simultaneous masking of p46, another recently identified NK-specific triggering surface molecule. These data strongly suggest that NKp44 functions as a triggering receptor selectively expressed by activated NK cells that, together with p46, may be involved in the process of non-MHC-restricted lysis. Finally, we show that p46 and NKp44 are coupled to the intracytoplasmic transduction machinery via the association with CD3ζ or KARAP/DAP12, respectively; these associated molecules are tyrosine phosphorylated upon NK cell stimulation.

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.

Limited information is available on the surface molecules that are involved in natural killer (NK) cell triggering. In this study, we selected the BAB281 monoclonal antibody (mAb) on the basis of its ability to trigger NK-mediated target cell lysis. BAB281 identified a novel NK cell–specific surface molecule of 46 kD (p46) that is expressed by all resting or activated NK cells. Importantly, unlike the NK cell antigens identified so far, the expression of p46 was strictly confined to NK cells. Upon mAb-mediated cross-linking, p46 molecules induced strong cell triggering leading to [Ca2+]i increases, lymphokine production, and cytolytic activity both in resting NK cells and NK cell clones. The p46-mediated induction of Ca2+ increases or triggering of cytolytic activity was downregulated by the simultaneous engagement of inhibitory receptors including p58, p70, and CD94/NKG2A. Both the unique cellular distribution and functional capability of p46 molecules suggest a possible role in the mechanisms of non-major histocompatibility complex–restricted cytolysis mediated by human NK cells.

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.

SummaryHuman CD3-16 + 56 § natural killer (NK) cells have been shown to display a donaUy distributed ability to recognize major histocompatibility complex (MHC) class I alleles. Opposite to T lymphocytes, in NK cells, specific recognition of MHC class I molecttles appears to induce inhibition of cytolytic activity and, thus, to protect target cells. Since a precise correlation has been established between the expression of the NK-specific GL183 and EB6 surface molecules (belonging to the novel p58 molecular family) and the specificity of NK clones, we analyzed whether p58 molecules could function as receptors for MHC in human NK cells. NK clones displaying the previously defined "specificity 2" and characterized by the GL183 + EB6 + phenotype, specifically recognize the Cw3 allele and thus fail to lyse the Fc"/R + P815 target cells transfected with Cw3. On the other hand, NK clones displaying "specificity 1" and expressing the GL183-EB6 + phenotype failed to lyse Cw4 + target cells. Addition of the F(ab')2 fragments of either GL183 or EB6 mAb as well as the XA141 mAb of IgM isotype (specific for the EB6 molecules) completely restored the lysis of Cw3-transfected P815 cells by the Cw3-specific NK clones EX2 and EX4. Similarly, both the entire EB6 mAb, its F(ab')z fragment and the XA141 mAb reconstituted the lysis of C1R, a Fc'yR-target cell expressing Cw4 as the only serologically detected class I antigen. Thus, it appears that masking of different members of p58 molecules prevents recognition of "protective" MHC class I alleles and thus the delivering of inhibitory signals. Further support to the concept that p58 molecules represent a NK receptor delivering a negative signal was provided by experiments in which the entire anti-p58 mAbs (of IgG isotype) could inhibit the lysis of unprotected Fc'yR + P815 target cells, thus mimicking the inhibitory effect of MHC class I molecules. ecent data have substantially modified the general concept that human CD3-CD16 + CD56 + NK cells (1, 2) represent a homogeneous cytolytic lymphoid population displaying nonspecific, non-MHC-restricted functions. Thus, it has been shown that NK cells are capable of specific functions since they can discriminate among different normal allogeneic target cells (3,4). This ability is clonally distributed as different NK clones derived from single individuals display unique cytolytic patterns against allogeneic target cells (5-7). Thus, five distinct groups of alloreactive clones (each displaying a different specificity) have been identified (7). Genetic analysis (6, 7) and the use of cell transfectants (8) indicated that the target molecules recognized by NK clones are represented, at least in some instances, by HLA class I alleles (8-11). Whereas specific recognition of MHC molecules by alloreactive cytolytic T lymphocytes leads to triggering of their cytolytic function, in the case of NK lymphocytes, a given HLA allele (Cw3) was found to confer specific protection from lysis by a group of NK clones displaying a defined specificity (specific...

In humans, natural killer (NK) cell function is regulated by a series of receptors and coreceptors with either triggering or inhibitory activity. Here we describe a novel 60-kD glycoprotein, termed NTB-A, that is expressed by all human NK, T, and B lymphocytes. Monoclonal antibody (mAb)-mediated cross-linking of NTB-A results in the induction of NK-mediated cytotoxicity. Similar to 2B4 (CD244) functioning as a coreceptor in the NK cell activation, NTB-A also triggers cytolytic activity only in NK cells expressing high surface densities of natural cytotoxicity receptors. This suggests that also NTB-A may function as a coreceptor in the process of NK cell activation. Molecular cloning of the cDNA coding for NTB-A molecule revealed a novel member of the immunoglobulin superfamily belonging to the CD2 subfamily. NTB-A is characterized, in its extracellular portion, by a distal V-type and a proximal C2-type domain and by a cytoplasmic portion containing three tyrosine-based motifs. NTB-A undergoes tyrosine phosphorylation and associates with the Src homology 2 domain–containing protein (SH2D1A) as well as with SH2 domain–containing phosphatases (SHPs). Importantly, analysis of NK cells derived from patients with X-linked lymphoproliferative disease (XLP) showed that the lack of SH2D1A protein profoundly affects the function not only of 2B4 but also of NTB-A. Thus, in XLP-NK cells, NTB-A mediates inhibitory rather than activating signals. These inhibitory signals are induced by the interaction of NTB-A with still undefined ligands expressed on Epstein-Barr virus (EBV)-infected target cells. Moreover, mAb-mediated masking of NTB-A can partially revert this inhibitory effect while a maximal recovery of target cell lysis can be obtained when both 2B4 and NTB-A are simultaneously masked. Thus, the altered function of NTB-A appears to play an important role in the inability of XLP-NK cells to kill EBV-infected target cells.

SummaryThe natural killer (NK) cell-specific p58 molecules EB6 and GL183 have been shown to represent the putative surface receptors for two distinct groups of human histocompatibility leukocyte antigen (HLA) C alleles. Interaction between p58 receptors and class I molecules expressed on target cells results in inhibition of the NK-mediated cytolytic activity and thus in target cell protection. In the present study, we show that EB6 molecules may also act as receptors mediating NK cell triggering. Activatory EB6 molecules were fouiad to be confined only to certain donors. Moreover, in these donors, only a fraction of EB6 + NK clones expressed the activatory form of EB6 molecules, while the remaining clones expressed the conventional inhibitory form. Biochemical analysis of the activatory EB6 molecules revealed a molecular mass of "~50 kD (pS0), thus differing from the 58-kD inhibitory form. This difference was not due to differential glycosylation of the same protein, as revealed by deglycosylation experiments on isolated EB6 molecules. Treatment of purified p58 or pS0/EB6 molecules with proteolytic enzymes, including V8-protease, chymotrypsin, and papain, showed only minor differences in the resulting peptides. Treatment with pepsin followed by two-dimensional peptide mapping demonstrated that, although the majority of peptides migrated in identical positions, differences between the two forms could be detected for at least one major peptide. Anti-EB6 monoclonal antibody (mAb)-mediated crosslinking of p50 molecules was required to trigger the cytolytic activity and the intracellular calcium ([Ca++]i) increases in appropriate NK clones. Likewise, mAb-mediated cross-linking of the p58 EB6 molecules was needed to inhibit the cytolytic activity; however, in this case, no [Ca + + ]i increases could be detected. In NK clones expressing the inhibitory p58 EB6 receptors, soluble anti-EB6 mAb prevented recognition of protective Cw4 molecules and reconstituted target cell lysis. In contrast, in clones expressing the activatory p50/EB6 receptor, EB6 masking frequently resulted in partial inhibition of the cytolytic activity against Cw4 + target cells. Therefore, it appears that NK clones expressing the p50/EB6 receptors are induced to lyse Cw4 + target cells upon specific interaction with Cw4 molecules. This concept was further substantiated by experiments in which target cells were represented by the HLA-negative LCL721.221 cell line transfected with the Cw4 allele. Phenotypic and functional analysis of a large number of NK clones showed that clones expressing the activatory p50/EB6 molecules consistently coexpressed inhibitory receptors for other HLA class I alleles. Taken together, our data indicate that recognition of dass I molecules may result either in inhibition or in activation of NK-mediated cytolysis. However, the inhibitory pathway appears to dominate the activatory one, thus preventing lysis of class I-protected autologous normal cells. ecent data in mouse and in humans have provided direct evidence that NK c...

In this study, in an attempt to identify neuroblastoma-associated surface antigens, we generated mAbs against the ACN neuroblastoma cell line. A mAb was selected (5B14) that reacted with all neuroblastoma cell lines analyzed and allowed detection of tumor cell infiltrates in bone marrow aspirates from neuroblastoma patients. In cytofluorimetric analysis, unlike anti-disialoganglioside mAb, 5B14 mAb did not display reactivity with normal bone marrow hematopoietic cell precursors, thus representing a highly specific marker for identifying neuroblastoma cells. Molecular analysis revealed that the 5B14 mAb-reactive surface glycoprotein corresponded to the recently identified 4Ig-B7-H3 molecule. Remarkably, mAb-mediated masking of the 4Ig-B7-H3 molecule on cell transfectants or on freshly isolated neuroblastoma cells resulted in enhancement of natural killer-mediated lysis of these target cells. These data suggest that 4Ig-B7-H3 molecules expressed at the tumor cell surface can exert a protective role from natural killer-mediated lysis by interacting with a still undefined inhibitory receptor expressed on natural killer cells.